Аннотация:Aim of the work: to study the biological properties of a matrix from cryogenically structured hydrogel in the form of a resorbable macroporous gelatin sponge, as well as the possibility of creating cellular engineering constructs on its basis.Materials and methods. The main components of the cryogenically structured hydrogel were gelatin (type A) obtained from pig skin collagen, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide, (EDC) and urea (all – Sigma-Aldrich, USA). The morphology of the surface was studied using scanning electron microscopy. The degree of swelling in the water of the samples was determined by the gravimetric method. Cytotoxicity was studied on NIH 3T3 mouse fibroblasts and human adipose tissue mesenchymal stromal cells (ADSCs) using IncuCyte ZOOM (EssenBioscience, USA). The metabolic activity of ADSCs was evaluated using PrestoBlue ™ reagents (Invitrogen ™ , USA). To create a cellular engineering constructs (CEC), we used ADSCs, hepatocellular carcinoma HepG2 cells or human umbilical vein endothelial cells of the EA.hy926 line. The albumin content in the culture medium was determined by enzyme immunoassay. The rate of ammonia metabolism was evaluated after 90 minutes of incubation with 1 mM ammonium chloride (Sigma-Aldrich, USA) diluted in culture medium on the 15th day of the experiment.Results. The production of cryogenically structured hydrogel in the form of a macroporous gelatin sponge included freezing of an aqueous solution of a mixture of gelatin and urea, removal of polycrystals of frozen solvent by lyophilization, extraction of urea with ethanol and treatment of cryostructurate with an ethanol solution of EDC. Scanning electron microscopy made it possible to distinguish three types of pores on the surface of the carrier: large (109±17 microns), medium (39±10 microns) and small (16± 6 microns). The degree of swelling in the water of the matrix samples was 3.8± 0.2 g of H2O per 1 g of dry polymer. The ability of macroporous gelatin sponge in the composition of CEC to maintain adhesion and proliferation of ADSCs, endothelial cells of the human umbilical vein line EA.hy926 and HepG2 for 28, 15 and 9 days, respectively, was established. The presence of albumin secretion and ammonia metabolism during the cultivation of HepG2 cells on a gelatin sponge has been proven.Conclusion. Using the example of the cellular engineering design of the liver, the prospects of using a macroporous cryogenically structured gelatin-based hydrogel to create tissue engineering products are shown.
