Construction Of Artificial Porcine Intestine-on-A-Chip Based On Microfluidics

The intestine is the most important organ in the digestive system of animals and is the main place for nutrient digestion and absorption.It is also an important part of immune system and endocrine system and there is two-way communication between it and other organs.So the intestinal health is the foundation of healthy pig breeding.Currently,the main approaches for studying the pig intestine include in vivo research models(animal testing)and in vitro research models(two-dimensional cell culture models and organoid culture models).But both of them have certain limitations.Microfluidics has facilitated the development of in vitro organ simulation technology and provided new ideas for intestinal research.Organ-on-a-chip refers to the construction of organ physiological microenvironment containing complex factors such as multiple living cells,functional tissue interfaces,biofluids and mechanical force stimuli on a microfluidic chip to simulate the main structure and function of tissues and organs,which has the advantages of rich measurement information,small size and realistic simulation of in vivo physiological environment.Therefore,we constructed a 3D hydrogel microfluidic chip with a perfusable intestinal lumen and a crypt-like structure to simulate the three dimensional structure and the microenvironment such as extracellular matrix of intestine and optimized the parameters in the fabrication process in this study.And we cultured IPECJ2 in this chip to construct a bionic porcine artificial intestine verified the permeability barrier function and absorption transport function of this porcine artificial intestine.The main experiments and results are as follows:Experiment 1 Controlled construction of three-dimensional hydrogel microfluidic chipTo construct 3D hydrogel microfluidic chips with perfusable intestinal lumen and cryptlike structures,we have tried three construction methods: laser microdissection,height difference method and gelatin template method.While the height difference method can’t produce complex crypt-like structures,the laser microdissection method and gelatin template method can successfully produce 3D hydrogel scaffold with crypt-like structures.But the laser microdissection method has the limitations of long time consuming and low success rate.So the optimal method is the gelatin template method and its preparation parameters are optimized as follows:(1)In the chip design,the upper PDMS chip was designed to snap to hold the gelatin in place.(2)During the gelatin template fabrication process,vacuum treatment of gelatin chips to facilitate the formation of crypt structures.Treatment of gelatin chips with 2% Pluronic F-127 and treatment of PDMS negatives with 1% PEI and 0.1% GA facilitates the complete release of gelatin templates.Gelatin templates containing 15% gelatin and 150 μm height with the best physical form were selected for the test.(3)The morphology and swelling of the 3D hydrogel scaffold with a solidification time of 3 min for collagen gels was superior to that of the 1 min and 5 min groups.(4)Under optimal conditions,the 3D hydrogel scaffold swells with a swelling ratio of about 1.5 for channel width,1.6 for crypt width,and 1.4 for crypt depth compared to the gelatin template.Experiment 2: Construction of porcine artificial intestine based on 3D hydrogel scaffold and verification functionBased on the three-dimensional hydrogel microfluidic chip constructed by the three methods in Experiment 1,the IPEC-J2 cells were cultured on the microfluidic chip and the cells could adhere,proliferate and grow normally with good activity.The barrier function and absorption-transport function of porcine artificial intestine were verified by immunofluorescence staining of F-actin(actin filaments),determination of permeability coefficient and determination of m RNA levels of tight junction and channel proteins.The main results are as follows:(1)IPEC-J2 cells in porcine artificial intestine can grow into tubular lumen-like channels with crypt-like structures along a three-dimensional hydrogel scaffold.(2)The porcine artificial intestine cultured up to 5 days formed an intestinal barrier function.(3)By comparing cells grown in hydrogel chip(porcine artificial intestine),monolayer chip,and well plate,the m RNA expression of ZO-1 and Occludin genes in hydrogel chip IPEC-J2 cells was not significantly different from that in well plate(P > 0.05),and the m RNA expression of E-cadherin gene was higher than that in monolayer chip(P < 0.05).(4)By comparing the m RNA expression of nutrient transporters in cells grown in hydrogel chip and well plate,the m RNA expression of AQP1,GLUT2 and SLC1A1 in hydrogel chip IPEC-J2 cells was not significantly different from that in well plates,demonstrating that the porcine artificial intestine has absorption and transport function(P >0.05).In summary,the method of constructing three-dimensional hydrogel microfluidic chips with perfusable intestinal lumen ducts and crypt-like structures was explored,and the optimal method was the gelatin template method on balance.IPEC-J2 cells were cultured in the 3D hydrogel scaffold to construct a porcine artificial intestine,and the model had good barrier function and absorption and transport function.Therefore,the construction of porcine artificial intestine model based on gelatin template method can reproduce the barrier function,which is expected to be used to establish damage model and screen repair substances and provide a new in vitro research tool for this porcine intestine research.