This work was funded by the National Institutes of Health (OD/NICHD DP2HD083961 and NHBLI R01HL148104). Leo Q. Wan is a Pew Scholar in Biomedical Sciences (PEW 00026185), supported by the Pew Charitable Trusts. Haokang Zhang is supported by American Heart Association Predoctoral Fellowship (20PRE35210243).

1. Fabrication of polydimethylsiloxane (PDMS) stamps16
<ol>
	<li>Draw an array of microscale rings using CAD software, with an inner diameter of 250 &#181;m and an outer diameter of 450 &#181;m. The pattern used in this protocol is a 10 x 10 array with an 850 &#181;m distance between rings.</li>
	<li>Print a transparency mask of the pattern at the desired resolution using a microfabrication company&#39;s mask printing service (see Table of Materials). 
	NOTE: The provided dimensions of the ring have been proven to work for many cell types7.</li>
	<li>Conduct ultraviolet (UV) photolithography using the mask containing desired features to make a negative photoresist mold (such as SU-8)8.
	<ol>
		<li>Cover a silicon wafer spin-coated with photoresist by the transparency mask fabricated in steps 1.1-1.2.</li>
		<li>On a UV contact aligner, polymerize the photoresist under transparent regions of the mask by UV light. 
		NOTE: After further wash and development, the master mold with desired features is fabricated8. Operational details depend on the specific equipment used.</li>
	</ol>
	</li>
	<li>Prepare PDMS elastomeric prepolymers by mixing with its curing agent at a 10:1 ratio and then cast the mixture onto the mold in a Petri dish.</li>
	<li>Place the dish in a vacuum chamber for 30 min to remove air bubbles in PDMS and bake in an oven at 60 &#176;C for at least 2 h.</li>
	<li>After complete curing of PDMS, cut the pattern arrays into stamps. 
	&#8203;NOTE: Thicker stamps (about 1 cm) may be preferred due to the ease of handling at the later steps of microcontact printing.</li>
</ol>
2. Coating of glass slides
<ol>
	<li>Clean the glass slides. Soak the slides in a 100% ethanol bath, sonicate for 5 min, and then rinse with water.</li>
	<li>Repeat this step first with acetone, then with isopropanol, followed by drying in a nitrogen stream.</li>
	<li>Use an electron beam (E-beam) evaporation equipment to coat titanium and gold layers on glass slides. 
	NOTE: For the titanium layer, the desired thickness is 15 &#197; (1 &#197; = 10-10 m). For the gold layer, the desired thickness is 150 &#197;. Coat titanium first and then gold, as the titanium layer serves as an adhesive between gold and the glass slide.</li>
	<li>Place the cleaned slides into the evaporation chamber and insert gold and titanium crucibles under the shutters.</li>
	<li>Pump down the chamber to create a vacuum lower than 10-5 Pa before melting metals. Focus the electron beam onto the metal and adjust the power so that the depositing rate is appropriate for controlling the deposition thickness.</li>
	<li>Open the shutter to start the metal deposition. Close the shutter when the desired thickness is reached.</li>
	<li>Switch crucibles between sequential evaporation of two metals.</li>
	<li>Wait for the crucibles to cool down as required by the equipment, vent the chamber, and take the gold-coated glass slides out.</li>
	<li>Pump down the chamber again for equipment maintenance. 
	&#8203;NOTE: Operational details may depend on a specific E-beam evaporator. Having a well-controlled, low depositing rate of titanium is important. A thick titanium layer often leads to poor visibility, especially for fluorescence imaging of the cells. After coating, the titanium/gold-coated glass slides can be stored in a dry vacuum chamber at room temperature for at least 1 month.</li>
</ol>
3. Microcontact printing
<ol>
	<li>Cut the titanium/gold-coated slide into smaller pieces, if desired. A square shape of 12 mm x 12 mm is recommended for a typical application (Figure 1B).</li>
	<li>Use a glass cutter to slide across the surface to leave a dented track, then hold the glass slide at both sides and bend at the dent to break into small quarters. Avoid touching the surface coated with gold. 
