This study is financially supported by the National Key R&#38;D Program of China (2021YFA1101100), the National Natural Science Foundation of China (32000971), the Fundamental Research Funds for the Central Universities (No. 2021FZZX001-42), and the Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study (Grant No. SN-ZJU-SIAS-004).

1. Assembling the magnet sets
<ol>
	<li>Assemble the magnet sets for strip patterns.
	<ol>
		<li>Choose flat rectangular magnets, as depicted in Figure 1A. The dimensions of the rectangular magnets used for this demonstration are 1.5 mm &#215; 10 mm &#215; 35 mm (thickness &#215; height &#215; length) (see Table of Materials). The thickness of the magnets determines the gaps between the cell stripes.</li>
		<li>Cut 2 mm-thick silicone plates (see T...

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

<ol>
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J., Moffett, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+human+trophoblast+invasion+in+vitro.">Investigation of human trophoblast invasion in vitro.</a> Hum Reprod Update. 26 (4), 501-513 (2020).</li><li>Park, M., 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=Modulation+of+heterotypic+and+homotypic+cell-cell+interactions+via+zwitterionic+lipid+masks.">Modulation of heterotypic and homotypic cell-cell interactions via zwitterionic lipid masks.</a> Adv Healthc Mater. 6 (15), 1700063 (2017).</li><li>Ren, T., Chen, P., Gu, L., Ogut, M. G., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+ring-shaped+cellu-robots+with+simultaneous+locomotion+in+batches.">Soft ring-shaped cellu-robots with simultaneous locomotion in batches.</a> Adv Mater. 32 (8), e1905713 (2020).</li><li>Ren, T., Steiger, W., Chen, P., Ovsianikov, A., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+cell+packing+in+buckyballs+by+acoustofluidic+activation.">Enhancing cell packing in buckyballs by acoustofluidic activation.</a> Biofabrication. 12 (2), 025033 (2020).</li><li>Ge, 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=Magnetic+levitation+in+chemistry,+materials+science,+and+biochemistry.">Magnetic levitation in chemistry, materials science, and biochemistry.</a> Angew Chem Int Ed Engl. 59 (41), 17810-17855 (2020).</li><li>Puluca, N., 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=Levitating+cells+to+sort+the+fit+and+the+fat.">Levitating cells to sort the fit and the fat.</a> Adv Biosyst. 4 (6), e1900300 (2020).</li><li>Ren, T., 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=Programing+cell+assembly+via+ink-free,+label-free+magneto-archimedes+based+strategy.">Programing cell assembly via ink-free, label-free magneto-archimedes based strategy.</a> ACS Nano. 17 (13), 12072-12086 (2023).</li><li>Li, Y. C., 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=Programmable+laser-assisted+surface+microfabrication+on+a+poly(vinyl+alcohol)-coated+glass+chip+with+self-changing+cell+adhesivity+for+heterotypic+cell+patterning.">Programmable laser-assisted surface microfabrication on a poly(vinyl alcohol)-coated glass chip with self-changing cell adhesivity for heterotypic cell patterning.</a> ACS Appl Mater Interfaces. 7 (40), 22322-22332 (2015).</li><li>Chliara, M. A., Elezoglou, S., Zergioti, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioprinting+on+organ-on-chip:+Development+and+applications.">Bioprinting on organ-on-chip: Development and applications.</a> Biosensors (Basel). 12 (12), 1135 (2022).</li><li>Moncal, K. K., Yaman, S., Durmus, N. 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The Mag-Arch-based cell patterning provides a user-friendly solution for most biomedical laboratories. This method advances parallel to characters of ink-free, label-free, substrate-independent, and the ability for high-throughput patterning8,13. For mono-type cell patterning, it patterns cells in a one-step way. The procedure finishes simply by refreshing culture mediums.
Previous studies have used magnetic particles to label cells an...

