Construct On Of Caco-2 Cell Model Based On Electrospun Nanofiber Membranes And Absorption Mechanism Of Quercetin

Caco-2 cell is a kind of human colon adenocarcinoma cell with good homology and strong vitality.After culture,this kind of cell can form a continuous cell monolayer and differentiate into a state similar to that of mature small intestinal epithelial cells in morphology and function.Therefore,Caco-2 cells are widely used in the construction of intestinal models in vitro.Due to the attachment-dependent property of Caco-2 cells,monolayer culture model is one of the most widely used Caco-2 cell models in vitro.Till now,Caco-2 cell monolayer cultured on the inserted semi-permeable membrane in the traditional Transwell is the most widely used intestinal models in vitro.However,the cell culture time in this model is as long as 21 days.Besides,there are some other problems such as complex operation,high cost and low screening flux.In order to construct a new Caco-2 cell model to study absorption and transport of functional components in gut,this work intends to use nanofiber membrane as the culture material of Caco-2 cells to construct an intestinal model in vitro.It was hoped that Caco-2 cells cultured on the electrospun nanofiber membranes can fully express the unique physiological function of intestinal epithelium to predict the oral absorption behavior of nutriments and drugs by using in vitro cell culture technology and based on the application advantages of nanofiber membrane in tissue engineering.For this purpose,the electrospun polylactic acid nanofiber membrane suitable for Caco-2 cell attachment and growth was first screened out from four kings of electrospun nanofiber membranes.The morphology and permeation experiments of Caco-2 cells in the Caco-2 cell model based on electrospun polylactic acid nanofiber membranes showed that Caco-2 cell can form a tight cell monolayer after 4 days of culture.The integrity of monolayer cells showed that Caco-2 cells had already differentiated and matured after 7 days of culture.The activity of alkaline phosphatase and P-glycoprotein of Caco-2 cells after 9 days of culture in the model with electrospun nanofiber membranes was higher than that of Caco-2 cells after 21 days of culture in the traditional Transwell model.The culture period of this modified model is much shorter than that of the traditional culture model.Subsequently,the Caco-2 cell model based on electrospun polylactic acid nanofiber membrane was used to predict quercetin absorption.The result showed that quercetin transport across membrane was pH-dependent.But it was necessary to verify whether quercetin was the substrate of P-gp and whether there was efflux effect by adding P-gp inhibitor in the model.At the same time,it was demonstrated that the transport mechanism of quercetin across the Caco-2 cell monolayer membrane at high concentration is mainly passive diffusion.In general,the Caco-2 cell model based on electrospun polylactic acid nanofiber membrane can form a complete and compact cell fusion monolayer within 4 days,and the model can be used for absorption experiments after being cultured for 7 days.Therefore,the Caco-2 cell model based on electrospun polyactic acid nanofiber membrane can be constructed in a short period and has the ability to evaluate the absorption of nutrients and drug components in the small intestine in vitro.