ognizant Communication Corporation


VOLUME 13, NUMBER 2, 2004

Cell Transplantation, Vol. 13, pp. 93-101, 2004
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Clonal Population of Adult Stem Cells: Life Span and Differentiation Potential

Mitchel Seruya,1 Anup Shah,2 Dawn Pedrotty,3 Tracey du Laney,3 Ryan Melgiri,4 J. Andrew McKee,4 Henry E. Young,5 and Laura E. Niklason3,6

1Columbia University College of Physicians and Surgeons, New York, NY 10032
2Stanford University School of Medicine, Palo Alto, CA 94305
3Department of Biomedical Engineering, Duke University, Durham, NC 27708
4Duke University Medical School, Durham, NC 27708
5Departments of Basic Medical Sciences and Pediatrics, Mercer University School of Medicine, Macon, GA 31207
6Department of Anesthesia, Duke University, Durham, NC 27710

Adult stem cells derived from bone marrow, connective tissue, and solid organs can exhibit a range of differentiation potentials. Some controversy exists regarding the classification of mesenchymal stem cells as bona fide stem cells, which is in part derived from the limited ability to propagate true clonal populations of precursor cells. We isolated putative mesenchymal stem cells from the connective tissue of an adult rat (rMSC), and generated clonal populations via three rounds of dilutional cloning. The replicative potential of the clonal rMSC line far exceeded Hayflick's limit of 50-70 population doublings. The high capacity for self-renewal in vitro correlated with telomerase activity, as demonstrated by telomerase repeat amplification protocol (TRAP) assay. Exposure to nonspecific differentiation culture medium revealed multilineage differentiation potential of rMSC clones. Immunostaining confirmed the appearance of mesodermal phenotypes, including adipocytes possessing lipid-rich vacuoles, chondrocytes depositing pericellular type II collagen, and skeletal myoblasts expressing MyoD1. Importantly, the spectrum of differentiation capability was sustained through repeated passaging. Furthermore, serum-free conditions that led to high-efficiency smooth muscle differentiation were identified. rMSCs plated on collagen IV-coated surfaces and exposed to transforming growth factor-b1 (TGF-b1) differentiated into a homogeneous population expressing a-actin and calponin. Hence, clonogenic analysis confirmed the presence of a putative MSC population derived from the connective tissue of rat skeletal muscle. The ability to differentiate into a smooth muscle cell (SMC) phenotype, combined with a high proliferative capacity, make such a connective tissue-derived MSC population ideal for applications in vascular tissue construction.

Key words: Mesenchymal stem cell; Differentiation; Smooth muscle cell; Growth factors

Address correspondence to Laura E. Niklason, M.D., Ph.D., Assistant Professor, Departments of Anesthesia and Biomedical Engineering, Duke University, Room 136 Hudson Hall, Research Dr. at Science Dr., Durham, NC 27708. Tel: (919) 660-5149; Fax: (919) 681-7022; E-mail: nikla001@mc.duke.edu

Cell Transplantation, Vol. 13, pp. 103-111, 2004
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Transplantation of Adipose Tissue-Derived Stromal Cells Increases Mass and Functional Capacity of Damaged Skeletal Muscle

Francis Bacou,1 Ramzi Boubaker el Andalousi,1 Paul-André Daussin,1,2 Jean-Paul Micallef,3 Jonathan M. Levin,1 Michel Chammas,2 Louis Casteilla,4 Yves Reyne,1 and Jean Nouguès1

1UMR 866 Différenciation cellulaire et Croissance, INRA, Montpellier Cedex 1, France
2Service de Chirurgie Orthopédique 2 et Chirurgie de la Main, Hôpital Lapeyronie, CHU Montpellier, France
3INSERM ADR 08, Montpellier Cedex 5, France
4UMR 5018 UPS CNRS, Toulouse Cedex, France

