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Extraction and culture method of human umbilical cord mesenchymal stem cells

A technology of mesenchymal stem cells and culture methods, applied in the direction of animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of limited quantity and limited application, and achieve the effect of maintaining the ability of proliferation and differentiation

Inactive Publication Date: 2013-03-13
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the limited number of mesenchymal stem cells derived from adult tissues and the influence of factors such as patient age and disease, their clinical application is limited.

Method used

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  • Extraction and culture method of human umbilical cord mesenchymal stem cells
  • Extraction and culture method of human umbilical cord mesenchymal stem cells
  • Extraction and culture method of human umbilical cord mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Human umbilical cord mesenchymal stem cell medium preparation: add 25ml fetal bovine serum, 2.5μg human fibroblast growth factor, 2.5μg epithelial cell growth factor, 0.5μg cell transcription factor, 5μg cholesterol, 5ml antibiotic to 500ml low-glucose medium, 0.22 Sterilize with a μm filter and store at 4°C for later use.

[0042] Extraction and culture of human umbilical cord mesenchymal stem cells

[0043] 1. Take the umbilical cord of a healthy, naturally delivered fetus and cut it into 3cm pieces first, then wash it with PBS several times to wash off the residual blood, remove two arteries and one vein, and cut it to 1mm 3 Little pieces left and right.

[0044] 2. Add PBS to wash, centrifuge at 800rmp for 5 minutes to remove supernatant, and repeat washing twice.

[0045] 3. Add 20ml type I collagenase to digest for 2 hours, and mix the tissue pieces intermittently during the digestion process to promote digestion.

[0046] 4. After the digestion is completed, c...

Embodiment 2

[0050] Human umbilical cord mesenchymal stem cell medium preparation: add 50ml fetal bovine serum, 5μg human fibroblast growth factor, 5μg epithelial cell growth factor, 1μg cell transcription factor, 10μg cholesterol, 5ml antibiotics to 500ml low-glucose medium, filter with 0.22μm filter Sterilize and store at 4°C for later use.

[0051] Extraction and culture of human umbilical cord mesenchymal stem cells

[0052] 1. Take a 9cm healthy and natural fetal umbilical cord and cut it into 3 sections of 3cm each, then wash with PBS several times to wash off the residual blood, remove two arteries and one vein, and cut them to 1mm 3 Little pieces left and right.

[0053] 2. Add PBS to wash, centrifuge at 800rmp for 5 minutes to remove supernatant, and repeat washing twice.

[0054] 3. Add 20ml type I collagenase to digest for 2 hours, and mix the tissue pieces intermittently during the digestion process to promote digestion.

[0055]4. After the digestion is completed, centrifug...

Embodiment 3

[0059] Detection of cell proliferation ability:

[0060] 1. Take human umbilical cord mesenchymal stem cells cultured at passage 3, passage 7 and passage 10, and add 1ml of 0.25% trypsin to digest the cells.

[0061] 2. Count the cells with a hemocytometer and dilute the cell suspension to 1×10 5 pieces / ml.

[0062] 3. Use three 12-well cell culture plates, inoculate the third generation cells 1×10 in the first row of four wells of the first culture plate 5 cells / ml, the second row was inoculated with 1×10 cells of passage 7 5 cells / ml, the third row was inoculated with 1×10 cells of passage 10 5 cells / ml, add 2ml of cell suspension to each well, and use the same procedure for the other two culture plates.

[0063] 4. After 24 hours, take out the first culture plate, and count the number of cells in 4 wells of each type of cells.

[0064] 5. Take out the second culture plate after 36 hours, and count the number of cells in 4 wells of each type of cells.

[0065] 6. After...

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Abstract

The invention discloses an extraction and culture method of human umbilical cord mesenchymal stem cells. The umbilical cord tissue is cut up and digested by collagenase I and then transferred into a culture bottle containing a culture medium for continuous culture. The culture medium is a low-sugar DMEM (Dulbecco modified eagle medium) and is added with fetal bovine serum, fibroblast growth factor, epithelial cell growth factor, cell transcription factor and cholesterol. The extraction and culture method disclosed by the invention can culture human umbilical cord mesenchymal stem cells for a long time while maintaining the activity of the stem cells. Through the invention, the problems of excessively fast cell aging and differentiation in current culture of human umbilical cord mesenchymal stem cells are solved, and the human umbilical cord mesenchymal stem cells with the characteristics of stem cells can be obtained for a long time.

Description

technical field [0001] The invention relates to a method for extracting and culturing human umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells derived from mesoderm and ectoderm, and belong to adult stem cells. Mesenchymal stem cells have the ability of self-renewal and multi-directional differentiation potential, and can differentiate into bone, cartilage, fat, nerve, cardiac muscle and other tissue cells under specific induction conditions. Mesenchymal stem cells exist in a variety of tissues, and mesenchymal stem cells have been successfully obtained from bone marrow, fat, muscle and other tissues. Due to the limited number of mesenchymal stem cells derived from adult tissues and the influence of factors such as patient age and disease, their clinical application is limited. Umbilical cord and umbilical cord blood are an ideal source of mesenchymal stem cells because of their non-invasive way of ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 肖忠党孟宪会孙博胡飞虎梁高峰
Owner SOUTHEAST UNIV
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