Generating suspended cell monolayers for mechanobiological studies

Journal name:
Nature Protocols
Volume:
8,
Pages:
2516–2530
Year published:
DOI:
doi:10.1038/nprot.2013.151
Published online

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.

At a glance

Figures

  1. Schematic diagrams of the culture devices.
    Figure 1: Schematic diagrams of the culture devices.

    (a) Generating suspended monolayers, a profile view schematic of the device before (top) and after (bottom) removal of the collagen scaffold. The collagen scaffold (red) can be removed by enzymatic digestion, leaving the monolayer (green) freely suspended between the test rods (black circles). (b,c) Schematics of monolayer stretching devices. On both diagrams, the monolayer is represented in green and the glue for fixing the device to the Petri dish is shown in black. (b) Simple device. (c) Force measurement device. (d) Simple device after application of a constant extension, showing the interaction of the prong and manipulator with the test rods. The Petri dish is represented by a circle. (e) The L-shaped wire prong can be detached from the micromanipulator arm and glued to the rim of the Petri dish, enabling application of a constant strain. (f) Position of the micromanipulators and L-shaped wire prongs in force measurements. The Petri dish is represented by a circle.

  2. Experimental setup for measuring monolayer mechanics.
    Figure 2: Experimental setup for measuring monolayer mechanics.

    (a) Line diagram of the mechanical testing equipment. Two micromanipulators, one manual and one motorized, are mounted onto the platen of an inverted microscope. They are held securely in position on the metal stage by magnetic feet (Supplementary Fig. 2b). The position of the test rods in the device can be imaged with a top-down macroscope (green light path) aligned such that the flexible wire is visible within its field of view (FOV; green area in the inset device diagram, bottom left). The Petri dish containing the device is positioned such that the monolayer can be imaged with the inverted microscope (red light path and red area in the inset device diagram, bottom left). Acquisition and mechanical testing are controlled by a computer. (b,c) Side views of the macroscope and the micromanipulators. L-shaped wire prongs attached to the custom-made micromanipulator arms (Supplementary Fig. 2a) are positioned between the two test rods and are used to pry them apart (Fig. 1d–f).

  3. Assembly of simple stretching devices.
    Figure 3: Assembly of simple stretching devices.

    On all panels, each small subdivision represents 1 cm. (a,b) Glass capillary bent into a U shape (a) and cut to size (b) by using the black marks shown in a. (c) A glass capillary, a metallic wire and a U-shaped device ready for assembly. (d) The glass capillary is threaded onto the metal wire, leaving ∼0.5 cm protruding on either side. (e) The wire and glass capillary are then inserted into the short arm of the U-shaped capillary. (f) The device is secured to a Petri dish with hot glue. A bent capillary serves as temporary stand while the glue hardens to ensure that the test rods do not contact the bottom surface of the Petri dish.

  4. Assembly of force-measurement devices.
    Figure 4: Assembly of force-measurement devices.

    On all panels, each small subdivision represents 1 cm. (a) Glass capillary bent into a U shape. Marks used for cutting appear in black. (b) The flexible arm is cut to size. (c) Tygon tubing, glass sleeve, metal wires and U-shaped device ready for assembly. (d) One metal wire has been threaded into the long arm of the U-shaped capillary (top arm), leaving ∼0.5 cm protruding from the extremity. (e) The short glass sleeve is threaded onto the second metal wire. The wire-sleeve assembly is then fixed to the short arm of the U-shaped capillary (bottom arm). (f) The Tygon tubing is threaded to the end of the two arms to act as a substrate for culture. (g) The device is secured to a Petri dish using hot glue. A bent capillary (arrowhead) serves as a temporary stand while the glue hardens to ensure that the test rods do not contact the bottom surface of the Petri dish. (h) A small PDMS spacer (arrowhead) is inserted between the test rods to maintain their position during cell culture.

  5. Deposition of the scaffold and seeding of the cells.
    Figure 5: Deposition of the scaffold and seeding of the cells.

    (a) A drop of collagen in reconstitution buffer is deposited between the two test rods and left to polymerize in a clean dry incubator. See Supplementary Video 1 for the deposition technique. (b) High-magnification image of the collagen drop. Ensure that the drop spans between the test rods, covering part of each test rod. (c) Collagen scaffold after polymerization. (d) Rehydration of the scaffold with a droplet (white arrowhead, see Supplementary Video 2 for the deposition technique). Scant medium is also added to the dish to prevent drying (black arrowhead). (e) High-magnification image of the monolayer during rehydration. (f) High-magnification image of the cells on the rehydrated collagen scaffold submerged in culture medium. Cells appear as bright features against the darker background of the collagen scaffold (white arrowhead). Scale bars, 2 mm.

