This research was funded by National Institute of Health grant numbers R01NS050452 and R01EB009429. Fabrication of the cantilever chips was performed externally by collaborators at the NanoFabrication Facility located at Cornell University. All equipment used in the cantilever fabrication process was located in this facility. Special thanks to Mandy Esch and Jean-Matthieu Prot for their assistance with cantilever micro-fabrication. Video animation of cantilever functionality was generated by Charles Hughes, Alex Zelenin and Eric Imperiale from the Synthetic Reality Lab at UCF.

1. Cantilever Chip Fabrication
Illustrated details of the described fabrication steps are provided in Figure 1.
<ol>
	<li>Place Silicon-On-Insulator (SOI) wafers in an oven and bake at 125 &#176;C for 20 min&#160;to dehydrate them.</li>
	<li>Deposit a 1.5 &#181;m thick layer of silicon oxide onto the handle layer of the dehydrated SOI wafer using a Plasma Enhanced Chemical Vapor Deposition (PECVD) tool.</li>
	<li>Place the wafer on the spin coater chuck with the device layer facing up. Ensure wafer is centered, dispense 2 ml&#160;of P20 primer on the center of the wafer, and spin at 3,000 rpm for 60 sec&#160;at an acceleration of 1,000 rpm/sec. P20 primer promotes the adhesion of photoresist to the device layer.</li>
	<li>While the wafer is still on the spin coater chuck, dispense 2 ml&#160;of S1818 photoresist on the center of the wafer, and spin at 3,000 rpm for 60 sec&#160;at an acceleration of 1,000 rpm/sec to obtain a 1.5 &#181;m thick layer of photoresist.</li>
	<li>Place the wafer on a hot plate and perform a soft bake at 115 &#176;C for 1 min.</li>
	<li>Perform a hard contact UV exposure using a photolithography contact mask aligner in order to transfer the pattern from the cantilever mask to the photoresist on the device layer. Calculate the exposure time based on exposure energy value of 125 mJ/cm2 for S1818 photoresist.</li>
	<li>Develop the photoresist by immersing the wafer in a 726 MIF photoresist developer for 1 min&#160;while gently shaking the wafer back and forth.</li>
	<li>Rinse the wafer with de-ionized (DI) water and dry it, preferably using a spin rinse dryer (SRD) tool.</li>
	<li>Remove the photoresist from the edge of the wafer (up to 5 mm from the edge) using a cotton swab dipped in acetone.</li>
	<li>Load the wafer in a Deep Reactive Ion Etch (DRIE) tool, with the photoresist side facing up, and run a recipe to etch the patterned silicon layer through the device layer. The buried oxide layer functions as an etch stop, so perform an etch with 50% to 100% more cycles than is expected to be necessary to ensure a complete etch.</li>
	<li>Place the wafer in a hot photoresist bath solution for 20 min&#160;to strip the photoresist. Transfer the wafer to a quick-dump-rinser (QDR) to rinse the wafer with DI water. Following the photoresist strip, load the wafer in a SRD tool to rinse and dry the wafer. Inspect the wafer under a microscope to ensure the cantilever pattern appears as expected.</li>
	<li>Deposit a 1 &#181;m thick layer of un-doped silicon oxide on the device layer of the wafer using a PECVD tool. This oxide layer functions as a protective layer for the cantilevers during further processing steps.</li>
	<li>Place the wafer on the spin coater chuck with the handle layer facing up to begin processing of the back side of the wafer. Ensure wafer is centered, dispense 2 ml&#160;of P20 primer on the center of the wafer, and spin at 3,000 rpm for 60 sec&#160;at an acceleration of 1,000 rpm/sec.</li>
	<li>While the wafer is still on the spin coater chuck, dispense 2 ml&#160;of SPR220-4.5 photoresist onto the center of the wafer, and spin at 2,000 rpm for 45 sec&#160;at an acceleration of 1,000 rpm/sec to obtain a 6.5 &#181;m thick layer of photoresist.</li>
	<li>Place the wafer on a proximity hot plate to perform a soft bake at 115 &#176;C for 2 min.</li>
	<li>Using a photolithography contact aligner capable of front/back alignment, align the backside window mask to the front side cantilever pattern and perform a hard contact UV exposure in order to transfer the pattern from the backside window mask to the photoresist on the handle layer. Use an exposure time calculated based on exposure energy value of 480 mJ/cm2 for SPR220-4.5 photoresist.</li>
	<li>After UV exposure, let the wafers remain in a dark area for 30 min&#160;before the next processing step.</li>
	<li>Develop the photoresist by immersing the wafer in a 726 MIF photoresist developer for 2 min&#160;while gently shaking the wafer back and forth.</li>
	<li>Rinse the wafer with de-ionized (DI) water and dry it, preferably using a spin rinse dryer (SRD) tool.</li>
	<li>Place the wafers in an oven at 90 &#176;C for 12 hr.</li>
	<li>Etch the silicon oxide layer on the backside of the wafer using an RIE system with fluorinated gases and a recipe for etching oxide. The patterned oxide on the backside of the wafer acts as an etch mask for further processing. Inspect the wafers under a microscope to ensure that the silicon oxide in the exposed window is completely etched.</li>
	<li>Remove the photoresist on the edge of the wafer (up to 5 mm from the edge) using a clean room swab dipped in acetone.</li>
	<li>Load the wafer in a DRIE tool with and run a recipe to etch the patterned handle layer to a depth of 500 &#181;m with the buried oxide layer functioning as an etch stop. Split this etch into multiple runs to prevent excessive heating of the wafer, which causes inconsistent etching of the silicon. This etch step completely removes the silicon underneath the cantilevers.</li>
	<li>Perform a wet etching step using 25% dilute HF etchant, to strip the buried oxide layer below the cantilevers and the protective oxide layer on top of the cantilevers. 
	NOTE: This step releases the bottom surface of the silicon cantilevers and also opens a window underneath to provide an access to probe the cantilever with the laser.</li>
