|ognizant Communication Corporation|
The Regenerative Medicine Journal
VOLUME 18, NUMBER 1, 2009
Cell Transplantation, Vol. 18, pp. 1-12, 2009
0963-6897/09 $90.00 + 00
Copyright © 2009 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Extracellular Matrix in Pancreatic Islets: Relevance to Scaffold Design and Transplantation
John C. Stendahl,1,2* Dixon B. Kaufman,1,3** and Samuel I. Stupp1,2,4,5**
1Institute for BioNanotechnology in Advanced Medicine, Northwestern
University, Chicago, IL, USA
2Department of Materials Science and Engineering, Northwestern University, Evanston, IL, USA
3Department of Surgery, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
4Department of Chemistry, Northwestern University, Evanston, IL, USA
5Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
Intrahepatic islet transplantation provides a potentially more benign alternative to pancreatic transplantation. However, islet transplants are associated with limited engraftment potential. This inefficiency is likely at least partially attributable to the isolation process, which removes islets from their native environment. Isolation not only disrupts the internal vascularization and innervation of islets, but also fundamentally changes interactions between islet cells and macromolecules of the extracellular matrix (ECM). Signaling interactions between islet cells and ECM are known to regulate multiple aspects of islet physiology, including survival, proliferation, and insulin secretion. Although it is highly likely that disruptions to these interactions during isolation significantly affect transplant outcomes, the true implications of these conditions are not well understood. The following article reviews current understandings and uncertainties in islet-ECM interactions and explains their potential impact on posttransplant engraftment. Topics covered include matrix and receptor compositions in native islets, effects of isolation and culture on islet-ECM interactions, and potential for postisolation restoration of islet-ECM interactions. Greater understanding in these areas may help to reduce isolation and transplantation stresses and improve islet engraftment.
Key words: Islet transplantation; b-Cells; Extracellular matrix; Basement membrane; Integrins
Address correspondence to Samuel I. Stupp, Department of Materials Science and Engineering, Northwestern University, 2220 Campus Dr., Evanston, IL 60208. Tel: 847-491-3002; Fax: 847-491-3010; E-mail: email@example.com
*Present address: Diabetes Institute for Immunology and Transplantation,
University of Minnesota, Minneapolis, MN 55455, USA.
**These contributors are co-senior authors for the review.
Toward Improving Human Islet Isolation From Younger Donors: Rescue Purification Is Efficient for Trapped Islets
A. Miki,1 C. Ricordi,1,2,3 S. Messinger,4 T. Yamamoto,1 A. Mita,1 S. Barker,1 R. Haetter,1 A. Khan,1 R. Alejandro,1,5 and H. Ichii1,2,3
1Cell Transplant Center, Diabetes Research Institute, University
of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
2DeWitt Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
3Jackson Memorial Hospital Transplant Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
4Department of Epidemiology and Public Health, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
5Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
Several reports suggest that islets isolated from younger donor pancreata are of better quality for clinical islet transplantation. The relative inefficiency of the continuous gradient purification process (CGP) is one of the major obstacles to the utilization of these younger donor pancreata. This study demonstrates the benefits of utilizing an additional purification step, rescue gradient purification (RGP), to recover trapped islets and examines the possible superiority of these rescued islets. Seventy-three human islet isolations purified by RGP following CGP were divided into two groups based on age, and the isolation results were retrospectively analyzed (group I: age <=40, group II: age >40). The quality of islets from both CGP and RGP were assessed by b-cell fractional viability (bFV) and ADP/ATP ratio. Significant increases in the percent islet recovery from RGP and the percent trapped islets in group I compared to group II were observed. Donor age correlated negatively to the percent islets recovered from RGP (R = 0.440) and to the percent of trapped islets (R = 0.511). RGP islets had higher bFV and better ADP/ATP ratio compared to CGP islets. In conclusion, RGP improved the efficiency in the purification of trapped islets, which often come from younger donor pancreata. The better quality of b-cells in RGP islets encourages us to perform RGP, considering the higher quality as well as the quantity of remaining islets.
