ognizant Communication Corporation

CELL TRANSPLANTATION

ABSTRACTS
VOLUME 10, NUMBERS 4/5

Cell Transplantation, Vol. 10, pp. 353-361
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Selective Repopulation of Mice Liver After Fas-Resistant Hepatocyte Transplantation

Masayuki Fujino,1,3 Xiao-Kang Li,1 Yusuke Kitazawa,1 Naoko Funeshima,1 Lei Guo,1 Torayuki Okuyama,2 Takashi Amano,3 Hiroshi Amemiya,1 and Seiichi Suzuki1

1Department of Experimental Surgery and Bioengineering and 2Genetics, National Children's Medical Research Center, Tokyo, Japan
3Department of Zootechnical Science, Tokyo University of Agriculture, Tokyo, Japan

Hepatocyte transplantation has been proposed as a potential therapeutic method to treat irreversible liver failure and inherited hepatic disorders, although transplanted cells do not easily reconstruct the liver tissue under intact conditions. This study was aimed at modulating the recipient liver conditions to promote repopulation of the liver after hepatocyte transplantation. Hepatocytes isolated from male MRL-lpr/lpr (lpr) mice with a mutation of Fas antigen were transplanted in a number of 1 x 106 cells in female MRL-+/+ (wild-type mice) by intrasplenic injection. An agonistic anti-Fas antibody (0.15 mg/kg) was administered intravenously 24 h after cell transplantation. We also administrated the antibody at 0.3 mg/kg 1 week after grafting and at 0.6 mg/kg 2 weeks after transplantation. The liver specimens were taken at different time intervals for histological examination. The reconstructed male lpr hepatocytes in the female wild-type mice were determined by a real-time quantitative PCR assay using the primers and probe for the sry gene. The pathologic findings of the recipient livers after treatment with anti-Fas antibody revealed a large number of apoptotic hepatocytes. The grafted lpr hepatocytes were observed to reconstruct as much as 6.9% of the recipient liver in the anti-Fas antibody-treated group 3 months after transplantation. In contrast, we observed the transplanted cells at lower than 0.1% in the nontreated livers. These findings demonstrated that repeated induction of apoptosis in recipient hepatocytes shifts the environment of the liver to a regenerative condition. This method may be useful to promote the reconstruction of transplanted hepatocytes in a recipient liver.

Key words: Fas; Hepatocyte transplantation; Real-time quantitative PCR; sry gene

Address correspondence to Xiao-Kang Li, M.D., Ph.D., Department of Experimental Surgery & Bioengineering, National Children's Medical Research Center, 3-35-31 Taishido Setagaya-ku, Tokyo, 154-8509 Japan. Tel: 81-3-3414-8121; Fax: 81-3-3414-3208; E-mail: sri@nch.go.jp




Cell Transplantation, Vol. 10, pp. 363-371
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Development of a Cryopreservation Procedure Employing a Freezer Bag for Pancreatic Islets Using a Newly Developed Cryoprotectant

Masaaki Miyamoto,1 A. N. Balamurugan,1 Yuka Nozawa,1 Tomonori Sakurai,1 Baoyou Xu,1 Shigehiro Yoshimura,2 Tsuneo Tanaka,2 Toshihiro Tohyama,3 Junji Miyakoshi,4 and Kazutomo Inoue1

1Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
2Gas Application Department of Technical Developent, Taiyo Toyo Sanso Co., Ltd. Osaka, Japan
3Osaka Research Laboratory, Nihon Pharmaceutical Co., Ltd. Osaka, Japan
4Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Kyoto, Japan

One of the most important requirements for success in clinical islet transplantation is the use of a large number of viable donor islets. To achieve this, the ability to cryopreserve islets and to establish an islet bank are critical. Previously, we developed a two-step cryopreservation procedure with freezing tubes utilizing low and high concentrations of dimethyl sulfoxide (DMSO) and using a fully automated cryomachine for human pancreatic islets and porcine islet-like cell clusters (ICCs). Based on these experiments, we developed a simple and efficient cryopreservation procedure of a freezer bag for isolated islets using a fully automated computer-controlled cryomachine with a newly developed cryoprotectant consisting of ethylene glycol (EG) instead of DMSO for decreasing injury of the islets by freezing. A 250 ml Cryocyte blood freezer bag and our newly developed cryoprotectant containing ethylene glycol (EG) were used in the freezing procedure. The islets were frozen by a fully automated computer-controlled cryomachine (GE 9,000) with our original program of slow cooling. Nucleation occurred at -8°C, and the frozen islets were stored at -196°C in a liquid nitrogen tank. The frozen-stored islets were subsequently rapidly thawed in a 37°C water bath and cultured before viability testing. In vitro function, the stimulation index of insulin release during the static incubation test for rat islets cryopreserved in a freezer bag vs. nonfrozen islets as control, was 2.13 ± 0.42 and 2.02 ± 0.38 (94.8% compared with control), respectively (n = 5, p = NS). The islet recovery compared with the nonfrozen control group was 85% (n = 5) in insulin content. When 1000 rat islets cryopreserved in a freezer bag were transplanted into the renal capsule of diabetic athymic mice, all the mice became normoglycemic within 7 days from transplantation. Before nephrectomy, the intravenous glucose torelance test (IVGTT) was performed. The fractional decay constant of the glucose level (K value) of the frozen-thawed group was 0.42 ± 0.06%/min. A histological study of renal subcapsular grafts demonstrated the morphological integrity of the islets. These results demonstrate the utility of our cryopreservation procedure of a freezer bag for isolated islets using a fully automated computer-controlled cryomachine with a newly developed cryoprotectant for the maintenance of viability and function of frozen-stored islets both in culture and after transplantation. Cryopreservation using freezer bags with the new cryoprotectant is an effective and simple method for making an islet bank for clinical trials of islet transplantation.

Key words: Cryopreservation; Islet transplantation; Freezer bag; Cryoprotectant

Address correspondence to Prof. Kazutomo Inoue, Department of Organ Reconstruction, Field of Clinical Application, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin Sakyo-ku, Kyoto 606-8507, Japan. Tel: 81-75-751-4850; Fax: 81-75-751-4145; E-mail: miyamoto@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 10, pp. 373-376
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Insertion of a Suicide Gene Into an Immortalized Human Hepatocyte Cell Line

Naoya Kobayashi,1 Hirofumi Noguchi,1 Toshinori Totsugawa,1 Takamasa Watanabe,1 Toshihisa Matsumura,1 Toshiyoshi Fujiwara,1 Masahiro Miyazaki,2 Kenichi Fukaya,2 Masayoshi Namba,2 and Noriaki Tanaka1

1First Department of Surgery and 2Department of Cell Biology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan

For developing a bioartificial liver (BAL) device, an attractive alternative to the primary human hepatocytes would be the use of highly differentiated immortalized human hepatocytes with a safeguard. To test the feasibility, the primary human hepatocytes were immortalized by a plasmid SV3neo encoding simian virus 40 large T antigen (SV40Tag) gene. A highly differentiated hepatocyte line OUMS-29 was established. A suicide gene of herpes simplex virus-thymidine kinase (HSV-TK) was retrovirally introduced into OUMS-29 cells as a safeguard for clinical application. One of the resulting HSV-TK-positive cell lines, OUMS-29/tk, grew in chemically defined serum-free medium with the gene expression of differentiated liver functions. OUMS-29/tk cells were 100 times more sensitive to ganciclovir compared with unmodified OUMS-29 cells in in vitro experiments. We have established a tightly regulated immortalized human hepatocyte cell line. Essentially unlimited availability of OUMS-29/tk cells may be clinically useful for BAL therapy.