	NOTE: To visually determine which side of the glass is the coated surface in case of accidental tipping over, compare the reflection of light on both sides. The gold-covered surface will show a brighter reflection, whereas the non-coated side will have a dimmer reflection (from the titanium layer on the other side of the glass). An alternative way is to observe the edges of the slide, and the coating side can be determined from the vertical cutting surface.</li>
	<li>Clean the slides by soaking in 100% ethanol in a Petri dish with the gold side up on an orbital shaker for at least 10 min. Aspirate out the ethanol, then dry each slide by blowing with a nitrogen stream.</li>
	<li>Clean the PDMS stamps with soapy water and then 100% ethanol. Dry the stamps with nitrogen gas.</li>
	<li>Prepare 2 mM octadecanethiol (C18) solution by dissolving 5.74 mg of C18 powder in 10 mL of 100% ethanol. 
	NOTE: Seal and store C18 solution at room temperature. Printing patterns with C18 creates an adhesive self-assembled monolayer for which the fibronectin and cells will preferentially attach.</li>
	<li>Soak the PDMS stamps in C18 solution with the patterned surface facing down for 10 s and gently dry with nitrogen gas for 60 s. 
	NOTE: When drying, place the stream away from the stamp first (about 1 m) until most liquid is evaporated and then slowly move the stream closer to fully dry the stamp. This is to avoid C18 solution being blown away instead of drying on the stamp.</li>
	<li>Lay the stamp face down onto the gold slides for 60 s before removal.</li>
	<li>To ensure proper stamping, gently tap the tweezers onto the stamps to properly transfer the patterns. Do not push down too hard, and the weight of tweezers is typically sufficient. 
	NOTE: Visual inspection of the stamp during tapping will ensure that the stamp and glass slide have made contact uniformly.</li>
	<li>Prepare humidity chambers.
	<ol>
		<li>Pipette 1 mL of 70% ethanol into an inverted Petri dish lid and lay down a piece of parafilm to cover the surface (covered face up).</li>
		<li>Use tweezers to lift, lay down parafilm to ensure no air bubbles, and remove excess ethanol if needed.</li>
	</ol>
	</li>
	<li>Prepare 2 mM HS-(CH2)11-EG3-OH (EG3) solution: Dilute 5 &#181;L stock solution in 5 mL of 100% ethanol. 
	NOTE: The diluted EG3 solution can be sealed and stored at 4 &#176;C, and undiluted EG3 stock should be stored at -20 &#176;C. The EG3 treatment is used to make the glass region without C18 non-adhesive to cells.</li>
	<li>Micropipette 40 &#181;L droplets of EG3 for each quarter glass slide onto the parafilm in the humidity chamber, leaving enough space between droplets to account for the spacing of the gold slides.</li>
	<li>Use tweezers to place C18-printed gold slides face down onto the droplets and ensure no bubbles under the slides. Gently push the slides together without lifting them to minimize evaporation. 
	NOTE: Place one edge of the glass slides down near the droplet, and then slowly lowering the other end of the glass slide down. If a bubble is present, push the gold slide without lifting it until the bubble is no longer present.</li>
	<li>Seal the Petri dish tightly with parafilm and leave it at room temperature for at least 3 h. 
	NOTE: If extensive incubation is desired, double seal the Petri dish and leave it for up to 24 h.</li>
	<li>In a biosafety cabinet, place gold slides face up in a Petri dish, soak and rinse 3 times in 70% ethanol to remove EG3, then leave in ethanol for 10 min for sterilization.</li>
	<li>Aspirate out the ethanol and replace it with sterile PBS.</li>
	<li>Prepare another humidity chamber as described in step 3.9, with PBS instead of ethanol. 
	NOTE: Due to the difference in surface tension, it is easier to first add 4-5 mL of PBS to lay down parafilm and remove bubbles, then aspirate out the excess.</li>
	<li>Prepare the 50 &#181;g/mL fibronectin solution (other proteins, if desired) in sterile PBS and micropipette 50 &#181;L droplets of fibronectin solution for each quarter slide onto the parafilm, leaving some space between droplets. 
	NOTE: Fibronectin coating facilitates cell adhesion to patterned surfaces.</li>
	<li>Use wafer tweezers to place slides face down onto the droplets and ensure no bubbles under the slides. Gently push the slides together without lifting them. 
	NOTE: Glass slide tweezers or wafer tweezers with straight wide tips are recommended for handling the slides.</li>
	<li>Leave the Petri dish in the biosafety cabinet for 30 min.</li>
