METHOD FOR IN VITRO DETERMINATION OF BIOCOMPATIBILITY OF SCAFFOLDS FOR NEUROTRANSPLANTATION Russian patent published in 2022 - IPC G01N33/52 C12N5/79 

FIELD: medicine.

SUBSTANCE: present invention relates to the field of cellular and tissue biology, medicine and neurobiology, in particular to an in vitro method for determining the biocompatibility of scaffolds for neurotransplantation. To implement the method, primary neuronal cultures with an initial cell density of 27,000 cells/cm² obtained from the brain tissue of mouse embryos of the SHK line, with scaffolds on the surface of the constructs, are co-cultured. Next, viability indicators, morphometric parameters and indicators of the functional activity of cells in culture are determined, the levels of cytotoxicity of the scaffold material, its adhesive properties and the optimality of the scaffold architectonics are evaluated. At the same time, viability assessment is carried out using fluorescent dyes propidium iodide and bis-benzimide, as well as qualitatively using a cytotoxicity scale, according to which the scaffold material does not exhibit toxic effects on nerve cells in the presence of 90-100% of viable cells in culture. The morphometric parameters of the cell culture are evaluated using scanning microscopy and the ImageJ program, according to the results of which, with a decrease in the Feret diameter and the surface area of cells 1.5 times or more, it is believed that the material of the construct has a destructive effect on the cell culture. The analysis of spontaneous calcium activity is carried out using a specific calcium dye Oregon Green 488 BAPTA-1 AM and a confocal laser scanning microscope, according to which the scaffold is considered biocompatible with the number of cells exhibiting calcium activity of at least 80%, the duration of calcium events 8-10 seconds, the frequency of calcium oscillations 1.5-2 numbers of calcium events/min. The adhesive properties of the scaffold are estimated by the surface area of cellular conglomerates and their number, according to which the scaffold material has good adhesive properties for nerve cells when at least 7 cellular conglomerates/200 mcm² with a surface area of at least 8000 mcm² are formed on the scaffold.

EFFECT: present invention makes it possible to increase the efficiency and accuracy of assessing the risks of developing pathological reactions that can lead to transplant rejection.

2 cl, 3 dwg, 5 tbl, 2 ex