Objective To select a host cell line with traceable monoclonal origin and good growth status by serum-free sus-pension domestication culture of CHO-K1 cells,and verify its expression.Methods Adherent CHO-K1 cells were domesti-cated into suspension cells suitable for serum-free medium by gradually decreasing serum concentration.Monoclonal cells were selected by cell seeding and imaging system Verified In-Situ Plate Seeding(VIPS system for short),and the parame-ters such as doubling time,agglomeration rate and cell diameter of monoclonal cells were comprehensively evaluated in continuous culture to determine the candidate clones.The candidate clones were transfected with plasmids contained green and red fluorescent proteins respectively,and one clone was identified as the final host cell of the engineering cell line according to the expression of fluorescent proteins.Results The obtained CHO-K1 cell lines were adapted to serum-free suspension culture,and the doubling time was 20-24 h.The monoclonal cell line identified by monoclonal screening and yield evaluation had clear monoclonal origin and could be traced back.At an inoculation density of 0.5 × 106cells/mL,the maximum growth density reached 8.27 × 106 cells/mL through continuous cultivation of 7 d,the viability was not lower than 80％with the mean doubling time of 20.31 h,and the exogenous gene was effectively expressed.Conclusion Through serum-free domestication culture,CHO-K1 cell line with traceable monoclonal origin,good growth status and high protein expression was successfully obtained.
CHO-K1 cellsSerum-free cultureCell domesticationMonoclonal screeningExogenous protein expression
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