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Human myocardial cell separation reagent, culture medium and separation and culture methods

A technology of cardiomyocytes and culture methods, applied in cell dissociation methods, cell culture active agents, cell culture supports/coatings, etc., capable of solving problems such as high fluorescence, low water solubility, phototoxicity and cytotoxicity

Active Publication Date: 2020-10-16
青岛百洋智心科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, it is known that (-)-Blebbistatin has some unfavorable chemical properties, such as instability to light, phototoxicity and cytotoxicity, high fluorescence and low water solubility, etc., and the solubility of (-)-Blebbistatin is only 10.9±0.9μM
Because of these unfavorable properties, researchers are biased against the application of (-)-Blebbistatin. So far, there has been no report on the use of (-)-Blebbistatin for the isolation or culture of human cardiomyocytes, especially primary human cardiomyocytes.

Method used

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  • Human myocardial cell separation reagent, culture medium and separation and culture methods
  • Human myocardial cell separation reagent, culture medium and separation and culture methods
  • Human myocardial cell separation reagent, culture medium and separation and culture methods

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Experimental program
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Effect test

Embodiment approach 1

[0058] Embodiment 1: Human cardiomyocyte separation test

[0059] The cardiomyocyte isolation process generally includes tissue transfer and sectioning, decalcification, enzymatic digestion and cell collection. Among them, the calcium-free bench-top solution (calcium-free solution) is the main reagent used in the separation process, which is used for decalcification, enzyme solution digestion and cell collection. According to literature (Liang Chen Guang-ran Guo, Man Rao, Kai Chen, Jiang-ping Song* and Sheng-shou Hu*, "A Modified Method for Isolation of Human Cardiomyocytes to Model Cardiac Diseases.", Journal of Translational Medicine ( 2018).) The cardiomyocyte isolation experimental protocol described in the operation. Specific steps are as follows.

[0060] Immediately place the isolated left atrial appendage in organ preservation solution (manufactured by the University of Wisconsin, USA) (about 4°C), quickly transfer it to a 60mm cell culture dish in a clean bench, a...

Embodiment approach 2

[0077] Embodiment 2: Human cardiomyocyte culture test

[0078] (1) Effects of different concentrations of (-)-Blebbistatin and para-aminoblebbistatin on the rod rate of human cardiomyocytes

[0079] The medium obtained by adding 10% fetal bovine serum, 100 U / ml penicillin and 100 µg / ml streptomycin to M199 was used as a basal medium. The experiment was divided into 9 groups, and 0, 2.5, 5, 10, 20 μM (-)-Blebbistatin and 5 μM, 10 μM, 20 μM, 50 μM para-aminoblebbistatin were added to the basal medium respectively. The isolated human cardiomyocytes were divided into 9 groups, and the cells were plated on 48-well cell culture plates coated with 200 μg laminin in advance using 9 kinds of media, and placed in a 37°C cell culture incubator (5% CO 2 , the relative saturated humidity is 95%) and cultured for 7 days, and the rod-shaped rate of cardiomyocytes was calculated, and the results were as follows figure 2 shown.

[0080] Depend on figure 2 A shows that the concentration...

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Abstract

The invention relates to a human myocardial cell separation reagent, culture medium and separation and culture methods. The human myocardial cell separation reagent disclosed by the invention contains(-)-Blebbistatin, and the human myocardial cell culture medium contains (-)-Blebbistatin or para-aminoblebbistatin. By the adoption of the reagent, the culture medium and the separation and culture methods, cells can be kept in the optimal state and the morphology, survival rate and purity of the cells are kept in separation and culture.

Description

technical field [0001] The present invention relates to separation reagents and separation methods for isolating human cardiomyocytes. The present invention also relates to a culture medium and a culture method for culturing human cardiomyocytes. Background technique [0002] The death caused by cardiovascular disease accounts for more than 40% of the total number of deaths due to disease, which is higher than that of tumors and other diseases, and is the biggest killer of human life and health. Therefore, the prevention and treatment of cardiovascular diseases is very important to human health, and the importance of scientific research on the prevention and treatment of cardiovascular diseases is self-evident. [0003] To study the prevention and treatment of any disease, we must have a good cell model. However, unlike other somatic cells, cardiomyocytes, the research model of cardiovascular diseases, have the characteristics of strong motility and high oxygen consumption...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2509/00C12N2500/30C12N2533/52C12N2533/90C12N2501/73C12N2500/34C12N2500/46
Inventor 胡盛寿周冰莹侯永凤师珣唐晓丽
Owner 青岛百洋智心科技有限公司
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