|ognizant Communication Corporation|
VOLUME 10, NUMBER 7
Cell Transplantation, Vol. 10, pp. 561-571
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
John M. Baust, Martin J. Vogel, Robert Van Buskirk, and John G. Baust
Institute of Biomedical Technology, State University of New York, Binghamton, NY 13902
The requirement for more effective cryopreservation (CP) methodologies in support of the emerging fields of cell bioprocessing and cell therapy is now critical. Current CP strategies appropriately focus on minimizing the damaging actions of physicochemical stressors and membrane disruption associated with extra- and intracellular ice formation that occurs during the freeze-thaw process. CP protocols derived from this conceptual paradigm, however, yield suboptimal survival rates. We now provide the first report on the identification of delayed-onset cell death following CP and the significance of modulating molecular biological aspects of the cellular responses (apoptosis) to low temperature as an essential component to improve postthaw outcome. In this study we quantitatively examined the molecular basis of cell death associated with CP failure in a canine renal cell model. In addition, we report on the significant improvement in CP outcome through the modulation of these molecular mechanisms by the utilization of an organ preservation solution, HypoThermosol®. Further, the utilization of HypoThermosol® as the preservation medium and the modulation of molecular-based cell death have led to a paradigm shift in biologic preservation methodologies. The recognition of molecular mechanisms associated with CP-induced cell death offers the promise of improved CP of more complex and/or fragile biological systems such as stem cells, engineered tissues, and human organs.
Key words: Apoptosis; Hypothermia; HypoThermosol®; CryoStor; Annexin V; Cryopreservation
Address correspondence to John M. Baust, Institute of Biomedical Technology, Science III Suite 144, Binghamton University, Binghamton, NY 13902-6000. Tel: (607) 777-4415; Fax: (607) 777-2803.
Jan Koopmans,1 Ineke HogenEsch,2 Sjef Copray,3 Berrie Middel,4 Henk van Dijk,5 Kian-Gwan Go,1 and Michiel Staal1
1Department of Neurosurgery, University Hospital Groningen,
Hanzeplein 1, 9700 RB Groningen, The Netherlands
2Department of Neurology, Fylkessjukehuset, N-5500 Haugesund, Norway
3Department of Medical Physiology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
4Department of Health Sciences, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
5Department of Veterinary Anatomy and Physiology, University of Utrecht, Yalelann 1, 3584 CL, The Netherlands
In this study we examined the efficacy of cryopreserving porcine fetal mesencephalic tissue. After microscopical dissection of the ventral mesencephalon (VM) from E28 pig fetuses, the collection of explants was randomly divided into two equal parts. One part was directly prepared as cell suspension. The other part was stored in hibernation medium for less than 2 days and then cryopreserved as tissue fragments and stored in liquid nitrogen. After 2 weeks up to 1 year, these tissue fragments were thawed and processed as cell suspensions. After cell counting and assessment of viability, these cell suspensions were used to examine survival, morphology, and neurite formation of the dopaminergic neurons in cell culture as well as after intrastriatal implantation in 6-OHDA-lesioned rats. Comparison of cryopreserved with fresh VM cell suspensions showed no significant difference with respect to cell viability and the average number of living cells per VM explant. The morphology of cultured dopaminergic neurons after cryopreservation was identical to that of fresh cells. After intrastriatal implantation, survival and outgrowth of cryopreserved dopaminergic neurons as well as functional effects did not differ from those of fresh cells. In conclusion, the cryopreservation technique we used proves to be a reliably effective method for storing porcine fetal VM tissue.
