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Young pig islet cell culture medium and application method thereof

A technique of islet cells and culture medium, applied in the field of islet cell culture medium of young pigs, can solve the problems of islet cell mass easily broken, islet cell mass prone to apoptosis, difficult to continue culturing, etc., to increase cell recovery rate, suitable for transplantation , the effect of complete coating

Active Publication Date: 2019-06-14
HUNAN XENO LIFE SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, as the culture time prolongs, the islet cell clusters are more likely to be broken, thus forming smaller islet cell clusters (diameter <50um) that are more prone to apoptosis and difficult to continue culturing
Up to now, there is no method and medium for culturing young pig islet cells with high purity, activity and stable yield, so how to establish the optimal medium composition and culture method has become the most urgent problem for young pig islet cell culture

Method used

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  • Young pig islet cell culture medium and application method thereof
  • Young pig islet cell culture medium and application method thereof
  • Young pig islet cell culture medium and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0031] Implementation case 1, culture medium and culture method test of the present invention

[0032] In the present invention, 10IU / kg heparin sodium is injected intraperitoneally before pancreas operation in young pigs. The inoculation concentration of the present invention is 5000-10000IEQ / 25-30mL for isolated and purified young pig islet cells in the medium, and the culture medium (M199 medium Add glutathione 25mM, glutamine 25mM, ITS0.5%, nicotinamide 20mM, water-soluble vitamin E 9μM, heparin 60U / L, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride 0.25 mM, Exenatide 0.1μM. And porcine serum accounting for 10% of the volume of the medium) in a 15cm non-adherent culture dish, placed at 37 ° C, 5% CO 2 , Cultured in a 95% air incubator, the prepared cells were replaced on the first day with the culture dish and the medium, and then replaced every other day, and the cells were collected on the 5th and 7th day of culture for counting and related detection.

Embodiment example 2

[0034]In the present invention, 10IU / kg heparin sodium is injected intraperitoneally before pancreas operation in young pigs. The inoculation concentration of the present invention is 5000-10000IEQ / 25-30mL for isolated and purified young pig islet cells in the medium, and the culture medium (M199 medium Add glutathione 50mM, glutamine 50mM, ITS 1%, nicotinamide 45mM, water-soluble vitamin E 20μM, heparin 50U / L, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride 0.01mM , Exenatide 0.1μM. And porcine serum accounting for 10% of the medium volume percentage) in a 15cm non-adherent culture dish, placed at 37 ° C, 5% CO 2 , Cultured in a 95% air incubator, the prepared cells were replaced on the first day with the culture dish and the medium, and then replaced every other day, and the cells were collected on the 5th and 7th day of culture for counting and related detection.

Embodiment example 3

[0036] In the present invention, 10IU / kg heparin sodium is injected intraperitoneally before pancreas operation in young pigs. The inoculation concentration of the present invention is 5000-10000IEQ / 25-30mL for isolated and purified young pig islet cells in the medium, and the culture medium (M199 medium Add glutathione 2mM, glutamine 2mM, ITS 0.06%, nicotinamide 2mM, water-soluble vitamin E 0.1μM, heparin 100U / L, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride 0.2 mM, exenatide 0.1μM and porcine serum accounting for 10% of the volume of the medium) in a 15cm non-adherent culture dish, placed at 37°C, 5% CO 2 , Cultured in a 95% air incubator, the prepared cells were replaced on the first day with the culture dish and the medium, and then replaced every other day, and the cells were collected on the 5th and 7th day of culture for counting and related detection.

[0037] result:

[0038] Different concentration culture medium of the present invention has shown better cu...

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Abstract

The invention discloses a young pig islet cell culture medium and an application method thereof. The culture medium is added into a basic culture medium (M199): 1-50 mM of glutathione, 1-50 mM of glutamine, ITS 0.01-1 % of ITS, 1-50 mM of nicotinamide, 0.1-20 micro-M of water-soluble vitamin E, 1-100 U / L of heparin, 0.01-0.5 mM of 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride and 0.01-0.1 mM of exenatide. Compared with the common media (1640, F10 and EGM2), the viability and maturation rate of islet cells in young pigs can be significantly improved.

Description

technical field [0001] The invention belongs to the technical field of animal islet cell culture, and in particular relates to a young pig islet cell culture medium and a use method thereof. Background technique [0002] Type I diabetes is recognized as one of the most threatening chronic metabolic diseases in the world. It is characterized by selective immune-mediated destruction of pancreatic β-cells, which makes patients need exogenous insulin therapy for life. The outbreak of various complications in the late stage of the disease will lead to aggravated life and economic burden and high mortality of patients. Since the successful implementation of the Edmonton protocol, islet transplantation has attracted worldwide attention. After summarizing the basis of islet transplantation research results around the world, promoting the improvement of transplantation methods and immunosuppressive regimens brings hope to patients with type 1 diabetes. Human pancreatic islet allogr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 王维王佳谷星石李桑徐畅
Owner HUNAN XENO LIFE SCI
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