Methods of obtaining stem cells of the myocardium of cat
Abstract
Year 2003 may be considered as the beginning of the study of “stem cells of the adult heart”, when Beltrami A.P. with his colleagues finally confirmed the presence of the latter in the heart. Later, Makkar R.R. and others proved the efficacy of these cells for treatment of myocardial ischemia. However, despite the positive effect of the use of “stem cells of the adult heart” for myocardial ischemia, an obstacle to their global clinical use are the ethical issues and difficulty in obtaining. Given the above, the aim of our work was to determine the optimal method of obtaining stem cells from the myocardium of cat. The experiments on animals were performed in accordance with the requirements of the “General ethical principles for Experiments on Animals” adopted by the first National Congress on bioethics (dated 20.09.04, Kyiv, Ukraine). To obtain heart tissues the frozen fetuses of kittens, that remained after providing the birth assistance, were used (with the consent of the owners). In order to determine the optimal method for obtaining a stem cells culture of the myocardium of cat we compared the explant method and enzymatic treatment methods of tissues using different combinations of enzymes: 2 mg/ml of collagenase (type II); 2 mg/ml of collagenase (type II) supplemented with 5% bovine serum; 1 mg/ml of collagenase (type II), 1 mg/ml of hyaluronidase and 5% BSA; 2.5% of trypsin; 2.5% of trypsin, 0.5 mg/ml of collagenase (type II) and 0.5 mg/ml of hyaluronidase. In order to determine the optimal method for obtaining a cells culture of the myocardium the counting of the number of cells after the formation of monolayer in one of the experimental groups was carried out (day 5 since the beginning of the research). The counting of number of cells was performed in the Gorjaev's chamber under 200 magnification of microscope in all the squares. The most effective method for obtaining the stem cells culture from the myocardium is to use a combination of enzymes 2.5% of trypsin, 0.5 mg/ml of collagenase (type II) and 0.5 mg/ml of hyaluronidase, which allows to obtain 986.0 ± 9.3 thousand cells capable for proliferation with 10 mg of tissue. The least effective was the explant method (without use of enzymes), because the number of cells attached to the cultural plastic of the cells during its use on day 5 of cultivation was amounted 35.7 ± 6.2 thousand. The proposed method is optimal for obtaining the stem cells culture of the myocardium of the cat, which will be used later in our studies in the treatment of diseases of the heart muscle.
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