Science and Innovations in MedicineScience and Innovations in Medicine2500-13882618-754XFSBEI of Higher Education SamSMU of Ministry of Health of the Russian Federation4379310.35693/2500-1388-2019-4-4-68-72Research ArticleNew approaches to the study of cell vital activity cultivated in different growing conditions with analysis of oxygen in the mediumVolovaLarisa T.<p>PhD, Professor, the Head of Biotechnology Department of IEMB, director of Samara Tissue Bank</p>evgenesius@mail.ruhttps://orcid.org/0000-0002-8510-3118PugachevEvgeni I.<p>research associate of the Biotechnology Department of IEMB</p>evgenesius@mail.ruhttps://orcid.org/0000-0002-3594-0874RiazanovaTatyana K.<p>PhD, the Head of the laboratory of sanitary methods of Institute of Hygiene</p>evgenesius@mail.ruhttps://orcid.org/0000-0002-4581-8610NefedovaIrina F.<p>the Head of the Experimental morphology Department of IEMB</p>evgenesius@mail.ruhttps://orcid.org/0000-0003-3521-0748BoltovskayaVioletta V.<p>PhD, senior research associate of the Biotechnology Department of IEMB</p>evgenesius@mail.ruhttps://orcid.org/0000-0003-3457-8524MaksimenkoNatalya A.<p>Deputy director of IEMB, senior specialist of Samara Tissue Bank</p>evgenesius@mail.ruSamara State Medical University1512201944687208092020Copyright © 2019, Volova L.T., Pugachev E.I., Riazanova T.K., Nefedova I.F., Boltovskaya V.V., Maksimenko N.A.2019<p><strong>Objectives</strong> to develop new approaches to the study of morphofunctional state of chondroblasts, cultured at 37C on a 3D carrier in different environments: in a CO<sub>2</sub>incubator with 5% of CO<sub>2</sub>and in a thermostat in an air-proof tube.</p>
<p><strong>Material and methods.</strong>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<sup></sup>. The resulting tissue-engineered structures were grown in a complete cell culture medium at 37С under different conditions: in a closed system in thermostat and in an open system in CO<sub>2</sub>incubator (5% CO<sub>2</sub>). To assess the morphofunctional state of the cells on the surface of the 3D carrier, the picrosirius red staining, a LIVE/DEAD<sup></sup>fluorescent dye kit, and scanning electron microscopy were used. The oxygen concentration in the culture medium was evaluated by the modified Winkler titration method.</p>
<p><strong>Results.</strong>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 CO<sub>2</sub>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 CO<sub>2</sub>incubator.</p>
<p><strong>Conclusion.</strong>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 CO<sub>2</sub>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.</p>tissue-engineered structuresspongiosa3D carrierchondroblaststitrimetryhistological researchтканеинженерная конструкцияаллоспонгиоза3D-носитель для культур клетокхондробластытитриметриягистологическое исследование[Saburina IN, Repin VS. 3D-cultivation: from cells to regenerative tissue. Kletochnaya transplantologiya i tkanevaya inzheneriya. 2010;5(2):75–86. (In Russ.). [Сабурина И.Н., Репин В.С. 3D-культивирование: от отдельных клеток к регенерационной ткани. Клеточная трансплантология и тканевая инженерия. 2010; 5(2):75–86].][Krishna L, Dhamodaran K, Jayadev C, et al. Nanostructured scaffold as a determinate of stem cell fate. Stem Cell Res Ther. 2016 Dec 30;7(1):188. doi: 10.1186/s13287-016-0440-y][Surguchenko VA, Ponomareva AS, Kirsanova LA, et al. Development of tissue engineering constructs of cartilage tissue in vitro. 2013;15(3):66–72. (In Russ.). [Сургученко В.А., Пономарева А.С., Кирсанова Л.А. и др. Формирование тканеинженерной конструкции хрящевой ткани в условиях in vitro. Вестник трансплантологии и искусственных органов. 2013;15(3):66–72].][Shishackaya EI, Nikolaeva ED, Shumilova AA, et al. Cultivation of multipotent mesenchymal stromal bone marrow cells on resorbable Bioplastotan. Kletochnaya transplantologiya i tkanevaya inzheneriya. 2013;8(1):57–65. (In Russ.). [Шишацкая Е.И., Николаева Е.Д., Шумилова А.А. и др. Культивирование мультипотентных мезенхимальных стромальных клеток костного мозга на носителях из резорбируемого биопластотана. Клеточная трансплантология и тканевая инженерия. 2013;8(1):57–65].][Ho MH, Kuo PY, Hsieh HJ, Hsieh TYa, et al. Preparation of porous scaffolds by using freeze-extraction and freeze-gelation methods. Biomaterials. 2004;25:129–138. doi: 10.1016/S0142-9612(03)00483-6][Cao W, Wang A, Jing D, Gong Y, et al. Novel biodegradable films and scaffolds of chitosan blended with poly (3-hydroxybutyrate). Jornal of Biomaterials’ Science Polymers. 2005;16(11):1379–94. doi: 10.1163/156856205774472308][Shi DH, Cai DZ, Zhou CR, et al. Development and potential of a biomimetic chitosan/type II collagen scaffold for cartilage tissue engineering. Chin Med J (Engl). 2005 Sep 5;118(17):1436–43.][Pugachev EI. Comparative analysis of the proliferative activity of chondroblasts on a 3D bio-carrier under different cultivation conditions. Materialy konferencii. Samara, 2017. (In Russ.). [Пугачев Е.И. Сравнительный анализ пролиферативной активности хондробластов на 3D-бионосителе в разных условиях культивирования. Материалы конференции. Самара, 2017]. http://www.samsmu.ru/files/news/2017/201017/asp_read_2017.pdf]