ognizant Communication Corporation

CELL TRANSPLANTATION
The Regenerative Medicine Journal

ABSTRACTS
VOLUME 15, NUMBER 2, 2006

Cell Transplantation, Vol. 15, pp. 89-104, 2006
0963-6897/06 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
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Pig-to-Nonhuman Primate Islet Xenotransplantation: A Review of Current Problems

P. P. M. Rood,1,2,3 L. H. Buhler,4 R. Bottino,3 M. Trucco,3 and D. K. C. Cooper1

1Starzl Transplantation Institute, University of Pittsburgh Medical Center, Pittsburgh, PA, USA
2Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands
3Division of Immunogenetics, Department of Pediatrics, Children's Hostpital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
4University Hospital Geneva, Geneva, Switzerland

Islet allotransplantation has been shown to have potential as a treatment for type 1 diabetic patients. Xenotransplantation, using the pig as a donor, offers the possibility of an unlimited number of islets. This comprehensive review focuses on experience obtained in pig-to-nonhuman primate models, particularly with regard to the different types of islets (fetal, neonatal, adult) and isolation procedures used, and the methods to determine islet viability. The advantages and disadvantages of the methods to induce diabetes (pancreatectomy, streptozotocin) are discussed. Experience in pig-to-nonhuman primate islet transplantation studies is reviewed, including discussion of the possible mechanisms of rejection and the immunosuppressive regimens used. The research carried out to date has led to workable animal models to study islet xenotransplantation, but several questions regarding methodology remain unanswered, and details of these practicalities require to be adequately addressed. The encouraging porcine islet survival reported recently provides an indicator for future immunosuppressive regimens.

Key words: Diabetes mellitus; Porcine islets; Nonhuman primates; Streptozotocin; Xenotransplantation

Address correspondence to D. K. C. Cooper, M.D., Ph.D., FRCS, Thomas E. Starzl Transplantation Institute, University of Pittsburgh Medical Center, Biomedical Science Tower, E1550A, 200 Lothrop Street, Pittsburgh, PA 15261, USA. Tel: 412-383-6961; Fax: 412-624-6666; E-mail: cooperdk@upmc.edu




Cell Transplantation, Vol. 15, pp. 105-119, 2006
0963-6897/06 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
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Coinhibitory T-Cell Signaling in Islet Allograft Rejection and Tolerance

Wayne Truong,1 Wayne W. Hancock,2 Colin C. Anderson,1 Shaheed Merani,1 and A. M. James Shapiro1

1Department of Surgery, Faculty of Medicine, University of Alberta, Edmonton, AB, Canada
2Department of Pathology and Laboratory Medicine, Joseph Stokes, Jr. Research Institute and Biesecker Pediatric Liver Center, Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA, USA

Autoaggressive T cells directed against insulin secreting pancreatic β-cells mediate the development of type 1 diabetes. Islet transplantation offers superior glycemic control over exogenous insulin, but chronic immunosuppression limits its broad application. Pathogenic T cells are also important in allograft rejection. Inducing and maintaining antigen-specific peripheral T-cell tolerance toward b-cells is an attractive strategy to prevent autoimmune disease, and to facilitate treatment of diabetes with islet allografts without long-term immunosuppression. Recent efforts have focused on blocking costimulatory T-cell signals for tolerance induction. Although costimulatory blockade can prolong graft survival, true immunological tolerance remains elusive. Costimulatory signals may even be required for the maintenance of peripheral tolerance. The discovery of novel coinhibitory T-cell pathways, including CTLA-4, PD-1, and BTLA, offers an alternative approach. Stimulating negative T cell cosignals alone or in combination may help induce tolerance. The focus of this review is to summarize the strategies directed at turning off the immune response by exploiting these negative cosignaling pathways in tolerance induction in islet transplantation. Activating several coinhibitory pathways together may be synergistic in preventing pathogenic T-cell responses. Tolerance induction will likely rely on understanding the balance of positive and negative signals affecting the state of T-cell activation.

