Long-term culture of intestinal tissue-derived epithelial cells became possible in 2009 following a report from the Hans Clevers laboratory that established the critical culture conditions (Sato et al. Nature, 2009). Major advantages of this technique are that non-transformed, non-tumorigenic epithelial cell lines can be established and studied. Since then, we (and others) have adapted the method by Sato et al. in a variety of ways to probe epithelial cell function.
We use conditioned medium from the L-WRN cell line, engineered by the Stappenbeck laboratory (Miyoshi et al. Science, 2012; Miyoshi & Stappenbeck. Nat Protoc, 2013), to support the long-term growth of human and mouse tissue-derived epithelial stem cells as spheroids in a 3D matrix (Miyoshi & Stappenbeck. Nat Protoc, 2013; VanDussen et al. Gut, 2015). The L-WRN conditioned medium has reproducible stem cell-supporting activity from batch-to-batch (VanDussen et al. Stem Cell Res, 2019) and the resulting spheroids contain nearly all proliferating cells (Miyoshi et al. Science, 2012). Because of this, we are able to rapidly expand the number of intestinal epithelial cells to facilitate patient-based assays (VanDussen et al. Gut, 2015). Spheroid epithelial cell lines can be established from intestinal biopsy tissues collected during routine endoscopy procedures and stored long-term in biobanks from essentially any individual, including Crohn’s disease patients. These cell lines hold great promise for use in personalized medicine approaches.
With the L-WRN conditioned medium spheroid system, we can investigate epithelial cells grown on a variety of platforms and in a variety of cell states. Epithelial cells can be grown in a 2D monolayer on Transwell membranes to provide access to the apical membrane (Moon et al. Mucosal Immunol 2014; VanDussen et al. Gut, 2015); with 3D spheroids, the apical membrane faces the inner lumen and therefore is difficult to access. We can also induce the spheroid epithelial cells to differentiate to post-mitotic wound-associated epithelial cell and enterocyte lineages by altering the medium composition (Miyoshi & VanDussen et al. EMBO J, 2017). Importantly for us, the spheroid enterocytes develop a mature microvilli brush border in 48 hours.