The Construction Of Stamp-like Spheroidized Culture Tool And Its Application In Individualized Drug Treatment Of Liver Cancer

Background:Primary liver cancer is one of the most common malignant tumors in the world.At present,chemotherapy is still an important treatment for advanced liver cancer.Due to the potential liver dysfunction and the resistance of liver cancer cells to traditional chemotherapy drugs,the tolerance of HCC patients to chemotherapy is generally poor.Therefore,it is an urgent problem to select effective new anti-hepatoma drugs and make personalized treatment for HCC patients.However,the traditional tumor models can’t rebuild the three-dimensional structure and tumor microenvironment of cells in vivo,which makes the ability of drug sensitivity test to predict clinical drug efficacy poor.The three-dimensional spheroid culture is a mature method of cell culture in vitro,which can imitate the three-dimensional structure of tumor cells and reconstruct the cell microenvironment.Three-dimensional tumor model is more reliable than two-dimensional tumor model in vitro drug sensitivity experiment,so it is recommended to use three-dimensional tumor model for drug sensitivity experiment in individualized treatment.At present,the commercialized Three-dimensional cell culture system has some disadvantages,such as high price and uneven spheroidization.Therefore,it is of practical significance to develop a new cheap and uniform three-dimensional culture model,which is helpful to promote the individualized treatment of liver cancer.Objective:to develop a low-cost,simple and effective three-dimensional cell culture tool for the individualized treatment of liver cancer.Method:Chapter I Construction and evaluation of the stamp-like spheroidized culture toolConstruction of culture system:using commercialized three-dimensional cell culture systems for reference,we innovatively designed a stamp-like spheroidizing culture tool,and then drew it into a graph with Solidworks 2016 software.The mold of the three-dimensional culture system was printed by the 3D printing technology,and the agarose in 96 plates was molded by the mold to form the agarose micro-well spheroidized culture plate.Culture system evaluation:four kinds of common HCC cells were inoculated into the agarose micro-well spheroidized culture plate and ultra-low attachment round bottom 96 well plate,respectively,to observe the morphological changes and cell viability changes of hepatocarcinoma cells in the two culture systems in different time periods.In addition,the hepatoma cell lines were inoculated into plate culture flask and new three-dimensional culture system respectively,and the expression differences of drug metabolism related genes in the two culture methods were compared.Chapter II Three-dimensional drug sensitivity test of human primary hepatoma cellsThe primary human hepatoma cells were isolated and purified by collagenase digestion and Percoll density gradient centrifugation.The primary cells were inoculated into the agarose micro-well spheroidized culture plate.Some of the primary cell spheroids were collected for Arg-1 immunohistochemical staining,and compared with the original tumor tissue.At the same time,four groups of antitumor drugs were added to the rest of the three-dimensional cultured cells for co-culture Then ATP method was used to measure the cell viability and calculate the inhibition rate of drugs on tumor cells.Finally,the results were analyzed by sensitivity classification to evaluate the sensitive drugs.Result:Chapter I Construction and evaluation of the stamp-like spheroidized culture toolThe stamp-like three-dimensional culture mold made by 3D printing has smooth surface,complete structure.The structure of agarose matrix is intact after molding.Compared with the ultra-low attachment 96 well plate,the four kinds of hepatoma cells in the new three-dimensional culture system formed spheres faster and more uniform.Liver cancer cells can be cultured in the agarose micro-well spheroidized culture plate for more than 14 days.The results of cell function test showed that the expression level of drug metabolism related genes in three-dimensional hepatocytes was higher than that in two-dimensional hepatocytes.Chapter II Three-dimensional drug sensitivity test of human primary liver cancer cellsFour human primary liver cancer cells were successfully isolated,and all of them were able to aggregate into spheres in the agarose micro-well spheroidized culture plate.The results of drug sensitivity showed that the primary liver cancer cells had different responses to four groups of anti-tumor drugs.Compared with the other three groups of anti-tumor drugs,doxorubicin showed better inhibition effect.Conclusion:In this project,a new three-dimensional cell culture system was constructed by using 3D printing technology and agarose.In this system,liver cancer cells can form spheres rapidly,the spheres are uniform in size,and the three-dimensional structure of liver cancer cells is good.It was found that the function of three-dimensional culture was better than that of two-dimensional culture.The three-dimensional drug sensitivity test of primary liver cancer cells showed that different patients’ liver cancer cells had different sensitivity to antitumor drugs.The three-dimensional culture system has the advantages of low cost,good 3D culture effect and easy to change liquid.This experiment provides a certain reference for the individualized treatment of liver cancer patients.