Effect Of Elastic Modulus Of Gelatin/nanofiber Composite Scaffold On 3D Intestinal Epithelium Cell Monolayer Model And The Assessment Of Probiotics Immune Functions

Intestinal epithelium is the important place for the digestion and absorption of nutrients and the interaction with intestinal flora.Since the successful culture of mouse small intestinal organoids reported by Hans Clevers in 2009,the understanding of intestinal stem cell physiology has greatly enhanced and enabled the generation of complex intestinal models in vitro.However,the intestinal epithelium of 3D organoid is enclosed inside the spherical structure,making it difficult to access.It is necessary to construct 3D intestinal epithelium model with an open lumen.The most important thing is to prepare scaffolds promoting intestinal epithelial cells proliferation and differentiation and to gain in vitro 3D intestinal epithelial cell model with cell composition function close to living tissue.In this work,gelatin/nanofiber composite porous scaffolds were prepared with biomimetic physical and chemical structures of crypts(gelatin chemical composition,nanofibrous network structure and crypt-like porous structure).Intestinal crypts grew and differentiated on gelatin/nanofiber composite scaffolds,forming a fully differentiated and expressed 3D intestinal cell monolayer in vitro.With the constructed 3D intestinal cell monolayer model,the roles of pathogens and probiotics in intestinal inflammation were evaluated and the potency of this model to be used as in vitro intestinal epithelium immunoreaction assessment tool was verified.The main results are as follows:1.Gelatin(Gel)was composited with electrospun polylactic acid nanofibers or oxidized bacterial cellulose nanofibers(TOBC)to prepare porous scaffolds with uniform pore distribution,suitable pore size,and nanofibrous networks bio-mimicing collagen nanofibers in extracellular matrix.After EDC/NHS crosslinking,Gel/TOBC composite scaffolds had better water stabilities and higher elastic modulus.2.Replacing EDC/NHS crosslinking with crosslinked by Maillard reaction,the elastic modulus of Gel/TOBC composite scaffolds were further increased.With 20 wt% glucose content,Gel/TOBC composite scaffold had the best water stability(weight loss rate <5%),the highest porosity(88.30±0.79%),and the highest elastic modulus(99.71±5.58 KPa).The results of Caco-2 cell culture demonstrated that Gel/TOBC composite scaffolds with Maillard reaction crosslinking had no cytotoxicity and good cytocompatibility.The increased elastic modulus could promote Caco-2 cells proliferation on Gel/TOBC composite scaffolds.3.Compared with the traditional polycarbonate Transwell,Gel/TOBC composite scaffolds crosslinked by Maillard reaction was suitable for proliferation and differentiation of primary intestinal cells.Gel/TOBC composite scaffolds with high elastic modulus facilitated intestinal cell differentiation and the gene expression(including ZO-1,SI,Mucin-2,VILLIN,CHGA and Lgr5)and protein expression(Mucin-2,CHGA and SI)related to cell differentiation were closer to original organization than Gel/TOBC composite scaffolds with low elastic modulus.4.In vitro 3D intestinal cell monolayer model based on Gel/TOBC composite scaffolds could be used as an analytical tool to evaluate the immunoreaction of intestinal epithelium.The roles of probiotics in inhibiting the intestinal inflammatory caused by Escherichia coli infection were assessed.The results showed that probiotics significantly inhibited the increase of TNF-α expression in primary intestinal cells caused by E.coli,and enhanced IL-10 expression.In addition,probiotics protected intestinal cells from E.coli induced apoptosis of intestinal epithelial cells.