METHOD FOR OBTAINING DECELLULARISED MATRIX OF AMNIOTIC MEMBRANE FOR FURTHER RECONSTRUCTION OF TISSUE DEFECTS

摘要

FIELD: medicine.;SUBSTANCE: invention relates to medicine, particularly to biomedicine, namely to regenerative medicine and transplantology, tissue engineering, for obtaining a decellularised matrix of the amniotic membrane for further reconstruction of tissue defects due to thermal, chemical and radiation burns, ulcers, etc. The method for obtaining a decellularised matrix of the amniotic membrane for further reconstruction of tissue defects as a coating with intact structural components of the extracellular matrix includes the following: the donor placenta is transferred to a laminar flow cabinet of the biological hazard class II, the internal fetal amniotic membrane is separated from the chorion, wherein approximately 300 cm² of the amniotic membrane is isolated from one placenta. The isolated fragments of the amnion with the area of 50 cm² are thoroughly washed from blood in a phosphate-buffered saline solution with the addition of 250 U/ml of penicillin and 250 mcg/ml of streptomycin three times within 24 hours, the solution is replaced every 3 hours. The isolated fragments of the amniotic membrane with the area of 50 cm² are then subjected to detergent-enzymatic decellularisation using: (1) 0.5% sodium deoxycholate + 0.5% TritonX100 - 24 hours, 400 rpm; (2) 0.05% Trypsin-EDTA - 1 hour, 200 rpm; (3) DMEM, 10% FBS, 1% penicillin-streptomycin - 24 hours, 400 rpm; (4) 300 U/ml DNAse I, 40 mM Tris-HCl, 10 mM MgCl₂, 10 mM NaCl - 16 hours, 400 rpm; (5) PBS, 1% penicillin-streptomycin - 48 hours, 400 rpm. The matrix is then analysed for the presence/absence of cell nuclei or shadow nuclei, the integrity of the main components of the extracellular matrix (collagen, laminin, fibronectin) and the level of the residual genomic DNA.;EFFECT: obtained is decellularised matrix of the amniotic membrane not causing inflammatory fibrosis and macrophage-lymphocytic infiltration, for the purpose of healing the affected surface by increasing the reparative and immune processes, restoring trophicity, remodeling fibrous connective tissue.;1 cl