Cell differentiation medium composition, high secretion insulin-producing cells and preparation method thereof

A preparation method for a high secretion insulin-producing cell, which comprises,

• (a) cell proliferation step:
• culturing stem cells at least 3 days for cell proliferation by using a proliferation medium;
• the proliferation medium consists of keratinocyte serum-free media, fetal bovine serum, N-acetyl-L-cysteine, L2 ascorbic acid, and phosphate;
• (b) cell attachment step:
• pour a solution containing the stem cells into a cell culture container and let stand for at least 24 hours for cell attachment, so that the quantity of stem cells attached to the peripheral wall of the culture container is in a range of 6,000 and 15,000 cells/cm2;
• (c) cell differentiation step:
• removing the solution from the culture container, putting a cell differentiation medium composition into the culture container, inducing the stem cells into high-secreting insulin-producing cells under the conditions of an ambient temperature of 35.5˜39.5° C. and a CO2 concentration of 5%, and then collecting the high-secreting insulin-producing cells after differentiating for at least 2 days;
• wherein
• the cell differentiation medium composition is antibiotic-free and is a serum-free DMEM/F12 medium comprising at least 5˜25 mM glucose, 5˜15 mM nicotinamide, 1˜10 μM activin-A, 5˜20 nM exendin-4, 80˜120 nM hepatocyte growth factor, 2˜20 nM pentagastrin, and a 0.1˜5% N-2 Supplement;
• the morphology of the high secretion insulin-producing cell is spindle-shaped;
• and
• the insulin secretion of each 100 thousand of the high secretion insulin-producing cells per day is above 1,000 mIU/L.