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Preparation method and application of mesenchymal stem cell exosome

A technology of stem cells and exosomes, which is used in the field of preparing biological products to improve mitochondrial damage and prevent cell apoptosis.

Inactive Publication Date: 2018-10-09
章毅 +7
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Some researchers directly inject stem cells or cardiomyocytes expanded and cultured in vitro into the heart, which can repair the damage caused by myocardial ischemia-reperfusion and improve heart function. It is a new strategy for heart repair after myocardial infarction, but the use of cell therapy will bring Many challenges such as: immune rejection, dedifferentiated growth of cells, etc.

Method used

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  • Preparation method and application of mesenchymal stem cell exosome
  • Preparation method and application of mesenchymal stem cell exosome
  • Preparation method and application of mesenchymal stem cell exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Isolation and culture of placental mesenchymal stem cells and collection and purification of exosomes

[0026] (1) Isolation and culture of rat placental mesenchymal stem cells

[0027] Two-week pregnant Sprague Dawley rats were taken, ether inhaled anesthesia, cervical vertebrae were dissected and sacrificed, soaked in 75% ethanol for disinfection, and under aseptic conditions in an ultra-clean table, the abdominal cavity of the pregnant rat was cut to take out the uterus, the uterus was cut open, and the amnion was peeled off. Disconnect the placenta, collect all rat placentas, rinse with sterile phosphate buffer and set aside.

[0028] Use tweezers and scissors to remove residual umbilical cord and amniotic membrane tissue, and cut placental tissue to about 1mm 3 ~2mm 3 Small tissue pieces, the smaller the piece, the better the post-digestion effect. Add 0.1% type I collagenase collagen (Sigma-Aldrich), 0.25% trypsin (GIBICO), digest at 37°C for about 14...

Embodiment 2

[0036] Example 2 Application of Exosomes Prepared from Placental Mesenchymal Stem Cells in Improving Heart Function

[0037] (1) Establishment of myocardial cell model of ischemia-reperfusion injury in rats

[0038] Rat cardiomyocyte cell line H9C2 was cultured and passaged in DMEM high glucose medium (GIBICO) + 10% fetal bovine serum (Hyclone). Adjust the oxygen concentration of the three-gas incubator to 1% to prepare a hypoxic environment, replace the medium with DMEM medium without glucose, take out the cells at different times under the condition of hypoxia and glucose deficiency, and observe the cell morphology under an inverted microscope The cell survival rate was detected by CCK-8 colorimetry, and the growth inhibition rate was compared. The results showed that under the condition of hypoxia and glucose deficiency, the growth of rat cardiomyocytes was significantly inhibited (p figure 2 .

[0039] (2) The effect of exosomes on myocardial cells in rats with ischemia-...

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Abstract

The invention relates to a preparation method of a mesenchymal stem cell exosome. The preparation method comprises the following steps: culturing mesenchymal stem cells for several hours by using a serum-free conditioned culture medium, centrifuging and removing the cells, collecting a liquid supernatant crude product, then re-centrifuging, filtering, and removing the residual cells as well as cell debris to prepare liquid supernatant containing an exosome; preparing the exosome by stepwise centrifugation of polyethylene glycol adopted by the liquid supernatant containing the exosomes. Exosome-expressed specific proteins CD63 prepared by the preparation method provided by the invention can effectively improve the mitochondria damage of the myocardial cells, prevent the cell apoptosis and improve the functions of the myocardial cells and are used for preparing a drug for treating myocardial ischemia reperfusion injury.

Description

technical field [0001] The invention relates to a method for preparing biological products, in particular to a method for preparing stem cell exosomes, and its application in the preparation of therapeutic drugs for treating myocardial ischemia-reperfusion injury. Background technique [0002] Myocardial ischemia-reperfusion (ischaemia / reperfusion) injury refers to the process of progressive and more serious pathological damage to the original ischemic myocardial tissue when the coronary arteries supplying blood to the heart are partially or completely blocked for a short period of time and the blood vessels are unblocked again. Severe cardiac arrhythmias occurred leading to sudden death. The damage manifests itself in the death of cardiomyocytes, arrhythmia, cardiac dysfunction, and ultimately heart failure. A series of damaging changes in myocardial cell structure, function and energy metabolism caused by myocardial ischemia are more prominent after recanalization. [00...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61K35/28A61P9/00A61P9/10
CPCA61P9/00A61P9/10A61K35/28C12N5/0668C12N2509/00
Inventor 章毅陈亮陆薇黄海
Owner 章毅
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