Application of silicone coating to optimize the process of obtaining cellular spheroids by the hanging drop method

Purpose of the study. To study the effect of SIEL 159–330 coating on the cell clusters formation rate in a hanging drop method in combination with the use of methylcellulose (MC) and collagen as cell aggregation improving agents.
Materials and methods. BT20 breast cancer cells were cultured in drops of 20 μL (104  cells per drop) on the lid of a polystyrene Petri dish coated with SIEL 159–330 silicone elastomer (GNIIKHTEOS, Moscow, Russia) or without coating. The study tested three concentrations of MC (0.1 %, 0.25 % and 0.4 %) and collagen (150 µg/ml, 300 µg/ml and 600 µg/ml). The rate of formation of cell conglomerates was assessed by evaluating their area after 4, 24, 48, and 72 hours of cultivation.
Results. The use of SIEL 159–330 coating made it possible to obtain spheroids of the same size as the addition of 0.4 % MC over a time interval of 72 hours. The silicone coating additionally reduced the size of cell spheroids in the medium with 0.1 % MC at all time points; however, this effect disappeared with increasing concentration of MC. In addition, the use of SIEL 159–330 reduced the relationship between the size of cellular spheroids and the concentration of MC, which allows us to consider the use of this coating as an alternative to MC or a way to reduce its concentration. In the experiment with the addition of collagen to the culture medium, the sizes of cell conglomerates formed on the silicone coating were significantly smaller than on uncoated plastic in all variants of the experiment and time points. The effect was more pronounced for a collagen concentration of 600 μg/ml. The use of SIEL 159–330 coating, in addition, reduced the variability in the size and shape of the resulting cell conglomerates.
Conclusion. Accelerated aggregation of cells and fibers of the extracellular matrix in hanging drops, as well as a reduction in the variability in the size and shape of the resulting cell clusters on SIEL 159–330, allows us to reduce the time of experiments and material costs, as in experiments with the addition of substances that accelerate the formation of spheroids (MC and collagen), as well as in their absence.

1. Kit OI, Shatova JuS, Novikova IA, Vladimirova LJu, Uljanova EP, Komova EA, et al. P53 and BCL2 expression in different breast cancer subtypes. Fundamental Research. 2014;(10-1):85–88. Available at: https://fundamental-research.ru/ru/article/view?id=35219, Accessed: 16.03.2022. (In Russ.).

2. Timofeeva SV, Shamova TV, Sitkovskaya A.O. 3D bioprinting of tumor microenvironment: recent achievements. Biology Bulletin Reviews. 2021;82(5):389–400. (In Russ.). https://doi.org/10.31857/S0044459621050067

3. Liu D, Chen S, Win Naing M. A review of manufacturing capabilities of cell spheroid generation technologies and future development. Biotechnol Bioeng. 2021 Feb;118(2):542–554. https://doi.org/10.1002/bit.27620

4. Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng. 2003 Jul 20;83(2):173–180. https://doi.org/10.1002/bit.10655

5. Ivascu A, Kubbies M. Diversity of cell-mediated adhesions in breast cancer spheroids. Int J Oncol. 2007 Dec;31(6):1403–1413. https://doi.org/10.3892/ijo.31.6.1403

6. Leung BM, Lesher-Perez SC, Matsuoka T, Moraes C, Takayama S. Media additives to promote spheroid circularity and compactness in hanging drop platform. Biomater Sci. 2015 Feb;3(2):336–344. https://doi.org/10.1039/c4bm00319e

7. Huang H, Wright S, Zhang J, Brekken RA. Getting a grip on adhesion: Cadherin switching and collagen signaling. Biochim Biophys Acta Mol Cell Res. 2019 Nov;1866(11):118472. https://doi.org/10.1016/j.bbamcr.2019.04.002

8. Shamova TV, Sitkovskaya AO, Rostorguev EE, Kuznetsova NS, Kavitskiy SE. Preparation of Primary Glial Tumor Cell Lines. Perm Medical Journal. 2020;37(5):79–89. https://doi.org/10.17816/pmj37579-89

9. Dalir Abdolahinia E, Jafari B, Parvizpour S, Barar J, Nadri S, Omidi Y. Role of cellulose family in fibril organization of collagen for forming 3D cancer spheroids: In vitro and in silico approach. Bioimpacts. 2021;11(2):111–117. https://doi.org/10.34172/bi.2021.18

10. Oliveira MB, Neto AI, Correia CR, Rial-Hermida MI, Alvarez-Lorenzo C, Mano JF. Superhydrophobic chips for cell spheroids high-throughput generation and drug screening. ACS Appl Mater Interfaces. 2014 Jun 25;6(12):9488–9495. https://doi.org/10.1021/am5018607

11. Fu JJ, Lv XH, Wang LX, He X, Li Y, Yu L, et al. Cutting and Bonding Parafilm® to Fast Prototyping Flexible Hanging Drop Chips for 3D Spheroid Cultures. Cell Mol Bioeng. 2021 Apr;14(2):187–199. https://doi.org/10.1007/s12195-020-00660-x

12. Kuo CT, Wang JY, Lin YF, Wo AM, Chen BPC, Lee H. Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array. Sci Rep. 2017 Jun 29;7(1):4363. https://doi.org/10.1038/s41598-017-04718-1

13. Nanushyan SR. Organosilicon Materials of Rapid Curing: History of Trend Development. Chemical Industry Journal Today. 2015;(11):21–26. (In Russ.).

14. Filippova SYu, Sitkovskaya AO, Vashchenko LN, Kechedzhieva EE, Dashkova IR, Ausheva TV, et al. Application of Silicone Coating for Obtaining Cell Spheroids Using the Hanging Drop Method. Journal of Medical and Biological Research. 2022;10(1):44–51. https://doi.org/10.37482/2687-1491-Z089

15. Ware MJ, Colbert K, Keshishian V, Ho J, Corr SJ, Curley SA, et al. Generation of Homogenous Three-Dimensional Pancreatic Cancer Cell Spheroids Using an Improved Hanging Drop Technique. Tissue Eng Part C Methods. 2016 Apr;22(4):312–321. https://doi.org/10.1089/ten.TEC.2015.0280