CELL-DISLODGING METHODS UNDER SERUM-FREE CONDITIONS

In this work, a BHK21 clone producing a fusion protein consisting of a recombinant human IgG molecule with a cytokine tail, growing in a pro tein-free medium, was used to test several alternatives to avoid the u se of serum for trypsin inactivation, currently used in cell dislodgin g. These included (1) trypsin inactivated with soybean trypsin inhibit or (STI); (2) cell dissociation solution instead of trypsin; (3) dispa se instead of trypsin; (4) trypsin inactivated with fetal calf serum ( positive control); (5) non-inactivated trypsin (negative control). Use of a centrifugation step was also tested for each alternative. Result s indicate that the best method regarding cell growth, viability and a dherent fraction is to use trypsin inactivated with STI followed by a centrifugation step. For all methods tested, the utilization of a cent rifugation step always led to improved results. The optimal proportion for total trypsin inactivation is 1:1 trypsin (0.2% w/v) to STI (1 mg ml(-1)), equivalent to 2 mg trypsin to 1 mg STI. No toxic effect was observed for STI at the concentrations used. Longterm subculturing wit h this new, alternative dislodging method did not affect cell growth, viability and productivity.