Key words: Parkinson's disease; Transplantation; Cryopreservation; Porcine; Fetal; Ventral mesencephalon
Address correspondence to J. Koopmans, Department of Neurosurgery, University Hospital Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands. Tel: +31-50-3613218; Fax: +31-50-3611715; E-mail: J.Koopmans@nchir.azg.nl
Islet Cryopreservation Using Intracellular Preservation Solutions
Jonathan R. T. Lakey,1,2 Ray V. Rajotte,1,2,3 Christine A. Fedorow,1 and Michael J. Taylor4,5
1Surgical-Medical Research Institute, Departments of 2Surgery
and 3Medicine, University of Alberta, Edmonton, Alberta, Canada
4Organ Recovery Systems Inc., Charleston, SC
5Allegheny University of the Health Sciences, Pittsburgh, PA
Cryopreservation of islets adds great flexibility to clinical islet transplant programs. Methods of islet cryopreservation have traditionally utilized permeating cryoprotectants contained within isotonic solutions without specifically addressing issues of ionic balances, buffering capacity, or oxygen free radicals that occur during hypothermic stresses. These factors may become significant issues during low-temperature storage and during the freezing and thawing process. Since its development in the early 1980s, the University of Wisconsin (UW) organ preservation solution has become the standard vascular flush and preservation solution. Recently, Hypothermosol preservation solution (HTS) was developed as a hypothermic blood substitute. The unique characteristics and composition of these preservation solutions may be important when developing solutions specific for the cryopreservation of cells and tissues. It was the aim of this study to evaluate these two hypothermic preservation solutions as the media used in cryopreservation of islets. Groups of canine islets [5000 islet equivalents (IE)/group] were cryopreserved using the standard protocol of stepwise addition of dimethyl sulfoxide (DMSO) to 2 M, controlled nucleation, slow cooling (0.25°C/min), and rapid thawing (200°C/min). The cryopreservation solutions were made with 1) UW solution, 2) HTS solution, or 3) Medium 199 solution with 10% fetal calf serum (FCS). Additional control groups included islets cryopreserved using 4) HTS, 5) UW solution, and 6) Medium 199 alone, without DMSO. Recovery of islets immediately following thawing was equivalent between the groups with the exception of the islets cryopreserved without DMSO (groups 4-6, p < 0.05). After 48 h of postcryopreservation tissue culture, islet recovery was highest in the groups frozen with UW and HTS (mean ± SEM) (79.8 ± 1.9% and 82.5 ± 1.5%, p < 0.05 vs. group 3, 69.1 ± 3.3%, p < 0.05, ANOVA). Less than 15% of the islets were recovered when they were cryopreserved without the cryoprotectant DMSO (groups 4-6). Functional viability was assessed by measuring the glucose-stimulated insulin secretion during static incubation after 48-h culture. The stimulation indexes were 4.6 ± 1.0, 4.2 ± 0.8, 3.6 ± 1.2, 0.6 ± 0.5, and 0.4 ± 0.2 for islets in groups 1-5, respectively. This study demonstrates that postcryopreservation survival can be improved using intracellular-based preservation solutions, including UW or HTS, in conjunction with DMSO.
Key words: Cryopreservation; Pancreatic islets
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) 492-3077; Fax: (780) 492-6335; E-mail: firstname.lastname@example.org
Survival of Macroencapsulated Allogeneic Parathyroid Tissue One Year After Transplantation in Nonimmunosuppressed Humans
Annika Tibell,1 Ehab Rafael,1 Lars Wennberg,1 Jörgen Nordenström,4 Mats Bergström,3 Robin L. Geller,5 Thomas Loudovaris,5 Robert C. Johnson,5 James H. Brauker,5 Steven Neuenfeldt,5 and Annika Wernerson2
Departments of 1Transplantation Surgery, 2Pathology,
3Clinical Chemistry, and 4Surgery, Karolinska Institutet,
Huddinge University Hospital, Stockholm, Sweden
5Baxter Healthcare Corporation, Round Lake, IL
The use of immunoisolation devices may allow transplantation without need for immunosuppression and could widen the indications for cell transplantation. In this study, we evaluated the survival of encapsulated parathyroid tissue in nonimmunosuppressed humans. Autologous parathyroid implants: Seven patients undergoing parathyroidectomy had devices containing small pieces of their own parathyroid tissue implanted SC. These devices were explanted after 2-4 weeks for histological evaluation. Allogeneic parathyroid implants: Four patients with chronic hypoparathyroidism were transplanted with one to three large (40 ml) and one small (4.5 ml) device filled with meshed parathyroid tissue and implanted SC. The small devices were explanted at 4 weeks, while the large ones were explanted 8.5 to 14 months after implantation. In both studies, control implants were placed in nude mice. Autologous study results: At explantation, the grafts consisted of 22 ± 6% endocrine tissue and 63 ± 7% fibrosis, while 15 ± 5% of the grafts were necrotic. Allogeneic study results: In devices explanted from the patients at 4 weeks, fibrosis dominated and only 1%, 5%, and 23% of the grafts consisted of endocrine tissue. A similar histological appearance was found in grafts from nude mice. In devices explanted at 8.5-14 months, histologically intact endocrine tissue was found in all patients. However, nearly all the tissue consisted of fibrosis. There was no detectable increase in the parathormone (PTH) level in all patients. Macroencapsulated human allogeneic parathyroid tissue can survive up to 1 year after transplantation into nonimmunosuppressed patients. However, marked fibroblast overgrowth occurred, especially in the allogeneic implant study, using meshed parathyroid tissue. This was probably not related to the allo-response, because similar findings were observed in the nude mouse implants. In future studies, better tissue preparation and improvements in the physiological milieu inside the device may help to reduce fibroblast overgrowth and increase survival of the parathyroid cells.
