微囊牛肾上腺髓质细胞的冻存及其活性评价

Cryopreservation of Microencapsulated Bovine Chromaffin Cells

目的 建立一种微囊牛肾上腺髓质细胞 (bovinechromaffincell,BCC)的冻存方法 ,为临床推广微囊人工生物细胞技术移植异种细胞奠定基础。方法 微囊化牛肾上腺髓质细胞冻存以二甲基亚砜为保护剂 ,循序缓慢降温 (4℃ ,1h ,- 2 0℃ 2h ,- 5 0℃过夜 ,液氮 ) ,复苏时迅速将冻存管放入 37℃水浴中。通过检测其基础儿茶酚胺分泌量和在高钾 (5 8mmol/L)、乙酰胆碱 (10 -4mol/L)刺激下的分泌量来观察其功能活性。结果 冻存复苏后微囊BCC保留了儿茶酚胺的分泌功能 ,基础与刺激分泌量分别为 (3.2 0 7± 0 .35 0 )ng/ml和 (12 .4 96± 2 .30 2 )ng/ml,大约为冻存前微囊牛肾上腺髓质细胞分泌量的 80 % ;同时刺激后儿茶酚胺分泌增加 (P <0 .0 1)。结论 建立一种微囊牛肾上腺髓质细胞的冻存方法 。

Objective To develop an effective method for cryopreserving alginate-polylysine-alginate microencapsulated bovine chromaffin cells(BCC),in order to lay the foundation of spreading the clinical application of microencapsulated artificial cells in xenograft transplantation. Methods Bovine chromaffin cells microencapsulated in alginate-polylysine- alginate were cryopreserved with Dimethyl Sulfoxide as preservative agent and gradually lowering temperature in liquid nitrogen. Then rewarmed in 37 ℃ water bath. The functional activity of BCC was monitored by assessing its catacolamine secreation spontaneously and in the condition existing high concentration of potassium and acetylcholine respectively. Results The rewarmed cryopreserved BCCs still remained 80 % ability of secreting cetecholamine both spontaneously and in response to high concentration of high potassium and acetylcholine. The secreations were increased after responses (P<0.01). Conclusions It is identified by functional determinations that the established cryopresevative technique for BCC is effective and successful.