| As an important research technology in the field of life science,the key of cell culture in vitro lies in how to build a cell microenvironment similar to in vivo growth.Cell microenvironment is a three-dimensional ecological environment,which is affected by many dynamic physical and chemical factors.The change of its characteristics directly affects the growth state and behavior of cells.Traditional cell culture in vitro can not reproduce the growth of cells in three-dimensional space.In recent years,microfluidic chip has become an important experimental platform for cell in vitro research because of its advantages of low reagent consumption and high detection efficiency.However,there are few researches on dynamic and variable three-dimensional cell microenvironment,which need to be improved.In this study,a new cell culture microfluidic chip with three-dimensional differential microenvironment is designed.The chip uses a bio-hydrogel material to build three-dimensional scaffolds for micro environment and microstructures are designed to make the hydrogel accurately pattern on the chip.In view of the effect of chip design on the encapsulation performance of hydrogel solution and whether the chip design can meet the requirements of differentially three-dimensional microenvironment,the following studies are carried out in this paper:(1)The structure design of microfluidic chip for differential three-dimensional micro environment.According to the flow principle of liquid in microchannel,the size of microchip is analyzed and designed.According to the patterning effect of the hydrogel solution on the chip is affected by the surface tension and capillary force,the material of the chip is selected and the injection process of the hydrogel solution in the chip is simulated by finite element simulation.The encapsulation effect of the trapezoidal,hexagonal,octagonal,and circular(external circle is 150 ?m)microcolumn is compared and the best circular microcolumn is selected.The three-dimensional modeling and simulation analysis of the differential microenvironment on chip are carried out.The simulation results meet the requirements of chip design.In addition,the stability of the velocity field of the microenvironment in chip is verified by simulation.(2)Chip fabrication and differential microenvironment construction.The soft etching method is used to make the chip and the manufacturing process of SU-8 glue mold is designed with MEMS processing technology.The casting mold is used to complete the production of PDMS structure sheet.Through surface treatment,the whole chip is fabricated by bonding.The three-dimensional microenvironment of different concentrations of small molecules on the chip is verified by the design of the prepolymerized collagen solution encapsulation experiment and the diffusion experiment of small molecules of fluorescein sodium.The collagen solution can be encapsulated in the microcolumn gap to form a stable gas-liquid interface without overflow so as to achieve the purpose of patterning hydrogels on the chip.The molecular motion of fluorescein sodium mimics the diffusion of biochemical factors in the chip.After 2.5 hours,a three-dimensional micro environment with stable distribution of differential molecular concentration of fluorescein sodium can be formed on both sides of the chip.(3)Biological experiment application of the chip.PC12 cells are cultured on the chip in three dimensions.The growth of PC12 cells on the chip results in the growth of pseudo foe,which can migrate through the microcolumn gap and present the trend of growth in a high glucose three-dimensional gel microenvironment with unilateral side channel.The survival rate of cells on the chip is 92.21%.In the three-dimensional micro environment on both sides of the chip,the stable concentration of 46-47 mmol/L high glucose and 14-15 mmol/L low glucose can be formed by continuous measurement of glucose concentration during cell growth,which achieves the design goal of differential cell micro environment. |
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