基于蚕丝蛋白的低温保护剂用于梅山猪耳成纤维细胞的低温保存研究

细胞低温保存为临床治疗和科学研究提供优质的细胞。体积分数为10%二甲基亚砜(DMSO)和体积分数为20%胎牛血清(FBS)是目前冻存细胞常用的保护剂。但DMSO对细胞具有毒性损伤,FBS存在携带病毒、感染疾病的风险。本文以梅山猪耳成纤维细胞作为模型细胞进行冻存实验,将蚕丝蛋白用于低温保护剂中,用不同质量浓度的丝胶蛋白和不同体积分数的丝素蛋白来分别替代FBS和DMSO,验证其在细胞低温保存中的有效性。解冻复苏后用台盼蓝染色法、MTT法、24 h贴壁率等检测细胞的存活率和生长活力,筛选出最佳的基于天然蚕丝蛋白的低温保护剂配方。结果表明:含质量浓度为1%丝胶蛋白和含体积分数为20%FBS的低温保护剂对猪耳细胞的冻存效果无显著差异,说明丝胶蛋白能有效替代FBS。将体积分数为10%DMSO浓度降至5%,添加体积分数为10%丝素蛋白后,细胞的存活率和贴壁率与对照组相比无明显差异,说明丝素蛋白能降低DMSO的浓度。将质量浓度为1%丝胶蛋白与体积分数为10%丝素蛋白联用后,能达到较好的冻存效果。

Research on the Cryopreservation of Meishan Pig Ear Fibroblast Cells using Silk Protein

Cell cryopreservation provides high quality cells for clinical application and scientific studies. Specifically, 20% (v/v) FBS supplemented with 10% (v/v) DMSO are widely used in cryoprotectants. However, high concentrations of DMSO are cytotoxic to cells, and FBS exhibits problems related to diseases such as bovine spongiform encephalopathy and viral infections. Silk protein is a type of natural macromolecule fiber protein with good biocompatibility. The aim of the study involved testing the effectiveness of the silk protein as cryoprotectant and developing a novel FBS-free and low concentration DMSO freezing medium. Meishan pig ear fibroblast cells were cryopreserved by using a freezing medium in the presence of various mass concentrations of silk sericin and various volume fractions of silk fibroin. The preservation efficiency was evaluated by trypan blue dye exclusion test, MTT assay, and the 24 h adherent rate after thawing. The results indicate that the survival rate of cryopreserved cells does not significantly differ when the cells are cryopreserved with a freezing medium containing 1% (w/v) sericin and 20% (v/v) FBS, and this indicates that sericin can substitute for FBS in the freezing medium. A significant difference is absent between 10% (v/v) fibroin protein with 5% (v/v) DMSO and 10% (v/v) DMSO, and thus the fibroin decreases the concentration of DMSO. Finally, 5% (v/v) DMSO supplemented with 1% (w/v) sericin and 10% (v/v) fibroin exhibit similar results as a control group.