The regenerating skeletal muscle environment is capable of inducing uncommitted progenitors to terminally differentiate. The aim of this work was to determine whether adipose tissue-derived stromal cells were able to participate in muscle regeneration and to characterize the effect on muscle mass and functional capacities after transplantation of these cells. Adipose tissue stromal cells labeled with Adv cyto LacZ from 3-day-old primary cultures (SVF1) were autotransplanted into damaged tibialis anterior muscles. Fifteen days later, b-galactosidase staining of regenerated fibers was detected, showing participation of these cells in muscle regeneration. Two months after SVF1 cell transfer, muscles were heavier, showed a significantly larger fiber section area, and developed a significantly higher maximal force compared with damaged control muscles. These results are similar to those previously obtained after satellite cell transplantation. However, SVF1 transfer also generated a small amount of adipose tissue localized along the needle course. To minimize these adipose contaminants, we transferred cells from 7-day-old secondary cultures of the SVF1, containing only a small proportion of already engaged preadipocytes (SVF2). Under these conditions, no adipose tissue was observed in regenerated muscle but there was also no effect on muscle performances compared with damaged control muscles. This result provides further evidence for the existence of progenitor cells in the stromal fraction of freshly isolated adipose tissue cells, which, under our conditions, keep some of their pluripotent properties in primary cultures.

Key words: Skeletal muscle; Cell therapy; Stromal cells; Adipose tissue; Rabbits

Address correspondence to Francis Bacou, UMR 866 Différenciation cellulaire et Croissance, INRA, 2 Place Pierre Viala, 34060 Montpellier Cedex 1, France. Tel: (33) 04 99 61 24 07; Fax: (33) 04 67 54 56 94; E-mail: francis.bacou@ensam.inra.fr

Cell Transplantation, Vol. 13, pp. 113-122, 2004
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Intraspinal Transplantation of CD34+ Human Umbilical Cord Blood Cells After Spinal Cord Hemisection Injury Improves Functional Recovery in Adult Rats

Zong Mao Zhao,1,2* Hong Jun Li,1* Hai Ying Liu,1,2* Shi Hong Lu,1 Ren Chi Yang,1 Qing Jun Zhang,2 and Zhong Chao Han1

1National Research Center for Stem Cell Engineering & Technology, State Key Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, People's Republic of China
2Department of Neurosurgery, The Second Hospital of Hebei Medical University, Shijiazhuang, People's Republic of China

The present study was designed to compare the functional outcome of the intraspinal transplantation of CD34+ human umbilical cord blood (CB) cells with that of human bone marrow stromal (BMS) cells in adult rats with spinal cord injury. Sixty adult Wistar rats were subjected to left spinal cord hemisection, and then divided into three groups randomly. The control group received an injection of PBS without cells, while the two other groups of rats received a transplantation of 5 x 105 CD34+ CB or BMS cells, respectively. Functional outcome was measured using the modified Tarlov score at days 1, 7, 14, 21, and 28 after transplantation. A statistically significant improvement in functional outcome and survival rate in the experimental groups of rats was observed compared with the control group. Rats that received CD34+ CB cells achieved a better improvement in functional score than those that received BMS cells at days 7 and 14 after transplantation. Histological evaluation revealed that bromodeoxyuridine (BrdU)-labeled CD34+ CB and BMS cells survived and migrated into the injured area. Some of these cells expressed glial fibriliary acidic protein (GFAP) or neuronal nuclear antigen (NeuN). Our data demonstrate for the first time that intraspinal transplantation of human CD34+ CB cells provides benefit in function recovery after spinal cord hemisection in rats and suggest that CD34+ CB cells may be an excellent choice of cells as routine starting material of allogenic and autologous transplantations for the treatment of spinal cord injury.

Key words: Spinal cord injury; Bone marrow stromal cells; Human umbilical cord blood cells; Transplantation; Stem cell plasticity; Rats

*Z. M. Zhao, H. J. Li, and H. Y. Liu contributed equally in this study and should be considered as co-first authors.

Address correspondence to Dr. Zhong Chao Han, Professor, Institute of Hematology, Chinese Academy of Medical Sciences, Tianjin 300020, PR China. Tel: 86 22 27317276; Fax: 86 22 27317273; E-mail: tihzch@public.tpt.tj.cn

Cell Transplantation, Vol. 13, pp. 123-136, 2004
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Transplanted hNT Cells ("LBS Neurons") in a Rat Model of Huntington's Disease: Good Survival, Incomplete Differentiation, and Limited Functional Recovery

