Construction And Application Of Liver/vessel Cell Chips Based On Nonswellable Hydrogel

Cell chips are defined as chip microreactors containing living cells in two-dimensional(2D)or three-dimensional(3D)culture including two categories,microfluidic cell chips and non-fluidic cell chips such as microwell chips and microarray chips.As a soft and wet material,hydrogels have become an ideal choice for cell chip constructions owing to their similar physical properties with human tissues and organs.However,conventional hydrogels swell significantly in aqueous solutions,which greatly impairs their mechanical properties and destroys the chip structure.Based on the above facts,we prepared a nonswellable hydrogel,F127-DA hydrogel at first.It is a cross-linking triblock polymer hydrogel of polyethylene glycol and polypropylene glycol with high mechanical strength and good cell compatibility.Subsequently,a liver-vessel microfluidic chip was constructed using this nonswellable hydrogel to simulate the structure and function of the human liver lobules.Finally,for the evaluation of a complex substance,we simplified the microfluidic chip into a hepatic/vascular spheroid microwell chips and then it was applied as a high throughout screening(HTS)platform to evaluate the heath properties of various Chinese liquors.The results obtained in this thesis are as follows:First,the F127-DA hydrogel was prepared by ultraviolet irradiation,and then we approved that its non-swellability in an aqueous solution at 37? wouldn't be affected by the ionic strength and pH.Moreover,it still maintained good mechanical properties(tensile strength>300 kPa,tensile strain at break>1100%;compressive strength>4.6 MPa,compressive strain at break is>93%)after swelling.And the hydrogel structure remained stable for more than 21 days.The above physical properties made it suitable for the construction of hydrogel microfluidic chips.Therefore,we constructed a liver-vessel microfluidic chip to mimick the hepatic lobule structure with coculture of human hepatoma cells(HepG2)and human hepatic stellate cells(LX-2)in the microwells of the outer channel and monolayer culture of human umbilical vein endothelial cells(IIUVEC)at the bottom of the inner channel.Subsequently,the inner channel was perfused while the outer channel was kept in static condition to simulate the mass transfer of nutrition in the liver through intravascular blood flow.After perfusion for 6 days,HUVEC grew and covered the surface of the inner channel with expression of endothelial function such as vascular endothelial growth factor.And the hepatic spheroids in the outer channel formed within 2 days and maintained high cell viability on the 6th day.What's more,the albumin and urea expression were always higher than those under static condition.Compared with present hepatic spheroid microfluidic chips,this liver-vessel microfluidic chip better simulates the structural characteristics and mass transfer mode in the hepatic lobules,which is expected to be an ideal platform to study the pathophysiology of liver.Secondly,a microwell cell chip based on the nonswellable hydrogel was constructed for HTS.By retaining the hepatic spheroids and removing the perfusion device of the liver-vessel microfluidic chip,we successfully constructed statically cultured spheroid microwell chips of hepatic and endothelial cells.The study revealed that:(1)Coculture of HepG2 and LX-2 and adding collagen could accelerate the formation of uniform hepatic spheroids within 2 days and maintain the structure and function for at least 10 days;(2)Coculture of HUVEC and human fibroblasts promoted the formation of vascular spheroids that expressed endothelial functions such as nitric oxide,PGI2 and TFPI and formed vascular networks.Since each cell chip requires only about 106 cells and is cultured in a 24-well plate statically,it basically meet the requirements of HTS experiments.Finally,the health properties of 4 Chinese liquors and 2 vodkas were evaluated via the hepatic/vascular spheroid microwell chips.It was found that Chinese liquors could cause a decrease in cell viability and was not significantly different from that of ethanol and vodkas,indicating their impairment to human health as an alcoholic beverage However Chinese liquors could also lower the ROS level and reduce the damage of cell functions demonstrating less damage than ethanol and vodkas which was probably due to the abundant bioactive compounds.In summary,the microfluidic and microwell cell chips based on nonswellable hydrogels are expected to have good potential in the study of organ physiology and pathology as well as drug toxicity analysis and food health evaluation.