Culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and inducing method and application of culture solution

A technology of islet-like cells and stromal cells, which is applied in the field of culture medium of functional islet-like cell clusters, can solve the problems of increasing the risk of clinical application, inducing long-term residual factors, preventing cells from agglomerating into clusters, etc., and reducing clinical application. risk, improve the efficiency of inducing differentiation, and improve the effect of insulin secretion

Active Publication Date: 2012-08-01
UNION STEMCELL & GENE ENG
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

Due to the strong adherence of mesenchymal stem cells, it hinders the aggregation of cells during the induction process, so the induction rate is generally low
The two-part method or the three-step met...

Method used

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  • Culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and inducing method and application of culture solution
  • Culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and inducing method and application of culture solution
  • Culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and inducing method and application of culture solution

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Embodiment

[0043] Example: Preparation of adipose-derived mesenchymal stem cells

[0044] 1.1 Preparation of adipose-derived mesenchymal stem cells

[0045] Under sterile conditions, the by-product of clinical surgery or plastic surgery—adipose tissue is taken, and the adipose tissue is washed 2-3 times in PBS in a biological safety cabinet to completely remove red blood cells and tissue fragments. Then add preheated 0.1% type I collagenase solution, put in a 37°C water bath and shake for 1h. Centrifuge at 300g for 5min to remove the upper fat cells and collagenase solution. Wash the bottom pellet with PBS+1% BSA, centrifuge at 300g for 5min, add an appropriate amount of DMEM containing 10%FBS to resuspend the cells, filter through a 100μm cell sieve, adjust the cell density and inoculate into a cell culture flask, and store at 37°C, 5% CO 2 cultured in an incubator. After 48 hours, the medium was changed to remove non-adherent cells and residual red blood cells. The medium was chang...

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Abstract

The invention discloses a culture solution for inducing mesenchymal stem cells to differentiate into islet-like cells, and an inducing method and application of the culture solution. The culture solution comprises the following materials: niacinamide, Conophylline, cell growth factor, betacellulin and a base medium. The base medium contains 97% of high glucose DMEM (Dulbecco Modified Eagle Medium), 2% of B-27 and 1% of N-2. The inducing method comprises the following steps of: preparing an inducing culture solution; preparing the human mesenchymal stem cells; taking the human mesenchymal stem cells, inoculating the human mesenchymal stem cells into a six-hole ultralow absorption culture plate by 1.5-2*105cells/hole, adding 3ml inducing culture medium into each hole and carrying out suspended induction; and changing liquid at every 3 days, collecting cell supernatant at the ninth day, and storing the cell supernatant at the temperature of -20 DEG C. The culture solution disclosed by the invention has the advantages that the human mesenchymal stem cells are induced to differentiate into the islet-like cells by utilizing the combination of the niacinamide and the Conophylline, so that the inducing cycle is shortened, the suspension cells are beneficial to being clustered to form cell clusters similar to natural islets, further the induced differentiation efficiency is obviously increased and the clinical application risk is reduced; and the function of inducing the secretion of the cell insulin is obviously improved.

Description

technical field [0001] The present invention relates to a culture medium, an induction method and an application for efficiently inducing human adipose stem cells to differentiate into functional islet-like cell clusters by using the novel plant inducer Conophylline and nicotinamide. Background technique [0002] Type 1 diabetes, also known as insulin-dependent diabetes, is an autoimmune disease mediated by primary T cells, which eventually leads to massive destruction of β cells, insufficient insulin secretion, and unbalanced regulation of blood sugar concentration, seriously endangering human health. Islet transplantation is the most effective way to treat diabetes, but there is a serious shortage of donor cells, so it is necessary to find new substitutes for islet transplantation. Mesenchymal stem cells are a kind of cells with multi-lineage differentiation potential, which can be directional induced into various cells such as islet-like cells and chondrocytes. Moreover,...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 黄家学杨萍陈晓波韩红起
Owner UNION STEMCELL & GENE ENG
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