Key words: Parathyroid transplantation; Macroencapsulation; Autograft; Allograft
Address correspondence to Annika Tibell, M.D., Ph.D., Department of Transplantation Surgery, Huddinge University Hospital, S-141 86 Huddinge, Sweden. Tel: + 46 8 58 58 76 64; Fax: + 46 8 774 31 91; E-mail: Annika.Tibell@karo.ki.se
R. Sarkis,1,2 J. Honiger,1 N. Chafai,1 M. Baudrimont,3 K. Sarkis,1 R. Delelo,1 L. Becquemont,4 S. Benoist,1,5 P. Balladur,1,2 J. Capeau,1 and B. Nordlinger1,5
1Research Unit 402, INSERM, Paris, France
Departments of 2Surgery, 3Pathology, and 4Pharmacology, Hôpital Saint-Antoine, Paris, France
5Department of Surgery, Hôpital Ambroise Paré Boulogne-Billancourt, France
We have previously demonstrated that fresh or cryopreserved xenogeneic hepatocytes manually macroencapsulated in AN69 polymer and transplanted intraperitoneally in rats were able to improve the survival rate after 95% hepatectomy without immunosuppression. In addition, we developed a semiautomatic device where porcine hepatocytes were coextruded with AN69 hydrogel in order to macroencapsulate large amounts of cells. The purpose of the present study was to 1) test whether transplanted porcine hepatocytes macroencapsulated in this device remained functional as evaluated by their ability to prevent death from acute liver failure, and 2) compare the efficiency of cryopreserved or freshly isolated hepatocytes. Fresh or cryopreserved porcine hepatocytes were macroencapsulated in the semiautomatic device by coextrusion in AN69 polymer in 2-m minitubes containing 6 x 107 cells. Acute liver failure was induced in rats by two-step 95% hepatectomy. At the time of completion of liver resection, rats were either not transplanted with minitubes (control group I, n = 13), or were implanted with two minitubes containing culture medium (control group II, n = 11), hepatocytes killed by heat treatment (control group III, n = 10), coextruded fresh hepatocytes (group IV, n = 11), or coextruded cryopreserved hepatocytes (group V, n = 11), without immunosuppression. The survival rate at day 7 was between 0% and 31% in the three control groups. By contrast, coextruded fresh hepatocytes significantly improved the survival rate (group IV, 82%) as did cryopreserved cells (group V, 91% survival). In surviving rats, minitubes were explanted after 20 days: either fresh or cryopreserved hepatocytes appeared morphologically viable and their ultrastructure was preserved. Their detoxification capacities evaluated by the activity of the cyt P450 CYP3A4 were partly maintained. In conclusion, porcine hepatocytes macroencapsulated by coextrusion using a semiautomatic device and transplanted without immunosuppression were able to prevent death from acute liver failure in rats. Cryopreserved cells were as efficient as fresh hepatocytes.
