INFLUENCE OF THEORETICALLY ESTABLISHED CRYOPRESERVATION REGIMEN ON THE FUNCTIONAL STATE OF DECONSERVATED CELLULAR SPHEROIDS

Moisieiev A. I., Bozhok G. A.

BIOLOGY

Scentific article

Search and optimization of methods of cryopreservation of multicellular objects, in particular spheroids (SF), is an urgent issue, the solution of which will allow more efficient application of SF in biological research, biotechnology, regenerative medicine and tissue engineering. In the present study, a cryopreservation regime was used, which included temperature and cooling rate parameters, and SF saturation time in a cryoprotective medium, previously determined by physical and mathematical modeling based on the permeability coefficients of water molecules and dimethyl sulfoxide cryoprotectant (DMSO). The goal of the work is to study the functional state of SF from L929 cells after cryopreservation according to the regimen established on the basis of physical and mathematical modeling. Experimental studies were performed on SF from L929 cells after cryopreservation according to the regime determined on the basis of theoretical calculations (exposure in a cryoprotective medium for 65 sec, cooling rate 2°C/min to – 80°C followed by immersion into liquid nitrogen ( – 196°C)) in comparison with empirically established regimens (exposure in a cryoprotective medium for 10 minutes, cooling rate 1°C/min to – 40°C or to – 80°C followed by immersion into liquid nitrogen). Post thawing, SF was placed in culture conditions, after which the ability of SF to adhere and their metabolic activity in the MMT assay, the proliferative activity of cells that migrated from SF, and the migratory – proliferative potential in the monolayer “scratch assay” were studied. It was found that all the above indicators, except for the ability of SF to adhesion, were higher when using SF cryopreserved in a theoretically determined regimen, compared with SF cryopreserved in empirically selected regimens. Thus, cryopreservation according to the regimen established on the basis of physical and mathematical modeling leads to more effective preservation of functional properties (cell migration and proliferation) and metabolic state (MTT – assay) of SF after thawing compared to other regimes.

spheroid, L929 cell line, cryopreservation, migration, proliferation, MTT test.

1. Cui X, Hartanto Y, Zhang H. Advances in multicellular spheroids formation. J.R. Soc. Interface. 2017;(127):20160877. DOI: https:// doi.org/10.1098/rsif. 2016.0877.
2. Pinto B, Henriques AC, Silva PM, Bousbaa H. Three‑dimensional spheroids as in vitro preclinical models for cancer research. Pharmaceutics [Internet]. 2020;12:1186. Available from: https://microbialcellfactories.biomedcentral. com/articles/10.1186/ s12934–015–0383–5.
3. Lee JH, Jung DH, Lee DH, Park JK, Lee SK. Effect of spheroid aggregation on susceptibility of primary pig hepatocytes to cryopreservation. Transplant Proc. 2012;44:1015–7.
4. Moisieiev AI, Kovalenko IF, Kovalenko SYe, Bozhok GA, Gordiyenko OI. Dynamika pronykannya dymetylsulʹfoksydu v klityny liniyi L929 i sferoyidy z nykh. Probl Cryobiol Cryomed. 2021;31(4):316–325. DOI: https://doi. org/10.15407/cryo31.04.316. [in Ukrainian].
5. Moisieiev AI, Kovalenko IF, Bozhok GA, Gordiyenko OI. Teoretychni pidkhody do vyznachennya optymalʹnykh rezhymiv kriokonservuvannya klitynnykh sferoyidiv riznykh terminiv kulʹtyvuvannya. Biofi zychnyy visnyk. 2021;46:7–22. DOI: https://doi. org/10.26565/2075–3810–2021–46–01. [in Ukrainian].
6. Moisieiev AI, Bozhok GA, Gorina OL. Porivnyalʹna kharakterystyka tryvymirnoho ta monosharovoho kulʹtyvuvannya pereshcheplyuvanoyi liniyi fi broblastiv L929. Dopovidi Natsionalʹnoyi Akademiyi Nauk Ukrayiny. 2019;8:93–101. DOI: https://doi. org/10.15407/dopovidi2019.08.093. [in Ukrainian].
7. Bozhok GA, Moisieiev AI, Gorina OL, Bondarenko ТP. Morfofunktsionalʹni osoblyvosti fi broblastiv liniyi L929 v umovakh 3D‑kulʹtyvuvannya. Fiziolohichnyy zhurnal. 2019;65(3):34–40. DOI: https://doi.org/10.15407/ fz65.03.034. [in Ukrainian].
8. Tarusin DN, Kireyev VA, Kovalenko SYe, Kovalenko IF, Rozanov LF, Petrenko AYu. Selection of protocols to cryopreserve mesenchymal stromal cells in suspension and alginate microspheres by studying their osmotic responses in 1M DMSO. Probl Cryobiol Cryomed. 2016;26(2):133–144. DOI: https://doi.org/10.15407/cryo26.02.
9. Denker SP, Barber DL. Cell migration requires both ion translocation and cytoskeletal anchoring by the Na- H exchanger NHE 1. J Cell Biol. 2002;159:1087–96. DOI: http://www.jcb.org/cgi/doi/10.1083/jcb.20020805.
10. Salas‑ Oropeza J, Jimenez‑ Estrada M, Perez‑ Torres A, Castell‑ Rodriguez AE, Becerril‑ Millan R, Rodriguez‑ Monroy MA, еt al. Wound healing activity of the essential oil of Bursera morelensis, in mice. Molecules. 2020;25(8):1795. DOI: https://doi: 10.3390/ molecules25081795.

«Bulletin of problems biology and medicine» Issue 2 Part 1 (164), 2022 year, 100-108 pages, index UDK 57.086.13:576.5.016

10.29254/2077-4214-2022-2-1-164-100-108