Easy and Accurate Mechano-profiling on Micropost Arrays

Podsumowanie

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Streszczenie

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts.

The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow “mechano-profiling” of cell phenotypes.

A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Wprowadzenie

Mechano-sensitive cell-based assays allow for investigating adherent cells, with a broad range of applications that reflect the central role that mechanics can play in cell biology. These applications often focus on the underlying mechanisms that drive subcellular processes or whole-cell behavior. On the one hand, external environmental factors such as extra cellular matrix composition or matrix stiffness can dramatically affect the mechanical and biological response of a cell.¹ The same can be observed after use of many classes of pharmaceutically active compounds, the effects of which are often characterized using cell culture models.² On the other hand genotypic properties, such as those caused by spontaneous or experimentally induced genetic mutations, can induce marked changes in cell phenotype that are associated with alterations in the cytoskeleton structure and function.³ These examples are just a few of the many possible topics for which mechanical phenotyping of cells is relevant, and all of these have been usefully investigated with micropost arrays.

At time of this writing, approximately 200 articles have been published describing cell-micropost interaction. These works discuss theoretical aspects of micropost deflection principles as well as practical instructions on their manufacture. The first article describing the interaction of cells and flexible micropost arrays was published by Tan and colleagues in 2003.⁴ In contrast to classic traction force microscopy (TFM) where continuous soft substrates are used to estimate nanonewton-scale cell contractility, Tan et al. described a method using multiple closely spaced vertical beams made of silicone elastomer. The main advantages of this technique emerge from two major features. First in order to change the cell-apparent substrate stiffness one only needs to change the micropost dimensions while keeping the substrate composition otherwise constant and thus avoiding differences in surface topology and chemistry. Second microposts act like individual springs that can be discretely analyzed with force and spatial resolutions on the order of individual focal adhesions and can reduce the analytical challenges that are inherent to analogous analysis by standard TFM.

Today the range of applications for micropost arrays greatly exceeds just the mapping of forces for a few single cells. For example, Akiyama reports the use of an isolated dorsal vessel tissue from a moth caterpillar as an actuator for a micropost array, in order to develop an insect muscle-powered autonomous micro-robot.⁵

However, most published applications of microposts have focused on studies of medical conditions like infection or cancer. For instance, micropost arrays have been used to study the force generation of bundled type IV pili of Neisseria gonorrhoea colonies that is associated with signal cascades enhancing infection.⁶ Others have used microposts to study breast cancer cells treated with pharmaceutical compounds targeting the cytoskeleton.⁷

Deflection of a micropost is often described using classical beam theory for a cantilever with an end load assuming the cell attaches only to the very tip of the micropost. Here the applied force F that causes a deflection δ depends on the micropost’s “bending stiffness” k and is calculated by:

(1)

with E, I, and L being the Young’s modulus, area moment of inertia and beam length respectively. However, results from this equation only give a general approximation of the forces at work since beam shearing and bending as well as substrate warping are not taken into account. Considering that microposts are typically made from soft materials like polydimethylsiloxane (PDMS)-based silicone rubber these factors need to be included. Schoen et al. demonstrated that there is such a correction factor based on the aspect ratio of the micropost (L/D) and the corresponding polymer’s Poisson ratio v.⁸ It is given by:

(2)

With T_tilt(v) being a tilting coefficient that includes fitting parameter a = 1.3 as can be found in the same article:

(3)

That means a micropost’s corrected stiffness k_corr is the product of the pure bending stiffness k=k_bend and the correction factor corr given by:

(4)

Therefore, cell force calculations should be performed using the more refined variation of equation (1) now reading:

(5)

The impact of the correction becomes more obvious as soon as typical values for micropost dimensions are used. For example, a 15-micron long micropost with a circular cross section and a diameter of 5 µm made of PDMS-based silicone rubber leads to a correction factor of 0.77 and therefore an uncorrected calculation would overestimate the exerted cell forces by 23%. This becomes even more severe for microposts with smaller aspect ratios.

Traditionally, micropost image analysis has also been based on idealized beam bending theory. In 2005 the group that pioneered the use of micropost arrays published an image analysis software suited for micropost analysis.⁹ The software requires a software license and the user must take three images for each position; one each from the micropost’s top and bottom planes in transmission mode and another one in fluorescence mode with the stained cell. After comparing the top and bottom positions for each micropost the software determines a force vector field and calculates related parameters like force per post. Other software packages exist and their analysis principles are briefly mentioned in the corresponding articles that describe them, but these analytical software packages are generally not publicly available.^10,11

