METHOD OF MODELLING BIOENGINEERED HEART MATRIX IN EXPERIMENT ON RAT

摘要

FIELD: medicine.;SUBSTANCE: method of modelling a bioengineered heart matrix in experiment on a rat includes the introduction of an anticoagulant to the rat, the organ exposure, cleaning it from surrounding adipose tissue, cannulation of the aorta, realisation of decellularisation by perfusion in a bioreactor, as well as control of the quality of the obtained matrix for biocompatibility and viability. The anticoagulant heparin is introduced to the rat intraperitoneally in a dose of 100 U before sampling the heart-lungs organ complex. The aorta is cannuled above the level of the left subclavian artery deviation with the following ligation of the aortic arch branches. Ligation of the orifice of hollow veins is realised, lungs are cut off. Perfusion for decellularisation is realised for 28 hours through the aorta under atmospheric pressure and the rate of the flow of reagents through the organ of 2.4-3.6 ml/min. Perfusion with a phosphate buffer with the addition of 1% of penicillin-streptomycin and deionised water is carried out for 1.5 hours. After that a 4% solution of sodium deoxycholate in a combination with 0.002 M of Na2-EDTA is used for 3.5 hours. The phosphate buffer with the addition of 1% of penicillin-streptomycin is used for 1 hour, porcine pancreatic DNA-ase-I 2000 U/200 ml of the phosphate buffer with calcium and magnesium - for 2.5 hours. Decellularisation is completed with the phosphate buffer with the addition of 1% penicillin-streptomycin with the replacement of the solution every 6 hours. Viability of cells on the obtained matrix is determined by the presence of the differential coloration of live and dead cells, by the ability of the live cells dehydrogenases to recover non-coloured forms of 3-4,5-dimethylthiazol-2yl-2,5-diphenylterarazole to light blue crystalline formazan, soluble in dimethylsulphoxide.;EFFECT: method makes it possible to reduce the time of exposition of perfusion solutions, reduce the probability of bacterial contamination, increase the quality of the obtained matrix in comparison with other methods for the same purpose, estimate the biocompatibility and viability of cells, inoculated on the matrix.;6 dwg