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Isolation and culture method of placenta mesenchyma precursor stem cells

A technology for isolating and culturing stem cells, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc. It can solve the problems of low proliferation, influence effect and application scope, and decrease in the positive rate of CD271, and achieve the ability to proliferate and differentiate. strong effect

Inactive Publication Date: 2015-05-20
JILIN JIHUI BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some patents (201010178277.5; CN100344757C) apply for the preparation method of placental mesenchymal stem cells mainly adopt tissue block method or enzymatic digestion, and then adhere to culture medium containing fetal bovine serum or other serum, and use fine adherent property to separate Culture placental mesenchymal stem cells or placental stem cells. The mesenchymal stem cells obtained by these methods express the markers of mesenchymal stem cells, but most of them are actually relatively mature mesenchymal progenitor cells, which have the limitations of mesenchymal stem cells, such as Low proliferation, limited life, gradually loses the characteristics of stem cells during in vitro expansion, does not express nestin, and the positive rate of CD271 also decreases, which greatly affects their effect and application range in tissue engineering

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  • Isolation and culture method of placenta mesenchyma precursor stem cells
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  • Isolation and culture method of placenta mesenchyma precursor stem cells

Examples

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Embodiment 1

[0024] Example 1: Isolation and culture method of placental mesenchymal precursor stem cells

[0025] 1. Aseptically collect the placenta of full-term infants, and wash the blood clot with 2 L of phosphate buffered saline containing gentamicin (100 U / ml) and heparin (50 U / ml);

[0026] 2. Separate the chorion and amniotic membrane, mechanically remove the decidua tissue with tissue forceps, rinse the chorion and blood vessels with PBS, and cut into pieces (2mm×2mm);

[0027] 3. Digest with 0.1% collagenase Ⅰ and tissue dispase Ⅱ by shaking at 37°C for 1 hour;

[0028] 4. Then digest with 0.05% trypsin for 0.5 hours;

[0029] 5. Dilute with a large amount of phosphate buffer, and filter tissue residue with a 200-mesh filter. Centrifuge at 2000rpm for 10min, resuspend in phosphate buffer;

[0030] 6. Discontinuous density gradient centrifugation: Add 50%, 30%, 20%, 10% Percoll solution in sequence, centrifuge at 2500rpm for 20min in discontinuous density gradient, and absorb ...

Embodiment 2

[0041] 1) Isolation of placental tissue: Aseptically collect the placenta, using toothed forceps and a scalpel to separate the chorion and amniotic membrane by conventional methods, wash the chorion and blood vessels with PBS, and cut into 2mm×2mm pieces;

[0042] 2) Combined enzyme digestion: 1% collagenase Ⅰ and 1% tissue dispase Ⅱ, 37 ° C for 6 hours, then digested with 0.25% trypsin for 1 hour; dilute with 5LPBS or αMEM, and filter the tissue residue with a 200-mesh filter .

[0043] 3) Discontinuous density gradient separation: use 50% Percoll solution, centrifuge at a density gradient of 2500rpm for 20min, absorb single cells between 30%Percoll, and wash twice by PBS centrifugation;

[0044] 4) Add neurotrophic factor receptor mouse anti-human CD271 monoclonal antibody, incubate for 30 minutes, wash twice with PBS, centrifuge at 1500 rpm for 5 minutes; add magnetic bead-labeled secondary antibody anti-mouse IgG1, incubate at 2-8°C for 15 minutes, wash twice with PBS, Ce...

Embodiment 3

[0047] 1) Isolation of placental tissue: Aseptically collect the placenta, using toothed forceps and a scalpel to separate the chorion and amniotic membrane by conventional methods, wash the chorion and blood vessels with PBS, and cut into 2mm×2mm pieces;

[0048] Combined enzyme digestion: 0.5% collagenase Ⅰ and 0.5% tissue dispase Ⅱ, 37 ° C shaking digestion for 3 hours, and then digested with 0.15% trypsin for 0.5 hours; dilute with 3.5 LPBS or αMEM, and filter the tissue residue with a 200-mesh filter;

[0049] 2) Discontinuous density gradient separation: use 50% Percoll solution, centrifuge at a density gradient of 2500rpm for 20 minutes, absorb single cells between 30% Percoll, and wash twice with PBS;

[0050] 3) Add neurotrophic factor receptor mouse anti-human CD271 monoclonal antibody, incubate for 30 min, wash twice with PBS, centrifuge at 1500 rpm for 5 min; add magnetic bead-labeled secondary antibody anti-mouse IgG1, incubate at 2-8°C for 15 min, wash twice with ...

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Abstract

The invention relates to an isolation and culture method for placenta mesenchyma precursor stem cells. The method includes the processes of placental tissue isolation, combined enzyme digestion, discontinuous gradient density isolation, immunomagnetic bead isolation and mesenchymal precursor stem cell suspension culture. The method concretely comprises the following steps: carrying out placental tissue separation: aseptically collecting placenta through a conventional technology; carrying out combined enzyme digestion; diluting by using 2-5L of PBS or alpha-MEM, and filtering tissue residues by using a 200 mesh filter screen; carrying out discontinuous gradient density isolation; adding a neurotrophic factor receptor mouse anti-human CD271 monoclonal antibody, sorting through a magnetic separation column, and collecting eluted marked cells; carrying out placenta precursor stem cell culture; adding a fresh medium every 2-3d; and allowing placenta precursor stem cells to grow in an embryoid body-like sphere manner within 10-14d. The mesenchyma precursor stem cells obtained in the invention express mesenchymal stem cell signs, also express neural stem cell marker nestin and CD271, and have the characteristics of strong self updating and differentiation capability, and wide application prospect.

Description

technical field [0001] The invention relates to a method for separating and culturing placental mesenchymal precursor stem cells, belonging to the field of biotechnology. Background technique [0002] The cultivation, expansion and transplantation of stem cells have become a hot spot in current regenerative medicine. Utilizing the ability of stem cells to differentiate into a variety of cells to promote the repair and regeneration of damaged tissues and organs in the process of body injury and disease recovery, and to improve or restore the function of damaged tissues and organs has become the focus of biology, basic medicine and clinical medicine. hot spot. According to reports, about tens of millions of people around the world suffer from various forms of trauma every year, and millions of people lose their functions due to fibrosis of vital organs during the recovery process of diseases, and hundreds of thousands of people are eager for various treatments. organ transpl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 魏斯溧徐冲吴杨
Owner JILIN JIHUI BIOTECH CO LTD
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