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

在接下来的十年里，细胞疗法可能会成为一个价值2000多亿美元的全球市场。目前的研究大多集中在间充质基质细胞(MSCs)上。目前评估MSC培养活性的最佳实践要求对细胞进行染色以检测某些细胞成分的存在。然而，这种方法有缺点，可能不能代表细胞的真实状态。理想的监测技术将允许在没有污渍或标签的情况下评估整个细胞培养。该项目将开发成像技术来监控无污渍的培养。这项技术可以用于电池制造过程。该项目还将通过研究生院为K-12的学生提供细胞制造方面的培训。该项目将使用多光子成像方法。它将研究和合成参数，以创建与成骨分化相关的多个细胞特征的同时成像。这些指标包括使用氧化还原比和NAD/FAD寿命的细胞代谢、细胞形状和细胞器含量以及胶原蛋白的产生。2-D培养成像的验证将使用多元技术。这些技术将提取细胞和亚细胞细胞器的形状、代谢活动和细胞外基质的产生。这些技术还将在3D成骨微珠和水凝胶微载体上创建小规模培养的3D重建。实验将探索MSC对成骨介质配方的分化反应和结果，作为细胞年龄和扩张液的函数。最后，本项目将评估这些无标记多光子成像策略，以检测MSC分化的差异。建议的方法有可能显著改变目前对染色或重新镀膜的MSC样本进行成像和分选的做法。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.