ognizant Communication Corporation

CELL TRANSPLANTATION

ABSTRACTS
VOLUME 8

Cell Transplantation, Vol. 8 pp. 11-23, 1999
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[3H]CNQX and NMDA-Sensitive [3H]Glutamate Binding Sites and AMPA Receptor Subunit RNA Transcripts in the Striatum of Normal and Weaver Mutant Mice and Effects of Ventral Mesencephalic Grafts

Adamantia Mitsacos,1 Masahiko Tomiyama,3 Kalliope Stasi, Panayiotis Giompres,2 Elias D. Kouvelas, Roser Cortés, José M. Palacios,3*Guadalupe Mengod, and Lazaros C. Triarhou4

1Department of Physiology, Faculty of Medicine, and 2Department of Human and Animal Physiology, School of Biological Sciences, University of Patras, 26500 Patras, Greece
3Department of Neurochemistry, Instituto de Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigaciones Científicas, 08034 Barcelona, Spain
4Department of Pathology and Laboratory Medicine and Program in Medical Neurobiology, Indiana University School of Medicine, Indianapolis, IN 46202

Levels of excitatory amino acid receptors were studied in the weaver mouse model of DA deficiency after unilateral intrastriatal transplantation of E12 +/+ mesencephalic cell suspensions. Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM [3H]GBR 12935 (average increase in grafted dorsal striatum compared to nongrafted side, 60%). Autoradiography of 80 nM [3H]CNQX and 100 nM NMDA-sensitive [3H]glutamate binding was carried out to visualize the topography of non-NMDA and NMDA receptors, respectively, in +/+ mice and in recipient weaver mutants 3 months after grafting. Increases of 30% or more were found for [3H]CNQX binding in the dorsal nongrafted weaver striatum compared to +/+, and a further 69% increase in grafted weaver compared to nongrafted side. The added increase of non-NMDA receptors in the transplanted striatum might be explained by a presence of such receptors on DA presynaptic endings of graft origin. A 20% increase in NMDA-sensitive [3H]glutamate binding was measured in the dorsal nongrafted weaver striatum compared to +/+. NMDA-sensitive [3H]glutamate binding in the transplanted side of weaver mutants tended to be slightly higher in all areas of the striatal complex compared to the nongrafted side, without reaching conventional levels of statistical significance. Using in situ hybridization histochemistry with synthetic 32P-labeled oligonucleotide probes, we investigated RNA transcripts encoding the four AMPA receptor subunits. RNA transcripts in the striatum are seen with a decreasing signal intensity in the following order: GluRB > GluRA > GluRC > GluRD. The weaver caudate-putamen shows a 12% increase in GluRA subunit mRNA compared to +/+, whereas mesencephalic neuron transplantation leads to slight increases (3%) in the levels of GluRB mRNA in the nucleus accumbens. The results are placed in the context of the important interaction between the converging glutamatergic corticostriatal and the DAergic nigrostriatal pathways in controlling the functional output of the basal ganglia in Parkinson's disease and in experimental models of DA deficiency.

Key words: Mesostriatal dopamine projection system; Weaver mutant mouse; Neural transplant; NMDA receptors; Non-NMDA receptors; Autoradiography; In situ hybridization histochemistry

Address correspondence to Dr. Lazaros C. Triarhou, Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 635 Barnhill Drive MS-A128, Indianapolis, IN 46202-5120. Tel: (317) 274-7254; Fax: (317) 278-2018.

*Permanent address: Research Center, Almirall Prodesfarma, 08024 Barcelona, Spain.



Cell Transplantation, Vol. 8 pp. 25-36, 1999
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GDNF Increases the Density of Cells Containing Calbindin But Not of Cells Containing Calretinin in Cultured Rat and Human Fetal Nigral Tissue

Morten Meyer,1,2 Jens Zimmer,2 Rolf W. Seiler,1 and Hans R. Widmer1

1Department of Neurosurgery, University of Bern, CH-3010 Bern, Switzerland
2 Department of Anatomy and Cell Biology, University of Odense, DK-5000 Odense C, Denmark

Among the dopaminergic neurons in substantia nigra pars compacta and in the ventral tegmental area, subpopulations express the calcium-binding proteins calbindin (CB) and calretinin (CR), and the CB-containing neurons are supposed to be less prone to degeneration in Parkinson's disease. Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for nigrostriatal dopaminergic neurons. Using free-floating roller-tube (FFRT) cultures derived from fetal rat (E14) ventral mesencephalon we found that GDNF (10 ng/ml) significantly increased the number of surviving tyrosine hydroxylase (TH)-immunoreactive neurons. The possible effects of GDNF treatment on CB-immunoreactive (CB-ir) and CR-ir neurons in such cultures were examined in the present study. The neuronal cell densities were measured by quantifying the numbers of CB-ir and CR-ir neurons in areas of sections through the most extensive parts of the spherical cultures. In 4-day-old and 8-day-old cultures GDNF treatment increased the density of CB-ir neurons by 50% and 59%, respectively. Partial co-existence of TH and CB was shown using the method of double immunolabeling. The density of CR-containing neurons was unaffected by GDNF treatment as confirmed by Western blotting for CR. Parallel effects of GDNF treatment were obtained for cultures of human fetal ventral mesencephalon (8 weeks postconception). In conclusion, our findings identify GDNF as a potent factor for fetal rat and human nigral CB-ir neurons able to promote their survival in culture. Referring to a suggested neuroprotective role of CB, the results may be of relevance in the context of neuronal transplantation of patients suffering from severe Parkinson's disease.

Keywords: Calcium-binding proteins; Glial cell line-derived neurotrophic factor; Mesencephalic cell cultures; Parkinson's disease

Address correspondence to Hans R. Widmer, Ph.D., Department of Neurosurgery, University of Bern, Freiburgstrasse, CH-3010 Bern, Switzerland. Tel: ++41(0) 31 632 2770; Fax: ++41(0) 31 382 2414; E-mail: hanswi@insel.ch



Cell Transplantation, Vol. 8 pp. 37-45, 1999
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Intranigral Transplantation of Fetal Substantia Nigra Allograft in the Hemiparkinsonian Rhesus Monkey

Philip A. Starr,1 Thomas Wichmann,2 Craig van Horne,3 and Roy A. E Bakay4

1Department of Neurological Surgery, University of California, San Francisco, 505 Parnassus Ave., San Francisco, CA 94143
2Department of Neurology, Emory University School of Medicine, 1639 Pierce Drive, Atlanta, GA 30322
3Department of Neurosurgery, Brigham and Women's Hospital, 75 Francis Street, Boston MA 02115
4Emory Clinic Neurosurgery, 1365B Clifton Road, Atlanta, GA 30322

Current clinical protocols for fetal cell transplantation for Parkinson's disease (PD) have focused on restoring dopamine in the striatum. However, there are now a number of human transplant recipients who have had robust innervation of the striatum by dopaminergic grafts (documented by positron emission tomography or by autopsy), but only a partial improvement in parkinsonian motor signs. Thus, there is a need for improved transplant strategies. In animal models of PD, there is recent evidence that restoring dopamine in the substantia nigra, instead of or in addition to thestriatum, may be important to correct abnormal motor behavior. This pilot study examined the morphological features and behavioral effects of fetal dopaminergic neuronal allografts placed into the substantia nigra of three 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated hemiparkinsonian rhesus monkeys. We show that grafts can survive in host substantia nigra. Characteristics of the graft host interface were variable. Inone animal, reinnervation of host substantia nigra was observed, and this animal showed behavioral improvement in a reach-and-retrieval task.

Key words: Fetal cell transplantation; Substantia nigra; Parkinson's disease; Rhesus monkey

Address correspondence to Philip Starr, M.D., Ph.D., Department of Neurological Surgery, University of California, San Francisco, 779 Moffitt, 505 Parnassus Ave, San Francisco, CA 94143. Tel: (415) 502-3744; Fax: (415) 753 1772; E-mail: starrp@itsa.ucsf.edu



Cell Transplantation, Vol. 8 pp. 47-58, 1999
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Biocompatibility of Poly (DL-Lactide-co-Glycolide) Microspheres Implanted Into the Brain

Dwaine F. Emerich, Mark A. Tracy, Kevin L. Ward, Maria Figueiredo, Rulin Qian, Carroll Henschel, and Raymond T. Bartus

Alkermes, Inc., 64 Sidney Street, Cambridge, MA 02139

The delivery of therapeutic molecules to the brain has been limited inpart due to the presence of the blood brain barrier. One potential solution is the implantation of biodegradable polymers with sustained release of drugs. Poly (DL-lactide-co-glycolide) (PLG) is a bioerodible polymer with a long and successful history of use as a suture material. More recently, PLG has been investigated for localized and sustained delivery of molecules into both peripheral sites and the brain. Despite its well-defined safety profile for parenteral applications, little informationexists concerning the safety of PLG when implanted into the brain. To further characterize the biocompatibility of PLG in the brain, we examined the gliotic response following implants of PLG into the brains of rats. As a control, each animal received an injection of the suspension medium into the contralateral hemisphere. Following implantation, PLG was well tolerated. GFAP-positive astrocytes were observed throughout the cerebral cortex and striatum on both the implanted and control sides, with the reaction being greatest within the heavily myelinated fiber tracts of the corpus callosum. Quantitative analyses revealed that this reaction occurred within 1 h postsurgery, reached its peak at 1 week following surgery, andthen decreased markedly by 1 month postsurgery. A minimal gliotic reaction was still present 1 year postsurgery but was localized to the needle tract. No differences in GFAP reactivity were seen between the polymer-implanted and control sides at any time point. Histological analysis determined that the majority of the PLG disappeared between 1 and 4 weeks. A setof parallel studies in which PLG samples were retrieved from the brain at various time points corroborated these findings and determined that themajority of PLG degraded within 2 weeks following implantation. Together, these results demonstrate that PLG is well tolerated following implantation into the CNS and that the astrocytic response to PLG is largely a consequence of the mechanical trauma that occurs during surgery. The biocompatibility of PLG implanted into the CNS provides further support for itsuse in a wide range of new therapeutic applications for sustained and localized drug delivery to the brain.

Key words: Polymer; GFAP; Astrocyte; Sustained release; Microsphere

Address correspondence to Dwaine F. Emerich, Ph.D., Department of Pharmacology, Alkermes, Inc., 64 Sidney Street, Cambridge, MA 02139. Tel: (617) 494-0171; Fax: (617) 494-9263.



Cell Transplantation, Vol. 8 pp. 59-73, 1999
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Neuron-Enriched Second Trimester Human Cultures: Growth Factor Response and In Vivo Graft Survival

Michael G. White,1 Robert R. Hammond,2 Virginia J. Sanders,3 Eugene A. Bonaroti,1 Anil P. Mehta1, Guoji Wang,1 Clayton A. Wiley,1 and Cristian L. Achim1

1University of Pittsburgh School of Medicine, Department of Pathology,Division of Neuropathology, Pittsburgh, PA 15213
2University of Western Ontario, Department of Pathology, London, Canada
3University of California at San Diego, Department of Neurosciences, La Jolla, CA 92093

Grafts of first trimester fetal tissue show limited survival and integration in the adult CNS. Alternative grafting strategies have been soughtfor treatment of neurodegenerative disease. We have developed cultures of human second trimester fetal tissues to study neuronal differentiation.Grafted into the SCID mouse striatum, aggregates of these cultures formed neuron-rich xenografts for at least 8 months. We examined the influenceof various neurotrophic factors, including basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), transforming growth factor-beta 1 (TGF-b1), and hepatocyte growth factor (HGF), on the growth and differentiationof neuronal and glial cell populations. BDNF promoted the survival and differentiation of second trimester neurons whereas bFGF exhibited a strong proliferative effect on precursors and the astroglial population. Our data suggest that second trimester human fetal cultures contain neuroprogenitor cells that can be directed to the neuronal lineage. This process may be amplified by treatment with BDNF, which we hypothesize could improvethe long-term in vivo survival of neuron-enriched grafts.

Keywords: Human; Neuron; Neurotrophic; Precursor; Transplantation

Address correspondence to Dr. Cristian L. Achim, UPMC-A515, Division of Neuropathology, 200 Lothrop St., Pittsburgh, PA 15213. Tel: (412) 383-7816; E-mail: achim@np.awing.upmc.edu



Cell Transplantation, Vol. 8 pp. 75-85, 1999
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Effects of Osteogenic Protein-1 (OP-1) Treatment on Fetal Spinal Cord Transplants to the Anterior Chamber of the Eye

A. Ch. Granholm,1,3,4 L. A. Sanders,1 B. Ickes,1 D. Albeck,1,3,4 B. J. Hoffer,3,4 D. A. Young,2 and P. L. Kaplan5

Departments of  1Basic Science, 2Biometrics, and  3Pharmacology, and the 4Neuroscience Training Program, University of Colorado Health Sciences Center, Denver, CO 80262
5Creative Biomolecules Inc., Hopkinton, MA

Spinal cord injury represents a serious medical problem, and leads to chronic conditions that cannot be reversed at present. It has been suggested that trophic factor treatment may reduce the extent of damage and restore damaged neurons following the injury. We have tested the effects of osteogenic protein-1 (OP-1, also known as BMP-7), a member of the transforming growth factor-b superfamily of growth factors, on developing spinal cord motor neurons in an intraocular transplantation model. Embryonic day 13 or 18 spinal cord tissue was dissected, incubated with OP-1 or vehicle, and injected intothe anterior chamber of the eye of adult rats. Injections of additional doses of OP-1 were performed weekly, and the overall growth of the grafted tissue was assessed noninvasively. Four to 6 weeks postgrafting, animals were sacrificed and the tissue was processed for immunohistochemistry using antibodies directed against choline acetyltransferase, neurofilament, and the dendritic marker MAP-II. We found that OP-1 treatment stimulated overall growth of spinal cord tissue when dissected from embryonic day 18, but not from embryonic day 13. OP-1 treatment increased cell size andextent of cholinergic markers in motor neurons from both embryonic stages. The neurons also appeared to have a more extensive dendritic network in OP-1-treated grafts compared to controls. These findings indicate that OP-1 treatment may reduce the extent of axotomy-induced cell death of motor neurons, at least in the developing spinal cord.

Key words: Spinal cord; Development; Transplantation; Trophic factors;Bone morphogenic factors; Acetylcholine; Motor neurons; Transforming growth factor-b

Address correspondence to Ann-Charlotte Granholm, Professor, Department of Basic Science Box C286, University of Colorado HSC, Denver, CO 80262 Tel: (303) 315-4698; Fax: (303) 315-3013; E-mail: granholm@uchsc.edu



Cell Transplantation, Vol. 8 pp. 87-101, 1999
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Transplants of Neuronal Cells Bioengineered to Synthesize GABA Alleviate Chronic Neuropathic Pain

M. J. Eaton,1,2,3 J. A. Plunkett,1 M. A. Martinez,1 T. Lopez,1 S. Karmally,1 P. Cejas,1 and S. R. Whittemore1,2,3

1The Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, FL 33136
2Department of Neurological Surgery, University of Miami School of Medicine, Miami, FL 33136
3Department of Physiology and Biophysics, University of Miami Schoolof Medicine, Miami, FL 33136

The use of cell lines utilized as biologic "minipumps" to provide antinociceptive molecules, such as GABA, in animal models of pain is a newly developing area in transplantation biology. The neuronal cell line, RN33B, derived from E13 brain stem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat GAD67 cDNA (glutamate decarboxylase, the synthetic enzyme forGABA), and the GABAergic cell line, 33G10.17, was isolated. The 33G10.17cells transfected with the GAD67 gene expressed GAD67 protein and synthesized low levels of GABA at permissive temperature (33°C), when the cells were proliferating, and increased GAD67 and GABA during differentiation at nonpermissive temperature (39°C) in vitro, because GAD67 protein expression was upregulated with differentiation. A control cell line, 33V1, transfected with the vectoralone, contained no GAD67 or GABA at either temperature. These cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and tactile hyperalgesia, were evaluated after CCI in the affected hind paw. When 33G10.17 and 33V1 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after CCI, they survived greaterthan 7 weeks on the pia mater around the spinal cord. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced during the 27-week period after grafts of 33G10.17 cells. The maximal effect on chronic pain behaviors with the GABAergic grafts occurred 23 weeks after transplantation. Transplants of 33V1 control cells had no effect on the allodynia and hyperalgesia induced by CCI. These data suggest that a chronically applied, low local dose of GABA presumably supplied by transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver inhibitory neurotransmitters, such as GABA, in a model of chronic pain offers anovel approach to pain management.

Key words: GABA; Neural cell lines; Transplantation; Chronic constriction injury

Address correspondence to M. J. Eaton, Ph.D., The Miami Project to Cure Paralysis, University of Miami School of Medicine, 1600 N.W. 10th Avenue (R-48), Miami, FL 33136. Tel: (305) 243-6226; Fax: (305) 243-4427; Email: meaton@miamiproj.med.miami.edu



Cell Transplantation, Vol. 8 pp. 103-109, 1999
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Purification of Adrenal Chromaffin Cells Increases Antinociceptive Efficacy of Xenotransplants Without Immunosuppression

P. Michalewicz,1 C. E. Laurito,2 G. D. Pappas,1 Y. Lu,1 and D. C. Yeomans1,2

1Department of Anatomy and Cell Biology and 2Department of Anesthesiology, University of Illinois at Chicago, Chicago, IL 60612

We have found that immunosuppression is necessary for the survival of xenogeneic adrenal medullary transplants. Because chromaffin cells are essentially nonimmunogenic, it is likely that the highly immunogenic "passenger" cells in the transplant preparation bring about rejection. This article describes a procedure that produces an essentially pure preparation of chromaffin cells for transplantation. Bovine adrenal medullary cells were isolated and differentially plated, resulting in a semipurified preparation of chromaffin cells. Ferromagnetic beads were added to the cell suspension, some of which were phagocytized by endothelial cells, which allowed their removal by exposure to a magnet. The remaining cells were thenexposed to ferromagnetic beads coated with isolectin B4 from Griffonia simplicifolia and once again to a magnetic field. The "semipurified" preparation contained approximately 90% chromaffin cells, whereas the "highly purified" preparation was > 99.5% chromaffin cells as determined immunohistochemically. The immunogenicity of the two cell preparations was assessed in vitro by determining their capacity to evoke lymphocyte proliferation. Rat spleen lymphocytes were mixed with either a highly purified or semipurified population of bovine chromaffin cells. The results of this assay demonstrated that the highly purified preparationwas a much weaker stimulant of lymphocyte proliferation than was the semipurified preparation and may demonstrate better graft survival in vivo. Transplantation via intrathecal catheter of either 80,000 or 250,000 cells from the highly or partially purified preparations onto the lumbar spinal cord of nonimmunosuppressed and nonnicotine-stimulated rats produced acell number-dependent antinociception for both Ad and C fiber-mediated thermonociception at 6 days after transplantation. After 6 days and up to 28 days, only the "highly purified" preparation showed antinociception. These results suggest that nearly complete purification of bovine chromaffin cells minimizes immunorejection of xenogeneic transplants of these cells.

