Kenji Ohnishi1, Makoto Murakami, Mitsuhiro Morikawa, Akio Yamaguchi. 1. First Department of Surgery, Faculty of Medicine, University of Fukui, School of Medicine, 23 Shimoaiduki, Matsuoka, Eiheiji-cho, Fukui 910-1193, Japan. kenjidr8170228@yahoo.co.jp
Abstract
BACKGROUND/ PURPOSE: Cryopreservation is necessary for the long-term storage of islet cells and to increase the practicality of clinical islet transplantation. Fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) is generally used as a freezing medium for islet cells. However, FBS should ideally be avoided in cell culture and transplantation because of recent animal health problems, such as bovine spongiform encephalopathy and viral infections. The aim of this study was to develop a new serum-free freezing medium by examining the effectiveness of the silk protein sericin, which is produced by Bombyx mori. METHODS: Islets prepared from Lewis rats by collagenase digestion and Histopaque gradient centrifugation, followed by culture in medium containing 0.1% sericin for 3 days, were cryopreserved using 0.1, 0.5, 1, 2, and 5% sericin or FBS. DMSO (1, 4, 7, 10, and 15%) was added to the medium as a cryoprotectant. After thawing, on days 1, 4, 7, and 14, viable islets were counted in order to evaluate their survival. Insulin secretion was measured in vitro by a static incubation test on day 4. The in vivo function of cultured islets was tested by syngeneic transplantation. Islets were evaluated histologically and immunohistochemically after transplantation. RESULTS: There were no significant differences between freezing medium containing 1% sericin and that containing 10% FBS with regard to the survival rate of islets and stimulated insulin secretion. Following transplantation, islets rapidly reversed hyperglycemia and maintained normal glycemic control. In addition, the use of 7% DMSO as a cryoprotectant with sericin showed the same results as higher DMSO concentrations with FBS. CONCLUSION: The present results showed that serum-free medium containing sericin is useful for both cryopreservation and cell culture.
BACKGROUND/ PURPOSE: Cryopreservation is necessary for the long-term storage of islet cells and to increase the practicality of clinical islet transplantation. Fetal bovine serum (FBS) supplemented with 10% dimethyl sulfoxide (DMSO) is generally used as a freezing medium for islet cells. However, FBS should ideally be avoided in cell culture and transplantation because of recent animal health problems, such as bovine spongiform encephalopathy and viral infections. The aim of this study was to develop a new serum-free freezing medium by examining the effectiveness of the silk protein sericin, which is produced by Bombyx mori. METHODS: Islets prepared from Lewis rats by collagenase digestion and Histopaque gradient centrifugation, followed by culture in medium containing 0.1% sericin for 3 days, were cryopreserved using 0.1, 0.5, 1, 2, and 5% sericin or FBS. DMSO (1, 4, 7, 10, and 15%) was added to the medium as a cryoprotectant. After thawing, on days 1, 4, 7, and 14, viable islets were counted in order to evaluate their survival. Insulin secretion was measured in vitro by a static incubation test on day 4. The in vivo function of cultured islets was tested by syngeneic transplantation. Islets were evaluated histologically and immunohistochemically after transplantation. RESULTS: There were no significant differences between freezing medium containing 1% sericin and that containing 10% FBS with regard to the survival rate of islets and stimulated insulin secretion. Following transplantation, islets rapidly reversed hyperglycemia and maintained normal glycemic control. In addition, the use of 7% DMSO as a cryoprotectant with sericin showed the same results as higher DMSO concentrations with FBS. CONCLUSION: The present results showed that serum-free medium containing sericin is useful for both cryopreservation and cell culture.
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