Key words: Immoralized human hepatocytes; Suicide gene; Herpes simplex virus-thymidine kinase

Address correspondence to Naoya Kobayashi, M.D., Ph.D., First Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: (+81) 86-235-7259; Fax: (+81) 86-235-8775; E-mail: immortal@md.okayama-u.ac.jp




Cell Transplantation, Vol. 10, pp. 377-381
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Successful Retroviral Gene Transfer of Simian Virus 40 T-Antigen and Herpes Simplex Virus-Thymidine Kinase Into Human Hepatocytes

Naoya Kobayashi,1 Hirofumi Noguchi,1 Karen A. Westerman,2 Takamasa Watanabe,1 Toshihisa Matsumura,1 Totsugawa Toshinori,1 Toshiyoshi Fujiwara,1 Philippe Leboulch,2 and Noriaki Tanaka1

1First Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
2Harvard-Massachusetts Institute of Technology, Division of Health Sciences and Technology, Cambridge, MA 02139

Current clinical reports have indicated that hepatocyte transplantation (HTX) could be used in patients with liver failure and in children with liver-based metabolic diseases. One of the major limiting factors of HTX is a serious shortage of donor livers for hepatocyte isolation. To address this issue, we immortalized adult human hepatocytes with a retroviral vector SSR#69 expressing the genes of simian virus 40 large T antigen and herpes simplex virus-thymidine kinase simultaneously. One of the resulting clones, NKNT-3, grew steadily in chemically defined serum-free medium without any obvious crisis and showed the gene expression of differentiated liver functions. Under the administration of 5 mM ganciclovir, NKNT-3 cells stopped proliferation and died in in vitro experiments. We have established a tightly regulated immortal human hepatocyte cell line. The cells could allow the need for immediate availability of consistent and functionally uniform cells in sufficient quantity and adequate quality.

Key words: Immortalized human hepatocyte; Retroviral gene transfer; Simian virus 40 large T antigen

Address correspondence to Naoya Kobayashi, M.D., Ph.D., First Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan. Tel: (+81) 86-235-7259; Fax: (+81) 86-221-8775; E-mail: immortal@md.okayama-u.ac.jp

A more comprehensive review of the work can be found in Science 287: 1258-1262; 2000.




Cell Transplantation, Vol. 10, pp. 383-386
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Cre/loxP-Based Reversible Immortalization of Human Hepatocytes

Naoya Kobayashi,1 Hirofumi Noguchi,1 Karen A. Westerman,2 Takamasa Watanabe,1 Toshihisa Matsumura,1 Toshinori Totsugawa,1 Toshiyoshi Fujiwara,1 Philippe Leboulch,2 and Noriaki Tanaka1

1First Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
2Harvard-Massachusetts Institute of Technology, Division of Health Sciences and Technology, Cambridge, MA 02139

An ideal alternative to the primary human hepatocytes for hepatocyte transplantation would be to use a clonal cell line that grows economically in culture and exhibits the characteristics of differentiated, nontransformed hepatocytes following transplantation. The purpose of the present studies was to establish a reversibly immortalized human hepatocyte cell line. Human hepatocytes were immortalized with a retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40Tag) gene flanked by a pair of loxP recombination targets. One of the resulting clones, NKNT-3, showed morphological characteristics of liver parenchymal cells and expressed the genes of differentiated liver functions. NKNT-3 cells offered unlimited availability. After an adenoviral delivery of Cre recombinase and subsequent differential selection, efficient removal of SV40Tag from NKNT-3 cells was performed. Here we represent that elimination of the retrovirally transferred SV40Tag gene can be excised by adenovirus-mediated site-specific recombination.

Key words: Reversible immortalization; Human hepatocytes; Cre/loxP system; Simian virus 40 large T antigen

Address correspondence to Naoya Kobayashi, M.D., Ph.D., First Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan. Tel: (+81) 86-235-7259; Fax: (+81) 86-221-8775; E-mail: immortal@md.okayama-u.ac.jp

A more comprehensive review of the work can be found in Science 287: 1258-1262; 2000.




Cell Transplantation, Vol. 10, pp. 387-392
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Rapidly Functional Immobilization of Immortalized Human Hepatocytes Using Cell Adhesive GRGDS Peptide-Carrying Cellulose Microspheres

Naoya Kobayashi,1 Takehito Taguchi,2 Hirofumi Noguchi,1 Teru Okitsu,1 Toshinori Totsugawa,1 Takamasa Watanabe,1 Toshihisa Matsumura,1 Toshiyoshi Fujiwara,1 Haruo Urata,3 Nobuyuki Kishimoto,3 Nobuyuki Hayashi,4 Shuhei Nakaji,4 Takuro Murakami,2 and Noriaki Tanaka1

1First Department of Surgery, 2Second Department of Anatomy, 3Central Research Laboratory, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
4Department of Medical Products, Kuraray Co., LTD. 1621 Sakazu, Kurashiki, Okayama, 710-8662 Japan

With the development of biotechnology, hepatic support by a hybrid artificial liver (HAL) using hepatocytes has been given much attention. Because the availability of human livers is limited, we have established a tightly regulated immortal human hepatocyte cell line, NKNT-3, for developing HAL. Because high-density cell culture allows the compactness of the HAL device and its easy use under emergency circumstances, we have developed cell adhesive GRGDS peptide-containing cellulose microspheres (GRGDS/CMS). The GRGDS/CMS efficiently immobilized NKNT-3 cells within 24 h in a stirred suspension culture. Electron microscopic examinations demonstrated glycogen granules and well-developed endoplasmic reticulum and mitochondria in NKNT-3 cells attached to the GRGDS/CMS. The cells showed ammonia clearance activity, whereas HepG2-transformed human liver cells did not remove the loaded ammonia. An efficient adenoviral delivery of the lacZ reporter gene was performed in GRGDS/CMS-immobilized NKNT-3 cells. In this study we present rapid immobilization of NKNT-3 immortal human hepatocytes using cellulose microspheres carrying GRGDS peptides. These microspheres satisfied immediate preparation of NKNT-3 cells in sufficient quantity and of adequate quality.