Keywords: Lymphocyte; Proliferation; Ferromagnetic; C-fiber

Address correspondence to David C. Yeomans, Ph.D., Department of Anatomy and Cell Biology (M/C 512), University of Illinois at Chicago, 808 S. Wood St., Chicago, IL 60612. Tel: (312) 996-7223; Fax: (312) 413-0354; E-mail: yeomans@uic.edu



Cell Transplantation, Vol. 8 pp. 111-129, 1999
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Pig Fetal Septal Neurons Implanted Into the Hippocampus of Aged or Cholinergic Deafferented Rats Grow Axons and Form Cross-Species Synapses in Appropriate Target Regions

Terrence Deacon, Brandi Whatley, Celeste LeBlanc, Ling Lin, and Ole Isacson

Neuroregeneration Laboratory, McLean Hospital, Harvard Medical School,Belmont, MA 02178

The anatomical specificity of axon growth from fetal pig septal xenografts was studied by transplanting septal cells from E3035 pig fetuses into cholinergic deafferented (192-IgG-saporin-infused) rats or into aged rats (>18 months). Cell suspensions (100,000 cells/ml) were injected bilaterally into the dorsal and ventral hippocampus of immunosuppresed rats (10 mg/kg/day cyclosporine A). To assess axonal growth and synapse formation, acetylcholinesterase histochemistry, an antibodyto choline acetyltransferase (ChAT), and three pig-positive/rat-negativeantibodies: bovine 70kD neurofilament (NF70), human low-affinity NGF receptor (hNGFr), and human synaptobrevin (hSB) were used. In rats with surviving grafts at 6 months, NF70 axonal labeling was more extensive than either ChAT or hNGFr labeling. All three markers demonstrated graft axons extending selectively through the hippocampal CA fields and the molecular layer of the dentate gyrus. Graft axons did not extend into adjacent entorhinal cortex or neocortex. The distribution of pig hSB-positive synapsescorrelated with AChE-positive fiber outgrowth in to the host. Electron microscopic analysis of hSB-immunostained hippocampal sections revealed pig presynaptic terminals in contact with normal rat postsynaptic structures in the CA fields and the dentate gyrus. These data demonstrate target-appropriate growth of pig cholinergic axons and the formation of cross-species synapses in the deafferented or aged rat hippocampus.

Key words: Fetal pig xenografts; Rat hippocampus; Cross-species synapses

Address correspondence to Dr. Ole Isacson, Neuroregeneration Laboratories, McLean Hospital, MRC 119, Belmont, MA 02178. Tel: (617) 855-3283; Fax: (617) 855-3284; E-mail: isac@harvarda



Cell Transplantation, Vol. 8 pp. 131-142, 1999
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Morris Water Maze Analysis of 192-IgG-Saporin-Lesioned Rats and Porcine Cholinergic Transplants to the Hippocampus

C. J. LeBlanc,1 T. W. Deacon,1 B. R. Whatley,1 J. Dinsmore,2 L. Lin,1 and O. Isacson1

1Neuroregeneration Laboratories, Harvard Medical School, McLean Hospital, 115 Mill St., Belmont, MA 01278
2Diacrin Inc., Charlestown, MA 02129

Adults rats were lesioned with 192-IgG-saporin, an immunotoxin that targets cholinergic neurons in the basal forebrain expressing the low-affinity nerve growth factor receptor (p75). One month later, rats received E3035 porcine cholinergic neurons bilaterally into the hippocampus, and were tested in the Morris water maze and the passive avoidance task 4.56 months after transplantation (in two experiments, rats were retested inthe water maze) followed by histological and cellular analyses. The 192-IgG-saporin-lesioned animals displayed clear cognitive deficits in the Morris water maze. In all experiments the lesioned animals had spatial probe deficits on day 5 testing. A large variance was found among the transplanted animals, with individual animals exhibiting improved performance, but little overall improvement when compared to lesion-alone animals as a group. The relationships between behavioral performance and graft cholinergic factors were established by histological analyses. Grafted animals exhibited an increase in cholinergic innervation of the dentate gyrus (DG) region of the dorsal hippocampus when compared to lesion-alone animals. There was a significant correlation between the level of cholinergic innervation in the dentate gyrus and spatial navigation performance (latency and spatial probe) in the Morris water maze task. These data provide evidence of memory and spatial deficits following cholinergic denervation, and of target-specific growth of xenogeneic cholinergic neurons into the hippocampus. The lack of a clear treatment (transplant) effect in the behavioral measures leads us to believe that functional restoration of cognitive function would require cholinergic reinnervation of both the hippocampus and the neocortex in this 192-IgG-saporin animal model.

Key words: 192-IgG-saporin; Cholinergic neurons; Hippocampus; Morris water maze

Address correspondence to Dr. Ole Isacson, NeuroRegeneration Laboratory, Harvard Medical School, McLean Hospital, 115 Mill St., Belmont, MA 01278. Tel: (617) 855-3243; Fax: (617) 855-3284; E-mail: isacson@helix.mgh.harvard.edu



Cell Transplantation, Vol. 8 pp. 143-151, 1999
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Neural Transplantation of hNT Neurons for Huntington's Disease

Marc S. Hurlbert, Rocio I. Gianani, Cynthia Hutt, Curt R. Freed, and Farida G. Kaddis

Departments of Medicine & Pharmacology, Division of Clinical Pharmacology and Toxicology, University of Colorado Health Sciences Center, Denver, CO 80262

Fetal striatal tissue transplants have been shown to restore motor deficits in rat and monkey models of Huntington's disease (HD). In the present study, using rats with unilateral striatal lesions, we compared fetal striatal tissue transplants to transplants of human NT (hNT) neurons. hNTneurons are terminally differentiated cells derived from the human NTera-2 cell line. In vitro, we have found that purified hNT neurons have a biochemical phenotype similar to that of human fetal striatal tissue. Both hNT neurons and fetal striatal tissue express mRNAs for glutamic acid decarboxylase, choline acetyltransferase, and the D1 and D2 dopamine receptors. Grafts of either hNT neurons or fetal striatal tissue into unilateral quinolinic acid-lesioned rat striatum improved methamphetamine-induced circling behavior. Sham controls showed no changes in methamphetamine-induced circling behavior. In the staircase test for skilled forelimb use, both transplant groups showed partial recovery in skilled use of the paw contralateral to the side of lesion, whereas the control animals showed continued deficits. These findings suggest that transplantation of hNT neurons may be an alternative to transplantation of fetal striatal tissue in the treatment of HD.

Key words: Neural transplantation; hNT neurons; Fetal striatal tissue;Huntington's disease

Address correspondence to Curt R. Freed, M.D., Box C237, 4200 E. 9th Avenue, Denver, CO 80262. Tel: (303) 315-8455; Fax: (303) 315-3272; E-mail: curt.freed@uchsc.edu



Cell Transplantation, Vol. 8 pp. 153-159, 1999
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Cyclosporine A-Induced Hyperactivity in Rats: Is it Mediated by Immunosuppression, Neurotrophism, or Both?

Cesario V. Borlongan,1 Christine E. Stahl,2 Tomoko Fujisaki,3 PaulR. Sanberg,2 and Shigeru Watanabe3

1National Institutes of Health, National Institute on Drug Abuse, Intramural Research Program, Cellular Neurobiology, 5500 Nathan Shock Drive, Baltimore, MD 21224
2Department of Surgery and Program in Neuroscience, University of South Florida College of Medicine, 12901 Bruce B. Downs Blvd., Tampa, FL 33612
3Department of Psychology, Keio University, 2-15-45 Mita, Minato-ku,Tokyo 108, Japan

Cyclosporine A (CsA) immunosuppressive treatment has become an adjunctive therapy in neural transplantation of dopamine-secreting cells for treatment of Parkinson's disease (PD). Recently, CsA and its analogues have been shown to promote trophic effects against neurodegenerative disorders, and therefore CsA may have direct beneficial effects on dopaminergic neurons and dopamine-mediated behaviors. The present study examined the interaction between the reported CsA-induced hyperactivity and the possible alterations in nigral tyrosine hydroxylase (TH)-immunoreactive neurons inrats with damaged blood brain barrier. CsA was administered at a therapeutic dose (10 mg/kg/day, IP, for 9 days) used in neural transplantationprotocol for PD animal models. CsA-treated animals displayed significantly higher general spontaneous locomotor activity than control animals at drug injection days 7 and 9. Histological assays at day 9 revealed that there was a significant increase in TH-immunoreactive neurons in the nigraof CsA-treated rats compared to that of the vehicle-treated rats. The nigral TH elevation was accompanied by suppressed calcium-phosphotase calcineurin activity, indicating an inhibition of host immune response. This is the first report of CsA exerting simultaneous immunosuppressive and neurotrophic effects, as well as increasing general spontaneous locomotor behavior. These results support the utility of CsA as a therapeutic agent for PD and other movement disorders.

Key words: Immunosuppression; Dopamine; Substantia nigra; Calcineurin; Locomotor activity; Neuroprotection; Parkinson's disease

Address correspondence to Cesario V. Borlongan, National Institutes of Health, National Institute on Drug Abuse Intramural Research Program, Cellular Nuerobiology, 5500 Nathan Shock Drive, Baltimore, MD 21224. Tel: (410) 550-1754; Fax: (410) 550-1645; E-mail: cborlong@intra.nida.nih.gov



Cell Transplantation, Vol. 8 pp. 219-232, 1999
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Characterization and Evaluation of Detoxification Functions of a Nontumorigenic Immortalized Porcine Hepatocyte Cell Line (HepLiu*)

Jin Liu,1 Jing Pan,1 Sharda Naik,1 Henry Santangini,1 Donna Trenkler,1 Nancy Thompson,2,3 Abdalla Rifai,1 Jayanta Roy Chowdhury,4 and Hugo O. Jauregui1,3

1Department of Pathology, Rhode Island Hospital, Providence, RI 02903
2Department of Medical Oncology, Rhode Island Hospital and Brown University, Providence, RI 02906
3Department of Pathology and Laboratory Medicine, Division of Biology and Medicine, Brown University, Providence, RI 02906
4Department of Medicine and Molecular Genetics and The Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461

Primary porcine hepatocytes (PPH) are currently used in research and therapeutic applications as the biological component of extracorporeal liver assist devices to overcome the shortage of human hepatocytes. However, their finite life span and typically rapid loss of functions limit their utility. An immortalized, nontumorigenic, highly differentiated porcine hepatocyte cell line was developed in our laboratory to resolve these disadvantages. PPH were transfected with simian virus 40 (SV40) T antigen under the control of the SV40 early promoter. From the established 69 clones, 23 clones displaying hepatocyte-like morphology were screened for diazepam metabolism. One clone, HepLiu D63, has been maintained in culture for >2 years, through more than 60 passages and 240 divisions. Albumin protein, present in early passages, was lost at later passages, but albumin transcript still was detectable in later passages. Carbamoyl phosphate synthetase, a gateway enzyme of the urea cycle, was consistently detectable in HepLiu cells. Cytokeratin 18, a characteristic marker of primary hepatocytes, was detected by both immunofluorescent staining and Western blot in HepLiu cells. Furthermore, maintenance of P450 functions in HepLiu cells was evidenced by diazepam and 7-ethoxycoumarin metabolites measured by HPLC. Phase II conjugative function was measured as acetaminophen glucuronidation. P450 dealkylase was demonstrated microscopically by the conversion of a nonfluorescent substrate to a fluorescent product. Both Northern blot analysis and immunofluorescent staining showed SV40 T antigen expression in the nuclei of HepLiu cells. No tumor formation occurred when HepLiu cells were injected into severe combined immunodeficient (SCID) mice nor was the TA1 (a tumor marker) mRNA expressed, even in later passages. This immortalized, nontumorigenic, highly functional cell line may provide a valuable tool for drug/toxicological studies, liver biologic regulation studies, and artificial liver support systems.

Key words: Immortalized porcine hepatocytes; Drug metabolism; Cytochrome P-450 mono-oxygenase; Urea

Address correspondence to Hugo O. Jauregui, M.D., Ph.D., Department of Pathology and Laboratory Medicine, Aldrich Bldg.-Room 501, Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903. Tel: (401) 444-5153; Fax: (401) 444-8741.
*U.S. patent #5,869,243.



Cell Transplantation, Vol. 8 pp. 233-246, 1999
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Enhanced Cytochrome P450 IA1 Activity of Self-Assembled Rat Hepatocyte Spheroids

Florence J. Wu,1 Julie R. Friend,1 Rory P. Remmel,2 Frank B. Cerra,3 and Wei-Shou Hu1

Departments of 1Chemical Engineering and Materials Science, 2Medicinal Chemistry, and 3Surgery, University of Minnesota, Minneapolis, MN 55455-0132

Primary rat hepatocytes can self-assemble to form multicellular spheroids when plated onto Primaria petri dishes. Spheroids have been observed to exhibit enhanced liver-specific functions and differentiated ultrastructure compared to monolayer cultures on dry collagen. With confocal scanning laser microscopy, CYP1A1 activity was evaluated in situ by detecting resorufin. This highly fluorescent molecule is the P450-mediated product of 7-ethoxyresorufin O-dealkylation (EROD). Significantly higher P450 activity was observed in spheroids compared to monolayers on collagen upon induction with 50 mM b-naphthoflavone (BNF), a CYP1A inducer. This was confirmed by measuring microsomal EROD activity. The distribution of CYP1A1 activity within spheroids was heterogeneous, with higher activity localized to the hepatocytes in the interior. During the process of spheroid formation, cells were initially seen to attach and spread out as a monolayer. This stage was associated with relatively low CYP1A1 activity. As cells formed multicellular structures and aggregated into spheroids, the level of CYP1A1 activity increased over time. At least a fivefold higher fluorescence intensity was observed in spheroids compared to that of monolayers maintained on collagen. The higher P450 activity within spheroids may be associated with their ability to maintain a greater degree of differentiation compared to monolayers. These studies demonstrate the potential of hepatocyte spheroids as a model system for investigating drug metabolism, tissue engineering, and tissue self-assembly.

Key words: Spheroid; Hepatocyte; Cytochrome P450; Confocal microscopy

Address correspondence to Wei-Shou Hu, 421 Washington Ave. SE, Minneapolis, MN 55455-0132. Tel: (612) 625-0564; Fax: (612) 626-7246; E-mail: wshu@cems.umn.edu



Cell Transplantation, Vol. 8 pp. 247-258, 1999
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Extracorporeal Circulation as a New Experimental Pathway for Myoblast Implantation in mdx Mice

Y. Torrente,2 M. G. D'Angelo,1 R. Del Bo,3 A. DeLiso,1 R. Casati,2 R. Benti,2 S. Corti,1 G. P. Comi,1,2 P. Gerundini,2 A. Anichini,4 G. Scarlato,1,2 and N. Bresolin1,2

1Centro Dino Ferrari, Institute of Clinical Neurology, University of Milan, Milan, Italy
2IRCCS Ospedale Maggiore Policlinico, Milan, Italy
3IRCCS Eugenio Medea, Bosisio Parini, Milan, Italy
4Istituto Nazionale per lo studio e la cura dei tumori, Divisione Oncologia Sperimentale D, Milan, Italy

The deficiency of dystrophin, a sarcolemmal associated protein, is responsible for Duchenne muscular dystrophy (DMD). Gene replacement is attractive as a potential therapy. In this article, we describe a new method for myoblast transplantation and expression of dystrophin in skeletal muscle tissue of dystrophin-deficient mdx mouse through iliac vessels extracorporeal circulation. We evaluated the extracorporeal circulation as an alternative route of delivering myoblasts to the target tissue. Two series of experiments were performed with the extracorporeal circulation. In a first series, L6 rat myoblasts, transfected with LacZ reporter gene, were perfused in limbs of 15 rats. In the second series, the muscle limbs of three 68-week-old mdx were perfused with myoblasts of donor C57BL10J mice. Before these perfusions, the right tibialis anterior (TA) muscle of the rats and mdx was injected three times at several sites with bupivacaine (BPVC) and hyaluronidase. The ability of injected cells to migrate in the host tissue was assessed in rats by technetium-99m cell labeling. No radioactivity was detected in the lungs, bowels, and liver of animals treated with extracorporeal circulation. The tissue integration and viability of the myoblasts were ultimately confirmed by genetic and histochemical analysis of LacZ reporter gene. Following a single extracorporeal perfusion of myoblasts from donor C57BL10J, sarcolemmal expression of dystrophin was observed in clusters of myofibers in tibialis anterior muscles previously treated with BPVC and hyaluronidase. Furthermore, large clusters of dystrophin-positive fibers were observed in muscles up to 21 days after repeated treatments. These clusters represented an average of 4.2% of the total muscle fibers. These results demonstrate that the extracorporeal circulation allows selective myoblast-mediated gene transfer to muscles, opening new perspectives in muscular dystrophy gene therapy.

Key words: Gene therapy; Myoblast transplantation; Extracorporeal circulation

Address correspondence to Prof. Nereo Bresolin, Institute of Clinical Neurology, University of Milan, Padiglione Ponti, Ospedale Policlinico, via Francesco Sforza 35, 20122 Milan, Italy. Tel: 0039-02-55033817; Fax: 0039-02-55190392.



