Cell monolayers line most of the surfaces and cavities in the human body. During development and normal physiology, monolayers sustain, detect and generate mechanical stresses, yet little is known about their mechanical properties. We describe a cell culture and mechanical testing protocol for generating freely suspended cell monolayers and examining their mechanical and biological response to uniaxial stretch. Cells are cultured on temporary collagen scaffolds polymerized between two parallel glass capillaries. Once cells form a monolayer covering the collagen and the capillaries, the scaffold is removed with collagenase, leaving the monolayer suspended between the test rods. The suspended monolayers are subjected to stretching by prying the capillaries apart with a micromanipulator. The applied force can be measured for the characterization of monolayer mechanics. Monolayers can be imaged with standard optical microscopy to examine changes in cell morphology and subcellular organization concomitant with stretch. The entire preparation and testing protocol requires 3–4 d.
Subscribe to Journal
Get full journal access for 1 year
only $21.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Heisenberg, C.-P. & Bellaïche, Y. Forces in tissue morphogenesis and patterning. Cell 153, 948–962 (2013).
Martin, A.C., Gelbart, M., Fernandez-Gonzalez, R., Kaschube, M. & Wieschaus, E.F. Integration of contractile forces during tissue invagination. J. Cell Biol. 188, 735–749 (2010).
Simpson, C.L., Patel, D.M. & Green, K.J. Deconstructing the skin: cytoarchitectural determinants of epidermal morphogenesis. Nat. Rev. Mol. Cell Biol. 12, 565–580 (2011).
Lai-Cheong, J.E., Arita, K. & McGrath, J.A. Genetic diseases of junctions. J. Invest. Dermatol. 127, 2713–2725 (2007).
Getsios, S., Huen, A.C. & Green, K.J. Working out the strength and flexibility of desmosomes. Nat. Rev. Mol. Cell Biol. 5, 271–281 (2004).
Levine, E., Lee, C.H., Kintner, C. & Gumbiner, B.M. Selective disruption of E-cadherin function in early Xenopus embryos by a dominant-negative mutant. Development 120, 901–909 (1994).
Tambe, D.T. et al. Collective cell guidance by cooperative intercellular forces. Nat. Mater. 10, 469–475 (2011).
Marinari, E. et al. Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding. Nature 484, 542–545 (2012).
Eisenhoffer, G.T. et al. Crowding induces live-cell extrusion to maintain homeostatic cell numbers in epithelia. Nature 484, 546–549 (2012).
Aigouy, B. et al. Cell flow reorients the axis of planar polarity in the wing epithelium of Drosophila. Cell 142, 773–786 (2010).
Harris, T.J. & Tepass, U. Adherens junctions: from molecules to morphogenesis. Nat. Rev. Mol. Cell Biol. 11, 502–514 (2010).
Matter, K. & Balda, M.S. Signalling to and from tight junctions. Nat. Rev. Mol. Cell Biol. 4, 225–236 (2003).
Kunda, P. et al. PP1-mediated moesin dephosphorylation couples polar relaxation to mitotic exit. Curr. Biol. 22, 231–236 (2012).
Moeendarbary, E. et al. The cytoplasm of living cells behaves as a poroelastic material. Nat. Mater. 12, 253–261 (2013).
Kajita, M. et al. Interaction with surrounding normal epithelial cells influences signalling pathways and behaviour of Src-transformed cells. J. Cell Sci. 123, 171–180 (2010).
Terry, S.J. et al. Stimulation of cortical myosin phosphorylation by p114RhoGEF drives cell migration and tumor cell invasion. PLoS ONE 7, e50188 (2012).
Harris, A.R. & Charras, G.T. Experimental validation of atomic force microscopy-based cell elasticity measurements. Nanotechnology 22, 345102–345102 (2011).
Blanchard, G.B. et al. Tissue tectonics: morphogenetic strain rates, cell shape change and intercalation. Nat. Methods 6, 458–464 (2009).
Trzewik, J. et al. Evaluation of lateral mechanical tension in thin-film tissue constructs. Ann. Biomed. Eng. 32, 1243–1251 (2004).
Liu, Z. et al. Mechanical tugging force regulates the size of cell-cell junctions. Proc. Natl. Acad. Sci. USA 107, 9944–9949 (2010).
Mertz, A.F. et al. Cadherin-based intercellular adhesions organize epithelial cell-matrix traction forces. Proc. Natl. Acad. Sci. USA 110, 842–847 (2013).
Chu, Y.-S. et al. Force measurements in E-cadherin–mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42. J. Cell Biol. 167, 1183–1194 (2004).
