Culturing explanted colon crypts highly improves viability of primary non-transformed human colon epithelial cells.

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.

Butyrate suppresses mRNA increase of osteopontin and cyclooxygenase-2 in human colon tumor tissue.

The short chain fatty acid (SCFA) butyrate, a product of fermentation of dietary fiber in the human colon, is found to exert multiple regulatory processes in colon carcinogenesis. The aim of this study was to find out whether butyrate affects the tumor-promoting genes osteopontin (OPN) and cyclooxygenase (COX)-2, their respective proteins and/or their functional activity in matched normal, adenoma and tumor colon tissues obtained from 20 individuals at colon cancer surgery. Quantitative real-time polymerase chain reaction experiments showed increased levels of OPN and COX-2 messenger RNA in tumor tissues when compared with the adjacent normal samples (P < 0.001). The addition of butyrate reduced OPN and COX-2 mRNA expression in all tissue types compared with the related medium controls (tumor: P < 0.05). In tumor samples, a downregulation of up to median 35% (COX-2) and 50% (OPN) was observed, respectively. Thereby, tumors with lower levels of OPN basal expression were more sensitive to inhibition and vice versa for COX-2 in normal tissue. At the protein and enzyme level, which were determined by using western blot and enzyme immunometric assays, the impact of the SCFA was not clearly visible anymore. The active proteins of OPN and COX-2 (determined by prostaglandin E(2)) were found to correlate with their respective mRNA expression only in 50-63% of analyzed donors. For the first time, our data reveal new insights into the chemoprotective potential of butyrate by showing the suppression of OPN and COX-2 mRNA in primary human colon tissue with the strongest effects observed in tumors.