The micropost arrays designed for mapping cell forces can be classified as either being in an orthogonal micropost layout or a hexagonal one, the latter of which have the advantage of equidistant gaps between all neighbor microposts. Typical microposts have a circular cross section and their dimensions range from 1.0 µm to 10 µm in diameter and 2 to 50 µm in length. ⁴ However, microposts with elliptic or square cross section have also been reported.^12,13

The use of PDMS-based silicone mixtures as micropost material allows for adding nanoparticles into the mixture. For example adding cobalt nano-rods enables a magnetic activation of the micropost and thus gives another degree of freedom to potential experimental designs.¹⁴ Most groups produce their micropost arrays on flat rigid substrates like cover glass or inside a Petri dish. However, Mann and co-workers recently reported a micropost array formed on a stretchable membrane.¹⁵ This allows the application of cell stretching forces to adherent cells while studying live-cell subcellular dynamic responses in terms of cell contractility.

The widely employed and most established process for making micropost arrays is based on soft lithography as described in the insightful protocols of Sniadecki and colleagues.^16-18 In short standard cleanroom processes are used to generate the microstructures on top of a silicon wafer using SU8 photoresist. This is followed by a copying process wherein the silicone rubber is cast over the structures transferring them into molds. In a second step these molds are used to replicate the initial microstructure using silicone rubber on top of a chosen substrate. However despite the large and growing number of publications related to their application, establishing a manufacturing process for microposts takes considerable amount of time even for micro-engineering experts; there are many process steps that require optimization and adaptation to the specific lab environment and micropost layout to yield an acceptable quality level.

Commercial micropost arrays are now available in a ready-to-use (“off-the-shelf”) format with a consistently high quality. As such they are an alternative to the complex and lengthy manufacturing process required for on-site production. In this paper a commercially available micropost array was used for mapping cellular forces using a single bright-field microscopy image. More importantly this article describes and documents a fully-functional open-source software named MechProfiler, which is available for download as supplementary material to this manuscript. An actively maintained version of the software can also be found at http://www.orthobiomech.ethz.ch.

The combination of an “off-the-shelf” assay and a compatible open-source analysis software markedly lowers the entry hurdle to achieve accurate TFM experiments. Researchers without access to either clean room facilities or software development expertise can analyze cellular forces successfully. It enables a user to focus on the mechanosensitivity assay output rather than the technology itself, and makes traction force measurements available to a broader community. Furthermore, this is an important step to pave the way towards fully automatic screening of micropost arrays.

The MechProfiler analysis software processes images in file format tiff, png, bmp and jpg. The images can be taken using fluorescence, phase contrast or bright-field light microscopy. The standalone program runs together with the free Matlab Compiler Runtime (available at: Figure 12) and underlying algorithms allow for streamlined image processing, which enables the user to process images with single or multiple cells in about 1 min. Further, these cells may either be living or “fixed”.

The MechProfiler software is able to greatly increase data analysis throughput by relying on reproducibility of quality commercial micropost arrays, more specifically, the default “non-deflected” position of each post in the array can be presumed against an ideal grid (manufacturing deviations for the grid in the arrays used for this study were less than 100 nm).

In short one opens a selection of image files for analysis, crops them to the region of interest, defines the posts covered by cells or which need to be discarded, determines the post positions, calculates the deflections/forces against the ideal grid, and finally saves all cell-specific data with a possibility for export, including to a standard office spreadsheet.

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Protokół

1. Culturing Cells on Micropost Arrays

Note: All steps must be performed in a biosafety cabinet to ensure sterility. The volumes given here are for T25 cell culture flasks. The cell culture medium recipe and cell seeding density are optimized for bone cancer cell lines HuO9 and M132.