Rosemary A. Fricker-Gates,1 Janice A. Muir,2 and Stephen B. Dunnett1

Schools of 1Biosciences and 2Psychology, Cardiff University, Cardiff, Wales, UK

A variety of immortalized cell lines have been proposed to exhibit sufficient phenotypic plasticity to allow them to replace primary embryonic neurons for restorative cell transplantation. In the present experiments we evaluate the functional viability of one particular cell line, the hNT cells developed by Layton Bioscience, to replace lost neurons and alleviate asymmetrical motor deficits in a unilateral excitotoxic lesion model of Huntington's disease. Because the grafts involved implantation of human-derived cells into a rat host environment, all animals were immunosuppressed. Cyclosporin A and FK-506 were similar in providing effective immunoprotection of the hNT xenografts, and whereas the lesions induced a marked inflammatory response in the host brain, this was not exacerbated by the presence of xenograft cells. The presence of grafted cells was determined with the human-specific antigen HuNu, and good graft survival was demonstrated in almost all animals up to the longest survival examined, 16 weeks posttransplantation. Although the cells exhibited progressively greater maturation and differentiation at 10-day, 4- and 16-week time points, staining for the mature neuronal marker NeuN was at best very weak, and we were unable to detect unequivocal staining with any markers of mature striatal phenotype, including DARPP-32, calbindin, parvalbumin, choline acetyl transferase, or NADPH diaphorase (with in all cases positive control provided by good staining on the intact contralateral side of the brain). Nor were we able to detect any differences between rats with lesions alone and rats with grafts in the contralateral motor deficits exhibited in a test of skilled paw reaching or cylinder placing. These results suggest that further and more extensive studies should be undertaken to assess whether hNT neurons can show more extensive and appropriate maturation and be associated with recovery in appropriate behavioral models, before they may be considered a suitable replacement for primary embryonic cells for clinical application in Huntington's disease.

Key words: Striatal lesions; Striatal grafts; Immortalized cells; Paw reaching; Neuronal differentiation; Functional recovery

Address correspondence to Rosemary A. Fricker-Gates, School of Biosciences, Cardiff University, Museum Avenue Box 911, Cardiff CF10 3US, Wales, UK. Tel: +44 2920 875188; Fax: +44 2920 876749; E-mail: gatesra@cf.ac.uk

Cell Transplantation, Vol. 13, pp. 137-143, 2004
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Natural Antibodies Prevent In Vivo Transmission of Porcine Islet-Derived Endogenous Retrovirus to Human Cells

Brice W. McKane,1* Sabarinathan Ramachandran,1* Xiao-Chun Xu,1 Barbara J. Olack,1 William C. Chapman,1 and T. Mohanakumar1,2

Departments of 1Surgery and 2Pathology & Immunology, Washington University School of Medicine, St. Louis, MO 63110

The discovery of porcine endogenous retroviruses (PERV) has raised concerns regarding the safety of porcine xenotransplantation. However, transmission of PERV had not been observed in humans exposed to porcine tissue. We examined whether PERV derived from porcine pancreatic islet cells could infect human cells in vivo and the role of natural antibodies in inhibiting PERV infection. In vivo infective potential of PERV was studied in SCID mice reconstituted with human peripheral blood leucocytes. Porcine islets were transplanted under the kidney capsule. PERV infection was determined by analyzing PERV gene expression in graft infiltrating lymphocytes (GIL) harvested 21 days posttransplantation. Mice were administered normal human serum prior to and 2 days posttransplantation to study their role in protection of human cells against PERV infection. PERV genes were expressed in all porcine tissues examined, including purified porcine islets. PERV expression was detected in GILs from three of five human-SCID mice. Administration of human serum blocked PERV infection in GILs in five of five human-SCID mice. These results indicate that PERV from porcine islets can infect human cells in vivo. Normal human serum blocks transmission of retrovirus in vivo, suggesting that natural xenoreactive antibodies can prevent PERV infection.

Key words: Porcine endogenous retrovirus (PERV); Porcine islets; Natural antibodies; Immunity; Xenotransplantation; Real-time PCR

*Equal contribution by both authors.

