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Spontaneous establishment and characterization of mouse keratinocyte cell lines in serum-free medium

  • Rapid Communications in Cell Biology
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Editor's Statement Keratinocytes are useful and important models for studies of carcinogenesis and tumor promotion and differentiation. This paper provides a solid in vitro basis for examination of the cellular endocrinology of these phenomena in vitro, and implicates TGF beta as a regulator of these cells.

Summary

Mouse keratinocytes cultures readily develop into established cell lines without undergoing a “crisis” in a newly-developed serum-free medium, LEP/MK2. LEP/MK2 consists of calcium-free MEM with non-essential amino acids supplemented with 8 factors. Two lines, MK1 and MKDC4, have been isolated and have now doubled more than 400 and 200 times respectively. In MK1 cells, Giemsa banding has revealed significant karyotypic changes as early as the 4th passage, leading to a near-tetraploid karyotype with random loss and gain of individual chromosomes. Minute chromosomes, but no stable markers have been observed. After these initial changes, examination of cultures at several passage levels has shown that the karyotype has remained essentially stable. The MKDC4 line, also sub-tetraploid at the 7th passage, had 4 marker chromosomes by the 47th passage. The rapid increase in chromosome number may have contributed to the “immortalization” of these lines.

The response of these established keratinocyte lines to growth factors and serum-derived inhibitors changed with increasing passage level. Most notable of these changes were a reduction in the requirement for bovine pituitary extract (an absolute requirement for growth of secondary MK1 cells) and a decreased sensitivity to serum and serum-derived inhibitors, e.g., transforming growth factor-β. The established lines, like primary and secondary keratinocytes, remain responsive to calcium-induced terminal differentiation and are non-tumorigenic in athymic, nude mice. This serum-free system is suitable for transformation studies with oncogenes and chemical carcinogens.

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Author notes

  1. M. E. Kaighn

    Present address: Laboratory of Human Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, 20892, Bethesda, MD

  2. R. F. Camalier

    Present address: Program Development Research Group, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, 21701, Frederick, MD

  3. F. Bertolero

    Present address: Department of Toxicology, Karolinska Institute, S-10401, Stockholm, Sweden

Authors and Affiliations

  1. Laboratory of Experimental Pathology, Division of Cancer Etiology, National Cancer Institute, Bldg. 41, Rm. C-105, 20892, Bethesda, MD

    M. E. Kaighn, R. F. Camalier, F. Bertolero & U. Saffiotti

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  1. M. E. Kaighn
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  2. R. F. Camalier
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  3. F. Bertolero
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  4. U. Saffiotti
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Kaighn, M.E., Camalier, R.F., Bertolero, F. et al. Spontaneous establishment and characterization of mouse keratinocyte cell lines in serum-free medium. In Vitro Cell Dev Biol 24, 845–854 (1988). https://doi.org/10.1007/BF02623657

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