Key words: T cell; Cosignaling; Coinhibition; Islet; Allograft; Tolerance

Address correspondence to Dr. A. M. James Shapiro. M.D., Ph.D., FRCS(Eng), FRCSC, Wyeth-Ayerst Canada/CIHR Clinical Research Chair in Transplantation, Director, Clinical Islet Transplant Program, University of Alberta, Roberts Centre, 2000 College Plaza, Edmonton, Alberta Canada T6G 2C8. Tel: (780) 407 7330; Fax: (780) 407 6933; E-mail: amjs@islet.ca




Cell Transplantation, Vol. 15, pp. 121-133, 2006
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Copyright © 2006 Cognizant Comm. Corp.
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Viability and Functionality of Bovine Chromaffin Cells Encapsulated Into Alginate-PLL Microcapsules With a Liquefied Inner Core

T. Moustafa,1 S. Girod,2 F. Tortosa,1 R. Li1 J. C. Sol,1 F. Rodriguez,2 R. Bastide,1 Y. Lazorthes,1 and B. Sallerin1

1Laboratoire Douleur et Thérapie Cellulaire, Faculté de médecine Rangueil, 133 route de Narbonne, 31 062 Toulouse Cedex, France
2GEFSOD, EA 2631, 35 chemin des Maraîchers, 31 062 Toulouse Cedex, France

Implantation of adrenal medullary bovine chromaffin cells (BCC), which synthesize and secrete a combination of pain-reducing neuroactive compounds including catecholamines and opioid peptides, has been proposed for the treatment of intractable cancer pain. Macro- or microencapsulation of such cells within semipermeable membranes is expected to protect the transplant from the host's immune system. In the present study, we report the viability and functionality of BCC encapsulated into microcapsules of alginate-poly-L-lysine (PLL) with a liquefied inner core. The experiment was carried out during 44 days. Empty microcapsules were characterized in terms of morphology, permeability, and mechanical resistance. At the same time, the viability and functionality of both encapsulated and nonencapsulated BCC were evaluated in vitro. We obtained viable BCC with excellent functionality: immunocytochemical analysis revealed robust survival of chromaffin cells 30 days after isolation and microencapsulation. HPLC assay showed that encapsulated BCC released catecholamines basally during the time course study. Taken together, these results demonstrate that viable BCC can be successfully encapsulated into alginate-PLL microcapsules with a liquefied inner core.

Key words: Bovine chromaffin cells; Alginate encapsulation; Pain therapy; Cell viability; Cell functionality

Address correspondence to B. Sallerin, Laboratoire Douleur et Thérapie Cellulaire, Faculté de médecine Rangueil, 133 route de Narbonne, 31 062 Toulouse Cedex, France. E-mail: sallerin@cict.fr




Cell Transplantation, Vol. 15, pp. 135-145, 2006
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One-Step Induction of Neurons From Mouse Embryonic Stem Cells in Serum-Free Media Containing Vitamin B12 and Heparin

Hironori Yamazoe,1* Masato Kobori,2* Yoshinobu Murakami,1 Keiichi Yano,2 Mitsuo Satoh,2 Kenji Mizuseki,3 Yoshiki Sasai,3 and Hiroo Iwata1

1Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo, Kyoto 606-8507, Japan
2Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahimachi, Machida, Tokyo 194-8533, Japan
3Organogenesis and Neurogenesis Group, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan

We present a simple method for neural cell fate specification directly from mouse embryonic stem cells (ES cells) in serum-free conditions in the absence of embryoid body formation. Dissociated ES cells were cultured in serum-free media supplemented with vitamin B12 and heparin, but without any expensive cytokines. After 14 days in culture, b-tubulin type III (TuJ1) and tyrosine hydroxylase (TH)-positive colonies were detected by immunocytochemical examinations. In addition, specific gene analyses by RT-PCR demonstrated expression of an early central nerve system, mature neuron, and midbrain dopaminergic neuron-specific molecules (i.e., nestin, middle molecular mass neurofilament protein, Nurr1, and TH, respectively). Dopamine was also detected in the culture media by reverse-phase HPLC analysis. These facts indicate that addition of vitamin B12/heparin to serum-free culture media induced neurons from ES cells, which included cells that released dopamine. Other supplements, such as putrescine, biotin, and Fe2+, could not induce neurons from ES cells by themselves, but produced synergistic effects with vitamin B12/heparin. The rate of TuJ1+/TH+ colony formation was increased threefold and the amounts of dopamine released increased 1.5-fold by the addition of a mixture of putrescine, biotin, and Fe2+ to vitamin B12/heparin culture media. Our method is a simple tool to differentiate ES cells to dopaminergic neurons for the preparation of dopamine-releasing cells for the cell transplantation therapy of Parkinson's disease. In addition, this method can facilitate the discovery of soluble factors and genes that can aid in the induction of the ES cell to its neural fate.

