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Myocardial cell resuscitation method

A technology of cardiomyocytes and human serum albumin, applied in the field of cardiomyocyte resuscitation, can solve the problems of cardiomyocyte myocardial contractility reducing electrophysiological activity, affecting the stability of cardiomyocytes, and no method of cryopreservation or resuscitation of cardiomyocytes has been found. Achieve stable electrophysiological activity and high activity rate

Pending Publication Date: 2022-07-19
HELP STEM CELL INNOVATIONS CO LTD
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Problems solved by technology

[0005] The document Effects of Cryopreservation on Human Induced Pluripotent StemCell Derived Cardiomyocytes for Assessing Drug Safety Response Profiles discloses a cryopreservation medium comprising 90% fetal bovine serum and 10% DMSO to cryopreserve cardiomyocytes differentiated from induced pluripotent stem cells, but frozen The myocardial contractility and electrophysiological activity of the resuscitated cardiomyocytes were changed in different degrees compared with those of the cardiomyocytes without cryopreservation. Stability of cardiomyocytes during storage
[0006] At present, no cardiomyocyte cryopreservation or resuscitation method has been found that combines the cell viability and electrophysiological characteristics after cardiomyocyte cryopreservation and resuscitation

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Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1 Preparation of cardiomyocyte cryopreservation solution

[0023] 1: Prepare a cardiomyocyte cryopreservation solution containing 90% fetal bovine serum and 10% DMSO;

[0024] 2: Dissolve 0.8 g of hydroxyethyl starch in 70 mL of physiological saline with a concentration of 0.9%, and then add 0.3 mL of DMSO and 30 mL of human serum albumin after it is completely dissolved;

[0025] 3: Dissolve 1 g of hydroxyethyl starch in 75 mL of physiological saline with a concentration of 0.9%, and then add 0.5 mL of DMSO and 25 mL of human serum albumin respectively after it is completely dissolved;

[0026] 4: Dissolve 5g of hydroxyethyl starch in 80mL of physiological saline with a concentration of 0.9%, and then add 10mL of DMSO and 10mL of human serum albumin respectively after it is completely dissolved;

[0027] 5: Dissolve 1 g of hydroxyethyl starch, 0.1 μmol of ROCK inhibitor Y27632, and 0.25 g of glucose in 70 mL of physiological saline with a concentration of 0.9%...

Embodiment 3

[0042] Example 3 The effect of cryopreservation on myocardial cell viability

[0043] The myocardial cell viability was calculated by PI staining method for the cryopreserved and resuscitated cardiomyocytes. Table 1 shows the effects of different cardiomyocyte cryopreservation solutions for 6 months on the viability of cardiomyocytes differentiated to the 8th day:

[0044] Table 1: Cell viability of cardiomyocytes on day 21 of differentiation in different cardiomyocyte cryopreservations for 6 months

[0045] cryopreservation 1 2 3 4 5 6 7 8 cell viability 47.25％ 65.5％ 72.5％ 72.75％ 83.25％ 81.75％ 91.5％ 93.25％

[0046] It can be seen that, compared with the cardiomyocyte cryopreservation solution of group 1 in Example 1 of the present invention, groups 2 to 8 provided in the embodiment of the present invention significantly improved the viability of cardiomyocytes. Cardiomyocytes were frozen in the cardiomyocyte cryopreservation solution of group...

Embodiment 4

[0048] Example 4 The effect of cryopreservation on the electrophysiological activity of cardiomyocytes

[0049] Patch-clamp detection and recording of electrophysiological properties of beating cardiomyocytes

[0050] The same batch of cardiomyocytes with the same maturity were stored in the cryopreservation solution provided in Example 1 for 12 months, then recovered and cultured in cardiomyocyte culture medium, and pulsating myocardium was selected after culturing for 48h. The cells were also subjected to patch-clamp experiments, in which the cardiomyocyte culture medium was a purchased commercial cardiomyocyte culture medium.

[0051] figure 1 It is the action potential of unfrozen cardiomyocytes, showing that its action potential duration APD is 416.10 ± 32.61, and the action potential duration APD of the cardiomyocytes frozen in the first group of cryopreserved solution in Example 1 is 265 ± 36.98. In Example 1, the action potential durations of the cardiomyocytes cryop...

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Abstract

The invention provides a myocardial cell resuscitation method which comprises the following steps: pre-treating cryopreserved myocardial cells in a water bath kettle, and after the cryopreserved myocardial cells are converted into liquid from solid, adding a human serum albumin solution with the concentration of less than 20% into the cryopreserved myocardial cells for resuscitation. According to the myocardial cell resuscitation method provided by the invention, the cryopreserved myocardial cells can have relatively high cell viability and stable electrophysiological activity after being resuscitated.

Description

technical field [0001] The invention relates to the technical field of cell resuscitation, in particular to a method for resuscitating cardiomyocytes. Background technique [0002] Cardiovascular disease causes approximately 17 million deaths each year and is the leading cause of death from disease worldwide. Cardiovascular disease is currently difficult to prevent due to complex pathogenic mechanisms. In 2006, the team of Japanese scientist Shinya Yamanaka used viruses to transfer 4 transcription factors into differentiated fibroblasts to obtain induced pluripotent stem cells. The development of induced pluripotent stem cell technology avoided the ethical problems encountered by embryonic stem cells and provided a good solution for related diseases. research and treatment possibilities. [0003] Induced pluripotent stem cells have the potential to differentiate into various functional cells. Similarly, induced pluripotent stem cells can generate cardiomyocytes with contrac...

Claims

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Application Information

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IPC IPC(8): C12N5/077A01N1/02
CPCC12N5/0657A01N1/0221C12N2501/998
Inventor 张月辉陈涛涛王倩王嘉显
Owner HELP STEM CELL INNOVATIONS CO LTD
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