Maître, J.-L. et al. Adhesion functions in cell sorting by mechanically coupling the cortices of adhering cells. Science 338, 253–256 (2012).
Farhadifar, R., Röper, J.-C., Aigouy, B., Eaton, S. & Jülicher, F. The influence of cell mechanics, cell-cell interactions, and proliferation on epithelial packing. Curr. Biol. 17, 2095–2104 (2007).
Bonnet, I. et al. Mechanical state, material properties and continuous description of an epithelial tissue. J. R. Soc. Interface 9, 2614–2623 (2012).
Wiebe, C. & Brodland, G.W. Tensile properties of embryonic epithelia measured using a novel instrument. J. Biomech. 38, 2087–2094 (2005).
Harris, A.R. et al. Characterizing the mechanics of cultured cell monolayers. Proc. Natl. Acad. Sci. USA 109, 16449–16454 (2012).
Hoffman, B.D., Grashoff, C. & Schwartz, M.A. Dynamic molecular processes mediate cellular mechanotransduction. Nature 475, 316–323 (2011).
del Rio, A. et al. Stretching single talin rod molecules activates vinculin binding. Science 323, 638–641 (2009).
Yonemura, S., Wada, Y., Watanabe, T., Nagafuchi, A. & Shibata, M. α-Catenin as a tension transducer that induces adherens junction development. Nat. Cell Biol. 12, 533–542 (2010).
le Duc, Q. et al. Vinculin potentiates E-cadherin mechanosensing and is recruited to actin-anchored sites within adherens junctions in a myosin II–dependent manner. J. Cell Biol. 189, 1107–1115 (2010).
Taguchi, K., Ishiuchi, T. & Takeichi, M. Mechanosensitive EPLIN-dependent remodeling of adherens junctions regulates epithelial reshaping. J. Cell Biol. 194, 643–656 (2011).
Palsson, E. A three-dimensional model of cell movement in multicellular systems. Future Generation Computer Systems 17, 835–852 (2001).
Rejniak, K.A. & Dillon, R.H. A single cell-based model of the ductal tumour microarchitecture. Comput. Math. Methods Med. 8, 51–69 (2007).
Ranft, J. et al. Fluidization of tissues by cell division and apoptosis. Proc. Natl. Acad. Sci. USA 107, 20863–20868 (2010).
Jones, G.W. & Chapman, S.J. Modelling apical constriction in epithelia using elastic shell theory. Biomech. Model. Mechanobiol. 9, 247–261 (2010).
Conte, V., Muñoz, J.J. & Miodownik, M. A 3D finite element model of ventral furrow invagination in the Drosophila melanogaster embryo. J. Mech. Behav. Biomed. Mater. 1, 188–198 (2008).
Conte, V. et al. A biomechanical analysis of ventral furrow formation in the Drosophila melanogaster embryo. PloS ONE 7, e34473 (2012).
Brodland, G.W. et al. Video force microscopy reveals the mechanics of ventral furrow invagination in Drosophila. Proc. Natl. Acad. Sci. USA 107, 22111–22116 (2010).
Ishihara, S. et al. Comparative study of non-invasive force and stress inference methods in tissue. Eur. Phys. J. E 36, 1–13 (2013).
Fung, Y.C. Biomechanics: Mechanical Properties of Living Tissues (Springer, 1993).
Fritzsche, M., Lewalle, A., Duke, T., Kruse, K. & Charras, G. Analysis of turnover dynamics of the submembranous actin cortex. Mol. Biol. Cell 24, 757–767 (2013).
Shimamoto, Y. & Kapoor, T.M. Microneedle-based analysis of the micromechanics of the metaphase spindle assembled in Xenopus laevis egg extracts. Nat. Protoc. 7, 959–969 (2012).
Trepat, X. et al. Universal physical responses to stretch in the living cell. Nature 447, 592–595 (2007).
Edelstein, A., Amodaj, N., Hoover, K., Vale, R. & Stuurman, N. Computer control of microscopes using μManager. Curr. Protoc. Mol. Biol 92, 14.20.1–14.20.17 (2010).
Kollmannsberger, P. & Fabry, B. Linear and nonlinear rheology of living cells. Annu. Rev. Mater. Res. 41, 75–97 (2011).
