This work was supported by the Committee Technology and Innovation (CTI) Switzerland grant 14796-PFLS-LSCTI. MicroPost arrays were generously provided by MicroDuits GmbH.

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.
<ol>
	<li>Prepare a cell culture for seeding onto a micropost array.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Add 5 ml cell culture medium (Dulbecco&#8217;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&#8217;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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.&#160;</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Replace the DI-water with PBS-buffer in the same manner by adding and aspirating approximately 1 ml. Repeat this step 3 times.</li>
		<li>Replace the PBS-buffer with medium by adding and aspirating approximately 1 ml of medium. Repeat this step 3 times.</li>
	</ol>
	</li>
	<li>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 &#176;C). Let the cells adhere and grow on top of microposts for 6&#160;-7 hr.</li>
	<li>Inspect the adhesion process occasionally by using a light a microscope. Check that most of the cells appear spread out across multiple microposts.</li>
</ol>
2. Fixing &#38; 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.
<ol>
	<li>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.</li>
	<li>Fix the cells with 0.5 ml 3.7% buffered formaldehyde solution for 5 min.</li>
	<li>Replace the formaldehyde solution by washing the micropost array 2x with 1 ml sterile DI water.</li>
	<li>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&#160;ml sterile DI water. Add 1 ml DI water to micropost array.</li>
	<li>Check staining result using a light microscope. Repeat the staining step, if cell body is too faint to be visualized.</li>
	<li>Store micropost arrays with the stained cells on top in a refrigerator at 4 &#176;C. Ensure to keep them under water at all times.</li>
</ol>
3. Cell Imaging
Note: Step 4 only applies to microscopes without infinity corrected optics.
<ol>
	<li>Add 2 ml DI-water to a Petri dish with a thin glass bottom for high resolution imaging.</li>
	<li>Transfer micropost array using a pair of tweezers into the imaging dish with the microposts facing up.</li>
	<li>Place the imaging Petri dish on a moveable stage of a light microscope.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#181;m.</li>
	<li>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.</li>
</ol>
4. Image Analysis with Open Source Software &#8220;MechProfiler&#8221;
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.
<ol>
	<li>Start image analysis software MechProfiler and open a range of images that need to be analyzed by clicking &#8220;Open&#8221;.</li>
	<li>Insert all parameters into the &#8220;Settings&#8221; 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.</li>
	<li>Analyze images one-by-one by cropping them to the area of interest by using &#8220;Crop&#8221; (click and drag with the mouse button pressed down). Double click inside the drawn rectangle to finish this action.</li>
	<li>Marking each visible cell outline one-by-one by clicking on &#8220;Draw cell outlines&#8221; and use the cross hair cursor for drawing including all microposts the cell is attached to.</li>
	<li>Discard any unwanted microposts by clicking &#8220;Discard Posts&#8221; 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.</li>
	<li>Start the software subroutine &#8220;Find Centroids&#8221; by mouse click. Adjust the filter setting right next to the &#8220;Find Centroids&#8221; 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.</li>
	<li>Use the manual editing function &#8220;Manual Edit&#8221; 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.</li>
	<li>Find the ideal micropost grid by activating the &#8220;Generate Grid&#8221; function with a mouse click. Ensure the position corresponds with the true micropost head, shown by a blue ring, inside the drawn cell outline.</li>
	<li>Correct any misplaced grid maker inside the cell area where needed using the corresponding &#8220;Manuel Edit&#8221; 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.</li>
	<li>Use the button &#8220;Calculate Deflections&#8221; by mouse click to get a histogram of the calculated deflection values based on the difference between a micropost&#8217;s position inside the image section and the generated ideal grid.</li>
	<li>Save the complete analysis including the tables of values by a mouse click on &#8220;Save&#8221;.</li>
	<li>Continue the image analysis either by clicking on &#8220;Reset View&#8221; and analyze another image section or mouse click on &#8220;Next Image&#8221;, which conveniently can be done using the right keyboard curser key.</li>
</ol>
5. Data Analysis &#8212; Mechanoprofiling
<ol>
	<li>Open the file &#8220;results.xls&#8221; 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.</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+biomechanical+gradients+for+directed+bone+marrow+stromal+cell+differentiation+toward+tendon+and+bone.">Biochemical and biomechanical gradients for directed bone marrow stromal cell differentiation toward tendon and bone.</a> Biomaterials. 31 (30), 7695-7704 (2010).</li><li>Suresh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+and+biophysics+of+cancer+cells.">Biomechanics and biophysics of cancer cells.</a> Acta Biomater. 3 (4), 413-438 (2007).</li><li>Bartalena, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+assessing+adherent+single-cell+stiffness+in+tension:+design+and+testing+of+a+substrate-based+live+cell+functional+imaging+device.">A novel method for assessing adherent single-cell stiffness in tension: design and testing of a substrate-based live cell functional imaging device.</a> Biomed Microdevices. 13 (2), 291-301 (2011).</li><li>Tan, J. 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The lead author (NG) is co-founder of MicroDuits GmbH, the commercial supplier of the micropost arrays used in this study. No other authors have any potential conflicts of interest with this work.

