cell sorting and 3) significantly expand organoids as either enriched for stem cells or as differentiated cells that retain a site-specific program of differentiation and are free from mesenchymal contamination. Here we present an improved isolation and culture method that addresses these needs. METHOD: Human gastrointestinal epithelial cells were cultured using an adaptation of our published mouse epithelium culturing system. This system utilizes conditioned media collected from an L cell line (L-WRN) engineered to secrete the factors required for epithelial stem cell growth (Wnt3a, R-spondin3 and Noggin). Importantly, cultures were established from biopsy tissue (2-3 biopsies with area of 6-24 mm2) collected during routine endoscopy. Cell sorting enrichment was not required. Epithelial cells were maintained as 3-dimensional organoids in basement membrane matrix (Matrigel) with 50% conditioned media (1:1 dilution with fresh crypt culture media). Analysis of cellular markers of proliferative and differentiated cells was performed using qRT-PCR, immunofluorescence and flow assisted cell sorting. RESULTS: Human epithelial cultures were established from multiple regions of the gastrointestinal tract, including small intestine (n=9), colon (n=23), stomach (n=2) and Barrett's esophagus (n=2). Biopsy donors included healthy and IBD patients. Mesenchymal cells carried over from the isolation process were eliminated during initial passages. Subsequent studies focused on intestinal organoids, which were maintained for.40 passages (.120 days) and were able to be stored as frozen stocks. In 50% conditioned media, the majority of cells were proliferative and expressed high levels of the stem cell markers, LGR5 and OLFM4. Accordingly, organoids expanded at a rapid rate and were split 1:3 every 3 days. An intestine-specific program of differentiation was achieved using lower percentage conditioned media plus additional factors, including the Notch inhibitor DAPT. CONCLUSIONS:We have generated a culture system that will allow for large-scale, functional experiments of human intestinal epithelial cells. This system will enable us to create a biobank of human epithelial cell lines. Moreover, because cultures can be established from endoscopic biopsies, tissue donors could potentially be pre-selected based on genotype to facilitate studies of the interactions between genotype and environmental factors.