Cardiomyocyte preparation and preparation method therefor and use thereof
Provided are a cardiomyocyte preparation, a preparation method therefor and the use of the preparation in treating heart failure. The cardiomyocyte preparation comprises cardiomyocytes and fibroblasts, differentiated from pluripotent stem cells, wherein the concentration of the cardiomyocytes in the cardiomyocyte preparation is 0.75×10 7 -1.0×10 9 cells/mL, and the content of the cardiomyocytes is higher than 80% while the content of the fibroblasts is not higher than 20%. The cardiomyocyte preparation can improve cardiac ejection fraction and left ventricular fractional shortening, and increase the thickness of the left ventricular inner wall.
1. A cardiomyocyte formulation comprising cardiomyocytes and fibroblasts differentiated from pluripotent stem cells, wherein the concentration of the cardiomyocytes in the cardiomyocyte formulation is 0.75×10 7 to 1.2×10 8 cells/mL, and the content of the cardiomyocytes is higher than 80% and the content of the fibroblasts is not higher than 20%.
2. The cardiomyocyte formulation of claim 1 , wherein residual amount of the pluripotent stem cells in the cardiomyocyte formulation is not more than 1%.
3. The cardiomyocyte formulation of claim 2 , wherein residual amount of the pluripotent stem cells in the cardiomyocyte formulation is not higher than 0.3%.
4. A method for preparing the cardiomyocyte formulation of claim 1 , wherein the cardiomyocytes are prepared from pluripotent stem cells by steps of:
1) Pretreating the pluripotent stem cells: seeding the pluripotent stem cells into a pluripotent stem cell culture medium at a density of 0.9×10 5 to 3.0×10 5 cells/cm 2 , culturing the cells at 37° C. with 5% CO 2 until cell density exceeds 40%;
2) differentiating to cardiomyocytes: removing the pluripotent stem cell culture medium by suction, adding cardiomyocyte differentiation medium containing 0.01 to 0.09 mM CHIR99021, culturing for 48 hours; replacing the medium with cardiomyocyte differentiation medium containing 0.01 to 0.05 mM IWR-1, continuing to culture the cells for 48 hours; continuing to culturing with the cardiomyocyte differentiation medium, and replacing the cardiomyocyte differentiation medium according to cell growth state; and
3) Purifying cardiomyocytes: continuing to culture the cardiomyocytes via the step of differentiating to cardiomyocytes until emergence of observable beating, seeding the cells in the cardiomyocyte differentiation medium at a density of 6.0×10 5 to 9.0×10 5 cells/cm 2 , and adjusting the content of fibroblasts to less than 20% by using a cardiomyocyte purification liquid.
5. The method for preparing the cardiomyocyte formulation of claim 4 , wherein the pluripotent stem cells include induced pluripotent stem cells and embryonic stem cells.
6. The method for preparing the cardiomyocyte formulation of claim 4 , wherein the step of purifying cardiomyocytes further comprises culturing the cells in a cardiomyocyte culturing medium containing 0.75-5 μM STF-31 for 2-5 days.
7. The method for preparing the cardiomyocyte formulation of claim 4 , further comprising preparing a cell suspension with a cardiomyocyte concentration of 0.75×10 7 to 1.2×10 8 cells/mL by using the cells obtained in the step of purifying cardiomyocytes and physiological saline under sterile conditions.
8. A method of treating heart failure, comprising administering the cardiomyocyte formulation of claim 1 to a subject in need thereof.
9. The method according to claim 8 , wherein the cardiomyocyte formulation is used to treat heart failure with reduced ejection fraction (HFrEF).
10. The method according to claim 8 , wherein the cardiomyocyte formulation is injected through epicardium via a syringe into a scar or a thinned myocardial wall region.