Key words: Embryonic stem cell; Differentiation; Vitamin B12; Heparin; Dopaminergic neuron

Address correspondence to Hiroo Iwata, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. Tel/Fax: +81-75-751-4119; E-mail: iwata@frontier.kyoto-u.ac.jp

*Both authors contributed equally to this work.




Cell Transplantation, Vol. 15, pp. 147-160, 2006
0963-6897/06 $20.00 + 00
E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
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Engrafted Neural Progenitor Cells Express a Tissue-Restricted Reporter Gene Associated With Differentiated Retinal Photoreceptor Cells

Thien N. Sam,1 Jing Xiao,2 Heidi Roehrich,1 Walter C. Low,2 and Dale S. Gregerson1

1Department of Ophthalmology, University of Minnesota, Minneapolis, MN 55455, USA
2Department of Neurosurgery, University of Minnesota, Minneapolis, MN 55455, USA

Neural progenitor cells (NPCs) have shown ability to repair injured CNS, and might provide precursors to retinal neurons. NPCs were isolated from the brains of 14 day murine embryos of transgenic mice, which express b-galactosidase (b-gal) on the arrestin promoter, which specifically directs expression to retinal photoreceptor cells. NPCs were transferred to adult, syngeneic mice via inoculation into the anterior chamber of the eye, the peritoneal cavity, or the brain. At 14 weeks postgrafting, tissues were collected and examined to determine if differentiated NPC progeny were present in retina based on histochemical detection of b-gal. Four of six anterior chamber-inoculated recipients showed Bluo-gal-stained cells in retina, indicating the presence of transferred NPCs or their progeny. Because the progenitor cells do not express b-gal, positive staining indicates differentiation leading to activation of the arrestin promoter. Two recipients inoculated by the intraperitoneal route also exhibited Bluo-gal staining in retina. The NPCs did not express b-gal if inoculated into brain, but survived and dispersed. Most recipients, regardless of inoculation route, were PCR positive for b-gal DNA in extraocular tissues, but no Bluo-gal staining was found outside of the retina. Injury to the retina promoted, but was not required, for progenitor cell engraftment. b-Gal-positive cells were concentrated in the outer layers of the retina. In summary, a reporter gene specifically expressed in differentiated retinal photoreceptor cells due to the activity of the arrestin promoter was expressed in recipient mouse retina following transfer of NPCs prepared from the b-gal transgenic mice. The presence of b-gal DNA, but not Bluo-gal staining, in spleen and other tissues revealed that the cells also migrated elsewhere and took up residence in other organs, but did not undergo differentiation that led to b-gal expression.

Key words: Stem cell transplantation; Progenitor cell transplantation; Retina; Cell differentiation; Neurons; Mice, transgenic; Photoreceptor cells; Neural proteins; Immunofluorescence; Immunohistochemistry

Address correspondence to Dale S. Gregerson, Ph.D., Department of Ophthalmology, University of Minnesota, Lions Research Building, Rm. 314, 2001 6th St. SE, Minneapolis, MN 55431-2404, USA. Tel: 612-626-0772; Fax: 612-626-0781; E-mail: grege001@umn.edu




Cell Transplantation, Vol. 15, pp. 161-168, 2006
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E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
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Subnormothermic Preservation Maintains Viability and Function in a Porcine Hepatocyte Culture Model Simulating Bioreactor Transport

