The Study Of Screening Of Serum Substitutes Based On Yeast Extracts

Objective: To establish a method for the detection of endotoxin in yeast extracts and provide guidance for the application of yeast extracts in serum-free media.Based on this,screening serum substitutes suitable for growth of animal cells.Methods: The yeast extract of FM 902 produced by Angel Yeast Co.,Ltd.was used as the test object.According to the "Pharmacopoeia of the People’s Republic of China"and the State Food and Drug Administration,"Bacterial endotoxin test method routine monitoring and skipping batch The relevant requirements of the “Inspection” industry standard require the use of dry heat program validity test,sensitivity review test,FM 902 gel interference test and FM 902 gel semi-quantitative test to detect the content of endotoxin in yeast extracts;CHO cells were selected as the study subjects.Four kinds of yeast extracts and 11 cytokines were screened by CCK-8 method to determine the serum-free medium or low-serum medium suitable for the growth of CHO adherent cells.HEK 293 cells were used as the research object.Batch culture methods were used to test the effect of various yeast hydrolysates on the growth of suspension cells.Results: The endotoxin contained in the FM 902 extract was 5 EU in accordance with the dry heat program validity test,sensitivity review test,FM 902 gel interference test,and FM 902 gel semi-quantitative test.Pharmacopoeia(2015 version)of the limited standard.The results showed that it is feasible to properly add yeast extract FM 902 in serum-free medium;by screening different yeast extracts FM 888,FM 502,YI 0201,FP103 and cytokines,two pairs of adherent CHO cell growth were determined.There are booster yeast extracts: FP 103 and yeast extract formulations(FM 888,YI 0201)with concentrations of 1 g/L and 0.125 g/L,respectively.At the same time,it was confirmed that epidermal growth factor and transferrin are essential additives for the growth of adherent CHO cells,which can replace serum concentrations of 0.5% FBS and concentrations of 1 ng/m L and 10 mg/L,respectively;In the method,the cell density of the suspension cell HEK293 was measured every 24 h,and the cell viability was monitored.The yeast extract FP 103 was found to be the best additive,and the optimal concentration was 3 g/L.conclusion:1.The endotoxin content of Angel Yeast extract meets the limit standard of the“Pharmacopoeia of the People’s Republic of China”(2015 edition)and can be used as a serum-free medium supplement;2.The primary formula for maintaining the growth of CHO cells was: basic culture medium(DMEM/F12)+ FP 103 + epidermal growth factor + transferrin + yeast mixture(FM 888,YI 0201).This formulation allows passage of CHO cells for two generations;3.Angel Yeast extract FP 103 is the best additive to promote suspension cell HEK 293.