Key words: Immortal hepatocytes; Cellulose microspheres; Artificial liver

Address correspondence to Naoya Kobayashi, M.D., Ph.D., First Department of Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: (+81) 86-235-7259; Fax: (+81) 86-235-8775; E-mail: immortal@md.okayama-u.ac.jp




Cell Transplantation, Vol. 10, pp. 393-396
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Clonal Expansion of Hepatic Stem/Progenitor Cells Following Flow Cytometric Cell Sorting

Atsushi Suzuki,1 Yun-wen Zheng,1 Katashi Fukao,1 Hiromitsu Nakauchi,2 and Hideki Taniguchi1

1Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
2Department of Immunology, Institute of Basic Medical Sciences, University of Tsukuba, and CREST (Japan Science and Technology Corporation), 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan

Although hepatic stem cells are believed to exist and play a critical role in developing and regenerating liver, little is known about their cell surface specificity or differentiation capabilities. To make prospective studies of hepatic stem cells possible, we established an in vitro culture system for identification and characterization of hepatic stem/progenitor cells. By combining this culture system with fluorescence activated cell sorting (FACS), a population of cells that were capable of forming large colonies and providing their descendants for relative longer period was isolated from fetal mouse livers. These data suggest that hepatic stem/progenitor cells with high proliferative potential are existent in the developing mouse liver, and that they are enriched by using flow cytometry.

Key words: Hepatocyte; Stem cell; Flow cytometry; Integrin

Address correspondence to Hideki Taniguchi, M.D., Ph.D., Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. Tel: 81-298-53-3221; Fax: 81-298-53-3222; E-mail: rtanigu@igaku.md.tsukuba.ac.jp




Cell Transplantation, Vol. 10, pp. 397-401
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Grafting of Encapsulated Genetically Modified Cells Secreting GDNF Into the Striatum of Parkinsonian Model Rats

Isao Date, Tetsuro Shingo, Hideyuki Yoshida, Kenjiro Fujiwara, Kazuki Kobayashi, Akira Takeuchi, and Takashi Ohmoto

Department of Neurological Surgery, Okayama University Medical School, Okayama, Japan

In order to deliver glial cell line-derived neurotrophic factor (GDNF) into the brain, we have established a cell line that produces GDNF in a continuous fashion by genetic engineering. These cells were encapsulated and grafted into parkinsonian model rats that had received unilateral intrastriatal injection of 6-hydroxydopamine 2 weeks earlier. Neurochemical analysis showed that GDNF has been produced from the capsule for 6 months after grafting and histological analysis revealed good survival of GDNF-producing cells in the capsule 6 months after grafting. The density of nigrostriatal dopaminergic fibers in the striatum as well as the number of dopaminergic cell bodies in the substantia nigra recovered significantly after GDNF-producing cell grafting. These results suggest the possible application of GDNF-producing cell grafting for the treatment of Parkinson's disease.

Key words: Neural transplantation; Glial cell line-derived neurotrophic factor (GDNF); Encapsulation; Parkinson's disease

Address correspondence to Isao Date, M.D., Department of Neurological Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: 81-86-235-7336; Fax: 81-86-227-0191; E-mail: idate333@med.okayama-u.ac.jp




Cell Transplantation, Vol. 10, pp. 403-408
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Functional Comparison of the Single-Layer Agarose Microbeads and the Developed Three-Layer Agarose Microbeads as the Bioartificial Pancreas: An In Vitro Study

Baoyou Xu,1 Hiroo Iwata,2 Masaaki Miyamoto,2 A. N. Balamurugan,2 Yoshinobu Murakami,2 Wanxing Cui,1 Masayuki Imamura,1 and Kazutomo Inoue2

1First Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Japan
2Institute for Frontier Medical Sciences, Kyoto University, Japan

In this study, the insulin secretory characteristics of the microencapsulated hamster islets were studied during long-term culture. The hamster islets were encapsulated as single-layer agarose microbeads or three-layer agarose microbeads with agarose and agarose containing poly(styrene sulfonic acid) (PSSa), respectively. The influence of PSSa on the function of the rat islets microencapsulted in three-layer microbeads was primarily monitored. The aim of this study was to examine the influence of the PSSa on the in vitro function of the islets encapsulated in the agarose/PSSa microbeads compared with single-layer agarose microbeads during long-term culture. The microbeads were cultured for 30 days in medium of Eagle's MEM at 37°C in 5%CO2 and 95% air. The basal insulin secretion into the culture medium was measured daily during the first 12 days and two times per week until 30 days. The microbeads were subjected to static incubation test on the 10th, 20th, and 30th day during culture. The basal insulin secretion level of the agarose/PSSa microbeads was significantly higher than that of single-layer agarose microbeads. The static incubation tests revealed a similar pattern of insulin secretion from both microbeads when they were exposed to high glucose challenge. In the static incubation test, both could significantly increase insulin release to more than 6.61 times (stimulation index) in response to high glucose stimulation and could significantly decrease when glucose concentration returned from high glucose to low glucose on the 10th, 20th, and 30th day of culture. This study demonstrated that the hamster islets enclosed in agarose/PSSa hydrogel not only continuously secreted basal amounts of insulin, but also maintained their response to high glucose stimulation similar to the agarose microbeads. The above results together with those of our previous in vivo study suggest that the three-layer microbeads (agarose/PSSa) are well suitable for xenotransplantation of islets for the clinical application.

Key words: Biohybrid artificial pancreas; Agarose microbeads; Agarose/PSSa microbeads; Hamster islets

Address correspondence to Kazutomo Inoue, M.D., Professor, Department of Organ Reconstruction, Institute for Frontier Medical Sciences, kyoto University, 53 Kawaracho Shogoin, Sakyo-ku, Kyoto 606, Japan. Tel: (075) 751-4850; Fax: (075) 751-4145; E-mail: xubaoyou@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 10, pp. 409-412
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Acute Lethal Injury of Lung and Liver in Mice Transplanted With Ex Vivo-Expanded CTLs

Tadayuki Sato,1 Kiyoshi Ando,1,2 Masayuki Oki,1,2 Hiroko Miyatake,1,2 Hideyuki Matsuzawa,1,2 Tomomitsu Hotta,1,2 and Shunichi Kato1,3

1Research Center for Genetic Engineering and Cell Transplantation, 2Department of Hematology, and 3Department of Pediatrics, Tokai University School of Medicine, Isehara, Kanagawa, Japan

Clinical application of cytotoxic T lymphocytes (CTL) induced in vitro is extensively used for the treatment of viral infection and malignant diseases. We produced anti H-2d CTL in vitro from C57BL/6 (B6) splenocytes presensitized with (B6 x DBA/2) F1 (BDF1) splenocytes to establish a model system of CTL therapy. The specificity and cytotoxic activity were high enough (E/T ratio 1:1 = 38.8%) to induce graft versus host reaction. Though the total number of B6 splenocytes decreased by 0.27 during the 4 days of culture, the number of CD8+ lymphocytes increased 1.3-fold. When more than 5 x 106 cells of H-2d-reactive CTL were transplanted into BDF1 mice, mice died within 2 days postinduction. This lethal effect was not seen in the mice induced with ConA-stimulated T cells. Histological examination of the lungs and liver revealed massive infiltration of neutrophils in alveoli and the necrosis of hepatocytes. Therefore, this protocol was shown to be effective to produce alloantigen-specific CTLs and applicable to in vitro manipulation such as retrovirus-mediated gene transfer.