Cell Transplantation, Vol. 8 pp. 259-264, 1999
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Outcome of Xenografted Fetal Porcine Pancreatic Tissue Is Superior in Inbred scid (C.B-17/Icr-scid/scid) Compared to Outbred Nude (CD-1-nu/nu) Mice

Bernard E. Tuch and Frances M. Casamento

Pancreas Transplant Unit, Department of Endocrinology, Prince of Wales Hospital, Sydney, Australia

Nude mice are used as recipients of foreign tissue because of their inability to reject these grafts. Our experience has been that there is variable rejection of fetal porcine insulin-producing tissue transplanted into CD-1 (athymic) outbred nude mice. To examine the suitability of this line of nude mouse as a recipient of the tissue, fetal porcine pancreas was grafted either into these outbred animals or into an inbred mutant strain of mice, the more immunocompromised severe combined immunodeficient (scid) mouse. Eight weeks after transplantation grafts were recovered from recipients and assayed for insulin content. Mean insulin levels were not significantly different between the two groups of mice, but a wider range of values was obtained from grafts recovered from nude (CD-1-nu/nu) mice. Reversal of diabetes in hyperglycemic recipients was achieved in 4 of 8 nude mice and 8 of 8 scid (C.B-17/Icr-scid/scid) mice. The time taken to achieve this was longer in the nudes than the scid mice, 121 12 vs. 44 2 days, the grafts increasing in size at a slower rate in the nude mice. Time taken for the weight of the grafts to double in size was 94 17 vs. 32 1 days, respectively. Histologically the grafts in the scid mice contained mostly epithelial cell clusters, a majority of which were insulin containing. In the nude mice that achieved normoglycemia, a similar pattern was observed and, as well, there was a localized lymphoid infiltrate. In those nude mice that remained diabetic fibrous tissue predominated together with a lymphoid infiltrate. In summary, fetal porcine pancreatic tissue grows and develops more efficiently when xenografted into scid rather than outbred nude mice.

Key words: Nude mice; scid mice; Fetal; Porcine insulin; Diabetes; Transplantation

Tel: 61-2-938Address correspondence to Bernard Tuch, FRACP, Ph.D., Department of Endocrinology, Prince of Wales Hospital, Randwick, New South Wales 2031, Australia. 2 4814; Fax: 61-2-9382 4826; E-mail: b.tuch@unsw.edu.au



Cell Transplantation, Vol. 8 pp. 265-276, 1999
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A Preliminary Study of the Activation of Endogenous Pancreatic Exocrine Enzymes During Automated Porcine Islet Isolation*

S. A. White, H. Djaballah, D. P. Hughes, D. L. Roberts, H. H. Contractor, S. Pathak, and N. J. M. London

Department of Surgery, University of Leicester, Leicester, UK

The activation of endogenous pancreatic enzymes during automated pancreas digestion may be detrimental to islet isolation. In this report we assessed the activation of trypsin, chymotrypsin, elastase, carboxypeptidases A and B, phospholipase A2, and lipase using a porcine model Four islet isolations were examined. Duplicate aliquots were taken from the automated circuit at 5-min time intervals up to the completion of pancreas digestion (approx 60 min). One aliquot was activated in vitro with exogenous trypsin in order to convert the enzymes into their active non-"proform," with the exception of trypsinogen, which was activated with exogenous enterokinase. This was done to assess the percentage activation of each individual enzyme (total potentially activatable enzyme release). The extent of activation between isolations was extremely variable. During the closed (recirculating) circuit phase of pancreas digestion there were both gradual and rapid increases in the levels of enzymes released. Peak activity of enzyme activation varied from 13 to 30 min; similarly, total potentially activatable peaks occurred between 13 and 38 min. Lipase and carboxypeptidase B showed greater than 70% activation, chymotrypsin, carboxypeptidase A, and phospholipase A2 between 50% and 70% activation, and trypsin and elastase less than 20%. There were up to 30-fold differences between the four islet preparations. In summary, it is unlikely that poor islet yields are soley explained by variations between collagenases; the variable activation of endogenous pancreatic exocrine enzymes is also likely to be influential to porcine islet yields.

Key words: Swine; Pancreas; Islets of Langerhans; Protease; Collagenase; Trypsin

Address correspondence to Steven A. White, M.D., FRCS, Lecturer in Surgery, Department of Transplantation Surgery, University of Leicester, Leicester General Hospital, Gwendolen Road, Leicester, UK. Tel: 0116 4490490; Fax: 0116 2523179.
*Presented at the 15th Artificial Insulin Delivery Systems Pancreas and Islet Transplantation Congress, Igles, Austria, 1996.



Cell Transplantation, Vol. 8 pp. 277-284, 1999
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Osmotic Tolerance Limits of Canine Pancreatic Islets

Michael A. J. Zieger,1 Erik J. Woods,2 Jonathan R. T. Lakey,3 Jun Liu,2 and John K. Critser2

1Methodist Research Institute, Inc., Clarian Health Partners, Inc., Indianapolis, IN 46206
2Cryobiology Research Institute, Wells Research Center, Indiana University Medical School, Indianapolis, IN 46202
3Comprehensive Tissue Center, Department of Surgery, Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada

Future improvements in the recovery and function of pancreatic islets following cryopreservation will require a more precise quantification of the stresses that occur at each stage of the cryopreservation protocol. Changes in solution osmolality during the addition and dilution of cryoprotectants and during freezing and thawing induce changes in islet volume that may exceed tolerable limits. The aim of this study was to determine the range of solution osmolalities that results in significant changes in islet function. Islets were isolated from canine pancreases by collagenase digestion and Euro-Ficoll purification. Following 12-h culture at 37°C, islets were counted and dispensed into multiwell plate inserts. Islet function was assessed in each well immediately before and 24 h following a 10-min osmotic challenge with hypo- or hyperosmotic solutions of PBS (0, 75, 150, 300, 600, 1200, or 2300 mOsm/kg) at 22°C. Canine islets reached their osmotic equilibrium within 10 min. Duplicate wells were used for each osmolality treatment for each of six donors (n = 12). No significant differences in basal or glucose-stimulated insulin secretion were found between wells prior to the osmotic challenge (3.35 ± 0.45 and 20.98  ± 3.36 mIU/IE/h, respectively). Following the osmotic challenge and 24-h in vitro tissue culture, a significant increase in basal secretion was observed for islets exposed to 0 and 75 mOsm/kg solutions and a significant decrease for islets exposed to 2300 mOsm/kg solution. Islets exposed to 0 and 2300 mOsm/kg solutions showed significant decreases in the stimulated insulin secretion when compared to controls. Solution osmolalities of 150-1200 mOsm/kg appear to be tolerated by canine islets with no significant deviations in insulin secretion. The corresponding tolerable volume range was 152.6 ±  6.8% to 60 ±  5.1% of the isotonic islet volume. The minimum critical volume was used in a theoretical analysis of the islet volumes that would result from equilibrium freezing in dimethyl sulfoxide (DMSO) The calculations show that 1.5 mol/l DMSO is sufficient to prevent damage to islets due to excessive shrinkage. Further refinement of cryoprotectant addition and dilution protocols, and cooling and warming protocols for canine islets, are now possible.

Key words: Pancreatic islet; Cryopreservation; Diabetes; Osmotic tolerance limits

Address correspondence to Michael A. J. Zieger, Ph.D., Methodist Research Institute, Inc., Clarian Health Partners, Inc., 1701 N. Senate Blvd., P.O. Box 1367, Indianapolis, IN 46202-1367. Tel: (317) 929-6940; Fax: (317) 929-5954; E-mail: MZieger@Clarian.com



Cell Transplantation, Vol. 8 pp. 285-292, 1999
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Intraductal Collagenase Delivery Into the Human Pancreas Using Syringe Loading or Controlled Perfusion

Jonathan R. T. Lakey, Garth L. Warnock, A. M. James Shapiro, Gregory S. Korbutt, Ziliang Ao, Norman M. Kneteman, and Ray V. Rajotte

Department of Surgery and the Surgical-Medical Research Institute, University of Alberta, Edmonton, Canada

Effective intraductal delivery of the enzyme collagenase into the pancreas is crucial to the subsequent ability to isolate viable islets. Most clinical islet transplant centers load the enzyme into the pancreas by retrograde injection using a syringe following cannulation of the pancreatic duct. An alternative approach is to perfuse the pancreas via the pancreatic duct with collagenase solution using a recirculating perfusion device system. This provides control over perfusion pressures and collagenase temperature. This study reports on our evaluation of the delivery of LiberaseÔ -HI into the pancreas of 14 consecutive adult multiorgan cadaveric donors. Alternate glands were procured and processed using an identical protocol with the exception of collagenase delivery. The first group of pancreases was loaded using the perfusion technique where cold (4°C) LiberaseÔ-HI was perfused at 80 mmHg for 5 min after which the pressure was increased to 180 mmHg. The collagenase solution was then slowly warmed to 35°C, transferred to the dissociation chamber and mechanically dissociated, and then purified using discontinuous gradients of Ficoll. Pancreases in the second group were loaded with collagenase (28-32°C) using the syringe technique before mechanical dissociation and purification. There were no significant differences in pancreas cold ischemia, donor age, body mass index, maximum blood glucose, or serum amylase of the donors between the two groups. Mean collagenase digestion time in the digestion chamber was not different between the two groups; however, the amount of undigested tissue remaining after dissociation was significantly higher in the syringe-loaded group (15.3 ± 2.6 g vs. 4.6 ± 2.1 g, mean SEM, p < 0.05). Postdigestion recovery of islets was 471 ± 83  ´ 103 IE in the perfusion group compared with 391± 57 ´ 103 IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 ± 45 vs. 251 ± 28 ´ 103 IE, p < 0.05, two-tailed paired t-test). No difference in in vitro islet viability was observed between the two groups following glucose perifusion with the calculated stimulation index of 4.6 ± 0.6 for the perfusion group and 4.2 ± 0.7 for the syringe-loaded group. Controlled perfusion via the pancreatic duct allows the effective delivery of the enzyme achieving maximal distension to all regions of the pancreas leading to an increased recovery of the islets with no detrimental effect on subsequent in vitro islet function.

Key words: Diabetes; Pancreas; Islet; Collagenase

Address correspondence to Dr. Ray V. Rajotte, Director Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Building, University of Alberta, Edmonton, Alberta T6G 2N8, Canada. E-mail: rrajotte@gpu.srv.ualberta.ca



Cell Transplantation, Vol. 8 pp. 293-306, 1999
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In Vitro and In Vivo Performance of Porcine Islets Encapsulated in Interfacially Photopolymerized Poly(Ethylene Glycol) Diacrylate Membranes

Gregory M. Cruise,2,3* Orion D. Hegre,4 Francis V. Lamberti,4 Steven R. Hager,4 Ron Hill,4** David S. Scharp,4 and Jeffrey A. Hubbell1

1Department of Materials and Institute for Biomedical Engineering, Swiss Federal Institute of Technology Zürich and University of Zürich, Zürich, Switzerland
2Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125
3Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712
4Neocrin Company, 31 Technology, Suite 100, Irvine, CA 92618

The usefulness of interfacial photopolymerization of poly(ethylene glycol) (PEG) diacrylate at a variety of concentrations and molecular weights to form hydrogel membranes for encapsulating porcine islets of Langerhans was investigated. The results from this study show in vitro and in vivo function of PEG-encapsulated porcine islets and the ability of PEG membranes to prevent immune rejection in a discordant xenograft model. Encapsulated islets demonstrated an average viability of 85% during the first week after encapsulation, slightly but significantly lower than unencapsulated controls. Encapsulated porcine islets were shown to be glucose responsive using static glucose stimulation and perifusion assays. Higher rates of insulin release were observed for porcine islets encapsulated in lower concentrations of PEG diacrylate (10-13%), not significantly reduced relative to unencapsulated controls, than were observed in islets encapsulated in higher concentrations (25%) of PEG diacrylate. Perifusion results showed biphasic insulin release from encapsulated islets in response to glucose stimulation. Streptozotocin-induced diabetic athymic mice maintained normoglycemia for up to 110 days after the implantation of 5,000-8,000 encapsulated porcine islet equivalents into the peritoneal cavity. Normoglycemia was also confirmed in these animals using glucose tolerance tests. PEG diacrylate-encapsulated porcine islets were shown to be viable and contain insulin after 30 days in the peritoneal cavity of Sprague-Dawley rats, a discordant xenograft model. From these studies, we conclude that PEG diacrylate encapsulation of porcine islets by interfacial photopolymerization shows promise for use as a method of xenoprotection toward a bioartifical endocrine pancreas.

Key words: Islets of Langerhans; Encapsulation; Poly(ethylene glycol); Hydrogel

Address correspondence to Jeffrey A. Hubbell, Department of Materials and Institute for Biomedical Engineering, Swiss Federal Institute of Technology Zürich and University of Zürich, Moussonstrasse 18, CH-8044 Zürich, Switzerland. Tel: +41 1 632 4575; Fax: +41 1 632 1214; E-mail: hubbell@biomed.mat.ethz.ch
*Present address: CardioSynopsis, Inc., 735 Palomar Ave., Sunnyvale, CA 94086..
**Present address: Genset Corporation, 875 Prospect St., LaJolla, CA 92037.



Cell Transplantation, Vol. 8 pp. 307-315, 1999
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The Rescue Effect of 15-Deoxyspergualin on Intraperitoneal Microencapsulated Xenoislets

Brend Ray-Sea Hsu,1 Fu-Hsiung Chang,2 Jyuhn-Huarng Juang,1 Yu-Yao Huang,1 and Shin-Huei Fu1

1Division of Endocrinology and Metabolism, Chang-Gung Memorial Hospital, Lin-Kou Medical Center, Tao-Yuan Hsien, Taiwan
2Department of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan

Because the development of surface neogrowth composed mainly of macrophages and fibroblasts precedes the recurrence of hyperglycemia in treated diabetic animals, the pericapsular macrophages may adversely affect the graft function of IP alginate-poly-L-lysine-alginate (A-P-A) microencapsulated islets. In order to clarify the role of pericapsular macrophages on late islet xenograft dysfunction, we investigated whether 15-deoxyspergualin (15-DSG), a macrophage inhibitor, has a rescue effect on the recurrent hyperglycemia in streptozotocin-induced diabetic mice that had been treated with IP transplantation of A-P-A microencapsulated rat islets. The mean duration of normoglycemia (whole blood glucose level below 8.3 mmol/l) in streptozotocin-induced diabetic mice treated with implantation of about 2200-2400 of A-P-A microencapsulated rat islets was 75 days. When the blood glucose levels were higher than 11.1 mmol/l for two consecutive determinations, 15-DSG at a dose of 0.625 mg/kg body weight or isotonic sodium chloride solution (control group) was given daily SC. The blood glucose levels decreased significantly from 13.9 ± 0.5 mmol/l to 11.0 ± 1.3 mmol/l (n = 18, p < 0.05) at the fourth day and to 7.6 ± 1.0 mmol/l (n = 18) at the 14th day of 15-DSG administration. That was not significantly different from the mean glycemic level during the normoglycemic period (7.6 ± 1.0 vs. 7.0 ± 1.7 mmol/l, n = 18, p = NS). Isotonic sodium chloride solution injections did not reduce glycemic levels of mice in the control group. As another control, 10 streptozotocin-induced diabetic mice were given the same daily doses of 15-DSG for 14 days. 15-DSG did not decrease the blood glucose levels of diabetic mice in the control group. We further studied the effect of 15-DSG on the expression of interleukin-1b (IL-1b) in peritoneal exudate mononuclear cells (PEMCs) using reverse transcription-polymerase chain reaction. It was found that the mRNA of IL-1b was undetectable in PEMCs of 15-DSG-treated diabetic mice even after those cells were stimulated by lipopolysaccharides in vitro. Administration of 15-DSG at a daily dose of 0.625 mg/kg body weight from the 22nd to the 28th day after transplantation and 7 consecutive days every 3 weeks thereafter did not prolong graft survival of IP microencapsulated rat islets. Our data suggest that 15-DSG has a rescue effect when A-P-A microencapsulated islets have induced cellular overgrowth that threatens the survival of the graft. It is possible that the surface overgrowth composed of macrophages is involved in the pathophysiology of late failure of A-P-A microencapsulated xenogeneic islets.

Key words: Microencapsulation; Islet; 15-Deoxyspergualin; Interleukin-1b

Address correspondence to Brend Ray-Sea Hsu, M.D., Ph.D., Division of Endocrinology & Metabolism, Chang-Gung Memorial Hospital, Lin-Kou Medical Center, 5 Fu-Shin St. Kwei-Shan County, Tao-Yuan Hsien, Taiwan. Tel: 886-3-328-1200, Ext. 2500; Fax: 886-3-328-8257; E-mail: metafu@mail.cgu.edu.tw



Cell Transplantation, Vol. 8 pp. 317-326, 1999
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In Vivo Evaluation of Glucose Permeability of an Immunoisolation Device Intended for Islet Transplantation: A Novel Application of the Microdialysis Technique

E. Rafael,1 A. Wernerson,3 P. Arner,2 G. S. Wu,1 and A. Tibell1

Departments of *Transplantation Surgery, 2Internal Medicine, and 3Pathology, Karolinska Institute, Huddinge Hospital, Stockholm, Sweden

Immunoisolation devices consist of semipermeable membranes chosen to protect the islets from the immune system but still allow sufficient passage of nutrients, oxygen, and the therapeutic products, insulin. The exchange between the device and the microcirculation will influence the survival of the graft as well as the metabolic efficacy of the islet implant. Glucose is the important trigger factor for insulin secretion. In this study, we evaluate the in vivo glucose permeability of the TheracyteÔ immunoisolation device at various times after implantation. Empty devices were implanted SC in rats. The glucose kinetics in the device was compared to that in the SC tissue during IV glucose tolerance tests (IVGTTs), using the microdialysis technique. In rats studied on day 1, or 1, 2, and 4 weeks after implantation, the peak glucose levels (Cmax) were significantly lower, the times-to-peak (TTP) were significantly longer, and the areas under the curve during the first 40 min (AUC0-40) were significantly smaller in the device than in the SC fat. However, at 3 months all parameters improved and Cmax, TTP, and AUC0-40 in the device did not differ significantly from those measured in the SC fat. Thus, during the first 4 weeks the device constitutes a significant diffusion barrier, but at 3 months the exchange between the lumen of devices and the blood stream improves. Our data indicate that implantation of the device several months before transplantation of the cellular graft would improve the exchange across the membrane during the early posttransplant period. This should have positive effects on graft survival and function. We also suggest that microdialysis is a useful tool for evaluating the in vivo performance of macroencapsulation devices.