Key words: Xenogeneic transplantation; Porcine hepatocytes; Cryopreservation; Semiautomatic encapsulation; Hepatectomy; Survival rate; Ultrastructure; cyt P450
Address correspondence to Pr Jacqueline Capeau, INSERM U.402, Faculté de Médecine Saint Antoine, 27 rue Chaligny, 75571 Paris Cedex 12, France. Fax: (33) 1-40-01-13-32; E-mail: email@example.com
Partha Roy, Junji Washizu, Arno W. Tilles, Martin L. Yarmush, and Mehmet Toner
Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, and Shriners Hospitals for Children, Boston, MA 02114
Ethoxyresorufin-o-deethylation (EROD) can be used as a sensitive measure of hepatic detoxification function. In this study, we employed a fluorescence assay based on EROD to study the effect of varying Peclet number (or flow) on hepatic function in a microchannel flat-plate bioartificial liver (BAL) reactor containing a coculture of hepatocytes and fibroblasts. Static culture and reactor flow experiments established that: 1) a pseudo-steady-state detoxification rate could be attained at each Peclet number, 2) the steady-state detoxification rate increased nonlinearly with Peclet number (ranging from 167 to 2500), 3) the uptake rate of substrate was a linear function of cell surface substrate concentration (<1 mM), and 4) a shear stress of 10 dyne/cm2 did not adversely affect hepatic function for at least 12 h. A convection-diffusion-reaction model supports the conclusion that increased convective mass transfer of substrate to the cell surface is the primary cause of the observed increase in EROD rate with Peclet number. Our results suggest that detoxification rates can be enhanced by an order of magnitude by choosing an appropriate Peclet number. For our bioreactor configuration, this optimum corresponds to a Peclet number range of 1000-2000 at a Damkohler number of 0.55. The usefulness of the mathematical model is discussed in the context of scale-up to a clinical BAL reactor for human application.
Key words: Bioartificial liver; Cytochrome P4501A1; Detoxification; Flow; Mathematical model
Address correspondence to Mehmet Toner, Ph.D., Center for Engineering in Medicine, Massachusetts General Hospital, Bigelow 1401, 55 Fruit Street, Boston, MA 02114-2696. Tel: (617) 371-4876; Fax: (617) 371-4950; E-mail: firstname.lastname@example.org
Wassim Y. Almawi,1 Joumana W. Assi,2 Dagmara M. Chudzik,2 Maroun M. Abou Jaoude,4 and Michael J. Rieder3
1Department of Medical Biochemistry, Arabian Gulf University,
Departments of 2Medicine and 3Pharmacology & Toxicology, University of Western Ontario, London, Ontario, Canada
4Renal Transplantation Unit, St. Georges-Orthodox Hospital, Beirut, Lebanon
Insofar as it exerted its immunosuppressive effect by inhibiting cytokine expression, we assessed the effect of FK506 (Tacrolimus) on cytokine-stimulated T-cell activation. Human T cells, treated with FK506, or controls were stimulated with the mitogens PHA + PMA, Con A, and the "CD3-bypass" stimulation regimen, PMA + ionomycin. T-cell proliferation was quantitated by measuring the uptake of tritiated thymidine, and mRNA expression was assessed by RT-PCR. FK506, in a concentration-dependent fashion, inhibited T-cell proliferation and steady-state mRNA expression of IL-2 and IL-7; half-maximal suppression was obtained at 107 to 5 x 10-8 M. We tested whether FK506 antiproliferative effect could be overcome with exogenously reconstituted rIL-2 and/or rIL-7. Neither rIL-2 nor rIL-7, individually in conjunction with suboptimal concentrations of PHA or Con A, or in combination without any costimulus, was capable of abrogating FK506 antiproliferative effect, indicating that FK506 also acted by inhibiting cytokine-stimulated T-cell activation. To confirm this, T cells were treated with FK506 and stimulated by rIL-2 and rIL-7, individually in conjunction with suboptimal concentration of PHA and Con A. In addition, T cells were stimulated with rIL-2 and rIL-7 without any costimuli. FK506 inhibited T-cell activation stimulated by rIL-2 and by rIL-7, individually and in combination. This confirms that, in exerting its antiproliferative effect, FK506 acts at two levels, by inhibiting cytokine availability and by suppressing cytokine effect on target cells, and explains the beneficial effect of FK506 in attenuating ongoing immune responses.
