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Serum-free medium for inducing differentiation of umbilical cord mesenchymal stem cells into insulin secretory-like cells and preparation method and application thereof

A serum-free medium, insulin secretion technology, applied in cell culture active agents, biochemical equipment and methods, animal cells, etc., can solve the problems of prohibitive cost and limited number of organs, achieve high-efficiency solutions, and avoid cell growth process. Instability, the effect of eliminating the risk of spreading xenogeneic pathogens

Pending Publication Date: 2017-05-17
里程 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number of organs available for transplantation is limited after all, and the cost of organ transplantation is often prohibitive, and organ transplantation often causes strong immune rejection. These problems have always plagued doctors and patients

Method used

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  • Serum-free medium for inducing differentiation of umbilical cord mesenchymal stem cells into insulin secretory-like cells and preparation method and application thereof
  • Serum-free medium for inducing differentiation of umbilical cord mesenchymal stem cells into insulin secretory-like cells and preparation method and application thereof
  • Serum-free medium for inducing differentiation of umbilical cord mesenchymal stem cells into insulin secretory-like cells and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Screening of medium composition

[0061] Basal medium: DMEM (glucose content 4.5g / L)+5%SR+1%NEAA+2%B27(50X)+1%N-2(100X)+10ng / ml HGF+10ng / ml EGF+10ng / ml b-FGF+10mmol / L Nicotinamide;

[0062] Screening components: add 100ng / ml vinblastine III (Conophylline), 1 volume part of ITS, 1ug / ml heparin (Heparin), and 10ng / ml betacellulin (betacellulin) to the basal medium ), sialidin 4 (Exendi-4), insulin-like growth factor (IGF-1), and pentagastrin. The groupings are listed in Table 3 below.

[0063] table 3

[0064]

[0065] In a biological safety cabinet, the third-generation hUC-MSCs isolated from the Wharton's glue tissue of the umbilical cord of spontaneously delivered neonates were collected at 5 × 10 4 cells / cm 2 The density was inoculated into the ultra-low adsorption six-well plate, and 2 ml of the test medium shown in Table 3 was added to each well to observe the cell growth and the number of cell clusters.

[0066] Results: In the first group of ...

Embodiment 2

[0067] Example 2 Screening of the Content of Medium Components (Vinblastine Ⅲ)

[0068] Test medium: 89 parts by volume of high glucose DMEM (glucose content 4.5g / L), 5 parts by volume of serum replacement (SR), 2 parts by volume of B27 (50X), 1 part by volume of ITS, 1 part by volume of non-essential amino acids (NEAA), 1 volume of N-2 (100X), heparin (Heparin) at a final concentration of 1ug / ml, nicotinamide at a final concentration of 10mmol / L, recombinant human alkaloids at a final concentration of 10ng / ml Fibroblast growth factor (b-FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), pentagastrin and concentration gradients of 1, 10, 50, 100, 200, 300 ng / ml Vinblastine III (Conophylline).

[0069] Human umbilical cord mesenchymal stem cells were induced to differentiate and cultured using the above-mentioned media with different concentrations of vinblastine III (Conophylline).

[0070] RESULTS: In the two groups of test media with the concentration o...

Embodiment 3

[0071] Example 3. Screening of the content of medium components (pentagastrin)

[0072] Test medium: 89 parts by volume of high glucose DMEM (glucose content 4.5g / L), 5 parts by volume of serum replacement (SR), 2 parts by volume of B27 (50X), 1 part by volume of ITS, 1 part by volume of non-essential amino acids (NEAA), final concentration of 1ug / ml heparin (Heparin), 1 volume of N-2 (100X), 100ng / ml vinblastine III (Conophylline), 10mmol / L nicotinamide, Recombinant human basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF) with a final concentration of 10ng / ml and a concentration gradient of 1, 2, 5, 10, 20 , 30, 50ng / ml of pentagastrin.

[0073] The above-mentioned media with different concentrations of pentagastrin were used to induce differentiation and culture of human umbilical cord mesenchymal stem cells. 4 A) the amount of insulin released in each group, and the cells in each group were compared. The sugar stimulat...

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Abstract

The invention discloses a novel serum-free medium. The medium includes high-glucose DMEM (glucose content is 4.5g / L), B27, recombinant human basic fibroblast growth factor (b-FGF), nicotianamine, N-2, vinblastine III (Conophylline), non-essential amino acid (NEAA), heparin (Heparin), epidermal growth factor (EGF), hepatocyte growth factor (HGF), serum replacement (SR), an insulin-transferrin-selenium compound (ITS), and pentagastrin. By the utilization of the medium of the invention, mesenchymal stem cells can be differentiated into insulin secretory-like cells within 6 days by a one-step method.

Description

technical field [0001] The invention relates to the research field of stem cells, in particular to a novel and efficient serum-free medium suitable for inducing differentiation of mesenchymal stem cells into insulin-secreting-like cells, and a preparation method and application thereof. Background technique [0002] Diabetes has become a worldwide epidemic in the 21st century. It is the third most serious chronic non-communicable disease that threatens human health after tumor and vascular disease. It has the characteristics of high mortality, high disability and high medical expenses. . At present, the number of diabetic patients in China has reached 114 million, ranking first in the world, accounting for about one-third of the total number of diabetics in the world. Therefore, diabetes has become a serious public health problem faced by countries all over the world, especially China. [0003] After the middle of the 20th century, with the rise and development of human or...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0602C12N2500/25C12N2500/30C12N2500/32C12N2500/34C12N2500/90C12N2501/11C12N2501/115C12N2501/12C12N2501/345C12N2506/1392
Inventor 郭镭
Owner 里程
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