Application of population doubling level in evaluation of passage stability of HEK293 cells in suspension culture
Objective To evaluate the passage stability of HEK293 cells in suspension culture by using population doubling level(PDL)and verify the method,in order to provide experimental basis for the industrial production and culture of this cells.Methods The working seed lot of HEK293 cells were subcultured continuously for 60 d,one generation every 2 d,and the cell stability was evaluated when PDL increased to 10-60.The calculation of related research results showed that when HEK293 cells were cultured to 2 500 L,the PDL of each generation should be controlled within 2±0.2.The working seed lot of HEK293 cells were cultured in different stages of shaking flask(125,500,1 000 and 3 000 mL,four generations),cell expansion system(CES)(25,25 and 50 L,three generations)and bioreactor(100,500 and 500,three generations).The PDL of each generation was controlled within 2±0.2.Totally three batches of cells were cultured and analyzed for the indicators such as cell density,viability,agglomeration rate and diameter.After culture for 72 h in a 500 L bioreactor,the HEK293 cells were inoculated with the working seed lot of adenovirus at a MOI of 5-10,cultured for 2 d,then the virus liquid was harvested and detected for the number of virus particles.Results HEK293 cells in the working seed lot in serial passage maintained high cell density and viability when the PDL reached 60.When PDL was controlled in the range of 2±0.2,the density of three batches of HEK293 cells in the shaking flask,CES and bioreactor was all greater than 2.0 × 106 cells/mL,the viability was all greater than 96％,and the cell diameter was about 17 μm.The agglomeration rates were all lower than 35％.The three batches of HEK293 cells cultured in the 500 L bioreactor were inoculated with virus for 2 d,and the number of virus particles reached 11.68 × 1010,12.55 × 1010 and 9.38 × 1010 vp/mL,respectively.Conclusion It is feasible to evaluate the stability of passage of HEK293 cells by PDL,which can reflect the growth status of passage cells more scientifically.
Population doubling level(PDL)HEK293 cellsSuspension culturePassageStability
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