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 Table of Materials) into 2 mm &#215; 8 mm &#215; 30 mm rectangles. Ensure that the latter two dimensions of these silicone plates are slightly smaller than those of the rectangular magnets mentioned above to optimize the assembly. The thickness of the silicone plates determines the width of the cell stripes.</li>
		<li>Assemble the rectangular magnets and the silicone plates layer by layer, as illustrated in Figure 1A. Each magnet set consists of 4 magnets and 3 silicone plates. Ensure that the poles of each adjacent magnet are opposite so that the magnet sets can automatically align due to the intrinsic attractive force.</li>
		<li>Sterilize the magnets before use. 
		NOTE: It is recommended to wash the magnet sets with pure water and then immerse them in 75% ethanol for 10 min. The overall dimensions of these magnet sets are 12 mm &#215; 10 mm &#215; 35 mm, designed to fit standard 6-well cell culture plates.</li>
	</ol>
	</li>
	<li>Assemble the magnet sets for dot-array patterns
	<ol>
		<li>Choose miniature cylindrical magnets, as shown in Figure 1B. The dimensions of the cylindrical magnets used for this demonstration are &#934;1.5 mm &#215; 10 mm (diameter &#215; height), magnetized in the axial direction.</li>
		<li>Assemble the cylindrical magnets as indicated in Figure 1B. Ensure that each magnet set consists of 36 cylindrical magnets arranged in a 6 &#215; 6 grid. Also, ensure that the poles of each adjacent magnet are opposite to allow the magnet sets to align automatically due to the intrinsic attractive force.</li>
		<li>Sterilize the magnets before use, following the same procedure as in step 1.1.4. The overall dimensions of these magnet sets are 9 mm &#215; 10 mm &#215; 9 mm, designed to fit standard 6-well cell culture plates.</li>
	</ol>
	</li>
</ol>
2. Cell patterning on glass slides
<ol>
	<li>Prepare the cell culture device.
	<ol>
		<li>Use 2 mm-thick silicone plates to create silicone molds. Employ a puncher to create a 1 cm &#215; 1 cm rectangular hole in the plate. Trim the silicone plate around the hole to craft a round mold (diameter = 25 mm) with the hole at the center, as depicted in Figure 1C.</li>
		<li>Thoroughly clean the silicone molds using an ultrasonic cleaner. Sterilize the molds by washing them with pure water for 10 min, followed by replacing the water with 75% ethanol for another 10 min. Finally, dry the molds in a sterilizer box at 65 &#176;C for 60 min.</li>
		<li>Attach the sterilized silicone mold to a &#934;25 mm round glass cell slide and gently press it so that the silicone adheres to the glass, as shown in Figure 1C. The resulting cell culture device will have a glass underside and a 200 &#181;L culture cavity. The dimensions of the device are &#934;25 mm &#215; 2.15 mm, making it suitable for standard 6-well cell culture plates. 
		NOTE: The cell culture device described above can be replaced with other Petri dishes with undersides thinner than 0.5 mm, such as confocal dishes.</li>
		<li>Place the magnet sets on a 6-well plate. Non-magnetic tweezers are recommended for ease of operation. 
		NOTE: All subsequent steps should be performed under sterile conditions from this point onward until the observation of cell patterns.</li>
		<li>Position the cell culture device on top of the magnet set in the well of the 6-well plate. Ensure that the cell culture device is placed horizontally, and the underside is in close contact with the magnet set.</li>
	</ol>
	</li>
	<li>Prepare the cell culture medium containing Gd-DTPA
	<ol>
		<li>Prepare commercially available Gd-DTPA injection (see Table of Materials), usually with a concentration of 500 mM. Dilute the injection by adding 30 &#181;L of Gd-DTPA injection to 470 &#181;L of complete cell culture medium to achieve a target concentration of 30 mM. 
		NOTE: Depending on local regulations and availability, other gadolinium-based contrast agents (GBCAs) with the same target concentration may yield similar results11.</li>
	</ol>
	</li>
	<li>Create the cell patterns.
	<ol>
		<li>Harvest cells for patterning. In this demonstration, human umbilical vein endothelial cells (HUVECs, see Table of Materials) were used for patterning (Figure 2). Centrifuge the cells for 5 min at 200 x g (at room temperature) to collect them from suspension and resuspend them in the Gd-DTPA-containing medium. Count the cell density.</li>
		<li>Adjust the cell density to approximately ~2 &#215; 105 cells/mL with Gd-DTPA-containing medium. Gently mix the cell suspension and add 200 &#181;L to the cavity of each cell culture mold to create a brim-full liquid surface.</li>
		<li>Carefully transfer the plate into a cell culture incubator and incubate for 3-6 h until the cells adhere well to the substrate. Avoid slamming the incubator door to prevent interference with the assembly of cell patterns. For cells with poor adhesion rates, overnight treatment with GBCAs is generally safe12.</li>
		<li>Cell patterning is considered complete when cells adhere to the bottom of the cavity of the cell culture device. Replace the Gd-DTPA-containing medium with complete culture medium.</li>
		<li>Remove the cell culture device from the magnet set. One can either observe the cell pattern immediately or transfer the device into a new culture plate for further cultivation.</li>
	</ol>
	</li>
</ol>
3. Co-culture patterning by magnet sideways: fabrication of the moving template
NOTE: The following procedure is presented to take advantage of Mag-Arch-based cell patterning and explore the possibility of more applications.
