Design And Preliminary Study Of A Microfluidics-Based Gut Simulator

The small intestine plays a vital role in digestion,adoption,secretion,and immunity.The gut simulator has been an emerging important tool for intestine research.Microfluidics is a cutting-edge technology broadly adopted in chemistry,physics,biology,and medicine which exhibits prominent merits.The gut simulator is developed based on microfluidics,and it has great potentials for simulating the structure and functions of the small intestine as well as in-depth understanding of host-gut microbiota interactions.Experiment 1:Manufacture of the in vitro gut simulator and microfluidics for cell culture.After printing the designed channel graphics into Filin mask,the channel graphics are etched on the silicon wafer by soft lithography to create a chip mold.The configured PDMS is poured into the mold,after baking,cutting,punching,plasma washer processing,bonding and other steps,the microfluidics can be put into use after sterilization and disinfection.Poly-L-lysine(0.1mg/mL)or collagen ?(0.1mg/mL)was selected to encrust the channel,comparing the results with chips that were not encrusted.At 2,4,6 h,cells were dyed and counted.0.5×106,1.0×106 and 2.0×106/mL were set for testing the influence of inoculation density,living cells were dyed by using acridine orange and the number of that were counted in dl and d2.Then,the cell proliferation rate was calculated under different inoculation density.The main results are as follows:(1)The number of adhesive cells in the Poly-L-lysine group was significantly larger than that in the collagen ? group at 2 and 4 h(P<0.05),and there was no significant difference in the number of adhesive cells between the three groups at 6 h(P>0.05).(2)1.0×106/mL is the appropriate cell seeding density on microfluidic chip.Experiment 2:Functional verification of the gut simulator and construction of the host-microbiota crosstalk model.ZO-1 was verified by immunofluorescence.After the crosstalk between IPEC-J2 cells and the intestinal microorganisms,the microfluidic chip was placed under an inverted fluorescent microscope to observe the planting of Lactobacillus acidophilus and Bacillus subtilis on IPEC-J2 cells.Metabolites and metabolic pathway were analyzed by untargeted metabolomic analysis.Main results are as follows:(1)The image of immunofluorescence shows that ZO-1 can be observed between IPEC-J2 cells after five days,which represents the epithelial barrier formed on microfluidic chip.(2)A large number of living bacteria can be observed in the Lactobacillus acidophilus group,indicating that Lactobacillus acidophilus has a better ability to adhere to IPEC-J2 cells,even under the condition of microflow.However,Bacillus subtilis is less likely to adhere to IPEC-J2 cells.(3)After the analysis of the main components,three groups show obvious differences in both positive and negative ions modes.After classifying the metabolites identified,the metabolites in the positive and negative ion modes are mainly benzene and its derivatives,amino acids,peptides,organic acids,carbohydrates,pyridine,phenols,alcohols,amines,and fatty acids.(4)In the positive ion mode,there are 1540 differential metabolites between the Lactobacillus acidophilus group and the control group,of which 380 differential metabolites are significantly up-regulated and 1160 differential metabolites are significantly down-regulated;there are 1593 differential metabolites between the Bacillus subtilis group and the control,of which 459 differential metabolites are significantly up-regulated,and 1134 differential metabolites are significantly down-regulated.In the negative ion mode,there are 373 differential metabolites between the Lactobacillus acidophilus group and the control group,of which 90 differential metabolites are significantly up-regulated,and 283 differential metabolites are significantly down-regulated;between the Bacillus subtilis group and the control group,there are 394 differential metabolites,of which 105 differential metabolites were significantly up-regulated,and 289 differential metabolites were significantly down-regulated.(5)Based on the KEGG database,most significant differential metabolites of the two microorganisms are the metabolites of amino acid metabolism,the unsaturated fatty acid metabolism,carbohydrate metabolism,vitamin metabolism,lipid metabolism,membrane transport,and signaling pathway.In summary:(1)Poly-L-lysine has a better adhesive growth ability than collagen ?;1.0×106/mL is the appropriate cell seeding density on microfluidic chip.(2)By verifying ZO-1,it is shown that the in vitro gut simulator model has the function of intestinal epithelial barrier.(3)Lactobacillus acidophilus has a better ability to adhere to IPEC-J2 cells,Bacillus subtilis is less likely to adhere to IPEC-J2 cells in the fluid environment.The main metabolites of these two microorganisms are benzene and its derivatives,amino acids,peptides,organic acids,etc.The path of metabolic pathway enrichment with more differential metabolites are the amino acid metabolism and the unsaturated fatty acid metabolism.