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 ensu...

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 border="0" cellpadding="0" cellspacing="0" width="690">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:299px;">Name of material/ equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:108px;">Catalog number</td>
			<td style="width:156px;">Comments/ Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle growth medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurobasal medium</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">21103-049&#160;</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">B27 (50x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17504044</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glutamax (100x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">35050061</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">G5 supplement</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17503-012&#160;</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glial-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRG400B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Brain-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRB600B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Ciliary Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC400A</td>
			<td dir="LTR" style="width:156px;">40 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-3</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN500B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-4</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN501B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Acidic Fibroblast Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">13241-013&#160;</td>
			<td dir="LTR" style="width:156px;">25 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vascular Endothelial Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">P2654</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Cardiotrophin-1</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC700B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Heparin Sulphate</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">D9809&#160;</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Leukemia Inhibitory Factor</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">L5158&#160;</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vitronectin</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">V0132</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle differentiation medium</td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">NB Activ 4</td>
			<td dir="LTR" style="width:128px;">Brain Bits LLC</td>
			<td dir="LTR" style="width:108px;">NB4-500</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 red diode laser</td>
			<td>Newport</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Photo-detector</td>
			<td>Noah Industries</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Model 2100 Pulse stimulator</td>
			<td>A-M systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Multiclamp 700B Digitizer</td>
			<td>Axon Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Patch clamp microscope and stage</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Delta T4 culture dish controller</td>
			<td>Bioptechs</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Axoscope software</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LabVIEW software</td>
			<td>National Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">37oC, 5% CO2 incubator</td>
			<td>NAPCO</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 microbiological flow hood</td>
			<td>Labconco</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipettes and tips</td>
			<td>Eppendorf</td>
			<td>N/A</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 ...

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

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Bioengineering

10.2K ビュー.  University of Central Florida. このプロトコルは、 インビトロで筋細胞の収縮性を測定するための柔軟な培養表面としてマイクロスケールのシリコンカンチレバーの使用を記載している。細胞収縮を測定記録し、 インビトロで収縮機能を測定するための非侵襲的かつスケーラブルなシステムを提供し、力の読み取り値に変換することができるカンチレバー屈曲を引き起こ...

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

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Measuring and Modeling Contractile Drying in Human Stratum Corneum

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both cleansing agents and daily variations in ambient moisture; both of which can alter the water content of the tissue. Reductions in water content from severe barrier dysfunction or low humidity environments can alter SC stiffness and cause a build-up of drying stresses. In extreme conditions, these factors can cause mechanical rupture of the tissue. We have established a high throughput method of quantifying dynamic changes in the mechanical properties of SC upon drying. This technique can be employed to quantify changes in the drying behavior and mechanical properties of SC with cosmetic cleanser and moisturizer treatments. This is achieved by measuring dynamic variations in spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. In-plane radial displacements acquired during drying are azimuthally averaged and fitted with a profile based on a linear elastic contractility model. Dynamic changes in drying stress and SC elastic modulus can then be extracted from the fitted model profiles.

This article describes a method of quantifying the dynamic drying behavior and mechanical properties of stratum corneum by measuring spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. This technique can be used to measure how different chemical treatments alter drying and tissue mechanical properties.

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.