Abstract
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.
Highlights
IntroductionFeeder Growth Arrest Failure growth arrested Swiss mouse embryonic 3T3 dermal fibroblasts [1]
Large quantities of cultured epithelial autografts (CEA) for clinical use in the treatment of extensively burned patients are speedily grown from the adult epidermal keratinocytes over the PLOS ONE | DOI:10.1371/journal.pone.0122056 March 23, 2015Feeder Growth Arrest Failure growth arrested Swiss mouse embryonic 3T3 dermal fibroblasts [1]
The growth arrested 3T3 fibroblasts reportedly survived in CEA and elicited immunogenicity in recipient resulting in complete graft breakdown [4]
Summary
Feeder Growth Arrest Failure growth arrested Swiss mouse embryonic 3T3 dermal fibroblasts [1]. These cells are superior in supporting the growth of other target cells as well [2, 3]. The growth arrested 3T3 fibroblasts reportedly survived in CEA and elicited immunogenicity in recipient resulting in complete graft breakdown [4]. The viable feeders can result either from the mitotically inactive yet surviving feeders or the proliferating ones. There is evidence of proliferation in other growth arrested mouse embryonic feeders, but there are no specific studies to link the persistence of the viable 3T3 feeders with the failure of growth arrest [5]
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