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A method for the isolation and culture of human colonic crypts in collagen gels

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Summary

Little is known concerning the biological factors that control the proliferation of the stem cells of the colonic mucosa. In part this is due to a lack of systems suitable for studying the proliferation of this mucosa in vitro. We describe a simple technique for the isolation of single viable intact crypts which are free of stroma and which can then be cultured for periods of at least 16 d using a collagen gel culture method. This method of crypt isolation was efficient with the mean yield of viable intact crypts being 1.4 ±1.2×104 (\(\bar X\) ± SD) crypts/cm2 of mucosa. In culture, mucosal cells only survived for extended periods when the crypts were cultured in collagen gels over a feeder layer of bovine aortic endothelial cells. Cells containing mucus were present in the cultured crypts at all stages of the culture; however we have not been able to demonstrate alkaline phosphatase activity in these crypts. Studies of DNA synthesis after 7 d in culture, using a 18-h pulse label with bromodeoxyuridine (BUdR) has shown that DNA synthesis, as measured by incorporation of BUdR into nuclei, is still occurring in these cultured crypts.

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Authors and Affiliations

  1. Ludwig Institute for Cancer Besearch, Melbourne Tumor Biology Branch, P. O. Royal Melbourne Hospital, 3050, Victoria, Melbourne, Australia

    Robert H. Whitehead, Anthony Brown & Prithi S. Bhathal

  2. Department of Anatomical Pathology, Royal Melbourne Hospital, 3050, Melbourne, Australia

    Robert H. Whitehead, Anthony Brown & Prithi S. Bhathal

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  1. Robert H. Whitehead
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  2. Anthony Brown
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  3. Prithi S. Bhathal
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Whitehead, R.H., Brown, A. & Bhathal, P.S. A method for the isolation and culture of human colonic crypts in collagen gels. In Vitro Cell Dev Biol 23, 436–442 (1987). https://doi.org/10.1007/BF02623860

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  • DOI: https://doi.org/10.1007/BF02623860

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