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Culture method of rat myocardial cell

A culture method and cardiomyocyte technology, which is applied in the field of cell culture, can solve the problems of long time consumption, high culture cost, and complicated culture procedures, and achieve the effects of simplifying operation steps, reducing usage, and shortening culture time

Active Publication Date: 2014-10-08
国家(上海)新药安全评价研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The technical problem to be solved by the present invention is aimed at the high cost, time-consuming and complicated culture procedures of the existing rat cardiomyocyte culture method, and the use of penicillin and Brdu and other drugs that may have potential impact on the follow-up experiment process during the culture process However, to provide a new method for culturing rat cardiomyocytes with low cost, short time consumption, simplified procedures and no use of drugs such as penicillin and Brdu

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. 10 SD rats born 1 day old, the apex of the heart was taken out with forceps under sterile conditions, placed in pre-cooled DMEM, rinsed 3 times to remove blood vessel tissue, and cut into 1mm 3 Debris, discard supernatant.

[0033] 2. Add 1.5mL of 0.7g / L trypsin and 0.5g / L Type II collagenase digestive enzyme mixed solution, place in a 37°C incubator for 10 times of digestion (8min each time), and discard the digestion solution.

[0034] 3. Add DMEM medium containing 10% FBS, pipette the residual tissue pieces until the tissue pieces basically disappear, collect the supernatant and put it in a 50mL centrifuge tube. Centrifuge at 800×g for 5 min, discard the supernatant, and add 5 mL of DMEM medium containing 10% FBS to resuspend the cells. The resuspended cardiomyocytes were passed through a 200-mesh cell sieve and collected into a new 50mL centrifuge tube. Finally, the cell suspension was transferred to a culture bottle for differential attachment twice, 45min each...

Embodiment 2

[0037] 1. Twelve SD rats born 2 days old, the apex of the heart was taken out with forceps under sterile conditions, placed in pre-cooled DMEM, rinsed twice to remove blood vessel tissue, and cut into 2mm 3 Debris, discard supernatant.

[0038] 2. Add 1.0g / L trypsin and 0.8g / L Type II collagenase digestive enzyme mixed solution to submerge the myocardial tissue, place it in a 36.5°C incubator for 12 times of digestion (6min each time), discard the digestion liquid.

[0039]3. Add DMEM medium containing 12% FBS, pipette the remaining tissue pieces until the tissue pieces basically disappear, collect the supernatant and put it in a 50mL centrifuge tube. Centrifuge at 800×g for 5 min, discard the supernatant, and add DMEM medium containing 12% FBS to resuspend the cells. The resuspended cardiomyocytes were passed through a 180-mesh cell sieve and collected into a new 50mL centrifuge tube. Finally, the cell suspension was transferred to a culture bottle for differential attachme...

Embodiment 3

[0042] 1. 10 SD rats born 1 day old, the apex of the heart was taken out with forceps under sterile conditions, rinsed once in pre-cooled DMEM to remove vascular tissue, and cut into 1mm 3 Debris, discard supernatant.

[0043] 2. Add 1.5mL of 0.7g / L trypsin and 0.5g / L type II collagenase digestive enzyme mixed solution, place in a 37.5°C incubator for 10 times of digestion (10min each time), and discard the digestion solution.

[0044] 3. Add DMEM medium containing 8% FBS, pipette the remaining tissue pieces until the tissue pieces basically disappear, collect the supernatant and put it in a 50mL centrifuge tube. Centrifuge at 800×g for 5 min, discard the supernatant, and add 5 mL of DMEM medium containing 8% FBS to resuspend the cells. The resuspended cardiomyocytes were passed through a 220-mesh cell sieve and collected into a new 50mL centrifuge tube. Finally, the cell suspension was transferred to a culture bottle for differential attachment twice, 50 min each time.

[0...

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Abstract

The invention discloses a culture method of rat myocardial cell. The culture method of the rat myocardial cell comprises the following steps: (1) soaking rat myocardial tissue in digestive enzyme mixed liquid, carrying out fractional digestion, removing digestion liquid until the myocardial tissue is loose and soft, then adding the loose and soft myocardial tissue into a DMEM (Dulbecco Modified Eagle Medium) culture medium, blowing and beating the loose and soft myocardial tissue and taking supernatant for centrifuging so as to obtain a cell sediment; (2) re-suspending the cell sediment by using the DMEM culture medium, passing a cell suspension through a sieve with 180-220 meshes, subsequently transferring the cell suspension to a culture flask and carrying out differential attachment 2-3 times for 40-50 minutes per time; (3) inoculating the myocardial cell in a culture vessel with a density of (1.5-2.5)*10 <5>cells / mL, and putting the culture vessel in a culture box for culturing after inoculating. The method is low in cost, short in time and simple in process; drugs such as penicillin, Brdu and the like are not used, so that the potential impact of penicillin and Brdu on the subsequent research can be avoided.

Description

technical field [0001] The invention belongs to the technical field of cell culture, in particular to a method for culturing rat cardiomyocytes. Background technique [0002] Cardiomyocyte culture refers to giving the nutrients needed for the growth of cardiomyocytes under suitable conditions in vitro to make them grow stably. However, primary cardiomyocytes are generally susceptible to external conditions and the survival rate is often low. Cardiomyocyte culture technology has been widely used in cardiovascular research at the molecular level, which helps to study the pathogenesis and treatment of cardiovascular diseases at the cellular and molecular levels. At present, the common cardiomyocyte culture method in this field is to adopt trypsin and collagenase to digest heart cells and then inoculate and cultivate them. This method uses more experimental materials, such as trypsin and type II collagenase. Penicillin and Brdu (5-bromodeoxyuridine) will be used in the culture ...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 马璟汪溪洁王淑颜惠涛涛周国民程一帆欧红梅
Owner 国家(上海)新药安全评价研究中心
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