Key words: Islet transplantation; Purification; Viability; Trapped islet; Young donor
Address correspondence to Hirohito Ichii, M.D., Ph.D., Cell Transplant Center,Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, 1450 NW 10th Avenue (R-134), Miami, FL 33136, USA. Tel: (305) 243-3700; Fax: (305) 243-4404; E-mail: firstname.lastname@example.org
Beneficial Role of Pancreatic Microenvironment for Angiogenesis in Transplanted Pancreatic Islets
Joey Lau,1 Caroline Kampf,1,2 Göran Mattsson,1 Daniel Nyqvist,3 Martin Köhler,3 Per-Olof Berggren,3 and Per-Ola Carlsson1,4
1Department of Medical Cell Biology, Uppsala University,
2Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden
3The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden
4Department of Medical Sciences, Uppsala University, Uppsala, Sweden
Pancreatic islets implanted heterotopically (i.e., into the kidney, spleen, or liver) become poorly revascularized following transplantation. We hypothesized that islets implanted into the pancreas would become better revascularized. Islets isolated from transgenic mice expressing enhanced yellow fluorescent protein (EYFP) in all somatic cells were cultured before they were implanted into the pancreas or beneath the renal capsule of athymic mice. Vascular density was evaluated in histological sections 1 month posttransplantation. EYFP was used as reporter for the transgene to identify the transplanted islets. Islet endothelial cells were visualized by staining with the lectin Bandeiraea simplicifolia (BS-1). Capillary numbers in intrapancreatically implanted islets were only slightly lower than those counted in endogenous islets, whereas islets implanted beneath the renal capsule had a markedly lower vascular density. In order to determine if this high graft vascular density at the intrapancreatic site reflected expansion of remnant donor endothelial cells or increased ingrowth of blood vessels from the host, also islets from Tie2-green fluorescent protein (GFP) mice (i.e., islets with fluorescent endothelial cells) were transplanted into the pancreas or beneath the renal capsule of athymic mice. These islet grafts revealed that the new vascular structures formed in the islet grafts contained very few GFP-positive cells, and thus mainly were of recipient origin. The reason(s) for the much better ingrowth of blood vessels at the intrapancreatic site merits further studies, because this may help us form strategies to overcome the barrier for ingrowth of host vessels also into islets in heterotopic implantation sites.
Key words: Engraftment; Bandeiraea simplicifolia; Islet graft; Angiogenesis; Pancreas
Address correspondence to Joey Lau, Department of Medical Cell Biology, Uppsala University, Husargatan 3, Box 571, SE-751 23 Uppsala, Sweden. Tel: +46 18 471 4440; Fax: +46 18 471 4059; E-mail: Joey.Lau@mcb.uu.se
The Effect of Composite Pig Islet-Human Endothelial Cell Grafts on the Instant Blood-Mediated Inflammatory Reaction
Hyoung-Il Kim,1,2,3 Jae Eun Yu,1,2 Song Yi Lee,1,2 A. Young Sul,1,2 Min Seok Jang,1,2 M. A. Rashid,1,4 Sang Gyu Park,2,5 Sang Jun Kim,1,2,6 Chung-Gyu Park,1,2,4 Jae Hyeon Kim,2,5,7 and Kyong Soo Park2,5,8
1Xenotransplantation Research Center, Seoul 110-744, Republic
2Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-799, Republic of Korea
3Department of Surgery, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea
4Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul 110-799, Republic of Korea
5Innovative Research Institute for Cell Therapy, Seoul National University Children's Hospital, Seoul 110-744, Republic of Korea
6Department of Surgery, Seoul National University College of Medicine, Seoul 110-744, Republic of Korea
7Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Republic of Korea
8Department of Internal Medicine, Seoul National University College of Medicine, Seoul 110-744, Republic of Korea
Instant blood-mediated inflammatory reaction (IBMIR) causes rapid islet loss in portal vein islet transplantation. Endothelial cells are known to protect against complement-mediated lysis and activation of coagulation. We tested composite pig islet-human endothelial cell grafts as a strategy to overcome IBMIR. Porcine islets were cocultured with human endothelial cells in specially modified culture medium composed of M199 and M200 for 1-9 days. A positive control group, negative control group, and the endothelial cell-coated group were examined with an in vitro tubing loop assay using human blood. The endothelial cell-coated group was subdivided and analyzed by degree of surface coverage by endothelial cells (<=50% vs. >50%) or coculture time (<5 days vs. >=5 days). Platelet consumption and complement and coagulation activation were assessed by platelet count, C3a, and thrombin-antithrombin complex (TAT), respectively. After 60-min incubation in human blood, the endothelial cell-coated group showed platelet consumption inhibition and low C3a and TAT assay results compared to uncoated controls. When the endothelial cell-coated group was subdivided by degree of surface coverage, the <=50% coated group showed less platelet consumption and less activation of complement and coagulation compared with the positive control (uncoated) group. On analysis by coculture time, only the subgroup cocultured for <5 days showed the same protective effect. Human endothelial cell-coated pig islets, especially the partially coated and short-term cocultured pig islet-human endothelial cell composites, reduced all components of IBMIR. If the optimal endothelial cell-islet coculture method could be identified, human endothelial cell coating of pig islets would offer new strategies to improve xenogenic islet transplantation outcomes.
