Mesenchymal stem cells (MSCs) are of great interest in cell therapies due to their immunomodulatory properties and other effects they have on recipients after autologous or allogeneic transplantation. In the majority of clinical applications, a high number of MSCs is required; for this reason, isolated MSCs have to be expanded in the cell culture until the desired number is reached. Determining the MSC count from different tissue origins preceding cell expansion is not widely implemented. Phenotyping using flow cytometry and MSCs enumeration is usually performed only during or after their expansion. Counting freshly isolated cells is challenging due to their rareness and heterogeneity. Heterogeneity is noticeable among donors, tissues, and cell subpopulations. The identification of MSCs from different tissues is also complex because there is no consensus on the uniform cell surface marker panel. There are also differences in the concentration of cells which may be influenced by donor age, health status, isolation methods, and various other factors. As MSC applicability is continuously growing and developing, there is a need to implement and standardise counting methods for freshly isolated MSCs. With the introduction of a uniform procedure to count MSCs right after tissue harvest, we could predict the number of passages needed for cell expansion and reduce overall cell manufacturing costs. For new methodologies, where uncultured cells are used in therapy with what are referred to as one-step procedures, determining the number of freshly isolated MSCs is even more important
