Summary
Techniques are described for the dissociation, fractionation through Percoll, and in vitro maintenance of mucosal epithelia from the human or guinea pig large bowel. Tissue recovered after surgery is predigested with trypsin-citrate and treated subsequently with a mixture of trypsin, citrate, and collagenase. The resulting suspension of single cells, cell clusters, and partially digested crypts is suspended over a Percoll solution and enriched in multicellular elements by two sequential centrifugations. The recovered multicellular complexes are inoculated to specially treated culture vessels in a serum-free medium supplemented with epidermal growth factor, insulin, transferrin, selenium, and bovine pituitary extract. Epithelia, characterized as such by transmission electron microscopy, adhere to the substrate, form colonies, and can be maintained routinely for study for at least 10 wk.
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Thompson, A.A., Dilworth, S. & Hay, R.J. Isolation and culture of colonic epithelial cells in serum-free medium. Journal of Tissue Culture Methods 9, 117–122 (1985). https://doi.org/10.1007/BF01797782
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DOI: https://doi.org/10.1007/BF01797782