Abstract:
Trypsin is an essential tool enzyme for adherent cell passaging. The effects of porcine trypsin, two kinds of bovine trypsin and fully synthetic trypsin TrypLE on the digestion of Vero cells and KMB17 cells were compared, and the optimal digestion process of cultured cells in vaccine production and the substitution of porcine trypsin in Halal verification in the process of providing biological products to Islamic countries and people were evaluated in this study. First, the cells were digested with porcine derived trypsin, bovine-derived trypsin-1, bovine-derived trypsin-2, and total synthetic trypsin TrypLE reagents with well configured viability units all ranging from 1400 to 1500 U/ml at 37 ℃, respectively, and the fluorescence counter was used for counting and activity detection. Then, the obtained cell suspension was diluted to 2×10
5 cells/mL and inoculated into 6-well plate and 96 well plate, and cell growth curves were drawn at different times of culture to detect cell proliferation and toxicity. After screening to alternative digestive enzymes, 10 replicate experiments were performed. The results showed that the digestive capacity of porcine derived trypsin was relatively good compared with other pancreatic enzymes in the Vero cell digestion experiment, in which bovine derived trypsin-2 had the greatest effect on cell proliferation and toxicity after digestion of the cells. In the KMB-17 cell digestion experiment, there was no significant difference in the digestive capacity of any of the four digestive enzymes with significant difference in the digestion time (
P<0.05), and the effects of the four digestive enzymes on the proliferation and toxicity of KMB-17 cells were significantly different after 48 hours. The CV value of Vero cells digested by porcine trypsin, bovine trypsin-1 and TrypLE were 1.08%, 1.12% and 1.22%, respectively. The CV of KMB-17 cells digested by porcine trypsin and bovine trypsin-2 were 1.15% and 1.04%, respectively. To meet the demand for vaccine and biologic products used in Islamic countries and populations worldwide, bovine derived trypsin-1 and TrypLE can be used instead of porcine derived trypsin to digest Vero cells, and bovine trypsin-2 can be used to replace porcine trypsin to digest KMB-17.