Key words: Diffusion chamber; Cell transplantation; Macroencapsulation; Glucose; Permeability; Microdialysis

Address correspondence to Dr. Ehab Rafael, Department of Transplantation Surgery, Karolinska Institute, Huddinge Hospital, S-141 86 Huddinge, Sweden. Tel: + 46 8 58582549; Fax: + 46 8 774 31 91.



Cell Transplantation, Vol. 8 pp. 327-337, 1999
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Studies on Smaller (~315 mM) Microcapsules: IV. Feasibility and Safety of Intrahepatic Implantations of Small Alginate Poly-L-Lysine Microcapsules

François A. Leblond,1 Ginette Simard,1 Nathalie Henley,1 Bernard Rocheleau,2 P.-Michel Huet,2 and Jean-Pierre Hallé1

1Centre de Recherche Guy-Bernier, Hôpital Maisonneuve Rosemont, Université de Montréal and 2Centre de Recherche André-Viallet, Hôpital St-Luc, Université de Montréal, Montréal, Québec, Canada

The most successful transplantation site of nonencapsulated islets of Langerhans is the liver. Because usual alginate poly-L-lysine microcapsules were too large (700-1200 mm diameter) for intravascular implantations and were almost exclusively implanted intraperitoneally, the question of the preferred implantation site of microencapsulated islets has received little attention. The feasibility of implanting smaller (~315 mm) alginate poly-L-lysine microcapsules into the liver and the effect of such implantations on portal pressure and liver histology was evaluated in Wistar rats. A bolus of 10,000 microcapsules of 315 mm diameter was injected intraportally (group 1; n = 22). The portal pressure increased from 6.4 ± 1.8 mmHg to a maximum of 19 mmHg, returned to basal levels within 2 h, and remained normal after 2 months. In group 2 (n = 3), following the injection of 10,000 larger microcapsules (420 mm), the portal pressure increased to > 60 mmHg and two out of the three rats died within 3 h. When 5,000 microcapsules of 420-mm diameter were injected (group 3; n = 5), the portal pressure peaked to 30 ±  8 mmHg and remained elevated after 4 h (12 3 mmHg), but returned to normal (8  ± 1 mmHg) after 2 weeks. Histological studies showed normal hepatic architecture without collagen deposition into portal tracts occupied by microcapsules. Conclusion: intrahepatic implantations of ~315-mm alginate poly-L-lysine microcapsules are feasible and safe. These results justify further investigation of this potential implantation site for microencapsulated islets.

Key words: Microencapsulation; Intrahepatic implantation; Microcapsule size; Portal pressure

Address correspondence to Dr. Jean-Pierre Hallé, Centre de Recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont, 5415 boul. de l'Assomption, Montréal, Qc, Canada, H1T 2M4. Tel: (514) 252-3400, Ext. 3373; Fax: (514) 252-3430.



Cell Transplantation, Volume 8, pp. 339-344, 1999
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Direct Effects of Cyclosporin A on Proliferation of Hematopoietic Stem and Progenitor Cells

S. Scott Perry, MiJung Kim, and Gerald J. Spangrude

Departments of Oncological Sciences, Pathology, and Medicine, University of Utah Medical Center, Salt Lake City, UT 84132

Cyclosporin A (Cy A) has been reported to both stimulate and inhibit bone marrow colony assays in a dose-dependent manner. The observation that anti-g-IFN antibodies stimulate hematopoiesis to the same degree as Cy A has led several groups to propose that the stimulatory effects of Cy A are due to inhibition of g-IFN production by T cells. In this study we observed that cultures of highly enriched hematopoietic stem/progenitor cells (HSPC), devoid of CD3/5/8+ T cells, also exhibit enhanced cloning efficiency when cultured in the presence of Cy A. Normal bone marrow cells or Thy-1.1lowSca-1+Linneg HSPC were incubated in methylcellulose cultures and stimulated with various combinations of steel factor (SF), interleukin (IL)-3, IL-6, granulocyte colony stimulating factor (G-CSF), and erythropoietin (EPO) in the presence of increasing concentrations of Cy A. HSPC cultures with SF, IL-3, and IL-6 stimulation and low Cy A concentrations had from 24% to 78% higher cloning efficiencies than did parallel cultures without Cy A, and did not fall below control levels until the Cy A concentration was increased to more than 1.25 mg/ml. The addition of EPO and G-CSF abrogated the Cy A stimulation observed with SF, IL-3, and IL-6. These results were reflected in whole bone marrow, but with a higher range of variability. Cultures in which FK-506 replaced Cy A showed no consistent stimulation or inhibition of colony formation. These studies show that Cy A can stimulate hematopoietic stem cell growth independent of mediation by T cells. Consequently, these results argue for a direct positive effect of Cy A on the signal transduction pathways in HSPC.

Key words: Cyclosporin A; Hematopoietic stem cells; Bone marrow transplantation; Signal transduction

Address correspondence to Gerald J. Spangrude, Department of Oncological Sciences Room 5C334SOM, 50 North Medical Drive, Salt Lake City, UT 84132. Tel: (801) 585 5544; Fax: (801) 585 3778; E-mail: drblood@medschool.med.utah.edu



Cell Transplantation, Volume 8, pp. 345-350, 1999
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Development of Poly(Propylene Fumarate-co-Ethylene Glycol) as an Injectable Carrier for Endothelial Cells

Laura J. Suggs and Antonios G. Mikos

Cox Laboratory for Biomedical Engineering, Institute of Biosciences and Bioengineering and Department of Chemical Engineering, Rice University, Houston, TX 77005

Poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)] hydrogels were examined as in situ polymerizable carriers for endothelial cells. The temperature increase from 37°C during cross-linking was measured. The maximum temperature did not increase above 38.3°C for any copolymer formulation. The temperature profiles also appeared to be independent of the amount or molecular weight of poly(ethylene glycol). These materials were polymerized in situ in a subcutaneous rat model and evaluated for initial biocompatibility. A normal wound-healing response was seen with formation and subsequent maturity of a fibrous capsule. Endothelial cells were embedded in vitro during the cross-linking process and their proliferation was assessed over the first 24 h. There was significant DNA synthesis by the embedded endothelial cells during this time period. These data suggest that P(PF-co-EG) hydrogels could be developed for use as injectable cell carriers.

Key words: Hydrogel; Poly(ethylene glycol); Poly(propylene fumarate); Cell carrier; Tissue engineering

Address correspondence to Dr. Antonios G. Mikos, Institute of Biosciences and Bioengineering, Rice University, 6100 S. Main, MS-144, Houston, TX 77005. Tel: (713) 285-5355; Fax: (713) 285-5353; E-mail: mikos@rice.edu



Cell Transplantation, Volume 8, pp. 351-364, 1999
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Design Engineering of a Bioartificial Renal Tubule Cell Therapy Device

Janeta Nikolovski,1 Erdogan Gulari,2 and H. David Humes3

Departments of  1Biomedical Engineering, 2Chemical Engineering, 3Internal Medicine and VA Medical Center, University of Michigan, Ann Arbor, MI 48109-2136

The use of a bioartificial renal tubule device composed of renal proximal tubule cells grown within a hollow fiber cartridge is a first step in engineering a bioartificial kidney to provide more complete replacement therapy of renal function than is available today. In this study, the feasibility of two designs for a tubule device were investigated: one with cells grown on microcarrier beads densely packed within the extracapillary space of a hollow fiber cartridge, and the other with cells grown as a confluent monolayer within the hollow fibers themselves. First, the oxygen requirements of porcine renal proximal tubule cells were determined, both attached to microcarriers and in suspension and compared to that of proximal tubule segments. The basal rate of cell respiration was found to be 2.29 ± 0.53 nmol O2/106 cells/min for our cultured proximal tubule cells in suspension and no significant difference was seen with attached cells. Proximal tubule segments displayed significantly higher respiratory rates. Cells were also found to be responsive in the presence of mitochondrial inhibitors or uncouplers, and their respiratory rates remained constant, despite multiple passaging. The resultant cell oxygen consumption parameter was used in models describing oxygen concentration profiles within the two device configurations. From these models, it was found that cells within our proposed device designs could theoretically be sustained and remain viable, with respect to oxygen limitations. Finally, flow visualization studies were performed to assess fluid flow distribution and determine optimal device configuration and geometry to decrease areas of low or stagnant flow.

Key words: Bioartificial kidney; Proximal tubule cells; Flow visualization; Modeling; Oxygen consumption

Address correspondence to H. David Humes, Department of Internal Medicine, University of Michigan Health System, 3101 Taubman Center, Ann Arbor, MI 48109-0368. Tel: (734) 936-4495; Fax: (734) 936-7024; E-mail: dhumes@umich.edu



Cell Transplantation, Volume 8, pp. 365-373, 1999
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Analysis of the Th1/Th2 Paradigm in Transplantation: Interferon-g Deficiency Converts Th1-Type Proislet Allograft Rejection to a Th2-Type Xenograft-Like Response

Charmaine J. Simeonovic,1 Michelle J. Townsend,1 Gunasegaran Karupiah,2 J. Dennis Wilson,4 Jodie C. Zarb,1 David A. Mann,3 and Ian G. Young3

1Division of Molecular Medicine, 2Division of Immunology and Cell Biology, 3Division of Biochemistry and Molecular Biology, The John Curtin School of Medical Research, The Australian National University, Canberra, A.C.T., 2601, Australia
4Department of Endocrinology, The Canberra Hospital, Woden, A.C.T., 2606, Australia

The rejection mechanisms for fetal proislet allografts and pig proislet xenografts in mice are characterized by different intragraft cytokine mRNA profiles and cellular responses. Allograft rejection is predominantly CD8 T-cell-dependent and is associated with a Th1-type cytokine pattern (i.e., IFN-g , IL-2 but no IL-4 or IL-5 mRNA). In contrast, xenograft rejection is CD4 T-cell-dependent and is accompanied by a strong Th2-type response (i.e., enhanced expression of IL-4 and IL-5 mRNA) and by marked eosinophil accumulation at the graft site. We have now examined and compared the regulatory role of IFN-g in both proislet allograft and xenograft rejection processes. The histopathology and intragraft cytokine mRNA profile of BALB/c (H-2d) proislet allografts were examined in IFN-g -deficient and wild-type C57BL/6J recipient mice. The survival of pig proislet xenografts was also assessed in IFN-g -/- and wild-type hosts. Both proislet allografts and xenografts were acutely rejected in IFN-g -/- and wild-type mice. Unlike the conventional allograft reaction, which lacks eosinophil infiltration, the rejection of proislet allografts in IFN-g -deficient hosts correlated with intragraft expression of IL-4 and IL-5 mRNA (i.e., a Th2-type response) and eosinophil recruitment. The rejection of proislet allografts and xenografts can therefore occur by IFN-g -independent pathways; IFN-g , however, regulates the pathology of the allograft reaction but not the xenograft response. The immune destruction of proislet allografts is not prevented by Th2 cytokine gene expression; instead, the latter correlated with the recruitment of unconventional inflammatory cells (eosinophils), which may play an accessory role in effecting graft injury. Significantly, the Th1-to-Th2-like switch resulted in the novel conversion of an allograft rejection reaction into a xenograft-like rejection process.

Key words: Allograft; Xenograft; Proislet; Rejection; IFN-g deficient

Address correspondence to Dr. C. J. Simeonovic, Division of Molecular Medicine, The John Curtin School of Medical Research, The Australian National University, G.P.O. Box 334, Canberra, A.C.T., 2601, Australia. Tel: 61-2-6249-4709; Fax: 61-2-6249-0413; E-mail: Charmaine.Simeonovic@anu.edu.au



Cell Transplantation, Volume 8, pp. 375-381, 1999
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Regulatory T Cells Maintain Peripheral Tolerance to Islet Allografts Induced by Intrathymic Injection of MHC Class I Allopeptides*

David V. Saborio,1 Nepal C. Chowdhury,1 Ming-Xing Jin,1 Anil Chandraker,2 Mohamed H. Sayegh,2 and Soji F. Oluwole1

1Department of Surgery, College of Physicians and Surgeons of Columbia University, New York, NY
2Laboratory of Immunogenetics and Transplantation, Brigham and Women's Hospital, Harvard Medical School, Boston, MA

Although transplantation remains the treatment of choice for diabetes mellitus, immunological rejection of allografts continues to be a major problem. The search for strategies to prevent graft rejection led us to examine if the fate of developing T cells may be influenced by the presence of allo MHC class I peptides in the thymus because T cell receptor-MHC class I/self-peptide interaction regulates thymocyte development. We studied the effects of intrathymic (IT) injection of a short segment of a synthetic immunogenic MHC class I peptide (peptide 2, residues 67-85) of the hypervariable domain of RT1.A derived from WAG rat (RT1U) on islet graft survival in the WF(RT1U)-to-ACI combination. Adult diabetic male recipients were treated with IT injection of a single WAG-derived MHC class I peptide 7 days before intraportal islet transplantation. Long-term unresponsive islet recipients were examined for the development of alloantigen (Ag)-specific regulatory cells. The results showed that while IT injection of 150 mg peptide 2 on day -7 did not prolong graft survival in naive recipients [median survival time (MST) of 14.0 days vs. 9.6 in controls], IT injection of 300 or 600 mg peptide 2 led to normoglycemia and permanent islet survival (>200 days) in 4/6 and 3/5 STZ-induced diabetic ACI recipients, respectively. IT injection of 150, 300, or 600 mg peptide 2 combined with 0.5 antilymphocyte serum (ALS) immunosuppression on day -7 led to 100% permanent islet allograft survival (>200 days) compared to MST of 15.0 ± 2.3 days in ALS alone-treated controls. Similarly prepared animals rejected third-party Brown Norway (BN) islets in an acute fashion, thus demonstrating donor specificity. Intravenous injection of 300 mg peptide 2 combined with 0.5 ml ALS did not prolong islet allograft survival. The long-term unresponsive islet allograft recipients challenged with second set grafts accepted permanently 100% donor-type cardiac allografts while rejecting third-party (BN) hearts without rejecting the primary Wistar Furth (WF) islets. In analyzing the underlying mechanisms of acquired systemic tolerance, we found no suppressor/regulatory cells in adoptive transfer studies in tolerant animals at 30 days after IT injection of allopeptides. In contrast, adoptive transfer of 5 ´ 107 unseparated spleen cells from tolerant animals at 60 and 100 days after islet transplantation into lightly irradiated [200 rad total body irridation (TBI)] ACI recipients led to donor-specific permanent islet graft survival in 2/3 and 4/5 secondary recipients, respectively, compared to an MST of 13.8 days in lightly irradiated ACI given unmodified syngeneic spleen cells. In addition, adoptive transfer of 2 ´ 107 purified T cells obtained from long-term functioning islet recipients led to permanent donor-specific islet survival in secondary recipients. The finding that IT injection of a short segment of a synthetic immunodominant MHC class I peptide derived from WAG that shares the RT1.AU domain with the graft donor is capable of inducing acquired systemic tolerance to WF islets suggests that linked recognition or epitope suppression may be involved in the induction of unresponsiveness. Generation of peripheral Ag-specific regulatory cells that suppress Ag-specific alloreactive T cells is, in part, responsible for the maintenance of tolerance in this model.

Key words: MHC class I allopeptides; Islet allografts; Regulatory T cells

Address correspondence to Soji F. Oluwole, M.D., Department of Surgery, College of Physicians and Surgeons, Columbia University, 630 West 168th. Street, New York, NY 10032. Tel: (212) 305-3948; Fax: (212) 305-4061; E-mail: so5@columbia.edu
*Presented at the 24th Annual Meeting of the American Society of Transplant Surgeons, May 13-15, 1998, Chicago, IL.



Cell Transplantation, Volume 8, pp. 383-388, 1999
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Forskolin-Stimulated Adenylylcyclase Activity: A Marker to Assess Islet Cell Viability Following Cold Storage in Different Solutions and to Predict Islet Cell Function Following Transplantation

Shigeo Kanazawa,1 Tsukasa Tsunoda,2 Masakazu Murakami,2 Sueharu Iwamoto,2 Masatoshi Kimoto,2 Eishi Onuma,2 Toshimitsu Majima,2 and Yasuhisa Yamamoto2

1Department of Surgery, Mitsugi General Hospital, Hiroshima, Japan
2Department of Surgery, Division of Gastroenterological Surgery, Kawasaki Medical School, Kurashiki, Japan

For clinical islet cell transplantation, short-term storage of islet cells is likely to be necessary, and it is imperative that the islet cells be kept as viable as possible during the period. However, there are little data on which preservative solutions are most suitable for the storage of islet cells after isolations or before transplantation. To estimate islet cell viability and transplantation success rate in the present study, adenylylcyclase activity was measured with a rapid new fluorometric assay in rat islet cells prior to transplantation, because cAMP plays an essential role in determining islet b-cell viability and responsiveness to various hormonal stimuli. Adenylylcyclase activity was measured in islet cells stored for different periods of time (0, 3, 16, 24, 48, 96 h) and in different preservative solutions. Approximately 1,000 islet cells from each preservation group using University of Wisconsin (UW) solution were transplanted to streptozotocin-induced diabetes mellitus (DM) rats. Transplant success was evaluated by measuring blood glucose levels. Preoperative adenylylcyclase activity was compared with posttransplant islet cell function. The adenylylcyclase activity of UW solution was significantly higher than that of Euro-Collins solution and lactate-Ringer's solution through the different preservation time periods. Preoperative adenylylcyclase activity correlated well with posttransplant islet cell function in a rat model of DM. We conclude that adenylylcyclase activity can be used as a marker to assess islet cell viability as well as differences in preservation media and may predict islet cell transplant success.

Key words: Diabetes mellitus; cAMP; Enzymatic cycling; Islet cell transplantation; Cell viability

Address correspondence to Shigeo Kanazawa, Department of Surgery, Mitsugi General Hospital, 124, Ichi, Mitsugi-cho, Mitsugi-gun, Hiroshima, 722-0311, Japan. Tel: 08487-6-1111; Fax: 08487-6-3002.