Key words: Parent into F1 model; Allo-reactive CTLs; Lethal effect; Lung injury

Address correspondence to Dr. Kiyoshi Ando, M.D., Research Center for Genetic Engineering and Cell Transplantation, Tokai University School of Medicine, Isehara, Kanagawa, Japan, 259-1193. Tel: +81-463-93-1211, Ext. 2700; Fax: +81-463-92-4750; E-mail: andok@keyaki.cc.u-tokai.ac.jp




Cell Transplantation, Vol. 10, pp. 413-417
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The Influence of the Anticomplement Synthetic Sulfonic Polymers on the Function of Pancreatic Islets: An In Vitro Study

Baoyou Xu,1 Yuanjun Gu,2 Masaaki Miyamoto,2 A. N. Balamurugan,2 Wanxing Cui,1 Masayuki Imamura,1 Hiroo Iwata,2 and Kazutomo Inoue2

1First Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Japan
2Institute for Frontier Medical Sciences, Kyoto University, Japan

In a previous experiment, we demonstrated the anticomplementary efficacy of poly(stryrene sulfonic acid) (PSSa) and poly(2-acrylamido-2-methyl propane sulfonic acid) (PAMPS). The aim of this study was to examine their influence on the function of pancreatic islets in vitro. In this study, after culturing the rat islets with RPMI-1640 culture medium containing different concentrations of soluble PSSa or PAMPS for 24 h at 37°C, we performed morphological and functional examination of the rat islets. We found that the islets maintained their normal morphology regardless of whether they were in the PSSa or PAMPS groups when the concentrations of soluble PSSa or PAMPS in the media were below 1 g/dl. In the static incubation study, the islets cultured in the PAMPS groups showed significantly high insulin secretory response to glucose challenge but those in the PSSa groups lost the response when the concentrations of soluble PSSa or PAMPS in the media were below 1 g/dl. The PAMPS not only has strong anticomplementry effect, but also maintained the good insulin secretory capacity of the islets. These results indicated that PAMPS is a promising bioartificial material for future clinical application of biohybrid artificial pancreas preparation. It is well suitable for xenotransplantation experiments.

Key words: Anticomplement synthetic sulfonic polyers; Rat islets; Poly(styrene sulfonic acid) (PSSa); Poly(2-acrylamido-2methylpropane sulfonic acid) (PAMPS)

Address correspondence to Kazutomo Inoue, M.D., Professor, Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaracho Shogoin, Sakyo-ku, Kyoto 606, Japan. Tel: (075) 751-4850; Fax: (075) 751-4145; E-mail: xubaoyou@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 10, pp. 419-422
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Neurotrophic Factor-Secreting Cell Grafting for Cerebral Ischemia: Preliminary Report

K. Fujiwara, I. Date, T. Shingo, H. Yoshida, K. Kobayashi, A. Takeuchi, T. Tamiya, and T. Ohmoto

Department of Neurological Surgery, Okayama University Medical School, Okayama, Japan

In this experiment, we examined a possible protective effect of encapsulated neurotrophic factor-secreting cell grafting for ischemic injury. We established a basic fibroblast growth factor (bFGF)-secreting cell line by genetic manipulation. We enveloped these cells into polymer capsules, which consist of a semipermeable membrane, and implanted them into the right striatum of rats. At 6 days after implantation, these rats received right middle cerebral artery occlusion (MCAO) using interluminal suture technique. At 24 h after MCAO, rats were sacrificed and their cerebral infarction volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining and image analysis. We found approximately 30% reduction in infarct volume in the encapsulated bFGF-secreting cell grafting groups vs. the encapsulated naive BHK cell grafting group or the without implantation group. We measured bFGF secretion from encapsulated bFGF-secreting cells using enzyme-linked immunosorbent assay (ELISA). The retrieved capsules continued to secrete bFGF. There was no significant difference of bFGF secretion between the capsules before and after transplantation. A large number of viable BHK-bFGF cells were observed within the full length of the retrieved capsule. These results indicate that encapsulated bFGF-secreting cell grafting exerts a protective effect on ischemic injury.

Key words: Basic fibroblast growth factor (bFGF); Encapsulation; Cell grafting; Ischemia

Address correspondence to Kenjiro Fujiwara, M.D., Department of Neurological Surgery, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel: 81-86-235-7336; Fax: 81-86-227-0191; E-mail: kenjiro@gb3.so-net.ne.jp




Cell Transplantation, Vol. 10, pp. 423-427
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Cytological Examination of Rat Amniotic Epithelial Cells and Cell Transplantation to the Liver

Tatsuya Nakajima,1 Shin Enosawa,1 Tasuku Mitani,1 Xiao-Kang Li,1 Seiichi Suzuki,1 Hiroshi Amemiya,1 Osamu Koiwai,2 and Norio Sakuragawa3

1National Children's Medical Research Centre, 2Science University of Tokyo, and 3National Institute of Neuroscience, Tokyo, Japan

It is hoped that amniotic epithelial cells can be useful in cell-mediated gene therapy. We report here an experimental cell transplantation model of amniotic cells in rats. There is an anatomical difference between human and rodent embryos. We established a method to isolate amniotic cells that are equivalent to human amniotic epithelial cells. An amniotic membrane distinct from the yolk sac was carefully collected and teased in saline containing deoxyribonuclease and hyaluronidase, followed by collagenase digestion. The cell yield was approximately 106 cells per pregnant female (105 cells per fetus), roughly in proportion to the age of fetus used, and 60% of the isolated cells were attached to the dish under culture conditions. Telomerase activity was higher in the cells isolated from fetuses in the middle stage (day 13.5 to 15.5) than in the late stage (day 17.5 to 21.5). Adherent cells exhibited two to three times more cell division, resulting in a ninefold increase in the number of cells. Immunohistochemical analysis revealed that approximately half of the adherent cells were albumin positive and formed clusters. The senescent cells survived for 2 months without apparent morphological changes. The adherent cells were able to be stored in liquid nitrogen and had a viability of 70% when thawed. Gene transduction with adenovirus vector was highly effective for rat amniotic cells. Transplantation of lacZ transfected amniotic cells into syngeneic rat liver resulted in the integration of the transplanted cells in the liver structure and the cells survived for at least 30 days.

