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Method for culturing islet cells suitable for clinical application

A clinical application technology of islet cells, which is applied in the field of biomedicine, can solve the problems of transplantation scheme, transplantation amount, expected therapeutic effect cannot be evaluated, islet cell glucose stimulation response varies greatly, druggability cannot be effectively detected and evaluated, etc., to achieve no miscellaneous Effects of cell residue, improved cell transformation rate, and small batch-to-batch differences

Pending Publication Date: 2022-04-05
王意忠
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are some problems in the reports and patents on the differentiation of MSCs into IPCs at home and abroad. First, the efficiency and degree of differentiation of the induced islet-like cells to secrete insulin are low, which cannot meet the actual clinical needs. Secondly, in terms of the selection of induction reagents, domestic and foreign Mainly focus on chemical reagents such as β-mercaptoethanol, etc., which are toxic to the human body and cannot be used clinically
At the same time, due to the large difference in induction between different batches of islet cell culture, it is difficult to effectively detect and evaluate the risk due to unexpected differentiation in terms of druggability, and it is difficult to form a standardized large-scale IPC production
In particular, islet cells cultured in different ways and batches vary greatly in response to glucose stimulation, making it impossible to evaluate the transplantation scheme, transplantation volume, and expected therapeutic effect

Method used

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  • Method for culturing islet cells suitable for clinical application
  • Method for culturing islet cells suitable for clinical application
  • Method for culturing islet cells suitable for clinical application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Obtaining islet cells by induction and culture of umbilical cord mesenchymal stem cells

[0039] The full-term umbilical cords were selected and processed and cultured within 24 hours. First, cut the umbilical cord into pieces of 8-16mm 3 The small pieces were cultured by the tissue block culture method, and the culture medium was selected from the serum-free medium of mesenchymal stem cells. After 18 days of culture, the cells were subcultured until the cells were congested until the mesenchymal stem cells were cultured to the fifth generation. The cells were tested for bacteria, fungi, and mycoplasma. The results were all negative, and the cell viability was 95%. The surface markers of mesenchymal stem cells were detected, and the test results were CD73, CD90, CD105, CD44, CD166≥95%, CD11b, CD19, CD79a, CD34, CD45, HLA-DR≤2%. Nucleic acid / antibody detection method was used to detect HIV-1 / 2, HBV, HCV, HTLV-1 / 2, EBV, CMV, TP, B19, HPV, HHV and other path...

Embodiment 2

[0049] Example 2: Using adipose-derived mesenchymal stem cells to induce and culture islet cells

[0050]Adipose tissue from healthy individuals was collected and processed within 24 hours. First, the adipose tissue was washed with a sufficient amount of normal saline, centrifuged at 1000rpm / min for 20min, the middle adipose layer tissue was taken, digested with type I collagenase for 30min, centrifuged at 1000rpm / min for 10min, the pelleted cells were taken and washed twice with PBS. According to the cell volume of 10000 / ml, inoculated in T175 culture flasks, and after 14 days, the cells were congested and passaged until the mesenchymal stem cells were cultured to the fifth generation. Bacteria, fungi, and mycoplasma were tested on the fifth-generation cells, and the results were negative, and the cell viability was 95%. Flow cytometry was used to detect the surface markers of mesenchymal stem cells, and the test results were CD73, CD90, CD105, CD44, CD166≥95%, CD11b, CD19...

Embodiment 3

[0060] Example 3: Detection of pancreatic islet cells

[0061] 1. Detection of the purity of islet cells

[0062] Add 50 mg of dithizone (SIGMA-ALORCH, product number: 43820-10G) to 5 ml of DMSO, take 1 ml of the above solution, add 20 ml of Hank's containing 2% fetal bovine serum, mix well and filter with a 0.22 μm filter, take 3 0.5 ml of each independent batch of islet cell suspension was placed in a 24-well plate, added dithizone staining solution, stained for 30 s, observed under an inverted microscope, and the purity of islet cells was calculated.

[0063] Islet cell purity = (number of positive islets stained with dithizone / total number of purified cell clusters) × 100%. The test results showed that the three batches of islet cells were all stained in strong brownish red, and the purity of the islet cells was 100%. Figure 9 shown.

[0064] 2. Transformation rate of islet cells

[0065] Three independent batches of islet cell induction culture were carried out. ...

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Abstract

According to the method, the islet cells are obtained through induced differentiation by taking mesenchymal stem cells as an initial raw material, and the method comprises the following steps: S1, carrying out starvation culture on the mesenchymal stem cells, so that the cell cycle is retarded in a G0 / G1 phase; step S2, carrying out first-stage induction culture so as to generate islet-like bulk cells; s3, carrying out second-stage induction culture, so that the ratio of the insulin positive cells to the nest protein positive cells reaches a preset value; and S4, carrying out third-stage induction culture to obtain mature islet cells. The islet cells prepared by the method can be used for preventing, relieving and treating type 1 diabetes and type 2 diabetes.

Description

technical field [0001] The invention belongs to the field of biomedicine, and more specifically, the invention relates to a method for cultivating pancreatic islet cells suitable for clinical application. Background technique [0002] Diabetes mellitus is a metabolic syndrome characterized by hyperglycemia due to the relative insufficiency of insulin secretion by pancreatic β cells due to multiple factors. Long-term metabolic disorders can lead to complications in various systems of the body. The current conventional treatment for diabetes is only oral hypoglycemic drugs or insulin therapy. The best state is to make blood sugar control stable, but the dosage is gradually increasing, and the function of pancreatic islets is gradually failing. At the same time, the occurrence of severe hypoglycemia, significant weight gain, and changes in diet and lifestyle seriously affect the quality of life of patients. [0003] A large number of research results have confirmed that islet ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/0775A61K35/39A61P3/10
Inventor 王意忠张社毅孙岳
Owner 王意忠
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