	<li>After 30 min, place the gold slides face up in PBS after rinsing for 3 times. 
	&#8203;NOTE: Fibronectin-patterned slides can be stored in PBS at 4 &#176;C for about a month.</li>
</ol>
4. Seeding cells onto micropatterned slides
<ol>
	<li>Warm up the cell culture media and trypsin in a 37 &#176;C water bath. 
	NOTE: The following description of subculture is for NIH/3T3 cells. Culture conditions may vary depending on cell types.</li>
	<li>(Optional) Soak patterned slides in culture media in a 12-well plate, warm up to 37 &#176;C in an incubator prior to trypsinization of cells for better cell attachment.</li>
	<li>Trypsinize the cells, neutralize with FBS-containing media, centrifuge down at 100 x g for 3 min, and then resuspend by mixing well with fresh media. 
	NOTE: Make sure that the cells are fully dissociated from each other.</li>
	<li>Count the cells, dilute to 200,000 cells/mL and add 0.5 mL of the cell suspension to each well containing one gold slide.</li>
	<li>Gently shake the well plate a few times for uniform cell seeding and store it in an incubator for 15 min to allow for cell attachment.</li>
	<li>After 15 min, check cell attachment under a microscope. Allow additional time, if needed. 
	NOTE: The attached cells will spread out on the surface with visible filopodium or flattening and will not move from the surface when the well plate is gently tapped or moved.</li>
	<li>Aspirate out the media containing unattached cells from each well and add 1 mL of culture media. For drug treatment, supplement additional reagents to culture media at this step.</li>
	<li>Culture the cells in the incubator for 24 h and check confluency to determine if chirality has formed. 
	&#8203;NOTE: Above 75% confluency is recommended for most cell types, and over-confluency may affect chirality characterization.</li>
</ol>
5. Image collection
<ol>
	<li>Fix the cells upon confluency.
	<ol>
		<li>Remove culture media from the well plate and rinse once with PBS.</li>
		<li>Add 4% paraformaldehyde solution, incubate at room temperature for 15 min, and then rinse 3 times with PBS. 
		NOTE: If the fixation significantly changes cell morphology, imaging before fixation is recommended</li>
	</ol>
	</li>
	<li>Use a phase-contrast microscope with camera functionality, image each ring on the slides at high resolution (10x object lens is sufficient for analysis).</li>
</ol>
6. Cell chirality characterization (Figure 2)
<ol>
	<li>Download MATLAB code files for chirality characterization from the supplementary files (Supplementary File 1). 
	NOTE: The code has been tested to work with MATLAB_R2015b and later versions on both Windows and macOS systems. The circular statistics toolbox is also required to run the files, which can be found in reference17.</li>
	<li>Add the code folder and sub-folders (with circular statistics toolbox inside) to the MATLAB path and open the &#34;ROI_selection.m&#34; file. In line 4, change the directory to the desired data folder (For window users, switch &#34;/&#34; into &#34;\&#34; in lines 4 and 5).</li>
	<li>Change the image size in line 14, with the first two figures representing the inner circle size of the ring while the other two representing the outer. 
	NOTE: Make sure that the sizes of all images in a folder to be analyzed are identical.</li>
	<li>Click on the Run button to execute the MATLAB code &#34;ROI_selection.m&#34; to determine the region of interest (ROI) in phase-contrast images.</li>
	<li>Manually drag the selection square to fit the ring, then double click on the image to confirm the selection.</li>
	<li>Repeat this step for every image in the folder (the next image will automatically pop up after confirming the ROI selection of a previous image); a &#34;.mat&#34; file will be generated to store the ROI information for each image.</li>
	<li>Open &#34;Analysis_batch.m&#34; file and change the directory of the folder same as step 6.2. (For window users, for the first-time use, switch &#34;/&#34; into &#34;\&#34; in lines 5, 6, 124, and 130)</li>
	<li>Click on the Run button to execute the code &#34;Analysis_batch.m&#34; to determine the chirality of multiple cellular ring patterns. A &#34;datatoexcel.txt&#34; file will be generated, containing circular statistics for each ring as well as the numbers of clockwise, non-chiral, and anti-clockwise rings.</li>
</ol>