Key words: Rat; Amniotic cells; Cell transplantation; Gene carrier

Address correspondence to Dr. Shin Enosawa, National Children's Medical Research Centre, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8509, Japan. Tel: +81-3-3414-8121, Ext. 2776; Fax: +81-3-3411-7309; E-mail: senosawa@nch.go.jp




Cell Transplantation, Vol. 10, pp. 429-433
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In Vivo Estimation of Bioartificial Liver With Recombinant HepG2 Cells Using Pigs With Ischemic Liver Failure

Shin Enosawa,1 Tomoyuki Miyashita,1 Yuji Fujita,1 Seiichi Suzuki,1 Hiroshi Amemiya,1 Takeshi Omasa,2 Shinya Hiramatsu,2 Kenichi Suga,2 and Toshiharu Matsumura3

1National Children's Medical Research Center
2University of Osaka, Graduate School of Technology
3Meiji Institute of Health Science

Biological efficacy of a recombinant human hepatic cell line, glutamine synthetase transfected HepG2 (GS-HepG2), was examined with large-scale culture in a circulatory flow bioreactor and in pigs with ischemic liver failure. GS-HepG2 cells were cultured in a circulatory flow bioreactor from 5 x 107 to 4 x 109 cells for 109 days. The cells showed ammonia removal activity even under substrate (glutamic acid)-free medium, suggesting that the GS catalyzed the activity using intracellular glutamic acid that had been pooled during conventional culture. When GS-HepG2 bioartificial liver (BAL) was applied to pigs with ischemic liver failure, survival time was prolonged to 18.8 ± 6.1 h (mean ± SD, n = 4) from 13.8 ± 5.4 h (n = 6) and 10.7 ± 4.1 h (n = 6) (groups treated with cell-free BAL and treated with plasma exchange and continuous hemodiafiltration, respectively). Laboratory data indicated the tendency for improvement in increase of blood ammonia level and decline of blood coagulation indices in the GS-HepG2 BAL-treated group. The advantages and potential for the cell line as a bioreactor in BAL is also discussed, comparing to those of isolated porcine hepatocytes.

Key words: Ammonia removal; Glutamine synthetase; HepG2; Bioartificial liver

Address correspondence to Dr. Shin Enosawa, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8509 Japan. Tel: +81-3-3414-8121, Ext. 2776; Fax: +81-3-3411-7309; E-mail: senosawa@nch.go.jp




Cell Transplantation, Vol. 10, pp. 435-439
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Widespread Distribution of Adenovirus-Transduced Monkey Amniotic Epithelial Cells After Local Intracerebral Injection: Implication for Cell-Mediated Therapy for Lysosome Storage Disorders

Motomichi Kosuga,1,4 Satoru Takahashi,3 Akiko Tanabe,1,5 Masayuki Fujino,2 Xiao-Kang Li,2 Seiichi Suzuki,2 Masao Yamada,1 Kohji Kakishita,6 Fumiko Ono,7 Norio Sakuragawa,3 and Torayuki Okuyama1,4

Departments of 1Genetics and 2Experimental Surgery, National Children's Medical Research Center, Tokyo, Japan 154-8509
3Department of Inherited and Metabolic Diseases, National Institute of Neuroscience
4Department of Pediatrics, Keio University School of Medicine
5Department of Science and Technology, Science University of Tokyo 6Department of Neurological Surgery, Wakayama Medical College
7The Corporation for Production and Research of Laboratory Primates

Cell-mediated therapy for mucopolysaccharidosis type VII (MPSVII) was studied using monkey amniotic epithelial cells (mAEC). The cells were transduced with a recombinant adenovirus expressing human \GK\b-glucuronidase (GUSB), and cells overexpressing GUSB were generated. The cells expressed 2000-fold higher activities than the endogenous GUSB activities of nontransduced mAEC, demonstrating that mAEC were successfully transduced with adenoviral vectors. These cells also secreted high levels of GUSB. To clarify the cross-correction of GUSB secreted from mAEC, the conditioned medium containing high levels of GUSB was added into the medium for culturing human or murine fibroblasts established from an MPSVII patient or a mouse model of the disease. Dramatic increases in GUSB activities were observed in both fibroblasts. We then transplanted the cells transduced with an adenovirus expressing LacZ into the caudate-putamen of monkey brain. Survival and distribution of the transplanted cells 1 month after the treatment were evaluated. Histochemical analysis showed that LacZ-positive cells were widely distributed in the brain, suggesting that the transplanted cells had migrated and were distributed even at regions far from the implantation site. These findings suggest that local intracerebral engraftment of genetically engineered amniotic epithelial cells is favorable for the treatment of lysosome storage disorders, whose pathological abnormalities are not restricted to specific regions of the brain.

Key words: Mucopolysaccharidosis type VII; Monkey amniotic epithelial cells; Cell-mediated therapy; Lysosomal storage disorders

Address correspondence to Torayuki Okuyama, Department of Genetics, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku Tokyo 154-8509, Japan. Tel: +81-3-3414-8121, Ext. 2752; Fax: +81-3-3414-3208; E-mail: tora@nch.go.jp




Cell Transplantation, Vol. 10, pp. 441-445
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Formation of Human Fibroblast Aggregates (Spheroids) by Rotational Culture

Katsuko S. Furukawa,1,2 Takashi Ushida,1,3 Yasuyuki Sakai,4 Motoyuki Suzuki,4 Junzo Tanaka,2,5 and Tetsuya Tateishi1,3

1Tissue Engineering Laboratory, Graduate School of Engineering, University of Tokyo, Bunkyo-ku, Tokyo, Japan
2CREST, Japan science and Technology Corporation, Japan
33 Dimensional Cell & Tissue-Engineering Group, National Institute for Interdisciplinary Research, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Tsukuba, Ibaraki, Japan
4Institute of Industrial Science, University of Tokyo, Minato-ku, Tokyo, Japan
5National Institute for Research in Inorganic Materials, Japan

In the current study, we attempted to form aggregates of fibroblasts by rotationally shaking, declining fibroblast-material interactions, and augmenting cell-cell interactions. In addition, to promote cell-cell interactions, the medium was supplemented with insulin, dexamethasone, and basic fibroblast growth. Under such improved culture conditions, normal neonatal human dermal fibroblasts formed spheroidal aggregates within 1 day of rotation on a rotational shaker. The aggregates that formed had irregular shapes and were composed from only several cells after 12 h. However, they became nearly spheroidal after 24 h of shaking. The aggregates were approximately 240 mm in diameter. After 36 h of shaking, their shape became more rounded and their surfaces became smoother. No evidence of necrosis in the center of the aggregates was observed, although a small number of dead cells was scattered throughout the aggregates. After 24-36 h, aggregates of normal human fibroblasts were collected and reinoculated onto a scaffold composed of polyglycolic acid, which is used commercially as a scaffold for artificial skin, coated with collagen. The aggregates were successfully trapped to the mesh of polyglycolic acid and became attached within 24 h. Therefore, the aggregates could provide an alternative method for seeding fibroblasts to scaffold for an artificial skin, such as a mesh of polyglycolic acid.

