Facilitate your research on
Gastrointestinal Epithelium Cells with Organoid Cultures
The in vitro studies of gastrointestinal (stomach, small intestinal and colonic) epithelium has long been hampered by the lack of suitable culture systems. Although a variety of organ/organoid and epithelial cell culture models have been developed, most are restricted to short-term application due to the rapid apoptosis that ails intestinal cells once they have been removed from the basement membrane and underlying stroma.
The ability to source normal epithelial cell lines from the stem cells found in the base of intestinal crypts has allowed for the stablishment of longterm systems for intestinal epithelium cultures. This advancement has allowed for the exploitation of stem cells in tissue regenerative therapies, and the development of treatment models targeting degenerative disorders of the digestive tract.
The model for a robust, long-term small intestinal epithelium organoid culture system was developed in 2009 . Lgr5+ stem cells isolated from murine crypts were cultured with ROCK inhibitor (Y-27632) and the ENR growth factor bundle of EGF (Epidermal Growth Factor), Noggin, and R-Spondin-1. This culture system mimicks normal intestinal epithelial growth and differentiation, and is able to maintain these characteristics for more than eight months.
Subsequently, a protocol for long-term organoid culturing of human small intestinal epithelium, and both murine and human colonic epitheliums was established. This system added the necessary signaling protein Wnt-3A to the above mentioned ENR growth factor bundle (WENR).
In the case of human small intestinal and colonic crypt cultures, the further addition of p38 MAPK Inhibitor (SB 202190) and TGF-β inhibitor (A 83-01) was required . Currently, these protocols are routinely used in studies involving human or mouse intestinal crypt cultures [3, 4].
The addition of FGF-10 (Fibroblast Growth Factor-10) to the WENR bundle (WENRF) allowed researchers to establish long-term gastric gland and human small intestinal epithelium cultures [5,6,7,8].
Small molecules can also be an integral component of intestinal cultures, either as a means of directing culture fate or a tool for for studying signaling pathways. The GSK3β inhibitor CHIR99021 and the Wnt production inhibitor IWP-2 were employed in the study of how Wnt/β-catenin signaling influences the differentiation of paneth cells .
The development of a new primary tissue model based on gut organoids has allowed for the retention of physiological characteristics in culture. These gut organoids expanded from healthy, human small intestinal crypts on Matrigel ECM (Extra Cellular Matrix) with the assistance of the ENR growth factor bundle plus Wnt-3A, Y-27632, Gastrin, Nicotinamide, A 83-01, SB 202190 and LY2157299 .
PeproTech, along with the PeproTech Brand BioGems, are proud to be able to support your research by providing you with cytokines and small molecules required for gastrointestinal epithelium cell culture protocols.
|A83-01||Potent inhibitor of activin receptor-like kinase (ALK)|
|CHIR99021||Selective and potent inhibitor of glycogen synthase kinase 3 (GSK-3) and activator of the WNT pathway|
|Gastrin I||Peptide hormone responsible for regulating gastric acid secretion and promoting gastric mucosal growth|
|IWP-2||Wnt pathway inhibitor|
|LY2157299||Potent inhibitor of TGF-β receptor type I (ALK5)|
|Nicotinamide||Active form of vitamin B3 and a component of the coenzyme NAD|
|SB202190||Selective and potent inhibitor of p38 MAP Kinases (MAPK)|
|Y-27632 Dihydrochloride||Selective inhibitor of Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) family of protein kinases that selectively competes with ATP to bind to the catalytic site|
1. Sato, T, et al. (2009) “Single Lgr5 stem cells build crypt villus structures in vitro without a mesenchymal niche.” Nature 459.7244: 262-265.
2. Sato, T, et al. (2011) “Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s
epithelium.” Gastroenterology 141.5: 1762-1772.
3. Schwank, G, et al. (2013) “Generation of BAC transgenic epithelial organoids.” PloS One 8.10: e76871.
4. Grabinger, T, et al. (2014) “Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in
intestinal epithelial cells and enteropathy.” Cell death & disease 5.5: e1228.
5. Lahar, N, et al. (2011) “Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium.”
PLoS One 6.11: e26898.
6. Jabaji, Z, et al. (2014) “Type I collagen as an extracellular matrix for the in vitro growth of human small intestinal epithelium.” PloS One
7. Barker, N, et al. (2010) “Lgr5+ ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.” Cell Stem Cell
8. Stange, D, et al. (2013) “Differentiated Troy+ chief cells act as reserve stem cells to generate all lineages of the stomach epithelium.”
Cell 155.2: 357-368.
9. Koo, B-K, and Clevers, H, (2014) “Stem cells marked by the R-spondin receptor LGR5.” Gastroenterology 147.2: 289-302.
10. Cramer, J, et al. (2015) “Distinct human stem cell populations in small and large intestine.” PloS One 10.3: e0118792.
11. Farin, H. F., Van Es, J. H., & Clevers, H. (2012) “Redundant sources of Wnt regulate intestinal stem cells and promote formation of Paneth
cells.” Gastroenterology 143.6: 1518-1529.
12. Schweinlin, M, et al. (2016). “Development of an advanced primary human in vitro model of the small intestine.” Tissue Engineering
Part C: Methods