Department of Nutritional Toxicology, Institute of Nutrition, Friedrich-Schiller-University Jena, Dornburger Strasse 24, 07743 Jena, Germany.
Toxicol In Vitro. 2012 Feb;26(1):133-41. doi: 10.1016/j.tiv.2011.10.008. Epub 2011 Oct 21.
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 (2-24h). To avoid cells from anoikis, also intact colon crypts were isolated to maintain cell interactions. These crypts were incubated with gut fermentation products (24h) 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 2h to 20%. Intact colon crypts cultured as pellets showed a stable viability up to 24h (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 24h and represents a promising approach to study early events in carcinogenesis and chemoprevention as well as other diseases of the colon.
营养化合物的化学保护作用通常在细胞系中进行研究。由于缺乏关于原代人结肠细胞培养的既定方法，因此使用原代人结肠细胞的研究受到限制。因此，我们优化了从个体供体中分离和培养非转化人上皮细胞的方法，以富集活细胞和足够数量的完整 RNA。分离的上皮细胞被播种在不同包被的细胞培养皿中，并结合几种培养基（2-24 小时）。为了避免细胞发生凋亡，还分离了完整的结肠隐窝以维持细胞间的相互作用。这些隐窝与来自不可消化碳水化合物的肠道发酵产物（24 小时）孵育。在没有包被的（纤连蛋白、层粘连蛋白）细胞培养皿中，分离的上皮细胞没有附着。这些细胞在悬浮液中的数量在 2 小时后已经减少到 20%。作为小球培养的完整结肠隐窝显示出稳定的活力，持续 24 小时（91±4%），并且适合获得足够数量的 RNA。使用适当的细胞培养基培养结肠隐窝可以将肠上皮细胞的寿命从 12 小时延长至 24 小时，并为研究癌变和化学预防以及结肠的其他疾病的早期事件提供了有前途的方法。