<ol>
	<li>Wan, L. Q., Chin, A. S., Worley, K. E., Ray, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+chirality:+emergence+of+asymmetry+from+cell+culture.">Cell chirality: emergence of asymmetry from cell culture.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 371, 20150413 (2016).</li><li>Rahman, T., Zhang, H., Fan, J., Wan, L. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+chirality+in+cardiovascular+development+and+disease.">Cell chirality in cardiovascular development and disease.</a> APL Bioengineering. 4 (3), (2020).</li><li>Inaki, M., Liu, J., Matsuno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+chirality:+Its+origin+and+roles+in+left-right+asymmetric+development.">Cell chirality: Its origin and roles in left-right asymmetric development.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 371 (1710), 20150413 (2016).</li><li>Utsunomiya, S., 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=Cells+with+broken+left-right+symmetry:+Roles+of+intrinsic+cell+chirality+in+left-right+asymmetric+epithelial+morphogenesis.">Cells with broken left-right symmetry: Roles of intrinsic cell chirality in left-right asymmetric epithelial morphogenesis.</a> Symmetry. 11 (4), 505 (2019).</li><li>Tamada, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+neuronal+motility:+The+missing+link+between+molecular+chirality+and+brain+asymmetry.">Chiral neuronal motility: The missing link between molecular chirality and brain asymmetry.</a> Symmetry. 11 (1), 102 (2019).</li><li>Tee, Y. 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=Cellular+chirality+arising+from+the+self-organization+of+the+actin+cytoskeleton.">Cellular chirality arising from the self-organization of the actin cytoskeleton.</a> Nature Cell Biology. 17 (4), 445-457 (2015).</li><li>Wan, L. Q., 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=Micropatterned+mammalian+cells+exhibit+phenotype-specific+left-right+asymmetry.">Micropatterned mammalian cells exhibit phenotype-specific left-right asymmetry.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (30), 12295-12300 (2011).</li><li>Fan, J., 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=Cell+chirality+regulates+intercellular+junctions+and+endothelial+permeability.">Cell chirality regulates intercellular junctions and endothelial permeability.</a> Science Advances. 4 (10), 2111 (2018).</li><li>Singh, A. V., 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=Carbon+nanotube-induced+loss+of+multicellular+chirality+on+micropatterned+substrate+is+mediated+by+oxidative+stress.">Carbon nanotube-induced loss of multicellular chirality on micropatterned substrate is mediated by oxidative stress.</a> ACS Nano. 8 (3), 2196-2205 (2014).</li><li>Zhang, H., Fan, J., Zhao, Z., Wang, C., Wan, L. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Alzheimer's+disease-related+proteins+on+the+chirality+of+brain+endothelial+cells.">Effects of Alzheimer's disease-related proteins on the chirality of brain endothelial cells.</a> Cellular and Molecular Bioengineering. 14, 231-240 (2021).</li><li>Chen, T. 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=Left-right+symmetry+breaking+in+tissue+morphogenesis+via+cytoskeletal+mechanics.">Left-right symmetry breaking in tissue morphogenesis via cytoskeletal mechanics.</a> Circulation Research. 110 (4), 551-559 (2012).</li><li>Ray, P., 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=Intrinsic+cellular+chirality+regulates+left-right+symmetry+breaking+during+cardiac+looping.">Intrinsic cellular chirality regulates left-right symmetry breaking during cardiac looping.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (50), 11568-11577 (2018).</li><li>Fan, J., Zhang, H., Rahman, T., Stanton, D. N., Wan, L. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+organelle-based+analysis+of+cell+chirality.">Cell organelle-based analysis of cell chirality.</a> Communicative and Integrative Biology. 12 (1), 78-81 (2019).</li><li>Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometric+control+of+cell+life+and+death.">Geometric control of cell life and death.</a> Science. 276 (5317), 1425-1428 (1997).</li><li>Liu, W., 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=Nanowire+magnetoscope+reveals+a+cellular+torque+with+left-right+bias.">Nanowire magnetoscope reveals a cellular torque with left-right bias.</a> ACS Nano. 10 (8), 7409-7417 (2016).</li><li>Wan, L. Q., 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=Geometric+control+of+human+stem+cell+morphology+and+differentiation.">Geometric control of human stem cell morphology and differentiation.</a> Integrative Biology. 2 (7-8), 346-353 (2010).</li><li>Circular Statistics Toolbox. MathWorks Available from: <a target="_blank" href="https://www.mathworks.com/matlabcentral/fileexchange/10676-circular-statistics-toolbox-directional-statistics">https://www.mathworks.com/matlabcentral/fileexchange/10676-circular-statistics-toolbox-directional-statistics</a> (2021)</li><li>Chin, A. S., Worley, K. E., Ray, P., Kaur, G., Fan, J., Wan, L. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+chirality+revealed+by+three-dimensional+spontaneous+rotation.">Epithelial cell chirality revealed by three-dimensional spontaneous rotation.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (48), 12188-12193 (2018).</li><li>Worley, K. E., Chin, A. S., Wan, L. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage-specific+chiral+biases+of+human+embryonic+stem+cells+during+differentiation.">Lineage-specific chiral biases of human embryonic stem cells during differentiation.</a> Stem Cells International. 2018, 1848605 (2018).</li></ol>

