Construction And Application Of In Vitro Intestinal Model Based On The Patterned Electrospun Nanofiber Membranes

In all the in vitro intestinal model,Caco-2 cell model is the most used and classic model.However,Caco-2,a human colon carcinoma cell line,is quite distinct from healthy intestinal epithelial cells.For this reason,intestinal epithelial stem cells were isolated from the intestinal tract and cultured in vitro to form organoids which reproduce natural intestinal epithelium to in vitro mimic the physiological functions of intestine.However,intestinal epithelial stem cells are difficult to separate and the culture cost is very high.In addition,the closed structures make organoids be not easy to carry out the experiment of absorption,transport,and interaction.Therefore,We need to develop the in vitro intestinal model from 3D organoids model to 2D intestinal interface model.By controlling the fluids on both sides of the 2D intestinal interface model,this kind of model could mimic the real intestinal tract better.Compared with3 D organoids model,the 2D intestinal interface model will be more suitable to study the intestinal physiological phenomenon including the adsorption,transformation,and interaction of nutrients,drugs and microbes in vitro.In this work,the patterned electrospun polylactic acid nanofiber membranes were used to simulate crypt structure of intestinal epithelium,on which the primary intestinal cells were cultured to construct intestinal model in vitro.The primary intestinal cells formed 2D cell layers on the patterned electrospun polylactic acid nanofiber membranes and fully differentiated.This in vitro intestinal model was used to evaluate the colonization properties of probiotics in intestinal epithelium and the effect of probiotics on the intestinal epithelial cells.The detailed works concentrated on the following three aspects:1.Several kinds of patterned polylactic acid nanofiber membranes were prepared.The most suitable nanofiber membranes were chosen as the scaffold materials for theculture of primary intestinal cells.SEM pictures demonstrated that dense cell layers were formed on the material surfaces after 2-3 days culture and showed high cell vitality.2.Based on the in vitro intestinal model with patterned electrospun polylactic acid nanofiber membranes as the scaffold materials,the influences of culture time and Surface modification condition of Matrigel on the cell counting and vitality on the 2D cell layers were studied.In addition,cell differentiation and gene expression on the membrane surface were also analyzed.Compared with the traditional Transwell,the modified Transwell with patterned electrospun polylactic acid nanofiber membranes is more conducive to the growth and differentiation of the primary intestinal cells.3.The adhesion and colonization performance of Lactobacillus casei(from Yakult),Lactobacillus plantarum,Bifidobacterium infantis and Lactobacillus reuteri were evaluated on the constructed in vitro 2D intestinal interface model.Furthermore,the effects of commercial probiotics containing multiple probiotics on intestinal cell were also investigated.The results showed that Bifidobacterium infantis had a stronger adhesion ability on primary intestinal cell surface than the other three probiotics.The commercial probiotics containing multiple probiotics had little effect on the expression of the duodenal cells,but did on the expression of primary colon cells.Probiotics made the expression of the cultured colon cells closer to the real colon tissue.By contrast,the gene expression composition of Caco-2 cells in the Caco-2 cell model was far different from that of the real intestinal tissue.