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A method for isolating human placental chorionic mesenchymal stem cells

A technology of mesenchymal stem cells and separation methods, applied in the field of human placental chorionic mesenchymal stem cells, can solve the problems of decreased differentiation ability and cell number, strong immune rejection of allogeneic transplantation, and high chance of virus infection, etc., to reduce manual processing time and manpower consumption, increase the area of ​​tissue adhering to the wall, and reduce the effect of contamination opportunities

Active Publication Date: 2020-09-29
GUANGZHOU WOMEN AND CHILDRENS MEDICAL CENTER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the mesenchymal stem cells used are mainly bone marrow mesenchymal stem cells, which are convenient and quick to source, but the mesenchymal stem cells from this source have the following defects: 1. The content in bone marrow is low (about 0.1-0.01‰), and with As the age increases, the expansion, differentiation ability and cell number show an obvious downward trend; 2. There are ethical issues and the source is limited; 3. The donor feels uncomfortable and is not easy to accept; 4. The chance of virus infection is high; 5. Allogeneic transplantation strong immune rejection
These methods inevitably have the disadvantages of tissue block method or enzyme digestion method, some methods also increase the operating procedures, and some methods also add other chemical reagents or biological products

Method used

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  • A method for isolating human placental chorionic mesenchymal stem cells
  • A method for isolating human placental chorionic mesenchymal stem cells
  • A method for isolating human placental chorionic mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0047] 1) Human placental chorionic tissue was taken, cut into 1-3 cm strips, and washed repeatedly with normal saline until there was no blood;

[0048] 2) Use a hand-held electric homogenizer to process the chorion to 0.1-0.3cm 3 For fine particles, wash again with normal saline until clear;

[0049] 3) Process again with a homogenizer to 0.1-0.5mm 3 After left and right small particles, centrifuge at 300r / min for 5min to remove the upper layer of blood cells;

[0050] 4) Add the same volume of trypsin and collagenase II to the precipitation, and digest at 37°C for 0.5h;

[0051] 5) After the digest was centrifuged and washed twice with normal saline, the culture medium was added to the precipitate and placed at 37°C, 5% CO 2 Cultivated in an incubator;

[0052] 6) Discard the tissue pieces and culture medium on the 7th day, rinse the bottom of the culture bottle with normal saline, add fresh medium, and then replace the medium every 3 days until the cell confluence reac...

Embodiment 2

[0054] 1) Human placental chorionic tissue was taken, cut into 1-3 cm strips, and washed repeatedly with normal saline until there was no blood;

[0055] 2) Use a hand-held electric homogenizer to process the chorion to 0.1-0.3cm 3 For fine particles, wash again with normal saline until clear;

[0056] 3) Process again with a homogenizer to 0.1-0.5mm 3 After left and right small particles, centrifuge at 500r / min for 5min to remove the upper layer of blood cells;

[0057] 4) Add the same volume of trypsin and collagenase II to the precipitate, and digest at 37°C for 1 hour;

[0058] 5) After the digest was centrifuged and washed twice with normal saline, the culture medium was added to the precipitate and placed at 37°C, 5% CO 2 Cultivated in an incubator;

[0059] 6) Discard the tissue pieces and culture medium on the 7th day, rinse the bottom of the culture bottle with normal saline, add fresh medium, and then replace the medium every 3 days until the cell confluence reac...

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Abstract

The invention discloses a human placenta chorionic mesenchymal stem cell separation method, which comprises: 1) taking healthy human placenta chorionic tissue, cutting into strip blocks, and completely rinsing with physiological saline; 2) adding a proper amount of physiological saline, chopping the chorion into fine particles by using a homogenizer, and rinsing with physiological saline until the solution is clarified; 3) treating again with the homogenizer to achieve micro-particles, and carrying out centrifugation to remove the upper layer blood cells; 4) adding an enzyme to the precipitate, digesting, carrying out centrifugation, and separating the precipitate; 5) carrying out centrifugal washing with physiological saline twice, adding a culture medium to the precipitate, inoculating, and culturing; and 6) discarding the tissue blocks and the culture medium at the 7th day, rinsing the bottom of the culture flask by using physiological saline, adding a fresh culture medium, changing the culture medium every 3 days until the cell fusion degree achieves 80-90%, and carrying out digestion passage with trypsin to obtain the human placenta chorionic mesenchymal stem cells. According to the present invention, the operation is simple and rapid, and the high-quality human placenta chorionic mesenchymal stem cells can be obtained.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for separating human placental chorionic mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells are multipotent adult stem cells commonly found in different tissues. Mesenchymal stem cells have been isolated and cultured from bone marrow, fat, dental pulp, amniotic membrane, chorion, decidua, umbilical cord, umbilical cord blood and other tissues. Studies have shown that mesenchymal stem cells have strong differentiation ability, short doubling time, immunoregulatory effect, low immunogenicity, and easy transfection characteristics. They are an ideal seed cell and gene therapy carrier for regenerative medicine. Stem cells are a hotspot in clinical transformation application research, but how to easily and quickly isolate a large number of primary mesenchymal stem cells in vitro is the basis for mesenchymal stem cell research and appl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12N5/0735
CPCC12N5/0605C12N5/0668C12N2509/00
Inventor 吴韶清欧志英金宇林李发涛唐婕吴洁莹陈劲松谢闺娥陆琰梁绮华刘东
Owner GUANGZHOU WOMEN AND CHILDRENS MEDICAL CENTER
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