Abstract
The Caco-2 monolayer is the most widely used in vitro model of the human intestinal mucosa to study absorption. However, models lack communication from other cells present in the native intestine, such as signals from fibroblasts in the lamina propria. In this study, we have investigated the effects of fibroblasts upon the Caco-2 epithelium through two mechanisms: indirect signaling from fibroblasts and direct contact with fibroblasts. Culture of Caco-2 cells with paracrine signals from fibroblasts, through the use of conditioned media, did not induce a significant change in epithelial cell morphology or function. To examine the effects of direct contact between the epithelium and fibroblasts, we developed novel, humanized three-dimensional (3D) co-culture models whereby Caco-2 cells are grown on the surface of a subepithelial-like tissue construct containing intestinal or dermal fibroblasts. In our models, we observed endogenous extracellular matrix production from the fibroblasts that provides support to the above epithelium. The Caco-2 epithelium displayed morphological changes in 3D co-culture including enhanced polarization and the formation of a basement membrane-like attachment to the underlying stromal compartment. An important structural alteration was the significantly straightened lateral membrane that closely mimics the structure of the in vivo intestinal mucosa. This enhanced lateral membrane phenotype, in correlation with an reduction in TEER to levels more similar to the human intestine, is thought to be responsible for the increased paracellular permeability observed in 3D co-cultures. Our results demonstrate that direct contact between epithelial and mesenchymal cells results in an enhanced epithelial barrier. The in vitro models described herein have the potential to be used for studying intestinal epithelial-fibroblast interactions and could provide more accurate tools for drug permeability studies.
Highlights
The study of intestinal function greatly relies on animal models
CCD-18co cells are one of the few commercially available intestinal fibroblast lineages and are widely used in the literature in co-cultures with Caco-2 cells that mimic the small intestinal function. They were selected as they are derived from the human colon and as such are a close match for Caco-2 cells which are colonic in origin
Dermal fibroblasts are often used as a comparison to intestinal fibroblasts and their non-intestinal origin was used to test the importance of tissue-specific fibroblasts
Summary
With an increasing demand to replace animal use in science, coupled with a lack of availability of ex vivo human tissue, in vitro intestinal alternatives are utilized to understand complex cellular processes such as drug-permeability and toxicity in a simplified format. The current gold-standard in vitro model used extensively throughout academia and industry for over 30 years is the Caco-2 monolayer. This Transwell R -based culture system is one of the most extensively studied in vitro cell models due to its ability to form well-differentiated and polarized cell monolayers as surrogates for the human intestinal epithelium (Hidalgo et al, 1989). The models have many limitations which emanate from its adenocarcinoma origin and the simplicity of a monolayer culture system that lacks the complexity found in human tissue. Other limitations of the monolayer include abnormal cuboidal cell morphology as well as significantly heightened TEER compared to that of the normal human intestine
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
