Generating suspended cell monolayers for mechanobiological studies

Abstract

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.

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Figure 1: Schematic diagrams of the culture devices.
Figure 2: Experimental setup for measuring monolayer mechanics.
Figure 3: Assembly of simple stretching devices.
Figure 4: Assembly of force-measurement devices.
Figure 5: Deposition of the scaffold and seeding of the cells.
Figure 6: Simple data analysis.
Figure 7: Examples of expected results.

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Acknowledgements

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.

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Authors

Contributions

A.R.H., A.J.K. and G.T.C. designed the study. A.R.H. designed and constructed the culture and testing system. N.K. and T.W. contributed technical improvements to the system. A.R.H., J.B., T.W. and B.B. designed methods for immunostaining experiments and live imaging. A.R.H., N.K., T.W., A.J.K. and G.T.C. wrote the protocol.

Corresponding author

Correspondence to Guillaume T Charras.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Mechanical terminology.

(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.

Supplementary Figure 2 Custom made parts.

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.

Supplementary Figure 3 Calibration of the wire.

(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.

Supplementary information

Supplementary Figure 1

Mechanical terminology. (PDF 461 kb)

Supplementary Figure 2

Custom made parts. (PDF 357 kb)

Supplementary Figure 3

Calibration of the wire. (PDF 221 kb)

Depositing unpolymerized collagen onto a culture device.

Technique for depositing a droplet of collagen between the test rods and spreading it to create a temporary cell culture scaffold. (AVI 4956 kb)

Depositing medium onto a collagen scaffold for rehydration.

Technique for depositing a droplet of medium on top of the dehydrated collagen scaffold for rehydration prior to cell culture. (AVI 9189 kb)

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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

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