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Preparation method of human adult cardiac mesenchymal-like stem cells

A stem cell and adult technology, applied in the field of stem cells and regenerative medicine, can solve the problems of multi-operation steps, long culture time, etc., and achieve high cell expansion efficiency, easy operation, easy large-scale operation and standardized control effects

Pending Publication Date: 2021-11-19
大连市中心医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the purpose of cell sphere formation is to enrich stem cells, but this also requires more operation steps and longer culture time

Method used

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  • Preparation method of human adult cardiac mesenchymal-like stem cells
  • Preparation method of human adult cardiac mesenchymal-like stem cells
  • Preparation method of human adult cardiac mesenchymal-like stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054]The human adult cardiac-derived mesenchymal-like stem cells in this example were prepared by the following method:

[0055] 1. Atrial appendage tissue collection.

[0056] Select tissue providers who have been diagnosed with valvular heart disease combined with atrial fibrillation and have the indications for surgical treatment of atrial fibrillation, regardless of gender, aged 18-80 years old. Exclusion criteria: those with cardiomyopathy or other myocardial diseases such as myocarditis, hepatitis B, syphilis, AIDS positive or other known major active infections; those with serious primary diseases such as liver, kidney and hematopoietic system; those who abused drugs, drugs or alcohol historian. Sign the informed consent form before tissue collection. During cardiac surgery, the specimen provider aseptically collected the resected auricle tissue, and immediately placed it in a 2ml cryopreservation tube containing 1ml of calcium-magnesium-free PBS solution (phosphate-...

Embodiment 2

[0074] The human adult cardiac-derived mesenchymal-like stem cells in this example were prepared by the following method:

[0075] 1. Atrial appendage tissue collection: Same as in Example 1.

[0076] 2. Isolation of the atrial appendage tissue: the method is basically the same as in Example 1, except that the total mass of the atrial appendage tissue is 1.7 g, and the isolated comb muscle is 90 mg.

[0077] 3. Tissue digestion: the method is basically the same as in Example 1, the difference is that the isolated comb-shaped muscle tissue is evenly divided into two 50ml centrifuge tubes, and 20ml of tissue digestion solution is added to each centrifuge tube to carry out the same as in Example 1. Same digestion steps.

[0078] 4. Cell culture: basically the same as the method in Example 1, the difference is that the number of primary cells is 2×10 6 , all the above-mentioned primary cells were inoculated into a 10cm culture dish, and 8ml of complete medium was added, and the ...

Embodiment 3

[0082] The human adult cardiac-derived mesenchymal-like stem cells in this example were prepared by the following method:

[0083] 1. Atrial appendage tissue collection: Same as in Example 1.

[0084] 2. Isolation of the atrial appendage tissue: the method is basically the same as in Example 1, except that the total mass of the atrial appendage tissue is 0.6 g, and the isolated comb muscle is 25 mg.

[0085] 3. Tissue digestion: Same as Example 1.

[0086] 4. Cell culture: the method is basically the same as in Example 1, the difference is that the number of primary cells is 4×10 5 , On day 14, P0 generation cells grew like clones. Other cell culture processes are the same as in Example 1.

[0087] 5. Characterization identification: P7 human adult cardiogenic mesenchymal-like stem cells were collected to analyze the expression of surface marker molecules. Same as Example 1, the only difference is that the positive rate of CD90 is 32.57%. Same as Example 1, detect the gen...

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Abstract

The invention provides a preparation method of human adult cardiac mesenchymal-like stem cells, and belongs to the technical field of stem cells and regenerative medicine. The method comprises the following steps: 1, carrying out intraoperative collection of human adult atrial appendage tissues; 2, separating pectinate muscles from auricle tissues; 3, separating cardiac primary cells by an enzyme digestion method; 4, carrying out primary cell culture, passage and amplification; and 5, identifying the characteristics of the cells to obtain the human adult cardiogenic mesenchymal-like stem cells. According to the method, the cardiac stem cell preparation method which is simple and convenient to operate, easy to expand in scale, safe and controllable is provided for overcoming the defects that more operation steps and longer culture time are needed in an existing cardiac bulbous stem cell separation method. The risks of phenotype drift, tumorigenic variation and the like obtained by long-term in-vitro culture of cells can be reduced, and the stability of the in-vitro culture process is improved while the cell preparation efficiency is improved. The method can be used for establishing an in-vitro stem cell model and a stem cell bank and providing alternative seed cells for clinical individualized treatment.

Description

technical field [0001] The invention relates to the technical field of stem cells and regenerative medicine, in particular to a method for preparing human adult cardiogenic mesenchymal stem cells. Background technique [0002] Myocardial injury, repair and regeneration have always been a major problem in contemporary medicine. Traditional therapy can save the surviving myocardial tissue, but as the disease progresses, it will inevitably develop into heart failure. In recent years, regenerative medicine represented by stem cell therapy has brought new hope to the field of heart regeneration. Among the stem cell types in cardiac regeneration research, toti / pluripotent stem cells represented by embryonic stem cells and induced pluripotent stem cells can theoretically differentiate into cells with atrial, ventricular, and nodal characteristics. However, it has been reported in practice that pluripotent stem cell-derived cardiomyocytes are structurally and functionally immature...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/077
CPCC12N5/0668C12N5/0657C12N2509/00C12N2509/10C12N2501/115
Inventor 纪军高雁艳张利
Owner 大连市中心医院
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