Cardiomyocyte isolation reagent and isolation method

A technology of cardiomyocytes and separation methods, applied in the direction of cell dissociation methods, biochemical equipment and methods, animal cells, etc., can solve the problems of human cardiomyopathy research and experiment limitations, can not fully simulate human physiological and pathological conditions, and achieve Improvement of cell viability and quality, shortening of operation time

Active Publication Date: 2021-12-14
深圳百洋智心医学研究有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These factors have led to no mature and reliable method for isolating human cardiomyocytes so far. The current common practice is to use the myocardium of mice or rats for cardiomyocyte isolation experiments.
However, neither mice nor rats can completely simulate the physiological and pathological conditions of human beings, so research and experiments on human myocardial diseases are always limited

Method used

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  • Cardiomyocyte isolation reagent and isolation method
  • Cardiomyocyte isolation reagent and isolation method
  • Cardiomyocyte isolation reagent and isolation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1: Effect of using BLEB in the preprocessing step

[0102] In the traditional myocardial cell separation process, after the myocardial tissue is removed, the tissue is placed in organ preservation solution (UW solution) for slicing and shredding. The operation time in UW solution is about 1.5 hours. In this example, the effect of adding BLEB to the UW solution used in the traditional method on the separation effect in the pretreatment step was studied. Specifically, myocardial tissue was collected from the left atrial appendage of 4 male patients (age 75±7 years old) undergoing heart disease surgery, and the pretreatment solution was obtained by adding 10 μM BLEB to the UW solution, and the removed tissue was divided into two equally, respectively Proceed to the slicing and shredding steps. After shredding, decalcification, digestion and recalcification were performed according to the literature method (Guo et al., Amodified method for isolation of human cardio...

Embodiment 2

[0103] Example 2: Effect of presence or absence of an oxygenation step

[0104] First, prepare calcium-free liquid according to the following formula:

[0105] Element concentration Element concentration Sodium chloride 126mM taurine 2mM potassium chloride 4.4mM Creatine 5mM Magnesium chloride hexahydrate 5mM sodium pyruvate 5mM Sodium dihydrogen phosphate 5mM penicillin 200U / ml 4-Hydroxyethylpiperazineethanesulfonic acid 5mM streptomycin 200μg / ml glucose 22mM BLEB 10μM Deionized water

[0106] The above-mentioned calcium-free solution was divided into two groups for experimentation, and the calcium-free solution of one group was continuously filled with oxygen for 20 minutes before use (the oxygenated group), and the calcium-free solution of the other group was not filled with oxygen (the non-oxygenated solution). group), directly used for the isolation of cardiomyocytes. Myocard...

Embodiment 3

[0108] Embodiment 3: the comparison of cleaning decalcification method and gradient decalcification method

[0109]Myocardial tissues were collected from the left atrial appendages of 4 heart disease patients (aged 50-70 years old), and were equally divided into two groups after the pretreatment step of Example 1. The first group carries out the gradient decalcification operation known in the prior art, uses the calcium-free liquid used in embodiment 2 to divide and carry out gradient decalcification 3 times, and the decalcification time is 2, 3, 4 minutes respectively, during this period constantly with sequential Shake the Erlenmeyer flask clockwise to make the tissue fully contact with the calcium-free solution, so that the calcium ions in the cells in the tissue can continuously flow out. After each timing, filter out the calcium-free solution in the bottle, and replace it with a new calcium-free solution for the next decalcification. The second group did not perform the ...

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Abstract

The present invention relates to separation reagents and separation methods for mammalian cardiomyocytes, particularly human cardiomyocytes. Specifically, the present invention relates to a method for separating cardiomyocytes, which includes the following steps: a pretreatment step, a cleaning and decalcification step, a digestion step, an equilibration step, and a drip recalcification step, wherein at least An operating fluid contains BLEB and / or PAB or a physiologically acceptable salt thereof. Therefore, the present invention can significantly reduce the operation time of cardiomyocyte separation, and at the same time improve the viability and quality of the separated cells.

Description

[0001] The scientific research project involved in this application is a medical and health science and technology innovation project of the Chinese Academy of Medical Sciences, and the project number is 2017-I2M-1-003. technical field [0002] The present invention relates to separation reagents and separation methods for mammalian cardiomyocytes, particularly human cardiomyocytes. Background technique [0003] In order to study the physiological characteristics of cells and the influence of various factors (including drug action) on cells, so as to meet the needs of scientific research and new drug development, it is often necessary to extract and separate individual cells from living tissues. Generally speaking, cell separation techniques include mechanical dispersion and digestion separation. Among them, the mechanical separation method is suitable for tissues with less fibrous components, such as brain tissue or some embryonic tissues; the digestion and separation metho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2509/00
Inventor 胡盛寿周冰莹师珣唐晓丽
Owner 深圳百洋智心医学研究有限公司
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