3D Printed Microfluidic Chips For Construction Of 3D Cell Model

The three-dimensional cell culture model,which can simulate tissue function,has great potential in predicting the biological reactions such as cell growth,proliferation,migration,metabolism and the occurrence and development of diseases in the real microenvironment.It provides the possibility to study complex physiological and pathophysiological processes in vitro.However,the three-dimensional cell culture system under macro conditions is still difficult to achieve the physiological related screening analysis in life science.Micro-fabrication technologies and microfluidic system were used to creatively design and make “organ on chips” that support tissue differentiation and recapitulate tissue-tissue interface,and provide the spatial-temporal distribution gradient of substances simulating physiological conditions and the microenvironment around living organs,allowing the development of new three-dimensional cell disease models in specific tissue organ microenvironment,and studying the physiological and pathological evolution closer to the actual situation of human body.In view of this,three kinds of personalized,novel microfluidic devices were prepared by one-step 3D printing technology in this paper.On these chips,liver cancer cells,neural cells and bone marrow mesenchymal stem cells were used as seed cells to construct three-dimensional cell culture models.Combining with enzyme-linked immunosorbent technology or electrochemical biosensor technology,the response of cells to drug stimulation in threedimensional microenvironment was monitored.The potential application fields of threedimensional cell model under microsystem are expanded,which is helpful to develop other more accurate three-dimensional cell model for biomedical research.The content of this paper mainly includes the following three parts:Part I: Construction and intervention of insulin resistance in vitro cell model on concentration gradient chipIn this chapter,an open microfluidic chip with controllable concentration gradient was designed,which used the capillary action of ?-cellulose to realize the mixing of substances and the formation of concentration gradient.At the same time,a threedimensional cell culture area was set up on the chip.Therefor,a high-throughput drug screening platform for three-dimensional cell culture system was successfully constructed.In order to achieve a relatively stable concentration gradient distribution,the performance of 3D printing supplies and the morphology of ?-cellulose were characterized,respectively.Results of colorimetric experiment showed that the open chip achieved the formation of concentration gradient without external pump and had good reproducibility.On the basis of this study,Hep G2 cell was used as the research object for the construction of insulin resistance three-dimensional cell model in vitro.And agarose hydrogel was used as a three-dimensional scaffold to simulate the microenvironment of cell growth on the platform.The insulin sensitivity and glucose consumption rate of insulin resistant cell models under the intervention of pioglitazone were investigated and were supported by cellular active oxygen imaging experiments.The results implied that the cell response to external stimuli such as insulin and pioglitazone was similar in tendency and different in degree between 2D monolayer cell culture and 3D cell culture on the chip.Pioglitazone could slightly improve the insulin sensitivity of insulin resistance cell models in threedimensional culture,far less than that in monolayer culture under the same conditions.Part II: Construction and intervention of the in vitro cell model of Alzheimer's disease on PLA-paper integrated chipIn this chapter,we used 3D printing and paraffin printing technology to fabricate a robust PLA-paper hybrid chip,and build an electrochemical integrated three-dimensional cell culture platform with electrochemical biosensor.The platform was used to construct PC12 three-dimensional cell injury models induced by ?-amyloid protein,donepezil and bone marrow mesenchymal stem cells(BMSCs).Dopamine level was monitored in realtime to evaluate cell activity via electrochemical sensors.Compared with PC12 cells and BMSCs grown on two-dimensional interface,PC12 cells and BMSCs seeded on PLApaper integrated chip had the characteristics of uniform distribution,good adhesion,good proliferation and reduced roundness,which proved that the proposed chip can be used for long-term cell culture and complex operation.PC12 cells cultured on the paper scaffolds were divided into five groups including control group,AD model group,donepezil intervention group,BMSCs supernatant intervention group and BMSCs intervention group.After treatment with various drugs,the dopamine secretion level of the five groups for model cells was detected with nano-gold modified screen printing electrodes to obtain the cell survival rate of each group.The results showed that the donepezil,BMSCs supernatant and BMSCs had a strong protective effect on the cell damage induced by ?-amyloid oligomer,and could significantly improve the cell viability.It is worth noting that different types of paper-based scaffolds,such as filter paper and printing paper,will lead to different positive reactions of cells to drugs.Part III: Preparation of bone marrow mesenchymal stem cell microspheres with fused deposition microchamber array chipIn this chapter,a series of microchamber arrays with different shapes and sizes were printed for the preparation of cell microspheres.The chips were ingeniously designed using the printing characteristics of the fused deposition modeling 3D printer in which the filling aperture of the three-dimensional structure was directly determined by the infill percentage.The key factors affecting the aperture of chip,such as geometry and size of the chip,layer thickness and printing materials and centrifugal conditions of cell capture were investigated.The two 3D printers from different manufacturers were compared,implying that the key factors affecting the chip aperture of printers from different manufacturers were different which suggested that researchers must optimize the printing parameters of printers to achieve the best modeling effect.Furthermore,cell microspheres with the gradient volume was successfully obtained by 3D printed gradient size microchamber array chips through centrifugation.