High-migration mesenchymal stem cell and preparation method and application thereof

A mesenchymal stem cell and single cell technology, applied in the field of stem cells, can solve the problem of lack of targeted migration ability of CXCR4 expression level of mesenchymal stem cells, and achieve the effect of strong cell migration ability, small size, and enhanced migration ability

Active Publication Date: 2021-07-13
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Three-dimensional (3D) hanging drop culture is also used, for example: WO2009134532A2 discloses a hanging drop culture method, although this method can form cell aggregates, but this method is a static culture method, and the expression level of CXCR4 in mesenchymal stem cells obtained by it is and the ability to target migration to ischemic tissue is still insufficient

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  • High-migration mesenchymal stem cell and preparation method and application thereof
  • High-migration mesenchymal stem cell and preparation method and application thereof
  • High-migration mesenchymal stem cell and preparation method and application thereof

Examples

Experimental program
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Embodiment 1

[0037] This embodiment provides a method for preparing high-migratory mesenchymal stem cells, which specifically includes the following steps:

[0038] (1) Using the method of cell culture spinner bottle rotation culture, the mesenchymal stem cells derived from human placenta were divided into 3 × 10 5 Cells / mL seeding density was suspended in mesenchymal stem cell culture medium, and the culture medium was placed in a cell spinner bottle at 37°C, 5% CO 2 Stirring under certain conditions to achieve flowing liquid culture, the stirring speed is 60rpm, so that the culture medium is in a flowing state in the incubator, and the culture is continuous for 60 hours. During the culture, the mesenchymal stem cells aggregate to form cell balls of uniform size, and the diameter of the cell balls is 200μm about. After the cultivation, the cell spheres were collected by centrifugation;

[0039] Among them, the mesenchymal stem cell culture medium contains 5% human platelet lysate (PLUS ...

Embodiment 2

[0076] Example 2: Comparative experiments on the effects of different culture methods on the migration ability of mesenchymal stem cells and the role of CXCR4 in them

[0077] The migration ability of the mesenchymal stem cells cultured by the different methods of the above-mentioned Example 1, Comparative Example 1 and Comparative Example 2 was measured with a Transwell chamber of a 24-well plate with a pore size of 8 μm. Specifically:

[0078] Add 600 μL of 0.5% BSA-DMEM or 50% mouse myocardial infarction tissue extract-DMEM (mouse acute myocardial infarction 24h, hydrolyzate of infarcted and ischemic myocardium) into the lower chamber of the orifice plate, and add 100 μL of Differently treated mesenchymal stem cell suspensions, the cell density was 2×10 5 cells / mL.

[0079] The mesenchymal stem cells were grouped as follows: human placental mesenchymal stem cells of passage 5 in 2D adherent culture, mesenchymal stem cells cultured in hanging drops for 60 hours, and mesenc...

Embodiment 3

[0081] Example 3: Comparative experiment on pulmonary obstruction of mice injected intravenously with mesenchymal stem cells by different culture methods

[0082] The human placental mesenchymal stem cells of the 5th generation of adherent culture in Comparative Example 1 were transfected with luciferase lentivirus (pLV-Luc), and some of the cells were cultured in the spinner bottle of Example 1 for 60 h and the suspension of Comparative Example 2. Drop culture for 60 hours; the mesenchymal stem cells cultured by the three methods were digested with trypsin to form a single cell suspension, and the cell density was 1×10 6 cells / 200 μL. Will 1×10 6 The mesenchymal stem cells cultured by the above-mentioned different methods were slowly injected into BALB / C mice (8 weeks, body weight 18-22 grams) through the tail vein. After 24 hours, the mice were anesthetized, intraperitoneally injected with luciferin (D-luciferin, 150 mg / kg), and the Bruker small animal imaging system colle...

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Abstract

The invention provides a high-migration mesenchymal stem cell and a preparation method and application thereof. The preparation method of the high-migration mesenchymal stem cells comprises the following steps: inoculating and suspending mesenchymal stem cells in a mesenchymal stem cell culture solution, carrying out flowing liquid culture in a cell culture rotary bottle or a stirring type fermentation tank without adding a microcarrier in the culture process, collecting cell balls after culture is finished, washing and digesting to form single cells, and obtaining the high-migration mesenchymal stem cells. The prepared high-migration mesenchymal stem cells are small in size, the average diameter is 11-14 microns, the cell surface CXCR4 expression rate reaches up to 30% or above, the cell migration ability is high, the lung retention phenomenon is basically avoided through intravenous injection, and the number of the mesenchymal stem cells reaching ischemic myocardium is remarkably increased.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to a high-migratory mesenchymal stem cell and its preparation method and application. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells, or mesenchymal stromal cells, MSC) is a kind of adult stem cells with multi-directional differentiation ability, which can differentiate into various tissue cells and participate in tissue regeneration by secreting a large number of growth and immune regulatory factors Microenvironment, and promote tissue damage repair. Mesenchymal stem cells are widely present in various tissues of the body, but their number decreases significantly with age. A large number of studies have proved that supplementing mesenchymal stem cells can promote the regeneration and repair of various tissues. [0003] Mesenchymal stem cells come from a wide range of sources and can be isolated from placenta, umbilical cord, bone marro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61P9/10A61P25/28A61P29/00A61P37/02A61P37/06A61P43/00
CPCC12N5/0662A61K35/28A61P25/28A61P43/00A61P37/02A61P37/06A61P9/10A61P29/00C12N2509/10C12N2509/00Y02A50/30
Inventor 吴耀炯莫妙华
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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