The small intestine is lined by a monolayer of polarized epithelial cells organized in protrusions called villi and invaginations named crypts. The intestinal epithelium is completely replaced every 3–6 days, making this tissue the most actively self-renewing tissue in the body. Epithelial intestine regeneration is driven by a pool of stem cells localized at the crypt base. Stem cell division gives rise to transit amplifying cells that proliferate and differentiate into absorptive and secretory cells while migrate up to the villi tip where undergo apoptosis and exfoliate. Indirect evidence has shown that the processes of cell proliferation, differentiation, migration and positioning are driven by specific protein gradients. However, direct experimental manipulation of these gradients and the concentration range that promote these functions has been hampered by limitations of the in vitro systems available. Nowadays the gold standard tissue culture method for in vitro primary intestinal epithelial cells is the Matrigel®-base intestinal organoids culture. Intestinal organoids are 3D structures which recapitulates some of the tissue functionality in vivo such as distinct stem cell/differentiated cell compartments and a central lumen region. Although intestinal organoids culture has been a crucial advance in the in vitro study of the intestine biology, their spheroidal structures make the luminal compartment inaccessible limiting their use and culture of organoids embedded within 3D Matrigel® challenges to data gathering. Therefore, new intestinal stem cell culture methods that allows a more accurate study of the events regulating intestinal epithelial cells proliferation, differentiation, migration and positioning are necessary.
In the current work, we report the development of an in vitro setup to investigate the effects of protein gradients on intestinal stem cells proliferation positioning and differentiation. The experimental setup is a 2D culture platform that allows the patterning of a protein gradient and the culture of single cells coming from organoids. We believe our platform will contribute to understand more accurately the effect of specific protein gradients/concentrations to the primary intestinal epithelial and stem cells behavior.