Cultivation of cells in alginate drops as a high-performance method of obtaining cell spheroids for bioprinting

Purpose of the study. Testing the protocol of obtaining cell spheroids of breast cancer cell cultures for bioprinting by growing in alginate drops.

Materials and methods. Cells of breast cancer cell lines BT-20 and MDA-MB-453 were cultured in DMEM medium supplemented with 10 % FBS. Next, the cells were removed from the plastic using a trypsin-V ersene solution and resuspended in a sterile 2 % alginate solution in DPBS to the concentration of 10⁵ cells/ml. Then the alginate solution with the cells was slowly dripped through a 30G needle into a sterile cooled solution of calcium chloride (100 mM) from a height of 10 cm. After polymerization, alginate drops were washed in DMEM and cultured for two weeks in DMEM with the addition of 10 % FBS at 37 °C and 5.0 % CO₂.The spheroids formed in the alginate were photographed on the 3rd, 7th, 10th, and 14th days of cultivation, after which they were removed from the alginate by keeping in 55 mM sodium citrate solution with the addition of 20mM ethylenediaminetetraacetic acid (EDTA) and embedded in paraffin blocks according to the standard method, followed by histological examination.

Results. Cellular spheroids were formed in both cell cultures already on the 3rd day of cultivation. From the 3rd to the 10th day in both cultures, a uniform growth of cell spheroids was observed with a gradual slowdown in the increase in the size of spheroids by the 14th day of cultivation. On the 10th day the proportion of cells that formed clones (more than 500 μm² in size) was 25.2 % ± 7.1 % (n = 25) in the BT-20 culture and 38.5 % ± 9.9 % (n = 25) in MDA-MB-453 culture. On the 14th day, BT-20 culture was characterized by spheroids varying little in size and shape, with an average area of 1652 ± 175 µm², having a dense structure with smooth edges. The spheroids in MDA-MB-453 culture turned out to be more loose and easily deformed, their size and shape varied noticeably, the average area of the spheroids was 2785 ± 345 µm².

Conclusion. The production of spheroids in alginate drops is inferior in speed to the methods of forming cell conglomerates in hanging drops or on microwells, but it surpasses these methods in productivity, which is comparable to the production of spheroids by constant medium stirring on low-adhesive substrates. In addition, the clonal nature of the obtained spheroids leads to an increase in research costs and thus limits their scalability.

1. Afonin GV, Ragulin YuA, Gulidov IA. Accelerated regimens of adjuvant radiotherapy in the treatment of breast cancer. Research and Practical Medicine Journal. 2017;4(3):66–74. (In Russ.). https://doi.org/10.17709/2409-2231-2017-4-3-6, EDN: ZGIHQH

2. Vykhristyuk YuV, Roitberg GE, Dorosh JV, Karaseva NV, Akobova RA. Preventive measures against development of breast cancer. South Russian Journal of Cancer. 2021;2(1):50–56. https://doi.org/10.37748/2686-9039-2021-2-1-6, EDN: LXFIJG

3. Balykova LA, Inchina VI, Tarasova TV, Mosina LM, Gvozdikova EN, Khaydar DA, et al. The effectiveness of liposomal doxorubicin hydrochloride in combination with cyclophosphan in the treatment of breast cancer in an experiment. Research and Practical Medicine Journal. 2021;8(4):23–32. (In Russ.). https://doi.org/10.17709/2410-1893-2021-8-4-2, EDN: ALOTUB

4. Vladimirova LYu, Storozhakova AE, Snezhko TA, Strakhova LK, Abramova NA, Kabanov SN, et al. Hormone-positive HER2-negative metastatic breast cancer: decision making in real clinical practice. South Russian Journal of Cancer. 2020;1(2):46–51. https://doi.org/10.37748/2687-0533-2020-1-2-6, EDN: GKSYDZ

5. Roitberg GE, Dorosh JV, Anikeeva OYu. Treatment of thrice-negative breast cancer in a patient with metabolic syndrome. Research and Practical Medicine Journal. 2021;8(1):62–68. (In Russ.). https://doi.org/10.17709/2409-2231-2021-8-1-6, EDN: HKVAWJ