M.-P. van de Kerkhove,1 R. Hoekstra,1,3 F. C. van Nooijen,1 F. O. B. Spoelstra,1 B. M. Doorschodt,1 A. C. W. A. van Wijk,1 P. P. C. Poyck,1 R. A. F. M. Chamuleau,2 and T. M. van Gulik1

1Department of Surgery (Surgical Laboratory), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
2Department of Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
3AMC Liver Center, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

Bioartificial liver (BAL) systems have been developed to bridge patients with acute liver failure (ALF) to liver transplantation or liver regeneration. Clinical application of BAL systems is dependent on the supportive quality of cells used and direct availability of the whole system. Reliable transport of BAL systems from the laboratory to remote treatment centers is therefore inevitable. Subsequently, preservation conditions play a crucial role during transport of a BAL, with temperature being one of the most determining factors. In this study, we assessed the effect of subnormothermic preservation on freshly isolated porcine hepatocytes cultured in monolayer under oxygenation. Additionally, the effect of the University of Wisconsin (UW) preservation solution was compared with Williams' E (WE) culture medium at 4°C. The control group was cultured for 3 days at 37°C, whereas the transport groups were cultured at 4°C, 15°C, 21°C, or 28°C for 24 h at day 2. All groups were tested each day for cell damage and hepatic functions. Subnormothermic culture (i.e., 15°C to 28°C) for a period of 24 h did not reduce any hepatic function and did not increase cellular damage. In contrast, culture of hepatocytes in WE medium and preservation in UW solution at 4°C significantly reduced hepatic function. In conclusion, freshly isolated porcine hepatocytes can be preserved for 24 h at subnormothermic temperatures as low as 15°C. Future research will focus on the implementation of the AMC-BAL in an oxygenated culture medium perfusion system for transport between the laboratory and the hospital.

Key words: Bioartificial liver; Hepatocyte; Transport; Temperature; Cell function

Address correspondence to T. M. van Gulik, Department of Surgery, Surgical Laboratory, IWO-1, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands. Fax: 0031-20-6976621; E-mail: t.m.vangulik@amc.uva.nl




Cell Transplantation, Vol. 15, pp. 169-174, 2006
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E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
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Quantitative Assay for Quality Assurance of Human Cells for Clinical Transplantation

Steven T. Boyce,1,2 Benjamin A. Anderson,1 and Horacio L. Rodriguez-Rilo1

1Departments of Surgery and Biomedical Engineering, University of Cincinnati, Cincinnati, OH, USA
22Research Department, Shriners Burns Hospital, Cincinnati, OH, USA

Transplantation of human cells after isolation and culture has become an important alternative for treatment of acute or chronic skin wounds. To increase the efficacy and reduce cost for transplantation of skin cells, more efficient and accurate techniques for evaluation of cell proliferation are needed. Hemocytometer counts provide a valid assessment of cell proliferation and viability, but they are very labor intensive and require removal of the cells from their substrate. In this study, hemocytometer counts were compared with a fluorometric assay (n = 21 per condition) that uses the commercially available reagent alamarBlueTM, which is reduced to a fluorescent substrate by cellular dehydrogenases. Human epidermal keratinocytes were inoculated at 200, 600, 2000, and 6000 cells/cm2 incubated for 6 days in modified MCDB 153 medium. AlamarBlueTM was incubated with cells for 2 h at 37°C, and fluorescence was measured with a microplate reader at 590 nm. Hemocytometer counts (x10-4) from the respective cell inoculation densities were 0.30 ± 0.04, 1.07 ± 0.10, 6.37 ± 0.62, and 16.99 ± 0.96. Fluorescence values (x10-3) for the respective inoculation densities were 0.14 ± 0.01, 0.34 ± 0.02, 1.20 ± 0.09, and 1.79 ± 0.12. Regression analysis showed a statistical significant (p < 0.0001) correlation (r2 = 0.87) between cell counts and optical density from the alamarBlueTM assay. These data demonstrate that alamarBlueTM provides a valid substitute for cell counts to assess cell proliferation before clinical transplantation of engineered skin. AlamarBlueTM also allows repeated, nondamaging assessment of living cells over time. These advantages are expected to increase the validity and reliability of quality assurance standards for transplanted skin cells, and to increase the efficacy of healing of cutaneous wounds.

