Objective ·To establish an in vitro culture model mimicking tumor microenvironment using hydrogel microspheres and fresh primary pancreatic cancer cells. Methods ·The morphological distribution of the hydrogel microspheres was recorded, observed and photographed under an inverted fluorescence microscope. The diameter of the microspheres was calculated by Image J, and the particle size distribution map was obtained by statistics. Renal epithelial cells (293T), pancreatic cancer cells (8988), and normal pancreatic epithelial cells (HPNE) were grown in DMEM complete medium, and passaged when the cells were 80%?90% confluent. 293T cells were cultured in DMEM medium and microsphere extract, and the proliferation curve of 293T cells cultured in the two mediums was detected by CCK-8 method to explore the biocompatibility of hydrogel microspheres. Fresh pancreatic tumor tissue was cut in the ultra-clean workbench, and pancreatic cancer tumor tissue was lysed by hyaluronidase and collagenase Ⅰ, and digested into single cell with interval shaking in a 37 ℃ water bath. The hydrogel microspheres and pancreatic cells were co-cultured in DMEM complete medium for 3 d, 1/2 of the cells were fixed with 4% paraformaldehyde, and stained with phalloidin and DAPI, and the morphology and cell distribution of the microspheres were observed under a common fluorescence microscope. The remaining 1/2 cells were used for suspension and adherent cell counting. The hydrogel microspheres and primary cells were co-cultured in the medium for 7 d, and the cell composition in the established culture model of in vitro pancreatic cancer primary cells based on the hydrogel microspheres was observed by immunofluorescence method. The pancreatic cancer tissue was embedded in paraffin, followed by paraffin tissue sectioning, hematoxylin and eosin staining of the tissue section, and observation of the pancreatic cancer tissue structure with a microscope. Results ·The size of the hydrogel microspheres was uniform, and the diameter of the microspheres was about 200 μm. The proliferation curve of 293T cells showed that the hydrogel microspheres had good biocompatibility. The co-culture experiments of hydrogel microspheres and pancreatic cell lines showed that the surface of hydrogel microspheres had strong cell affinity, which could provide support points for pancreatic cells to adhere to the surface of the microspheres and to grow normally. Through co-culture of hydrogel microspheres with fresh pancreatic cancer cells, a 3D culture model of pancreatic cancer cells in vitro was successfully established. Composition of cell types in this model was similar to that in the corresponding primary tumor tissue, which included pancreatic ductal epithelial cells, tumor stem cells, endothelial cells, fibroblasts and other cells. Conclusion ·The in vitro 3D culture model of primary pancreatic cancer cells based on hydrogel microspheres has important characteristics of pancreatic cancer tumor microenvironment.