Key words: Endothelial cells; Humans; Islets of Langerhans; Swine; Transplantation; Heterologous
Address correspondence to Jae Hyeon Kim, M.D., Ph.D., Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul 135-710, Republic of Korea. Tel: 82-2-3410-1580; Fax: 82-2-3410-3849; E-mail: Jae HyeonKim@paran.com or Kyong Soo Park, M.D., Ph.D., Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-Dong Jongro-Gu, Seoul, 110-744, Republic Korea. Tel: +82-2-2072-1789; Fax: +82-2-3676-8309; E-mail: email@example.com
Cotransplantation of Mouse Embryonic Stem Cells and Bone Marrow Stromal Cells Following Spinal Cord Injury Suppresses Tumor Development
Ryosuke Matsuda,1 Masahide Yoshikawa,2 Hajime Kimura,1 Yukiteru Ouji,2 Hiroyuki Nakase,1 Fumihiko Nishimura,1 Jun-ichi Nonaka,1 Hayato Toriumi,1 Shuichi Yamada,1 Mariko Nishiofuku,2 Kei Moriya,2 Shigeaki Ishizaka,2 Mitsutoshi Nakamura,3 and Toshisuke Sakaki1
1Department of Neurosurgery, Nara Medical University, Nara
2Department of Parasitology, Nara Medical University, Nara 634-8521, Japan
3Department of Pathology, Nara Medical University, Nara 634-8521, Japan
Embryonic stem (ES) cells are a potential source for treatment of spinal cord injury (SCI). Although one of the main problems of ES cell-based cell therapy is tumor formation, there is no ideal method to suppress tumor development. In this study, we examined whether transplantation with bone marrow stromal cells (BMSCs) prevented tumor formation in SCI model mice that received ES cell-derived grafts containing both undifferentiated ES cells and neural stem cells. Embryoid bodies (EBs) formed in 4-day hanging drop cultures were treated with retinoic acid (RA) at a low concentration of 5 × 10-9 M for 4 days, in order to allow some of the ES cells to remain in an undifferentiated state. RA-treated EBs were enzymatically digested into single cells and used as ES cell-derived graft cells. Mice transplanted with ES cell-derived graft cells alone developed tumors at the grafted site and behavioral improvement ceased after day 21. In contrast, no tumor development was observed in mice cotransplanted with BMSCs, which also showed sustained behavioral improvement. In vitro results demonstrated the disappearance of SSEA-1 expression in cytochemical examinations, as well as attenuated mRNA expressions of the undifferentiated markers Oct3/4, Utf1, Nanog, Sox2, and ERas by RT-PCR in RA-treated EBs cocultured with BMSCs. In addition, MAP2-immunopositive cells appeared in the EBs cocultured with BMSCs. Furthermore, the synthesis of NGF, GDNF, and BDNF was confirmed in cultured BMSCs, while immunohistochemical examinations demonstrated the survival of BMSCs and their maintained ability of neurotrophic factor production at the grafted site for up to 5 weeks after transplantation. These results suggest that BMSCs induce undifferentiated ES cells to differentiate into a neuronal lineage by neurotrophic factor production, resulting in suppression of tumor formation. Cotransplantation of BMSCs with ES cell-derived graft cells may be useful for preventing the development of ES cell-derived tumors.