	<li>Rinse the wafer using a series of DI water baths and carefully dry with nitrogen. Since the cantilevers are supported only at their bases, do not use forceful sprays of DI water or inert gas directly on the cantilevers.</li>
	<li>Cleave the individual chips from the wafer along the cleave lines produced during the handle layer etch step.</li>
</ol>
2. Cell Culture
<ol>
	<li>Prepare 13F coverslips according to previously published methods17. 
	NOTE: If 13F coverslips are not available, any hydrophobic surface with a contact angle above 95&#176; can be used.</li>
	<li>Sterilize cantilever chips and 13F coverslips in a 70% ethanol solution and allow to air dry in a flow hood.</li>
	<li>Place individual cantilever chips on top of 13F coverslips inside a standard 12 well plate.</li>
	<li>Coat the cantilevers with the biopolymer or surface modification optimized for the cell type being used according to the standard cell culture protocols.</li>
	<li>Re-suspend the cells in their specific growth medium to the desired concentration. 
	NOTE: This protocol will result in a substantial number of cells falling through the cantilever window and not adhering to the desired cantilever surface. Cell preparations should therefore be made 3-4x&#160;more concentrated than for standard coverslip preparations. For example, seeding of rat skeletal muscle satellite cells is typically carried out using a seeding density of 500 to 700 cells/square mm15,16, and are used with cantilever substrates at 2,000 cells/square mm. Optimization experiments should be carried out with new cell sources to ascertain an appropriate seeding density.</li>
	<li>Pipette 200 &#956;l&#160;of the cell suspension onto the cantilever chip surface, ensuring the bubble of medium covers the cantilever windows entirely. If the medium wicks through the window and doesn&#8217;t form a static bubble on top of the cantilevers replace the 13F coverslip and reattempt the plating.</li>
	<li>Transfer the plate containing the chips to an incubator and allow the cells to adhere for at least 1 hr (preferably 2-3 hr).</li>
	<li>After this plating period, use sterile forceps to transfer the chip to a clean well, without a 13F coverslip, and top up the culture with 1 ml&#160;of growth medium.</li>
	<li>Return the plate to the incubator.</li>
	<li>Maintain cells according to their standard protocol for in vitro maintenance on coverslips. For skeletal muscle cells, a switch of medium composition to induce myotube formation once cells become confluent will be necessary.</li>
</ol>
3. Setup of Hardware and Software for Cantilever Deflection Analysis
<ol>
	<li>Place a heated culture dish into the stage of an upright electrophysiology microscope.</li>
	<li>Add 3 ml&#160;of the feeding medium the cells are currently suspended in (+ 10 mM HEPES) to the heated microscope stage.</li>
	<li>Mount stainless steel electrodes on the inside of the heated culture dish at a separation distance of 15 mm and connect them to a pulse generator, capable of producing field stimulation pulses of varying intensity, frequency, and waveform, to allow the system to produce field stimulation of cells when appropriate.</li>
	<li>Bolt a Helium-Neon laser, mounted on XY translational stages, to the underside of the microscope table and adjust it so that the laser beam is directed through the base of the heated culture dish at a 30&#176; angle relative to the plane of the cantilever.</li>
	<li>Bolt a quadrant photo-detector module, mounted on XY translational stages, to the underside of the microscope stage and adjust the position so that the reflected laser beam lands in the center of the 4 quadrants. Figure 2 provides an overview of the hardware set up necessary for implementing the described protocol.</li>
	<li>Write a software program to control the linear actuators that scan across the cantilevers. Write the software program with reference to the flow chart provided in Figure 3. The graphical interface programmed for use with this system is provided in Figure 4.</li>
</ol>
4. Recording Cantilever Deflection Data
<ol>
	<li>Turn on the cantilever analysis hardware and associated software.</li>
	<li>Insert the heated stage thermistor into the medium and wait for it to read 37 &#176;C.</li>
	<li>Insert the cantilever chip into the stage with the cantilevers oriented toward the right hand side of the stage.</li>
	<li>Turn on the microscope light source.</li>
	<li>Focus the microscope to bring the edges of the cantilevers into view and use the laser/photo-detector control software to position the laser beam on the tip of cantilever 1. NOTE: Assuming the cantilevers are oriented to the right of the stage, cantilever 1 is the one positioned in the top left of the array and numbers run down to 16 in the bottom left. Cantilever 17 is in the top right position and runs to 32 in the bottom right (Figure 5A).</li>
	<li>Press &#8220;play&#8221; on the recording software.</li>
	<li>Position the photo-detector so that the signal reads &#8220;0&#8221; in both x and y frames by adjusting the stepper motors controlling the photo-detector.</li>
	<li>Set cantilever 1 position in the laser/photo-detector control software (Figure 4).</li>
	<li>Move the laser to the tip of cantilever 16, repeat step 4.7., and set cantilever 16 position in the laser/photo-detector control software.</li>
	<li>Move the laser to the tip of cantilever 32, repeat step 4.7., and set cantilever 32 position in the laser/photo-detector control software.</li>
	<li>Move the laser to the tip of cantilever 17, repeat step 4.7., and set cantilever 17 position in the laser/photo-detector control software.</li>
	<li>Turn off the microscope light source and the overhead light in the laboratory.</li>
	<li>Press &#8220;record&#8221; on the recording software.</li>