  6. Simple data analysis.
    Figure 6: Simple data analysis.

    (a) Macroscope image of a force-measurement device before the application of stretch. The rigid reference rod is to the left and the flexible cantilevered rod is to the right. The monolayer is outside of the field of view at the top, whereas the U-bend is situated out of the field of view at the bottom. The removable L-shaped wire attached to the micromanipulator is in contact with the glass reference sleeve and is visible in the bottom right of the image. The red dotted line delineates the unstressed position and is extrapolated from the reference sleeve (red circle, bottom). Before the two test rods have been pried apart, the extrapolated unstressed position and the actual cantilevered wire position coincide well. Points P1 and P2 measured in the data analysis are indicated on the image. (b) Macroscope image of a force-measurement device after the application of stretch. The unstressed position (red dotted line) and the actual wire position no longer coincide. Points P1, P2 and P3 used in the data analysis are indicated on the image. (c) Schematic diagram of a stretched monolayer indicating the distances to be measured for estimation of the applied force. L indicates the length of the monolayer, w the cross-sectional area of the monolayer (Supplementary Fig. 1); d indicates the deflection of the wire from the unstressed position; and y indicates the distance from P3 to the reference sleeve. Parts of the device not in the field of view of (a,b) are grayed out in the diagram.

  7. Examples of expected results.
    Figure 7: Examples of expected results.

    (a) Image of cell boundaries visualized using cells stably expressing E-cadherin–GFP before the application of stretch. Scale bar, 10 μm. (b) Image of the same area as in a after application of 50% stretch along the horizontal axis (gray arrows). All cells become elongated in the direction of stretch. In a and b, the same cell before and during stretch is indicated by a white arrowhead. (c) Representative force relaxation curve for an MDCK monolayer. After the application of a 25% strain, the monolayer relaxes to equilibrium in ∼25 s. (d) Averaged force extension curve for an MDCK monolayer. The slope of the curve allows measurement of the monolayer stiffness in the linear portion of the curve (dotted gray line).

  8. Mechanical terminology.
    Supplementary Fig. 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.

  9. Custom made parts.
    Supplementary Fig. 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.

  10. Calibration of the wire.
    Supplementary Fig. 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.

Videos

  1. Depositing unpolymerized collagen onto a culture device.
    Video 1: 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.
  2. Depositing medium onto a collagen scaffold for rehydration.
    Video 2: 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.

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Author information

Affiliations

  1. London Centre for Nanotechnology, University College London, London, UK.

    • Andrew R Harris,
    • Nargess Khalilgharibi,
    • Tom Wyatt &
    • Guillaume T Charras
  2. Medical Research Council Laboratory of Molecular Cell Biology (MRC-LMCB), University College London (UCL), London, UK.

    • Julien Bellis &
    • Buzz Baum
  3. Centre de Recherche de Biochimie Macromoléculaire, Montpellier, France.

    • Julien Bellis
  4. Centre for Mathematics and Physics in the Life Sciences and Experimental Biology (CoMPLEX), UCL, London, UK.

    • Nargess Khalilgharibi &
    • Tom Wyatt
  5. Engineering Department, University of Cambridge, Cambridge, UK.

    • Alexandre J Kabla
  6. Department of Cell and Developmental Biology, UCL, London, UK.

    • Guillaume T Charras

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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Author details

Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Mechanical terminology. (101 KB)

    (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.

  2. Supplementary Figure 2: Custom made parts. (81 KB)

    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.

  3. Supplementary Figure 3: Calibration of the wire. (34 KB)

    (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.

Video

  1. Video 1: Depositing unpolymerized collagen onto a culture device. (4.83 MB, Download)
    Technique for depositing a droplet of collagen between the test rods and spreading it to create a temporary cell culture scaffold.
  2. Video 2: Depositing medium onto a collagen scaffold for rehydration. (8.97 MB, Download)
    Technique for depositing a droplet of medium on top of the dehydrated collagen scaffold for rehydration prior to cell culture.

PDF files

  1. Supplementary Figure 1 (472.8 KB)

    Mechanical terminology.

  2. Supplementary Figure 2 (365.7 KB)

    Custom made parts.

  3. Supplementary Figure 3 (226.9 KB)

    Calibration of the wire.

Additional data