Quantifying the effects of decellularized extracellular matrix and photobiomodulation on cell characteristics with label-free nucleus position detection

Background:Cell migration assay is crucial for assessing the migratory capability of cells. Traditional methods typically rely on observing changes in scratched regions, which may introduce inherent errors. Decellularized extracellular matrix (dECM) and photobiomodulation (PBM) are recognized as effective wound repair methods that promote cell migration, proliferation, and differentiation. dECM, rich in fibronectin and collagen, provide good environment for cell, while PBM enhances cell movement by increasing mitochondrial ATP production.
Objective:This study aims to develop a novel quantitative method to investigate the synergistic effects of dECM and PBM on cell migration and evaluate their impact on migratory capabilities. Traditional assessment methods based on scratched area changes can be influenced by cell volume alterations. Therefore, we adopted a new quantitative approach by evaluating changes in the number of cell nuclei within specified regions for accurate assessment. To avoid potential cell damage and limitations in continuous observation associated with traditional fluorescent staining, we integrated a label-free method to mark cell nucleus positions.
Materials and Methods:Firstly, cells were cultured in Culture-Insert 2 Well for 24 hours, followed by application of dECM to a defined 500 µm cell-free gap area. Subsequently, cells were subjected to PBM irradiation at a dosage of 7.5 J/cm². Next, a U-Net model was trained to assist in generating images of cell nucleus positions. To assess cell migration capability, we utilized Residual U-Net and proprietary algorithms to quantify changes in cell nucleus numbers within the specified region.
Results:Experimental results demonstrate that PBM irradiation at a dose of 7.5 J/cm² significantly enhances the migratory ability of NIH3T3 cells. However, in our experiments, the application of dECM to the cell-free gap area did not significantly enhance cell migration compared to PBM irradiation alone at a dose of 7.5 J/cm². Additionally, efficient image segmentation of cell nucleus fluorescent staining images was achieved using U-Net, with the applied algorithm achieving 96.91% accuracy in cell nucleus counting.
Conclusion:This study confirms the effective role of PBM in promoting cell migration, while dECM did not perform as anticipated in enhancing cell migration. Evaluating changes in the number of cell nuclei within cell-free gap areas improves the accuracy of assessing cell migration capabilities. Despite potential issues such as cell toxicity and photobleaching associated with traditional fluorescent staining, our integration of a label-free method successfully mitigates potential cell damage associated with fluorescent staining, enabling more reliable observations.