Address correspondence to T. Mohanakumar, Ph.D., Washington University School of Medicine, Department of Surgery, Box 8109-3328 CSRB, 660 S. Euclid Avenue, St. Louis, MO 63110. Tel: (314) 362-8463; Fax: (314) 747-1560; E-mail: kumart@msnotes.wustl.edu

Cell Transplantation, Vol. 13, pp. 145-152, 2004
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Polyphenol, an Extract of Green Tea, Increases Culture Recovery Rates of Isolated Islets From Nonhuman Primate Pancreata and Marginal Grade Human Pancreata

Guangming Zhang,1 Shinichi Matsumoto,1,2 Suong-Hyu Hyon,3 Sabrina A. Qualley,1 Lisa Upshaw,1 D. Michael Strong,1 and Jo-Anna Reems1,4

1Northwest Tissue Center at the Puget Sound Blood Center, Seattle, WA 98104
2Kyoto University Hospital Transplant Unit, Kyoto, 606-8507 Japan
3Kyoto University, Institute for Frontier Medical Sciences, Kyoto, Japan
4University of Washington, Department of Medicine, Seattle, WA

Investigations indicate that an extract of green tea, polyphenol, can significantly increase the culture survival rate of rat islets without deteriorating their functionality. In this study, we examined the effect of adding polyphenol to islets isolated from human pancreata and nonhuman primate pancreata. Islets were isolated from human pancreata that did not meet criteria for clinical transplantation (n = 6) and from nonhuman primate pancreata (n = 5). The islets were cultured in CMRL-1066 + 10% FCS with the addition of 0, 30, 60, 125, 250, or 500 mg/ml of polyphenol. After 24 or 48 h of culture, islet yield, viability, purity, morphology, and stimulation index was assessed. RT-PCR and Western blot analysis were also performed to assess the expression levels of the apoptotic related genes, Bcl-2 and BAX. After 24 h of culture, islet yields were significantly higher in cultures supplemented with 30-250 mg/ml of polyphenol than in cultures without polyphenol. After 48 h of culture, significant differences in islet numbers were observed with polyphenol concentrations of 125 mg/ml (p < 0.01) and 250 mg/ml (p < 0.01). However, no significant differences were noted in islet viability, purity, morphology, and stimulation index at each time point with or without polyphenol. RT-PCR and Western blot analysis of the islets indicated that Bcl-2 levels increased by 2.5-fold and BAX levels decreased by twofold in cultures supplemented with polyphenol. This resulted in BAX/Bcl-2 ratios that were lower in polyphenol-supplemented cultures than with control cultures. Polyphenol increases culture recovery rates by precluding islet apoptosis.

Key words: Polyphenol; Islet; Preservation; Recovery rate; Apoptosis

Address correspondence to Dr. Shinichi Matsumoto, Kyoto University Hospital Transplant Unit, 54 Kawara-cho Shogoin, Sakyo-ku, Kyoto, 606-8507 Japan. Tel: 81-75-751-4324; Fax: 81-75-751-4348; E-mail: shinichi@kuhp.kyoto-u.ac.jp

Cell Transplantation, Vol. 13, pp. 153-160, 2004
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Endogenous Pancreatic Enzyme Activity Levels Show no Significant Effect on Human Islet Isolation Yield

Natisha L. Rose,1 Monica M. Palcic,2 A. M. James Shapiro,1 and Jonathan R. T. Lakey1

1Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Centre, University of Alberta, Edmonton, Alberta, T6G 2N8, Canada
2Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada

Despite advances in human islet isolation, islet yield remains inconsistent and unreliable. In recent studies, it has been suggested that serine proteases, in particular trypsin, have been shown to have a damaging effect on islet yield. This study evaluated enzyme activity levels throughout 42 human islet isolation procedures. Trypsin, chymotrypsin, and elastase activity was determined spectrophotometrically using suitable chromophoric substrates. The results of the islet isolations were rated as successful (n = 19) or unsuccessful (n = 23) based on the islet yield and functionality. The enzyme activity profiles of the isolations were compared. No significant differences in donor-related variables were found in this study. However, in the successful isolations, a significantly greater amount (85.6 ± 1.9%; p = 0.0017) of the pancreas was digested in a significantly shorter digestion time (19.7 ± 0.6 min; p = 0.0054) compared with 74.8 ± 2.5% of digested tissue in 22.6 ± 0.7 min in the poor isolations. This study showed no significant effect of serine protease levels on the outcome of islet isolations, regardless of enzyme inhibitor supplementation. These data suggest that serine protease activity does not sufficiently affect islet yield. However, the data show that the most successful human islet isolations are achieved when the maximum amount of tissue is digested in the shortest amount of time. This suggests that further understanding of the isolation process should focus on the role of the collagenase digestion solution in the dissociation of the endocrine-exocrine tissue connection.

