Purpose: Mesenchymal stem cells (MSCs) have demonstrated clinically relevant therapeutic effects for treatment of trauma and chronic diseases. The proliferative potential, immunomodulatory characteristics, and multipotentiality of MSCs in monolayer culture is reflected by their morphological phenotype. Standard techniques to evaluate culture viability are subjective, destructive, or time-consuming. We present an image analysis approach to objectively determine morphological phenotype of MSCs for prediction of culture efficacy. Approach: The algorithm was trained using phase-contrast micrographs acquired during the early and mid-logarithmic stages of MSC expansion. Cell regions are localized using edge detection, thresholding, and morphological operations, followed by cell marker identification using H-minima transform within each region to differentiate individual cells from cell clusters. Clusters are segmented using marker-controlled watershed to obtain single cells. Morphometric and textural features are extracted to classify cells based on phenotype using machine learning. Results: Algorithm performance was validated using an independent test dataset of 186 MSCs in 36 culture images. Results show 88% sensitivity and 86% precision for overall cell detection and a mean Sorensen-Dice coefficient of 0.849 ± 0.106 for segmentation per image. The algorithm exhibited an area under the curve of 0.816 ( CI 95 = 0.769 to 0.886) and 0.787 ( CI 95 = 0.716 to 0.851) for classifying MSCs according to their phenotype at early and mid-logarithmic expansion, respectively. Conclusions: The proposed method shows potential to segment and classify low and moderately dense MSCs based on phenotype with high accuracy and robustness. It enables quantifiable and consistent morphology-based quality assessment for various culture protocols to facilitate cytotherapy development.

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