3D stromal tissue equivalent affects intestinal epithelium morphogenesis in vitro

Abstract

Current in vitro models of human intestine commonly fail to mimic the complex intestinal functions and features required for drug development and disease research. Here, we deeply investigate the interaction existing between epithelium and the underneath stroma, and its role in the epithelium morphogenesis. We cultured human intestinal subepithelial myofibroblasts (ISEMFs) in two different 3D configurations: 3D-collagen gel equivalent (3D-CGE) and 3D cell-synthetized stromal equivalent (3D-CSSE). The 3D-CGEs were obtained by means of the traditional collagen-based cell technique and the 3D-CSSE were obtained by bottom-up tissue engineering strategy. The biophysical properties of both 3D models with regard to cell growth and composition (via histological analysis, immunofluorescence, and multiphoton imaging) were assessed. Then, human colorectal adenocarcinoma cell line (CaCo-2) was cultured on both the 3D constructs in order to produce the intestinal model. We identified higher levels of matrix-associated proteins from ISEMFs cultured in 3D-CSSE compared to 3D-CGE. Furthermore, multiphoton investigation revealed differences in the collagen network architecture in both models. At last, the more physiologically relevant stromal environment of the 3D-CSSE drove the CaCo-2 cell differentiation toward the four different type of intestinal epithelial cells (absorptive, mucus-secretory, enteroendocrine, and Paneth) phenotype and promotes, in contrast to the 3D-CGE, the production of the basement membrane. Taken together, these results highlight a fundamental role of the 3D stromal environment in addressing a correct epithelium morphogenesis as well as epithelial-stromal interface establishment.

Intestine‐on‐chip device increases ECM remodeling inducing faster epithelial cell differentiation

“…The TEER values evidenced a significant difference (p < .001) between static culture for 21 days (379 7 22.5 Ohm cm 2 ) and In-OC (357.1 ± 4.44 Ohm cm 2 ) samples, see Figure 5a. The latter presented a value close to the TEER value of native intestinal epithelium (De Gregorio et al, 2018a). The TEER measurements on the 3D HIE at 5 days of static culture were not reported, as they were very low (<50 Ohm cm 2 ), due the absence of contrast to 15-21 days, as reported in the literature (Navabi et al, 2013) and in our experiments.…”

“…The dynamic environment experienced by 3D‐HIE in the In‐OC affects the maturation and remodeling of the endogenous ECM inducing a fast production of the basement membrane as well as the terminal differentiation of the Caco‐2 cells. A bottom up approach was used to fabricate the 3D intestinal stromal equivalents (3D‐ISEs) provided with endogenous ECM obtained by molding the intestinal microtissue precursors (intestine‐µTP) in a maturation chamber, as previously reported (De Gregorio, Imparato, Urciuolo, & Netti, ). The combination of such physiological relevant 3D‐HIE with microfluidic technology, allowed us to qualitatively and quantitatively detect the epithelial polarization and differentiation as well as barrier function and to monitor ECM remodeling during the culture time.…”

“…The dynamic environment experienced by 3D-HIE in the In-OC affects the maturation and remodeling of the endogenous ECM inducing a fast production of the basement membrane as well as the terminal differentiation of the Caco-2 cells. A bottom up approach was used to fabricate the 3D intestinal stromal equivalents (3D-ISEs) provided with endogenous ECM obtained by molding the intestinal microtissue precursors (intestine-µTP) in a maturation chamber, as previously reported (De Gregorio, Imparato, Urciuolo, & Netti, 2018a).…”

A three‐dimensional microfluidized liver system to assess hepatic drug metabolism and hepatotoxicity

“…Among the biomaterials used for this application, gelatin micro-carries acting as microscaffolds are used in many 3D tissue models (Brancato et al, 2017;F Urciuolo et al, 2016;Francesco Urciuolo, Imparato, Totaro, & Netti, 2013). This type of cell support system provides an ECM-like structure to the cells and, when used in combination with bioreactor culture, improves cell yield and promotes cell-matrix interaction (De Gregorio, Imparato, Urciuolo, & Netti, 2017Imparato, Urciuolo, Casale, & Netti, 2013;Katt, Placone, Wong, Xu, & Searson, 2016;Nibourg et al, 2012;Rafiq, Coopman, Nienow, & Hewitt, 2016;Tan et al, 2014). Herein, we provide an effective set up able to recapitulate the key morphological and functional features of the liver by coupling a 3D tissue model with a microfluidic device.…”

Patient-derived small intestinal myofibroblasts direct perfused, physiologically responsive capillary development in a microfluidic Gut-on-a-Chip Model

“…The overarching goal in this study was to develop an ex vivo model of SI angiogenesis, and to incorporate ISEMFs and perfused vasculature into a more physiologically relevant GOC system. ISEMFs have multiple important roles in GI mucosal physiology 13,14,50 . As such, the relevance of their inclusion in ex vivo GI culture systems is inherent.…”