The authors have nothing to disclose.

The ring-shaped patterning assay described here provides an easy-to-use tool for quantitative characterization of multicellular chirality, capable of producing highly reliable and repeatable results. Rapid generation of identical defined microenvironments and unbiased analysis enables automated high-throughput processing of large size of samples. This protocol discusses the fabrication of the ring micropatterns, cell patterning, and automatic analysis of the biased cell alignment and directional motion. This method is co...

Left-right (LR) asymmetry of the cell, also known as cellular handedness or chirality, describes the cell polarity in the LR axis and is recognized to be a fundamental, conserved, biophysical property1,2,3,4,5. Cell chirality has been observed both in vivo and in vitro at multiple scales. Previous findings revealed chiral swirling of actin cytoskeleton in single cells seeded on circular islands6, biased migration and alignment of cells within confined boundaries7,8,9,10,11, and asymmetrical looping of chicken heat tube12.
At the multicellular level, cell chirality can be determined from directional migration or alignment, cellular rotation, cytoskeletal dynamics, and cell organelle positioning7,8,9,10,11,12,13. We have established a micropatterning-based14 assay to efficiently characterize the chiral bias of adherent cells7,8,9,10. With the ring-shaped micropatterns geometrically confining cell clusters, the cells collectively exhibit directional migration and biased alignment. A MATLAB program was developed to automatically detect and measure cell alignment in phase-contrast images of the ring. The direction of local cell alignment is quantified with a biased angle, depending on its deviation from the circumferential direction. Following statistical analysis, the ring pattern of cells is designated either as counterclockwise (CCW) biases or clockwise (CW) biases.
This assay has been used to characterize the chirality of multiple cell phenotypes (Table 1), and the LR asymmetry of cells has been found to be phenotype-specific7,11,15. Moreover, disruption to actin dynamics and morphology can result in a reversal of chiral bias7,8, and oxidative stress can alter cell chirality as well9. Because of the simplicity of the procedure and the robustness of the approach7,8,9,10, this 2D chirality assay provides an efficient and reliable tool for determining and studying multicellular chirality in vitro.
The purpose of this protocol is to demonstrate the use of this method to characterize cell chirality. This protocol describes how to fabricate patterned cellular arrays via microcontact printing technique and conduct chirality analysis in an automated fashion using the MATLAB program.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200 proof ethanol</td>
			<td>Koptec</td>
			<td>DSP-MD-43</td>
			<td></td>
		</tr>
		<tr>
			<td>BZX microscope system</td>
			<td>Keyence</td>
			<td>BZX-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium (DMEM), high glucose</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron beam evaporator</td>
			<td>Temscal</td>
			<td>BJD-1800</td>
			<td>Gold-titanum film coating</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>VWR</td>
			<td>89510-186</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin from bovine plasma</td>
			<td>Sigma</td>
			<td>F1141-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slides</td>
			<td>VWR</td>
			<td>10024-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tweezers</td>
			<td>Exelta</td>
			<td>390BSAPI</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold evaporation pellets</td>
			<td>International Advanced Materials</td>
			<td>AU18</td>
			<td></td>
		</tr>
		<tr>
			<td>HS-(CH2)11-EG3-OH (EG3)</td>
			<td>Prochimia</td>
			<td>TH 001-m11.n3-0.2</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>MATLAB_R2020b</td>
			<td></td>
		</tr>
		<tr>
			<td>NIH/3T3 cells</td>
			<td>ATCC</td>
			<td>CRL-1658</td>
			<td></td>
		</tr>
		<tr>
			<td>OAI contact aligner</td>
			<td>OAI</td>
			<td>200</td>
			<td>UV photolithography</td>
		</tr>
		<tr>
			<td>Octadecanethiol (C18)</td>
			<td>Sigma</td>
			<td>O1858-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>VWR</td>
			<td>89032-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Research product international</td>
			<td>P32080-100T</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane Sylgard 184</td>
			<td>Dow Corning</td>
			<td>DC4019862</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>University Wafer</td>
			<td>ID#809</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo fisher scientific</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3050 photoresist</td>
			<td>MicroChem</td>
			<td>Y311075 0500L1GL</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium evaporation pellets</td>
			<td>International Advanced Materials</td>
			<td>TI14</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparency mask (with feature)</td>
			<td>Outputicity.com</td>
			<td>N/A</td>
			<td>Mask printing service</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Thermo fisher scientific</td>
			<td>25200-072</td>
			<td></td>
		</tr>
	</tbody>
</table>