We thank D. Farquharson and his team at the UCL Biosciences Mechanical Workshop for technical assistance. We acknowledge the UCL Comprehensive Biomedical Research Centre for generous funding of microscopy equipment. This work was supported by a Royal Society Equipment Grant to G.T.C. and a UK Biotechnology and Biological Sciences Research Council (BBSRC) tools and development fund grant to G.T.C. and A.J.K. (BB/K013521). G.T.C. was supported by a Royal Society University Research Fellowship. During the development of this technique, A.R.H. was part of the Molecular Modelling and Materials Science M3S Engineering Doctorate program funded by the UK Engineering and Physical Sciences Research Council (EPSRC). N.K. and T.W. are part of the CoMPLEX Doctoral Training Program funded by the EPSRC. N.K. was supported by a UCL Overseas Research Scholarship and the UCL Graduate school fellowship fund.
The authors declare no competing financial interests.
Integrated supplementary information
(a) The engineering strain ɛ is a measure of a material's deformation from a reference shape, as defined above for a material of length L0 stretched to a length L by an external force. The engineering stress σ is a measure of the tension exerted within a material. It is a force per unit area, as defined above where W0 is the cross-sectional area of the material at rest. (b) Elastic materials are characterized by a reversible relationship between stress and strain regardless of their deformation history. Materials are linear elastic when the stress varies linearly with the strain. In that case, the slope of the stress-strain relationship is a measure of a material's stiffness. (c) Many materials, including cells and tissues, exhibit time-dependent responses following application of deformation, something often referred to as a visco-elastic behavior. Gels, and to some extent living cells and tissues, can be described to the first order using standard viscoelastic solid models, though many more complex behaviours have been documented46. One classic mechanical test is known as a stress relaxation test. In response to a step deformation, a viscoelastic material relaxes with a characteristic time τ above which the stress reaches an equilibrium value from which a stiffness can be defined. In contrast to viscoelastic materials, elastic materials subjected to a step deformation do not display relaxation. (d) Stress-strain relationship for a thin sheet of linear elastic material (PDMS) affixed to our force measurement device. (e) Stress-relaxation test for a thin sheet of PDMS affixed to our force measurement device. As PDMS is linear elastic, no relaxation can be detected following deformation.
On all panels, small subdivisions are 1cm and large subdivisions 5cm. (a) Micromanipulator arm – top view. This consisted of three parts: a plate for fastening to the micromanipulator (left), an arm, and an L-shaped wire prong fastened by inset screws to the arm (white arrows). The L-shaped wire prong (pointing towards the observer in the main image and downward in the inset) was used to interact directly with the test rods. The L-shaped prong is fastened to the arm using inset screws. In experiments necessitating application of a constant stretch, the L-shaped wire prong can be detached from the micromanipulator arm and glued to the rim of the Petri dish (Fig. 1d-e). Inset: Side view of the micromanipulator arm. (b) Micromanipulator base plate. Magnets inserted into the base plate allow the micromanipulators to be secured to the metallic microscope stage cover shown in d. (c) White Perspex microscope insert. This part is used to increase contrast with the metal wire to facilitate image segmentation force measurement. The rectangular window is used to image the monolayer with the microscope. (d) Metallic microscope stage cover.
(a) Composite image of the wire position before and after loading. The right extremity of the wire is threaded into a glass capillary that is maintained horizontal. The left extremity is left free. Upon loading, a small mass of plasticine is added to the free extremity of the wire. The total length of the wire Lw, the point of loading y, and the deflection d of the wire following loading are indicated on the image. Scale bar = 5mm. (b) Force plotted as a function of flexural rigidity 6Id/(3Lw-y)y2. Force is given in 10-4 N. Flexural rigidity is given in 10-15 m2. Experimental data points are indicated by black dots. A straight line is fitted to the experimental data points and its slope is the elastic modulus of the wire.
Mechanical terminology. (PDF 461 kb)
Custom made parts. (PDF 357 kb)
Calibration of the wire. (PDF 221 kb)
Technique for depositing a droplet of collagen between the test rods and spreading it to create a temporary cell culture scaffold. (AVI 4956 kb)
Technique for depositing a droplet of medium on top of the dehydrated collagen scaffold for rehydration prior to cell culture. (AVI 9189 kb)
About this article
Cite this article
Harris, A., Bellis, J., Khalilgharibi, N. et al. Generating suspended cell monolayers for mechanobiological studies. Nat Protoc 8, 2516–2530 (2013). https://doi.org/10.1038/nprot.2013.151
Nature Materials (2020)
Royal Society Open Science (2020)
Physical Review E (2020)
Nature Reviews Physics (2020)
Measurement of junctional tension in epithelial cells at the onset of primitive streak formation in the chick embryo via non-destructive optical manipulation