不同多羟基化合物对小鼠胚胎干细胞低温贮存的影响

背景:胚胎干细胞的长期低温贮存成为胚胎干细胞研究和应用的重要环节。传统的低温贮存保护剂主要包含二亚基亚砜、甘油、动物血清等,对于许多细胞类型并不能有效保证细胞的存活率,甚至于损失了细胞的某些功能。目的:开发适用于小鼠胚胎干细胞的低温贮存试剂。设计:观察实验。单位:广州医学院从化学院生理教研室与科宇联合干细胞生物技术有限公司。材料:实验于2004-10/2005-09在科宇公司实验室完成。小鼠细胞系mES-1为军事医学科学院基础医学研究所细胞生物学研究室惠赠。方法:将mES-1维持培养收集的细胞低温贮存。实验以二亚基亚砜为主要的低温贮存保护剂。按低温保护液成分不同分4组:对照组低温保护液包含DMEM(高糖)、10%二亚基亚砜、10%胎牛血清、10mg/mL抗坏血酸、0.18mg/mL肌醇、0.44mg/mL叶酸,甘露糖组在对照组的基础上补充7.56mg/mL甘露糖,海藻糖组补充34.23mg/mL海藻糖,蔗糖组补充41.94mg/mL蔗糖。观察不同多羟基化合物对小鼠胚胎干细胞低温贮存后存活率和多向分化能力的影响。主要观察指标:胚胎干细胞集落形成率和拟胚体形成率。结果:①冻存后各低温保存剂组的胚胎干细胞集落形成率均比冻存前有显著性降低[对照组:(24.0±8.8)%,甘露糖组:(42.0±10.1)%,海藻糖组:(84.0±8.2)%,蔗糖组:(70.0±14.2)%,冻存前:(95.0±4.7)%,P均<0.05],其中海藻糖组的胚胎干细胞集落形成率明显高于其他低温保护剂组(P<0.05)。②海藻糖组的拟胚体形成率高于对照组和甘露糖组[(90.0±5.2)%,(80.0±6.9)%,(82.0±9.6)%,P均<0.05]。与蔗糖组相比,差异无显著性意义。结论:以海藻糖为核心低温保护剂成分可有效维持小鼠胚胎干细胞的自我更新能力和多向分化能力。

Effect of different polyols on the cryopreservation of mouse embryonic stem cells

BACKGROUND: The long-term cryopreservation of embryonic stem cells is an important link in its researches and application. Traditional protectants of cryopreservation often contain dimethyl sulfoxide, glycerine, animal serum, etc., but they cannot effectively ensure the survival rate of some cells, and even damage some functions of the cells.OBJECTIVE: To develop the suitable protectant for the cropreservation of mouse embryonic stem cells. DESIGN: An observational study.SETTINGS: Department of Physiology, Conghua College of Guangzhou Medical College; Sinocells Bio Technologies Co.,Ltd.MATERIALS: The experiment was carried out in the laboratory of Sinocells Bio Technologies Co.,Ltd. from October 2004 to September 2005. Mouse cell line mES-1 was given as a present by the Research Room of Cell Biology, Institute of Basic Medical Sciences, Academy of Military Medical Sciences of Chinese PLA.METHODS: The cells collected from cultured mES-1, a mouse embryonic stem cell line. Dimethyl sulfoxide was used as the main protectant for cryopreservation. There were four different groups according to different protectants. In the control group, the protectant for cryopreservation contained DMEM (high sugar), 10% dimethyl sulfoxide, 10% FBS,10 mg/mL ascorbicacid, 0.18 mg/mL inositol and 0.44 mg/mL folic acid; Besides, 7.56 mg/mL mannose in the mannose group, 34.23 mg/mL mycose in the mycose and 41.94 mg/mL saccharu in the saccharu group were supplemented respectively. The effects of different polyols on the survival rate and ability of multiple differentiation of mouse embryonic stem cells after cryopreservation were observed.MATN OUTCOME MEASURES: Formation rate of mouse embryonic stem cells colony; Formation rate of embryoid bodies.were all significantly decreased as compared with that before cryopreservation [control group: (24.0±8.8)%; mannose group: (42.0±10.1)%; mycose group: (84.0±8.2)%; saccharu group (70.0±14.2)%; before cryopreservation: (95.0±4.7)%,embryoid bodies in the mycose group was higher than those in the control group and mannose group [(90.0±5.2)%,(80.0±6.9)%, (82.0±9.6)%, P＜0.05], but had no significant difference as compared with that in the saccharu group.CONCLUSION: The protectant for cryopreservation by taking mycose as the main component can effectively maintain the abilities of self-renewing and multiple differentiation of mouse embryonic stem cells.