3D-CULTURE OF HEK293 TUMOR CELLS AS AN EXPERIMENTAL MODEL IN ONCOLOGY

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Relevance. Currently, oncological diseases are increasingly being detected in young and middle-aged people, jeopardizing both the prognosis for life and for working capacity. Studies of new molecules are aimed at revealing their activity against tumor tissue and achieving safety for healthy cells [1]. The use of classical animal models is an expensive and effective method, but today the development of cellular technologies allows us to use lines of tumor cells formed not only as a two-dimensional monolayer, but also in a three-dimensional form, which makes it possible to simulate the course of oncological processes and test new drugs under conditions closest to clinical [2].

The aim of the study is to evaluate a 3D culture of tumor cells of the HEK293 line when testing chemotherapy drugs, in comparison with a monolayer.

Materials and methods. The study was conducted on the basis of the Research Institute of Experimental Medicine, Kursk State Medical University. Two types of tumor cultures were obtained from HEK293 tumor cells. Two-dimensional - in the form of a monolayer at the bottom of the culture flask, and three-dimensional - in the form of spheroids, formed by seeding on a non-adhesive matrix based on agarose. On the first day of cultivation, the experimental groups were treated with chemotherapy drugs: doxorubicin and cisplanin at a concentration of 35 μM. The result of their antiproliferative effect in the monolayer was assessed by the number of cells per 1 ml using a Goryaev camera on the 7th day; in 3D culture, by the volume of spheroids, since the volume of a spheroid directly depends on the number of constituent cells [3]. The result of the cytotoxic effect in the monolayer was stained with trypan blue and evaluated by the number of living cells per 1 ml using a Goryaev camera. Two-dimensional and three-dimensional cultures were compared by assessing the resistance of cells in increasing concentrations of chemotherapy drugs in terms of ID50 and LD50. The results were statistically processed in Microsoft Excel 2016.

Results and discussion. When evaluating a two-dimensional culture of tumor cells of the HEK293 line in the control group, a concentration of 120.2±8.1 thousand cells/ml was obtained. The concentration of tumor cells in a two-dimensional culture after treatment with doxorubicin and cisplanin was 15.6±1.6 thousand cells/ml and 30.8±1.2 thousand cells/ml. Living cells when using doxorubicin 5.7±2.6 thousand cells/ml (36.4%), and when using cisplanin 13.5±4.2 thousand cells/ml (43.9%). Doxorubicin in flat culture has an antiproliferative effect, manifested to a greater extent due to pronounced cytotoxicity, compared with cisplanin. A comparison of two types of cell culture showed that the cells are more resistant to the action of chemotherapy drugs in the form of spheroids. Thus, the ID50 of doxorubicin in 3D culture was 0.12±0.03 µM, and in flat culture it was 0.02±0.01 µM. With respect to cisplanin, the trend is confirmed by ID50 of 8.1±0.02 µM and 1.7±0.04 µM, respectively. However, when evaluating the cytotoxic effect, it was found that the LD 50 of doxorubicin for spheroids and the monolayer differ slightly, 26 ± 3 μM and 24 ± 4 μM, LD 50 of cisplanin 120 ± 5 μM and 142 ± 10 μM, respectively.

Conclusions. Doxorubicin in flat culture has an antiproliferative effect, manifested to a greater extent due to pronounced cytotoxicity, compared with cisplanin. The 3D culture is more resistant to the action of chemotherapy drugs in relation to cell division, which is associated with its surface growth zone and the effect of the active substance on it, and at the same time, it is equally susceptible to the cytotoxic effect, like the two-dimensional culture.

About the authors

Vadim Vadimovich Tsymbalyuk

Author for correspondence.
Email: vadimz2552@mail.ru
ORCID iD: 0000-0003-1419-0127
SPIN-code: 6176-6903
Russian Federation, 3, K. Markx str., Kursk, 305000, Russia

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