Key words: Embryonic stem cells; Bone marrow stromal cells; Spinal cord injury; Cotransplantation; Tumor suppression
Address correspondence to Masahide Yoshikawa, M.D., Division of Developmental Biology, Department of Parasitology, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan. Tel: +81-744-29-8847; Fax: +81-744-29-8847; E-mail: firstname.lastname@example.org
Retentive Multipotency of Adult Dorsal Root Ganglia Stem Cells
Rabindra P. Singh,1 Ying-Hua Cheng,1 Paul Nelson,2 and Feng C. Zhou1,3
1Department of Anatomy & Cell Biology, Indiana University
School of Medicine, Indianapolis, IN, USA
2Department of Neurosurgery, Indiana University School of Medicine, Indianapolis, IN, USA
3Stark Neuroscience Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA
Preservation of neural stem cells (NSCs) in the adult peripheral nervous system (PNS) has recently been confirmed. However, it is not clear whether peripheral NSCs possess predestined, bona fide phenotypes or a response to innate developmental cues. In this study, we first demonstrated the longevity, multipotency, and high fidelity of sensory features of postmigrating adult dorsal root ganglia (aDRG) stem cells. Derived from aDRG and after 4-5 years in culture without dissociating, the aDRG NSCs were found capable of proliferation, expressing neuroepithelial, neuronal, and glial markers. Remarkably, these aDRG NSCs expressed sensory neuronal markers vesicular glutamate transporter2 (VGluT2-glutamate terminals), transient receptor potential vanilloid1 (TrpV1-capsaicin sensitive), phosphorylated 200 kDa neurofilaments (pNF200-capsaicin insensitive, myelinated), and the serotonin transporter (5-HTT), which normally is transiently expressed in developing DRG. Furthermore, in response to neurotrophins, the aDRG NSCs enhanced TrpV1 expression upon exposure to nerve growth factor (NGF), but not to brain-derived neurotrophic factor (BDNF). On the contrary, BDNF increased the expression of NeuN. Third, the characterization of aDRG NSCs was demonstrated by transplantation of red fluorescent-expressing aDRG NSCs into injured spinal cord. These cells expressed nestin, Hu, and b-III-tubulin (immature neuronal markers), GFAP (astrocyte marker) as well as sensory neural marker TrpV1 (capsaicin sensitive) and pNF200 (mature, capsaicin insensitive, myelinated). Our results demonstrated that the postmigrating neural crest adult DRG stem cells not only preserved their multipotency but also were retentive in sensory potency despite the age and long-term ex vivo status.
Key words: Long-term potency; Neural progenitor cells; Spinal cord injury; Sensory neurons; Brain-derived neurotrophic factor (BDNF); Nerve growth factor (NGF)
Address correspondence to Feng C. Zhou, Ph.D., Department of Anatomy
& Cell Biology, Indiana University School of Medicine, 635 Barnhill
Drive, MS 508, Indianapolis, IN 46202, USA. Tel: 317-274-7359; Fax: 317-278-2040;
Noninvasive Imaging of Liver Repopulation Following Hepatocyte Transplantation
Sarah Koenig,1 Petra Krause,1 Ali Seif Amir Hosseini,2 Christian Dullin,3 Margret Rave-Fraenk,2 Sarah Kimmina,4 Andrew Lee Entwistle,5 Robert Michael Hermann,2 Clemens Friedrich Hess,2 Heinz Becker,1 and Hans Christiansen2
1Department of General and Visceral Surgery, University Hospital
Goettingen, Goettingen, Germany
2Department of Radiotherapy, University Hospital Goettingen, Goettingen, Germany
3Department of Radiology, University Hospital Goettingen, Goettingen, Germany
4Department of Laboratory Animal Science, University Hospital Goettingen, Goettingen, Germany
5Department of Genetic Epidemiology, University Hospital Goettingen, Goettingen, Germany
Near infrared fluorescence (NIRF) optical imaging is a technique particularly powerful when studying in vivo processes at the molecular level in preclinical animal models. We recently demonstrated liver irradiation under the additional stimulus of partial hepatectomy as being an effective primer in the rat liver repopulation model based on hepatocyte transplantation. The purpose of this study was to assess optical imaging and the feasibility of donor cell expansion tracking in vivo using a fluorescent probe. Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with irradiation. Four days later, a partial hepatectomy was performed and wild-type (DPPIV+) hepatocytes were transplanted into recipient livers via the spleen. Repopulation by transplanted DPPIV+ hepatocytes was detected in vivo with Cy5.5-conjugated DPPIV antibody using the eXplore OptixTM System (GE HealthCare). Results were compared with nontransplanted control animals and transplanted animals receiving nonspecific antibody. Optical imaging detected Cy5.5-specific fluorescence in the liver region of the transplanted animals, increasing in intensity with time, representing extensive host liver repopulation within 16 weeks following transplantation. A general pattern of donor cell multiplication emerged, with an initially accelerating growth curve and later plateau phase. In contrast, no specific fluorescence was detected in the control groups. Comparison with ex vivo immunofluorescence staining of liver sections confirmed the optical imaging results. Optical imaging constitutes a potent method of assessing the longitudinal kinetics of liver repopulation in the rat transplantation model. Our results provide a basis for the future development of clinical protocols for suitable fluorescent dyes and imaging technologies.
