Abstract
We describe a gel-supported in vitro system for culturing skin samples in a three-dimensional native state. All the cell types of skin remain viable and maintain their native architecture for at least 10 days. The culture system is used for toxicity measurements by ascertaining cell viability using two fluorescent dyes: 2',7'-bis-(2-carboxyethyl)-5-(and -6)carboxyfluorescein acetoxymethyl ester, specific for living cells, and propidium iodide, specific for dead cells. Cell staining with the dyes is measured throughout the tissue block by confocal scanning fluorescence microscopy. The dose-response to three agents--ethanol, doxorubicin, and sodium hypochlorite--is shown and, in the case of sodium hypochlorite, compared to in vivo skin toxicity with a high correlation. We also demonstrate that the end point of [3H]thymidine incorporation measured by histological autoradiography can be used to measure toxicity. Our results with the [3H]thymidine end point demonstrate that the hair follicle cells are the most sensitive to doxorubicin. The native-state model for skin may be an effective replacement for animal systems and superior to the dispersed skin cell systems used previously. It can allow rapid, inexpensive measurements of the effect of manufactured products, drugs, and pollutants on skin.
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- Freeman A. E., Hoffman R. M. In vivo-like growth of human tumors in vitro. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2694–2698. doi: 10.1073/pnas.83.8.2694. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldberg M. T., Tackaberry L. E., Hardy M. H., Noseworthy J. H. Nuclear aberrations in hair follicle cells of patients receiving cyclophosphamide. A possible in vivo assay for human exposure to genotoxic agents. Arch Toxicol. 1990;64(2):116–121. doi: 10.1007/BF01974396. [DOI] [PubMed] [Google Scholar]
- Hoffman R. M., Connors K. M., Meerson-Monosov A. Z., Herrera H., Price J. H. A general native-state method for determination of proliferation capacity of human normal and tumor tissues in vitro. Proc Natl Acad Sci U S A. 1989 Mar;86(6):2013–2017. doi: 10.1073/pnas.86.6.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kao J., Patterson F. K., Hall J. Skin penetration and metabolism of topically applied chemicals in six mammalian species, including man: an in vitro study with benzo[a]pyrene and testosterone. Toxicol Appl Pharmacol. 1985 Dec;81(3 Pt 1):502–516. doi: 10.1016/0041-008x(85)90421-1. [DOI] [PubMed] [Google Scholar]
- LEIGHTON J. A sponge matrix method for tissue culture; formation of organized aggregates of cells in vitro. J Natl Cancer Inst. 1951 Dec;12(3):545–561. [PubMed] [Google Scholar]
- Vescio R. A., Connors K. M., Bordin G. M., Robb J. A., Youngkin T., Umbreit J. N., Hoffman R. M. The distinction of small cell and non-small cell lung cancer by growth in native-state histoculture. Cancer Res. 1990 Sep 15;50(18):6095–6099. [PubMed] [Google Scholar]
- Vescio R. A., Connors K. M., Youngkin T., Bordin G. M., Robb J. A., Umbreit J. N., Hoffman R. M. Cancer biology for individualized therapy: correlation of growth fraction index in native-state histoculture with tumor grade and stage. Proc Natl Acad Sci U S A. 1990 Jan;87(2):691–695. doi: 10.1073/pnas.87.2.691. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vescio R. A., Redfern C. H., Nelson T. J., Ugoretz S., Stern P. H., Hoffman R. M. In vivo-like drug responses of human tumors growing in three-dimensional gel-supported primary culture. Proc Natl Acad Sci U S A. 1987 Jul;84(14):5029–5033. doi: 10.1073/pnas.84.14.5029. [DOI] [PMC free article] [PubMed] [Google Scholar]