Facilitate Your Research On Gastrointestinal Epithelium Cells With Organoid Cultures

by | Nov 16, 2021 | Organoids, Stem Cell

The Intestinal Epithelium Organoid Cultures

  • Challenges in establishing long-term systems for intestinal epithelium cultures
  • Establishment of long-term gastrointestinal epithelium organoid culture
  • The use of small molecules in intestinal organoid culture
  • Building of gut organoids
  • Cytokines used in long-term gastrointestinal epithelium culture
  • Small molecules and peptides used in gastrointestinal culture

Challenges in establishing long-term systems for intestinal epithelium cultures

The in vitro studies of gastrointestinal (stomach, small intestinal and colonic) epithelium have 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 the short-term application due to the rapid apoptosis that ails intestinal cells once they have been removed from the basement membrane and underlying stroma.

Establishment of long-term gastrointestinal epithelium organoid culture

1. Intestinal epithelium organoid cultures

  • The ability to source normal epithelial cell lines from the stem cells found in the base of intestinal crypts has allowed for the establishment of long-term 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 [1].
      • 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 mimics normal intestinal epithelial growth and differentiation and can maintain these characteristics for more than eight months.

2. Human small intestinal epithelium and human colonic epithelial organoid cultures

  • Subsequently, a protocol for long-term organoid culturing of the human small intestinal epithelium, and both murine and human colonic epithelia was established.
      • This system added the necessary signalling protein Wnt-3a to the above-mentioned ENR growth factor bundle (WENR).

3. Human small intestinal and colonic crypt organoid cultures

  • In the case of human small intestinal and colonic crypt cultures, the further addition of p38 MAPK inhibitor (SB202190) and TGF-β inhibitor (A 83-01) was required [2].
  • Currently, these protocols are routinely used in studies involving human or mouse intestinal crypt cultures [3, 4].

4. Long-term gastric gland and human small intestinal epithelium organoid cultures

  • 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].

The use of small molecules in intestinal organoid cultures

Small molecules can also be an integral component of intestinal cultures, either as a means of directing culture fate or as a tool for studying signalling pathways.   • Whereas the addition of SB-202190 and A 83-01 can be used to establish long-term primary organoid cultures from human Lrg5+ cells, the addition of Y-27632 to intestinal cell cultures can be used to yield single cell cultures [9,10]. • The GSK3β inhibitor, CHIR 99021, and the Wnt production inhibitor, IWP2, were employed in the study of how Wnt/β-catenin signalling influences the differentiation of Paneth cells [11].

Building of gut organoids

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 extracellular matrix, with the assistance of the ENR growth factor bundle, plus Wnt-3a, Y-27632, Gastrin, Nicotinamide, A 83-01, SB 202190 and LY2157299 [12].

Cytokines used in long-term gastrointestinal epithelium culture

EGF

for human small intestine, colon and stomach epithelium culture
EGF is a potent growth factor that stimulates the proliferation of various epidermal and epithelial cells. It has been shown to inhibit gastric secretion and be involved in wound healing.
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Noggin

for human small intestine, colon and stomach epithelium culture
Noggin belongs to a group of diffusible proteins that bind to ligands of the TGF-β family and regulate their activity by inhibiting their access to signalling receptors                              
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R-Spondin-1

for human small intestine, colon and stomach epithelium culture
R-Spondin-1 is expressed in certain areas of the developing central nervous system, as well as in the adrenal glands, ovary, testis, thyroid, and trachea.                                                                                            
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Wnt-3a

for human small intestine, colon and stomach epithelium culture
Wnt-3a signalling is essential for various morphogenetic events, including embryonic patterning, cell determination, cell proliferation, CNS development, and cytoskeletal formation.   
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FGF-10

for human stomach epithelium culture, optional for human small intestine culture
FGF-10 is a heparin-binding growth factor that belongs to the FGF family. Proteins of this family play a central role during prenatal development, postnatal growth and regeneration of a variety of tissues.
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Small molecules and peptides used in gastrointestinal culture

A-83-01

Potent inhibitor of activin receptor-like kinase (ALK)                                                
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LY2157299

Potent inhibitor of TGF-β receptor type I (ALK5)          
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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 
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CHIR 99021

Selective and potent inhibitor of glycogen synthase kinase 3 (GSK-3) and activator of the WNT pathway      
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Gastrin I

Peptide hormone is responsible for regulating gastric acid secretion and promoting gastric mucosal growth      
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SB202190

Selective and potent inhibitor of p38 MAP Kinases (MAPK)        
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IWP-2

Wnt pathway inhibitor                                                                                                                         
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Nicotinamide

The active form of vitamin B3 and a component of the coenzyme NAD      
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References:

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 9.9: e107814. 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 6.1: 25-36. 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

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