Cell and Organ Transplantology. 2025; 13(1):32-44 (e2025131175).
DOI: 10.22494/cot.v13i1.175

Optimization of conditions for the production of small extracellular vesicles from human umbilical cord-derived mesenchymal stem cells

Gordiienko I.^1,2, Shamshur M.^1,3, Novikova S.³, Zlatskiy I.³, Zlatska A.^1,3

• ¹Biotechnology Laboratory, Medical Company “Good Cells”, Kyiv, Ukraine
• ²R. E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine
• ³Institute of Genetic and Regenerative Medicine, M. D. Strazhesko National Scientific Center of Cardiology, Clinical and Regenerative Medicine, National Academy of Medical Sciences of Ukraine, Kyiv, Ukraine

Abstract

Extracellular vesicles (EVs), derived from mesenchymal stem cells (MSCs), play crucial roles in the physiological functions of MSCs and their therapeutic effects. Compared with cellular products, the composition of EVs, their biological properties, mechanisms of action, and superior stability make them attractive candidates for regenerative medicine, heralding a new era in cell therapy.
Aim – to identify optimal conditions for obtaining small EVs from cultured human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) based on quantitative indicators of EVs yield and the morpho-functional characteristics of MSC cultures depending on cultivation conditions.
Materials and methods. The MSCs were isolated from the human umbilical cord through enzymatic digestion. The process of optimizing the cultivation medium was conducted as follows. The first ways – culture medium was replaced with fresh medium and maintained for the subsequent 24 hours. The second ways – culture continued in the unaltered medium. Then they were changed to two media: Hank’s solution and MEM alpha medium, both without devoid of xenogeneic serum and exogenous growth factors. Cultivation and extracellular vesicles production from hUC-MSCs under serum-free conditions were carried out for 24, 48, and 72 hours. All vesicles were examined to confirm their specific size distribution, determined by nanoparticle tracking analysis (NTA), and the presence of CD63 and CD81 markers, as assessed by ELISA.
Results. Replacing the growth medium in the hUC-MSC culture 24 hours before switching to serum-free medium led to a significantly higher yield of extracellular vesicles. Upon transitioning the culture to serum-free medium, we identified a dependency on both the composition of the serum-free medium and the duration of MSC cultivation in these conditions. The highest yield of extracellular vesicles was observed after 48 and 72 hours of cultivation, with no significant difference observed between the serum-free medium compositions. We attribute this result to the adaptation period of the culture during the first 24 hours of serum-free cultivation. Given that no significant difference in extracellular vesicle yield was found between 48 and 72 hours, we propose 48 hours as the optimal cultivation time.
Conclusion. The findings of this study indicate that the optimal conditions for the cultivation of umbilical cord mesenchymal stem cells for the production of small extracellular vesicles in serum-free medium are as follows: culturing hUC-MSCs in complete growth medium with regular medium exchanges every 2-3 days until the culture achieves 70 % confluence; replacing the growth medium 24 hours prior to transitioning to serum-free medium; cultivating the hUC-MSC culture for 48 hours in Hank’s solution or MEM, without the addition of xenogeneic serum.

Key words: mesenchymal stem cells; extracellular vesicles; exosomes; serum-free medium

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