Mesenchymal stem cells (MSCs) are of great interest in cell therapies due to their immunomodulatory and regenerative properties. However, their low concentration and heterogeneity make it challenging to analyze them directly after isolation. MSCs are typically expanded in cell culture to reach the required numbers for clinical applications. This review discusses the heterogeneity and concentration of MSCs, as well as strategies for phenotyping and counting freshly isolated MSCs. MSCs are found in various tissues, including bone marrow, adipose tissue, and placenta. Their concentration varies depending on the tissue source and the patient's age and health. MSCs have diverse functions, including immunomodulation, tissue repair, and secretion of bioactive molecules. However, their therapeutic effectiveness can be affected by their low concentration and phenotypic diversity. MSCs are often used in clinical trials, but there is a need for standardized methods to count and characterize them, especially in one-step procedures where cells are implanted immediately after isolation. The review highlights the challenges in MSC counting and the importance of developing standardized methods for their accurate identification and quantification. The study also discusses the impact of MSC isolation techniques, tissue sources, and cell culture conditions on their phenotypic and functional properties. The review emphasizes the need for further research to improve the standardization and clinical application of MSC-based therapies.Mesenchymal stem cells (MSCs) are of great interest in cell therapies due to their immunomodulatory and regenerative properties. However, their low concentration and heterogeneity make it challenging to analyze them directly after isolation. MSCs are typically expanded in cell culture to reach the required numbers for clinical applications. This review discusses the heterogeneity and concentration of MSCs, as well as strategies for phenotyping and counting freshly isolated MSCs. MSCs are found in various tissues, including bone marrow, adipose tissue, and placenta. Their concentration varies depending on the tissue source and the patient's age and health. MSCs have diverse functions, including immunomodulation, tissue repair, and secretion of bioactive molecules. However, their therapeutic effectiveness can be affected by their low concentration and phenotypic diversity. MSCs are often used in clinical trials, but there is a need for standardized methods to count and characterize them, especially in one-step procedures where cells are implanted immediately after isolation. The review highlights the challenges in MSC counting and the importance of developing standardized methods for their accurate identification and quantification. The study also discusses the impact of MSC isolation techniques, tissue sources, and cell culture conditions on their phenotypic and functional properties. The review emphasizes the need for further research to improve the standardization and clinical application of MSC-based therapies.