Name: a micropatterning assay for measuring cell chirality
Uploaded: 2022-03-11T13:00:00
Description: Watch this Scientific Journal Video about A Micropatterning Assay for Measuring Cell Chirality at JoVE.com

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 d...

<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 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...

Watch this Scientific Journal Video about A Micropatterning Assay for Measuring Cell Chirality at JoVE.com

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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