a micropatterning assay for measuring cell chirality

Chirality is an intrinsic cellular property, which depicts the asymmetry in terms of polarization along the left-right axis of the cell. As this unique property attracts increasing attention due to its important roles in both development and disease, a standardized quantification method for characterizing cell chirality would advance research and potential applications. In this protocol, we describe a multicellular chirality characterization assay that utilizes micropatterned arrays of cells. Cellular micropatterns are fabricated on titanium/gold-coated glass slides via microcontact printing. After seeding on the geometrically defined (e.g., ring-shaped), protein-coated islands, cells directionally migrate and form a biased alignment toward either the clockwise or the counterclockwise direction, which can be automatically analyzed and quantified by a custom-written MATLAB program. Here we describe in detail the fabrication of micropatterned substrates, cell seeding, image collection, and data analysis and show representative results obtained using the NIH/3T3 cells. This protocol has previously been validated in multiple published studies and is an efficient and reliable tool for studying cell chirality in vitro.

Fifteen minutes after the seeding of NIH/3T3 cells, cell adhesion on the ring pattern was visually confirmed by phase-contrast imaging. After subsequent culture of 24 h, cells on the patterns became confluent and elongated with clearly asymmetrical alignments, biased towards the clockwise direction (Figure 2). Directional migration of attached cells is recorded by time-lapse imaging, cell motility and morphogenesis can be quantified with further analyses of the video. To conduct chirality an...

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A Micropatterning Assay for Measuring Cell Chirality

We present a protocol for determining multicellular chirality in vitro, using the micropatterning technique. This assay allows for automatic quantification of the left-right biases of various types of cells and can be used for screening purposes.

Chirality is an intrinsic cellular property, which depicts the asymmetry in terms of polarization along the left-right axis of the cell. As this unique ...

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Bioengineering

2.3K Views.  Rensselaer Polytechnic Institute. We present a protocol for determining multicellular chirality in vitro, using the micropatterning technique. This assay allows for automatic quantification of the left-right biases of various types of cells and can be used for screening purposes.

Video: A Micropatterning Assay for Measuring Cell Chirality

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Mechanical  properties of cells and extracellular matrix (ECM) play important roles in many  biological processes including stem cell differentiation, ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...