Methods for amplifying and differentiating pancreatic cells, and application thereof

An amplifying method of pancreatic cells, comprising:

• S1:
• obtaining pancreatic tissue, derived from a mammalian pancreatic duct;
• S2:
• performing digestion, resuspension, and discontinuous density gradient centrifugation treatment to the pancreatic tissue in sequence, collecting the cell clusters at each density gradient junction, and then removing islet cells and acinar cells in the cell clusters to obtain primary cells;
• S3:
• performing an amplifying and adhering treatment to the primary cells with an amplifying medium to induce reprogramming of the primary cells to obtain pancreatic precursor-like cells;
• wherein a period of the amplifying treatment was at least 72 hours, and the amplifying medium was replaced every 48 hours after the cells are adhered to a culture dish by the adhering treatment;
• wherein the amplifying medium comprising a reprogramming substance composed of several small molecule compounds, and the reprogramming substance comprises growth factor, Rock signal pathway inhibitor, WNT signal pathway agonist and TGF-β signal pathway inhibitor,
• in the step S2, at least three demixing agents with different densities are used to perform the discontinuous density gradient centrifugation treatment, the adjacent demixing agents are a low-density demixing agent and a high-density stratification, and the density of the low-density demixing agent is decreased by 0.1-3% relative to the density of the high-density demixing agent, and wherein
• the highest density of the demixing agents is not higher than 1.1 g/ml, and the lowest density of the demixing agents is not less than 1.03 g/ml;
• wherein the pancreatic precursor like cells are formed in a single cell layer by adhering to the culture dish in the adhering treatment, and the pancreatic precursor like cells of the single cell layer can be proliferated until the 10th generation and be still in an exponential growth phase.