1. Prepare a cell culture for seeding onto a micropost array.
   1. Inspect the cell culture quality by placing the culture flask on a standard light microscope. Ensure that the cells show a typical growth and morphology by estimating how much of the culture flask bottom is covered. A coverage of 70%-80% reflects a healthy growth rate. Pay attention to the amount of floating cells as they represent dead cells and/or an overgrown culture.
   2. Trypsinize cells by removing medium and washing with 4 ml 1x Phosphate buffered saline (PBS) buffer. Add 0.8 ml 1x Trypsin/Ethylenediaminetetraacetic acid (EDTA) and incubate at RT until the cells dislodge, which takes about 2-4 min. Check the process with the microscope and occasionally tap the flask gently to support the dislodging.
   3. Add 5 ml cell culture medium (Dulbecco’s modified Eagle medium (DMEM)/F12 with 10% fetal bovine serum (FBS), 1% penicillin/streptavidin (Pen/Strep)) to stop the reaction and to wash off residual cells that still sit on the flask’s bottom. Subsequently pipette this suspension up and down several times to gain a homogenous mixture. Make sure to pipette all solutions across the original growth area of the flask to gain an effective dislodging.
   4. Transfer the cell suspension in to a 15 ml tube and centrifuge it using a table top centrifuge at 0.5 x g for 3 min.
   5. Remove the supernatant and re-suspend the cell pellet in 5 ml medium by pipetting up and down 5 times. Make sure to avoid the generation of bubbles while doing so.
   6. Determine cell density by using a cell counting chamber and a light microscope. Check the cell suspension in cell counting chamber for cell aggregates and ensure to have only single cells for the subsequent experiment. Pipette again up and down several times if the cells tend to form aggregates to separate them.
   7. Dilute the cell suspension with sufficient medium to gain a cell solution of 25,000 cells/ml. Mix it well by inverting the closed tube several times. 
2. Prepare a micropost array for cell seeding.
   Critical Note: Do not pipette onto the micropost array directly as this could potentially harm the microposts, especially when unwanted bubbles are introduced. Furthermore, make sure that the micropost array never dries out as occurring capillary forces lead to collapsing of the microposts.
   1. Place the glass substrate with the micropost array face up in a well of a 12-well plate by using a pair of tweezers and wet it by adding 1 ml ethanol (99%). Incubate at RT for 20-30 sec.
   2. Dilute the ethanol stepwise by adding approximately 1 ml sterile de-ionized water (DI water) on the well side and aspirating roughly 1 ml using a transfer pipette or aspiration device. Repeat this step at least 3 times.
   3. Replace the DI-water with PBS-buffer in the same manner by adding and aspirating approximately 1 ml. Repeat this step 3 times.
   4. Replace the PBS-buffer with medium by adding and aspirating approximately 1 ml of medium. Repeat this step 3 times.
3. Pipette 1 ml cell solution (25,000 cells) on top of each prepared micropost array. Close multi-well plate and transfer it to an incubator (CO₂ 5%, 37 °C). Let the cells adhere and grow on top of microposts for 6 -7 hr.
4. Inspect the adhesion process occasionally by using a light a microscope. Check that most of the cells appear spread out across multiple microposts.

2. Fixing & Staining Cells

Note: All steps and volumes given here are for a single well of a 12-well plate. It is recommended to process not more than four of the twelve wells at a time to avoid a drying out of the wells after aspiration of any liquid, which will result in a collapse of the microposts.

1. Aspirate medium from the well and wash cells 2x with 1 ml PBS-buffer. Make sure to apply a gentle force while washing with PBS to remove cell debris that accumulated on the micropost array during incubation and to detach dead cells.
2. Fix the cells with 0.5 ml 3.7% buffered formaldehyde solution for 5 min.
3. Replace the formaldehyde solution by washing the micropost array 2x with 1 ml sterile DI water.
4. Stain the cells with dye (0.05% Coomassie Brilliant Blue in 50% water, 40% ethanol and 10% acetic acid) for about 90 sec. Wash excess staining solution off 2x with 1 ml sterile DI water. Add 1 ml DI water to micropost array.
5. Check staining result using a light microscope. Repeat the staining step, if cell body is too faint to be visualized.
6. Store micropost arrays with the stained cells on top in a refrigerator at 4 °C. Ensure to keep them under water at all times.

3. Cell Imaging

Note: Step 4 only applies to microscopes without infinity corrected optics.

1. Add 2 ml DI-water to a Petri dish with a thin glass bottom for high resolution imaging.
2. Transfer micropost array using a pair of tweezers into the imaging dish with the microposts facing up.
3. Place the imaging Petri dish on a moveable stage of a light microscope.
4. Turn the compensation ring of the lens used for imaging until the number on the scale represents the total thickness of all materials along the optical path to compensate for the mismatch in refractive indices of the glass substrate, the silicone elastomer and the liquid on top. This should lead to a bright appearance of the microposts cores.
5. Close the iris on the illumination side down to 50% and remove any phase contrast rings from the optical path enabling an ordinary bright-field mode.
6. Align the micropost array that the microposts form a horizontal line across the observation field. Define a start point (e.g., top left) and move the Petri dish stepwise across the stage scanning the micropost array while taking numerous images.
7. Take all images with the highest resolution setting for the camera using a 20X or 40X objective. Aim to have a single cell in the center of the image.
8. Sweep along the z-axis through the substrate until the micropost tips are in focus. Use the fine tuning focus wheel of the microscope and turn it towards focusing on the micropost bottom for about 2-3 µm.
9. Establish a standard imaging procedure by taking series of test images of one array section sweeping through the imaging parameters one at a time (e.g., exposure time, iris setting, lamp settings etc.). Analyze images with MechProfiler and determine a suitable parameter set for a fast and reliable analysis.

4. Image Analysis with Open Source Software “MechProfiler”

Note: All software functions can be activated with a pointing device using the left mouse button. However, a trained operator will use the documented shortcut keys designed to be on the left half of the keyboard to enable an efficient two-handed image processing.

