Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells

Abstract

Background & AimsThree-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D) tissue cultured from primary colon cells has not been accomplished.MethodsThe surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified.ResultsThe monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells.ConclusionsThis study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies.

Congruence of location-specific transcriptional programs in intestinal organoids during long-term culture

“…Furthermore, porcine intestinal physiology is considered to closely resemble that of humans increasing the potential for translation of results to humans. Moreover, recent progress in developing robust methods for generating 2D monolayers from 3D organoid cultures facilitates apical exposure to test substances and also opens up possibilities for studying epithelial transport [11,42].…”

Organoids – New Models for Host–Helminth Interactions

“…New engineering-based approaches for control over organoid composition, size, spatial organization, luminal accessibility, chemical gradients, and biomechanical forces are being developed [1,3,15,53,57,58]. These include 3D-bioprinting, chemically programmed assembly and defined ECM, microwell arrays, droplet-based microfluidics, peristaltic pumps, and bioreactors [3,15,16,58,59]. These approaches offer increasing control over physiological conditions and will enable a better understanding of how tissue architecture influences the cellular responses during helminth infection.…”

“…Co-cultures of induced PSC-derived organoids and other cells, such as vascular and neural cell types, to simulate embryonic organogenesis, results in their own 3D self-organization promoting higher order function [53]. However, for organoids of other origin, permeable supports/transwell systems and novel microengineered culture scaffolds have the potential to increase the complexity of the organoid models by allowing stromal cells to be added, such as fibroblasts and nerves, as separate matrix layers in a sandwich-style model [6,58,59,65,67]. These systems also permit the creation of air and nutrient gradients and the co-culture with immune cells to address their interactions with intestinal epithelial cells during infection (Figure 1).…”

Rotational 3D mechanogenomic Turing patterns of human colon Caco-2 cells during differentiation

“…The colon lumen pattern looks similar to self-organizing spots under the skin, morphogenetic feedback mechanisms are suggested to regulate the topology architecture of tissues [8, 77]. One may speculate that application of mechanobiology culture methods to isolated human colon crypts maintained in a matrix environment will replace the 2D Caco-2 cell model [78]. We anticipate that application of the programmable “gut-on-a-chip” mechanobiology culture methodology to 2D cultures will demonstrate epithelium morphology textures similar to the human colon lumen surface with functional microbiome, this system can be used to study the topology of colon epithelium in response to morphogens including FGF2/glucocorticoids/microbiome etc.…”