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Human chorion mesenchymal stem cell isolated culture method

A technology for isolating and culturing stem cells, applied in the field of biomedicine, can solve the problems of limited effect of anti-pollution measures, increase the cost of culture, complicated steps, etc., achieve stable maintenance of cell stemness and cell viability, save enzyme digestion time, and operation process. simple effect

Inactive Publication Date: 2016-06-29
YUNNAN SHUNXI REGENERATION MEDICAL ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are two methods for isolating chorionic mesenchymal stem cells: tissue block culture and enzymatic digestion. Enzyme digestion is the most widely used separation method. There are many kinds of enzymes used. Most of the digestion methods using trypsin and collagenase step by step are time-consuming and cumbersome. The digestion process will cause great damage to the cells and weaken the cell viability. A variety of enzymes and serum introduced a variety of foreign substances, hindering the clinical application of mesenchymal stem cells
The mesenchymal stem cells obtained by the tissue block culture method have the advantages of high cell purity and strong cell viability, but the success rate of isolation is low, the culture period is long, and animal-derived components such as fetal bovine serum are added to the culture, which increases the culture cost and also The introduction of exogenous animal protein makes the cultured cells unsuitable for clinical application
The capillaries attached to the chorion will not be removed during chorion stripping, which makes the types of isolated cells complex, mixed with many endothelial cells and blood cells, and affects the normal growth process of mesenchymal stem cells
The existing technology of placenta collection and chorion separation still lacks effective measures to reduce pollution. The anti-pollution measures have limited effect, the pollution rate of primary culture is high, and there is a lack of methods to reduce pollution from the source. Inhibiting the reproduction of contaminating bacteria during the process cannot effectively reduce pollution; adding antibiotics to the culture system is not conducive to the clinical application of chorionic stem cells

Method used

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  • Human chorion mesenchymal stem cell isolated culture method
  • Human chorion mesenchymal stem cell isolated culture method
  • Human chorion mesenchymal stem cell isolated culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Stripping of human placental chorion

[0050] Before the operation, first wipe the collection container with 75% alcohol for disinfection, then take it into the biological safety cabinet, open the placenta collection container in a sterile environment, and wipe the blood around the mouth of the container with gauze soaked in 75% alcohol to avoid If it drips on the operating table to cause contamination, turn the placenta over with 14cm sterilized tweezers, turn the placenta on the side close to the fetus up, and use 10cm sterilized tweezers to gently peel off the amniotic membrane to expose the chorion layer of the subamniotic membrane. The ophthalmic scissors cut the chorion layer around the umbilical cord along the section of the umbilical cord. During the cutting process, cut along the chorion as much as possible to remove the villi and capillaries connected under the chorion, and place the obtained tissue in 0.9% sodium chloride Wash and remove the blood in the inje...

Embodiment 2

[0052] Primary culture of human chorionic mesenchymal stem cells

[0053] Put 2ml of detoxified, cleaned, and chopped tissue into a T-75 cell culture bottle, and spread the tissue on the bottom of the cell culture bottle with a Pasteur pipette. Make sure there is a gap between the tissues to prevent the tissue from crawling. A certain space for the cells to grow, the cell culture flask was placed upside down in 5% CO 2 , In a cell culture incubator at 37°C, place it for 30 minutes so that the tissue is firmly attached to the bottom of the cell culture bottle. Slowly add to the part of the culture bottle where there is no tissue adhesion, cover the lid, turn the cell culture bottle over and shake gently, so that the medium is evenly spread between the tissues, and place the cell culture bottle in 5% CO 2 , 37°C cell incubator for static culture, replace the serum-free medium of mesenchymal stem cells after 5 days, observe under the microscope after 7 days of culture, it can be...

Embodiment 3

[0055] Human chorionic mesenchymal stem cells passage

[0056] Remove the medium in the T-75 culture flask completely, add 15ml of PBS to wash the cells once (washing the cells with PBS is not necessary, it can be determined according to the actual situation), remove the PBS, add 3ml of recombinant cell trypsin solution, and put Digest in a 37°C incubator for about 2 minutes. During this process, observe the shrinkage of the adherent cells under an inverted microscope. After all the cells have detached from the bottom of the bottle, pat the side wall of the culture bottle with your palm, and observe under the microscope whether the cells have been digested into individual cells. For cells, add 15ml of mesenchymal stem cell serum-free medium to dilute the recombinant cell trypsin solution to stop the trypsin action, then transfer the cell suspension to a 50ml centrifuge tube, take 200ul of the cell suspension and stain with trypan blue to count the number of viable cells . The...

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Abstract

The invention relates to a method for separating and culturing human chorionic mesenchymal stem cells, belonging to the technical field of biomedicine. The present invention obtains mesenchymal stem cells and in vitro culture and freezing of mesenchymal stem cells through the collection of human placental chorionic tissue, stripping of chorionic tissue, disinfection, washing, shredding, primary separation, and the isolated cells are related detection. The invention can separate and obtain a large number of mesenchymal stem cells with uniform and stable phenotype from human placental chorionic tissue, which meets clinical application standards through testing. The number of cells that can meet cryopreservation or clinical use can be obtained by culturing in vitro expansion.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for separating and culturing clinical-grade human chorionic mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of primitive cells with self-renewal ability and multilineage differentiation potential under suitable microenvironment. It is first found in bone marrow and subsequently isolated in fat, bone, muscle, lung, liver, pancreas, and in umbilical cord, cord blood, and placental tissue. Because of its strong proliferation ability, strong differentiation ability, low immunogenicity and immune regulation function, its clinical application value has attracted more and more attention. [0003] As a cell product to be used in clinical cell therapy, it should have the characteristics of convenient tissue collection, wide range of sources, no ethical restrictions, and simple separation process. However, bone marro...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2509/00
Inventor 闫姗姗李一佳甘明川莫春红
Owner YUNNAN SHUNXI REGENERATION MEDICAL ENG CO LTD
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