Growth arrested Swiss mouse embryonic 3T3 cells are used as feeders to support the growth of epidermal keratinocytes and several other target cells. The 3T3 cells have been extensively subcultured owing to their popularity and wide distribution in the world and, as a consequence selective inclusion of variants is a strong possibility in them. Inadvertently selected variants expressing innate resistance to mitomycin C may continue to proliferate even after treatment with such growth arresting agents. The failure of growth arrest can lead to a serious risk of proliferative feeder contamination in target cell cultures. In this study, we passaged Swiss 3T3 cells (CCL-92, ATCC) by different seeding densities and incubation periods. We tested the resultant cultures for differences in anchorage-independent growth, resumption of proliferation after mitomycin C treatment and occurrence of proliferative feeder contaminants in an epidermal keratinocyte co-culture system. The study revealed subculture dependent differential responses. The cultures of a particular subculture procedure displayed unique cell size distribution and disintegrated completely in 6 weeks following mitomycin C treatment, but their repeated subculture resulted in feeder regrowth as late as 11 weeks after the growth arrest. In contrast, mitomycin C failed to inhibit cell proliferation in cultures of the other subculture schemes and also in a clone that was established from a transformation focus of super-confluent culture. The resultant proliferative feeder cells contaminated the keratinocyte cultures. The anchorage-independent growth appeared in late passages as compared with the expression of mitomycin C resistance in earlier passages. The feeder regrowth was prevented by identifying a safe subculture protocol that discouraged the inclusion of resistant variants. We advocate routine anchorage-independent growth assay and absolute confirmation of feeder disintegration to qualify feeder batches and caution on the use of fetal bovine serum.

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Feeder cell functionality following growth-arrest with the cost-effective Mitomycin C vis-à-vis irradiation is controversial due to several methodological variables reported. Earlier, we demonstrated variability in growth arrested Swiss 3T3 feeder cell life-span following titration of feeder cell densities with Mitomycin C concentrations which led to the derivation of doses per cell. Alternatively, to counter the unexpected feeder regrowth at high exposure cell density, we proposed titration of a fixed density with arithmetically derived volumes of Mitomycin C solution that corresponded to permutations of specific concentrations and doses per cell. We now describe an experimental procedure of inducing differential feeder cell growth-arrest by titrating with such volumes and validating the best feeder batch through target cell growth assessment. A safe cell density of Swiss 3T3 tested for the exclusion of Mitomycin C resistant variants was titrated with a range of volumes of a Mitomycin C solution. The differentially growth-arrested feeder batches generated were tested for short-term and long-term viability and human epidermal keratinocyte growth supporting ability. The feeder cell extinction rate was directly proportional to the volume of Mitomycin C solution within a given concentration per se. The keratinocyte colony forming efficiency and the overall growth in mass cultures were maximal with a median extinction rate produced by an intermediate volume, while the faster and slower extinction rates by high and low volumes, respectively, were suboptimal. The described method could counter the inadequacies of growth-arrest with Mitomycin C.