Key words: Cultured keratinocytes; Skin substitutes; Wound healing; Burns; Chronic wounds

Address correspondence to Steven Boyce, Ph.D., Department of Surgery, University of Cincinnati, P.O. Box 670558, Cincinnati, OH 45267-0558, USA. Tel: 513-872-6080; Fax: 513-872-6107; E-mail: boycest@uc.edu




Cell Transplantation, Vol. 15, pp. 175-180, 2006
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E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
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Comparison of Cooling Systems During Islet Purification

Sue Swift, Tatsuya Kin, Mohammadreza Mirbolooki, Richard Wilson, and Jonathan R. T. Lakey

Clinical Islet Transplant Program, University of Alberta and Capital Health Authority, Edmonton, Alberta, Canada

Islet isolation is a complex procedure that includes digestion and purification of pancreatic tissue. As we move towards clinical regulatory control and standardization, understanding of the detailed stages of the procedure have become increasingly important. Purification on a COBE 2991 density gradient allows human islets to be separated from a large volume of acinar tissue. Cooling the gradient and tissue is thought to be important to reduce metabolic activity but cooling systems for the gradient are expensive, with limited availability. In this study, the efficiency of cooling methods for the COBE 2991 cell separator has been investigated. The two cooling systems were: a) COBE 2991 modified internally to allow coolant (polyethylene glycol) from a chiller to circulate either side of the spindle and around the bowl (original system), and b) an air-cooled system using an air conditioner to blow cold air into the bowl from above (air cooler system). Cooling required 20 min for the original system and temperature was stabilized within 4-7°C. The air system cooled rapidly but was not stable. There was an increase in the temperature of the medium with using both systems during centrifugation because of heat generated by the COBE machine; however, the temperature of the medium after centrifugation with the air system was significantly higher than that with the original system (13.3 ± 0.2°C vs. 8.7 ± 0.7°C, p < 0.05). The original cooler system was found to be more efficient at reducing heat generated by the COBE machine than the air system. Further investigation of the importance of the recorded temperatures is required.

Key words: Density gradient; Islet purification; Coolant; Chiller; Temperature

Address correspondence to Jonathan R. T. Lakey, Ph.D., Associate Professor of Surgery, Director of Clinical Islet Laboratory, Department of Surgery, 1074 Dentistry/Pharmacy Building, University of Alberta, Edmonton, Alberta, Canada T6G 2N8. Tel: (780) 492-3077; Fax: (780) 492-6335; E-mail: jlakey@ualberta.ca




Cell Transplantation, Vol. 15, pp. 181-185, 2006
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E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
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Estimation of Pancreas Weight From Donor Variables

Tatsuya Kin, Travis B. Murdoch, A. M. James Shapiro, and Jonathan R. T. Lakey

Clinical Islet Transplant Program, University of Alberta and Capital Health Authority, Edmonton, Alberta, Canada

Previous studies have identified several donor factors affecting the outcome of islet isolation. Pancreas weight has not been considered as a donor selection criterion, because a value cannot be obtained prior to organ procurement. However, a larger pancreas will likely contain a higher number of islets. Therefore, the prediction of pancreas weight would be helpful in donor selection, benefiting cost and efficiency of the islet isolation laboratory. The purpose of this study was to investigate normal pancreas weight in cadaveric donors and identify pancreas weight predictors from demographic data of cadaveric organ donors. We retrospectively analyzed data on pancreas weight from 354 cadaveric donors with respect to gender, age, body weight, body height, body mass index (BMI), and body surface area (BSA). In men, pancreas weight correlated more closely with body weight than with age, height, or BMI. BSA was as strong a correlate of pancreas weight as body weight. In women, pancreas weight had a similar pattern of relationships, with generally lower correlation coefficients. On the basis of the observation of gender-specific pancreas weight difference in elderly donors, stepwise multiple linear regression analyses were conducted separately for younger (<40 years) and elderly (>41 years) donors. In younger donors, body weight and age were the major predictors of pancreas weight [pancreas weight (g) = 4.355 + 0.742 x body weight (kg) + 0.837 x age (years) (R2 = 0.564, p < 0.001)]. In contrast, pancreas weight of elderly donors was best predicted by BSA and gender [pancreas weight (g) = -17.624 + 60.036 x BSA (m2) - 7.152 x gender (R2 = 0.372, p < 0.001; "gender": 1 = female, 0 = male)]. Pancreas weight was found to be positively associated with pre- and postpurification islet yields. These formulae should contribute to the estimation of pancreas weight, and thus improve donor selection for islet isolation and transplantation.

