Establishment of Human Colonic Epithelial Cells in Long-Term Culture

doi:10.1006/jsre.1996.0323

Journal of Surgical Research

Volume 64, Issue 2, August 1996, Pages 161-169

https://doi.org/10.1006/jsre.1996.0323 Get rights and content

Abstract

Study of normal colonic function is important in understanding the cellular mechanisms of carcinogenesis and other diseases of the colon. However, colonic pathophysiological studies have been limited due to the lack of long-term cultures of normal human colonic epithelial cells. The purpose of the present study was to develop methods of isolating viable human colonic epithelial cells for the establishment of nontransformed colonic epithelial cell lines. Human colonic epithelial cells were isolated from surgically resected normal human colons. We found that the use of a short enzymatic digestion gave a consistently higher number (>90%) of viable human colonic epithelial cells. These isolated colonocytes were grown on plastic, collagen-coated filters, or feeder layers using different media formulations. Those colonocytes from the initial primary cultures that were most “epithelial” in appearance were cloned and passaged to establish long-term cultures of nontransformed human colonic epithelial cells. The epithelial nature and secretory function of these established cell lines were confirmed by morphological criteria (light microscopy, phase contrast microscopy, and electron microscopy). We found that the long-term cultures remained immunopositive to anti-cytokeratin antibodies and immunonegative to anti-vimentin antibodies. Using a soft agar assay we found that the colonocytes did not form colonies, suggesting that the long-term culturing did not cause these cells to become transformed. Under serum-free conditions, we found that epidermal growth factor and transforming growth factor-α were equally potent in their mitogenic effects for these colonocytes. Some of the subcultured cells could be maintained for at least 8 months and still retain their epithelial characteristics. We believe that this methodology will serve as a valuable tool for the isolation and culturing of human colonic epithelial cells for studies of normal and malignant colonic disease processes.

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After 48 h crypts detached and cells around the crypts were getting in suspension. The epithelial nature of the attached monolayer clusters was proven by the specific epithelial antibody pan-cytokeratin (Deveney et al., 1996; Vidrich et al., 1988). The majority of attached cells reacted positive with the antibody and only some cells showed a positive staining for vimentin indicating the presence of fibroblasts or other mesenchymal cells (Mahida et al., 1997).
Chemoprotective effects of nutritional compounds are usually studied in cell lines. Studies using primary human colon cells have been limited due to the lack of established methods regarding their culture. We therefore optimized isolation and culture of non-transformed human epithelial cells from individual donors to enrich viable cells and sufficient amounts of intact RNA. Isolated epithelial cells were seeded in different coated cell culture dishes combined with several media (2–24 h). To avoid cells from anoikis, also intact colon crypts were isolated to maintain cell interactions. These crypts were incubated with gut fermentation products (24 h) derived from indigestible carbohydrates. In none of the coated (fibronectin, laminin) cell culture dishes isolated epithelial cells did attach. The number of these cells remaining in suspension, decreased already after 2 h to 20%. Intact colon crypts cultured as pellets showed a stable viability up to 24 h (91 ± 4%) and were suitable to gain a sufficient quantity of RNA. The use of colon crypts with an appropriate cell culture medium could double the lifespan of intestinal epithelial cells from 12 up to 24 h and represents a promising approach to study early events in carcinogenesis and chemoprevention as well as other diseases of the colon.
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^☆: Presented at the Annual Meeting of the Association for Academic Surgery, Dearborn, Michigan, November 8–11, 1995.

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Regular ArticleEstablishment of Human Colonic Epithelial Cells in Long-Term Culture☆

Abstract

Regular Article
Establishment of Human Colonic Epithelial Cells in Long-Term Culture☆