On preparation of amniotic membrane as a scafold for cultivated cells to create corneal bioengineering constructs

Aim: to determine the preparation of amniotic membrane (AM) necessary for its use as a scafold for cultured cells to create bioengineered constructions (BEC). Material and Methods. Native AM was placed in a special clamping device and subjected to additional mechanical, thermal and enzymatic treatment: removal of mucus residues from its surface and cryoconservation of AM scafolds was performed at -80°C, - 20°C with subsequent decellularization with a 0.25 % Tripsin-EDTA mixture. The lifetime assessment of the morphology of cells cultivated on AM scafolds was performed using a Nikon Eclipse TS100 inverted microscope equipped with camera. The viability and metabolic activity of AM cells was determined by means of an MTT test using a UNIFLAN AIFR-01 tablet spectrophotometer (Picon, Russia) at a wavelength of 570 nm and a reference wavelength of 620 nm. Results. It has been established that the presence of mucous residueson the surface of native AM, which are not removed during standard mechanical processing, negatively afect the survival of the cell test system. The efects of cryoconservation of AM scafolds revealed the positive efects of this process together with enzymatic decellularization for improving the viability of cells cultured on scafolds. Conclusion. Standard mechanical processing of native AM does not guarantee complete cleaning of its surface from mucus residues that interfere with adhesion and even distribution of cultured cells. It is necessary to reliably control the removal of AM surface mucus before immobilization and further manipulations. Cryopreservation and subsequent decellularization of AM scafolds contributes to the increased viability of the cell test system. AM scafolds, purifed from amniotic mucus, cryopreserved at -80 ° C in a mixture of DMEM-F12 and DMSO (1:1) and enzymatic decellularization after thawing, turned out to be the best of the studied substrates for cell cultivation.