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

  • Method in Cell Science
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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.

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Abbreviations

MC:

Mitomycin C

ECN:

Exposure cell number

HEPES:

4-(2-Hydroxy ethyl)-1-piperazineethanesulfonic acid

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Acknowledgements

Corresponding author is grateful to the Indian Council of Medical Research (ICMR) New Delhi, India, for research Grant Number 53/3/2009.

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Authors and Affiliations

  1. Cell Biology Laboratory, National Institute of Pathology (ICMR), New Delhi, India

    Rishi Man Chugh, Madhusudan Chaturvedi & Lakshmana Kumar Yerneni

  2. Department of Medical Elementology and Toxicology, Jamia Hamdard, New Delhi, India

    Madhusudan Chaturvedi

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  1. Rishi Man Chugh
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  2. Madhusudan Chaturvedi
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  3. Lakshmana Kumar Yerneni
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Correspondence to Lakshmana Kumar Yerneni.

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Fig. S1

Differential growth arrest by volume titrations. Schematic representation of producing differential growth arrest in Swiss 3T3 cells by Mitomycin C through titrations of a constant exposure cell density with varied volumes (υ1 to υ10) of treating solution which were calculated from the specific permutations of concentration (4 or 5 μg/ml) and dose (15, 75, 150 or 450 pg/cell). Concentrations 3 and 10 μg/ml combined with doses of 10 and 30 pg/cell served as controls for comparison. The doses were calculated previously through exposure cell density titrations using various concentrations in a fixed volume. Supplementary material 1 (TIFF 169 kb)

Fig. S2

Selection of optimal seeding ratio for keratinocyte-feeder co-culture. Preliminary screening was performed to identify the optimally performing keratinocyte-feeder seeding ratios using the short-listed feeder groups. Keratinocyte-feeder ratio of 1:0.75 (a), by employing 7500 feeders per cm2, produced a typical low saturation density growth curve suggestive of week feeder action. The ratios of 1:1 (b) and 1:1.5 (c) produced ideal growth curves but resulted in a lower keratinocyte output against culture time. The ratio of 1:2 (d), attained by raising the seeding of feeders cell seeding to 15,000/cm2 produced a maximal keratinocyte output in 9 days which was comparable to the day 12 yield of 1:1 ratio. The asterisk indicates significant variance (P < 0.05) calculated by Kruskal–Wallis. Supplementary material 2 (TIFF 605 kb)

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Chugh, R.M., Chaturvedi, M. & Yerneni, L.K. An optimization protocol for Swiss 3T3 feeder cell growth-arrest by Mitomycin C dose-to-volume derivation strategy. Cytotechnology 69, 391–404 (2017). https://doi.org/10.1007/s10616-017-0064-9

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