Establishment Of 3D In Vitro Culture System Based On Microspheres And Its Effect On The Biological Behavior Of Osteosarcoma MG63 Cells

Aim:GelMA(Gelatin methacryloyl)microspheres were prepared by microfluidic device to investigate the effect of 3D microsphere environment on the biological behavior of osteosarcoma MG63 cells.Methods:1)GelMA microspheres were prepared by microfluidic device,the morphology of microspheres and MG63 cells were observed by scanning electron microscope,cell morphology was observed by DAPI and F-actin staining,and the biocompatibility of microspheres was determined by live/dead staining method.2)The expression of CSC(Cancer stem cells),migration,EMT(Epithelial-mesenchymal transition),apoptosis and osteoclast-related genes in MG63 cells grown on microspheres were assayed by quantitative PCR;The proliferation of MG63 cells grown on microspheres was assayed by CCK-8 method.;The cell cycle of MG63 cells in 3D microspheres was assayed by PI staining.The apoptosis of MG63 cells grown on microspheres was assayed by flow cytometry.The Migration of MG63 cells in 3D microspheres was assayed by Transwell and scratch test.The osteoclast differentiation of BMM(bone marrow mononuclear-macrophage)cells induced by MG63 supernatant in 3D microspheres was observed by TRAP staining and the osteoclast gene expression was determined by PCR.Results:(1)The GelMA microspheres were successfully prepared,they are highly monodisperse in size and can be effectively for cell attachment and proliferation.(2)PCR results showed that the CSC,migration,EMT,anti-apoptosis and osteoclast-related genes of MG63 cells were increased in the 3D microsphere environment.The CCK-8 results indicated that the 3D environment of the microspheres promoted the proliferation of osteosarcoma MG63 cells relative to the tissue culture plate.PI staining showed that the 3D microsphere environment promoted DNA synthesis and promoted cell cycle differentiation from the G1 phase to the S phase.Flow cytometry indicated that the 3D microsphere environment enhanced the anti-apoptotic properties of osteosarcoma MG63 cells and was associated with up-regulated expression of anti-apoptotic genes.TRAP staining showed that the 3D microsphere environment enhanced the ability of osteosarcoma MG63 cells to promote osteoclast differentiation.The PCR results showed that the expression of osteoclast-related genes was promoted.Conclusions:The GelMA microspheres prepared by microfluidic are biocompatible and suitable for in vitro culture models of osteosarcoma;The microsphere-based 3D environment enhances the malignant biological behavior of osteosarcoma MG63 cells,providing a new research tool for further study of the biological behavior and drug screening of osteosarcoma.