Preparation Of Cardiac Decellularized Matrix Sponge And Its Application In The Construction Of Three-dimensional Tumor Model In Vitro

In the field of tumor biology,researchers usually use the culture models in vivo or in vitro to explore the biological characteristics of tumor cells.The traditional research models mainly rely on the animal model in vivo and the two-dimensional culture model in the petri dish.Both of them provide very important experimental data for the study of tumor biology,and they are widely used by many researchers.However,with further research on tumor,researchers have gradually realized that both of them have their own defects and shortcomings.Although the animal model in vivo can provide a three-dimensional microenvironment for tumors growth,there is a big difference between the the experimental bodies,and the immune problems are very complex.Especially for human tumor cells,they often can not survive and grow well in animal models.The two-dimensional culture model is short of extracellular matrix,so that it can not form three-dimensional microenvironment,and can not explore the process of interaction between tumor cells and various factors in microenvironment.Therefore,the development of a new in vitro culture model with three-dimensional microenvironment has become an urgent need of researchers.At present,it is one of the hot topics in the study of three-dimensional construction of tumor tissue model to find three-dimensional scaffold materials which are more bionic in physical,chemical and biological characteristics.The development of decellular technology provides an opportunity for the construction of more bionic three-dimensional tumor tissue.With the maturation of the preparation method of cardiac decellularized matrix,the decellularied matrix exhibits excellent properties such as low sensitivity,near-natural three-dimensional structure and so on in the field of life science,attracting more and more researchers to participate in it.In the past decade,cardiac decellularized scaffolds obtained by decellular strategies have been widely used in many fields.Cardiac decellularized matrix can be processed into different material types according to its different application purposes,such as decellularized matrix hydrogel,decellularized matrix patch,decellularized matrix sponge and so on.The decellularized matrix sponge is formed by freeze-drying of decellularized matrix solution.It is a main form of natural biological scaffold material,with good function and characteristics.For example,spongy materials have sufficient internal surface area to provide adhesion and proliferation of inoculated cells,and rich porous structures are conducive to the transport of nutrients and cellular metabolites.In addition,it has the unique advantages of easy processing,sterilization and preservation,so it is regarded as one of the most promising biomaterials,and has a wide range of scientific research and clinical application value.However,there is no report on the process of decellularized matrix into decellularized matrix sponge,nor has there been any related research on the three-dimensional model construction of lung cancer in vitro which use cardiac decellularized matrix sponge as scaffold material.Based on the above analysis,this paper mainly from the following three aspects of research work:Part 1:Preparation and identification of cardiac decellularized matrixAim:Preparing the porcine cardiac decellularized matrix and detecting and evaluating the structure of cardiac decellularized matrix and the components of extracellular matrix.Methods:The decellularized matrix was obtained from porcine heart by SDS elution based on tissue mass.The removal of cell and nucleic acid components in cardiac decellularized matrix and the retention of main protein components in extracellular matrix were detected by means of histology,molecular biology and other methods.Results:The cell components were removed thoroughly after decellular treatment,and the main protein components in the natural extracellular matrix were preserved,the glycosaminoglycan in the decellularized matrix was dispersed evenly,and the extracellular matrix network was well preserved.Conclusion:The porcine cardiac decellularized matrix has been successfully obtained,which not only effectively cleared the cell composition,but also retained the network structure of the extracellular matrix of the heart and the main components of the extracellular matrix.Part two:Preparation and biocompatibility evaluation of cardiac decellularized matrix spongeAim:Preparing and evaluating the biocompatibility of porcine cardiac decellularized matrix sponge.Methods:The cardiac decellularized matrix sponge was prepared by different concentrations of decellularized matrix solution by freeze-drying method.The pore size of decellularized matrix sponge was observed by histology and scanning electron microscope(SEM).The biocompatibility was evaluated by inoculating the embryonic fibroblasts,and the content of endotoxin was determined.Results:The pore diameter of cardiac decellularized matrix sponge decreased with the increase of extracellular matrix concentration.The pore diameter of decellularized matrix sponge prepared by 20 mg/mL decellularized matrix was about 90-120?m,and the activity of mouse embryonic fibroblasts which are cultured in this scaffold was significantly higher than the scaffold which the concentration of cardiac decellularized matrix is 10 mg/mL and 30 mg/mL.The results showed that the sponge scaffold prepared by 20 mg/mL cardiac decellularized matrix had good biocompatibility.The results of endotoxin test showed that the content of endotoxin was in accordance with the standard.Conclusion:The decellularized matrix sponge scaffold with good biocompatibility was prepared successfully.Part three:The construction of three-dimensional tumor model in vitro based on cardiac decellularized matrix spongeAim:Evaluating the feasibility of cardiac decellularized matrix sponge used in the construction of three-dimensional tumor model in vitro.Methods:A three-dimensional lung cancer model was constructed with decellularized matrix sponge as scaffold and lung adenocarcinoma cell line A549 as seed cell.The expression patterns of matrix metalloproteinase family related genes and vascularized genes in A549 cells were studied by real-time quantitative PCR and Western Blotting.Results:The A549 cells grew well in decellularized matrix sponge.The expression of some genes related to MMP family in A549 cells growing in decellularized matrix sponge was significantly higher than that in two-dimensional culture group,but significantly lower than that in traditional Matrigel three-dimensional model group.In addition,the vascularization ability of A549 cells in cardiac decellularized matrix sponge was significantly higher than that in two-dimensional culture.Conclusion:The cardiac decellularized matrix sponge can be used as scaffold material for three-dimensional tumor model in vitro.In this study,a three-dimensional model of lung cancer based on cardiac decellularized matrix sponge was established,which is expected to provide a new research model for in vitro study of the development of lung carcer and drug screening.It can also provide guidance and basis for the design and development of new scaffold materials in tumor tissue engineering.