Key words: Aggregates; Normal human skin fibroblasts; Biodegradable materials; Tissue-engineered skin

Address correspondence to Katsuko S. Furukawa, Department of Mechanical Engineering, Graduate School of Engineering, University of Tokyo, 8th Building 713, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8656, Japan. Tel: +81-3-5841-6375; Fax: +81-3-5841-6442; E-mail: furukawa@tissue.t.u-tokyo.ac.jp




Cell Transplantation, Vol. 10, pp. 447-451
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Evaluation of Insulin Secretion of Isolated Rat Islets Cultured in Extracellular Matrix

Natsuki Nagata,1 Yuanjun Gu,1 Hiroshi Hori,1 A. N. Balamurugan,1 Maki Touma,1 Yoshiyuki Kawakami,1 Wenjing Wang,1 Tomomi T. Baba,3 Akira Satake,1 Masumi Nozawa,1 Yasuhiko Tabata,2 and Kazutomo Inoue1

1Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
2Department of Biomaterials, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
3Department of Oral Biochemistry, Nagasaki University school of Dentistry, Nagasaki, Japan

Islet isolation involves enzymatic digestion of the interstitial matrix and mechanical disruption of the tissue. It is possible that a fundamental change of islet biology resulting from the loss of critical factors required for islet function or survival will occur. Extracellular matrix (ECM) is one of the most important components of the islet microenvironment. Reconstruction of the cell-matrix relationship seems to be effective for improving the loss of differentiated islet structure and function. The purpose of this study was to characterize and compare the effects of collagen gel mixture or Matrigel on b-cell function and islet cell survival. After isolation by the collagenase digestion technique, rat islets were divided and cultured with various types of collagen gel mixture. They were assessed for their glucose-stimulated insulin secretion and cell viability. Glucose-induced insulin secretion of islets cultured with collagen type I gel or a mixture of collagen type I and IV was improved after 11 days in culture. In conclusion, a type of gel composed of collagen type I and/or type IV as an islet microenvironment is sufficient to maintain glucose responsiveness and may be useful for islet transplantation.

Key words: Extracellular matrix; Collagen Gel; Insulin responsiveness; Cell viability

Address correspondence to Kazutomo Inoue, M.D., Ph.D., Professor, Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaracho Shogoin, sakyo-ku, Kyoto 606-8507, Japan. Tel: 075-751-4850; Fax: 075-751-4145; E-mail: k-inoue@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 10, pp. 453-457
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Development of a New Method to Induce Angiogenesis at Subcutaneous Site of Streptozotocin-Induced Diabetic Rate for Islet Transplantation

Yuanjun Gu,1 Yasuhiko Tabata,1 Yoshiyuki Kawakami,2 Appakaalai N. Balamurugan,1 Hiroshi Hori,1 Natsuki Nagata,1 Akira Satake,1 Wanxing Cui,2 Meirigeng Qi,1 Yoko Misawa,2 Maki Toma,1 Masaaki Miyamoto,1 Masumi Nozawa,1 and Kazutomo Inoue1

1Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
2Department of Surgical Basic Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan
3University of Medical Sciences, Mie, Japan

The subcutaneous space is a potential site for clinical islet transplantation. Even though there are several advantages, poor blood supply at this site mainly causes failure of islet survival. In this study, angiogenesis was induced in advance at the diabetic rats subcutis for islet transplantation by implanting a polyethylene terephthalate (PET) mesh bag containing gelatin microspheres incorporating basic fibroblast growth factor (bFGF) (MS/bFGF) and a collagen sponge. The bFGF was incorporated into gelatin microspheres for controlled release of bFGF. As controls, a PET mesh bag with or without either collagen sponges or MS/bFGF was implanted at the subcutaneous site of diabetic rats. Macroscopic and microscopic examinations revealed the formation of capillary network in and around the PET mesh bag containing MS/bFGF and collagen sponges 7 days after implantation when compare with other control groups. When tissue hemoglobin level was also measured, a significantly high level of hemoglobin amount was observed compared with that of control groups. When allogeneic islets mixed with 5% agarose were transplanted into the prevascularized rat subcutis, normoglycemia was maintained for more than 40 days, while other control groups were ineffective. This study demonstrated that combination of gelatin microspheres incorporating bFGF and collagen sponges enabled the mesh to induce neovascularization even at the subcutaneous site of streptozotocin-induced diabetic rats, resulting in improved function of islet transplantation.

Key words: Angiogenesis; Subcutaneous site; Basic fibroblast growth factor (bFGF); Collagen sponge

Address correspondence to Kazutomo Inoue, M.D., Ph.D., Department of Organ Reconstruction, Institute for Frontier Medical Sciences,Kyoto University, 53 shogoin Kawaracho, Sakyo- ku, Kyoto, 606-8507, Japan. Tel: +8175-751-4850; Fax: +8175-751-4145; E-mail: yjgu@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 10, pp. 459-464
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
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Isolation, Culture and Characterization of Endocrine Cells From 6-Month-Old Porcine Pancreas

Hiroshi Hori,1 Yuan Jun Gu,1 Natsuki Nagata,1 Appakaalai N. Balamurugan,1 Akira Satake,1 Yoshihiko Morimoto,1 Wen Jing Wang,1 Yuko Misawa,1,2 Yuka Nozawa,1 Tomoko Nembai,3 Masaaki Miyamoto,1 Masumi Nozawa,1 and Kazutomo Inoue1

1Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
2Suzuka University of Medical Sciences, Mie, Japan
3Applied Biosystems Japan Ltd., Tokyo, Japan

Porcine endocrine cells were isolated from pancreas of 6-month-old pigs by two-step enzymatic digestion procedures. They were separated by the density gradient (isopycnic) centrifugation method using Histopaque-1077. Isolated cells were cultured and divided into two groups: suspension cells and adhesion cells. Suspension cells maintained their cell numbers on and after 7 days in culture. Approximately 1 x 107 cells were obtained from single pancreas of a 6-month-old pig. The cultured suspension cells took up dithizone (DTZ) staining 14 days after isolation in culture and indicated the presence of b-cells. In in vitro study, the suspension cells were capable of secreting insulin into the culture medium. The suspension cells were tested for insulin and glucagon staining by Western blot analysis. These results indicated the maintenance of endocrine cell function after isolation. However, cultured adhesion cells failed to maintain their function during culture. In in vivo study, the suspension cells were transplanted into diabetes-induced nude mice. Reduction in blood glucose level was obtained after transplantation. Intraperitoneal glucose tolerance test (IPGTT) results showed a normal pattern of blood glucose clearance. After 1 week, the transplanted endocrine cells were detected with anti-insulin antibody by immunostaining and it showed the presence of viable b-cells under the renal capsule of nude mice. Collectively, our results suggest that isolated and cultured suspension porcine endocrine cells maintained their endocrine function. These endocrine cells can be used as isolated islets for further study, including transplantation experiments.