Key words: FK506; Interleukin-2; Interleukin-7; T cells; Cytokines
Address correspondence to Dr. Wassim Y. Almawi, Department of Medical Biochemistry, College of Medicine and Medical Sciences, Arabian Gulf University, P. O. Box 22979, Manama, Bahrain. Tel: +973-929-2958; Fax: +973-271-090; E-mail: email@example.com
Stanislaw Moskalewski, Anna Osiecka-Iwan, and Anna Hyc
Department of Histology and Embryology, Medical University of Warsaw, Pl-02004 Warsaw, Poland
Cartilage produced in 2-week-old intramuscular transplants of syngeneic chondrocytes in rats did not display any signs of rejection. Cartilage produced by similar transplants in animals presensitized with intramuscular transplants of allogeneic chondrocytes was surrounded by infiltrations composed mainly of lymphocytes and was partially resorbed. Spleen mononuclear cells (SMC) from recipients of syngeneic transplants alone were not stimulated in mixed splenocyte-chondrocyte cultures by syngeneic or allogeneic chondrocytes. SMC from recipients of allogeneic and subsequent syngeneic transplants were strongly stimulated by both syngeneic and allogeneic chondrocytes, although stimulation by the latter was significantly more pronounced. Sera from naive rats usually contained cytotoxic antichondrocyte antibodies but their level varied considerably in various individuals. In rats chosen as transplant recipients on the basis of low antichondrocyte cytotoxicity of their sera, this toxicity was markedly raised after sensitization with allo- and syngeneic chondrocytes. Absorption with thymocytes or fibroblasts decreased but did not abrogate cytotoxicity. These observations support previous reports suggesting expression of tissue-specific antigen(s) by chondrocytes.
Key words: Chondrocyte transplantation; Syngeneic cartilage rejection; 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: firstname.lastname@example.org
Thierry Berney, R. Damaris Molano, Antonello Pileggi, Pierre Cattan, Caterina Vizzardelli, Camillo Ricordi, and Luca Inverardi
Diabetes Research Institute, University of Miami Medical School, Miami, FL 33136
A role of macrophage-mediated inflammatory events in early islet graft loss is increasingly acknowledged. Osteopetrotic mice (op/op) have a complete absence of CSF-1, and thus of most tissue macrophages. We have investigated whether the absence of CSF-1-dependent macrophages in the graft itself or at the transplant site could decrease the delay to function of a syngeneic marginal islet mass. Islets transplanted into op/op or control recipients reversed diabetes in 59 days vs. 10 days (p = 0.28, NS). Islets isolated from op/op or control mice reversed diabetes in 11 days vs. 10 days. IL-1 and TNF-a release by cultured islets was markedly decreased for op/op islets compared with control islets (IL-1: 0 vs. 4.2 pg/ml, p = 0.07; TNF-a: 67 vs. 311 pg/ml, p = 0.002). In contrast, IL-6 release by op/op islets was significantly increased (11.1 vs. 4.3 ng/ml, p = 0.006). CSF-1-dependent tissue macrophages may not be critical in the inflammatory insult to islet transplants. Alternate patterns of intraislet release of deleterious proinflammatory cytokines may exist.
