Objective To screen lyophilized protectant of recombinant porcine trypsin (rPT) , and to investigate the stability of cell dissociation solution and its application. Methods Different concentrations of different types of lyophilized protectants were added to rPT solution. The role of lyophilized protectants was evaluated by comparing the retention rate of the activity of lyophilized powder, physical appearance, solubility and color. The cell dissociation solution was prepared with sterile lyophilized rPT powder. The content of endotoxin in the prepared cell dissociation solution was less than 3 EU per mg rPT. Then the preparation method was optimized. The cell dissociation solution was applied in cell culture. Results Owing to adding 3% mannitol and 3% trehalose, the retention rates of the activity of lyophilized product were 101% and 86,5%. The cell dissociation was prepared with PBS buffer (containing 0.01% EDTA), digesting attached cell best and having better stability when stored at -20 ℃. Conclusion From the evaluation of physical appearance of lyophilized sample and retention rates of the activity, 3% mannitol can be used as the best protectant. The successful application of rPT cell dissociation with PBS buffer in cell culture can prove that rPT can substitute for extracted animal trypsin and avoid extrinsic contamination induced by virus-derived factors.%目的 筛选重组猪胰蛋白酶(rPT)冻干保护剂和保存方法,及配制细胞消化液的稳定性及其应用.方法 在rPT酶液中添加不同浓度不同种类的冻干保护剂,比较冷冻干燥后的活性保留率,并从赋型性、色泽度、溶解性方面评价冻干保护剂的作用；rPT酶液经过无菌过滤处理,内毒素含量检测合格后,冻干.然后配制成细胞消化液,优化细胞消化液组成,将细胞消化液应用于细胞培养.结果 添加3％甘露醇和3％海藻糖的rPT冻干品的酶活性保留率分别为101％和86.5％:用PBS缓冲液配制细胞消化液(含有0.01％ EDTA)消化贴壁细胞的综合效果最好,且保存在-20℃时稳定性较好.结论 从冻干后酶活性保留率和冻干品外观评价,3％甘露醇可作为rPT冻干过程中的最佳保护剂；rPT细胞消化液应用在细胞培养中的成功,证明rPT可以替代提取的猪/牛胰蛋白酶使用,消除了动物源性病毒等外源因子的污染.
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