Cell Transplantation, Volume 8, pp. 391-398, 1999
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Novel Method for Isolation of Adult Porcine Pancreatic Islets With Two-Stage Digestion Procedure

Wanxing Cui,1 Yuanjun Gu,2 Masaaki Miyamoto,3 Masaki Tanaka,3 Baoyou Xu,1 Masayuki Imamura,1 Hiroo Iwata,2 Yoshihioto Ikada,2 and Kazutomo Inoue2

1Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Japan
2Institute for Frontier Medical Sciences, Kyoto University, Japan
3The Second Surgical Department, Medical School, Kinki University, Japan

It is particularly difficult to isolate porcine islets (PI). Experience suggests that the success rate of porcine islet isolation (PII) is probably considerably influenced by the distension and digestion of the pancreas. In this study, we divided the digestion procedure into two stages and developed a new enzyme solution to improve both the distension and digestion procedures. As a result, we established a novel and stable method of large-scale adult porcine islet isolation (APII). The harvested pancreata of 2-year-old pigs weighing over 200 kg (n = 18) were distended by introducing our new enzyme solution gently and slowly through the pancreatic ducts. Two-stage digestion (cold, then warm) was then performed by first placing the distended pancreata on ice for 2 h to cause diffusion of the enzyme solution around the islets, and then by incubating the pancreata in a water bath at 37°C for 45 min without shaking. The islets were purified by a COBE 2991 cell processor on dextran T70 discontinuous density gradients. Histological study was performed on porcine pancreata sampled after 0, 15, 30, and 45 min of the second stage, and stained with H&E stain. Next, islet equivalent was calculated. Static incubation study was performed by stimulating the islets with 3.3 and 16.7 mM glucose in Krebs' Ringer bicarbonate buffer (KRBB) solution at 37°C for 1 h, and finally the insulin released was measured. The dilated acinar cells septa around the islets were observed at time 0. Destruction of the acinar cells around the islets by warm digestion was recognized at 15 and 30 min, and destroyed and separated acinar cells present around the islets at 45 min. During the entire course of the warm digestion, the islets remained intact. The number of isolated islets was 291,667 ± 240,452 IEQ/pancreas (n = 4) and 3,294 ± 2199 IEQ/g of pancreatic tissue. The purity of recovered porcine islets was over 90%. The concentration of the insulin secreted by 10,000 IEQ islets selected at random was 83.9 ± 13.4 mU/dish/h in response to 3.3 mM glucose and 104.1 ± 12.9 mU/dish/h in response to 16.7 mM glucose (n = 20). A success rate of approximately 80% was attained with APII. We demonstrated that this increase in the success rate was due to the improved distension and digestion provided by this method. This two-stage APII method with its new enzyme solution may facilitate the future use of porcine islets in clinical xenotransplantation trials.

Key words: Porcine islet isolation; Enzyme solution; Two-stage digestion; Success rate

Address correspondence to Kazutomo Inoue, M.D., Professor, Department of Organ Reconstruction, Institute for Frontier Medical Sciences, 53 Kawaracho Shogoin, Sakyo-ku, Kyoto 606, Japan. Tel. (075) 751-4850; Fax. (075) 751-4338; E-mail. wxcui@frontier.kyoto-u.ac.jp



Cell Transplantation, Volume 8, pp. 399-404, 1999
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Bioartificial Endocrine Pancreas (Bio-AEP) for Treatment of Diabetes: Effect of Implantation of Bio-AEP on the Pancreas

Sachiko Hirotani,1,2 Reiko Eda,1,3 Takako Kawabata,1,4 Shohei Fuchinoue,2 Satoshi Teraoka,2 Tetsuzo Agishi,2 and Hisako Ohgawara1

1Medical Research Institute and 2Department of Surgery III, Tokyo Women's Medical University, Japan
3Tokyo University of Agriculture, Japan
4Nippon Veterinary and Animal Science University, Japan

Recently, we described a diffusion chamber for a bioartificial endocrine pancreas (Bio-AEP). Pancreatic islet cells in the Bio-AEP device were isolated from the immune system of the host by an artificial barrier, while nutrients, electrolytes, oxygen, and bioactive secretory products were exchanged across this barrier. This experiment was designed to evaluate whether the diffusion chamber could be useful as a Bio-AEP in the treatment of diabetes. Six streptozotocin (STZ)-induced diabetic rats each received a diffusion chamber containing 8 ´ 106 MIN6 cells as a xenograft Bio-AEP. In the STZ diabetic rats with Bio-AEPs, a return to normoglycemia was observed up to 30 weeks after implantation, without the use of any immunosuppressant. A gradual increase in the body weight of the rats was also observed. In three STZ diabetic rats, diffusion chambers without MIN6 cells were implanted as a sham operation. The fasting blood glucose levels in these three rats remained higher than 600 mg/dl, after implantation, and they lost weight. Thirty-five weeks after implantation, the pancreata were removed from the rats that underwent xenoimplantation, those that had the sham operation, and the normal control rats. In the sham-operated animals, the exocrine tissues of the pancreata were vacuolated and pancreatic B cells were not seen in the islets. In contrast, in the pancreata from the xenoimplantation, the exocrine tissues were normal, and a few pancreatic B cells were seen in the islets. These results indicated that xenoimplantation using the Bio-AEP might retard the progress of diabetes.

Key words: Bio-AEP; Xenoimplantation; Sham operation; Pancreas; Exocrine tissue

Address correspondence to Hisako Ohgawara, Medical Research Institute, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel/Fax: +81-3-5269-7364.



Cell Transplantation, Volume 8, pp. 405-411, 1999
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Effectiveness of Acidic Oxidative Potential Water in Preventing Bacterial Infection in Islet Transplantation

Masaaki Miyamoto,1 Kazutomo Inoue,1 Yuanjun Gu,1 Masanori Hoki,2 Seiji Haji,2 and Harumasa Ohyanagi2

1Department of Organ Reconstruction, Field of Clinical Application, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
2Department of Surgery II, Kinki University School of Medicine, Osaka, Japan

At a number of points in the current procedures of islet isolation and islet culture after the harvesting of donor pancreata, microorganisms could potentially infect the islet preparation. Furthermore, the use of islets from multiple donors can compound the risks of contamination of individual recipients. Acidic oxidative potential water (also termed electrolyzed strong acid solution, function water, or acqua oxidation water), which was developed in Japan, is a strong acid formed on the anode in the electrolysis of water containing a small amount of sodium chloride. It has these physical properties: pH, from 2.3 to 2.7; oxidative-reduction potential, from 1,000 to 1,100 mV; dissolved chlorine, from 30 to 40 ppm; and dissolved oxygen, from 10 to 30 ppm. Because of these properties, acidic oxidative potential water has strong bactericidal effects on all bacteria including methicillin-resistant Staphylococcus aureus (MRSA), viruses including HIV, HBV, HCV, CMV, and fungi as a result of the action of the active oxygen and active chlorine that it contains. We conducted this study to evaluate the effect of acidic oxidative potential water irrigation on bacterial contamination on the harvesting of porcine pancreata from slaughterhouses for islet xenotransplantation by counting the number of pancreatic surface bacteria using the Dip-slide method, and on the results of islet culture; and to evaluate the direct effect on isolated islets when it is used to prevent bacterial contamination by the static incubation test and by morphorogical examination. Direct irrigation of the pancreas by acidic oxidative potential water was found to be very effective in preventing bacterial contamination, but direct irrigation of isolated islets slightly decreased their viability and function.

Key words: Islet transplantation; Acidic oxidative potential water (AOPW); Electrolyzed strong acid solution; Bacterial infection

Address correspondence to Prof. Kazutomo Inoue, Department of Organ Reconstruction, Field of Clinical Application, Institute for Frontier Medical Sciences, Kyoto University 53 Kawahara-cho, Shogoin Sakyo-ku, Kyoto 606-8507, Japan. Tel: 81-75-751-4848, 4856; Fax: 81-75-751-4848.



Cell Transplantation, Volume 8, pp. 413-417, 1999
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Beneficial Effects of Immunoisolated Fetal and Neonatal Pig Liver Fragments on Acute Liver Failure in a Large Animal

Nobuyuki Kanai,1,2 Masao Hagihara,1 Yukio Nagamachi,2 and Kimiyoshi Tsuji1

1Department of Transplantation Immunology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan
21st Department of Surgery, Gunma University School of Medicine, 3-39-22 Showa, Maebashi, Gunma 371-8511, Japan

Xenogeneic cell (fragment) transplantation may be used as an interim therapy until the organ allotransplanation. Immunologic rejection, however, constitutes the major hurdle. To overcome this problem, "xeno'' fetal and neonatal liver fragments (FLF, NLF) were encapsulated into separate micropore devices that protect them from immunological attack by the recipient. The FLF or NLF were then transplanted into beagles with hepatic failure to observe their biological effects. In Experiment 1 (n = 5) beagles were injected IV with D-galactosamine (D-gal, 1.0 g/kg) on day 0 and then received FLF grafts (0, 0.3, 0.8, 1.0, 2.0 g/kg). In Experiment 2 (n = 6) beagles received NLF grafts (1.8 g/kg) and on the following day were injected with D-gal (1.0 g/kg). In Experiment 1 only the high dose of xeno-FLF (2.0 g/kg) decreased the elevated ALT (GPT) and T.Bil. levels. Histologic examination showed that some of the hepatocytes of the host liver survived only in the high-dose graft. In Experiment 2, at 36 and 48 h after D-gal injection, the transplanted group had a significantly lower AST (GOT) level than the control. The grafted NLF survived for 14 days, according to histologic examinations. Thus, encapsulated FLF and NLF xenotransplantation can prevent liver dysfunction in a large animal hepatic failure model.

Key words: Immunoisolation; Xenotransplantation; Liver failure; Pig fetal/neonatal liver fragment

Address correspondence to Masao Hagihara at his present address: Department of Hematology and Rheumatology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, 259-1193, Japan. Tel: +81-463-93-1121, Ext. 2700; Fax: +81-463-92-4750.



Cell Transplantation, Volume 8, pp. 419-425, 1999
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Protective Effect of Nafamostat Mesilate on Injury of Porcine Hepatocytes by Human Plasma

Yasushi Kawazoe, Susumu Eguchi, Nozomu Sugiyama, Hiroyuki Yuzawa, Yujo Kawashita, Hikaru Fujioka, and Takashi Kanematsu

Department of Surgery II, Nagasaki University School of Medicine, Nagasaki, Japan

Nafamostat mesilate (NM), a protease inhibitor, possesses a cytoprotective effect and inhibits the activation of complement. The present study investigated whether NM has any protective effect against injury of porcine hepatocytes by human plasma in a bioartificial liver support system. Porcine hepatocytes were harvested and seeded at a density of 2 ´ 105 cells on a 35-mm collagen-coated plate in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum. Twenty-four hours later, the medium was replaced with human plasma with three concentrations of NM between 3.8 ´ 10-5 and 3.8 ´ 10-4 M and then cultured for 6 h. The viability of porcine hepatocytes, lactate dehydrogenase (LDH) levels, lidocaine clearance, porcine albumin production, and changes in complement (C3) levels were measured. The viability of porcine hepatocytes in human plasma decreased significantly to 37.7 ± 11.4% of that in DMEM. NM improved the viability of the hepatocytes, lowered the levels of LDH, and increased lidocaine clearance and albumin production in a concentration-dependent manner. The concentrations of C3, the marker of xenogeneic reactions, did not change significantly, indicating that no hyperacute xenogeneic reaction occurred in our series. Together, our results suggested that NM exerts favorable effects on porcine hepatocytes in human plasma through direct effect such as prevention of protease activity in the plasma membrane of porcine hepatocytes rather than inhibition of complement-dependent immunoreactions.

Key words: Nafamostst mesilate; Porcine hepatocytes; Human plasma; Complement

Address correspondence to Takashi Kanematsu, M.D., F.A.C.S., Department of Surgery II, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki, 852-8501 Japan. Tel: 81-95-849-7316; Fax: 81-95-849-7319.



Cell Transplantation, Volume 8, pp. 427-430, 1999
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Evaluation of Reaction of Primate Brain to Grafted PC12 Cells

Hideyuki Yoshida, Isao Date, Tetsuro Shingo, Kenjiro Fujiwara, Yasuyuki Miyoshi, Tomohisa Furuta, and Takashi Ohmoto

Department of Neurological Surgery, Okayama University Medical School, Okayama 700-8558, Japan

Intrastriatal implantation of polymer-encapsulated PC12 cells, which constitute a dopaminergic cell line derived from rat pheochromocytoma, has proved useful for ameliorating parkinsonian symptoms in several kinds of animals. In considering the clinical application of this technique, we should make sure that PC12 cells are rejected completely by the host immune system in case the capsule breaks. In the present study, unencapsulated PC12 cells were injected into the brain of Japanese monkeys (Macaca fuscata). Histological [hematoxylin-eosin (H&E), Nissl] and immunocytochemical [tyrosine hydroxylase (TH), and glial fibrillary acidic protein (GFAP)] analyses were performed 1, 2, 4, and 8 weeks after transplantation. Also, encapsulated PC12 cells were transplanted into the brain of another group of Japanese monkeys to investigate the host reaction to the capsule and to confirm that the encapsulated PC12 cells continue to survive in the host brain. H&E and GFAP staining were performed 2, 4, and 8 weeks after transplantation. L-DOPA and dopamine release from the explanted capsules was measured by high performance liquid chromatography. Magnetic resonance imaging was performed in both unencapsulated and encapsulated PC12 cell grafted groups. Although the xenografted unencapsulated cells formed a small cluster at 1 and 2 weeks after implantation, very few and no viable PC12 cells remained at 4 and 8 weeks, respectively. The reaction of the host towards the xenograft gradually decreased. Encapsulated PC12 cells retrieved from the host brain were found to release L-DOPA and dopamine continuously even 8 weeks after implantation. The host reaction to the PC12-loaded capsule was much weaker than that to the unencapsulated PC12 cells, and decreased with time. These results indicate that encapsulated PC12 cell transplantation is an effective and safe strategy for the treatment of Parkinson's disease.

Key words: Encapsulation; PC12 cells; Transplantation; Nonhuman primate

Address correspondence to Hideyuki Yoshida, M.D., Department of Neurological Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama-shi, Okayama-ken, 700-8558, Japan. Tel: 81-86-235-7336; Fax: 81-86-227-0191; E-mail: h_yosida@cc.okayama-u.ac.jp



Cell Transplantation, Volume 8, pp. 431-434, 1999
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Long-Term Functional Assessment of Encapsulated Cells Transfected With Tet-On System

Yasushi Hagihara,1 Youichi Saitoh,1 Norio Arita,1 Yutaka Eguchi,2 Yoshihide Tsujimoto,2 Toshiki Yoshimine,1 and Toru Hayakawa1

Departments of 1Neurosurgery and 2Medical Genetics, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

In our previous study, xenogeneic mouse neuroblastoma cells bearing the POMC gene, the precursor of ACTH and b-endorphin, were implanted within polymer capsules into the CSF space of rats. Although ACTH and b-endorphin were secreted, we were not able to control the amounts or times of hormone release. A promoter that is inducible by administration of tetracycline derivatives (Tet) was linked to the POMC gene to control its gene expression (Neuro2A-Tet-On-POMC; NTP). The results showed that POMC gene expression in the implanted encapsulated NTP cells could be regulated in a dose-dependent manner by Tet administration to the hosts. However, no analysis of gene control with the Tet-On system over a long period has been performed. In this study, encapsulated NTP cells were treated in vitro with doxycycline (Dox) (1.0, 10, 100, 1000 ng/ml) continuously for a month. On day 4, the amount of ACTH secretion was dependent on the Dox dose. But in the course of the experiment, the difference of ACTH secretion among those treated with Dox 10, 100, and 1000 ng/ml was eliminated. On the other hand, NTP cells, which were treated with Dox (1000 ng/ml) just on days 7, 14, 21, and 28, secreted almost the same amount of ACTH in 24 h. From these results, for clinical use, an NTP cell line that secretes enough opiate to reduce pain sensitivity without Dox should be established, and Dox could then be administered if necessary.

Key words: Tetracycline; ACTH; Endorphin; Pain

Address correspondence to Youichi Saitoh, Department of Neurosurgery, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel: +81-6-879-3652; Fax: +81-6-879-3659; E-mail: saitoh@nsurg.med.osaka-u.ac.jp



Cell Transplantation, Volume 8, pp. 435-441, 1999
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Differentiation and Angiogenesis of Central Nervous System Stem Cells Implanted With Mesenchyme Into Ischemic Rat Brain

Atsushi Fukunaga, Koichi Uchida, Koichi Hara, Yoshiaki Kuroshima, and Takeshi Kawase

Department of Neurosurgery, Keio University School of Medicine, Tokyo 160-8582, Japan

After cerebral infarction, necrosis in neural tissues is not usually repaired or reconstructed by the injured brain. We therefore examined the effects on postinfarction repair of implanting central nervous system (CNS) stem cells together with mesenchyme, because CNS stem cells can be expected to adapt and survive in the adult brain. Cerebral infarction was induced by the Koizumi-Longa method, using the adult male spontaneous hypertensive rat model. Reperfusion was performed an hour after middle cerebral artery occlusion. The rat mesencephalic neural plate at the early somite stage (embryonic day 10.5) together with the adjacent ventral mesenchymal tissues was dissected out under the microscope and immediately implanted into the ischemic rat striatum. One month later, the cognitive function was evaluated by the Morris water maze method. Histologic and immunohistochemical examinations of the graft were made with hematoxylin-eosin (H&E), neurofilament-200, and tyrosine hydroxylase (TH) stains. In the water maze study, mean latency times required to reach an escape platform in the implanted animals with surviving grafts were found to be shorter than in those without grafts, but longer than in normal animals. In the spatial probe trial, the number of animals seen to cross the area in the pool where the platform had been located was greater in the implanted rats with surviving grafts than in other groups. Multiple vascularization in the grafted area was observed histologically in H&E-stained tissues, and neurofilament-200-positive cells were recognized in the graft. TH staining revealed within the graft many immunoreactive neuron-like cell bodies with long dendrites. It was suggested that grafted CNS stem cells with mesenchyme may survive and differentiate into mature CNS tissue within the adult ischemic rat brain, constructing vessels in and around the grafts, and may therefore have the potential to be effective in the recovery of the cognitive function of the rat model.