Cell patterning is evolving into an intuitive and powerful technology for in vitro studies1. By manipulating cell positions in culture plates, it provides solutions for a variety of experiments, including cell migration2, biomimetic multicellular co-culture3, organoid assembly4, biomaterial studies5, and more. In most situations, an ink-free, label-free method is preferred for cell patterning because it offers ease of operation and high cell viability for subsequent investigations.
The Mag-Arch effect is a physical phenomenon wherein diamagnetic objects in paramagnetic liquids tend to move toward regions with weak magnetic fields6. Living cells are naturally diamagnetic, while cell culture media can be made paramagnetic by adding soluble paramagnetic elements, such as gadopentetate dimeglumine (Gd-DTPA), commonly used intravenously in nuclear magnetic resonance imaging as a contrast agent7. Consequently, cells are expected to be repelled by the surrounding paramagnetic medium and move toward regions where magnetic fields are weaker8. A patterned magnetic field can be easily generated using a set of neodymium magnets. Ideally, cell patterns are assembled in opposition to the magnet patterns. Technically, this is defined as a label-free method because the only additional reagent, Gd-DTPA, remains in the extracellular environment and does not bind to cells. Thus, potential influences on subsequent cell culture can be easily avoided by replacing the culture medium. Compared to other methods1,3,9,10, the Mag-Arch-based strategy does not require bio-ink components or the application of specific particles to positively label the cells. Furthermore, it has been shown to work on multiple substrates for cell adhesion and is capable of high-throughput cell patterning4.
This article presents a detailed protocol for cell patterning using the Mag-Arch-based method, covering everything from device fabrication to adjusting the cell pattern. In addition to the patterns we have demonstrated, users can easily create various cell patterns using magnets and Gd-DTPA solution. Furthermore, protocols for assembling complex co-culture patterns and manipulating cells in enclosed microfluidic chips, are also provided.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A2780 ovarian cancer cells</td>
			<td>Procell</td>
			<td>CL-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture medium (DMEM, high glucose)</td>
			<td>Gibco</td>
			<td>11995040</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slides</td>
			<td>Citotest Scientific</td>
			<td>80340-3610</td>
			<td>For fabricating microfluidics. Dimension: 24 mm &#215; 50 mm</td>
		</tr>
		<tr>
			<td>DiD</td>
			<td>MedChemExpress (MCE)&#160;</td>
			<td>HY-D1028</td>
			<td>For labeling cells with red fluorescence (Ex: 640 nm)</td>
		</tr>
		<tr>
			<td>DiI</td>
			<td>MedChemExpress (MCE)&#160;</td>
			<td>HY-D0083&#160;</td>
			<td>For labeling cells with orange fluorescence (Ex: 550 nm)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Biochannel</td>
			<td>BC-SE-FBS07</td>
			<td></td>
		</tr>
		<tr>
			<td>Gadopentetate dimeglumine (Gd-DTPA)</td>
			<td>Beijing Beilu Pharmaceutical&#160;</td>
			<td>H10860002</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma Aldrich</td>
			<td>V900863</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cell slides</td>
			<td>Citotest Scientific</td>
			<td>80346-2510</td>
			<td>Diameter: 25 mm; thickness: 0.19-0.22 mm</td>
		</tr>
		<tr>
			<td>Glass plates</td>
			<td>PURESHI hardware store</td>
			<td></td>
			<td>For fabricating microfluidics. Dimension: 40 mm &#215; 75 mm</td>
		</tr>
		<tr>
			<td>Human Umbilical Vein Endothelial Cells (HUVECs)</td>
			<td>Servicebio</td>
			<td>STCC12103G-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Neodymium-iron-boron magnets (N52)</td>
			<td>Lalaci</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Non-toxic glass plate coating (Gel Slick Solution)</td>
			<td>Lonza</td>
			<td>1049286</td>
			<td>For convenience of demolding when fabricating microfluidics</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Servicebio</td>
			<td>G4200</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>SANHOPTT</td>
			<td>PT-2S</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS) kit</td>
			<td>DOWSIL</td>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>For fabricating microfluidics</td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene (PFTE) mold</td>
			<td>PURESHI hardware store</td>
			<td></td>
			<td>Customized online, for fabricating microfluidics</td>
		</tr>
		<tr>
			<td>Silicon plate</td>
			<td>PURESHI hardware store</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Smooth Muscle Cells (SMC)</td>
			<td>Procell</td>
			<td>CL-0517</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>Sapeen</td>
			<td>CSA-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 

Cell Patterning Using Magnetic-Archimedes Strategy

Cell patterning is intuitive and powerful technology for in vitro studies. Our research aims to present a versatile, user-friendly, and inexpensive cell pattern method, which could be replicated in most biomedical laboratories. Compared to other methods, our protocol doesn't require specialized skills and equipment.Also, it advances parallel to characters of ink-free, label-free, substrate-independent, and the ability for high-throughput patterning. The protocol requires only two necessities:magnets and the gadolinium-based contrast agents, which are both easy to get at low prices. The magnetic-based cell pattern method provides convenient solutions for the first step of a variety of experiments, including cell migration, biomimetic multicellular co-culture, organoid assembly, biomaterial studies, and more.

Rectangular (1.5 mm &#215; 10 mm &#215; 35 mm) and cylindrical (&#934;1.5 m &#215; 10 mm) magnets were selected to create cell patterns as a demonstration. Users have the flexibility to modify the size and shape of magnets or assemble them differently to create diverse cell patterns. In Figure 1A,B, the magnets were assembled, with the magnetic poles depicted in blue (south) and red (north) for clarity. In this configuration, magnets attract each other laterally and align th...

Watch this Scientific Journal Video about Magnetic-Based Cell Patterning Method for High-Throughput Biomedical Applications at JoVE.com

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