临床级脐带间充质细胞制备及鉴定方法研究

Generation of clinical grade umbilical cord mesenchymal stromal cells

目的在GLP实验室中制备并鉴定临床级脐带间充质细胞(UC-MSC)。方法新鲜获取的脐带去除血管并进行充分清洗,获得的华通胶(Wharton’s jelly)机械分离后分别进行直接贴壁法和不同的酶消化法,比较获取UC-MSC的数量差别;用不同的无血清培养基进行培养比较细胞形态是否良好,得到最佳形态的UC-MSC。体外培养第3代后进行UCMSC质检,包括细胞活性,生长曲线,无菌检测,人类相关病毒、支原体、内毒素检测,染色体核型分析,FACS免疫表型检测及分化能力检测。不同消化方法获取细胞数之间比较采用两样本配对t检验。结果胶原酶Ⅱ消化获得的UC-MSC数(5.3×10~6±0.58个/ml)与胶原酶Ⅰ+0.25﹪胰酶消化法(2.53×10~5±0.03个/ml)及直接贴壁法(2.6×10~5±0.05个/ml)之间差异有统计学意义(P<0.05),与胶原酶Ⅰ消化法获取细胞数(5.1×10~6±0.57个/ml)之间差异无统计学意义(P=0.07),但培养视野最干净;Mesen Cult-ACF Medium培养获得的UC-MSC形态最佳;UC-MSC质检细胞活性冻存前达99.8﹪,冻存复苏后达99﹪;无细菌、支原体、乙肝病毒、丙肝病毒、梅毒螺旋体、艾滋病毒、肺炎支原体、EB病毒、巨细胞病毒污染;内毒素检测结果均<1 EU;CD73/CD90/CD10~5阳性率达98﹪,CD34/CD45/HLA-DR呈阴性,阳性率<2﹪;染色体核型分析无突变或缺失;UC-MSC具有成脂、成骨或成软骨方向分化的能力。结论通过优化分离和培养条件,采用无血清及无动物来源的培养系统,在GLP实验室内可获得临床级UC-MSC。

Objective To produce clinical grade umbilical cord mesenchymal stromal cells （UC-MSCs） with a umbilical cord in a GLP lab. Methods The umbilical cord was collected after delivery and the arteries, veins and blood cells were removed. Different treatment conditions were applied to compare the yield of UC-MSCs. The Wharton＇s jelly was cut to 1 mm^3 cubes, which were then either directly placed into an adhesive culture dish, or digested with different types of collagenases, followed by expansion in three types of serum free culture medium. After three passages in vitro, the cells were characterized and analyzed for cell viability, morphology, growth, sterility, endotoxin levels, chromosome karyotyping, surface antigen expression and differentiation capacity. Data analysis between different groups was performed with coupled t test. Results Digestion with collagenase Ⅱ or collagenase Ⅰ achieved the highest yield of MSCs （5.3×10^6±0.58/ml and 5.1 ×10^6±0.57/ml, respectively, P = 0.07）, both higher than treatment with collagenase Ⅰ ＋ 0.25 % trypsin （2.53×10^5±0.03/ml） and enzyme-free method. The culture medium appeared clear after collagenase Ⅱ digestion. MSCs cultured in Stemcell MesenCult-ACF Medium showed optimal cell morphology. Before freezing, the cell viability was 99.8 %, and 99 % after freezing and thawing. The obtained MSCs were negative for bacteria or mycoplasma contamination, hepatitis B virus （HBV）,hepatitis C virus （HCV）, human immunodeficiency virus （HIV）, Mycoplasma pneumonia （MP）, Treponema pallidum （TP）, EB virus （EBV）, and cytomegalovirus （CMV）. The endotoxin levels were less than 1 EU as tested by ELISA. FACS results showed that more than 98 % of the obtained cells were positive for CD73, CD90 and CD105, and more than 98 % of the cells negative for CD34, CD45 and HLA-DR. The cells showed a normal karyotype and could be differentiated into adipocytes, osteoblasts and cartilage cells. Conclusion Clinical grade MSCs are generated in a GLP lab with optimized isolation/cultu_re conditions.