	<li>Set the pulse generator hardware to 40 msec, 5 V pulses at a frequency of 1 Hz, and turn the machine on. 
	NOTE: Optionally, employ optimized stimulation protocols for specific cell sources at this point, the stated settings are guidelines based on collected data using human and rodent cell sources12,13,15,16.</li>
	<li>Using the laser/photo-detector control software, set the hardware to scan across the 32 cantilever array, stopping for 5 sec&#160;at each one.</li>
	<li>When the scan of the 32 cantilevers is complete, turn off the stimulator, then stop the recording software and bring up the data file.</li>
	<li>Examine the recorded trace from each cantilever for evidence of contractile activity. Make a note of each cantilever with positive responses. A contraction is defined as a peak if the deflection is at least 0.1 V above the baseline.</li>
	<li>Remove any non-responsive cantilevers from the scan protocol on the laser/photo-detector control software.</li>
	<li>The active cantilevers can then be rescanned without stimulation in order to get a reading of the cell&#8217;s spontaneous contractile activity.</li>
	<li>Run scans with or without broad field electrical stimulation, following addition of a therapeutic compound to the medium in order to observe its effect on the functional output of the cultured cells.</li>
	<li>Carry out fatigue assessments by electrically stimulating the cells for extended periods and scanning levels of contractility to measure how long it takes for peak force to drop below a specific threshold.</li>
	<li>In experiments where motor neurons are maintained in co-culture with muscle, measure&#160;neuromuscular junction formation through treatment of motoneuron-myotube cantilever co-cultures with a neuronal stimulant (such as glutamate) or synaptic inhibitor (e.g., D-tubocurarine) and scanning for increases and decreases in spontaneous activity respectively16.</li>
	<li>Perform specific scans as dictated by the needs of the planned experiments.</li>
</ol>
5. Analysis of Cantilever Deflection Data
<ol>
	<li>Use modified Stoney&#8217;s equations15 (detailed below) to convert raw cantilever deflection data (in Volts) into a readout of stress in the cell layer or myotube (in Pascals), and a direct measurement of cellular contractile force (in nano-Newtons) : 
	<img alt="Equation 1" fo:content-width="3in" width="400" src="/files/ftp_upload/51866/51866eq1.jpg" /> Equation 1 
	<img alt="Equation 2" fo:content-width="2in" width="300" src="/files/ftp_upload/51866/51866eq2.jpg" /> Equation 2</li>
	<li>Where &#948; = cantilever tip deflection and stress produced by the myotube, &#963;c = stress produced by the myotube, assuming a uniform thick film the full width of the cantilever, Cdetector = the system-specific coefficient relating voltage to laser position on the photo-detector, &#952; = the angle of the laser and detector relative to the plane of the cantilever, ESi = the elastic modulus of silicon, tSi =the thicknesses of the cantilever, tf= myotube thickness, vSi =poison&#8217;s ratio of silicon, L = cantilever length, P = path length of laser from cantilever tip to detector, and wSi = the width of the cantilever. A schematic explaining the terms used in these equations is provided in Figure 6.</li>
	<li>Assuming the myotube is a uniform film, the force in the myotube is equal to the force in the film, leading to Equation 3, by equating the calculation of force from stress and the assumed cell cross sectional area that was used for the application of Stoney&#8217;s equation. 
	<img alt="Equation 3" fo:content-width="1.5in" src="/files/ftp_upload/51866/51866eq3.jpg" /> Equation 3</li>
	<li>Following functional data collection, fix cantilever chips for immunocytochemical analysis or utilize cells for protein or DNA analysis using standard techniques.</li>
	<li>Optionally, return the cells to the incubator instead of preparing for molecular analysis in order to reassess functional performance at a later time-point.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial+Surface+patterning+of+self+assembled+monolayers+for+controlling+neuronal+cell+behavior.">Biomaterial Surface patterning of self assembled monolayers for controlling neuronal cell behavior.</a> International journal of biomedical engineering and technology. 2, 104-134 (2009).</li><li>Guo, X., Johe, K., Molnar, P., Davis, H., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+human+fetal+spinal+cord+stem+cell+line,+NSI-566RSC,+and+its+induction+to+functional+motoneurons.">Characterization of a human fetal spinal cord stem cell line, NSI-566RSC, and its induction to functional motoneurons.</a> Journal of Tissue Engineering and Regenerative Medicine. 4, 181-193 (2010).</li><li>Varghese, K., 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+new+target+for+amyloid+beta+toxicity+validated+by+standard+and+high-throughput+electrophysiology.">A new target for amyloid beta toxicity validated by standard and high-throughput electrophysiology.</a> PLoS ONE. 5, e8643 (2010).</li><li>Natarajan, A., 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=Patterned+cardiomyocytes+on+microelectrode+arrays+as+a+functional,+high+information+content+drug+screening+platform.">Patterned cardiomyocytes on microelectrode arrays as a functional, high information content drug screening platform.</a> Biomaterials. 32, 4267-4274 (2011).</li><li>Davis, H., 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=Rat+Cortical+Oligodendrocyte-Embryonic+Motoneuron+Co-Culture:+An+Axon-Oligodendrocyte+Interaction+Model.">Rat Cortical Oligodendrocyte-Embryonic Motoneuron Co-Culture: An Axon-Oligodendrocyte Interaction Model.</a> Journal of biomaterials and tissue. 2, 206-214 (2012).</li><li>Natarajan, A., DeMarse, T., Molnar, P., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+In+Vitro+Feed-Forward+Networks.">Engineered In Vitro Feed-Forward Networks.</a> J Biotechnol Biomater. 3, 2 (2013).</li></ol>

