组织工程化肌腱种子细胞深低温保存的实验研究

Experimental Study on Cryopreservation of Seeding Cells of Tissue Engineered Tendons

对肌腱种子细胞进行深低温保存,研究保存过程中多个环节的影响因素对细胞存活率的影响及深低温保存对种子细胞生物学特性、胶原分泌功能的影响。实验结果表明二甲基亚砜是肌腱种子细胞深低温保存中比较好的抗冻保护剂;冻存后使用与培养时不同的营养血清处理对细胞有损害,可降低细胞存活率;在冷冻保存过程中,细胞存活率与细胞浓度有一定关系,浓度太低可能降低细胞存活率;细胞在冷冻保存时,降温速度对细胞存活率有影响,慢速的分步降温组细胞存活率明显高于直接入液氮的快速降温组;采用10%二甲基亚砜加15%小牛血清加75%DM EM配方保存肌腱种子细胞,对其分泌胶原功能无明显影响,对其生长曲线、细胞周期及染色体众数无明显改变,适于肌腱种子细胞的保存。

This study sought to find out a good way for the cryopreservation of tendon seeding cells so as to facilitate the preparation of tissue engineering tendons as products, The related questions are how different factors affect cell survival rate at the procedure of preservation and whether cryopreservation affects seeding cells＇ biological characters as well as collagen secretive function. The results of experiment indicate that DMSO is a more effective cryoprotectant in cryopreservation of tissue engineered tendon seeding cells. Blood serum nourishment is very important in cell culture, preservation and treatment. The same sustenance after cryopreservation increases cell survival rate. In the process of cryopreservation, the concentration of cells is important to cell survival rate; cell survival rate will decrease when it is less than 1.0× 10^6/ml. In the process of cryopreservation, the cooling speed is also important to cell survival rate, slow cooling method achieves higher cell survival rate than does the rapid cooling method. Cryopreservation by use of 10%DMSO＋ 15%FCS＋ 75%DMEM does not affect seeding cells＇ collagen secretive function greatly and does not affect seeding cells＇ growth curve ,cell cycle and chromosome mode obviously. The prescription of 10%DMSO ＋ 15% FCS＋ 75%DMEM is suited for the cryopreservation of tendon seeding cells.