Key words: Islet isolation; Trypsin; Chymotrypsin; Elastase; Enzyme inhibition

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

Cell Transplantation, Vol. 13, pp. 161-168, 2004
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Fate of a Chimeric Joint Construct in an Ectopic Site in SCID Mice

Scott Warden,1 David J. Zaleske,1 and Julie Glowacki1,2

1Skeletal Biology Research Center, Massachusetts General Hospital and
2Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA

This study examines the use of a devitalized biological knee as a scaffold for repopulation with chondrocytes and tests the hypothesis that the devitalized scaffold would become repopulated with the foreign chondrocytes when placed in a suitable environment. Chimeric knee constructs were engineered in vitro and their ectopic in vivo fate was examined in SCID mice. The constructs were made by applying porous collagen sponges that contained viable bovine articular chondrocytes to shaved articular surfaces of devitalized embryonic chick knees. The chimeric joints were cultured for 1 week and were subsequently transplanted into dorsal subcutaneous pouches of 5-week-old mice. Specimens were prepared for histological analysis at 1, 3, 6, or 8 weeks after transplantation. Controls included empty collagen sponges, collagen sponges seeded with viable bovine chondrocytes, and devitalized chick knees without collagen sponge inserts. One week after in vitro incubation of the constructs, the porous collagen sponges with viable bovine chondrocytes were adherent to the shaved articular surfaces of the devitalized chick joints. There was abundant metachromatic neo-matrix around the chondrocytes in the collagen sponges. During maintenance of the constructs in vivo, the chimeric joints exhibited dramatic changes. Bovine chondrocytes proliferated in the collagen sponges and formed abundant new matrix. Bovine chondrocytes migrated into preexisting chick cartilage canals at 1 week. Subsequently, bovine chondrocytes invaded the matrix of the devitalized chick knees. Bovine neo-cartilage obliterated the interface between the collagen sponge and the devitalized chick cartilage. With time in vivo, the bovine neocartilage expanded and replaced the chick matrix. The devitalized cartilage appears to provide a framework for supporting chondrogenesis in a chimeric joint.

Key words: Bioengineering; Joints; In vivo; Knee

Address correspondence to Julie Glowacki, Ph.D., Orthopedic Research, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115. Tel: (617) 732-5397; Fax: (617) 732-6937; E-mail: jglowacki@partners.org

Cell Transplantation, Vol. 13, pp. 169-177, 2004
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Tissue-Engineered Grafts Matured in the Right Ventricular Outflow Tract

Tsukasa Ozawa,1 Donald A. G. Mickle,1 Richard D. Weisel,1 Keiji Matsubayashi,1 Takeshiro Fujii,1 Paul W. M. Fedak,1 Nobuya Koyama,2 Yoshito Ikada,3 and Ren-Ke Li1

1Department of Surgery, Division of Cardiovascular Surgery, Toronto General Research Institute, Toronto General Hospital, University of Toronto, Canada
2Toho University School of Medicine, Japan
3Suzuka University of Medical Science, Japan

Autologous smooth muscle cell (SMC)-seeded biodegradable scaffolds could be a suitable material to repair some pediatric right ventricular outflow tract (RVOT) cardiac anomalies. Adult syngenic Lewis rat SMCs (2 x 106) were seeded onto a new biodegradable copolymer sponge made of e-caprolactone-co-L-lactide reinforced with poly-L-lactide fabric (PCLA). Two weeks after seeding, the patch was used to repair a surgically created RVOT defect in an adult rat. At 8 weeks after implantation the spongy copolymer component was biodegraded, and SM tissue and extracellular matrices containing elastin fibers were present in the scaffolds. By 22 weeks more fibroblasts and collagen were present (p < 0.05). The number of capillaries in the grafts also increased (p < 0.001) between 8 and 22 weeks. The fibrous poly-L-lactide component of the PCLA scaffold remained. The 22-week grafts maintained their thickness and surface area in the RVOT. The SMCs prior to implantation were in a synthetic phenotype and developed in vivo into a more contractile phenotype. By 8 weeks the patches were endothelialized on their endocardial surfaces. Future work to increase the SM tissue and elastin content in the patch will be necessary before implantation into a pediatric large-animal model is tested.