The authors have nothing to disclose.

The critical steps in analyzing microscale cantilevers for evidence of cellular contraction are the placement of the cantilever chip within the microscope stage, and the subsequent alignment of the laser and photo-detector with the tip of the corner cantilevers in the array. If this is not done accurately, then the software will be unable to extrapolate the positions of the remaining cantilevers in the array, potentially leading to accumulation of false negatives during data collection. Operators should take care to ensure that the cantilever chip is lying flush with the bottom of the culture dish before calibrating the laser positions. If the chip sits at an angle in the dish, it will alter the path of the deflected laser beam and confound data collection.
A functional air-table must be employed to cancel out background vibrations during data collection. The small thickness (roughly 4 &#181;m) of the cantilevers used in this study provides high sensitivity to contractile activity, but has the side effect of increased sensitivity to vibrations. Operators should take care to move around the hardware as little as possible during data recording, to reduce background readings. Finally, when placing the cantilever chip in the stage, take care to ensure the chip does not come into contact with the stimulating silver electrodes in the culture well. Application of broad-field stimulation with the electrode touching the chip will alter the current path and negatively impact on the system&#8217;s ability to effectively stimulate the cultured cells.
In order to obtain more accurate readings of force output in skeletal muscle myotubes, it is highly recommended to perform immunostaining of the cultured cells once functional analysis is complete. The equations listed require input of a myotube&#8217;s cross-sectional area (CSA) in order to calculate force. While an assumed value can be used based on previous data, measurement of a myotube&#8217;s exact CSA using confocal imaging techniques will provide a more accurate estimation of the force exerted by the contracting myotube. Relating raw contraction data to the physical characteristics of the cell in question is valuable in normalizing results across multiple cantilevers and between experimental conditions15, and therefore critical for generating accurate predictions of whole tissue responses to drug treatment or disease. Stoney&#8217;s equations assume the myotubes examined span the entire length of the cantilever so effort should be made to only include data obtained from cells which comply with this assumption. If this is not possible, finite element analysis can be used to provide an accurate measurement of force from smaller myotubes as published previously15. This analytical method is far more labor intensive, and therefore ill-suited to higher throughput applications, but may be necessary if optimized cell cultures conforming to Stoney&#8217;s assumptions cannot be obtained.
As already stated, culture parameters can be transferred from standard coverslip preparations. However, it is advised to consider utilizing serum-free media compositions and non-biological substrates for use of this system in drug screening applications. Due to the undefined nature of animal sera, the effect of novel therapeutics may be confounded by the inclusion of such additives in the culture medium. Serum-free media formulations are available for both rodent and human skeletal muscle cultures18-21 and provide a more controlled, well defined environment for performing compound assessment. Similarly, the use of biological coatings (collagen, laminin, etc.) on cantilevers introduces variability to the cell preparations which are avoided by application of non-biological substrates. The deposition of silane self-assembled monolayers onto culture surfaces have been shown extensively to support the adherence and development of multiple cell types16,18,20,22-31, and represent the means to generate fully-defined surfaces in a reliable and repeatable manner.
Due to the range of contractile cells present in mammalian systems, the application of this model for in vitro assays is relatively broad. Although optimized using skeletal muscle cells, the system is potentially applicable to smooth muscle cells and cardiomyocytes as well, providing the means to perform the rapid assessment of dose responses to novel therapeutics in these cells in real-time. Current cantilever chips consist of an array of 32 cantilevers (Figure 5A), but could be easily altered to include many more. Analysis of such arrays produces a substantial number of data points from a single culture, thereby greatly increasing the statistical power of any observed differences. As evidenced in Figure 8, cells cultured in this system undergo fatigue over time, in response to continuous broad-field stimulation, allowing assessment of drug effects on the endurance levels of cells in vitro. Addition of innervating motor neurons to this culture model also allows assessment of synapse formation through measurement of muscle contraction in response to neuronal stimulants (Figure 9). While the measurement of baseline functional parameters in a multiplexed assay (Figure 7) has obvious advantages over standard biomolecular assessment of in vitro cultures, the ability to assess neuromuscular functionality and the endurance capacity of seeded cells greatly increases the applicability of this model for drug screening and disease modeling applications. The described technique offers investigators the means to perform rapid, low cost functional assessment of both healthy and diseased muscle cells in vitro. The versatility of the culture system, and the high level of functional detail obtainable from analysis of the raw data, should produce more accurate predictions of in vivo tissue responses to novel pathological and chemical challenges.

