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Method for separating and extracting myocardial cells of grown-up rats by adopting one-step enzyme digestion process

A technology of enzymatic digestion and cardiomyocytes, which is applied to animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of complex operation steps, low survival rate, and unstable separation and extraction methods, so as to improve the survival rate, The effect of simplifying the experimental steps and reducing the probability of calcium imbalance in cardiomyocytes

Inactive Publication Date: 2013-02-06
AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing separation and extraction methods such as instability, low survival rate and complicated operation steps, and provide a method for separating and extracting adult rat cardiomyocytes by one-step enzymatic digestion

Method used

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Embodiment Construction

[0027] 1. Experimental animals: clean adult SD rats (provided by the Experimental Animal Center of Third Military Medical University), male or female, weighing 200-350 g.

[0028] 2. Main reagents: Ethylene glycol bis(2-aminoethyl ether) tetraacetic acid (EGTA), carnitine, creatine, taurine, bovine serum albumin (BSA), HEPES, and laminin were all purchased from SIgma, USA Company, type II collagenase, M199 medium were purchased from Gibco, USA, rabbit anti-rat myocardial actin antibody, secondary antibody, DAB kit (Wuhan Boster Company). The remaining reagents were domestic analytical reagents.

[0029] 3. Main instruments: Langendorff perfusion device (ALC-HP) was purchased from Shanghai Alcott Biotechnology Co., Ltd. Constant temperature shaker, carbon dioxide incubator.

[0030] 4. Solution preparation

[0031] Perfusion mother solution: NaCl 130 mMol / L, KCl 5.4 mMol / L, HEPES 5 mMol / L, D-Glucose 10 mMol / L, MgCl 2 ·6H 2 O 3.5 mMol / L, NaH 2 PO 4 0.4 mMol / L, with O 2 ...

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Abstract

The invention discloses a method for separating and extracting the myocardial cells of grown-up rats by adopting a one-step enzyme digestion process. The method comprises the steps of: carrying out reverse perfusion through aorta by adopting a Langendorff perfusion device, sequentially perfusing by using a calcium-containing solution, a calcium-free EGTA (Ethylene Glycol Tetraacetic Acid) solution and a II collagenase solution, collecting a II collagenase circulating solution to be added into a 10% BSA (Bovine Serum Albumin) solution, placing the ventricle in a constant-temperature shaker for shaking and digesting, blowing and beating the circulating solution by using a suction tube until the circulating solution is completely digested to obtain a digestion solution, filtering, naturally settling the filtrate, collecting cell sediments, and suspending in an enzyme eluant and repeatedly settling to obtain the myocardial cells. 75-93 percent of the rat myocardial cells obtained by adopting the method have activity, and the yield of living cells of the heart of each rat can reach up to about (5-8)*10<8>. According to the invention, the myocardial cells are separated by adopting single digestive enzyme and are settled by adopting a step-by-step gradient recalcification method, thus the experimental steps are simplified and the survival rate of the myocardial cells is greatly increased. The method is simple and economic, and is used for effectively separating the myocardial cells of grown-up rats.

Description

technical field [0001] The invention relates to the technical field of cell separation and extraction, in particular to a method for separating and extracting adult rat cardiomyocytes by a one-step enzyme digestion method. Background technique [0002] With the continuous development of heart disease research, isolated heart experiments have been paid more and more attention, especially single cardiomyocytes with normal physiological functions have become an important basis for the study of cardiac metabolism, function, and pathophysiological mechanisms. At present, the extraction and culture of cardiomyocytes are mostly done in suckling rats or young rats, but there are differences in function and structure between suckling rat cardiomyocytes and mature cardiomyocytes. [0003] There are also many methods for the isolation and culture of adult rat cardiomyocytes. The perfusion enzymatic hydrolysis method has been used for the isolation of adult rat cardiomyocytes for more t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 梁贵友王峰汤全肖志超蔡庆勇
Owner AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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