Key words: Islet transplantation; Islet primary nonfunction; Macrophages; Osteopetrotic mice
Address correspondence to Luca Inverardi, M.D., Diabetes Research Institute, University of Miami School of Medicine, 1450 NW 10 Avenue, Miami, FL 33136. Tel: (305) 243-2954; Fax: (305) 243-4404; E-mail: email@example.com
Prolongation of Islet Allograft Survival in Mice by Combined Treatment With Pravastatin and Low-Dose Cyclosporine
Seiji Arita, Ali Kasraie, Satoshi Une, Satoki Ohtsuka, Craig V. Smith, and Yoko Mullen
Islet Transplant Program, Department of Surgery, UCLA School of Medicine and Veterans Affairs Medical Center/West Los Angeles, Los Angeles, CA
Pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, is known to have suppressive effects on immune and inflammatory cells. We have previously shown in mice and dogs that this agent prevents primary nonfunction of islet iso- and autografts by reducing inflammation at the graft site. The present study was designed to further investigate whether pravastatin has a synergistic effect with cyclosporine (Cs) to prolong islet allograft survival in mice. Unpurified 3000 BALB/c newborn islets were transplanted under the renal capsule of a streptozotocin-diabetic C57BL/6 mouse. Pravastatin and Cs were administered for 10 days starting on the day of grafting (day 0). Five groups were set up based on the treatment protocol: group 1, treatment with 40 mg/kg pravastatin; group 2, 30 mg/kg Cs; group 3, 50 mg/kg Cs; group 4, 40 mg/kg pravastatin and 30 mg/kg Cs; group 5, vehicle alone. Graft survival was indicated by blood glucose levels sustained at <200 mg/dl, and graft rejection by >250 mg/dl for 2 consecutive days. Hyperglycemia persisted in six of the eight (75%) mice and grafts were rejected in 3.6 ± 0.5 days (mean ± SD) in group 5. In group 1, grafts were also rejected in 3.8 ± 0.8 days, but blood glucose was transiently <200 mg/dl in three of the five mice. Despite Cs, grafts were rejected between 7 and 15 days (10.3 ± 2.4 days) in group 2. Among six mice in group 3, one maintained euglycemia for >60 days, the other rejected the graft on day 15, and the remaining four died with functioning grafts between 9 and 13 days due to Cs toxicity. A combination of a low dose of Cs and pravastatin (group 4) prolonged graft survival for >19 days in five of the eight mice, and for 7-13 days in the remaining three mice. Histological examination of the grafts in this group showed significantly reduced local inflammation. Results indicate a synergistic effect of pravastatin and Cs on prevention of islet allograft rejection.
Key words: Pravastatin; Mouse islet allografts; Primary islet graft nonfunction; Immunosuppressant synergy
Address correspondence to Dr. Yoko Mullen, City of Hope, Diabetes, Endocrinology and Metabolism, 1500 E. Duarte Rd., Duarte, CA 91010. Tel: (626) 359-8111, ext. 62343; E-mail: firstname.lastname@example.org
Dalit Milo-Landesman,1 Manju Surana,2 Irina Berkovich,1 Amelia Compagni,3 Gerhard Christofori,3 Norman Fleischer,2 and Shimon Efrat1
1Department of Human Genetics and Molecular Medicine, Sackler
School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978 Israel
2Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461
3Institute for Molecular Pathology, Dr. Bohr-Gasse 7, A-1030, Vienna, Austria
Pancreatic b cell lines may offer an abundant source of cells for b-cell replacement in type I diabetes. Using regulatory elements of the bacterial tetracycline (tet) operon for conditional expression of SV40 T antigen oncoprotein in transgenic mouse b cells, we have shown that reversible immortalization is an efficient approach for regulated b-cell expansion, accompanied by enhanced cell differentiation upon growth arrest. The original system employed the tet-off approach, in which the cells proliferate in the absence of tet ligands and undergo growth arrest in their presence. The disadvantage of this system is the need for continuous treatment with the ligand in vivo for maintaining growth arrest. Here we utilized the tet-on regulatory system to generate b cell lines in which proliferation is regulated in reverse: these cells divide in the presence of tet ligands, and undergo growth arrest in their absence, as judged by [3H]thymidine and BrdU incorporation assays. These cell lines were derived from insulinomas, which heritably developed in transgenic mice continuously treated with the tet derivative doxycycline (dox). The cells produce and secrete high amounts of insulin, and can restore and maintain euglycemia in syngeneic streptozotocin-induced diabetic mice in the absence of dox. Such a system is more suitable for transplantation, compared with cells regulated by the tet-off approach, because ligand treatment is limited to cell expansion in culture and is not required for long-term maintenance of growth arrest in vivo.