Key words: Pancreas weight; Cadaveric donor; Islet transplantation; Body surface area

Address correspondence to Jonathan R. T. Lakey, Ph.D., Associate Professor of Surgery, Director of Clinical Islet Laboratory, Department of Surgery, 1074 Dentistry/Pharmacy Building, University of Alberta, Edmonton, Alberta, Canada T6G 2N8. Tel: (780) 492-3077; Fax: (780) 492-6335; E-mail: jlakey@ualberta.ca




Cell Transplantation, Vol. 15, pp. 187-194, 2006
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E-ISSN 1555-3892
Copyright © 2006 Cognizant Comm. Corp.
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Ameliorating Injury During Preservation and Isolation of Human Islets Using the Two-Layer Method With Perfluorocarbon and UW Solution

Payam Salehi,1* Mohammadreza Mirbolooki,1,2* Tatsuya Kin,2 Toshiaki Tsujimura,3 A. M. James Shapiro,1,2 Thomas A. Churchill,1 and Jonathan R. T. Lakey1,2

1Surgical-Medical Research Institute, Department of Surgery, University of Alberta, Edmonton, Alberta, Canada
2Clinical Islet Isolation Laboratory, University of Alberta Hospital/Capital Health Authority, Edmonton, Alberta, Canada
3Department of Gastroenterological Surgery, Graduate School of Medicine, Kobe University, Kobe, Japan

This study assessed the effects of a two-layer method (TLM), using perfluorocarbon and UW solution, on the quality of human pancreata following storage and islet yield/function after isolation. In part A, TLM was applied immediately after procurement and the energetic profile was compared to a group treated with UW solution only (control) throughout 24-h storage. In part B, cadaveric human pancreata were procured and subjected to a TLM after cold storage in UW solution (TLM group) or UW solution (control group). Energetics, lipid peroxidation, and islet recovery/function were assessed after preservation at 4°C. In part A, after 9-h storage, the energetic profile (ATP, ATP/ADP, energy charge) for the TLM group was superior to controls. In part B, TLM treatment resulted in consistently greater ATP, ATP/ADP, and energy charge values than with storage in UW solution alone (p < 0.05). UW treatment resulted in 40% greater peroxidative damage than in the TLM group (p < 0.05). Islet recovery and functional viability were 30-40% higher following TLM treatment (p < 0.05). These data support the hypothesis that islet viability and yields can be significantly improved using a brief period of TLM treatment following conventional UW storage; reduced energetic and oxidative stress are implicated as potential mechanisms.

Key words: Islets; Preservation; Two-layer method; Perfluorocarbon; University of Wisconsin solution

Address correspondence to J. R. T. Lakey, Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Bldg., University of Alberta, Edmonton, Alberta, Canada T6G 2N8. Tel: (780) 492-3077; Fax: (780) 492-1627; E-mail: jonathan.lakey@ualberta.ca

*These two authors contributed equally to this study.




Cell Transplantation, Vol. 15, pp. 195-203, 2006
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E-ISSN 1555-3892
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Functional MR Microimaging of Pancreatic b-Cell Activation

Barjor Gimi,1,2* Lara Leoni,2,6,7* Jose Oberholzer,2,3 Mark Braun,3 Jose Avila,3 Yong Wang,3 Tejal Desai,4 Louis H. Philipson,5 Richard L. Magin,2 and Brian B. Roman2,6,7

1Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD, 21205, USA
2Department of Bioengineering, University of Illinois at Chicago, 851 S. Morgan St., Chicago, IL, 60607, USA
3Division of Transplantation, Hospital, University of Illinois at Chicago, 840 S. Wood St., Chicago, IL, 60612, USA
4Department of Biomedical Engineering, Boston University, 44 Cummington St., Boston, MA, 20215, USA
5Department of Medicine, Section of Endocrinology, University of Chicago, 5841 South Maryland Ave, Chicago, IL, 60637, USA
6Department of Physiology and Biophysics, Center for Cardiovascular Research, University of Illinois at Chicago, 840 S. Wood St., Chicago, IL, 60612, USA
7Department of Radiology, University of Chicago, 5841 South Maryland Ave, Chicago, IL, 60637, USA

The increasing incidence of diabetes and the need to further understand its cellular basis has resulted in the development of new diagnostic and therapeutic techniques. Nonetheless, the quest to noninvasively ascertain b-cell mass and function has not been achieved. Manganese (Mn)-enhanced MRI is presented here as a tool to image b-cell functionality in cell culture and isolated islets. Similar to calcium, extracellular Mn was taken up by glucose-activated b-cells resulting in 200% increase in MRI contrast enhancement, versus nonactivated cells. Similarly, glucose-activated islets showed an increase in MRI contrast up to 45%. Although glucose-stimulated Ca influx was depressed in the presence of 100 mM Mn, no significant effect was seen at lower Mn concentrations. Moreover, islets exposed to Mn showed normal glucose sensitivity and insulin secretion. These results demonstrate a link between image contrast enhancement and b-cell activation in vitro, and provide the basis for future noninvasive in vivo imaging of islet functionality and b-cell mass.

Key words: MRI; Islets; b-Cells; Manganese; Activation

Address correspondence to Brian B. Roman, Ph.D., Department of Radiology, University of Chicago, 5841 S. Maryland Ave (MC 2026), Chicago, IL 60637, USA. Tel: 773-702-6906; Fax: 773-834-4097; E-mail: broman@uchicago.edu

*These authors contributed equally to this work.




Cell Transplantation, Vol. 15, pp. 205-209, 2006
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E-ISSN 1555-3892
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Size-Dependent Revascularization of Transplanted Pancreatic Islets

Caroline Kampf,1,2 Göran Mattsson,1 and Per-Ola Carlsson1,3

1Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
2Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden
3Department of Medical Sciences, Uppsala University, Uppsala, Sweden

For their survival and optimal function, pancreatic islets depend posttransplantation on a rapid and adequate revascularization. Native islets display a marked size-dependent heterogeneity in both angioarchitecture and degree of blood perfusion. This study evaluated whether there also are differences in the degree of revascularization of islets of different size when transplanted. Mouse pancreatic islets were isolated by collagenase digestion, and cultured in vitro for 4-7 days before transplantation. Groups of 200 islets with a diameter either exceeding or being below 100 mm were implanted beneath the left renal capsule of syngeneic C57 BL/6 mice. One month posttransplantation, graft-bearing kidneys were removed. Histological specimens were prepared and stained for endothelium with the lectin Bandeiraea simplicifolia. Pancreata from nontransplanted control animals were prepared similarly. The vascular density in transplanted islets was markedly lower than in native islets. However, islet transplants composed of small islets (<100 mm in diameter) had a vascular density in the endocrine tissue twice  that in transplants of larger islets (>100 mm). The connective tissue stroma surrounding smaller islets was also more revascularized than in corresponding grafts with large islets. The vascular density in the connective tissue stroma surrounding the individual islets in the grafts was markedly higher than in the endocrine parts per se. These combined observations indicate that smaller islets have a higher capacity to stimulate regrowth of blood vessels following transplantation. Further studies on islet differences with regard to revascularization capacity may teach us strategies for treatment of transplanted islets to improve their revascularization.

Key words: Engraftment; Bandeiraea simplicifolia; Islet graft; Angiogenesis

Address correspondence to Per-Ola Carlsson, M.D., Ph.D., Department of Medical Cell Biology, Uppsala University, Husargatan 3, Box 571, E-751 23 Uppsala, Sweden. Tel: +46 18 471 4425; Fax: +46 18 471 40 59; SE-mail: Per-Ola.Carlsson@medcellbiol.uu.se