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

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.

Exposure cell number during feeder cell growth-arrest by Mitomycin C is a critical pharmacological aspect in stem cell culture system.

INTRODUCTION: Growth-arrested feeder cells following Mitomycin C treatment are instrumental in stem cell culture allowing development of regenerative strategies and alternatives to animal testing in drug discovery. The concentration of Mitomycin C and feeder cell type was described to affect feeder performance but the criticality of feeder cell exposure density was not addressed. We hypothesize that the exposure cell density influences the effectiveness of Mitomycin C in an arithmetic manner. METHODS: Three different exposure cell densities of Swiss 3T3 fibroblasts were treated with a range of Mitomycin C concentrations for 2h. The cells were replaced and the viable cells counted on 3, 6, 9, 12 and 20days. The cell extinctions were compared with doses per cell which were derived by dividing the product of concentration and volume of Mitomycin C solution with exposure cell number. RESULTS: The periodic post-treatment feeder cell extinctions were not just dependent on Mitomycin C concentration but also on dose per cell. Analysis of linearity between viable cell number and Mitomycin C dose per cell derived from the concentrations of 3 to 10µg/ml revealed four distinct categories of growth-arrest. Confluent cultures exposed to low concentration showed growth-arrest failure. DISCUSSION: The in vitro cell density titration can facilitate prediction of a compound's operational in vivo dosing. For containing the growth arrest failure, an arithmetic volume derivation strategy is proposed by fixing the exposure density to a safe limit. The feeder extinction characteristics are critical for streamlining the stem cell based pharmacological and toxicological assays.

An evaluation of the choice of feeder cell growth arrest for the production of cultured epidermis.

Growth arrested 3T3 cells have been used as feeder cells in human epidermal keratinocyte cultures to produce cultured epidermal autografts for the treatment of burns. The feeder cells were ideally growth-arrested by gamma-irradiation. Alternatively, growth arrest by mitomycin C treatment is a cost effective option. We compared the functional efficacy of these two approaches in keratinocyte cultures by colony forming efficiency, the net growth area of colonies, BrdU labeling and histological features of cultured epidermal sheets. The growth area estimation involved a semi-automated digital technique using the Adobe Photoshop and comprised of isolation and enumeration of red pixels in Rhodamine B-stained keratinocyte colonies. A further refinement of the technique led to the identification of critical steps to increasing the degree of accuracy and enabling its application as an extension of colony formation assay. The results on feeder cell functionality revealed that the gamma irradiated feeders influenced significantly higher colony forming efficiency and larger growth area than the mitomycin C treated feeders. The BrdU labeling study indicated significant stimulation of the overall keratinocyte proliferation by the gamma irradiated feeders. The cultured epidermal sheets produced by gamma feeders were relatively thicker than those produced by mitomycin C feeders. We discussed the clinical utility of mitomycin C feeders from the viewpoint of cost-effective burn care in developing countries.

Occurrence and control of sporadic proliferation in growth arrested Swiss 3T3 feeder cells.

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.

Pharmacological action of high doses of melatonin on B16 murine melanoma cells depends on cell number at time of exposure.

Melatonin has been reported to possess growth inhibitory action at certain physiological doses in cancer cell lines in vitro and oncostatic action under in vivo conditions. In an attempt to achieve a pharmacologically effective anticancer action of melatonin, dose-response studies with high concentrations of melatonin (10(-6) M, 10(-5) M, 10(-4) M and 10(-3) M) were conducted in the B16 murine melanoma cell line using three different numbers of exposed cells. A range of effects, including stimulatory, oncostatic and oncocidal action, were studied 3 days after exposure to melatonin. In order to standardize the results, the concentration of melatonin per cell was calculated from the amount of melatonin added to the culture, and compared with the growth patterns of the cells. Melatonin had a mild stimulatory effect on cell proliferation at the lower end of the dose spectrum and an oncostatic influence at intermediate concentrations, while the higher concentrations per cell demonstrated clear lethal (oncocidal) action. It is suggested that by using a pharmacologically appropriate dosage regimen, melatonin could be useful in the treatment of responsive cancers. Furthermore, calculation of the concentration of melatonin per cell is important in understanding the true pharmacological potential of melatonin.