Key words: Porcine pancreatic endocrine cells; Two-step digestion; Suspension cells; Endocrine function

Address correspondence to Kazutomo Inoue, M.D., Ph.D., Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawara-cho, shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel: +81-75-751-4856; Fax: +81-75-751-4145; E-mail: k-inoue@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 10, pp. 465-471
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
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Effect of Basic Fibroblast Growth Factor on Insulin Secretion From Microencapsulated Pancreatic Islets: An In Vitro Study

Wenjing Wang, Yuanjun Gu, Masaaki Miyamoto, Hiroshi Hori, Natsuki Nagata, A. N. Balamurugan, Maki Touma, Tomonori Sakurai, and Kazutomo Inoue

Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan

Microencapsulation of pancreatic islets represents a potentially effective method to prevent graft rejection in allotransplantation or xenotransplantation without the need of immunosuppression. Adequate insulin secretion and glucose responsiveness of microencapsulated pancreatic islets has been regarded as a prerequisite for successful transplantation. The microencapsulated pancreatic islets were respectively cultured in bFGF+ RPMI-1640 medium (bFGF+) or bFGF- RPMI-1640 medium (bFGF-) for 21 days. The functional activities of microencapsulated pancreatic islets were assessed by measuring basal insulin secretion and stimulated insulin release at different time points. The results revealed that microencapsulated pancreatic islets in the presence of bFGF demonstrated an increase in basal insulin secretion. Furthermore, microencapsulated pancreatic islets in the presence of bFGF demonstrated a marked stimulated insulin release and relative stability of stimulation indices (SI). The results in the perifusion study showed that microencapsulated pancreatic islets in the presence of bFGF maintained good glucose responsiveness over the course of culture period as well. These results indicate that bFGF has a beneficial effect on insulin secretion from microencapsulated pancreatic islets during in vitro culture. New strategies for preserving and improving function of microencapsulated pancreatic islets prior to transplantation may be developed by application of growth factors or other factors.

Key words: Basic fibroblast growth factor (bFGF); Effect; Insulin secretion; Microencapsulated pancreatic islets

Address correspondence to Kazutomo Inoue, M.D., Ph.D., Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin Kawara-cho, Sakyo-ku, Kyoto, 606-8507, Japan. Tel: +81-75-751-4856; Fax: +81-75-751-4145; E-mail: k-inoue@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 10, pp. 473-477
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Copyright © 2001 Cognizant Comm. Corp.
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Regulation of Cell Proliferation Using Tissue Engineering in MIN6 Cells

Naoko Kinoshita,1,2 Yoshiya Echigo,2 Sigeo Shinohara,3 Yuanjun Gu,1 Junichi Miyazaki,4 Kazutomo Inoue,1 and Masayuki Imamura2

1Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
2Department of Surgery & Surgical Basic Science, Graduate School of Medicine, Kyoto University, Kyoto, Japan
3Otsuka Pharmaceutical Co., Ltd., Otsu Laboratory for Skin Care Science,
4Department of Nutrition and Physiological Chemistry, Osaka University, Osaka, Japan

Pancreatic islet transplantation for patients with diabetes mellitus has been hindered by the problem of donor shortage, as is the case for transplantation of other organs. Among several measures to overcome this problem, cell transplantation using xenogenic cell lines has been considered. For the treatment of diabetic patients, a murine pancreatic b-cell line MIN6 is a potential source of cell transplant. In order to restrict otherwise unlimited proliferation of transplanted MIN6 cells, cells are rendered to form spheroidal aggregates (SMIN6) on nonadherent culture dishes. SMIN6 stopped its growth around day 7 with a diameter of 220 ± 40 mm and kept its size almost constant at least until day 28. SMIN6 cells, however, had reduced responsiveness of insulin secretion to glucose concentration compared with MIN6 cells cultured in a monolayer. On the other hand, spheroid MIN6 cells formed in the presence of extracellular matrix gel (SMIN6E) possessed the capacity for glucose-dependent insulin secretion comparable with conventional MIN6 cells. SMIN6E encapsulated in agarose beads (SMIN6E-B) was also viable for at least 1 month in vitro with a constant diameter and favorable glucose responsiveness. The development of spheroid-type MIN6 may contribute to the future clinical application of MIN6 or other b-cell lines for treatment of diabetes mellitus.

Key words: MIN6; Transplantation; Spheroid formation; Tissue engineering

Address correspondence to Naoko Kinoshita, 53 Shogoin Kawaharacho Sakyo-ku, Kyoto 606-8507, Japan. Tel: +81-075-751-4850; Fax: +81-075-751-4145; E-mail: naok@frontier.kyoto-u.ac.jp




Cell Transplantation, Vol. 10, pp. 479-483
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Copyright © 2001 Cognizant Comm. Corp.
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In Vitro Organization of Biohybrid Rat Liver Tissue Incorporating Growth Factor- and Hormone-Releasing Biodegradable Polymer Microcapsules

Yasuyuki Sakai,1 Katsuko Furukawa,2 Takashi Ushida,2,3 Tetsuya Tateishi,2,3 and Motoyuki Suzuki1

1Institute of Industrial Science, University of Tokyo, 4-6-1 Komabou, Meguro-ku, Tokyo 153-8505, Japan
23-D Cell Tissue Engineering Group, National Institute for Advanced Interdisciplinary Research, Agency of Industrial Science and Technology, 1-1-4 Higashi, Tsukuba 305-8562, Japan
3Tissue Engineering Division, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

To enhance the liver-specific functions of rat hepatocyte aggregates without the addition of exogenous growth factors, polylactic acid-polyglycolic acid (PLGA)/gelatin microcapsules that release insulin, dexamethasone, epidermal growth factors, and glucagon were prepared and incorporated into the hepatocyte aggregates in suspension culture. Precoating the capsules with fibronectin enhanced the incorporation of the microcapsules into the hepatocyte aggregates. In a growth factor- and hormone-free culture medium, these microcapsule-containing aggregates showed a sustained cell number and an ammonium detoxification capacity compared with two types of control culture. One was the culture of microcapsule-free aggregates with albumin-containing control capsules and the other was the culture of capsule-free aggregates that were supplied with the same factors and hormones from the culture medium at concentration levels expected from the release kinetics of the microcapsules. Our new methodology demonstrates that the performance and duration of bioartificial liver systems can be enhanced due to a more efficient maintenance of cell number by using such growth factor- and hormone-releasing microcapsules.

Key words: Biodegradable polymer microcapsule; Growth factor; Rat hepatocyte; Biohybrid liver tissue

Address correspondence to Yasuyuki Saki, Institute of Industrial Science, University of Tokyo, 4-6-1 Komabou, Meguro-ku, Tokyo 153-8505, Japan. Tel: +813-5452-6352; Fax: +813-5452-6353; E-mail: yasuyuki@iis.u-tokyo.ac.jp




Cell Transplantation, Vol. 10, pp. 485-491
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Copyright © 2001 Cognizant Comm. Corp.
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Molecular Cloning of the Porcine Insulin cDNA Using a Monolayer Culture of Pancreatic Endocrine Cells

Mariko Tsuchiya,1 Ken Tsuchiya,2 and Hisako Ohgawara3

1Institute of Geriatrics, Aoyama Hospital, 2Department of Medicine IV, and 3Medical Research Institute, Tokyo Women's Medical University, Tokyo, Japan

Porcine pancreatic endocrine cells are an attractive candidate for islet cell transplantation in view of the immunological properties and structural similarities of porcine insulin to human insulin. We recently established a method of isolation and a primary monolayer culture of porcine pancreatic endocrine cells. In this study, cloning of the porcine insulin cDNA was performed to clarify the genetic background of the purified isolated cells. A homology-based PCR cloning method was employed to determine the sequence using mRNA extracted from the monolayer-forming cells, and the candidate products were then determined by a homology search on the human insulin cDNA. According to the newly identified sequence, rapid amplification of cDNA ends was applied to the 5´ and 3´ ends, and the entire cDNA sequence was determined. Gene and protein expression was confirmed by Northern blotting, immunohistochemistry, and enzyme assay. To examine the transcriptional level, the cultured cells were incubated in a 20 mM D-glucose medium in the presence or absence of 5 mM forskolin. The porcine insulin cDNA exhibited a high homology to the human cDNA and showed 85% matching with the human amino acid sequence. D-Glucose at 20 mM stimulated the insulin secretion and mRNA expression, and further addition of 5 mM forskolin with the glucose was applied as the strongest stimulus in this culture system.

