Density gradient liquid kit for myocardial cell isolation and purification and use method thereof

Abstract

The present invention discloses a density gradient liquid kit for myocardial cell isolation and purification and a use method thereof, and relates to the technical field of biological cell culture. The kit includes a first reagent and a second reagent. The first reagent includes ultra pure water, Percoll liquid and a buffer solution in the volume ratio of 1:3:1; the second reagent includes ultra the pure water, the Percoll liquid and the buffer solution in the volume ratio of 2:2:1; the buffer solution of the first reagent and the second reagent includes 35.064-40.032 parts of NaCl, 1.975-2.013 parts of KCl, 0.615-1.107 parts of MgSO4 .7H2O, 4.865-5.226 parts of glucose, 0.301-0.384 parts of KH2PO4, 0.213-0.365 parts of Na2HPO4 and 23.830-29.788 parts of 4-hydroxyethylpiperazine ethane sulfonic acid.

Description

technical field [0001] The invention relates to the technical field of biological cell culture, in particular to a density gradient liquid kit for separating and purifying cardiomyocytes and a use method thereof. Background technique [0002] Various gene knockout and transgenic mice have been widely used in the establishment of animal models of cardiovascular diseases, and have made great contributions to the study of cardiovascular disease mechanisms. In vitro cardiomyocyte culture, as an in vitro experimental research model, can exclude the interference of nerves and body fluids and independently study the occurrence and development mechanisms of cardiovascular diseases at the cellular and molecular levels. Most of the current tool mice are from the mouse background, and cardiomyocytes The level of research mostly uses rat neonatal rat cardiomyocytes. Compared with the isolation and purification technology of rat myocardial primary cells and the relatively mature H9C2 cel...

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Problems solved by technology

[0003] The disadvantage of combined digestion with trypsin and collagenase is: trypsin has a certain damage to cardiomyocytes, which greatly affects the survival rate of cardiomyocytes. At present, although the contact time between cells and trypsin and the digestion time of trypsin are reduced as much as possible, but The effect on the survival rate of cardiomyocytes still exists, which is especially obvious in the process of isolation and purification of primary mouse myocardium

[0004] The disadvantage of using a single collagenase digestion is that although collagenase has a moderate effect and less damage to cells, it is necessary to use chemical inhibitors (such as 5-bromodeoxyuridine) to inhibit the growth of fibroblasts in order to purify the myocardium.

There are still defects such as low cell survival rate and low purity in the culture of primary mouse myocardial cells, and whether the presence of chemical inhibitors affects the function of cardiomyocytes remains to be proven, and the presence of chemical inhibitors will increase the impact of the experiment itself factors that can lead to unforeseen biases in the results

[0005] At the same time, the process of separation and purification of primary myocardial cells using the differential adherence method generally takes 3-3.5 hours, and the purity and survival rate are about 90%, and it is difficult to take into account the two aspects of purity and survival rate at the same time.

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