Robust microdroplet-based mechanical probes for wide-ranging mechanobiology applications

项目摘要 机械线索严重影响细胞行为，这些行为对胚胎发育、器官形成和发育至关重要。 维持组织结构和体内平衡。机械力和材料性能 细胞微环境（例如，刚度）是已知的指导干细胞分化以及改变 恶性表型在肿瘤进展过程中的进展。虽然人们普遍承认， 细胞微环境的机械力和材料特性在控制细胞的生长中起着关键作用。 体外细胞行为，缺乏在3D中进行力学定量测量的技术 细胞微环境已经大大阻碍了我们理解力学在更多方面的作用的能力。 生理相关的环境。PI Campas和同事最近报道了一种新的方法 它能够在3D细胞微环境中进行直接的体内和原位力学测量， 包括活体组织。虽然这些方法具有开创性，但它们利用了 作为机械传感器的全氟油的荧光标记的磁响应微滴， 致动器，由于当前用于制备微滴的化学物质，在范围上仍然受到很大的限制。 目前微滴的化学成分非常挑剔，这极大地限制了这项技术的应用范围， 阻碍了测量的可重复性，并妨碍了这些方法的传播， 广泛的生物和生物医学社区。在这个多PI技术开发赠款，坎帕斯，Sletten， 和Zink合作，通过开发新的鲁棒的微滴技术来解决现有的问题。 化学物质，包括氟化表面活性剂、荧光团和磁性纳米颗粒， 在广泛的生物系统中使用微滴进行机械测量， 器官到类器官、肿瘤和3D细胞培养。在目标1中，我们将开发表面活性剂和荧光团， 适当控制液滴的界面张力、细胞液滴相互作用及其在3D中的可视化 多细胞系统在目标2中，我们将开发稳定的全氟化碳基铁磁流体， 界面张力、细胞-液滴相互作用以及强磁性， 更大的力量。在目标3中，我们将测试新合成的化合物的功能性并评估它们的性能。 在成熟的3D多细胞系统中进行力学测量的性能，无论是在体外还是在 vivo.在完成这些目标后，我们将实现优化的微滴技术， 本发明的方法可用于商业化，并且可用于广泛的系统，包括活组织、类器官、胚状体、 身体，肿瘤和3D细胞培养，从而使这些新的工具，研究机械 线索（机械生物学）在体内的整个生物和生物医学界，并可能改变 我们对生物系统的理解。

PROJECT SUMMARY Mechanical cues critically affect cell behaviors that are central to embryonic development, organ formation and the maintenance of tissue architecture and homeostasis. Both mechanical forces and the material properties of the cellular microenvironment (e.g., stiffness) are known to direct stem cell differentiation as well as alter the progression of malignant phenotypes during tumor progression. While it is generally acknowledged that mechanical forces and the material properties of the cellular microenvironment play a key role in the control of cell behaviors in vitro, the lack of technologies to perform quantitative measurements of mechanics in 3D cellular microenvironments has considerably hindered our ability to understand the role of mechanics in more physiologically relevant environments. PI Campas and coworkers have recently reported a novel methodology that enables direct in vivo and in situ mechanical measurements within 3D cellular microenvironments, including living tissues, for the first time. While groundbreaking, these methods, which make use of fluorescently-labeled, magnetically-responsive microdroplets of perfluorinated oil as mechanical sensors and actuators, remain strongly limited in scope because of current chemistry used to prepare the microdroplets. The current finicky chemical composition of the microdroplets strongly limits the scope of the technique, hampers the reproducibility of the measurements and precludes the dissemination of these methods to the wide biological and biomedical communities. In this multi-PI technology development grant, Campas, Sletten, and Zink team up to solve the existing problems with the microdroplet technology by developing new robust chemistries, including fluorinated surfactants, fluorophores, and magnetic nanoparticles, to enable accurate mechanical measurements with microdroplets in a wide range of biological systems, from living tissues and organs to organoids, tumors and 3D cell culture. In Aim 1, we will develop surfactants and fluorophores to properly control the droplet’s interfacial tension, the cell droplet interactions and their visualization in 3D multicellular systems. In Aim 2, we will develop robust perfluorocarbon-based ferrofluids with controlled interfacial tension, cell-droplet interactions and with strong magnetic properties enabling the application of larger forces. In Aim 3, we will test the functionality of the newly synthesized compounds and assess their performance in mechanical measurements in well-established 3D multicellular systems, both in vitro and in vivo. Upon completion of these aims, we will achieve an optimized microdroplet technology that is ready for commercialization and can be used in a wide range of systems, including living tissues, organoids, embryoid bodies, tumors and 3D cell culture, thereby making accessible these new tools for the study of mechanical cues (mechanobiology) in vivo to the entire biological and biomedical communities and potentially transforming our understanding of biological systems.