New approaches to the study of cell vital activity cultivated in different growing conditions with analysis of oxygen in the medium

Abstract

Objectives - to develop new approaches to the study of morphofimctional state of chondroblasts, cultured at 37°C on a 3D carrier in different environments: in a CO2 incubator with 5% of CO2 and in a thermostat in an air-proof tube. Material and methods. The study cell culture - chondroblasts, isolated from the cartilage of the articular surfaces of the extra-fingers' phalanges. 3D carrier for cells - the demineralized lyophilized human spongiosa Lioplast®. The resulting tissue-engineered structures were grown in a complete cell culture medium at 37°С under different conditions: in a closed system in thermostat and in an open system in CO2 incubator (5% CO2). To assess the morphofunctional state of the cells on the surface of the 3D carrier, the picrosirius red staining, a LIVE/ DEAD® fluorescent dye kit, and scanning electron microscopy were used. The oxygen concentration in the culture medium was evaluated by the modified Winkler titration method. Results. The complex of morphological methods revealed the presence of living cells on the surface of human spongiosa within the 7-day period of cultivation. The cells either are fusiform or have a polygonal form and have a capacity to grow in 2 or more layers. The titrimetric analysis has demonstrated a decline in the concentration of dissolved oxygen in the medium with cellular tissue material in 7 days of cultivation. The concentration declined by 72.4% in a thermostat and by 63.5% in a CO2 incubator. In the tests tubes which contained only the medium and no cells, there was a similar decline in oxygen concentration by 47.3% in a thermostat and by 66.1% in a CO2 incubator. Conclusion. 1. A method of measuring the amount of dissolved oxygen in a culture medium, during the adhesive cell cultivation on a 3D carrier, was developed, based on the Winkler titration method. 2. A comparative analysis of the amount of dissolved oxygen in the medium in the process of chondroblast cultivation on a 3D human spongiosa carrier, both in a CO2 incubator and in a closed test tube, revealed an overall tendency to a decrease in the concentration of oxygen within 7 days of cultivation. 3. A decrease in oxygen concentration in the test tubes with human spongiosa samples (without cells), within the 7 days of cultivation, was registered. 4. An efficient and cost-saving method of graft manufacturing for the purposes of chondroplasty is the transfer of juvenile joint cartilage chondroblasts to 3D human spongiosa carriers and their further cultivation in air-proof test tubes copletely filled with medium within a period of 7 days.