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Preparation method of bone marrow mesenchymal stem cells

A technology for bone marrow mesenchymal and stem cells, which is applied in the field of preparation of bone marrow mesenchymal stem cells to achieve the effects of being beneficial to storage and application, improving cell purity and high purity

Inactive Publication Date: 2020-03-06
吉林新海生物科技开发有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to solve many problems existing in the existing preparation methods of bone marrow mesenchymal stem cells, the present invention provides a preparation method of bone marrow mesenchymal stem cells

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  • Preparation method of bone marrow mesenchymal stem cells

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preparation example Construction

[0034] Such as figure 1 As shown, a method for preparing bone marrow mesenchymal stem cells of the present invention mainly obtains high-purity bone marrow mesenchymal stem cells by using gradient centrifugation combined with two rounds of antibody screening, and mainly includes the following steps:

[0035] Mix bone marrow fluid with 0.9% normal saline, and collect mesenchymal stem cells from the lower layer of plasma through density gradient centrifugation; culture buffy coat cells on a cell culture plate coated with CD73 antibody; when the cells grow to 70-80%, Digest the cells with mild digestive enzymes for stem cells; use a cell culture plate coated with CD90 antibody to continue culturing bone marrow mesenchymal stem cells; when the cells grow to 70-80%, digest the cells with mild digestive enzymes for stem cells and collect high-purity bone marrow mesenchymal stem cells.

[0036] The present invention adopts the gradient centrifugation adherence combined with two roun...

Embodiment 1

[0038]Example 1 Preparation of high-purity bone marrow mesenchymal stem cells - gradient centrifugation combined with two rounds of antibody screening (the present invention)

[0039] (1) Dissolve CD73 antibody and CD90 antibody with 50mM phosphate buffer solution to make the concentration 10-20μg / ml, add 2ml / dish into 100mm cell culture dish, and place overnight at 4°C;

[0040] (2) After discarding the coating solution the next day, wash with PBS 3 times, add 2ml of human serum albumin to each dish, and block at 37°C for 1 hour;

[0041] (3) wash with PBS 3 times, save for later use;

[0042] (4) Take 5-6 mL of bone marrow fluid, put it into a 50-mL centrifuge tube containing 2-3 mL of heparin, and oscillate the centrifuge tube at the same time to quickly mix the heparin and thick medullary blood to prevent the appearance of small clots;

[0043] (5) Add an equal volume of normal saline to the bone marrow fluid, blow and mix with a straw to make a cell suspension;

[0044]...

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Abstract

The invention discloses a preparation method of bone marrow mesenchymal stem cells, and relates to the field of stem cell preparation. The method comprises the following steps: mixing marrow fluid with 0.9% normal saline, carrying out density gradient centrifugation, and collecting mesenchymal stem cells from the lower layer of plasma; culturing leucoderma cells by using a cell culture plate coated with a CD73 antibody; when the cells grow to 70-80%, digesting the cells with stem cell mild digestive enzymes; continuously culturing the bone marrow mesenchymal stem cells by using a cell cultureplate coated with a CD90 antibody; and when the cells grow to 70-80%, digesting the cells by using the stem cell mild digestive enzymes, and collecting high-purity bone marrow mesenchymal stem cells.According to the invention, the high-purity mesenchymal stem cells are separated from the marrow fluid by combining gradient centrifugation adherence with two rounds of antibody forward screening, a large number of miscellaneous cells do not exist in the culture process, the cells combined with the antibody cannot be eluted into the cells, and the cell function is not affected. The preparation method disclosed by the invention is safe, and the capability of purifying and amplifying the bone marrow mesenchymal stem cells is strong.

Description

technical field [0001] The invention relates to the technical field of stem cell preparation, in particular to a method for preparing bone marrow mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) are a kind of pluripotent stem cells, which have all the common characteristics of stem cells, that is, self-replication ability and multi-lineage differentiation ability, and can be derived from a wide range of sources, such as bone marrow, umbilical cord blood, umbilical cord tissue , placental tissue, and adipose tissue. Mesenchymal stem cells have three notable characteristics: 1. Mesenchymal stem cells cultured in vitro are adherent growth. It has a wide range of sources, is easy to extract, and can be amplified in large quantities. 2. Mesenchymal stem cells highly express CD73, CD90 and CD105, but do not express markers such as CD31, CD34, CD45, HLADR, CD14, CD19 and CD11b. 3. Under appropriate stimulating factors, m...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N2501/50C12N2501/599C12N2509/00C12N2509/10
Inventor 于淼章宏曹月王月汪业菊刘玉东赵利伟
Owner 吉林新海生物科技开发有限公司
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