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CN-122012381-A - Method for in-vitro amplification and passage of myocardial cells

CN122012381ACN 122012381 ACN122012381 ACN 122012381ACN-122012381-A

Abstract

The invention relates to the technical field of biology, in particular to a method for in vitro expansion and passage of myocardial cells, which comprises the following steps of S1, taking human induced pluripotent stem cells to culture until the cell confluence reaches 70% -80%, S2, differentiating the human induced pluripotent stem cells into myocardial cells, S3, observing the number of beating myocardial cells, selecting a hole with the beating percentage of 50% -90%, digesting and resuspending the myocardial cells in the hole, counting cells, plating the cells into a six-hole plate according to 1/20-1/10 of the total cell number, culturing for 24 hours by using an RPMI/B-27 insulin-free culture medium containing Y-27632, wherein the concentration of Y-27632 is 2-10uM, S4 is replaced by an RPMI/B-27 insulin-free culture medium containing Chir99021, the next time of expansion is arranged after the culture is 3-5 days to the cell density of 70% -80%, S5, repeating the steps S3 and S4 for expansion and passage according to the requirement, and the problem of large-scale expansion of the myocardial cells of human pluripotent stem cells is solved, and the problem of in vitro expansion is limited is solved.

Inventors

  • ZHENG HONG
  • Liang Zechuan
  • YANG XIANGYU
  • He Yingxing
  • ZHANG CAITING

Assignees

  • 广州源井生物科技有限公司

Dates

Publication Date
20260512
Application Date
20260204

Claims (8)

  1. 1. A method for in vitro expansion and passage of cardiomyocytes, comprising the steps of: s1, inoculating human induced pluripotent stem cells into a twelve-hole plate according to the density of 0.5-1.5 ten thousand cells/hole, and culturing until the cell confluence reaches 70-80%; s2, performing induction culture on the human induced pluripotent stem cells with the cell confluence of 70% -80%, so that the human induced pluripotent stem cells are differentiated into myocardial cells; S3, observing the number of beating myocardial cells, selecting a hole with the beating percentage of 50% -90%, digesting the myocardial cells which are resuspended in the hole, counting cells, plating the cells into a six-hole plate according to 1/20-1/10 of the total cell number, and culturing for 24 hours by using an RPMI/B-27 insulin-free medium containing Y-27632; wherein the concentration of Y-27632 is 2-10uM; S4, changing the culture medium into an RPMI/B-27 insulin-free culture medium containing Chir99021, changing the culture medium every day, culturing for 3-5 days until the cell density is 70% -80%, and then arranging the next passage and amplification; s5, repeating the steps S3 and S4 as required to amplify and passaging, and obtaining the myocardial cells with increased numbers.
  2. 2. The method for in vitro expansion and passaging of myocardial cells according to claim 1, wherein in step S4, the content of Chir99021 is 0.2-6. Mu.M.
  3. 3. The method according to claim 1, wherein in step S3, the operations of digesting the heavy centre of suspension myocytes are as follows: Absorbing the original culture medium, washing cells with phosphate buffer solution, incubating myocardial cells with TRYPLE SELECT digest for 13-17 min, adding RPMI-1640 culture medium, and mixing to obtain cell suspension; The cell suspension was transferred to a centrifuge tube, centrifuged at 200 Xg for 3 minutes, the supernatant was aspirated off, and the cell suspension was resuspended in RPMI/B-27 insulin-free medium containing 2-10uM of Y-27632.
  4. 4. The method for in vitro expansion and passaging of cardiomyocytes according to claim 1, wherein the step S2 of induction culture is performed by: (1) On day 0 of differentiation, the medium was aspirated off, the culture was performed with the addition of myocardial induction medium, and the recording time was started; the myocardial induction culture medium is RPMI/B-27 insulin-free culture medium added with activin A, basic fibroblast growth factor and KnockOut serum substitute; (2) On the 2 nd day of differentiation, the myocardial induction medium is sucked and removed, and RPMI/B-27 insulin-free medium containing BMP4 and basic fibroblast growth factor is added for culture; (3) On day 5 of differentiation, the old culture medium is sucked and removed, and RPMI/B-27 insulin-free culture medium is added for continuous culture; (4) The old culture medium is sucked and removed on the 7 th day of differentiation, the RPMI/B-27 complete culture medium is added for culture, the RPMI/B-27 complete culture medium is replaced every three days until the differentiation is within 10-11 days, and the cells are successfully differentiated when spontaneously beating, so that the required myocardial cells are obtained.
  5. 5. The method for in vitro expansion and passaging of myocardial cells according to claim 4, wherein the concentration of activin A in the myocardial induction medium is 50-200ng/ml, the concentration of basic fibroblast growth factor is 10-20ng/ml, and the volume percentage of KnockOut serum replacement is 0.5% -2%.
  6. 6. The method of in vitro myocardial cell expansion and passage according to claim 4, wherein in step (2), the BMP4 concentration is 5ng/ml, and the basic fibroblast growth factor concentration is 5ng/ml.
  7. 7. The method for in vitro expansion and passage of myocardial cells according to claim 1, wherein in step S1, the inoculation operation is as follows: Taking human induced pluripotent stem cells with the fusion degree of 80% -90%, sucking and discarding old culture medium, adding Ackutase digestive juice, standing and culturing for 8 minutes, adding mTESR1 culture medium, re-suspending and collecting all cells, performing cell counting, centrifuging and discarding supernatant, re-suspending cells by using mTESR1 culture medium containing Y27632 with the concentration of 5 mu M, and inoculating into a twelve-well plate according to the density of 0.5-1.5 ten thousand cells per well.
  8. 8. The method according to claim 1, wherein in step S1, the cultured cells are inoculated with mTESR1 medium containing 1. Mu.M of CHIR 99021.