Key words: b cell lines; Diabetes; Growth arrest; Insulin secretion; SV40 T antigen
Address correspondence to Dr. Shimon Efrat, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978 Israel. Tel: 972-3-640 7701; Fax: 972-3-640 9900; E-mail: email@example.com
C. Fedorow,1 L. E. McGann,2 G. S. Korbutt,1,3 G. R. Rayat,4 R. V. Rajotte,1,3 and J. R. T. Lakey1,3
1Department of Surgery, 2Department of Laboratory
Medicine and Pathology, 3Surgical-Medical Research Institute,
University of Alberta, Edmonton, Canada T6G 2N8
4Barbara Davis Center for Childhood Diabetes, Denver, CO
The development of effective protocols for the low-temperature banking of pancreatic islets is an important step in islet transplantation for the treatment of type I diabetes mellitus. We have been exploring the use of islets from the newborn pig as an alternative source of tissue for transplantation. Current cryopreservation protocols are empirically derived, but may be optimized by modeling osmotic responses during the cryopreservation process. This study determined the osmotic and cryoprotectant permeability parameters of cells isolated from the pancreas of newborn pigs. Key parameters are: the osmotically inactive fraction of cell volume, hydraulic conductivity, the permeability coefficients of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) at varying temperatures, and the activation energies of these transport processes. Newborn pig islets were dispersed into single cells and kinetic and equilibrium cell volumes were recorded during osmotic excursions using an electronic particle counter interfaced to a computer. Data were fitted to theoretical descriptions of the osmotic responses of cells, based on the Kedem-Katchalsky approach. The hydraulic conductivity (Lp) in the absence of cryoprotectant was calculated as 0.050 ± 0.005, 0.071 ± 0.006, and 0.300 ± 0.016 mm/min/atm at 4°C, 10°C, and 22°C, respectively (mean ± SEM, n = 7, 6, or 9). These values give an activation energy value of 16.69 kcal/mol when put into an Arrhenius plot. The solute permeability (Ps) values for 1 M DMSO were 0.89 ± 0.12, 1.86 ± 0.28, and 5.33 ± 0.26 mm/min at 4°C, 10°C, and 22°C, respectively (n = 11, 8, or 10) giving an activation energy of 15.98 kcal/mol. The Lp values for cells exposed to 1 M DMSO were 0.071 ± 0.006, 0.084 ± 0.008, and 0.185 ± 0.014 mm/min/atm at 4°C, 10°C, and 22°C, respectively. The activation energy for these values was 8.95 kcal/mol. The Ps values for 2 M DMSO were 1.11 ± 0.13, 1.74 ± 0.19, and 7.68 ± 0.12 mm/min for the same temperatures, with a calculated activation energy of 17.89 kcal/mol. The Lp values in the presence of 2 M DMSO were 0.070 ± 0.006, 0.085 ± 0.008, and 0.192 ± 0.009 mm/min/atm at 4°C, 10°C, and 22°C, respectively, with an activation energy of 9.40 kcal/mol. Solutions of 1 M EG gave Ps values of 1.01 ± 0.13, 1.45 ± 0.25, and 4.90 ± 0.48 mm/min at the three test temperatures. The resulting activation energy was 14.60 kcal/mol. The corresponding Lp values were 0.071 ± 0.007, 0.068 ± 0.006, and 0.219 ± 0.012 mm/min/atm with an activation energy of 10.96 kcal/mol. The solute permeabilities in the presence of 2 M EG for newborn pig islet cells were 1.03 ± 0.15, 1.42 ± 0.23, and 5.56 ± 0.22 mm/min; the activation energy was 15.70. The Lp values for cells in the presence of 2 M EG were 0.068 ± 0.008, 0.071 ± 0.006, and 0.225 ± 0.010 mm/min/atm; the activation energy for these values was 11.49 kcal/mol. These key cryobiological parameters permit the mathematical modeling of osmotic responses of intact islets during the cryopreservation process, which may lead to further improvements in the low temperature storage of islets from newborn pigs.
Key words: Islet cell; Cryopreservation; Basic cryobiology; Xenotransplantation; Newborn pig; Diabetes
Address correspondence to Jonathan R. T. Lakey, Ph.D., Assistant
Professor of Surgery, Director, Clinical Islet Isolation Laboratory, Surgical-Medical
Research Institute, 1074 Dentistry/Pharmacy Building, University of Alberta,
Edmonton, Alberta T6G 2N8 Canada. Tel: (780) 492-3077; Fax: (780) 492-6335;