Strategies for optimizing the isolation and expansion of sensitive patient-derived duodenoids, ileoids and colonoids .pdf
Jay R. Thiagarajah, Katlynn Bugda Gwilt
Abstract
These protocols are an optimization of previous protocols and the original work described in Sato et al., 2009 which provided a foundation for the development of primary duodenoids, ileoids and colonoids from human samples. Here, we optimized methods specifically for use with samples from pediatric patients with genetic intestinal epithelial disorders. These protocols apply specifically to enteroids generated from endoscopic biopsies from patients with Congenital Diarrheas and Enteropathies (CoDE) or Very-Early-Onset Inflammatory Bowel Disease (VEO-IBD) or age-matched patients without any intestinal disease and include samples from the duodenum (duodenoids), ileum (ileoids), and colon (colonoids). The duodenoids, ileoids and colonoids from these patients exhibit a range of cellular phenotypes that potentially impact enteroid growth and maintenance including increased apoptosis, defects in proliferation, polarity and vesicular trafficking. These characteristics make it potentially challenging for long-term culture and expansion, limiting the availability of cells for functional experiments. Using a modified culture media, an expanded initial culture time after cell isolation from patient biopsies, and gentle passaging techniques, we were able to successfully culture and expand several patient lines over multiple passages and maintain the cultures for >5 years. The media conditions and protocols described here allow for reproducible phenotypes as well as scaling for larger functional studies on patient lines. These protocols also provide a useful starting point for further optimization for generating and culturing enteroids from patients with novel disease pathophysiology.
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Dissolve all reagents to the appropriate concentration according to manufacturer’s protocols. A * indicates that there were no modifications to the reagent.
