A kind of improved neonatal porcine islet cell culture medium and using method thereof

Abstract

The invention discloses an improved culture medium for neonatal pig islet cells and a use method of the same. An apoptosis inhibitor Z-VAD-FMK, a human basic fibroblast growth factor hFGF-beta, a vascular endothelial growth factor VEGF, an insulin-like growth factor R3-IGF-1, a human epidermal growth factor hEGF, a hepatocyte growth factor hHGF, pig serum, sodium selenite, insulin, transferrin, ethanol amine, 1-methyl 3-isobutyl xanthine, nicotine, cortisol, penicillin and streptomycin are added in HAM'S F10 culture medium. According to the culture medium, apoptosis can be reduced and recovery rate can be increased; the cultured cells have a sensitive glucose stimulation response and more mature functions compared with generally-cultured islet cells; the insulin secretion amount of the islet cells with the same quantity is increased; the viability of islet beta-cells is improved; the breakages of islet cell clusters are reduced, and the envelops of the cell clusters are complete, so that the cell clusters are more suitable for being transplanted.

Description

technical field [0001] The invention belongs to the technical field of animal cell culture, and in particular relates to an improved newborn pig islet cell culture medium and a use method thereof. Background technique [0002] Human islet cell transplantation is an effective way to treat diabetes, but the shortage of human donors has led people to pay more and more attention to finding human islet substitutes. Porcine insulin is very similar to human insulin and has similar blood sugar regulation points. It is very suitable as a substitute for human islets. Adult porcine islets are very fragile and easy to disperse. Newborn porcine islets have characteristics such as low immunogenicity and β-cell division potential, and have good blood sugar stimulation response after maturity (Korbutt, G.S.; Z.;Smith,D.K.;War-nock,G.L.;Rajotte,R.V.Large scale isolation,growth,and function of porcine neonatal islet cells.J.Clin.Invest.97(9):2119–2129;1996), after a period The long-term in v...

AI Technical Summary

Problems solved by technology

However, these methods and the medium used always have many defects, such as: unstable culture results, the amount of purified islet cells with active functions varies (2000IEQ / g-4000IEQ / g), the purity of cultured islet cells There are also differences (70-90%); in the process of low-temperature culture, islet cells will be lost due to apoptosis, especially β cells, and the length of the culture process is difficult to choose. If the culture time is short, the β cells will not mature. If the time is too long, a large number of islet cells will be lost due to apoptosis, and the general recovery rate is only about 60%, and the insulin secretion of the same amount of islet cells will be reduced due to the apoptosis of β cells (insulin / DNA value <49.00mIU / g)

In addition, as the culture time prolongs, the islet cell clusters are easily broken, and the small islet cell clusters (diameter <50um) formed are more likely to die, which is difficult to culture, and makes it difficult to remove pollution during the culture process, which is easy to bring inflammation to the subsequent transplantation process reaction, which is very unfavorable to the therapeutic effect of later transplantation

So far, there is no islet cell culture method and medium that can provide high-purity, active and stable yield in large quantities, so how to establish and optimize the optimal cell separation scheme, medium composition and culture method has become the most urgent issue for islet cell culture

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