Method for acutely separating rat and suckling mouse cardiomyocytes

A technique for cardiomyocytes and rat suckling mice, applied in cell dissociation methods, animal cells, vertebrate cells, etc., to achieve the effect of high survival rate and good cell condition

Inactive Publication Date: 2019-04-05
武汉大学人民医院
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Problems solved by technology

[0005] However, there is no literature report on the application of Langendorff perfusion to the isolation of neonatal rat cardiomyocytes

Method used

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  • Method for acutely separating rat and suckling mouse cardiomyocytes
  • Method for acutely separating rat and suckling mouse cardiomyocytes
  • Method for acutely separating rat and suckling mouse cardiomyocytes

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Embodiment

[0036] A method for acutely isolating neonatal rat cardiomyocytes, the specific process is as follows:

[0037] 1. One-day-old suckling rats were intraperitoneally injected with low-molecular-weight heparin sodium (average molecular weight less than 8000 Daltons) at a dose of 200 U / rat. After anesthesia, cut the precardiac skin and ribs, free lungs and other tissues, and expose the heart , and then cut the aorta at 2-3mm above the aortic root, clamp the aortic stump of suckling rats, and take out the heart;

[0038] 2. Hold the heart with tweezers and wash it in a pre-cooled Petri dish filled with KR solution, cut off the excess tissue, find the broken port of the aorta under a stereo microscope, and put it into a 20ml syringe filled with oxygenated KR solution Slowly insert the connected 25G blunt needle into the aorta for 2-3mm. Do not insert the needle too deep into the aortic valve and ventricular cavity, so as to prevent the perfusate from pouring into the ventricular cav...

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Abstract

The invention discloses a method for acutely separating rat and suckling mouse cardiomyocytes. The method comprises the steps of preparing perfusate, performing suckling mouse aorta reverse perfusion,and separating cardiac muscle tissues. The method is characterized in that through the needle head connection, collagenase liquid is continuously and sufficiently perfused into the cardiac muscle tissues through an aorta-coronary artery system. The collagenase liquid is perfused into the cardiac muscle tissues through the aorta-coronary artery system. Compared with a tissue-enzyme digestion method used for most scientific research personnel for separating the rat and suckling mouse cardiomyocytes, the method has the advantages that the survival rate of the obtained cardiomyocytes is high; thecell state is good; after the separation, the original generation cardiomyocyte culture can be performed; the subsequent experiment operation of patch clamp, unicellular staining and confocal microscopy imaging, live cell station dynamic observation and parameter analysis and the like can be instantly performed. When the conventional tissue-enzyme digestion method is used for experiment operations of patch clamp and the like, the cells obtained through separation need to be purified and cultured for more than 48 hours so that the cardiomyocytes recover the original form.

Description

technical field [0001] The invention belongs to the applied technical field of cardiology research, in particular to a method for acutely isolating neonatal rat cardiomyocytes. Background technique [0002] At present, the isolation methods of rat cardiomyocytes can be divided into mechanical collection, Langendorff in vitro perfusion and tissue-enzyme digestion. Among them, Langendorff in vitro perfusion is more commonly used and is mainly used in the isolation of adult rat cardiomyocytes. Compared with the other two methods, the acute separation of adult rat cardiomyocytes by Langendorff isolated perfusion method can improve the survival rate of cardiomyocytes after separation. The cells are stable and functional, and the cells obtained by acute separation are closer to the physiological state of the living body. , the experimental results are more convincing. [0003] Rat suckling rats refer to rats born 0-7 days after birth, whose hearts are relatively small. At present...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2509/00
Inventor 徐林范稣圳
Owner 武汉大学人民医院
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