Description

Method for in-vitro amplification and passage of myocardial cells Technical Field The invention relates to the technical field of biology, in particular to a method for in-vitro amplification and passage of myocardial cells. Background The cardiomyocytes of adult mammals are considered as terminally differentiated cells, which have extremely limited ability to self-renew and regenerate, once damaged, myocardial tissue is often repaired by fibrotic scarring rather than regeneration of functional myocardium, and such irreversible damage severely impairs the pumping function of the heart and eventually evolves into heart failure, imposing a heavy burden on patients, households and society. Therefore, repairing or regenerating damaged myocardial tissue is one of the hot problems that researchers in the cardiovascular field and regenerative medicine are urgent to solve, and a core challenge in achieving this goal is how to obtain a large number of myocardial cells that are functional, safe and reliable. Although the traditional organ transplantation provides a living machine for patients with end-stage heart failure, the traditional organ transplantation always faces the bottleneck that the donor is seriously short, immune rejection, ethical constraint and the like are difficult to surmount, and the scientific community is driven to aim at the in-vitro expansion technology of myocardial cells, namely, the myocardial cells with mature functions are prepared on a large scale in a laboratory environment by utilizing stem cell biology, tissue engineering, regenerative medicine and other means. Therefore, the importance and necessity of efficient and large-scale in vitro expansion of cardiomyocytes are self-evident. First, either cell patch-based myocardial patch transplantation or direct intracardiac injection of a cell suspension requires hundreds of millions of functional cardiomyocytes as "seeds" without a stable, sufficient source of cells, and any cell replacement therapy would be the air pavilion. Secondly, myocardial cells derived from somatic cells of patients (such as induced pluripotent stem cells, iPSCs) can construct a highly personalized disease model for precisely analyzing pathological mechanisms, screening and evaluating the effectiveness and cardiotoxicity of novel drugs, greatly accelerating the development process of the novel drugs and promoting the development of personalized medicine. However, in vitro expansion of cardiomyocytes still faces a number of serious challenges, such as how to maintain the purity of the cells, how to induce them to reach a highly mature phenotype to compete with adult cardiomyocytes, how to ensure their survival rate after transplantation and electrophysiological integration capacity, etc. The resolution of these problems is highly dependent on our in-depth understanding and technological innovation of cardiac developmental biology, electrophysiology and biomaterials. For this reason, many studies have been focused on screening molecules that promote proliferation of PSCs-derived differentiated cardiomyocytes, but when these molecules are applied to induce in vitro expansion of human pluripotent stem cells (hiPSCs) -derived cardiomyocytes, they also have only a slight proliferation capacity and limited proliferation extent, thus limiting the application of cardiomyocytes in the direction of cell therapy. Disclosure of Invention Aiming at the defects, the invention aims to provide a method for in-vitro expansion and passage of myocardial cells, which solves the problem of limited mass preparation during in-vitro expansion of human pluripotent stem cell-derived myocardial cells. In order to achieve the purpose, the invention adopts the following technical scheme: a method for in vitro expansion and passaging of cardiomyocytes comprising the steps of: s1, inoculating human induced pluripotent stem cells into a twelve-hole plate according to the density of 0.5-1.5 ten thousand cells/hole, and culturing until the cell confluence reaches 70-80%; s2, performing induction culture on the human induced pluripotent stem cells with the cell confluence of 70% -80%, so that the human induced pluripotent stem cells are differentiated into myocardial cells; S3, observing the number of beating myocardial cells, selecting a hole with the beating percentage of 50% -90%, digesting the myocardial cells which are resuspended in the hole, counting cells, plating the cells into a six-hole plate according to 1/20-1/10 of the total cell number, and culturing for 24 hours by using an RPMI/B-27 insulin-free medium containing Y-27632; wherein the concentration of Y-27632 is 2-10uM; S4, changing the culture medium into an RPMI/B-27 insulin-free culture medium containing Chir99021, changing the culture medium every day, culturing for 3-5 days until the cell density is 70% -80%, and then arranging the next passage and amplification; s5, repeating the steps S3 and S4 as required to amplify and passa