EAGER: Nonlinear label-free microscopy for evaluation of MSC osteogenic potency in 3D culture systems

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Cell therapeutics could become a $200+ billion global market in the next ten years. Much of the current research focuses on mesenchymal stromal cells (MSCs). Current best practice in evaluating MSC culture activity requires staining the cells to detect the presence of certain cell components. However, this approach has shortcomings and may not represent the true state of the cells. The ideal monitoring technique would allow evaluation of the entire cell culture without stains or labels. This project will develop imaging techniques to monitor the culture without stains. This technology could be used in cell manufacturing processes. The project will also provide students at K-12 through graduate school training in cell manufacturing. The project will use a multiphoton imaging approach. It will investigate and synthesize parameters to create simultaneous imaging of multiple cell features associated with osteogenic differentiation. These include cell metabolism using redox ratio and NAD/FAD lifetime, cell shape and organelle content, and collagen production. Validation of imaging of 2-D cultures will use multivariate techniques. These techniques will extract the shape of cells and subcellular organelle organization, metabolic activity, and extracellular matrix production. These techniques will also create a 3-D reconstruction of small-scale cultures on 3-D osteogenic microbeads and hydrogel microcarriers. Experiments will explore MSC differentiation response and outcomes to osteogenic media formulations as a function of cell age and expansion media. Finally, this project will evaluate these label-free multiphoton imaging strategies to detect differences in MSC differentiation. The proposed approach has the potential to significantly transform the current practice of imaging and sorting stained or re-plated MSC samples.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.