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Preparation method of human adipose tissue-derived stromal cell exosome with high expression of IL-10 for treating myocardial infarction

A technology of mesenchymal stem cells and myocardial infarction, which is applied in the field of preparation of human adipose-derived mesenchymal stem cell exosomes, can solve the problems of rejection reaction affecting function, etc., and achieve the effect of simple and convenient process, stable effect and alleviating damage

Pending Publication Date: 2020-11-13
FUJIAN MEDICAL UNIV UNION HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the retention rate of exogenous IL-10 in the damaged area and the body's own rejection will affect its function. How to make IL-10 play a stable role in the damaged area of ​​​​myocardial infarction remains to be further studied

Method used

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  • Preparation method of human adipose tissue-derived stromal cell exosome with high expression of IL-10 for treating myocardial infarction
  • Preparation method of human adipose tissue-derived stromal cell exosome with high expression of IL-10 for treating myocardial infarction
  • Preparation method of human adipose tissue-derived stromal cell exosome with high expression of IL-10 for treating myocardial infarction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The purpose of this example is to isolate and culture human adipose-derived mesenchymal stem cells.

[0037] 1. Materials

[0038] Prepare human fat (normal human fat discarded after surgery, signed informed consent), PBS, BSA, type IV collagenase, fetal bovine serum, CD29 + , CD44 + , CD90 + , CD34 - and CD45 - of monoclonal antibodies.

[0039] 2. Method

[0040] 2.1 Isolation of human adipose-derived mesenchymal stem cells

[0041] (1) Collect about 15 mL of adipose tissue under sterile conditions, and remove the connective tissue capsule and blood vessels on the surface of the adipose tissue;

[0042] (2) Quickly cut into paste, add 3 mL of exosome-free medium, which contains 0.06-0.16% type IV collagenase and 0.015-0.02% BSA at final concentration , 37℃, constant temperature, 120r / min, oscillating digestion for 30-35min, observe the digestion status of the tissue at any time, take it out and mix it every 5-8min during the period, after the digestion is term...

Embodiment 2

[0061] The purpose of this example is to isolate and purify human adipose-derived mesenchymal stem cell exosomes, including the following steps:

[0062] 1. Material method

[0063] Prepare human fat (normal body fat discarded after surgery, signed informed consent), PBS, BSA, type IV collagenase, fetal bovine serum, CD9, CD63, CD81, α-actinin-4 and CD40 monoclonal antibodies and Dimethyloxalylglycine.

[0064] 2. Collection of human adipose-derived mesenchymal stem cell exosomes

[0065] (1) Collect human adipose-derived mesenchymal stem cells, inoculate them, and use exosome-free serum medium at 37°C, 5% CO 2 Start culturing under the conditions;

[0066] (2) After culturing for 12 hours, replace it with a serum medium containing 300 μmol / L dimethyloxalylglycine exosomes from human adipose-derived mesenchymal stem cells, continue culturing for 36 hours, and collect the cell culture medium;

[0067] (3) The cell culture solution obtained in step (2) was subjected to diffe...

Embodiment 3

[0082] The purpose of this example is to culture h-MSCs cell line.

[0083] 1. Materials

[0084] Prepare human fat (normal human fat discarded after surgery, signed informed consent), PBS, BSA, type IV collagenase, fetal bovine serum, CD29 + , CD44 + , CD90 + , CD34 - and CD45 - of monoclonal antibodies.

[0085] 2. Passage of h-MSCs

[0086] (1) Collect adipose tissue according to the method shown in Example 1, digest it with the digestion conditions of 0.1% type IV collagenase + 0.02% BSA group, and culture the cells according to the method described in Example 1 until 80% to 90% confluence , digested with 0.25% trypsin-0.02% EDTA, and centrifuged to obtain cell pellets;

[0087] (2) Resuspend the cell pellet in fresh exosome-free medium, passage according to 1:2~4, and culture to P10, each generation of cells is named P1-h-MSCs, P2-h-MSCs, P3-h-MSCs, respectively. h-MSCs, P4-h-MSCs, P5-h-MSCs, P6-h-MSCs, P7-h-MSCs, P8-h-MSCs, P9-h-MSCs and P10-h-MSCs;

[0088] (3)...

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Abstract

The invention provides a preparation method of human adipose tissue-derived stromal cell exosome with high expression of IL-10 for treating myocardial infarction, and relates to the technical field ofbiology. The preparation method comprises the following steps: culturing human adipose tissue-derived stromal cells over-expressing IL-10, and collecting a cell culture solution; and carrying out differential centrifugation on the cell culture solution, collecting the supernatant, carrying out ultracentrifugation on the supernatant to obtain target microspheres, and filtering, purifying and sterilizing the target microspheres to obtain the exosome. The preparation method has the advantage that by culturing human adipose tissue-derived stromal cells over-expressing IL-10, the human adipose tissue-derived stromal cell exosome capable of over-expressing IL-10 is prepared; the IL-10 overexpression human adipose tissue-derived stromal cell exosome contains a large amount of IL-10 and other various RNAs and cytokines, effectively repairs infarcted myocardial cells, enhances the cardiac function, reduces compensatory myocardial load, relieves heart failure, and provides a therapeutic drug ora therapeutic strategy for myocardial infarction.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing human adipose-derived mesenchymal stem cell exosomes highly expressing IL-10 for treating myocardial infarction. Background technique [0002] Coronary atherosclerotic heart disease is due to abnormal lipid metabolism. The lipids in the blood deposit on the smooth arterial intima, and gradually accumulate to form white plaques, causing vascular stenosis or blockage, which can lead to severe Coronary artery occlusion, blood flow interruption, partial necrosis of myocardium due to severe persistent ischemia, resulting in myocardial infarction. After myocardial infarction, some myocardial cells are difficult to repair due to ischemia and hypoxia, resulting in reduced cardiac function and increased compensatory myocardial load, which will lead to serious complications such as heart failure over time. During the onset of coronary heart disease, the coronary artery ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/10C12N15/85C12N15/24A61P9/10
CPCC12N5/0667C12N15/85C07K14/5428A61P9/10C12N2509/00C12N2510/00C12N2800/107
Inventor 陈良万柴天赐
Owner FUJIAN MEDICAL UNIV UNION HOSPITAL
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