Key words: Neural transplantation; Mesencephalic neural plate; Mesenchyme; Cerebral infarction

Address correspondence to Atsuchi Fukunaga, Department of Neurosurgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel: +81-3-3353-1211, Ext. 2329; Fax: +81-3-3354-8053; E-mail: fukunaga@med.keio.ac.jp



Cell Transplantation, Volume 8, pp. 465-476, 1999
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Increased Myogenic Potential and Fusion of Matrilysin-Expressing Myoblasts Transplanted in Mice

Nicolas J. Caron, Isabelle Asselin, Guy Morel, and Jacques P. Tremblay

Laboratoire de Génétique Humaine, Centre de Recherche du Centre Hospitalier de l'Université Laval, Université Laval, Ste-Foy, Qc, Canada G1V 4G2

The success of myoblast transplantation in clinical trials has been limited in part by the low dispersion of grafted cells outside the injection site. Our research group previously reported that the culture of myoblasts with concanavalin A, a stimulator of metalloproteinase production, increased their migration. Several lines of evidence also suggested that muscle cell fusion involves metalloproteinase-sensitive mechanisms. To determine whether the increased expression of metalloproteinases had an influence on myoblast fusion and dispersion through the muscle following transplantation, we generated a myoblast cell line expressing human matrilysin (MMP-7). The MMP-7-expressing myoblasts were obtained by the stable transfection of a matrilysin expression vector in a TnILacZ immortomouse myoblast clone. Matrilysin-expressing myoblasts showed a highly increased in vitro fusion index, forming seven times (p < 0.001) more myotubes than the control cell line and three times (p < 0.001) more myotubes than the Immortomyoblast parental clone. Single-site transplantation of matrilysin-expressing myoblasts generated more fibers (p < 0.001), over a greater surface (p < 0.001) than the control cell line. The cotransplantation of matrilysin-expressing myoblasts and of normal human myoblasts in SCID mice increased the number of human dystrophin-positive fibers and myotubes by sixfold. Although no significant increased migration of myoblasts outside the injection sites was observed, our results show that the metalloproteinase activity can improve the myogenic potential of myoblasts in vitro and the fusion of myoblasts with host fibers in vivo. MMP-7 expression may be useful in increasing myoblast transplantation success.

Key words: Myoblast transplantation; Matrilysin; Cell migration; Skeletal muscle

Address correspondence to Pr Jacques P. Tremblay, Laboratoire de génétique humaine, Centre de recherche du CHUL, 2705 boulevard Laurier, Ste-Foy, Qc, Canada G1V 4G2. Tel: (418) 654-2186; Fax: (418) 654-2207; E-mail: jacques-p.tremblay@crchul.ulaval.ca



Cell Transplantation, Volume 8, pp. 477-488, 1999
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Fabrication of Compliant Hybrid Grafts Supported With Elastomeric Meshes

Teisei Kobashi1,2 and Takehisa Matsuda1

1Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-1, Fujishiro-dai, Suita, Osaka 565-8565, Japan
2Second Department of Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan

We devised tubular hybrid medial tissues with mechanical properties similar to those of native arteries, which were composed of bovine smooth muscle cells (SMCs) and type I collagen with minimal reinforcement with knitted fabric meshes made of synthetic elastomers. Three hybrid medial tissue models that incorporated segmented polyester (mesh A) or polyurethane-nylon (mesh B) meshes were designed: the inner, sandwich, and wrapping models. Hybrid medial tissues were prepared by pouring a cold mixed solution of SMCs and collagen into a tubular glass mold consisting of an inner mandrel and an outer sheath and subsequent thermal gelation, followed by further culture for 7 days. For the inner model, the mandrel was wrapped with a mesh. For the sandwich model, a cylindrically shaped mesh was incorporated into a space between the mandrel and the sheath. The wrapping model was prepared by wrapping a 7-day-incubated nonmesh gel with a mesh. The inner diameter was 3 mm, irrespective of the model, and the length was 2.5-4.0 cm, depending on the model. The intraluminal pressure-external diameter relationship showed that nonmesh and inner models had a very low burst strength below 50 mmHg, while the sandwich model ruptured at around 110-120 mmHg; no rupturing below 240 mmHg was observed for the wrapping model, regardless of the type of mesh used. Compliance values of wrapping and sandwich models were close to those of native arteries. Pressure-dependent distensibility characteristics similar to native arteries were observed for a mesh A wrapping model, whereas a mesh B wrapping model expanded almost linearly as intraluminal pressure increased, which appeared to be due to elasticity of the incorporated mesh. Thus, design criteria for hybrid vascular grafts with appropriate biomechanical matching with host arteries were established. Such hybrid grafts may be mechanically adapted in an arterial system.

Key words: Compliance matching; Hybrid vascular graft; Smooth muscle cells; Collagen gel

Address correspondence to Takehisa Matsuda, Department of Bioengineering, Graduate School of Medicine, Kyushu University, 3-1-1, Maidashi, Higashiku, Fukuoka, 812, Japan. Tel: (+81) 92-642-6210; Fax: (+81) 92-62-6212; E-mail: matsuda@med.kyushu-u.ac.jp



Cell Transplantation, Volume 8, pp. 489-499, 1999
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Lack of Effect of Short-Term Depletion of Plasma Complement C3 on the Survival of Syngeneic Dopaminergic Neurons Following Grafting Into the Intact Rat Striatum

M. H. Khorooshi, M. Meyer, E. B. Pedersen, and B. Finsen

Department of Anatomy and Neurobiology, Odense University, Denmark

Metabolically compromised cells may be subject to complement-mediated cytotoxicity. The aim of this study was to clarify to what extent plasma complement C3 might contribute to the low survival (5-20%) of grafted dopaminergic neurons. The survival of intrastriatal cell suspension grafts of syngeneic dopaminergic, tyrosine hydroxylase (TH)-containing neurons was compared in rats subjected to short-term IV treatment with 1) cobra venom factor (CVF), or 2) placebo treatment. Depletion of plasma complement C3 by CVF was confirmed by crossed immunoelectrophoresis. With 159 ± 37 (mean ± SEM) TH-immunoreactive and 154 ± 40 TH mRNA-expressing neurons in the CVF-treated rats (n = 9), and 117 ± 34 TH-immunoreactive and 160 ± 49 TH mRNA-expressing neurons in placebo rats (n = 6), the CVF treatment did not increase the survival of the grafted dopaminergic neurons. Similarly, CVF had no apparent effect on the astroglial, microglial, or oligodendroglial cell response within and around the graft. The data indicate that depletion of plasma complement C3 at the time of grafting has no effect on the long-term survival of syngeneic ventral mesencephalic dopaminergic neuronal grafts.

Key words: Transplantation; Cobra venom factor; Complement system; Parkinson's disease

Address correspondence to Bente Finsen, Department of Anatomy and Neurobiology, Institute of Medical Biology, Odense University, Winsloewparken 21, DK-5000 Odense C, Denmark. Tel: +45 65503800; Fax: +45 65906321; E-mail: finsen@imbmed.ou.dk



Cell Transplantation, Volume 8, pp. 501-510, 1999
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Characterization of Iris Pigment Epithelial Cell for Auto Cell Transplantation

Toshiaki Abe, Hiroshi Tomita, Toshifumi Ohashi, Katsura Yamada, Yoshiyuki Takeda, Keiko Akaishi, Madoka Yoshida, Masami Sato, and Makoto Tamai

Department of Ophthalmology, Tohoku University, School of Medicine, Sendai 980-8574, Japan

To establish auto iris pigment epithelial (IPE) transplantation, we characterized the properties of IPE cells and the method of culture using auto serum. Monkey and human IPE cells were obtained and cultured in several conditions, using auto, mouse, rabbit, bovine, or human serum. Immunocytochemical study was performed to confirm that the cells were epithelial in origin. The proliferation rate of the IPE was also calculated from fresh human IPE cells, which were obtained during filtering glaucoma surgery. Proliferation rate was also compared to that of retinal pigment epithelial (RPE) cells. Reverse-transcriptase and polymerase chain reaction for melanogenesis was performed, and the amount of pigment in the IPE cells was also calculated. Mouse and rabbit sera were not effective for the monkey IPE cell culture. Conversely, the cells grew well in the medium with auto, bovine, or human serum. Human IPE cells grew exponentially by the described methods and reached to 60,000 cells after about 4-5 weeks. When we compared them by proliferation rate, IPE cells were less proliferative than RPE cells. The gene expression for melanogenesis and the amount of pigment in the IPE gradually decreased through successive passages. Transplantation has been tried for the treatment of age-related macular degeneration using RPE from fetus or from eye bank eyes. However, focal rejection may play an important role in the clinical results. The establishment of auto IPE cell transplantation may improve the problem of rejection. In the present study, we established auto IPE cell culture using auto serum. The cultured IPE cell showed pigment epithelial cell properties until around five passages in both human and monkey.

Key words: Iris pigment epithelium; Auto cell transplantation; Monkey; Rejection

Address correspondence to Toshaiki Abe, Department of Ophthalmology, School of Medicine, 1-1 Seiryomachi Aobaku, Sendai, Miyagi 980-8574, Japan. Tel: 81-22-717-7294; Fax: 81-22-717-7298; E-mail: toshi@oph.med.tohoku.ac.jp



Cell Transplantation, Volume 8, pp. 511-519, 1999
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High Variability in Rabbit Bone Marrow-Derived Mesenchymal Cell Preparations

Luis A. Solchaga,1 Brian Johnstone,2 Jung U. Yoo,2 Victor M. Goldberg,2 and Arnold I. Caplan1

Skeletal Research Center, Departments of 1Biology and 2Orthopaedics,
Case Western Reserve University, Cleveland, OH 44106-7080

The rabbit has been extensively used for preclinical models, especially in orthopedic applications. One of the more troubling features of this model is the high interindividual variability that is encountered and that requires a careful experimental design with sufficient sample size to make judgments valid. We have processed 241 individual preparations of rabbit bone marrow-derived mesenchymal progenitor cells (MPCs) over the last 3 years and have kept detailed records of the performance of these cells in various assays. This communication details the lack of correlation between the analyzed parameters. Bone marrow was harvested from 4-month-old rabbits; the cells were centrifuged, resuspended, and cultured. When cells reached 80% of confluence, they were removed from the plates with trypsin and assayed for their osteo- and chondrogenic potential. The average yield of the 241 individual MPC preparations exhibited a coefficient of variation of 77. An in vivo implantation assay with porous calcium phosphate ceramic cubes exhibited scores with a coefficient of variation of 65. Lastly, an in vitro assay of alkaline phosphatase enzyme activity exhibited the most variability with a coefficient of variation of 132. All of the cell preparations tested in an in vitro aggregate culture assay underwent chondrogenic differentiation. No relationships between any of these parameters were found. The variability of the results within the different assays is interpreted to be the result of the heterogeneity of the preparations. The lack of correlation between the parameters studied shows the importance of the conditions intrinsic to the different assays. These results serve to emphasize that any experimental design involving rabbit progenitor cells must include a sufficiently large sample size to allow statistically significant and rigorous conclusions.

Key words: Variability; Bone marrow; Progenitor cell; In vitro; In vivo

Address correspondence to Arnold I. Caplan, Department of Biology, Case Western Reserve University, 2080 Adelbert Road, Cleveland, OH 44106-7080. Tel: (216) 368-3562; Fax: (216) 368-4077; E-mail: las29@po.cwru.edu



Cell Transplantation, Volume 8, pp. 521-530, 1999
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Freezing Characteristics of Genetically Modified Lymphocytes for the Treatment of MPS II

A. Hubel, T. B. Darr, and J. A. Norman

Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455

The freezing characteristics of genetically modified lymphocytes obtained from a donor with mucopolysaccharidosis type II (MPS II) were determined using cryomicroscopy and controlled rate freezing studies to determine postthaw viability. The cells from a donor with MPS II used in this investigation were cultured and transduced with a retroviral vector for the iduronate-2-sulfatase (IDS) enzyme for clinical studies for human gene therapy. The water transport and intracellular ice formation (IIF) characteristics of the cells were determined after completion of the culture and transduction protocol. The water transport parameters, Lpg and Elp, for the cultured and transduced cells were determined to be 4.4 ± 1.3 x 10-14  m3/Ns and 173 ± 25 kJ/mol, respectively. The IIF nucleation parameters, k and W, were 5.5 x 1010 K5 and 3.5 x 1011 (1/m2 s), respectively. The postthaw viability of the genetically modified cells was less than the viability of the freshly isolated cells from the same donor. The postthaw viability of the cultured and transduced cells from a donor with MPS II was also less than that observed with cells from a normal donor that were frozen and thawed under the same conditions. These studies are essential in understanding the biophysical changes resulting from the ex vivo culture of cells and the manner in which these changes influence the ability of the cells to be cryopreserved.

Key words: Cryopreservation; Lymphocytes; Gene therapy

Address correspondence to A. Hubel, Ph.D., Department of Laboratory Medicine and Pathology, University of Minnesota, Box 609 UMHC, Minneapolis, MN 55455. Tel: (612) 626-4451; Fax: (612) 625-1121; E-mail: hubel001@maroon.tc.umn.edu



Cell Transplantation, Volume 8, pp. 531-541, 1999
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A New Bioartificial Liver Using Porcine Hepatocyte Spheroids in High-Cell-Density Suspension Perfusion Culture: In Vitro Performance in Synthesized Culture Medium and in 100% Human Plasma

Yasuyuki Sakai,1 Katsutoshi Naruse,2 Ikuo Nagashima,2 Tetsuichiro Muto,3 and Motoyuki Suzuki1

1Institute of Industrial Science, University of Tokyo, Tokyo 106-8558, Japan
2Department of Surgery, Unit of Artificial Organs and Transplantation and 3Unit of Tumor Surgery, Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan

A prototype of a bioartificial liver (BAL) based on suspension perfusion culture of porcine hepatocyte spheroids was developed at 150 ml scale. About 2% (4 x 109 cells) of whole human liver cells was immobilized. The cell density in the bioreactor was 2.7 x 107 cells/ml, which was almost comparable to that of presently developed packed-bed-type BALs. The bioreactor was perfused with culture medium while retaining spheroids. This was done using a rotating stainless filter (pore size 50 mm). In vitro 8-h perfusion experiments utilizing both synthesized culture medium and 100% human plasma demonstrated the spheroids in the bioreactor had almost the same functions on a unit/cell basis as those in small-scale rotational culture. This indicated that the functional deterioration often associated with scaling up had been minimized. Rapid spheroid aggregation and dysfunction in specific human plasma pool must be eliminated before clinical application, although this phenomenon seemed to be inherent to porcine hepatocyte-based BALs. This prototype shows promise in meeting present clinical demands by achieving maximal metabolic activities even in the short term.

Key words: Bioartificial liver; Porcine hepatocyte; Spheroid; Suspension culture; Human plasma

Address correspondence to Yasuyuki Sakai, Institute of Industrial Science, University of Tokyo, 7-22-1 Roppongi, Minato-ku, Tokyo 106-8558, Japan. Tel: +813-3402-6231, ext. 2413 or 2458; Fax: +813-3408-0593; E-mail: yasuyuki@cc.iis.u-tokyo.ac.jp



Cell Transplantation, Volume 8, pp. 543-548, 1999
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15AU81, A Prostacyclin Analog, Enhances Donor-Specific Hepatocytes to Prolong the Survival of Rat Heart But Not Small Bowel Allografts

Michael J. Boyle and Lynette J. Dumble

Department of Surgery, Division of Immunology and Organ Transplantation, University of Texas Medical School at Houston, 6431 Fannin, MSB 6.240, Houston, TX 77030

Prostacyclin analogs have previously been shown to have not only cytoprotective but also independent immunosuppressive effects. The effect of one such analog, 15AU81, to enhance the immunosuppressive effects of liver was investigated. We have previously demonstrated that cyclosporine (CsA) in conjunction with rapamycin (RAPA) potentiates class I+, class II- donor-specific hepatocytes to prolong rat cardiac and small bowel allograft survival. Brown Norway (BN; RT1n) hepatocytes alone (5 x 107/kg, administered intrasplenically) failed to prolong the survival of BN heart allografts in Wistar Furth (WFu; RT1u) recipients, beyond that of untreated controls (MST = 7.2 ± 0.8 days). Survival of BN hearts was increased to 11.4 ± 1.7 days in WFu recipients treated with BN hepatocytes and 50 mg/kg/day 15AU81 administered by continuous SC infusion for 14 days using osmotic pumps (p < 0.05). The further addition of RAPA 0.0075 mg/kg/day and CsA 0.375 mg/kg/day delivered for 14 days by continuous IV infusion (CIVI) using osmotic pumps (a combination that alone prolonged BN heart allografts in WFu hosts to 18.4 ± 1.3 days and in conjunction with BN hepatocytes prolonged survival to 27.2 ± 1.9 days) prolonged allograft survival to 35.2 ± 5.2 days. In contrast, the survival of small bowel allografts was not enhanced by 15AU81 administration. Survival of BN small bowel transplants in LEW recipients treated with hepatocytes alone (MST = 11.6 ± 1.5 days) or hepatocytes plus 15AU81 (MST = 10.0 ± 1.0 days) was similar to controls (MST = 10.2 ± 1.9 days). Treatment with hepatocytes and RAPA/CsA increased survival to 21.2 ± 1.5 days. The further addition of 15AU81 failed to augment this (MST = 17.0 ± 1.9 days). In vitro WFu lymphocyte proliferative responses from animals pretreated with BN hepatocytes, 15AU81, or both treatments, for 2 weeks prior to harvesting, exhibited a reduction of at least 50%, compared to untreated controls upon allostimulation with irradiated BN or ACI spleen cells. These findings demonstrate that 15AU81 interacts favorably with hepatocytes either alone or in conjunction with RAPA and CsA to enhance their immunosuppressive effects on rat heart allograft survival. The failure to enhance small bowel allograft survival may be explained by the inability at this low dosage of 15AU81 to influence the intense graft versus host reaction elicited by small bowel transplants.

