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/pubmed?term=((Cell+chirality%3A+emergence+of+asymmetry+from+cell+culture%5BTitle%5D)+AND+%22Philosophical+Transactions+of+the+Royal+Society+B%3A+Biological+Sciences%22%5BJournal%5D)">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/pubmed?term=((Cell+chirality+in+cardiovascular+development+and+disease%5BTitle%5D)+AND+%22APL+Bioengineering%22%5BJournal%5D)">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/pubmed?term=((Cell+chirality%3A+Its+origin+and+roles+in+left-right+asymmetric+development%5BTitle%5D)+AND+%22Philosophical+Transactions+of+the+Royal+Society+B%3A+Biological+Sciences%22%5BJournal%5D)">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/pubmed?term=((Cells+with+broken+left-right+symmetry%3A+Roles+of+intrinsic+cell+chirality+in+left-right+asymmetric+epithelial+morphogenesis%5BTitle%5D)+AND+%22Symmetry%22%5BJournal%5D)">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/pubmed?term=((Chiral+neuronal+motility%3A+The+missing+link+between+molecular+chirality+and+brain+asymmetry%5BTitle%5D)+AND+%22Symmetry%22%5BJournal%5D)">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/pubmed?term=((Cellular+chirality+arising+from+the+self-organization+of+the+actin+cytoskeleton%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Micropatterned+mammalian+cells+exhibit+phenotype-specific+left-right+asymmetry%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Cell+chirality+regulates+intercellular+junctions+and+endothelial+permeability%5BTitle%5D)+AND+%22Science+Advances%22%5BJournal%5D)">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/pubmed?term=((Carbon+nanotube-induced+loss+of+multicellular+chirality+on+micropatterned+substrate+is+mediated+by+oxidative+stress%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">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/pubmed?term=((Effects+of+Alzheimer%27s+disease-related+proteins+on+the+chirality+of+brain+endothelial+cells%5BTitle%5D)+AND+%22Cellular+and+Molecular+Bioengineering%22%5BJournal%5D)">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/pubmed?term=((Left-right+symmetry+breaking+in+tissue+morphogenesis+via+cytoskeletal+mechanics%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">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/pubmed?term=((Intrinsic+cellular+chirality+regulates+left-right+symmetry+breaking+during+cardiac+looping%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Cell+organelle-based+analysis+of+cell+chirality%5BTitle%5D)+AND+%22Communicative+and+Integrative+Biology%22%5BJournal%5D)">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/pubmed?term=((Geometric+control+of+cell+life+and+death%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Nanowire+magnetoscope+reveals+a+cellular+torque+with+left-right+bias%5BTitle%5D)+AND+%22ACS+Nano%22%5BJournal%5D)">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/pubmed?term=((Geometric+control+of+human+stem+cell+morphology+and+differentiation%5BTitle%5D)+AND+%22Integrative+Biology%22%5BJournal%5D)">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/pubmed?term=((Epithelial+cell+chirality+revealed+by+three-dimensional+spontaneous+rotation%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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>
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</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><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&#039;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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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

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Biology

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

A Novel Ink-Free, Label-Free Magnetic-Archimedes Cell Patterning Method

Cell Patterning Using Magnetic-Archimedes Strategy

Cell patterning, allowing precise control of cell positioning, presents a unique advantage in the study of cell behavior. In this protocol, a cell patterning strategy based on the Magnetic-Archimedes (Mag-Arch) effect is introduced. This approach enables precise control of cell distribution without the use of ink materials or labeling particles. By introducing a paramagnetic reagent to enhance the magnetic susceptibility of the cell culture medium, cells are repelled by magnets and arrange themselves into a pattern complementary to the magnet sets positioned beneath the microfluidic substrate.
In this article, detailed procedures for cell patterning using the Mag-Arch-based strategy are provided. Methods for patterning single-cell types as well as multiple cell types for co-culture experiments are offered. Additionally, comprehensive instructions for fabricating microfluidic devices containing channels for cell patterning are provided. Achieving this feature using parallel methods is challenging but can be done in a simplified and cost-effective manner. Employing Mag-Arch-based cell patterning equips researchers with a powerful tool for in vitro research.

Author Spotlight: Magnetic-Based Cell Patterning Method for High-Throughput Biomedical Applications 

This protocol describes an ink-free, label-free, substrate-independent, high-throughput cell patterning method based on the Magnetic Archimedes effect.

A Micropatterning Assay for Measuring Cell Chirality

Summary

Explore More Videos

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates

A Versatile Method of Patterning Proteins and Cells

Simple, Affordable, and Modular Patterning of Cells using DNA

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Pattern Generation for Micropattern Traction Microscopy

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

Cell Patterning Using Magnetic-Archimedes Strategy

A Micropatterning Assay for Measuring Cell Chirality

Summary

Explore More Videos

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates

A Versatile Method of Patterning Proteins and Cells

Simple, Affordable, and Modular Patterning of Cells using DNA

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Pattern Generation for Micropattern Traction Microscopy

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

Cell Patterning Using Magnetic-Archimedes Strategy

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates