ognizant Communication Corporation


VOLUME 9, NUMBER 3, 2000

Cell Transplantation, Vol. 9, pp. 297-305, 2000
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Isolation of Adult Porcine Islets of Langerhans

Christian Toso,1 Daniel Brandhorst,2 José Oberholzer,1 Frédéric Triponez,1 Léo Bühler,1 and Philippe Morel1

1Division of Surgical Research, Clinic for Digestive and Transplant Surgery, University Hospital, 1211 Geneva 14, Switzerland
2Third Medical Department, Justus Liebig University, 35385 Giessen, Germany

While human islet allotransplantation is limited due to the shortage of donors, pig islet xenotransplantation appears to be a possible alternative. Enzymatic digestion is the method most commonly used for adult pig islet isolation, but since the description of the semiautomated isolation technique in the late 1980s, yields of pig islet isolations have not increased significantly. This review article is intended to collate published information on adult pig islet isolation to provide the reader with a clear overview of the relevant issues in this field, and to detail the current state of the art.

Key Words: Adult porcine islets; Islet isolation; Allotransplantation; Xenotransplantation

Address correspondence to Christian Toso, M.D., Division of Surgical Research, Clinic for Digestive and Transplant Surgery, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. Fax: ++ 41 22 372 77 55; E-mail: christian.toso@medecine.unige.ch

Cell Transplantation, Vol. 9, pp. 307-317, 2000
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In Vivo Persistence of Donor Cells Following Adoptive Transfer of Allogeneic Dendritic Cells in HIV-Infected Patients

Michael H. Shapero,1 Smriti K. Kundu,2 Edgar Engleman,3 Reiner Laus,1 Wim C. A. van Schooten,1 and Thomas C. Merigan2

1Dendreon Corporation, Seattle, WA 98121
2Center for AIDS Research, Stanford University Medical Center, Stanford, CA 94305
3Stanford Medical School Blood Center, Stanford, CA 94034

Peripheral blood samples from HIV-seropositive individuals enrolled in a pilot clinical trial investigating the use of allogeneic dendritic cell therapy were evaluated for mixed chimerism. In this study, dendritic cells from HLA-identical, HIV-seronegative siblings were used. Patients received an infusion of dendritic cells pulsed with HIV MN gp160 protein or with peptides from HLA-A2 restricted epitopes of env, gag, and pol proteins every month for 6-9 months. Of the five allogeneic dendritic cell recipients, two showed increases in HIV antigen-specific immune responses. Allele-specific polymorphisms were identified in three sib-pairs that allowed infused donor cells to be detected using sensitive PCR-based molecular methods. Analysis of blood samples from patients showed similar patterns of donor cell persistence after the first infusion, in that cells were detectable for at least 1 week. Also, differences were observed in the kinetics of cell survival between the first and subsequent infusion cycles in all three patients. This suggests variation in HIV-specific immune responses detected among these three patients was not due to differences in persistence of infused donor cells.

Key words: Dendritic cells; Mixed chimerism; Allele-specific polymorphism; Cell survival

Address correspondence to Michael H. Shapero, Ph.D., at his current address: Affymax Research Institute, 4001 Miranda Avenue, Palo Alto, CA 94304. Tel: (650) 812-8871; Fax: (650) 424-0832; E-mail: michael_shapero@affymax.com

Cell Transplantation, Vol. 9, pp. 319-327, 2000
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In Vivo Expression of Human Growth Hormone by Genetically Modified Murine Bone Marrow Stromal Cells and its Effect on the Cells In Vitro

K. Suzuki,1 M. Oyama,1 L. Faulcon,1 P. D. Robbins,2 and C. Niyibizi1,3

1Musculoskeletal Research Center, Department of Orthopaedic Surgery, 2Molecular Genetics and Biochemistry, and 3Cell Biology and Physiology,
University of Pittsburgh School of Medicine, Pittsburgh, PA 15261

Human growth hormone (hGH) is frequently used clinically for growth abnormalities in children and also in adults with growth hormone deficiency. The hormone is usually administered to the individuals by frequent injections. In the present study we investigated the potential of bone marrow stromal cells as vehicles to deliver the GH in vivo by infusion of cells transduced with hGH cDNA into mice femurs. The effect of the hormone on the transduced cells in vitro was also assessed. Bone marrow stromal cells established from a mouse model of human osteogenesis imperfecta mice (oim) were transduced with a retrovirus containing hGH and neomycin resistance genes. The hGH-expressing cells were selected in a medium containing G418 and were then assessed for the hGH expression in vitro. The selected cells synthesized 15 ng/106 cells of hGH per 24 h in vitro and exhibited alkaline phosphatase activity when they were treated with the human recombinant bone morphogenetic protein 2 (rhBMP-2). The transduced cells also proliferated faster than the LacZ transduced cells but they did not exhibit a higher rate of matrix synthesis. When 2 x 106 hGH+ cells were injected into the femurs of mice, hGH was detected in the serum of the recipient mice up to 10 days after injection. The highest level of growth hormone expression, 750 pg/ml, was detected in the serum of the recipient mice 1 day after injection of the transduced cells. hGH was also detected in the medium conditioned by cells that were flushed from the femurs of the recipient mice at 1, 3, and 6 days after cell injection. These data indicate that bone marrow stromal cells could potentially be used therapeutically for the delivery of GH or any other therapeutic proteins targeted for bone. The data also suggest that GH may exert its effects on bone marrow stromal cells by increasing their rate of proliferation.

Key words: Growth hormone; Bone marrow; Gene therapy; Stromal cells

Address correspondence to Christopher Niyibizi, Ph.D., Collagen Biochemistry Laboratory, Musculoskeletal Research Center, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, 200 Lothrop St., PUH, RM C313, Pittsburgh, PA 15213. Tel: (412) 648-1091; Fax: (412) 648-8412; E-mail: niyi@pitt.edu

Cell Transplantation, Vol. 9, pp. 329-336, 2000
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Analysis of Multivariables During Porcine Liver Digestion to Improve Hepatocyte Yield and Viability for Use in Bioartificial Liver Support Systems

Lisheng Wang,1 Junhong Sun,1 Chuanmin Wang,1 Kevin Woodman,1 Li Li,1 Lujia Wu,1 Colin Harbour,2 Brendan Johnston,1 Liwei Shi,1 Margret Horvat,1 Nick Koutalistras,1 Xianfeng Luo,1 Jenny Watson,1 and A. G. R. Sheil1

1Australian National Liver Transplantation Unit, Royal Prince Alfred Hospital and Department of Surgery, and 2Department of Infectious Disease, University of Sydney, NSW 2006, Australia

In order to achieve optimal BALSS function, preparation of porcine hepatocytes with high yield, viability, and P450 activity is known to be important. To date hepatocyte yields have varied from 0.58 x 1010 to 3.45 x 1010 and viabilities from 75% to 95% within and between laboratories, even when using the same digestion methods and procedures, indicating that hepatocyte isolation during porcine liver digestion is not fully optimized. The aim of this work was to identify the critical parameters affecting cell recovery during porcine liver harvesting by investigating 21 variables involved in the process, including pig body and liver weight, different digestion times of perfusates, pH, a range of concentrations of sodium and chloride in EDTA, and collagenase perfusates. Univariate and multivariate analysis of a retrospective study (n = 23) revealed that low perfusate pH during the process of digestion had a positive effect on hepatocyte yield (p < 0.05), while high (relative) concentrations of sodium and chloride in the perfusates had significant negative effects on hepatocyte viability (both p < 0.05). Sodium and chloride had narrow optimal ranges for achieving a >90% viability. These findings were then tested in a prospective study (n = 10) and further verified. High hepatocyte viabilities (91.8 ± 1.6%, p = 0.036) and yields (2.56 ± 0.48 x 1010) were achieved consistently, and P450IA1 activity was increased after sodium and chloride concentrations and pH in the perfusates were controlled. The physiological mechanism by which sodium and chloride affects hepatocyte viability during porcine liver digestion is discussed.

Key words: Bioartificial liver; Porcine; Hepatocyte isolation; Viability; P450IA1

Address correspondence to Professor A. G. R. Sheil, Department of Surgery, University of Sydney, NSW 2006, Australia. Tel: 61-2-93512107; Fax: 61-2-93513361.

Cell Transplantation, Vol. 9, pp. 337-348, 2000
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Determination of the Prime Electrostatic Endothelial Cell Transplantation Procedure for e-PTFE Vascular Prostheses

Gary L. Bowlin,1 Stanley E. Rittgers,2,3 Steven P. Schmidt,1 Thomas Alexander,4 Daniel B. Sheffer,3 and Amy Milsted5

1Department of Biomedical Engineering, Virginia Commonwealth University, P.O. Box 980694, Richmond, VA 23298-0694
2William H. Falor Center for Vascular Studies, Akron City Hospital, Summa Health System, Akron, OH 44309
3Department of Biomedical Engineering, The University of Akron, Akron, OH 44325-0302
4Department of Pathology, Akron City Hospital, Summa Health System, Akron, OH 44309
5Department of Biology, The University of Akron, Akron, OH 44325

The purpose of this study was to evaluate the extent of cellular adhesion (density and morphological maturation), cellular membrane damage, and cellular viability after an electrostatic transplantation of human umbilical vein endothelial cells (HUVECs) onto 6-cm segments of 4-mm I.D. e-PTFE (GORE-TEX®) vascular prostheses using a prototype electrostatic endothelial cell transplantation device (EECTD). The electrostatic transplantation parameters evaluated were the apparatus-applied voltage and transplantation time. By our definition, the combination of applied voltage and transplantation time that met the a priori criteria of: 1) maximum transplanted cellular viability, 2) maximum transplantation density, 3) maximum morphological maturation (degree of cellular flattening), and 4) minimal cellular membrane damage would be the prime transplantation procedure. The results of the experimentation indicated that the prime conditions for HUVEC transplantation were obtained when +1.0 V was applied for a transplantation time of 16 min. These conditions achieved an average viable graft surface coverage of 97.4 ± 1.6% with an average transplantation density of 73,540 ± 8,514 HUVECs/cm2. Furthermore, the transplanted HUVECs were morphologically mature (flattened) with minimal apparent cellular membrane damage (lysis or pitting). The overall clinical significance of this study is that viable endothelial cell transplantation to synthetic vascular grafts can be accomplished at high cellular densities and morphological maturation in 16 min using the EECTD. With the promising in vitro transplantation results, the next logical investigations will include additional in vitro evaluations (cellular retention upon shear stress exposure and biochemical assays) followed by in vivo evaluations to examine thromboresistance and influence on intimal/anastomotic hyperplasia.

Key words: Electrostatic endothelial cell transplantation device; Endothelial cell transplantation technique; Vascular prosthesis preparation

Address correspondence to Gary L. Bowlin, Ph.D., Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA 23298-0694. Tel: (804) 828-2592; Fax: (804) 828-4454; E-mail: glbowlin@saturn.vcu.edu

Cell Transplantation, Vol. 9, pp. 349-357, 2000
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Mechanical Barrier as a Protection Against Rejection of Allogeneic Cartilage Formed in Joint Surface Defects in Rats

Stanislaw Moskalewski, Anna Osiecka-Iwan, Anna Hyc, and Jaroslaw Jozwiak

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

Cartilage formed in transplants of allogeneic chondrocytes into joint cartilage defects in rats was infiltrated by immune cells migrating from the bone marrow while the surface on the side of the joint cavity remained free of infiltrations. This suggested that immunization occurred via bone marrow and not via joint cavity. Because articular cartilage is nourished exclusively by the synovial fluid, we have attempted to prevent cartilage rejection by protecting transplants from the contact with bone marrow. Defects in articular surface were filled with bone cement and chondrocytes were transplanted into a cavity prepared within the bone cement plug. Cartilage formed within the cement shell remained free of infiltrations and did not evoke systemic immunological response. However, distribution of glycosaminoglycans in the matrix of protected transplants was irregular. Cultures of chondrocytes growing in vitro on cement contained less glycosaminoglycans than the controls. This suggests that some factor(s) released from the cement unfavorably influenced chondrocytes and matrix production in protected transplants.

Key words: Articular cartilage; Allogenic chondrocytes; Joint defects; Mechanical barrier; Bone marrow

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, Vol. 9, pp. 359-368, 2000
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Comparison of Benefits on Myocardial Performance of Cellular Cardiomyoplasty With Skeletal Myoblasts and Fibroblasts

Kelley A. Hutcheson,2 B. Zane Atkins,2 Matthew T. Hueman,2 M. Benjamin Hopkins,1 Donald D. Glower,2,3 and Doris A. Taylor1,2,3

1Departments of 1Medicine, 2Surgery, and 3Biomedical Engineering, Duke University Medical Center, Durham, NC 27710

Cellular cardiomyoplasty (CCM), or introduction of immature cells into terminally injured heart, can mediate repair of chronically injured myocardium. Several different cell types, ranging from embryonic stem cells to autologous skeletal myoblasts, have been successfully propagated within damaged heart and shown to improve myocardial performance. However, it is unclear if the functional advantages associated with CCM depend upon the use of myogenic cells or if similar results can be seen with other cell types. Thus, we compared indices of regional contractile (systolic) and diastolic myocardial performance following transplantation of either autologous skeletal myoblasts (Mb) or dermal fibroblasts (Fb) into chronically injured rabbit heart. In vivo left ventricular (LV) pressure (P) and regional segment length (SL) were determined in 15 rabbits by micromanometry and sonomicrometry 1 week following LV cryoinjury (CRYO) and again 3 weeks after autologous skeletal Mb or dermal Fb transplantation. Quantification of systolic performance was based on the linear regression of regional stroke work and end-diastolic (ED) SL. Regional diastolic properties were assessed using the curvilinear relationships between LVEDP and strain (e) as well as LVEDP and EDSL. At study termination, cellular engraftment was characterized histologically in a blinded fashion. Indices of diastolic performance were improved following CCM with either Mb or Fb. However, only Mb transplantation improved systolic performance; Fb transfer actually resulted in a significant decline in systolic performance. These data suggest that both contractile and noncontractile cells can improve regional material properties or structural integrity of terminally injured heart, as reflected by improvements in diastolic performance. However, only Mb improved systolic performance in the damaged region, supporting the role of myogenic cells in augmenting contraction. Further studies are needed to define the mechanism by which these effects occur and to evaluate the long-term safety and efficacy of CCM with any cell type.

Key words: Cell transplantation; Myocardial performance; Fibroblasts; Myoblasts; Myocardial injury; Cellular cardiomyoplasty

Address correspondence to Doris A. Taylor, Ph.D., Duke University Medical Center, Box 3345, Durham, NC 27710. Tel: (919) 684-4484; Fax: (919) 684-8907; E-mail: dataylor@duke.edu

Cell Transplantation, Vol. 9, pp. 369-377, 2000
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Enhancement of Adult Muscle Regeneration by Primary Myoblast Transplantation

John F. DeRosimo,1* Charles H. Washabaugh,1 Martin P. Ontell,1 Monica J. Daood,2 Jon F. Watchko,2 Simon C. Watkins,1 Bill T. Ameredes,1 and Marcia Ontell1

1Department of Cell Biology & Physiology, and 2Department of Pediatrics and Magee Women's Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261

Extensor digitorum longus muscles (EDL) of SCID mice were induced to undergo degeneration-regeneration subsequent to orthotopic, whole-muscle transplantation. Two days after transplantation some of these muscles received injections of primary myoblasts derived from EDL muscles of transgenic mice, which express nuclear localizing b-galactosidase under the control of the myosin light-chain 3F promoter and enhancer. Nine weeks after transplantation, regenerated muscles that received exogenous myoblasts were compared to similarly transplanted muscles that received no further treatment and to unoperated EDL muscles in order to determine the effect of myoblast transfer on muscle regeneration. Many myofibers containing donor-derived myonuclei could be identified in the regenerated muscles that had received exogenous myoblasts. The mass of the muscles subjected to transplantation only was significantly less (31% less) than that of unoperated muscles. The addition of exogenous myoblasts to the regenerating EDL resulted in a muscle mass similar to that of unoperated muscles. The absolute twitch and tetanic tensions and specific twitch and tetanic tensions of transplant-only muscles were 28%, 36%, 32%, and 41%, respectively, of those of unoperated muscles. Myoblast transfer increased the absolute twitch and tetanic tensions of the regenerated muscles by 65% and 74%, respectively, and their specific twitch and tetanic tensions were increased by 41% and 48%, respectively. These data suggest a possible role for the addition of exogenous, primary myoblasts in the treatment of traumatized and/or diseased muscles that are characterized by myofiber loss.

Key words: Striated muscle; Muscle regeneration; Primary myoblasts; Myoblast transfer; Functional capacity

Address correspondence to Marcia Ontell, Ph.D., Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, South BST-S313, Pittsburgh, PA 15261. Tel: (412) 648-9567; E-mail: montell@pitt.edu

*Present address: Department of Surgery, Albany Medical Center.

Cell Transplantation, Vol. 9, pp. 379-393, 2000
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Exposure to Tissue Culture Conditions Can Adversely Affect Myoblast Behavior In Vivo in Whole Muscle Grafts: Implications for Myoblast Transfer Therapy

Gayle M. Smythe and Miranda D. Grounds

Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Western Australia, 6907

The effects of tissue culture conditions on the viability of myoblasts in whole muscles transplanted in vivo were investigated. Whole male (SJL/J) donor muscles were exposed to various tissue culture reagents and proteolytic enzymes, and allografted into female (SJL/J) host mice. Desmin immunohistochemistry was used to assess the numbers of myogenic cells (as an index of myoblast viability and the extent of regeneration) in tissue sections of whole-muscle grafts sampled on days 7 and 14. DNA quantitation with a Y-chromosome-specific probe was used to determine the total Y-1 sequence DNA (as an index of myoblast survival and proliferation) in whole-muscle grafts sampled on days 1, 3, and 7. In grafts exposed to serum-free medium, there was a delay in myoblast fusion at 7 days that was recovered by 14 days, but exposure to serum (10% or 20%) had a prolonged adverse effect on myotube formation at 14 days. DNA quantitation demonstrated that either serum-free culture medium or 10% serum enhanced the number of male cells within whole-muscle grafts at 7 days. Proteolytic digestion (even for 5 min) of whole muscles prior to grafting was extremely detrimental to myoblast survival and viability at 7 and 14 days. The unexpected finding of adverse effects of tissue culture conditions on the regeneration of whole-muscle grafts in vivo appears to parallel the major problem of the rapid death of isolated cultured donor myoblasts after injection in myoblast transfer therapy. The use of whole-muscle grafts provides an alternative and sensitive model to analyze the crucial effects of various tissue culture components on the subsequent survival and proliferation of myogenic cells in vivo.

Key words: Myoblast transfer therapy; Myoblast; Myotube; Tissue culture; Whole-muscle graft

Address correspondence to Dr. Gayle M. Smythe, Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Western Australia, 6907. Tel: 61 8 9380 7127; Fax: 61 8 9380 1051; E-mail: gsmythe@anhb.uwa.edu.au

Cell Transplantation, Vol. 9, pp. 395-407, 2000
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The Morphology, Integration, and Functional Efficacy of Striatal Grafts Differ Between Cell Suspensions and Tissue Pieces

Colin Watts,1,2 Peter J. Brasted,1 and Stephen B. Dunnett1,3

1MRC Cambridge Centre for Brain Repair, and the Departments of 2Neurosurgery and 3Experimental Psychology, University of Cambridge, Cambridge, UK

In order to develop a surgical protocol for use in clinical trials of striatal transplantation in Huntington's disease (HD), the issues involved in the preparation and implantation of the embryonic striatal tissue must be addressed. Rodent models of HD offer the best experimental paradigm with which to study various aspects of striatal transplantation. In this article we present the results of an investigation of the role of trypsin and the process of trituration in the preparation of cell suspensions compared to the use of solid pieces of tissue. The embryonic material was derived from the lateral ganglionic eminence (LGE) and implanted into the excitotoxically lesioned striatum of the host rats. Twelve weeks following implantation, retrograde tracing of projections from the graft to the globus pallidus was performed. Grafts derived from cell suspensions triturated in the presence of trypsin contained larger quantities of striatal tissue within the graft and more DARPP-32-positive medium spiny neurons than grafts implanted as fragments of tissue. Afferent and efferent connectivity was also better in the trypsinized suspension graft group. Modest recovery in paw reaching was observed contralateral to the grafted side in animals implanted with solid fragments of embryonic striatal tissue. No relationship was observed between functional effect and the graft anatomy. These results suggest that local graft-host interaction may also be involved in graft-mediated functional recovery.

Key words: Striatal transplantation; Cell suspensions; Solid tissue fragments; Huntington's disease

Address correspondence to Colin Watts, MRC Cambridge Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 2PY, UK. Tel: (+44) 1223 331160; Fax: (+44) 1223 331174; E-mail: cwclw@msn.com

Cell Transplantation, Vol. 9, pp. 409-414, 2000
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Islet Cell Transplantation: In Vivo and In Vitro Functional Assessment of Nonhuman Primate Pancreatic Islets

Alessandra Ranuncoli, Nicola Cautero, Camillo Ricordi, Michele Masetti, Ruth D Molano, Luca Inverardi, Rodolfo Alejandro, and Norma S Kenyon

Diabetes Research Institute, Cell Transplant Center, University of Miami School of Medicine, Miami, FL

Transplantation of pancreatic islets of Langerhans as a therapeutic approach for treatment of type I diabetes offers an alternative to subcutaneous insulin injections. Normalization of blood glucose levels by transplanted islets may prevent the development of diabetes-related complications. Problems related to rejection, recurrence of autoimmunity, and local inflammation upon transplantation of islets into the liver need to be solved before the implementation of islet cell transplantation can be viewed as a justifiable procedure in a large cohort of patients. Islet cell isolation has been quite successful in small animals, but the translation of this approach to nonhuman primates has been less rewarding. One of the main problems encountered in nonhuman primate models is the difficulty of isolating an adequate number of functional islets for transplantation. The aim of the present study was to develop a method for isolating a sufficient number of viable islets from nonhuman primates to allow for reversal of diabetes. By implementing minor modifications in the automated method for human islet isolation we were able to obtain viable, functional islets that responded normally to glucose stimulation in vitro. These islets were also able to reverse diabetes in immunocompromised nude mice, rendered diabetic by streptozotocin. This method of islet cell isolation has enabled us to proceed with protocols of allogeneic islet cell transplantation in preclinical, nonhuman primate models.

Key words: Nonhuman primate islets; Assessment; Yield; In vitro; In vivo

Address correspondence to Norma Sue Kenyon, Ph.D., Diabetes Research Institute, University of Miami School of Medicine, 1450 NW 10th Avenue (R-134), Miami, FL 33136. Tel: (305) 243-5346; Fax: (305) 243-1042; E-mail: nkenyon@med.miami.edu

Cell Transplantation, Vol. 9, pp. 415-422, 2000
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Effects of Short-Term Hypoxia on a Transformed Cell-Based Bioartificial Pancreatic Construct

K. K. Papas,1* R. C. Long, Jr.,2 I. Constantinidis,2 and A. Sambanis1

1School of Chemical Engineering and Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332
2Frederik Philips Magnetic Resonance Research Center, Department of Radiology, Emory University School of Medicine, Atlanta, GA 30322

Hypoxia is an adverse condition that can jeopardize the function of a bioartificial pancreatic construct. In this study we have investigated the effects of short-term hypoxic exposure (up to 24 h) on the bioenergetic status, metabolism, and insulin secretion of perfused pancreatic constructs composed of alginate/poly-L-lysine/alginate (APA) encapsulated mouse insulinoma bTC3 cells. The bioenergetic status of the encapsulated cells was monitored noninvasively with the aid of 31P NMR spectroscopy, while glucose, lactate, and insulin concentrations were measured with off-line assays from media samples removed from the perfusion loop. Our results demonstrate that in freshly prepared constructs insulin secretion was not affected by the hypoxic conditions, although intracellular ATP concentration decreased and glucose consumption increased. Alternatively, in constructs that were maintained in our perfusion system for at least 10 days, identical hypoxic conditions resulted in a decreased insulin secretion concomitant to a decreased intracellular ATP concentration and increased glucose consumption. These results suggest that the effects of hypoxia on a transformed cell-based pancreatic construct are not constant throughout the duration of an in vitro culture. The observed differences are attributed to the significant cell growth and rearrangement that occurs with time during an in vitro culture of the constructs.

Key words: bTC3; 31P NMR; Hypoxia; Bioartificial pancreas; Alginate encapsulation

Address correspondence to Ioannis Constantinidis, Emory University, Department of Radiology, 1364 Clifton Rd. NE, Room AG12, Atlanta, GA 30322. Fax: (404) 712-9704; E-mail: iconsta@emory.edu

*Present address: Novartis Institute for Biomedical Research, Novartis Pharmaceuticals Corporation, Core Technologies/Analytics-BioNMR, Summit, NJ 07901.

Cell Transplantation, Vol. 9, pp. 423-430, 2000
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Pretransplant Induction of HSP-70 in Isolated Adult Pig Islets Decreases Early Islet Xenograft Survival

Daniel Brandhorst, Hans-Peter Hammes, Heide Brandhorst, Anke Zwolinski, Fariborz Nahidi, Alexandra Alt, and Reinhard G. Bretzel

Third Medical Department, Justus-Liebig-University, 35385 Giessen, Germany

The heat-induced HSP-70 expression protects rat islet single cells against lysis mediated by nitric oxide (NO), reactive oxygen, and streptozotocin. The present study was performed to investigate the potential anti-inflammatory effect of pretransplant heat shock in adult pig islets for subsequent early islet xenograft survival. Maximum HSP-70 expression in freshly isolated pig islets was induced by hyperthermia at 43°C for 90 min prior to islet regeneration at 37°C for 4-6 h. Heat-stressed and sham-treated islets were incubated in 0.6 mM H2O2 or 1.5 mM Na-nitroprusside at 37°C for 20 h. Early graft survival was evaluated in normoglycemic Lewis rats after simultaneous, contralateral transplantation of heat-shocked islets and sham-treated islets into the renal subcapsular space of the same recipient. Prior hyperthermia significantly reduced specific lysis of islets exposed to NO or H2O2, although protection was only marginal. No differences were observed between viability of heat-shocked and sham-treated islets after NO exposure. In contrast, prior heat shock increased islet viability after H2O2 treatment. The finding that hyperthermia reduced recovery of initially grafted pig insulin 48 h after transplantation by 30% compared to controls contrasted significantly with an increased insulin recovery in heat-exposed islets at the end of simultaneous 37°C culture. The observation, that the heat-induced HSP-70 expression decreases early islet xenograft survival as reflected by recovery of grafted insulin, implies an enhancement of islet immunogenicity and the induction of apoptosis. Future experiments aiming at augmentation of intrinsic defense mechanims should consider detrimental effects associated with induction of heat shock proteins.

