Occurrence and Control of Sporadic Proliferation in Growth Arrested Swiss 3T3 Feeder Cells

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.

Urothelial cell senescence is not linked with telomere shortening

“…Recent work has demonstrated a risk of sporadic proliferation in growth‐arrested J2 3T3 feeder cells due to development of resistance to the MC drug (Chugh, Chaturvedi, & Yerneni, ). In this study, we found presence of feeder layer cells in four out of six cultures (sample donors #M1, M3, M4, and M6) after Passage 2, which was therefore identified as contamination of still proliferating J2 3T3 cells.…”

“…In this study, we found presence of feeder layer cells in four out of six cultures (sample donors #M1, M3, M4, and M6) after Passage 2, which was therefore identified as contamination of still proliferating J2 3T3 cells. This problem could be avoided by employing culture and subculture protocols that identify and control the proliferation of this cell line (Chugh et al, ; Chugh, Chaturvedi, & Yerneni, ).…”

“…This problem could be avoided by employing culture and subculture protocols that identify and control the proliferation of this cell line (Chugh et al, 2015;Chugh, Chaturvedi, & Yerneni, 2017). However, two of the cell cultures established on feeder cells (donors #M2, and M5) remained pure with only human urothelial cells and had a high proliferative capacity.…”

Unrestricted somatic stem cells, as a novel feeder layer: Ex vivo culture of human limbal stem cells

“…Thus, these feeder cells provided a qualified environment in co‐culture with a variety of cell types, through intermediating different mechanisms including cell‐cell and cell‐extracellular matrix interactions, secretion of soluble growth factors as well as removing toxicants from culture medium . Latest study suggest that using mouse embryonic 3T3 feeder layer can greatly increase the number of stem cells and it is useful in supporting and maintenance of the target cells . However, the mouse feeder cells contains N‐glycolylneuraminic acid and non‐human sialic acid (Neu5Gc), an immune response could be induced when we use the cells for transplantation, since most people have Neu5Gc circulating antibodies…”

An optimization protocol for Swiss 3T3 feeder cell growth-arrest by Mitomycin C dose-to-volume derivation strategy

“…In practical terms, such permutations represent a range of volumes of treating MC solutions which are directly and indirectly proportional to the derived dose per cell and concentration per se, respectively. Because, the 3T3 cultures were known to gradually accumulate spontaneous variants with altered characteristics (Rubin and Xu 1989;Matthews 1993), the exposure cell number has to be set to such a constant level that the cell population does not accumulate MC-resistant variants through successive passaging (Chugh et al 2015a). Accordingly, the titration of a safe density of feeder cells with varied volumes of a MC solution of a less toxic but effective concentration would then enable identification of a specific concentration-dose permutation that produced ideal feeder cell extinction which in turn influenced optimal stem cell proliferation.…”