Clonning Selection Of BHK-21 Suspension Cell Line And Its Cultivation Process Optimize

BHK-21 cell line is a WHO recommended cell substrate for the production of veterinary vaccines,and the BHK-21 single cell suspension culture model has been widely used in large-scale production.Compared with the traditional culture technology of roller bottle,the suspension culture technology has significant advantages in yields and quality.Suspension cell adaptation,medium development and high density cultivation in bioreactor are the key factor and main orientation of future research of suspension culture process.Nowadays BHK-21 suspension cell already used in veterinary vaccines production but the cell density still is very low.Suspension type BHK-21 cells like adherent cells,there was a significant difference in single cell growth during cell population doubling process.Some cellsgrow fast while others grow slowly.some cells have long term cell viab-ility while ot-hers are the opposite.For single cell clones derived from the same ancestor cells,because of its strong homology,genetic stability and biological characters of homogeneous and stable cell lines,this study were undertaken to get suspension cell lines by single cell culture of single cells and expanded in culture,grow fast,long durability clones cell strains were frozen according to the PDT and MPC to select high density growth cell lines.In this study,the morphology,viability,growth curve,microbial contamination,chromosome karyotype,isoenzyme of BHK-21 suspension cells which is from Gansu Province Animal Cell Engineering Technology Research Center were identified.Then the qualified BHK-21 suspension cell lines were obtained,using limited dilution method seed 1 cells per well to 96 well U culture plate,96 well flat culture plate,and the 24 well culture plate.Single cell clones were isolated by limited dilution method.During the culture period till 14d at the bottom of the well the cells form a big cluster,22 clone cell lines were obtained and cell bank were established.All 22 clone cell lines were resuscitated and compared,4 of them had significant advantages over the other clones.Identification of biological characteristics of the 4 clone cell lone.And compared with the biological characteristics of BHK-21 cell suspension without cloning.The results showed that the biological characteristics of clone cells were stable,and no mutation was found.4 strains of clone cell the foot and mouth disease virus TCID50 CL18 is the optimal one,depend on the growth characteristics of CL18 best inoculation density was 6×105/mL,shaking flask batch-culture the MPC was 85×105/mL,;fed-batch culture MPC was 90×105/mL.5 Liter NBS bioreactor culture CL18 cells,batch-cultured 72h cell density can reach 84x105/mL,fed-batch culture inoculation cell density was 6×105/mL,the MPC was 90×105/mL.The results showed that the identified monoclonal BHK-21CL18 strain can be used for the production and application of foot and mouth disease vaccine.