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 &#8211; keeping in mind that a typical experiment may require the analysis of hundre...

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 &#948; depends on the micropost&#8217;s &#8220;bending stiffness&#8221; k and is calculated by:
<img alt="Equation 1" src="/files/ftp_upload/53350/53350eq1.jpg" /> (1)
with E, I, and L being the Young&#8217;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&#8217;s Poisson ratio v.8 It is given by:
<img alt="Equation 2" src="/files/ftp_upload/53350/53350eq2.jpg" /> (2)
With Ttilt(v) being a tilting coefficient that includes fitting parameter a = 1.3 as can be found in the same article:
<img alt="Equation 3" src="/files/ftp_upload/53350/53350eq3.jpg" /> (3)
That means a micropost&#8217;s corrected stiffness kcorr is the product of the pure bending stiffness k=kbend and the correction factor corr given by:
<img alt="Equation 4" src="/files/ftp_upload/53350/53350eq4.jpg" /> (4)
Therefore, cell force calculations should be performed using the more refined variation of equation (1) now reading:
<img alt="Equation 5" src="/files/ftp_upload/53350/53350eq5.jpg" /> (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 &#181;m 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&#8217;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 &#181;m to 10&#160;&#181;m in diameter and 2 to 50 &#181;m 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 (&#8220;off-the-shelf&#8221;) 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 &#8220;off-the-shelf&#8221; 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 &#8220;fixed&#8221;.
The MechProfiler software is able to greatly increase data analysis throughput by relying on reproducibility of quality commercial micropost arrays, more specifically, the default &#8220;non-deflected&#8221; 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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>T25 cell culture flasks</td>
			<td>Nunc</td>
			<td>156367</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS-buffer</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium DMEM/F12</td>
			<td>Sigma</td>
			<td>D8437</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS South America</td>
			<td>Life Technologies</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100x</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml centrifuge vials</td>
			<td>Sarstedt</td>
			<td>62.554.502&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropost array&#160;</td>
			<td rowspan="2">MicroDuits</td>
			<td rowspan="2">MPA-col1/FN</td>
			<td>Micropost dimensions:</td>
		</tr>
		<tr>
			<td>pre-coated with Collagen I/Fibronectin</td>
			<td>&#216;= 6.4 &#181;m, l=18.2 &#181;m; grid=&#160;13 &#181;m, kcorr= 2.87 nN/&#181;m</td>
		</tr>
		<tr>
			<td>Ethanol abs p.A.</td>
			<td>Merck</td>
			<td>100.983</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Nunc</td>
			<td>150628</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution, neutral buffered 10%</td>
			<td>Sigma</td>
			<td>HT5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Blue G-250</td>
			<td>Sigma</td>
			<td>27815</td>
			<td>Coomassie blue</td>
		</tr>
		<tr>
			<td>Methanol ACS p.A.</td>
			<td>Merck</td>
			<td>1.06009</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom dish</td>
			<td>WillCo Wells BV</td>
			<td>GWSb-3522</td>
			<td>35 mm diameter, aperture 22&#160;mm</td>
		</tr>
		<tr>
			<td>T-100 Eclipse</td>
			<td>Nikon</td>
			<td>n/a</td>
			<td>Inverted microscope</td>
		</tr>
		<tr>
			<td>D3-L3</td>
			<td>Nikon</td>
			<td>n/a</td>
			<td>Camera controler</td>
		</tr>
		<tr>
			<td>DS Fi2</td>
			<td>Nikon</td>
			<td></td>
			<td>Camera</td>
		</tr>
	</tbody>
</table>

easy and accurate mechano-profiling on micropost arrays

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 &#8220;mechano-profiling&#8221; 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.

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 ...

Watch this Scientific Journal Video about Easy and Accurate Mechano-profiling on Micropost Arrays at JoVE.com

Easy and Accurate Mechano-profiling on Micropost Arrays

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.

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

easy-and-accurate-mechano-profiling-on-micropost-arrays

Research

JoVE Journal

Bioengineering

11.1K visualizações.  Balgrist University Hospital. 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. 

Vídeo: Easy and Accurate Mechano-profiling on Micropost Arrays

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

A microfabricated device with sealable femtoliter-volume reaction chambers is described. This report includes a protocol for sealing cell-free protein synthesis reactants inside these chambers for the purpose of understanding the role of crowding and confinement in gene expression.

Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...