Key words: Hepatocyte; Prostacyclin; Transplantation

Address correspondence to Michael J. Boyle, M.D., University of Oklahoma Health Sciences Center, 2808 South Sheridan Road, Tulsa, OK 74129. Tel: (305) 744-2086; Fax: (918) 744-3651; E-mail: michael-boyle@ouhsc.edu



Cell Transplantation, Volume 8, pp. 549-559, 1999
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Water and Cryoprotectant Permeability Characteristics of Isolated Human and Canine Pancreatic Islets

Erik J. Woods,1,2 Jun Liu,1,2 Michael A. J. Zieger,3 Jonathan R. T. Lakey,2,4 and John K. Critser1,2

1Cryobiology Research Institute, Wells Center for Pediatric Research, Indiana University Medical Center, Indianapolis, IN
2Department of Veterinary Clinical Sciences, and Hillenbrand Biomedical Engineering Center, Purdue University, West Lafayette, IN
3Methodist Research Institute, Methodist Hospital of Indiana, Inc., Indianapolis, IN
4Comprehensive Tissue Centre, Department of Surgery, Laboratory Medicine and Pathology, University of Alberta, Alberta, Edmonton, Canada

Cryopreservation allows accumulation of the necessary islet transplantable mass as well as adequate time for tissue typing and infectious disease screening. Cryopreservation protocols may be optimized by modeling the osmotically induced volume excursions that occur during the addition and removal of cryoprotective agents (CPAs). To that end, three transport parameters were measured at 22°C in canine and human islets isolated by collagenase digestion and euroficoll purification: (i) the apparent hydraulic conductivity (Lp), (ii) the permeability coefficient of the CPA (Ps), and (iii) the associated reflection coefficient (s). The parameters were determined by volumetric analysis of islets upon abrupt exposure to 1, 2, and 3 M dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol (GLY), and propylene glycol (PG). The parameters were calculated using the Kedem-Katchalsky theory to describe islet volume excursion kinetics (assuming islets to be single equivalent osmotic units with the same volume and surface area of the actual islet) and a three-parameter curve fit was performed using the Marquardt-Levenberg method. It was determined that the permeability characteristics of pancreatic islets are species specific, and based upon the measured parameters, the highest Ps values for canine islets were observed following exposure to 2 M EG, and the highest Ps values for human islets were observed following exposure to 2 M PG. The permeability parameters were analyzed adjusting for islet radius using ANCOVA procedures to acquire least square means. For canine islets exposed to 2 M EG these values were determined to be 0.936 mm/min/atm, 2.47 mm/s, and 0.90 (for Lp, Ps, and j, respectively) and for human islets exposed to 2 M PG the values were determined to be 1.56 mm/min/atm, 3.48 mm/s, and 0.85 (for Lp, Ps, and s, respectively). These parameters were used in a model to calculate osmotically induced islet volumetric response upon addition/dilution of the optimum CPAs, taking into consideration critical volume excursion limits at which irreversible damage occurs.

Key words: Cryopreservation; Pancreatic islet; Permeability characteristics

Address correspondence to John K. Critser, Cancer Research Building, Wells Research Center, Room 454, 1044 West Walnut Street, Indianapolis, IN 46202. Tel: (317) 278-3215; Fax: (317) 274-8679; E-mail:jcritser@iupui.edu



Cell Transplantation, Volume 8, pp. 561-565, 1999
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Long-Term Treatment of Streptozotocin-Induced Diabetes by Continuous Insulin Minipump in the Syrian Hamster

William E. Fisher

Department of Surgery, Baylor College of Medicine, Houston, TX

It is difficult to normalize plasma glucose for a prolonged period of time by SC injection in experimental animals. The goal of this study was to determine the feasibility and the dosage of insulin needed to maintain 24-h normoglycemia in streptozotocin-diabetic Syrian hamsters with a SC-implanted osmotic minipump. The pumps, which release insulin at a constant rate, were replaced every 14 days with fresh pumps for as long as 52 days. A high insulin dose (1 U/kg/h) was required to normalize plasma glucose and fatty acid concentrations, water and food consumption, urine output, and body weight.

Key words: Streptozotocin; Insulin; Osmotic pump; Syrian hamster

Address correspondence to William E. Fisher, M.D., Department of Surgery, Baylor College of Medicine, Veterans Affairs Medical Center, Surgical Service (112), 2002 Holcombe Blvd., Houston, TX 77030. Tel: (713) 794-7139; Fax: (713)794-7352.



Cell Transplantation, Volume 8, pp. 583-591, 1999
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Xenografting Human T2 Sympathetic Ganglion From Hyperhidrotic Patients Provides Short-Term Restoration of Catecholaminergic Functions in Hemiparkinsonian Athymic Rats

D. M. Liu,1 S. Z. Lin,1 S. D. Wang,1 M. Y. Wu,1 and Yun Wang1,2

1Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
2National Institute on Drug Abuse, IRP, 5500 Nathan Shock Drive, Baltimore, MD

Previous studies have suggested that allografting peripheral sympathetic ganglia, such as superior cervical ganglia, partially relieves clinical or behavioral deficits in parkinsonian patients and animals. However, removal of these ganglia can cause Horner's syndrome, which limits the utilization of this approach. Hyperhidrosis, a disease of excessive sweating, is commonly seen in young Orientals. Treatment of hyperhidrosis often involves surgical removal of the second thoracic sympathetic ganglia (T2G), which contains catecholaminergic neurons. The purpose of our study was to investigate behavioral responses and tyrosine hydroxylase (TH) immunoreactivity in hemiparkinsonian rats at different time points after transplantation of human T2G from hyperhidrotic patients. Athymic Fisher 344 rats were injected unilaterally with 6-hydroxydopamine into the medial forebrain bundle to destroy the nigrostriatal dopaminergic (DA) pathway. The effectiveness of lesions was tested by measuring methamphetamine (MA)-induced rotations. These unilaterally lesioned rats were later transplanted with T2G or T2 fiber tract (T2F) obtained from adult hyperhidrotic patients. Animals grafted with T2G showed a reduction in MA-induced rotation by 2 weeks; however, rotation returned to the pregrafting levels by 3 months. Animals receiving T2F grafts did not show any reduction of rotation over a 3-month period. Animals were later sacrificed for TH immunostaining at different time points. Tyrosine hydroxylase-positive [TH(+)] cell bodies and fibers were found in the lesioned striatum 2-4 weeks after T2G grafting, suggesting the survival of transplants. Two to 3 months after grafting, TH(+) fibers were still found in almost all the recipients. However, TH(+) cell bodies were found in only three of seven rats studied. Animals receiving T2F grafting did not show any TH immunoreactivity in the lesioned striatum over the 3-month period. These data indicate that T2G transplants from adult hyperhidrotic patients can survive and provide transient normalization of the motor behavior in the hemiparkinsonian athymic rats. Because of the short-term improvement in behavior after grafting, the use of T2G in human trials should be cautious at the present time. Further laboratory research is required.

Key words: Sympathetic ganglion; Parkinson's disease; Transplantation; Hyperhidrosis; Athymic rats

Address correspondence to Yun Wang, M.D., Ph.D., National Institute on Drug Abuse, IRP, 5500 Nathan Shock Drive, Baltimore, MD 21224. Tel: (410) 550-1753; Fax: (410) 550-1645; E-mail: Ywang@intra.nida.nih.gov



Cell Transplantation, Volume 8, pp. 593-599, 1999
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Fetal Porcine Dopaminergic Cell Survival In Vitro and its Relationship to Embryonic Age

Roger A. Barker,1 Emma Ratcliffe,1Andrew Richards,2 and Stephen B. Dunnett1

1MRC Cambridge Centre for Brain Repair, Forvie site, Robinson way, Cambridge CB2 2PY, UK
2Imutran Ltd, Cambridge CB2 2AH, UK

One of the critical factors in the survival of embryonic neural grafts is the age at which the population of donor neurons is harvested. This is especially the case for the developing dopaminergic neurons in the embryonic ventral mesencephalon, which are used for neural grafts in Parkinson's disease (PD). The donor age for optimal harvesting of these cells has been well characterized in the mouse, rat, and marmoset, and to a lesser extent in humans. However, the best donor age for porcine ventral mesencephalic tissue has not been ascertained, even though the use of this tissue for xenografts has been explored both experimentally and clinically. In this study the effect of donor age on dopaminergic cell survival was assessed in vitro, from a range of fetal pigs aged from E24 to E35. The number of tyrosine hydroxylase (TH)-positive cells per ventral mesencephalon was then calculated after 1 and 7 days in culture. E26-E27 embryos gave the highest yield of such cells at both survival time points, suggesting that this will be the optimal age for harvesting tissues whether for experimental or clinical nigral xenograft programs.

Key words: Porcine ventral mesencephalon; Donor age; In vitro; Neural xenografts

Address correspondence to Roger A. Barker, MRC Cambridge Centre for Brain Repair, Forvie site, Robinson way, Cambridge CB2 2PY, UK. Tel: (+44) 01223 331160; Fax: (+44) 01223 331174; E-mail: rab46@cus.cam.ac.uk



Cell Transplantation, Volume 8, pp. 601-610, 1999
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Porcine Embryonic Brain Cell Cytotoxicity Mediated by Human Natural Killer Cells

Suchitra Sumitran,1 Per Anderson,2 Håkan Widner,2 and Jan Holgersson1

1Division of Clinical Immunology, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Huddinge, Sweden
2Department of Physiological Sciences, Wallenberg Neuroscience Center, Lund University, SE-223 62 Lund, Sweden

Intracerebral transplantation of porcine embryonic dopamine-producing neurons has been suggested as a method to treat patients with Parkinson's disease. Even though the brain is an immunologically privileged site, neuronal xenografts are usually rejected within a few weeks. T cells are important for this process, but the exact cellular events leading to rejection are poorly characterized. Brain cells from ventral mesencephalon of 26-27-day-old pig embryos were used as target cells in flow cytometry-assessed cytotoxicity assays using non-and IL-2-activated CD3-CD16+CD56+ human natural killer (NK) cells as effector cells. The ability of human NK cells to kill pig embryonic brain cells by antibody-dependent cellular cytotoxicity (ADCC) in the presence of nondepleted and anti-Gala1,3Gal antibody-depleted human blood group AB serum (AB serum) was evaluated using the same assay. Both nondepleted and anti-Gala1,3Gal antibody-depleted AB serum could mediate ADCC of pig embryonic VM cells when human NK cells were used as effector cells. Nonactivated NK cells did not show any direct cytotoxic effect on freshly isolated VM cells, whereas IL-2-activated NK cells killed approximately 50% of the VM cells at an effector-to-target ratio of 50:1 in a 4-h cytotoxicity assay. Activation of VM cells by TNF-a did not change their sensitivity to human NK cell cytotoxicity. Human NK cells may thus contribute to a cellular rejection of pig neuronal xenografts by ADCC, or following IL-2 activation, by a direct cytotoxic effect.

Key words: Brain tissue transplantation; Transplantation; Xenogeneic; Natural killer cells; Parkinson's disease

Address correspondence to Jan Holgersson, M.D., Ph.D., Division of Clinical Immunology, F79, Karolinska Institutet, Huddinge University Hospital, SE-141 86 Huddinge, Sweden. Tel: +46 8 585 81384; Fax: +46 8 585 81390; E-mail: jan.holgersson@cbt.ki.se



Cell Transplantation, Volume 8, pp. 611-616, 1999
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Viability of Periosteal Tissue Obtained Postmortem

Shawn W. O'Driscoll, Borna Meisami, Yasushi Miura, and James S. Fitzsimmons

The Cartilage and Connective Tissue Research Laboratory, Mayo Clinic, Mayo Foundation, Rochester, MN

Periosteal autografts have the potential to regenerate articular cartilage defects, but this potential is limited by the patient's age. Allograft transplantation from a young donor to an older recipient might bypass this limitation. The effect of the time delay, between death and harvesting of a periosteal graft, on the chondrogenic potential of periosteum is important not only for transplantation but also for studies dealing with tissues retrieved postmortem (i.e., including the periosteal explant model). The purpose of this study was to investigate the chondrogenic potential of periosteum obtained postmortem and a possible beneficial effect of hypothermia. Thirty NZ white rabbits (2 months old) were sacrificed and stored at room temperature or 4°C for 0, 4, 6, 8, 12, 16, 18, or 24 h. Periosteal explants were then obtained and a standard cartilage yield assay performed by culturing them for 6 weeks using the periosteal organ culture model as previous published. TGF-b1 (10 ng/ml) was added for the first 14 days of culture. Histochemical analysis and quantitative collagen typing were performed. In the explants from the animals kept for 4 h at room temperature growth and chondrogenesis were dramatically reduced. Little or no chondrogenesis was seen in explants from rabbits maintained at room temperature after 4-8 h (or more) postmortem. Cooling the rabbits to 4°C partially prevented this loss of viability and continued to do so for 24 h. Even storage at 4°C did not eliminate the decrease in chondrogenic potential, though it did permit partial preservation of chondrogenic potential. If periosteum is to be used for allograft transplantation, or if it is used for experimental study, its viability must be assured. This is best accomplished by harvesting it immediately postmortem. Preservation techniques, cryopreservation, or hypothermia might be useful in preserving periosteal chondrogenic potential.

Key words: Articular cartilage repair; Periosteum; Chondrogenesis transplant; Tissue engineering

Address correspondence to Shawn W. O'Driscoll, Ph.D., M.D., Director, Cartilage and Connective Tissue Research Laboratory, Mayo Clinic, Mayo Foundation, Rochester, MN 55905. Tel: (507) 284-2266; Fax: (507) 284-5075; E-mail: odriscoll.shawn@mayo.edu



Cell Transplantation, Volume 8, pp. 617-625, 1999
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Subcutaneous Transplantation of Bovine and Human Adrenocortical Cells in Collagen Gel in scid Mice

Nikolay K. Popnikolov and Peter J. Hornsby

Huffington Center on Aging and Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030

Adrenocortical cells of bovine origin and of adult and fetal human origin were transplanted subcutaneously (SC) in scid mice after being embedded in collagen gel. In this site the cells survived, became vascularized by invasion of host endothelial cells, and secreted steroids into the circulation. The animals' own adrenal glands were removed at the time of cell transplantation. Steroids secreted by the transplants replaced the essential functions of the animals' own adrenal glands. Adrenalectomized animals without transplanted cells died after several days, but most animals with transplanted bovine or adult human adrenocortical cells survived; fewer animals survived with transplanted fetal human adrenocortical cells. The histology of the tissues formed from transplanted cells resembled that of the normal adrenal cortex. A few proliferating cells were observed in tissue from bovine or adult human cells; there was a greater percentage of dividing cells in tissue derived from fetal cells. Subcutaneous transplantation of bovine or human primary adrenocortical cells in collagen provides a model for the study of the physiology, cell biology, and molecular biology of adrenocortical cells in a three-dimensional vascularized tissue structure in a host animal.

Key words: Adrenocortical cells; Collagen; Proliferation; Vascularization

Address correspondence to Peter J. Hornsby, Ph.D., Huffington Center on Aging, Baylor College of Medicine, 1 Baylor Plaza M320, Houston, TX 77030. Tel: (713) 798-7334; Fax: (713) 798-6688; E-mail: phornsby@bcm.tmc.edu



Cell Transplantation, Volume 8, pp. 627-636, 1999
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Immunosuppression and Rejection of Cartilage Formed by Allogeneic Chondrocytes in Rats

Anna Osiecka-Iwan, Anna Hyc, and Stanislaw Moskalewski

Department of Histology and Embryology, Medical University of Warsaw, Pl-02004 Warsaw, Poland

Rat syngeneic and allogeneic chondrocytes were transplanted intramuscularly or into defects prepared in articular cartilage (intracartilaginous transplants). Recipients of allogeneic transplants received cyclosporin A (CsA), cladribine (2-chlorodeoxyadenosine, 2-CdA), or both drugs in combination. Transplants were taken for examination after 5 weeks. Cartilage formed intramuscularly by syngeneic chondrocytes was ossified. Allogeneic cartilage was resorbed by infiltrating cells. CsA or 2-CdA partially suppressed, and both these agents in combination strongly suppressed, formation of infiltrations. Both syngeneic and allogeneic chondrocytes formed cartilage in joint surface defects but only allogeneic cartilage was attacked by infiltrating cells. CsA + 2-CdA treatment slightly decreased intensity of infiltrations but did not prevent cartilage resorption. Antichondrocyte response was studied by evaluation of spleen mononuclear cells (SMC) stimulation in mixed splenocyte-chondrocyte cultures and by detection of antichondrocyte cytotoxic antibodies. SMC stimulation index (SI) was calculated separately for syngeneic and allogeneic chondrocytes. Comparison of SMC SI for syngeneic and allogeneic chondrocytes indicated lack of stimulation of SMC from control or syngeneic transplant recipients and significant stimulation of SMC from recipients of allogeneic transplants. SMC from animals treated with CsA + 2-CdA were not stimulated. Additional experiments aiming at an explanation of the lack of stimulation of SMC from intact animals by syngeneic chondrocytes reported in this work and contrary to other findings disclosed that it was caused by the use of collagenase solution containing Na-p-tosyl-l-lysine chloromethyl ketone for chondrocyte isolation. Spontaneous antichondrocyte cytotoxic antibody activity was found in intact rats raised only in sera from recipients of allogeneic intramuscular transplants without immunosuppression. Thus, strong immunosuppressive treatment of rats with allogeneic chondrocyte transplants was more effective in relation to the general immunological response than to the local reaction.

