人外周血淋巴细胞培养基冻干工艺研究

目的对染色体分析用人外周血淋巴细胞培养基进行冻干工艺进行优化。方法首先以澄清度和细胞培养分裂相比例为检测指标确定冻干前培养液浓缩倍率。其次运用正交试验设计法L934法,以冻干粉外观、水分残留率、分裂相比例为检测指标,对冻干工艺过程的的预冻、升华干燥、解析干燥等三个阶段的主要参数进行考察和分析,获得主要参数最优组合的冻干曲线。最后,以优化的冻干工艺生产的三批冻干培养基进行适用性验证。结果确定冻干前培养液的浓缩倍率为3．O。正交实验分析表明：主干燥时间和主干燥真空压力对细胞分裂相比例的影响有显著的统计学意义（P〈0．05）。优化的冻干参数为预冻温度一45℃、时间2h;升华干燥阶段温度从-45℃升温至20℃,时间14h,真空压力35Pa;解析干燥阶段的温度维持于28℃,真空压力为1．0Pa,终点测试压力无变化时通入干燥无茵空气至冻干箱内压力为76kPa后压塞,再通入干燥无菌空气至箱体内压力为1个标准大气压时冻千结束。用优化冻干工艺生产的三批培养基的验证结果显示外观合格率、水分残留率、常温保存180d后的分裂相比例均符合质量标准要求。结论优化的淋巴细胞培养基冻干工艺可行,可用于制备常温贮存的人外周血淋巴细胞培养基。

STUDY ON FREEZE- DRYING PROCESS OF LYOPHILIZED HUMAN PERIPHERAL BLOOD LYMPHOCYTES CULTURE MEDIUM

Objective To optimize the freeze - drying process of lyophilized human peripheral blood lymphocytes culture medium for chromosome analysis. Methods Firsdy, concentration ratio was determined with clearance degree and mitotic phase rate as test index. Secondly, with the appearance of lyophilized powder, residual water and mitotic phase rate as the test index, the key parameters in pre - freezing, main drying and final drying were analyzed by means of the test of orthog- onal experimental design L934 in order to optimize the combination of key parameters of freeze -drying process. Finally, the optimized freeze - drying process was validated by 3 lots of lyophilized culture media. Results Concentration ratio was deter- mined to be 3.0. The results of orthogonal test showed the duration and vacuum pressure of main drying significantly effect the mitotic phase rate （p 〈 0.05 ）. The optimized parameters were as follows： -45 ~C ,2 h in pre -freezing phase; -45 ~C to 20 ~C for 14 h,at 35 Pa in main drying phase; 28 ~C at 1.0 Pa in final drying phase. Then the dry aseptic air was introduced in- to the vessel to reach to 76 kPa prior to capping. The freeze - drying process was completed after the post - capping pressure of vessel reached to 1 standard atmospheric pressure. The validation results demonstrated that 3 lots of lyophilized culture media manufactured by optimized freeze - drying process were all met with specifications including of the appearance passing rate, residual water rate and mitotic phase rate after 180 d. Conclusion Optimized freeze -drying process is applicable for lyophilized human peripheral blood lymphocytes culture medium stored at RM temperature.