Formation of arrays of planar intestinal crypts possessing a stem/proliferative cell compartment and differentiated cell zone
A method for producing tissue constructs comprising two or more distinct regions, each of which comprises a different cell population or lineage is described. The method involves providing a support substrate or substrate assembly comprising two or more physically distinct regions, wherein the two or more physically distinct regions of the support substrate or substrate assembly are different from each other; and depositing/positioning one or more cells on the support substrate or substrate assembly. The cells can form a continuous monolayer with at least two zones, e.g., a proliferative zone and a nonproliferative zone, that can act as in vitro intestinal models. The models are two-dimensional, thus facilitating rapid and facile imaging Systems comprising the tissue constructs and methods of using the constructs to study the effects of pharmaceuticals, uutraceuticais, and metabolites on intestinal cells are also described.
1 . A method for producing a planar tissue construct comprising a first epithelial cell population and a second epithelial cell population, the first epithelial cell population and the second epithelial cell population each comprising different cell populations, the method comprising:
(a) providing an apparatus, the apparatus comprising:
(i) a basal well comprising a first wall and at least one sidewall extending upwardly from the first wall; and
(ii) a luminal well held within the basal well, the luminal well comprising
(1) a planar second wall comprising a first region traversed by a plurality of microholes and a second region that is not traversed by the plurality of microholes,
(2) a planar porous layer for culture of epithelial cells overlaying the first region and the second region of the planar second wall, to form a first planar porous region and a second planar porous region, respectively, and
(3) at least one sidewall extending upwardly from the planar second wall, wherein the planar second wall comprises reduced porosity as compared to
the planar porous layer;
(b) providing a basal culture media in the basal well and a luminal culture media in the luminal well, wherein the basal culture media is different from the luminal culture media;
(c) generating a gradient of growth factors from the basal culture media that extends from a center of the plurality of microholes outward in a planar manner;
(d) depositing epithelial cells on the first planar porous region and the second planar porous region;
(e) proliferating, differentiating, or both proliferating and differentiating the epithelial cells on the first planar porous region into the first epithelial cell population in response to the growth factors delivered via the plurality of microholes; and
(f) proliferating, differentiating, or both proliferating and differentiating the epithelial cells on the second planar porous region into the second epithelial cell population in response to the luminal culture media,
wherein the first epithelial cell population is different from the second epithelial cell population, and the first epithelial cell population and the second epithelial cell population proliferate, differentiate, or both proliferate and differentiate as a monolayer on the first planar porous region and the second planar porous region, respectively,
thereby producing the planar tissue construct.
2 . The method of claim 1 , wherein the first and second epithelial cell populations comprise primary epithelial cells.
3 . The method of claim 1 , wherein the basal culture media and/or the luminal culture media comprise one or more stimuli, wherein the one or more stimuli are each selected from the group consisting of a drug, a nutraceutical, a signaling molecule, a toxin, an inflammatory mediator, and a microbe-derived compound.
4 . The method of claim 3 , further comprising detecting or determining an effect of the one or more stimuli, optionally wherein the detecting or determining comprises comparing one or more of cell differentiation and cell proliferation after exposure to the one or more stimuli to one or more of cell differentiation and cell proliferation prior to exposure to the one or more stimuli and/or in a comparable tissue construct not exposed to the one or more stimuli.
5 . The method of claim 1 , further comprising providing an expansion media in the basal well and in the luminal well before providing the basal culture media in the basal well and the luminal culture media in the luminal well.
6 . The method of claim 1 , wherein the basal culture media is an expansion media, and the luminal culture media is a differentiation media.
7 . The method of claim 1 , wherein the basal culture media is a differentiation media, and the luminal culture media is an expansion media.