The in vitro culture of muscle cells from both human and rodent sources has been possible for decades1,2. However, while standard coverslip preparations are useful for biochemical assessment, they do not facilitate analysis of the cell&#8217;s primary functional output (contractility), and therefore are of somewhat limited value as a means to assess cellular maturation and performance. In order to maximize the amount of data obtainable from such in vitro cultures, it is necessary to advance the development of systems capable of housing such cells in configurations that permit the real-time assessment of their functional performance. The establishment of a multitude of three dimensional muscle models has made some progress toward fulfilling this need, and such systems have been used in a number of publications as a means to assess the contractile capacity of cultured muscle cells in vitro3-5. While such systems are invaluable for tissue modeling and reconstruction studies, they are limited in their applicability for studies of single cell responses. In such cases where single fiber studies are necessary, complex and labor intensive ex vivo methodologies remain the only option6-10. Furthermore, current movement toward the development of complex, multi-organ platforms for drug development and screening protocols requires the establishment of systems which are non-invasive, easily scalable and which integrate readily with supporting cells and tissue models11. 
Microscale cantilevers offer a simple method for assessing the functional contractile capacity of single cells/small populations of cells12,13. The technique is based on modified Atomic Force Microscopy (AFM) technology14, and uses a laser and photo-detector system to measure microscale cantilever deflection in response to cultured myotube contractile activity. Modified Stoney&#8217;s equations are then used to calculate stress in the myotube, and the force exerted by the myotube in order to generate the observed substrate deflection15. A scanning program has been written which enables simultaneous assessment of multiplexed cantilever arrays, offering potential moderate to high through-put applications for drug toxicity/efficacy studies15,16. Such technology may prove invaluable in the development of functional, pre-clinical assays for predicting drug efficacy in vivo. Furthermore, fabrication of cantilever chips in silicon does not impede post analysis processing of cells for standard biomolecular assays such as immunostaining, western blotting and PCR. 
This manuscript provides detailed instructions on the fabrication and preparation of microscale silicon cantilevers, the hardware and software set-up, and the operating guidelines for assessing the functionality of contractile cells cultured on these chips. Standard cell culture techniques can be implemented for plating and maintenance of cells on these surfaces, hence any contractile cell type for which reliable culture parameters exist should be able to integrate with this device with ease. The relatively simple 2D culture parameters utilized in this system makes integration of other cell models or addition of cell types that can interact with muscle (such as innervating neurons) straight-forward, greatly increasing the applicability of this model in the development of more complex functional in vitro assays and multi-organ models of mammalian systems.