6. Zubareva EYu, Senchukova MA, Virich EV, Zubarev MR, Goncharova MA. Serum HIF-1α and TGF-β1 levels depending on the clinical and morphological characteristics of breast cancer and sensitivity of tumor to neoadjuvant chemotherapy. Research and Practical Medicine Journal. 2021;8(4):53–64. (In Russ.). https://doi.org/10.17709/2410-1893-2021-8-4-5, EDN: GLUUQD

7. Malignant neoplasms in Russia in 2021 (morbidity and mortality). Ed. by Kaprin AD, Starinsky VV, Shahzadova AO. Moscow: P. A. Herzen MNIOI – Branch of the National Medical Research Radiological Center. 2021, 252 p. Available at: https://oncology-association.ru/wp-content/uploads/2021/11/zis-2020-elektronnaya-versiya.pdf, Accessed: 29.12.2022. (In Russ.)

8. Galimova ES, Galagudza MM. Two-dimensional and three-dimensional cell culture models in vitro: pros and cons. Bulletin of Siberian Medicine. 2018;17(3):188–196. (In Russ.). https://doi.org/10.20538/1682-0363-2018-3-188-196, EDN: VJUHHB

9. Kit OI, Shatova JuS, Novikova IA, Vladimirova LJu, Ul’janova EP, Komova EA, et al. P53 and BCL2 expression in different breast cancer subtypes. Fundamental’nye Issledovaniya. 2014;(10-1):85–88. (In Russ.). EDN: SMJMCL

10. Timofeeva SV, Shamova TV, Sitkovskaya AO. 3D Bioprinting of Tumor Microenvironment: Recent Achievements. Biology Bulletin Reviews. 2021;82(5):389–400. (In Russ.). https://doi:10.31857/S0044459621050067, EDN: GMEORZ

11. Jubelin C, Muñoz-Garcia J, Griscom L, Cochonneau D, Ollivier E, Heymann MF, et al. Three-dimensional in vitro culture models in oncology research. Cell Biosci. 2022 Sep 11;12(1):155. https://doi.org/10.1186/s13578-022-00887-3

12. Zhuang P, Chiang YH, Fernanda MS, He M. Using Spheroids as Building Blocks Towards 3D Bioprinting of Tumor Microenvironment. Int J Bioprint. 2021;7(4):444. https://doi.org/10.18063/ijb.v7i4.444

13. Hamburger AW, Salmon SE. Primary bioassay of human tumor stem cells. Science. 1977 Jul 29;197(4302):461–463. https://doi.org/10.1126/science.560061

14. Filippova SYu, Sitkovskaya AO, Bondarenko ES, Novikova IA, Kharagezov DA, Pozdnyakova VV, et al. The Use of Alginate Encapsulation to Study EGF and FGF2 Effect on Colorectal Cancer Cells Proliferation in Low Adhesion Conditions in vitro. Tsitologiya. 2021;63(2):184–192. (In Russ.). https://doi.org/10.31857/S0041377121020024, EDN: QMRKCI

15. Johnson PA, Menegatti S, Chambers AC, Alibhai D, Collard TJ, Williams AC, et al. A rapid high throughput bioprinted colorectal cancer spheroid platform forin vitrodrug- and radiation-response. Biofabrication. 2022 Nov 2;15(1). https://doi.org/10.1088/1758-5090/ac999f

16. Filippova SYu, Timofeeva SV, Chembarova TV, Mezhevova IV, Gnennaya NV, Novikova IA, et al. Experience in obtaining a multicomponent three‑dimensional cellular model of tumor growth for molecular genetic studies of breast cancer. The Journal "Live and Bio-Abiotic Systems". 2022;(42). (In Russ.). https://doi.org/10.18522/2308-9709-2022-42-9, EDN: WMULYR

17. Aggarwal V, Miranda O, Johnston PA, Sant S. Three dimensional engineered models to study hypoxia biology in breast cancer. Cancer Lett. 2020 Oct 10;490:124–142. https://doi.org/10.1016/j.canlet.2020.05.030

18. Hompland T, Fjeldbo CS, Lyng H. Tumor Hypoxia as a Barrier in Cancer Therapy: Why Levels Matter. Cancers (Basel). 2021 Jan 28;13(3):499. https://doi.org/10.3390/cancers13030499