Key words: In vivo optical imaging; Near infrared dye Cy5.5; Irradiation; Rat liver repopulation; DPPIV
Address correspondence to Dr. med. Sarah Koenig, Department of General Surgery, University Hospital Goettingen, Robert-Koch-Str. 40, D-37099 Goettingen, Germany. Tel: 0049-551-398977; Fax: 0049-551-396107; E-mail: email@example.com
Wheat Proteins Enhance Stability and Function of Adhesion Molecules in Cryopreserved Hepatocytes
Mélanie Grondin, Francine Hamel, Diana A. Averill-Bates, and Fathey Sarhan
Département des Sciences biologiques, Université du Québec à Montréal, Montréal, Québec H3C 3P8, Canada
Cryopreserved hepatocytes with good hepatospecific functions upon thawing are important for clinical transplantation and for in vitro drug toxicity testing. However, cryopreservation reduces viability and certain hepatospecific functions, but the most pronounced change is diminished attachment efficiency of hepatocytes. Adhesion of cells to the extracellular matrix and cell-cell contacts are crucial for many aspects of cellular function. These processes are partly mediated and controlled by cellular adhesion molecules. The mechanisms responsible for reduced attachment efficiency of cryopreserved hepatocytes are not well understood. To address this question, we investigated the effect of a new cryopreservation procedure, using wheat proteins (WPs) or mixtures of recombinant forms of wheat freezing tolerance-associated proteins, on the stability of three important adhesion molecules (b1-integrin, E-cadherin, and b-catenin). Immunoblot analyses revealed that the levels of b1-integrin, E-cadherin, and b-catenin were much lower in cryopreserved rat hepatocytes, when compared to fresh cells. Protein expression of the adhesion molecules was generally lower in cells cryopreserved with DMSO, compared to WPs. Moreover, the stability of the adhesion molecules was not affected by cryopreservation to the same degree, with more pronounced decreases occurring for b1-integrin (62-74%) > b-catenin (51-58%) > E-cadherin (21-37%). However, when hepatocytes were cryopreserved with partially purified WPs (SulWPE, AcWPE) or with mixtures of recombinant wheat proteins, there was a clear protective effect against the loss of protein expression of b1-integrin, E-cadherin, and b-catenin. Protein expression was only 10-20% lower than that observed in fresh hepatocytes. These findings clearly demonstrate that WPs, and more particularly, partially purified WPs and recombinant wheat proteins, were more efficient for cryopreservation of rat hepatocytes by maintaining good expression of these adhesion molecules. These promising results could lead to a new and improved cryopreservation technology for applications such as clinical transplantation of hepatocytes.