<table><tbody><tr><td>Primary rat muscle growth medium</td><td></td><td></td><td></td></tr><tr><td>Neurobasal medium</td><td>Life Technologies</td><td>21103-049</td><td></td></tr><tr><td>B27 (50x)</td><td>Life Technologies</td><td>17504044</td><td>1x</td></tr><tr><td>Glutamax (100x)</td><td>Life Technologies</td><td>35050061</td><td>1x</td></tr><tr><td>G5 supplement</td><td>Life Technologies</td><td>17503-012</td><td>1x</td></tr><tr><td>Glial-Derived Neurotrophic Factor</td><td>Cell sciences</td><td>CRG400B</td><td>20 ng/ml</td></tr><tr><td>Brain-Derived Neurotrophic Factor</td><td>Cell sciences</td><td>CRB600B</td><td>20 ng/ml</td></tr><tr><td>Ciliary Neurotrophic Factor</td><td>Cell sciences</td><td>CRC400A</td><td>40 ng/ml</td></tr><tr><td>Neurotrophin-3</td><td>Cell sciences</td><td>CRN500B</td><td>20 ng/ml</td></tr><tr><td>Neurotrophin-4</td><td>Cell sciences</td><td>CRN501B</td><td>20 ng/ml</td></tr><tr><td>Acidic Fibroblast Growth Factor</td><td>Life Technologies</td><td>13241-013&amp;nbsp;</td><td>25 ng/ml</td></tr><tr><td>Cardiotrophin-1</td><td>Cell sciences</td><td>CRC700B</td><td>20 ng/ml</td></tr><tr><td>Heparin Sulphate</td><td>Sigma</td><td>D9809&amp;nbsp;</td><td>100 ng/ml</td></tr><tr><td>Leukemia Inhibitory Factor</td><td>Sigma</td><td>L5158&amp;nbsp;</td><td>20 ng/ml</td></tr><tr><td>Vitronectin</td><td>Sigma</td><td>V0132</td><td>100 ng/ml</td></tr><tr><td>Primary rat muscle differentiation medium</td><td></td><td></td><td></td></tr><tr><td>NB Activ 4</td><td>Brain Bits LLC</td><td>NB4-500</td><td></td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Class 2 red diode laser</td><td>Newport</td><td></td><td></td></tr><tr><td>Photo-detector</td><td>Noah Industries</td><td></td><td></td></tr><tr><td>Model 2100 Pulse stimulator</td><td>A-M systems</td><td></td><td></td></tr><tr><td>Multiclamp 700B Digitizer</td><td>Axon Instruments</td><td></td><td></td></tr><tr><td>Patch clamp microscope and stage</td><td>Olympus</td><td></td><td></td></tr><tr><td>Delta T4 culture dish controller</td><td>Bioptechs</td><td></td><td></td></tr><tr><td>Axoscope software</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>LabVIEW software</td><td>National Instruments</td><td></td><td></td></tr><tr><td>37&lt;sup&gt; o&lt;/sup&gt;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>NAPCO</td><td></td><td></td></tr><tr><td>Class 2 microbiological flow hood</td><td>Labconco</td><td></td><td></td></tr><tr><td>Pipettes and tips</td><td>Eppendorf</td><td></td><td></td></tr></tbody></table>