Key words: Porcine islet; HSP-70; Heat shock; Early islet graft survival

Address correspondence to Daniel Brandhorst, Ph.D., Third Medical Department, Justus Liebig University, Rodthohl 6, 35385 Giessen, Germany. Tel: +49-641-99-42847; Fax: +49-641-99-42849; E-mail: Daniel.Brandhorst@innere.med.uni-giessen.de

Cell Transplantation, Vol. 9, pp. 431-438, 2000
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Protection From Cell Death in Cultured Human Fetal Pancreatic Cells

Gillian M. Beattie,1 Gil Leibowitz,1,2 Ana D. Lopez,1 Fred Levine,1,2 and Alberto Hayek1

1Department of Pediatrics, the Whittier Institute for Diabetes, Cancer Center, and the 2Center for Molecular Genetics, UCSD La Jolla, CA

Endocrine cells from the human fetal pancreas will proliferate in vitro on extracellular matrix but lose hormone expression, and redifferentiation requires removal of the expanded cells from the matrix and reaggregation into cell aggregates. However, extensive cell death occurs during manipulation and culture. The mechanism of cell death was examined at each stage throughout the process under different experimental conditions to determine optimal protocols to increase cell viability. During shipment, the addition of trehalose to the media to prevent necrosis increased yield 17-fold, while during culture as islet-like cell clusters the apoptosis inhibitor Z-VAD increased yield 1.8-fold. Following disruption of cell-matrix interactions and reaggregation, there was marked evidence of apoptotic bodies by the TUNEL assay. Addition of nicotinamide or Z-VAD, or removal of arginine from the media during reaggregation, reduced the number of apoptotic bodies and the effect was additive. However, a combination of treatments was necessary to significantly increase the yield of viable cells. We conclude that cell death of human fetal pancreatic tissue in culture results from both necrosis and apoptosis and that understanding the mechanisms at the cellular level will lead to protocols that will improve cell viability and promote b-cell growth.

Key words: Apoptosis; Necrosis; Trehalose; Nicotinamide; Human fetal pancreas

Address correspondence to Dr. Alberto Hayek, The Islet Research Laboratory, Department of Pediatrics, UCSD, 9894 Genesee Ave., La Jolla, CA 92037. Tel: (858) 622-7298; Fax: (858) 558-3495; E-mail: ahayek@ucsd.edu

Cell Transplantation, Vol. 9, pp. 439-443, 2000
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Angiogenesis Induced by the Implantation of Self-Bone Marrow Cells: A New Material for Therapeutic Angiogenesis

Kimikazu Hamano,1 Tao-Sheng Li,1 Toshiro Kobayashi,1 Sei Kobayashi,2 Masunori Matsuzaki,3 and Kensuke Esato1

1First Department of Surgery, 2First Department of Physiology, and 3Second Department of Internal Medicine, Yamaguchi University School of Medicine, 1-1-1, Minamikogushi, Ube, Yamaguchi, 755-8505, Japan

Bone marrow contains various primitive cells that are thought to secrete several angiogenic growth factors and may also differentiate into endothelial cells. The present study was conducted to investigate the possibility that bone marrow cells could be a novel material to induce angiogenesis. The expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in rat bone marrow cells was examined by immunohistochemistry. The production of VEGF was compared in tissue culture supernatant under the conditions of normoxia and hypoxia. The process of angiogenesis that occurred following the implantation of bone marrow cells was determined using a rat cornea model. VEGF- and bFGF-positive cells were found in rat bone marrow. The production of VEGF from bone marrow cells was significantly more enhanced by hypoxic conditions than by normoxic conditions. The rat cornea model showed that bone marrow cell implantation created new vessels. The implantation of self-bone marrow cells is a novel and simple method of inducing angiogenesis.

Key words: Angiogenesis; Bone marrow cells; Ischemia

Address correspondence to Kimikazu Hamano, M.D., First Department of Surgery, Yamaguchi University School of Medicine, 1-1-1, Minamikogushi, Ube, Yamaguchi, 755-8505, Japan. Tel: +81-836-22-2261; Fax: +81-836-22-2260; E-mail: kimikazu @po.cc.yamaguchi-u.ac.jp