Key words: Congenital heart defects; Pediatric cardiac surgery; Myocardium; Tissue engineering; Smooth muscle cells; Phenotype; Biodegradable scaffold; Biomaterial

Address correspondence to Ren-Ke Li, M.D., Ph.D., Toronto General Hospital, NU-G108, 200 Elizabeth Street, Toronto, Ontario M5G 2C4 Canada. Tel: (416) 340-3361; Fax: (416) 340-4806; E-mail: renkeli@uhnres.utoronto.ca

Cell Transplantation, Vol. 13, pp. 179-185, 2004
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Assessment of Different Transfection Parameters in Efficiency Optimization

A. T. L. Young,1 R. B. Moore,1 A. G. Murray,2 J. C. Mullen,1 and J. R. T. Lakey1

1Department of Surgery, Surgical-Medical Research Institute, and 2Department of Medicine, University of Alberta, Edmonton, Canada T6G 2N8

Achieving optimal transfection efficiency is the most critical step in overcoming the primary obstacle to success in nonviral-mediated gene therapy. Several transfection parameters were being examined including the effects of different types of transfection media, glucose concentration, reporter DNA concentration, and incubation time in lipotransfectant. Efficiency of transfection obtained was highest for Opti-MEM I (29 ± 2.28%; p = 0.001) followed by M199 (24 ± 1.54%; p = 0.009), both of which performed significantly better than DMEM (14 ± 0.28%) as a transfection medium. The rate of transfection was affected by glucose levels in only DMEM with higher efficiency achieved using low glucose containing DMEM (17 ± 0.38%) than its counterpart. Furthermore, transfection rate and cell viability were severely hampered by lengthened exposure to transfection complexes, leading to an overall mean efficiency of 5 ± 0.87%. However, doubling the DNA content in the transfection mixture did not significantly change the mean rate of transfection in M199 medium (24 ± 1.54% to 27 ± 1.54%; p = 0.273). The overall range of mean efficiency acquired with our protocol under different transfection conditions was between 14% and 29%. Hopefully results from this study will further potential success in nonviral-mediated gene transfer.

Key words: Transfection efficiency; HUVECs; Effectene; Glucose; Transfection media

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

Cell Transplantation, Vol. 13, pp. 187-195, 2004
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Long-Term Function of Cryopreserved Cryopreserved Rat Hepatocytes in a Coculture System

Keishi Sugimachi,* Meindert N. Sosef,* John M. Baust, Alex Fowler, Ronald G. Tompkins, and Mehmet Toner

Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, and Shriners Hospital for Children, Boston, MA 02114

The goal of this study was to investigate postpreservation long-term function of cryopreserved primary rat hepatocytes using the hepatocyte/3T3-J2 fibroblast coculture system. The long-term function of thawed hepatocytes cocultured with fibroblasts was evaluated and compared with hepatocytes cultured without fibroblasts. Fresh isolated primary rat hepatocytes were frozen at a controlled rate (-1°C/min) up to -80°C, and then stored in liquid nitrogen for up to 90 days. Thawed hepatocytes were thereafter cocultured with 3T3-J2 murine fibroblasts and cocultivation was monitored for 14 days. The viability of fresh isolated hepatocytes was 91.4%, and that of cryopreserved hepatocytes was 82.1%. Cellular morphology and polarity, which were determined by the localization of actin filaments and connexin-32, were successfully maintained in cryopreserved hepatocytes following cryopreservation. Albumin and urea synthesis reached the maximum level and became stable after day 7 in coculture in both fresh and cryopreserved hepatocytes. Urea synthesis of cryopreserved hepatocytes was maintained 89.0% of nonfrozen fresh control, and albumin production of cryopreserved hepatocytes was 63.7% of control in coculture. Cytochrome P450 activity, which was measured by deethylation of ethoxyresorufin, was also maintained in cryopreserved hepatocytes at 88.6% of nonfrozen fresh control in coculture. The retention of synthetic and detoxification activities was verified to be well preserved during extended low-temperature storage (90 days). Both fresh control and cryopreserved hepatocytes cultured without fibroblast did not retain their synthetic and detoxification functions in long-term culture. These data illustrate that, through the utilization of our cryopreservation procedure, primary hepatocyte function was successfully maintained when placed into coculture configuration following thawing.

Key words: Cryopreservation; Primary hepatocyte; Coculture; Connexin-32; Albumin; Cytochrome P450; HypoThermosol

*The first two authors equally contributed to this work.

Address correspondence to Mehmet Toner, Ph.D., Shriners Hospital for Children in Boston, 51 Blossom Street, Boston, MA 02114. Tel: (617) 371-4883; Fax: (617) 371-4950; E-mail: mtoner@sbi.org