Vol 66, No 4 (2024)

In this study, the viability and adhesive features of BRO and SK-MEL-2 cell lines were evaluated using a melanoma model. It was revealed that in BRO cells, the development of apoptosis after exposure to dacarbazine was combined with the transition of the proportion of cells to the G₀ phase of the cell cycle, that corresponded to previously obtained results. The absence of apoptosis in 3D spheroids and the absence of exit from the cell cycle were observed in SK-MEL-2 melanoma cells. It was also revealed that in the control spheroids (cells without exposure) of the BRO and SK-MEL-2 melanoma lines, adhesion to fibronectin was higher compared with the cells of the control monolayer, which is explained by the three-dimensional structure requiring cell communication with the extracellular matrix. In spheroids formed by SK-MEL-2 cells, dacarbazine induced a decrease in adhesion to fibronectin, which may be associated with the development of drug resistance. An increase in the expression of integrins AV and β8 in BRO and SK-MEL-2, as well as integrin β5 in SK-MEL-2, was determined in cells after exposure to dacarbazine, which may indicate the involvement of aforementioned molecules in the exit from proliferative stage of tumor cells.

Mesenchymal stem/stromal cells (MSCs) isolated from equine adipose tissue (AT) represent a promising material for the creation of bioveterinary products for the prevention and treatment of many diseases. The production of these cells for clinical use requires improved serum-free culture conditions. The microenvironment can influence the properties of MSCs. It is believed that the requirements for culture conditions without animal blood serum are species specific. The purpose of this study was to evaluate the commercially available serum-free media (SFM) MesenCult (STEMCELL Technologies, USA), created for human MSCs, for the cultivation of equine MSC(AT). One part of the cells was propagated for 10 passages in the standard DMEM medium with a low glucose content (1 g/l) and 10% fetal bovine serum (FBS), and the second in SFM. The results show that the propagation of equine MSCs in MesenCult serum free, intended for the cultivation of human MSCs, is possible, since the cells adapt well to it and retain properties characteristic of cells that are cultured in DMEM with FBS: morphology, growth rate, doubling time and mitotic index, clone-forming abilities, diploid set of chromosomes, a large number of cells with the CD90 phenotype (90.8%) and low with the CD31 (0.8%), CD34 (0.9%) phenotype, as well as the potency for induction of differentiation into adipo-, osteo- and chondrogenic directions. Equine MSC(AT) showed stable characteristics after being cultured for 10 passages in SFM, providing a promising basis for their further use. Our results demonstrate that MesenCult media may be an alternative for serum-free culture of equine MSC(AT) for expansion in preclinical studies.

Scientists need cell models from human tissues to develop methods of gene therapy and genome editing for monogenic diseases. It is preferable to use minimally invasive methods to obtain samples; these tissues can be applied for further screening in order to select the most effective approach to restore the synthesis of the target protein. We used the CRISPR/Cas9-SAM transcriptional activation system, which ensures expression of the DYSF gene in HEK293Т cells, as well as in fibroblasts from patients with dysferlinopathy (c.2779delG (Ala927LeufsX21)). After targeted activation of DYSF, it was possible to detect the main gene products: mRNA and protein (HEK293Т_ТА) and mRNA (fibroblasts). Transcriptionally activated dysferlin-deficient fibroblasts and HEK293 cells can be used to evaluate the in vitro efficacy of gene therapy for dysferlinopathies.