1. Start image analysis software MechProfiler and open a range of images that need to be analyzed by clicking “Open”.
2. Insert all parameters into the “Settings” section given by the software developer. Determine the parameters that must be custom configured (i.e., contour threshold and minimal distance) according to the procedures described in detail by the software manual.
3. Analyze images one-by-one by cropping them to the area of interest by using “Crop” (click and drag with the mouse button pressed down). Double click inside the drawn rectangle to finish this action.
4. Marking each visible cell outline one-by-one by clicking on “Draw cell outlines” and use the cross hair cursor for drawing including all microposts the cell is attached to.
5. Discard any unwanted microposts by clicking “Discard Posts” and use the cross hair cursor for drawing. Enclose all microposts that belong to a cell outside of the image section or that are deflected for any other reason but not by the cell of interest.
6. Start the software subroutine “Find Centroids” by mouse click. Adjust the filter setting right next to the “Find Centroids” button until all microposts are registered, which is visible by a red cross in their center. If the filter setting is too low microposts will be ignored, if it is too high multiple positions will be marked for a single micropost. Keep the filter setting constant during an analysis session.
7. Use the manual editing function “Manual Edit” for centroids with multiple or missed micropost positions. Use the mouse cross hair to select the micropost in question by clicking and dragging it across. Double click inside the rectangle and place the missing red marker in the enlarged image section, which automatically closes. Discard such a micropost if it is outside the drawn cell outline (see step 4.5) before proceeding.
8. Find the ideal micropost grid by activating the “Generate Grid” function with a mouse click. Ensure the position corresponds with the true micropost head, shown by a blue ring, inside the drawn cell outline.
9. Correct any misplaced grid maker inside the cell area where needed using the corresponding “Manuel Edit” function with a mouse click. Use the cross hair to select the micropost in question by clicking and dragging it across. Double click inside the appearing rectangle and place the missing blue marker in the enlarged image section, which automatically closes.
10. Use the button “Calculate Deflections” by mouse click to get a histogram of the calculated deflection values based on the difference between a micropost’s position inside the image section and the generated ideal grid.
11. Save the complete analysis including the tables of values by a mouse click on “Save”.
12. Continue the image analysis either by clicking on “Reset View” and analyze another image section or mouse click on “Next Image”, which conveniently can be done using the right keyboard curser key.

5. Data Analysis — Mechanoprofiling

1. Open the file “results.xls” with an office spreadsheet program from the folder with the analyzed images. It contains the data with all calculated values including all micropost deflections and standard derivative measures such as work (i.e., substrate deformation energy) by the analyzed cell.

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Wyniki

The main advantage of the described technique lies in its simplicity and potential for rapid and effective integration into routine lab work. The combination of high quality commercial sensor arrays paired with open-source software provides information about mechano-sensitivity that would otherwise requires access to cleanroom facilities and in-depth knowledge of image analysis and software development. Figure 1 illustrates the workflow of the presented method. It starts with preparing and seeding cells ...

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Dyskusje

This work seeks to advance the field of traction force microscopy by substantially lowering technical and practical barriers to entry. These barriers come from two sides. First and foremost are the numerous non-trivial technical challenges that must be overcome to reproducibly manufacture and experimentally commission a micropost array. Second, is the similarly non-trivial need for a reliable semi-automated analysis of single cell forces – keeping in mind that a typical experiment may require the analysis of hundre...

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Materiały

Name	Company	Catalog Number	Comments
T25 cell culture flasks	Nunc	156367
1x PBS-buffer	Sigma	D8537
0.5% Trypsin-EDTA	Life Technologies	15400054
Medium DMEM/F12	Sigma	D8437
FBS South America	Life Technologies	10270106
Penicillin-Streptomycin 100x	Sigma	P4333
15 ml centrifuge vials	Sarstedt	62.554.502
Micropost array pre-coated with Collagen I/Fibronectin	MicroDuits	MPA-col1/FN	Micropost dimensions: Ø=6.4 µm, l=18.2 µm; grid=13 µm, kcorr=2.87 nN/µm
Ethanol abs p.A.	Merck	100.983
12-well plate	Nunc	150628
Formalin solution, neutral buffered 10%	Sigma	HT5011
Brilliant Blue G-250	Sigma	27815	Coomassie blue
Methanol ACS p.A.	Merck	1.06009
Acetic acid	Sigma	695092
Glass bottom dish	WillCo Wells BV	GWSb-3522	35 mm diameter, aperture 22 mm
T-100 Eclipse	Nikon	n/a	Inverted microscope
D3-L3	Nikon	n/a	Camera controler
DS Fi2	Nikon		Camera