Optimization of sorting conditions for the selection of stable, high-producing mammalian cell lines

The production of Green Fluorescent Protein in recombinant NIH3T3 mouse fib roblast cells was used as a model to determine the optimal conditions for t he rapid isolation of high-producing cell lines with a fluorescence-activat ed cell sorter. "Bulk sorting", that is, sorting of a large number of posit ive cells, did not result in a stable, high-producing cell line due to over growth of high-producing cells by low- or nonproducing cells. The productio n kinetics and expression of GFP during batch culture was found to differ b etween NIH3T3 cells and HepG2 hepatoma cells, even though the same plasmid was used for transfection. The kinetics of product formation need therefore to be determined from case to case to select the optimal timepoint for ana lysis and sorting. Subcloning of sorted cells into microtiter plates only r esulted in high-producing subclones when 1 or 2 cells were seeded per well. Higher seeding rates again resulted in overgrowth of low- or nonproducers. By subcloning, two high-producing cells lines could be isolated. They had a 10- and 15-fold higher fluorescent signal compared to the negative contro l. While one of these subclones started to decrease it's GFP expression aft er 2 months, the other clone stably expressed GFP for 4 months.