Introduction: Mesenchymal stromal cells (MSCs) are multipotent stem cells that can be established from various tissues including bone marrow, adipose tissue, and umbilical cords. MSCs can differentiate into various types of cells such as bone, fat, muscle, and nerve cells. Because undifferentiated MSCs possess tissue-repair and immunomodulatory properties, cell therapies using MSC are gaining popularity in various fields of regenerative medicine. On the other hand, MSCs are known to highly express tissue factor (TF) on their cell surfaces, which gives them a prothrombotic potential. It is thought that thrombus formation, such as pulmonary embolism, may be induced by intravenous administration of MSCs that express high levels of TF (BBRC. 431(2):203-9. 2013). It is also known that the TF expression level of MSCs is affected by the tissue of origin and culture conditions, such as serum concentration (Theranostics. 2018; 8(5): 1421-34). Therefore, we investigated the effect of the cell culture medium on the levels of TF expression and the prothrombotic potential of MSCs.

Methods: We evaluated the TF expression levels of human adipose-derived MSCs cultured in various media for two to three days using fluorescence microscopy and flow cytometry. Furthermore, we quantitatively evaluated the prothrombotic potential of the cells by calculating the clotting time of the mixture of the cells and normal human plasma after adding CaCl₂.

Results: Serum-free medium (Ex-MSC XF medium, Myoridge) that we developed markedly suppressed TF expression on MSCs while retaining cell proliferation potency that was comparable to or superior to that of the FBS-containing DMEM medium. Furthermore, the clotting time was significantly prolonged in the Ex-MSC-cultured MSCs compared to FBS-containing DMEM medium-cultured MSCs, suggesting their lower prothrombotic potential. Conversely, the addition of FBS or human platelet-derived factor to Ex-MSCs increased TF expression in a concentration-dependent manner. Additionally, our TF-suppressive supplements that we recently designed reduced the TF expression on MSCs even in the presence of FBS, and significantly prolonged the clotting time of MSC-mixed human plasma.

Conclusion: The composition of the medium significantly affects the TF expression and TF-induced prothrombotic potential of MSCs. When considering intravenous administration of MSCs, it may be safer and more effective to use this type of culture medium when culturing MSCs to avoid adverse thrombotic events and achieve more therapeutic effects.