utilization of microscale silicon cantilevers to assess cellular contractile function in vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Successful culture of contractile cells on cantilevers is a relatively straightforward procedure, utilizing standard cell culture techniques (Figure 5). The percentage of cantilevers supporting contracting cells will vary depending on cell type being examined and specific culture technique employed. Using primary embryonic cells derived from rat hind limbs, contractile activity was detected on 12% of cantilevers examined (n = 4). Analysis of contractile function using the laser and photo-detector system described provides accurate real-time data pertaining to the functional maturity of the seeded cells. Use of standard electrophysiological software can then be used to analyze the raw data, facilitating calculation of relevant functional properties, such as peak force, time to peak force, and time to half relaxation, as illustrated in Figure 7. Subsequent data collection from cultures treated with therapeutic compounds allows for comparison of functional properties with and without drug addition, thereby enabling assessment of compound activity and subsequent prediction of in vivo responses. Furthermore, extended stimulation protocols provide the means to assess rates of fatigue in cultured cells, thus broadening the level of physiological data obtainable from this system (Figure 8).
The cantilever culture can be modified to include motor neurons in the culture system with skeletal muscle myotubes16, so as to allow assessment of neuromuscular synapse formation in vitro (Figure 9). In such cultures, rates of spontaneous contractile activity are compared to rates of contraction in response to treatment with a neuron-specific stimulant, such as&#160;glutamate. Any observed glutamate induced increases in contraction rates suggest the activation of cultured neurons, leading to acetylcholine release and subsequent myotube activation. Treatment with synaptic inhibitors, such as the acetylcholine receptor blocker D-tubocurarine, leading to cessation of glutamate induced activity provide further evidence for the presence of functional neuromuscular synapses in these cultures16.
<img alt="Figure 1" src="/files/ftp_upload/51866/51866fig1highres.jpg" />
Figure 1.&#160;Schematic detailing cantilever fabrication process flow. The numbers listed in the flow diagram correlate to protocol steps in the methods section titled &#8220;Cantilever chip fabrication.&#8221; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51866/51866fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/51866/51866fig2highres.jpg" /> 
Figure 2.&#160;Hardware details of the laser/photo-detector system used to assess cellular contraction on cantilevers. (A)&#160;Photograph of the modified electrophysiological microscope used for cantilever assessment. The laser (i) and photo-detector (ii) are mounted beneath the stage on XY translational stages. A transparent culture dish is mounted on the stage (iii), which allows laser passage to the cantilever array through the underside of the stage. (B)&#160;Schematic &#8220;top-down&#8221; perspective of the microscope stage. XY translational stages allow movement of the laser and photo-detector in both X and Y planes (red arrows), facilitating movement of the laser to interrogate each cantilever in an array. Cantilever chips (iii) should be placed into the stage with the cantilevers facing toward the right of the stage in order for the system to operate correctly. (C)&#160;Close up photograph of the laser (i) and photo-detector (ii) mounted beneath the microscope stage. The laser is positioned at a 30&#176; angle relative to the plane of the cantilever chip (&#952;). (D)&#160;Close up photograph of the culture dish. Broad-field electrical stimulation is applied by means of a pair of silver electrodes positioned 15 mm apart (iv). A heating element (v) attached to the underside of the dish is used to maintain the culture at 37 &#176;C during analysis. Temperature control is dynamic, and regulated by means of a thermistor placed in the medium (not shown). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51866/51866fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/51866/51866fig3highres.jpg" /> 
Figure 3.&#160;Flow chart of logic underlying&#160;software&#160;to control scanning of the laser and photo-detector. Two software loops control motion: a main loop for user input and&#160;a loop that controls the scanning motion. Setup is performed while in the main loop, followed by activation of the second loop for medium- to high-throughput data collection.&#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51866/51866fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/51866/51866fig4highres.jpg" /> 
Figure 4.&#160;Graphical user interface of software used to control laser and photo-detector positions during cantilever assessment. (A)&#160;Buttons to manually control laser and photo-detector positions in both X and Y planes. (B)&#160;Controls for manually setting the positions of the 4 corner cantilevers (1, 16, 17, and 32), thereby allowing the software to extrapolate the positions of the remaining cantilevers in the array. (C)&#160;Controls allowing users to select which cantilevers to include in each scan. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51866/51866fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/51866/51866fig5highres.jpg" /> 
Figure 5.&#160;Images of a custom built cantilever chip for use in this system, and representative images of cells maintained on similar surfaces. (A)&#160;Photograph of a single, custom built cantilever chip. Each chip contains 32 cantilevers arranged in 2 rows of 16. Cantilever 1 is positioned in the bottom left of the image and cantilever 32 in the top right. Cantilever chips are 15 x 15 mm2. (B)&#160;Phase-contrast image of primary rat skeletal muscle cells grown on cantilevers. Each cantilever is 750 &#956;m long, 100 &#956;m wide and 4 &#956;m thick. (C)&#160;Immunocytochemical stain of a primary rat myotube maintained on a cantilever. Cells were stained using an antibody probe for Myosin Heavy Chain. Note the striated appearance of the cultured fiber, indicating maturation of the contractile machinery. Cantilever edges have been artificially highlighted in this image to provide an indication of scale. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51866/51866fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/51866/51866fig6highres.jpg" /> 
Figure 6.&#160;Schematic illustrating the terms used in Stoney&#8217;s equations for deriving force produced by myotubes on cantilevers. &#948; = cantilever tip deflection and stress produced by the myotube, Cdetector = the system-specific coefficient relating voltage to laser position on the photo-detector, &#952; = the angle of the laser and detector relative to the plane of the cantilever, ESi = the elastic modulus of silicon, tSi = the thicknesses of the cantilever, tf = myotube thickness, vSi = poison&#8217;s ratio of silicon, L = cantilever length, and P = path length of laser from cantilever tip to detector. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51866/51866fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/51866/51866fig7highres.jpg" /> 
Figure 7.&#160;Representative raw data and analysis of contractile form using the cantilever system described. (A)&#160;Example of a raw data trace from the broad-field electrical stimulation of primary rat myotubes on cantilevers. Top trace = laser deflection (in Volts) in the x-axis, indicating lengthwise strain on the cantilever. Middle trace = laser deflection (in Volts) in the y-axis, indicating torsional strain across the cantilever. Bottom trace = Indication of the temporal position of electrical pulses used to elicit myotube contraction in this system. (B)&#160;Using standard electrophysiology software, it is possible to measure peak force (PF), time to peak force (TTP) and time to half relaxation (1/2 RT) from the collected raw data. (C)&#160;Collated contraction data (n = 11) from primary rat skeletal muscle myotubes. Application of a modified Stoney&#8217;s equation allows calculation of cantilever stress from raw data readings in Volts. From this data, direct calculation of force (in Newtons) can also be generated. Published data reprinted with permission13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51866/51866fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/51866/51866fig8highres.jpg" /> 
Figure 8.&#160;Representative data demonstrating analysis of skeletal muscle fatigue in cantilever cultures. Raw data (in Volts) was converted to a measurement of myotube force (in nano-Newtons) and re-plotted. Data illustrates magnitude of myotube contractions on cantilevers in response to 1 Hz broad-field electrical pulses after 0 min&#160;(black trace) and 120 min&#160;(grey trace) of continuous stimulation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51866/51866fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/51866/51866fig9highres.jpg" /> 
Figure 9.&#160;Representative traces from the analysis of a skeletal muscle-motoneuron co-culture maintained on cantilevers, demonstrating the functional effects of motoneuron stimulation with and without addition of a neuromuscular blocker. Raw data (in Volts) was converted to a measurement of myotube force (in nano-Newtons) and re-plotted. (A)&#160;Measurement of spontaneous contractions by the cultured myotubes without neuronal stimulation. (B)&#160;Measurement of myotube contraction following neuronal stimulation via the addition of 200 &#956;M glutamate. (C)&#160;Measurement of myotube contraction following glutamate and 12.5 &#956;M D-tubocurarine treatment. Published data reprinted with permission16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51866/51866fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The following tables list the specific reagents used to culture cells in the validation experiments described, as well as the equipment necessary to perform the cantilever assessment. Please note that culture reagents used are not necessary and this system should be easily integrated with the culture protocols of any lab maintaining contractile cells in vitro. The listed reagents are provided should investigators wish to repeat the specific experimental results described.

