title: An optimization protocol for Swiss 3T3 feeder cell growth-arrest by Mitomycin C dose-to-volume derivation strategy.
abstract: 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.
authors.affiliation: Cell Biology Laboratory, National Institute of Pathology (ICMR), New Delhi, India.
authors.forename: Rishi Man
authors.initials: RM
authors.lastname: Chugh
authors.affiliation: Cell Biology Laboratory, National Institute of Pathology (ICMR), New Delhi, India.|Department of Medical Elementology and Toxicology, Jamia Hamdard, New Delhi, India.
authors.forename: Madhusudan
authors.initials: M
authors.lastname: Chaturvedi
authors.affiliation: Cell Biology Laboratory, National Institute of Pathology (ICMR), New Delhi, India. yernenilk@icmr.org.in.
authors.forename: Lakshmana Kumar
authors.initials: LK
authors.lastname: Yerneni
keywords: Colony forming efficiency; Feeder growth-arrest; Keratinocyte proliferation; Mitomycin C dosing; Swiss 3T3