METHOD FOR PRODUCTION OF CELL-FREE MATRIX OF DERMA FOR FURTHER RECONSTRUCTION OF EXTENSIVE DEFECTS OF SOFT TISSUES

摘要

FIELD: medicine.;SUBSTANCE: invention refers to medicine, particularly to biomedicine, and can be used in organ transplantation and tissue engineering for reconstruction of soft tissue defects. Method for producing decellularised cutaneous flaps involves using a perfusion module to be integrated into Ebers Teb 500 bioreactor assembled from two different-volume reservoirs interconnected by tubes and luer-fitings. Flap placed into the perfusion module is treated as follows: flap is washed by perfusion with 250 ml of PBS with antibiotics addition during a day; then skin flap is successively washed in solutions of 0.5 M NaCl, 1M NaCl and sterile purified water. After washing, the skin flap is treated with solution of 0.25 % trypsin – EDTA on a platform shaker in the working space of the bioreactor EBERS TEV 500 or incubator at 37 °C at rate of 500–600 rpm. Further, the flap is washed successively in sterile purified water and 100 % isopropyl alcohol. That is followed by placing a fixed flap into the perfusion module and perfusing 250 ml of fresh filtered solution of 1 % of 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (TritonX100) and 0.8 % sodium deoxycholate (NaDOC) for 45 hours , at that, after 24 hours, the solution is replaced with fresh solution and for perfusion washing, the perfusion module is not disassembled, and the dried perfusate reservoir is poured with 200-250 ml of sterile water with addition of 0.3 % sodium azide and 1 % ampicillin sodium, perfusion is carried out for 48 hours with three replacements for each day. After the perfusion is stopped, the flap is washed with 40 % ethanol, then the flap is perfused at rate of 20 ml/min for two hours. After perfusion, the flap is transferred into a new container filled with 125 ml of 70 % ethanol by +4 °C and stored for a day, and then flap is preserved in 200–250 ml of 7.14 % DMSO and 7.14 % PVP in DMEM and stored at temperature of -80 °C.;EFFECT: method enables complete removal of nuclei in all skin layers, including adipocyte nuclei, removal of molecules, which are antigenic determinants, absence of MHC-1 expression.;1 cl, 1 ex