First Department of Surgery, Faculty of Medicine, University of Fukui, School of Medicine, 23 Shimoaiduki, Matsuoka, Eiheiji-cho, Fukui 910-1193, Japan.
J Hepatobiliary Pancreat Sci. 2012 Jul;19(4):354-60. doi: 10.1007/s00534-011-0415-4.
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.
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.
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.
The present results showed that serum-free medium containing sericin is useful for both cryopreservation and cell culture.
背景/目的：胰岛细胞的长期储存和增加临床胰岛移植的实用性需要进行冷冻保存。通常使用胎牛血清（FBS）加 10％二甲基亚砜（DMSO）作为胰岛细胞的冷冻保存介质。然而，由于最近出现了动物健康问题，如牛海绵状脑病和病毒感染，因此在细胞培养和移植中理想情况下应避免使用 FBS。本研究的目的是通过检查丝蛋白丝胶的有效性来开发一种新的无血清冷冻保存介质，丝胶是由家蚕产生的。
用胶原酶消化和 Histopaque 梯度离心从 Lewis 大鼠中制备胰岛，然后在含有 0.1％丝胶的培养基中培养 3 天，使用 0.1％、0.5％、1％、2％和 5％丝胶或 FBS 进行冷冻保存。将 DMSO（1％、4％、7％、10％和 15％）添加到培养基中作为冷冻保护剂。解冻后，在第 1、4、7 和 14 天，计数有活力的胰岛，以评估其存活率。在第 4 天通过静态孵育试验测量体外胰岛素分泌。通过同基因移植测试培养胰岛的体内功能。移植后评估胰岛的组织学和免疫组织化学。
在胰岛存活率和刺激胰岛素分泌方面，含 1％丝胶的冷冻保存介质与含 10％FBS 的冷冻保存介质没有显着差异。移植后，胰岛迅速逆转高血糖并维持正常血糖控制。此外，使用 7％DMSO 作为丝胶的冷冻保护剂与 FBS 中较高 DMSO 浓度的结果相同。
本研究结果表明，含丝胶的无血清培养基可同时用于冷冻保存和细胞培养。