Key words: Joint surface defects; Chondrocyte transplantation; Immunosuppressive treatment; Antichondrocyte immunological response

Address correspondence to Stanislaw Moskalewski, Department of Histology and Embryology, Medical University of Warsaw, Chalubinskiego 5, PL-02004 Warsaw, Poland. Fax: 0048 22629-52-82; E-mail: smosk@ib.amwaw.edu.pl



Cell Transplantation, Volume 8, pp. 637-647, 1999
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Pretreatment With Glucocorticoids Enhances T-Cell Effector Function: Possible Implication for Immune Rebound Accompanying Glucocorticoid Withdrawal

Wassim Y. Almawi,1 David A. Hess,3 Joumana W. Assi,2 Dagmara M. Chudzik,2 and Michael J. Rieder3

1Section of Molecular Biology, Department of Laboratory Medicine, St. George-Orthodox Hospital, Beirut, Lebanon
Departments of 2Medicine and 3Pharmacology, University of Western Ontario, London, Ontario, Canada

Glucocorticoids (GCs) exert their immunosuppressive/antiproliferative effects largely through inhibition of cytokine expression, and paradoxically upregulate the expression of (proinflammatory) cytokine receptors on select nonlymphoid cells. Clinically, withdrawal of GCs was frequently associated with worsening of the outcome of heightened immunity disorders, thereby implicating enhanced cytokine and cytokine receptor expression as a possible consequence of acute/short-term GCs withdrawal. In view of the significance of this complication of GC therapy, we addressed the effect of GC withdrawal on cytokine receptor expression and subsequent T-cell effector function, using the proliferation of human T cells as biological readout. To mimic GC withdrawal, T cells were treated with GCs or controls, stimulated, and incubated for 16-20 h at 37°C, washed, and reactivated for a further 4-48 h. Surface marker expression was assessed by FACS analysis, and proliferation was determined by measuring the cellular uptake of tritiated thymidine. Dexamethasone (DEX) and prednisolone (PRED), in a concentration-dependent manner, inhibited T-cell proliferation induced by anti-CD28 Ab + PMA. However, pretreatment of T cells activated with mitogens, cross-linking antibodies, or PMA + ionomycin ("CD3-bypass" stimulation regimen), but not resting T cells, with DEX or PRED resulted in a marked increase in IL-1R, IL-2Ra, and IL-6R expression, which was accompanied by a significant enhancement in T-cell proliferation. This effect of GCs was neither stimulus specific nor did it result from alteration in cell viability, and was paralleled by augmentation in cytokine (rIL-2) effects on DEX-pretreated and preactivated T cells. Taken together, our results underline the dual effects of GCs in regulating T-cell activation and cytokine expression. In essence, GCs directly inhibited T-cell proliferation by suppressing cytokine production, and, by enhancing cytokine receptor expression, pretreatment with GCs augmented T-cell proliferation.

Key words: Glucocorticoids; T cells; Cytokines; Transplantation; Dexamethasone; Prednisolone

Address correspondence to Dr. Wassim Y. Almawi, Section of Molecular Biology, Department of Laboratory Medicine, St. George-Orthodox Hospital, P.O. Box 166378-6417, Beirut, Lebanon. Tel: +961-3-812861; Fax: +961-1-582560.



Cell Transplantation, Volume 8, pp. 649-659, 1999
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Xenogeneic Transplantation of Porcine Hepatocytes Into the CCl4 Cirrhotic Rat Model

Andreas M. Stefan,2 Sarah Coulter,1 Brian Gray,1 Wayne LaMorte,3 Sigfus Nikelaeson,4 Albert S. B. Edge,1 and Nezam H. Afdhal2

1Department of Molecular and Cellular Biology, Diacrin, Inc.
Departments of 2Medicine, 3Surgery, and 4Pathology, Boston Medical Center and Boston University School of Medicine, Boston, MA

Liver support using extracorporeal devices and hepatocyte transplantation has received renewed interest for the management of acute and chronic liver failure. The aim of this study was to determine whether xenogeneic porcine hepatocytes could integrate into the liver parenchyma of cirrhotic Lewis rats when administered by an intrasplenic route. Cirrhosis was induced by carbon tetrachloride (CCl4) inhalation and confirmed histologically. Freshly isolated porcine hepatocytes were infused directly into the splenic pulp at laparotomy over a 5-15-min interval. Using 111In-labeled hepatocytes, the degree of localization of porcine hepatocytes to the spleen and liver was found to be greater than 60% in both control and cirrhotic rats. Integration of porcine hepatocytes into the rat liver parenchyma was determined by immunohistochemical staining for porcine albumin in rat liver sections. Further confirmation was provided by in situ hybridization using a porcine-specific probe that binds to a distinct repetitive element (PRE) in porcine DNA. Evidence of integrated porcine hepatocytes was seen for over 50 days in animals under cyclosporine immunosuppression. These data demonstrate the integration of xenogeneic porcine hepatocytes into the liver of the cirrhotic rat and their ability to produce porcine albumin for up to 50 days.

Key words: Xenotransplantation; Albumin; Cyclosporine; Intrasplenic infusion

Address correspondence to Nezam H. Afdhal, M.D., Chief of Hepatology, Boston Medical Center, Evans 201, 88 E. Newton Street, Boston, MA 02118. Tel: (617) 638-8353; Fax: (617) 638-6529; E-mail: nafdhal@bu.edu



Cell Transplantation, Volume 8, pp. 661-671, 1999
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Efficient Gene Transfer and Expression in Islets by an Adenoviral Vector That Lacks All Viral Genes

Zhiguang Guo,1 Jikun Shen,1 Deepak Mital,1 Yuanze Hong,1 Ramon Alemany,2 Wei-Wei Zhong,2 Stephen C. Jensik,1 and James W. Williams1

1Department of General Surgery, Rush-Presbyterian-St. Lukes's Medical Center, Chicago, IL 60612
2Baxter Healthcare Corporation, Chicago, IL 60612

Although adenoviral vector-mediated gene transfer has significant potential for gene therapy, host immune responses to virally expressed proteins and small insert capacity may limit its clinical application. In order to overcome these disadvantages, a new adenoviral vector that lacks all viral genes has been developed. Using the green fluorescent (GFP) gene as a reporter gene, we investigated the efficiency of gene transfer by this all-viral-genes-deleted and minimal cis-element remaining adenoviral vector (miniAd-GFP) in islets in vitro and ex vivo, and compared it with the E1-deleted adenoviral vector (E1-GFP). One day after in vitro infection, GFP was expressed in both miniAd-GFP-and E1-GFP-infected islets. The percentage of GFP-positive single cells was not significantly different between miniAd-GFP-infected islets and E1-GFP-infected islets. When these islets were transplanted into syngeneic diabetic mice, both miniAd-GFP-and E1-GFP-infected islet grafts reversed diabetes, and normal blood glucose levels were maintained for over 20 weeks posttransplantation. Mild lymphocyte infiltration was found in all E1-GFP-infected islet grafts at all time points. However, this was not seen in most miniAd-GFP-infected islet grafts. Our results indicate that gene transfer by an adenoviral vector that lacks all viral genes is as efficient as E1-deleted adenoviral vector-mediated gene transfer in islets. Furthermore, this adenoviral vector might be less immunogeneic than the E1-deleted adenoviral vector.

Key words: Islet graft; Adenoviral vector; Gene transfer; Immune response

Address correspondence to Zhiguang Guo, Department of Surgery, Box 195, University of Minnesota, 420 Delaware Street S.E., Minneapolis, MN 55455. Tel: (612)-626-5471; Fax: (612)-626-5855; E-mail: zhiguang@tc.umn.edu



Cell Transplantation, Volume 8, pp. 673-689, 1999
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Differentiation and Expansion of Beta Cell Mass in Porcine Neonatal Pancreatic Cell Clusters Transplanted Into Nude Mice

Kun-Ho Yoon,* Robert R. Quickel,* Krystyna Tatarkiewicz, Thomas R. Ulrich, Jennifer Hollister-Lock, Nitin Trivedi, Susan Bonner-Weir, and Gordon C. Weir

Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA
Department of Medicine, Harvard Medical School

Neonatal porcine pancreas has considerable capacity for growth and differentiation, making it an attractive potential source of islet tissue for xenotransplantation. Pancreases from 1-3-day-old newborn pigs were digested with collagenase and cultured for 8 days. The resulting cellular aggregates are called porcine neonatal pancreatic cell clusters (NPCCs). The mean yield of NPCCs from a newborn pig was 28,200 ± 1700 islet equivalents. Cytokeratin 7 (CK7) was used as a marker for the immunostaining of pancreatic duct cells. In neonatal pancreas, 18% of the insulin-positive cells co-stained for CK7, thus being protodifferentiated. NPCCs also contained protodifferentiated cells; insulin/PP and insulin/somatostatin co-stained cells were more common than insulin/glucagon cells. Between 1 and 8 days of culture, the DNA content of the NPCCs fell to 16% and the insulin content to 33% of the starting value, mainly due to the preferential loss of exocrine cells. Transplantation of 2000 or 4000 NPCCs into diabetic nude mice typically normalized glucose values in 10-20 weeks. Mice with successful grafts had lower fasting blood glucose levels than normal mice and accelerated glucose clearance after an IP glucose load. The starting NPCCs consisted of 17% insulin-staining cells, but the grafts of mice with reversed diabetes consisted of 94% beta cells, with some co-stained for CK7, indicating that the grafts still contained immature cells. The mass of insulin-producing cells rose from 0.22 ± 0.08 mg 1 week after transplantation to 4.34 ± 0.27 mg in mice sacrificed at 27-35 weeks. In summary, NPCCs contain mostly islet precursor cells, which when transplanted into nude mice undergo striking differentiation and beta cell expansion.

Key words: Islet transplantation; Xenotransplantation; Neonatal pancreatic cell clusters; Porcine islets; Beta cell expansion; Islet differentiation

Address correspondence to Gordon C. Weir, M.D., Section of Islet Transplantation and Cell Biology, Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel: (617) 732-2581; Fax: (617) 732-2650; E-mail: Gordon.Weir@joslin.harvard.edu

*Drs. Quickel and Yoon are co-first authors and contributed equally to the work.



Cell Transplantation, Volume 8, pp. 691-698, 1999
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Secretion From Islets and Single Islet Cells Following Cryopreservation

Jonathan R. T. Lakey,1 Craig A. Aspinwall,2 Thomas J. Cavanagh,3 and Robert T. Kennedy2

1Surgical-Medical Research Institute, University of Alberta, Edmonton, Alberta, Canada
2Department of Chemistry, University of Florida, Gainesville, FL
3Roche Molecular Biochemicals, Indianapolis, IN

The ability to cryopreserve pancreatic islets has allowed the development of low-temperature banks that permit pooling of islets from multiple donors and allows time for sterility and viability testing. However, previous studies have shown that during cryopreservation and thawing there is a loss of islet mass and a reduction in islet function. The aim of this study was to measure and compare insulin secretion from cultured nonfrozen and frozen-thawed canine islets and b-cells. Canine islets were isolated from mongrel dogs using intraductal collagenase distention, mechanical dissociation, and EuroFicoll purification. One group of purified islets was cultured overnight before dissociation into single cells and subsequent analysis. Remaining islets were cultured overnight (22°C) and then cryopreserved in 2 M dimethyl sulfoxide (DMSO) solution using a slow stepwise addition protocol with slow cooling to -40°C before storage in liquid nitrogen (-196°C). Frozen islets were rapidly thawed (200°C/min) and the DMSO removed using a sucrose dilution. From a series of seven consecutive canine islet isolations, islet recovery following postcryopreservation tissue culture was 81.5 ± 4.8% compared to precryopreservation counts. In vitro islet function was equivalent between cultured nonfrozen and frozen-thawed islets with a calculated stimulation index of 10.4 ± 1.5 (mean ± SEM) for the frozen-thawed islets, compared with 12.4 ± 1.2 for the cultured nonfrozen controls (p = ns, n = 7 paired experiments). Amperometric detection of secretion from single b-cells in vitro has the sensitivity and temporal resolution to detect single exocytotic events and allows secretion to be monitored from single b-cells in real time. Secretion from single b-cells elicited by chemical stimulation was detected using a carbon fiber microelectrode. The frequency of exocytosis events was equivalent between the cultured nonfrozen and frozen-thawed b-cells with an average of 7.0 ± 1.32 events per stimulation for the cultured nonfrozen group compared with 6.0 ± 1.45 events from the frozen then thawed preparations (minimum of 10 cells per run per paired experiment, p = ns) following stimulation with tolbutamide. The average amount of insulin released per individual exocytosis event was equivalent for the cultured nonfrozen and frozen-thawed islets. In addition, b-cells responded to both tolbutamide and muscarinic stimulation following cryopreservation. It was determined that b-cells recovered following cryopreservation are capable of secreting insulin at levels and frequencies comparable to those of cultured nonfrozen islet preparations.

Key words: Insulin secretion; Islet function; Cryopreservation; Amperometry

Address correspondence to Jonathan R. T. Lakey, Ph.D., Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, Alberta T6G 2N8 Canada. Tel: (780) 492-3386; Fax: (780) 492-1627; E-mail: jonathan.lakey@ualberta.ca



Cell Transplantation, Volume 8, pp. 699-708, 1999
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The Effects of Microencapsulation on Pancreatic Islet Osmotically Induced Volumetric Response

Erik J. Woods,1,2 Jun Liu,1 Michael A. J. Zieger,3 Jonathan R. T. Lakey,4 and John K. Critser1,2

1Cryobiology Research Institute, Wells Center for Pediatric Research, Indiana University Medical Center, Indianapolis, IN
2Department of Veterinary Clinical Science, Purdue University School of Veterinary Medicine, West Lafayette, IN
3Methodist Research Institute, Inc., Clarian Health Partners, Inc., Indianapolis, IN
4Comprehensive Tissue Centre, Department of Surgery, Laboratory Medicine and Pathology, University of Alberta, Alberta, Edmonton, Canada

Microencapsulation of pancreatic islets has been proposed as a means to prevent allograft rejection and to protect islets during cryopreservation. The aim of this study was to investigate: 1) the effects of the cryoprotectants (CPAs) dimethyl sulfoxide (DMSO) and ethylene glycol (EG) on the volume of Ca2+ alginate microcapsules, and 2) the effects of microencapsulation on the volumetric response of human and canine pancreatic islets during CPA equilibration. Stock sodium alginate with a high mannuronic acid content (HM) or a high guluronic acid content (HG) was used to generate empty capsules (mean diameter 200 mm) with an electrostatic generator. The capsules were held in place by a holding pipette system and videotaped during the addition of 2 or 3 M CPA at 22°C. Islets (isolated from human cadaveric donors and mongrel dogs and then cultured overnight at 37°C) were encapsulated in alginate (HM), loaded into a microperfusion chamber, and the change in islet volume was videotaped after exposure to the same CPAs and concentrations. These were compared to the volume responses of nonencapsulated islets. Images were analyzed using a computerized image analysis system and the data were analyzed using ANOVA. HG microcapsules showed a significant (p < 0.05) increase in volume following exposure to EG but not to DMSO. HM microcapsule volume did not change significantly following exposure to either EG or DMSO and was therefore chosen as the substrate for islet encapsulation. Free, nonencapsulated canine and human islets responded to the osmotic challenge of the 2 M DMSO by shrinking to 70.00 ± 1.04% (mean ± SEM) and 70.11 ± 1.05%, and in 2 M EG to 72.89 ± 1.93% and 69.33 ± 1.38%, respectively, of the isotonic volume before returning to the original cell volume. Exposure to 3 M DMSO or EG resulted in a further dehydration to 65.89 ± 0.91.% and 67.67 ± 1.91% for canine and 62.22 ± 0.66.% or 65.89 ± 1.30% for human islets. Minimum volumes were reached within 30-40 s after exposure to the cryoprotectant. Encapsulated human islets reached 86.88 ± 1.47% of their original volume in 2 M and 80.33 ± 0.89% in 3 M DMSO, and 87.33 ± 1.86% in 2 M and 82.80 ± 1.57% in 3 M EG. This volume change was significantly less (p < 0.01) than that observed in corresponding free islets. Encapsulated canine islets reached 83.67 ± 2.13% of their original volume in 2 M and 78.22 ± 0.95% in 3 M DMSO, and 85.44 ± 1.92% in 2 M and 78.11 ± 2.01% in 3 M EG. As with human islets, this was significantly different than free islets (p < 0.01). These minimal volumes were reached within 30-50 s. These results demonstrate that there are cryoprotectant and alginate-specific interactions and that microencapsulation modulates the degree of osmotically induced shrinkage of islets. The development or modification of existing cryopreservation protocols to improve postcryopreservation recovery or function must account for these factors.

Key words: Encapsulation; Pancreatic islet; Osmotic effects; Cryopreservation

Address correspondence to John K. Critser, Ph.D., Cryobiology Research Institute, Wells Center for Pediatric Research, Rm. 454, Indiana University School of Medicine, Cancer Research Building, 1044 West Walnut St., Indianapolis, IN 46202. Tel: (317) 278-3216; Fax: (317) 274-8928.



Cell Transplantation, Volume 8, pp. 709-712, 1999
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Lessons From In Vitro Perifusion of Pancreatic Islets Isolated From 80 Human Pancreases

F. Bertuzzi,1 P. Garancini,2 C. Socci,1 R. Nano,1 M. V. Taglietti,3 M. Santopinto,1 V. Di Carlo,1 and A. M. Davalli4

Istituto Scientifico San Raffaele, 1Department of Surgery, 2Unit of Medical Statistic, 3Department of Internal Medicine, 4Unit of Metabolic Diseases, University of Milan, Milan, Italy

We report the average insulin response to acute glucose measured by in vitro perifusion of pancreatic islets isolated from 80 consecutive human organs. Different perifusion parameters were considered [basal release, stimulation index (SI), time to peak, incremental area under the curve (D-AUCa)], and the correlation among them was determined. SI positively correlated with D-AUCa (p < 0.001, r = 0.80) while negatively with time to peak (p < 0.05, r = -0.23). We also evaluated several variables of the isolation procedure that might affect responsiveness to glucose by human islets. Sex and age of pancreas donors, cold ischemia time, duration of the digestion, collagenase concentration, and lot characteristics (collagenase, trypsin, clostripain, and proteases activity), and final islet yield were considered. Multivariate regression analysis showed only an independent association between SI and the concentration of collagenase (p = 0.01).

Key words: Perifusion parameters; Insulin response; Pancreatic islets;

Address correspondence to Dr. Federico Bertuzzi, M.D., Istituto Scientifico San Raffaele, Via Olgettina 60, 20132, Milan, Italy. Fax: 39-02-26432961; E-mail: bertuzzi.federico@hsr.it