On-Chip Construction Of Three-dimensional Cell Culture Models And Their Applications In Biomedicine

Cells are the basic structural and functional units of organisms.It is known that all organisms except viruses are composed of cells.Since cells in any multicellular organisms exist in three-dimensional microenvironment including adjacent cells and extracellular matrix,it is essential to construct three-dimensional cell culture models in basic research,drug screening,tissue engineering and regenerative medicine.In recent years,with the cross-application of materials science,engineeringscience and other disciplines in the field of life sciences,a number of three-dimensional cell culture models based on microfabrication technologies and tissue engineering technologies have been proposed and widely used in cell therapy,drug development and regenerative medicineresearch.However,the current three-dimensional cell culture models still could not meet the needs of biomedical development.In view of this situation,four kinds ofon chip three-dimensional cell culture models based on new methods were proposed for drug screening and tissue engineering in this paper.The main research results are as follows:(1)A high-throughput3 D wound healing model had been constructed to study the three-dimensional growth process of cells in vitro,and a novel high-throughput SU-8 mesh chip had been fabricated based on microfluidic chip fabrication technology.Cells were firstseeded into each mesh of the chip to forma novel hollow 3D cell spheroid model,which was called 3D wound model.With the follow-up culture of the hollow cell spheroid,the cells would spontaneously grow into the 3D middle wound areas.Compared with the traditional two-dimensional wound healing model,this process was called 3D wound healing process in this study,which provided a platform closer to the in vivo microenvironment for the study of cell proliferation in vitro.Then,using endothelial growth factor and arginine vasopressin as model drugs to stimulate the growth of HUVEC and NIH-3T3 cells in the chip,the sensitivity of the platform to drug response was verified.In addition,we had further cultured cancer cell lines in this chip platformsuccessfully,which hadexpanded the potential application fields of the model.(2)We had developed a high-throughput angiogenesis-on-chip model based on the three-dimensional wound healing chip.The structure of SU-8 mesh chipwas first further optimized,then human umbilical vein endothelial cells(HUVEC)were seeded on the lateral surface of each SU-8 mesh to form three-dimensional hollow cell spheroid.After the cells had grown steadily into three-dimensional multi-layer cellspheroid in the SU-8 mesh,collagen was filled into the SU-8 mesh and the cells were embedded in the hydrogel.With the subsequent culture,the cells would sprout to the hydrogel to generate the tendency of angiogenesis.By adding different concentrations of vascular endothelial growth factor to the chip,we validated the sensitivity of the model to drugs that promoting angiogenesis.At the same time,by comparing the single-layer cell culture methods based on the chip platform,we had discussed the effect of 3D multi-layer vascular structure culture on angiogenesis,and had verified the superiority of three-dimensional multi-layer cell culture methods on angiogenesis.(3)We had proposed a method for constructing myocardial tissue engineering based on reduced graphene oxide foamchip.This method took nickel foam as template,and the reduced graphere oxide foam chip was obtainedthrough the redox reaction of nickel foam and graphene oxide solution.The structure,properties and biocompatibility of the reduced graphene oxide foamchipwas first tested to confirm its applicability forconstructing myocardial tissues.Thenwe seeded primary cardiomyocytes into the foam,after long-term culture,we had further confirmed that the foam with good biological functions such as cell adhesion,spreading,tissue activity and beating.In a word,we had demonstrated the potential application of thereduced graphene oxide foamchip in myocardial tissue engineering and heart-on-chipresearchin this chapter.(4)We had exploreda method to fabricate silica-biocomposite vascular chip based on vascular tissues for 3D multi-layer vascular assemblingin vitro.In this method,3D bifurcated vascular tissue in vivo was used as template.Firstly,the tissue was acellularized,and then the vascular chip structure was obtained by silica bioreplication technology.By characterizing the structure of the chip,we proved that it had retained the penetrating structure of vascular tissue,and with complex surface topological structure,which provided a structural basis for cell adhesion and nutrition supply.Then,a layer of HUVEC cells was reassembled on the surface of the vascular chip by collagen adhesion cell method,and induced the formation of new vascular network in collagen hydrogel.In addition,by incubating collagen solution again,we further explored the two-layer cell assembly method based on the principle of collagen adhesion cells,which provided a simple and feasible idea for the construction of multi-layer and three-dimensional tissue structure in vitro.In summary,based on the potential application of three-dimensional cell culture technology in drug screening and tissue engineering in vitro,this paper aims to establish new three-dimensional cell culture models with novel micochip methods,and apply them to high-throughput drug screening and tissue engineering.