Vsevolodska S. O.

FEATURES OF SPHEROID FORMATION BY NEURAL CELLS OF NEWBORN RATS IN DROPS

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About the author:

Vsevolodska S. O.

Heading:

BIOLOGY

Type of article:

Scientific article

Annotation:

The article discusses the development and optimization of a method for forming neural cells in the form of spheroids using the hanging drop method. Neural cells obtained from the brain tissue of newborn rats were cultivated in three-dimensional structures spheroids that can reproduce the complex physiological microenvironment of the brain in vitro. The use of the hanging drop method enabled the control of cell aggregation conditions, ensuring the formation of stable spheroids of optimal size, which is crucial for maintaining cell viability and facilitating the effective diffusion of oxygen and nutrients. Studies have shown that only pre-cultured neural cells that have undergone a recovery phase after enzymatic and mechanical isolation are capable of forming spheroids. Freshly obtained cells did not form stable three-dimen- sional structures due to damage to the cell membrane and loss of adhesion molecules. It was found that the optimal drop volume is 20 μl, which ensures the stability of the drop shape during cultivation. The size of spheroids directly depends on the initial concentration of neural cells in the drop and reaches optimal values at 2-8×10³ cells/drop. The cultivation time required to achieve a three-dimensional structure is 4-5 days. Spheroids obtained by the “hanging drop” method consist of viable cells capable of migration, differentiation, and formation of neuronal networks, confirming their functional suitability for modeling nervous tissue. The developed model is of great importance for further research into neurodegenerative diseases, toxicological analysis, and screening of pharmacological drugs. Thus, the “hanging drop” method for forming spheroids from neural cells is an effective tool for creating 3D models of nervous tissue with high biological relevance.

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Bibliography:

1. Fetah K, Tebon P, Goudie MJ, Eichenbaum J, Ren L, Barros N, et al. The emergence of 3D bioprinting in organ-on-chip systems. Prog Biomed Eng. 2019;1(1):0120011. DOI: 10.1088/2516-1091/ab23df.
2. Wang Y, Li W, Gu Y, Zhu Y, Qi J. Engineering stem cell-derived 3D brain organoids in a perfusable organ-on-a-chip system. RSC Adv. 2018;3:1677-1685. DOI: 10.1039/C7RA11714K.
3.  Hasan MF, Ghiasvand S, Wang H, Miwa JM, Berdichevsky Y. Neural layer self-assembly in geometrically confined rat and human 3D cultures. Biofabrication. 2019;11(4):045011. DOI: 10.1088/1758-5090/ab2d3f.
4. Zbigniev B, Česnaitis V, Šušnjev N, Stankevičius G, Dambrauskaitė K, Inčiūra H, et al. Cerebellar cells self-assemble into functional organ- oids on synthetic, chemically crosslinked ECM mimicking peptide hydrogels. Biomolecules. 2020;10(5):754. DOI: 10.3390/biom10050754.
5. Vinci M, Gowan S, Boxall F, Patterson L, Zimmermann M, Court W, et al. Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol. 2012;10:29. DOI: 10.1186/1741-7007-10-29.
6. Pampaloni F, Reynaud EG, Stelzer EHK. The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol. 2007;8(10):839-45. DOI: 10.1038/nrm2236.
7. Kim Y, Kang K, Yoon S, Kim JS, Park SA, Kim WD, et al. Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures. Organogenesis. 2018;14(1):1-12. DOI: 10.1080/15476278. 2018.1423931.
8. Petrenko AY, Sukach AN. Isolation of intact mitochondria and hepatocytes using vibration. Anal Biochem. 1991;194(2):326-9. DOI: 10.1016/0003-2697(91)90236-m.
9.  Sukach AN, Liashenko TD, Shevchenko MV. Properties of isolated neural cells from newborn rat in tissue in vitro. Biotechnol Acta. 2013;6(3):63-8. Available from: http://nbuv.gov.ua/UJRN/biot_2013_6_3_8.
10.  Sukach AN, Shevchenko MV, Liashenko TD. Comparative study on influence of fetal bovine serum and serum of adult rat on cultivation of newborn rat neural cells. Biopolym Cell. 2014;30(5):394-9. DOI: 10.7124/bc.0008B7.
11. Harley-Troxell ME, Dhar M. Assembling spheroids of rat primary neurons using a stress-free 3D culture system. Int J Mol Sci. 2023;24(17):13506. DOI: 10.3390/ijms241713506.
12. Zhou QZ, Feng XL, Jia XF, Mohd Nor NHB, Harun MH, Feng DX. Culture and identification of neonatal rat brain-derived neural stem cells. World J Stem Cells. 2023;15(6):607-616. DOI: 10.4252/wjsc.v15.i6.607.
13. Gonmanee T, Arayapisit T, Vongsavan K, Phruksaniyom C, Sritanaudomchai H. Optimal culture conditions for neurosphere formation and neuronal differentiation from human dental pulp stem cells. J Appl Oral Sci. 2021;29:e20210296. DOI: 10.1590/1678-7757-2021-0296.
14. Vilinski-Mazur K, Kirillov B, Rogozin O, Kolomenskiy D. Numerical modeling of oxygen diffusion in tissue spheroids undergoing fusion using functional representation and finite volumes. Sci Rep. 2025;15:5054. DOI: https://doi.org/10.1038/s41598-025-86805-2.

Publication of the article:

«Bulletin of problems biology and medicine», 2025 Issue 3,178, 97-106 pages, index UDC 611.018.82.082.2:567.7

DOI:

10.29254/2077-4214-2025-3-178- 97-106