Comparative analysis of enriched mesenchymal stem cells conditioned medium fractions obtained by ultrafiltration
Human mesenchymal stem/stromal cells (MSCs) secrete a wide range of biologically active molecules (the secretome), which can reduce tissue inflammation and enable its regeneration, making them extremely promising for application in regenerative medicine and immunotherapy. Furthermore, the MSC secretome, containing exosomes and biologically active molecules, can be used separately from the cells themselves as a promising, cell-free alternative to cell therapy, for which there are currently a number of legislative restrictions.
The research is dedicated to developing a fractionation method and characterizing the fractions of the mesenchymal stem cell (MSC) secretome produced by the cells into the culture medium (conditioned medium, CM).
To isolate functionally significant components of the secretome, sequential filtration of the CM was applied using filters with 0.2 μm pores and a 50 kDa molecular weight cutoff. This approach allows for the isolation of a fraction enriched with exosomes and medium-sized proteins. It is this fraction that contains the main biologically active part of the secretome (including growth factors, cytokines, exosomes, etc.). The proposed method effectively separates larger particles (cellular debris, microvesicles) and low-molecular-weight compounds (< 50 kDa), which can interfere with analysis or therapeutic use, and concentrates the target components. Using this method, fractions were obtained for CM containing a standard component of nutrient culture media — fetal bovine serum — and for CM obtained under serum-free conditions. A qualitative and quantitative analysis of the fraction composition was then conducted. The CM obtained in the presence of serum demonstrates a higher content of total protein, free RNA, exosomes, and extracellular vesicles, where some molecules are secreted by the MSCs themselves, and some represent residual serum components introduced with the culture medium. The CM obtained under serum-free conditions contains exclusively MSC secretion products, which eliminates the problem of contamination with xenogenic (foreign) serum components. This is critically important for clinical application, where maximum standardization and safety are required. Thus, the choice of CM production method should depend on the experimental goals: for fundamental research, where the priority may be the maximum yield of a wide spectrum of components (especially exosomes), serum-containing CM may be acceptable. For developing therapeutic products and pre-clinical/clinical research, the absolute priority is the use of serum-free CM.
The article emphasizes that for the use of the MSC secretome in therapy, standardized serum-free MSC culture protocols and effective, easily scalable methods for secretome purification/fractionation, such as ultrafiltration, are necessary to ensure a safe and reproducible product with a known composition.
Article authors: E. V. Andreev (FLNR JINR), E. V. Kravchenko (DLNP JINR), E. G. Zavyalova (Lomonosov Moscow State University, FLNR JINR), P. S. Eremin (Federal State Budgetary Institution "National Medical Research Center for Rehabilitation and Balneology" of the Ministry of Health of the Russian Federation), A. V. Rzyanina (DLNP JINR), P. A. Markov (Federal State Budgetary Institution "National Medical Research Center for Rehabilitation and Balneology" of the Ministry of Health of the Russian Federation), A. N. Nechaev (FLNR JINR).