Watch this Scientific Journal Video about Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro at JoVE.com

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

utilization-microscale-silicon-cantilevers-to-assess-cellular

Nanyang Technological University

Research

JoVE Journal

Bioengineering

10.2K Views.  University of Central Florida.  Dit protocol beschrijft het gebruik van microschaal silicium consoles als buigzaam cultuur oppervlakken voor het meten van de contractiliteit van spiercellen in vitro. Cellular samentrekking cantilever buigen, die kan worden gemeten, geregistreerd en omgezet in uitlezingen van kracht, die een non-invasieve en schaalbaar systeem voor het meten contractiele functie in vitro. 

Video: Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young&#39;s modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition.
Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Quantifying the contact area and force applied by an atomic force microscope (AFM) probe tip to a sample surface enables nanoscale mechanical property determination. Best practices to implement AFM cantilever-based nanoindentation in air or fluid on soft and hard samples to measure elastic modulus or other nanomechanical properties are discussed.

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by ...

Engineering

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

High-resolution Imaging of Nuclear Dynamics in Live Cells under Uniaxial Tensile Strain

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the effect of applied extracellular tensile strain on cell populations in vitro via biochemical assays, a device has previously been designed which can be fabricated simply and is small enough to fit inside tissue culture incubators, as well as on top of microscope stages. However, the previous design of the polydimethylsiloxane substratum did not allow high-resolution subcellular imaging via oil-immersion objectives. This work describes a redesigned geometry of the polydimethylsiloxane substratum and a customized imaging setup that together can facilitate high-resolution subcellular imaging of live cells while under applied strain. This substratum can be used with the same, earlier designed device and, hence, has the same advantages as listed above, in addition to allowing high-resolution optical imaging. The design of the polydimethylsiloxane substratum can be improved by incorporating a grid which will facilitate tracking the same cell before and after the application of strain. Representative results demonstrate high-resolution time-lapse imaging of fluorescently labeled nuclei within strained cells captured using the method described here. These nuclear dynamics data give insights into the mechanism by which applied tensile strain promotes differentiation of oligodendrocyte progenitor cells.&#160;

Using a previously designed device to apply mechanical strain to adherent cells, this paper describes a redesigned substratum geometry and a customized apparatus for high-resolution single-cell imaging of strained cells with a 100x oil immersion objective.

Extracellular mechanical strain is known to elicit cell phenotypic responses and has physiological relevance in several tissue systems. To capture the ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

Single Cell Durotaxis Assay for Assessing Mechanical Control of Cellular Movement and Related Signaling Events

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate underlying a cell must be manipulated. While hydrogels with graded stiffness and long-term migration assays have proven useful in durotaxis studies, immediate, acute responses to local changes in substrate tension allow focused study of individual cell movements and subcellular signaling events. To repeatably test the ability of cells to sense and respond to the underlying substrate stiffness, a modified method for application of acute gradients of increased tension to individual cells cultured on deformable hydrogels is used which allows for real time manipulation of the strength and direction of stiffness gradients imparted upon cells in question. Additionally, by fine tuning the details and parameters of the assay, such as the shape and dimensions of the micropipette or the relative position, placement, and direction of the applied gradient, the assay can be optimized for the study of any mechanically sensitive cell type and system. These parameters can be altered to reliably change the applied stimulus and expand the functionality and versatility of the assay. This method allows examination of both long term durotactic movement as well as more immediate changes in cellular signaling and morphological dynamics in response to changing stiffness.

Mechanical forces are important for controlling cell migration. This protocol demonstrates the use of elastic hydrogels that can be deformed using a glass micropipette and a micromanipulator to stimulate cells with a local stiffness gradient to elicit changes in cell structure and migration.

Durotaxis is the process by which cells sense and respond to gradients of tension. In order to study this process in vitro, the stiffness of the substrate ...

Biology

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this&#160;system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

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