Key words: Cryopreservation; Hepatocytes; Wheat proteins; E-cadherin; b1-Integrin; b-Catenin
Address correspondence to Dr. Fathey Sarhan, Département des Sciences biologiques, Université du Québec à Montréal, C.P. 8888, Succursale Centre-ville, Montréal, Québec H3C 3P8, Canada. Tel: (514) 987-3000, ext. 3363; Fax: (514) 987-4647; E-mail: firstname.lastname@example.org
Suppression of Carbon Tetrachloride-Induced Liver Fibrosis by Transplantation of a Clonal Mesenchymal Stem Cell Line Derived From Rat Bone Marrow
Marhaen Hardjo,1,4 Masahiro Miyazaki,1,5 Masakiyo Sakaguchi,1 Takuro Masaka,1,2 Sukaeni Ibrahim,2 Ken Kataoka,1 and Nam-ho Huh1
1Department of Cell Biology, Okayama University Graduate
School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558,
2Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan
3Department of Behavioral Pediatric Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan
4Department of Biochemistry, Medical Faculty of Hasanuddin University, South Sulawesi, Indonesia
5Department of Food and Nutrition, Faculty of Human Services, Okayama Gakuin University, Kurashiki 710-8511, Japan
Transplantation of hepatocytes or bone marrow-derived cells has been shown to ameliorate liver fibrosis in animal models, but no direct comparison of relative efficiency has been made. The aim of this study was to compare the efficiency of a bone marrow-derived clonal mesenchymal stem cell line established by us (rBM25/S3) with that of its adipogenic or hepatogenic differentiation derivative for suppression of rat liver fibrosis. After induction of differentiation of rBM25/S3 cells into adipogenic or hepatogenic cells in culture, we intrasplenically transplanted the three types of cells into rats (3 × 107 cells/rat) before and 4 weeks after initiation of carbon tetrachloride treatment (1 ml/kg body weight twice a week for 8 weeks) to induce liver fibrosis. Undifferentiated rBM25/S3 cells were the most effective for suppression of liver fibrosis, followed by the adipogenic cells and hepatogenic cells. Expression levels of MMP-2 and MMP-9 were also highest in undifferentiated rBM25/S3 cells. These results indicate that bone marrow-derived clonal mesenchymal stem cell lines are useful for further mechanistic studies on cell-mediated suppression of liver fibrosis and that such cell lines will provide information on an appropriate cell source for transplantation therapy for cirrhosis.
Key words: Liver fibrosis; Mesenchymal stem cells
Address correspondence to Nam-ho Huh, Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikatachou, Okayama 700-8558, Japan. Tel: +81-86-235-7393; Fax: +81-86-235-7400; E-mail: email@example.com
Improved Survival of Fulminant Liver Failure by Transplantation of Microencapsulated Cryopreserved Porcine Hepatocytes in Mice
Jie Mei,1,2* Antonino Sgroi,1* Gang Mai,1,3 Reto Baertschiger,1 Carmen Gonelle-Gispert,1 Véronique Serre-Beinier,1 Philippe Morel,1 and Leo H. Bühler1
1Surgical Research Unit, Department of Surgery, University
Hospital Geneva, Geneva, Switzerland
2Sichuan Provincial Hospital, Chengdu 610041, Sichuan Province, China
3Department of General Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
The aim of this study was to establish hepatocyte isolation in pigs, and to evaluate function of isolated hepatocytes after encapsulation, cryopreservation, and transplantation (Tx) in a mouse model of fulminant liver failure (FLF). After isolation, porcine hepatocytes were microencapsulated with alginate-poly-L-Lysinealginate membranes and cryopreserved. In vitro, albumin production of free and encapsulated hepatocytes were measured by enzyme linked-immunoadsorbent assay. In vivo, encapsulated hepatocytes were transplanted into different groups of mice with FLF and the following experimental groups were performed: group 1, Tx of empty capsules; group 2, Tx of free primary porcine hepatocytes; group 3, Tx of fresh encapsulated porcine hepatocytes; group 4, Tx of cryopreserved encapsulated porcine hepatocytes. In vitro, fresh or cryopreserved encapsulated porcine hepatocytes showed a continuous decreasing metabolic function over 1 week (albumin and urea synthesis, drug catabolism). In vivo, groups 1 and 2 showed similar survival (18% and 25%, respectively, p >0.05). In groups 3 and 4, Tx of fresh or cryopreserved encapsulated porcine hepatocytes significantly increased survival rate to 75% and 68%, respectively (p < 0.05). Primary porcine hepatocytes maintained metabolic functions after encapsulation and cryopreservation. In mice with FLF, Tx of encapsulated xenogeneic hepatocytes significantly improved survival. These results indicate that porcine hepatocytes can successfully be isolated, encapsulated, stored using cryopreservation, and transplanted into xenogeneic recipients with liver failure and sustain liver metabolic functions.
Key words: Hepatocyte transplantation; Fulminant liver failure; Xenotransplantation; Cryopreservation; Encapsulation
Address correspondence to Leo H. Bühler, M.D., Surgical Research Unit, Department of Surgery, University Hospital Geneva, 24, Rue Michelidu-Crest, 1211, Geneva 14, Switzerland. Tel: 41 22/3727698; Fax: 41 22/3727689; E-mail: firstname.lastname@example.org
*Contributed equally to this work.