1] School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA. [2] Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts, USA. [3] Vascular Biology Program, Children's Hospital, Boston, Massachusetts, USA. [4] Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, USA. [5].
Vascular Biology Program, Children's Hospital, Boston, Massachusetts, USA.
Nat Methods. 2014 Feb;11(2):183-9. doi: 10.1038/nmeth.2761. Epub 2013 Dec 8.
Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. Little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing three-dimensional (3D) tissues in vivo. We present a method to quantify cell-generated mechanical stresses exerted locally within living embryonic tissues, using fluorescent, cell-sized oil microdroplets with defined mechanical properties and coated with adhesion receptor ligands. After a droplet is introduced between cells in a tissue, local stresses are determined from droplet shape deformations, measured using fluorescence microscopy and computerized image analysis. Using this method, we quantified the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates, and we confirmed that these stresses (3.4 nN μm(-2)) are dependent on myosin II activity and are more than twofold larger than stresses generated by cells of embryonic tooth mesenchyme, either within cultured aggregates or in developing whole mouse mandibles.
细胞产生的机械力在胚胎组织形态发生和器官形成中起着关键作用。由于在体内无法测量发育中的三维 (3D) 组织内的细胞力，因此对于这些力如何塑造胚胎器官知之甚少。我们提出了一种方法，可以使用具有明确定义的机械性能的荧光、细胞大小的油微滴来量化活胚胎组织内局部产生的细胞机械应力，并将其涂覆有粘附受体配体。在组织中的细胞之间引入液滴后，使用荧光显微镜和计算机化图像分析测量液滴形状变形来确定局部应力。使用这种方法，我们量化了在 3D 聚集体中培养的乳腺上皮细胞产生的各向异性应力，并且证实这些应力（3.4 nN μm(-2)）依赖于肌球蛋白 II 活性，并且比在培养的聚集体内或在发育中的整个小鼠下颌骨中产生的胚胎牙齿间充质细胞产生的应力大两倍以上。
