METHOD FOR PREPARATION OF A CARDIOMYOCYTE MATRIX

摘要

The invention relates to biology and medicine, and specifically to cardiology and cardiac surgery, to cellular biotechnology and bioengineering, and to transplantology, and may be utilized for the culturing, proliferation and differentiation of stem cells into cardiomyocytes with the object of transplantation. The object of the invention is to improve culturing quality and stability, and the proliferative activity of pluripotent stem cells with differentiation thereof into cardiomyocytes, to eliminate cell microenvironment immunogenicity, and to ensure that a cellular matrix is obtained which is suitable for direct transplantation. The object is achieved in that a biomass of human embryonic stem cells of the feeder-free hESKM-05 line are grown using a basic mTeSR medium in vials coated with Matrigel and are passaged using 0.05% dispase solution, then for the formation of embryoid bodies from the embryonic stem cells the cells are transferred with the aid of dispase into koDMEM medium with the addition of 10% serum replacement SR, 100 µg/ml of kanamycin sulphate, 1 mM of L-glutamine solution and 1 mM of a solution of essential aminoacids, and also 2 µg/ml of a solution of 5-aza-2-desoxycytidine or 2 µM of sodium butyrate solution and are stimulated for 3 days, the embryoid bodies subsequently being transferred to a prepared collagen-chitosan matrix in the form of a sponge, film, microspheres, gel or fibres and are cultivated in koDMEM nutrient medium with the addition of 1 mM of a solution of essential aminoacids, 1 mM of L-glutamine solution, 10% of serum replacement SR, 10-7 M of retinoic acid and 10 ng/ml of ascorbic acid, with the medium changed every three days. Use of the method improves culturing quality and stability, and the proliferative activity of pluripotent stem cells with differentiation thereof into cardiomyocytes, eliminates cell microenvironment immunogenicity, and ensures that a cellular matrix is obtained which is suitable for direct transplantation.