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Oil droplets as force transducers Force induced growth
Measuring cellular forces in vivo and in situ back Top

Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. However, little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing 3D tissues in vivo and in situ.

Droplet as force transducer

Figure 1: Mesenchymal cells (red) of a mouse mandible (E13.5) squeezing an oil droplet (cyan). The deformation of the droplet allows us to obtain the squeezing forces generated by the cells around the droplet. Image by Otger Campàs

We have developed a method to quantify cell-generated mechanical stresses that are exerted locally within living embryonic tissues using fluorescent, cell-sized, oil microdroplets with defined mechanical properties and coated with surface integrin or cadherin receptor ligands. After introducing a droplet between cells in a tissue (Fig. 1), local stresses are determined from the droplet shape deformations, which are obtained via fluorescence microscopy and computerized image analysis (Fig. 2). Using this method, we quantified the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates and confirmed that these stresses are dependent on myosin II activity and more than two-fold larger than the stresses generated by cells of embryonic tooth mesenchyme when analyzed within similar cultured aggregates or in developing whole mouse mandibles. These results are published in the advance online publication of the journal Nature Methods.

Droplet stresses
Figure 2: Three-dimensional reconstruction of a droplet deformed by cellular forces. The stronger the cellular forces applied to the droplet, the larger its deformation. The value of the cellular stresses on the droplet's surface is color coded. Image by Otger Campàs

This technique is inspired on a previous project, in which vegetable oil droplets were used to measure the forces generated by growing actin networks. Unlike experiments with isolated molecules, force measurements involving living cells or tissues cannot be performed with vegetable oils as the lipids composing the droplets easily transfer to cell membranes, potentially causing toxicity or complicated side effects. In order to use oil droplets as force transducers within living tissues, we used fluorocarbon oils and designed specific coatings to control cell-droplet interactions. The technique was first tested in 3D cell aggregates and also in living mouse tissue (mouse mandibles) to demonstrate that it is possible to quantify cellular stresses in situ and in vivo.

However, the droplet technique can also be used to quantify stresses generated by single cells or cells grown in standard monolayer cultures. The combination of 3D droplet reconstruction and time-lapse fluorescence microscopy allows quantitative measurements of both tensional and compressional cellular stresses surrounding the droplet as well as their temporal changes. In addition, the ability to control the type and concentration of ligands on the surface of the droplet, as well as its interfacial tension, allows these force transducers to be adapted to a wide variety of experimental conditions. Therefore, the characteristics of this technique are well suited for any study that requires quantification of stresses generated by individual living cells or groups of cells in culture, embryonic tissues or adult organs. This technique should therefore enable quantitative analysis of the role of cellular forces in embryonic development, and potentially, in disease processes as well.

This technique was initially developed by Otger Campàs, Donald Ingber and coworkers in the Wyss Institute at Harvard University. Our group is currently developing this technique further to measure the patterns of cellular forces that shape embryonic tissues in fish, chicken and other species.

LinkMeasuring cell-generated mechanical forces within living embryonic tissues. O. Campàs, T. Mammoto, S. Hasso, R.A. Sperling, D. O'Connell, A.G. Bischof, R. Maas, D.A. Weitz, L. Mahadevan and D.E. Ingber. Nature Methods, in press (2013).

 

Related press coverage:

- May the cellular force be with you. UC Santa Barbara news.
- Measuring life's tugs and nudges. Harvard SEAS news.

- Novel method developed for measuring mechanical stresses within Tissues. Materials360 online.

Mechanical control of tissue growth back Top

 

Tissue morphogenesis
Scheme showing tension-driven tissue remodelling during normal
morphogenesis and its deregulation during tumour formation. From Huang & Ingber, Nature Cell Biology (1999).

In any process of morphogenesis, including embryo and tissue growth, there is clear physical aspect associated to the actual growth of the structure. Morphogens, growth factors and other signaling molecules are known to orchestrate developmental processes so that tissues are properly shaped into their functional morphology. However, it is well known that physical force can affect substantially growth patterns during development, meaning that the ultimate functional morphology of a tissue is governed by the combined action of signaling molecules and physical forces. We are interested in understanding quantitatively how physical force affects growth patterns. From a purely physics viewpoint, we would say that we want to understand the coupling between the stress and growth fields in development.

In order to study the influence of physical force on cell proliferation, we developed a microfluidic device that allows us to apply spatial and temporal force patterns on cell monolayers. Using this system we are studying how spatial force fields (force gradients or differentials) can generate large scales tissue morphologies through mechanically induced differential cell growth. We are also quantifying the relation between cell proliferation and physical force, and the way forces affect spindle axis orientation during cell division.

 

Experimental results and methodology will be published soon.