Key words: Porcine; Pancreatic b-cell; Molecular cloning; RACE (rapid amplification of cDNA ends)

Address correspondence to Dr. Hisako Ohgawara, Medical Research Institute, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel:/Fax: +81-3-5269-7364; E-mail: hisakooh@lab.twmu.ac.jp




Cell Transplantation, Vol. 10, pp. 493-498
0963-6897/01 $20.00 + 00
Copyright © 2001 Cognizant Comm. Corp.
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Effect of the Extracellular Matrix on Pancreatic Endocrine Cell Function and its Biocompatibility in Dogs

Kazuya Edamura,1,4 Hisako Ohgawara,4 Koko Nasu,4 Yukiko Iwami,4 Ayako Sato,4,5 Shiho Ishikawa,2 Naoaki Matsuki,2 Kenichiro Ono,2 Hiroyuki Ogawa,1 and Nobuo Sasaki3

Laboratories of 1Veterinary Emergency Medicine, 2Veterinary Clinical Pathobiology, and 3Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo
4Medical Research Institute, School of Medicine, Tokyo Women's Medical University
5Institute of Applied Biochemistry, University of Tsukuba

The effect of the synthetic extracellular matrix (ECM) in a diffusion chamber for a bioartificial endocrine pancreas (Bio-AEP) on pancreatic endocrine cells in vitro and its biocompatibility in dogs were investigated. Two different types of ECM were used: type I collagen treated with low antigen (type I LA), and reconstituted basement membrane matrix (Matrigel) derived from Englbreth-Holm-Swarm (EHS) mouse sarcoma. Matrigel contains growth and differentiation factors and cell adhesion molecules such as laminin, heparan sulfate proteoglycan, and entactin. Purified porcine pancreatic endocrine (PE) cells were suspended in type I LA or Matrigel and then placed into a 12-well culture plate (4 x 107 cells/ml · gel/well). The insulin accumulation from PE cells in Matrigel was significantly greater than that in type I LA (9.3 ± 3.6 mU/well vs. 2.3 ± 1.3 mU/well). When Bio-AEP with Matrigel and PE cells was implanted into the abdominal cavity of a pancreatectomized diabetic dog, the exogenous insulin requirement for maintaining normoglycemia was reduced for the first 4 weeks. However, after 6 weeks of implantation, fasting blood glucose levels suddenly increased. Laparotomy revealed encapsulated Bio-AEP with thick fibrous tissue. Following removal of the Bio-AEP from the abdominal cavity, another Bio-AEP containing type I LA and PE cells was implanted into the same dog. The exogenous insulin requirement was gradually decreased to almost half that of preimplantation levels. Bio-AEPs containing type I LA or Matrigel, but not PE cells, were implanted into the abdominal cavities of four healthy dogs. After 4 weeks of implantation, the Bio-AEP with Matrigel was encapsulated with fibrous tissue similar to that in the diabetic dog, but the Bio-AEP with type I LA was not. These results indicate that Matrigel may be incompatible with dogs and that the type I LA is more suitable for Bio-AEP.

Key words: Bioartificial endocrine pancreas; Biocompatibility; Diffusion chamber; Extracellular matrix

Address correspondence to Hisako Ohgawara, M.D., Medical Research Institute, School of Medicine, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. Tel: +81-3-5269-7364; Fax: +81-3-5269-7364; E-mail: hisakooh@ lab.twmu.ac.jp




Cell Transplantation, Vol. 10, pp. 499-502
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Copyright © 2001 Cognizant Comm. Corp.
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Tissue-Engineered Pancreatic Islets: Culturing Rat Islets in the Chitosan Sponge

Wanxing Cui,1 Do-Hoon Kim,2 Masayuki Imamura,1 Suong-Hyu Hyon,2 and Kazutomo Inoue2

1Department of Surgery and Surgical Basic Science, Graduate School of Medicine and 2Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

Subcutaneous islet transplantation has become an attractive modality. With development of tissue-engineering techniques, it is possible to rectify the disadvantage of poor blood supply in the subcutaneous site by reconstruction of the capillary network. According to reports, the Chitosan sponge (CS) could be used for reconstruction of in vitro capillary-like network and could be used in artificial skin equivalent. In this study, we cultured the islets in CS for future application. CSs, having 200-500 \GK\mm pore size, were prepared by freeze-drying method. Rat islets were isolated from the pancreas of Lewis rats (10 weeks old, 280-300 g, male) by collagenase digestion followed by discontinuous dextran gradient centrifugation method. Each 20 islets were seeded equally into the CSs and were cultured for 62 days with various culture media such as RPMI-1640, Dulbecco's modified Eagle's medium (DMEM), and Eagle's MEM. They contained 10% fetal bovine serum (FBS) and 5 ml/L antibiotic-antimycotic mixed stock solution in the culture dishes. Insulin concentration both inside and outside of the islet-seeded CS was measured during culture. Changes in the morphology of islets were also observed in this study. Freshly isolated islets had a loose appearance with an irregular border, and most were seen as a single islet. Occasionally a cluster, consisting of 2-4 islets ranging mainly from 150 to 250 mm in diameter, was observed. Islets cultured in the CSs in different culture media retained initial morphology, which had well-delineated smooth borders for at least 53 days. The insulin release behavior of islets cultured in the CS showed constant secretory capacities for 49 days. After that they exhibited a rapid and definitive decline from the initial insulin release. Until this stage, insulin concentration in the CS was well maintained. The properties were dependent on culture medium used and insulin diffusion released from islets. This experiment is a new study model for establishment of islet culture in a three-dimensional matrix. Also extension of this observation will provide new insights for islet transplantation at the subcutaneous site by a tissue-engineering approach.

Key words: Subcutaneous islet transplantation; Chitosan sponge; Tissue-engineering approach; Three-dimensional culture

Address correspondence to Kazutomo Inoue, M.D., Professor, Department of Organ Reconstruction, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaracho Shogoin, Sakyo-ku, Kyoto 606, Japan. Tel: (075) 751-4850; Fax: (075) 751-4145; E-mail: wxcui@frontier.kyoto-u.ac.jp