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Isolating culture method and application of human umbilical cord mesenchymal stem cells

A technology for separating and culturing stem cells, applied in cell culture active agents, animal cells, tissue culture, etc., can solve problems such as thrombus, and achieve the effect of reducing the risk of thrombus and improving immune system diseases

Inactive Publication Date: 2021-06-18
贾在美
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to the genetic mutation of stem cells, it is found in clinical practice that mesenchymal stem cells express tissue factor, which is involved in the coagulation process as coagulation factor 3. Therefore, during the clinical use of mesenchymal stem cells, intravenous injection of mesenchymal stem cells may lead to the occurrence of thrombus.

Method used

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  • Isolating culture method and application of human umbilical cord mesenchymal stem cells
  • Isolating culture method and application of human umbilical cord mesenchymal stem cells
  • Isolating culture method and application of human umbilical cord mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This embodiment is a method for isolating and culturing human umbilical cord mesenchymal stem cells. The method for isolating and culturing comprises the following steps:

[0041] (a) Collect standard umbilical cord tissue, place the isolated human umbilical cord tissue in DMEM containing antibiotics (the antibiotics are penicillin and streptomycin, and the final concentration is 100U / mL), 3mM melatonin and 1% trehalose In the culture medium, store and transport at 4°C, and process the tissue within 48 hours; then wash with PBS and use surgical scissors to remove the arteries and veins, and the remaining tissue is the umbilical cord Wharton's jelly;

[0042] (b) Put the umbilical cord Wharton's jelly in a petri dish, add 10 mL of serum-free medium containing 10 μM melatonin (serum-free medium is SFM basal medium), and then cut the umbilical cord Wharton's jelly with tissue scissors Shredded jelly to 1~3mm 3 , then, place the Petri dish in 2% O 2 , 5%CO 2 Cultivate ov...

Embodiment 2

[0046] This example is a study on the number and viability of cells in the culture process of human umbilical cord mesenchymal stem cells

[0047] The research methods are:

[0048] (a) The cells harvested in Example 1 are used as the experimental group, and while the stem cells are isolated and cultured in Example 1, the non-treated control group for stem cells is set, that is, the stem cells do not add melatonin to the culture medium in the isolation and culture process, and do not use low The cells were cultured in an oxygen mode, and the rest of the operations were the same as in Example 1. After 20 days of cell culture, the cells were harvested and frozen.

[0049] (b) Rapid recovery of cryopreserved stem cells in the experimental group and control group in a 37-degree water bath.

[0050] (c) Cell number and viability detection were obtained according to the method of automatic cell counter.

[0051] During the whole process of umbilical cord mesenchymal stem cell iso...

Embodiment 3

[0054] This example is flow cytometry detection of tissue factor and chemokine expression of human umbilical cord mesenchymal stem cells obtained by different culture methods:

[0055] (1) According to Table 1, take 1.5*10^6 cells and centrifuge at 1500rpm for 7min. Discard the supernatant, add 4mL PBS to resuspend the cells, and centrifuge at 1500rpm for 7min. Then add 1.5mL PBS to resuspend the cells, and use nylon mesh to filter the cell suspension.

[0056] (2) Take 4 flow-type test tubes, mark 1 to 4, and add the flow-type antibody combination in advance (use a pipette to add antibodies to different positions at the bottom of the flow-type test tubes to prevent flow-type antibodies from crossing).

[0057] (3) Add 100 μL of cells (at least 1.0*10^5 cells) to each test tube on the machine, filter the cells with a 300 mesh (40 μM) cell sieve, fully vibrate and mix the antibody and cell suspension, and centrifuge instantaneously to sediment the cells on the tube wall to th...

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Abstract

The invention discloses an isolating culture method and application of human umbilical cord mesenchymal stem cells. The isolating culture method comprises the following steps: separating umbilical cord Wharton's jelly from umbilical cord tissues; adding a serum-free culture medium containing melatonin into the umbilical cord Wharton's jelly, cutting the umbilical cord Wharton's jelly into pieces, placing the umbilical cord Wharton's jelly at the concentration of O2 of 1.5-2.5% for adherent culture, when the adherent cell confluence is 50-80%, discarding fragments of the umbilical cord Wharton's jelly and continuing to culture; and after the culture is finished, digesting and dispersing the cells, and then centrifuging and seeding for subculture to obtain the human umbilical cord mesenchymal stem cells. According to the isolating culture method, the harvested human umbilical cord mesenchymal stem cells can generate more immunomodulatory cell factors and chemotactic receptors, the activity of the human umbilical cord mesenchymal stem cells harvested after cryopreservation and recovery is higher, and the expression of stem cell tissue factors can be reduced, so that the thrombus risk caused by the process of stem cell intravenous injection is reduced.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for separating and culturing human umbilical cord mesenchymal stem cells and its application. Background technique [0002] The emergence of cell therapy has provided a breakthrough technology for diseases that currently have no effective treatments, such as Car-T cells for tumors and mesenchymal stem cells for immune abnormalities. Among them, mesenchymal stem cells produce a large number of immune regulatory cytokines through paracrine, and have outstanding curative effects on many immune abnormal diseases. [0003] During the conventional isolation and culture of mesenchymal stem cells, due to genetic instability and superoxide accumulation, the cells gradually accumulate gene mutations with the increase of cell generations. It has been reported that when stem cells are passaged to P8, mesenchymal Genetic mutations in stem cells will increase significantly. In ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61K35/51A61K9/00A61P37/02
CPCA61K9/0019A61K35/51A61P37/02C12N5/0668C12N2501/30
Inventor 贾在美
Owner 贾在美
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