Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays

Abstract

Objective The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualized medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (i.e., barrier function and host-microbial interactions). Design We created a large panel of human gastrointestinal epithelial cell lines (n = 65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays. Results We obtained epithelial lines from all accessible tissue sites within two weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3–1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarized monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli. Conclusion This culture system will facilitate the study of inter-individual, functional studies of human intestinal epithelial cells, including host-microbial interactions.

Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival

“…To confirm fasting-mediated protection of SI stem cells in the absence of tamoxifen-mediated lineage tracing, we monitored the viability of SI stem cells in fed and fasted mice following etoposide treatment by establishing cultures of fast-cycling, Lgr5 + stem cellenriched epithelial spheroids from treated mice (19,20). The culture conditions used conditioned medium containing Wnt3a, R-spondin 3, and Noggin to support the growth of mouse intestinal epithelial spheroids that are enriched for stem cells (20)(21)(22). Under these culture conditions, stem cells do not differentiate into the organoid/bud-like structures seen using protocols published by others (23).…”

Modeling infectious diseases and host-microbe interactions in gastrointestinal organoids

“…Specifically, organoids may provide a system that is able to address if an intrinsic defect in the intestinal epithelium contributes to disease pathogenesis. Recently, a biobank of organoids has been established from samples taken from throughout the gastrointestinal tract of IBD patients (VanDussen et al, 2015). Organoids from IBD patients have been sequenced and the results have confirmed known risk factors .…”

“…This and other engineered tissues may pose more complex models for infection biology in the future. On the other side of the spectrum, infection biologists may prefer to simplify the model and use organoids merely as a source of 2D culture material, rendering the addition of microorganisms to the medium on top of an epithelial cell layer more convenient than microinjection Schlaermann et al, 2016;VanDussen et al, 2015). Recently, a breakthrough was achieved by using 2D cultures from ASC-derived intestinal organoids: after decades of failed attempts to culture human norovirus in epithelial cell lines, Ettayebi and colleagues demonstrated that the virus can infect organoid-derived enterocytes .…”

“…Recently, a breakthrough was achieved by using 2D cultures from ASC-derived intestinal organoids: after decades of failed attempts to culture human norovirus in epithelial cell lines, Ettayebi and colleagues demonstrated that the virus can infect organoid-derived enterocytes . For this, ASC-derived organoids were cultured according to standard methods (Sato et al, 2011), then disrupted into single cells and seeded in 2D layers (VanDussen et al 2015). Fecal samples collected from patients harboring norovirus were filtered and added to the cells for one hour.…”

“…Genome wide association studies (GWAS) have identified risk factors such as cytokines and pattern recognition receptors (reviewed in Wroblewski et al, 2010), but the mechanism underlying susceptibility is still unclear. Uniquely, organoids can be grown from seemingly every patient, which has allowed the generation of "living biobanks" amenable to phenotypic or drug screens (Fujii et al, 2016;van de Wetering et al, 2015;VanDussen et al, 2015). Such biobanks will enable researchers to identify and better understand risk factors in infection-associated pathologies such as gastric cancer and IBD, eventually paving the way for personalized treatments.…”