<ol>
	<li>Prepare the device for stripe cell patterning following step 1 and step 2. However, reduce the cell density to 1 &#215; 105 cells/mL and replace the silicone plates that separate the magnets with thinner ones, making each stripe cell pattern thinner. 
	NOTE: This provides sufficient area for laying multiple cells in stripe patterns. As a reference, we suggest using 1 mm silicone plates instead of 2 mm to separate the magnets.</li>
	<li>Pattern the first type of cells as illustrated in steps 2.2-2.3. This step also requires 3-6 h for cell attachment. 
	NOTE: To distinguish different cell types in the co-culture system, users may label cells with fluorescent dyes such as DiI, DiD, or cell trackers (CMTPX, CMFDA, CMAC, etc.) before patterning. For example, for DiI and DiD staining, add 1 &#181;L of the stock solution (10 mM) (see Table of Materials) per 1 mL of FBS-free medium and mix well to obtain a working solution. Replace the culture medium with the working solution to cover adherent cells. After incubating for 30 min and washing with PBS three times, proceed with cell harvesting.</li>
	<li>After patterning the first type of cells, move the cell culture device 1 mm from the magnets below to the right, as illustrated in Figure 3Aii.</li>
	<li>Gently wash the glass slides with 200 &#181;L of PBS twice. Avoid letting the slides dry.</li>
	<li>Pattern the second type of cells in the same manner as illustrated in steps 2.2-2.3.</li>
	<li>Move the cell culture device 1 mm from the magnets below to the right, as illustrated in Figure 3Aiii, and wash the glass slides with 200 &#181;L of PBS again. Pattern the third type of cells in the same manner as illustrated in steps 2.2-2.3.</li>
	<li>After patterning all types of cells, gently wash the glass slides with 200 &#181;L of PBS twice and replace with complete cell culture medium.</li>
	<li>Remove the cell culture device from the magnet set. One can then observe the cell pattern or transfer the device to a new culture plate for further cultivation.</li>
</ol>
4. Co-culture patterning by adjusting the concentration of Gd-DTPA
NOTE: GBCAs do not significantly affect cell adhesion or subsequent growth at working concentrations (&#8804;75 mM). Additionally, cell patterns are influenced by the concentration of Gd-DTPA: higher concentrations result in smaller/thinner cell patterns. Thus, it is possible to create co-culture systems by simply adjusting the concentration of Gd-DTPA. This example demonstrates patterning concentric circular arrays.
<ol>
	<li>Pattern the first type of cells as illustrated in steps 2.2-2.3 using miniature cylindrical magnets from step 1.2. Label the cells with fluorescent dyes such as DiI, DiD, or cell trackers (see step 3) before harvesting them to distinguish different cell types in the co-culture system. 
	NOTE: One will need a higher concentration of Gd-DTPA (50 mM instead of 30 mM) and a lower cell density, approximately 1 &#215; 105 cells/mL.</li>
	<li>After patterning the first cell array, gently wash the glass slides with 200 &#181;L of PBS twice. Avoid allowing the slides to dry.</li>
	<li>Pattern the second type of cells following the same procedure as before. Keep the glass slides relatively motionless on the magnets to prevent dislocation. Attaching a silicone plate with a hollow that fits the magnet sets to the undersurface of the glass slide is recommended. 
	NOTE: Here, one will need a lower concentration of Gd-DTPA (20 mM instead of 30 mM) so that the second cell patterns are expected to be larger than the first, as illustrated in Figure 3B.</li>
	<li>After patterning all types of cells, gently wash the glass slides with 200 &#181;L of PBS twice and replace the medium with complete cell culture medium.</li>
	<li>Remove the cell culture device from the magnet set. One can then observe the cell pattern or transfer the device to a new culture plate for further cultivation.</li>
</ol>
5. Cell patterning in microfluidic chip
NOTE: The Mag-Arch-based method has been demonstrated to work in enclosed narrow chambers in our previous study8. Here is an example of patterning dot arrays in a microfluidic channel.
<ol>
	<li>Fabrication of the microfluid chip
	<ol>
		<li>Customize 40 mm &#215; 75 mm &#215; 20 mm Polytetrafluoroethylene (PTFE) molds (see Table of Materials) containing a rectangular cavity of 24 mm &#215; 50 mm &#215; 8 mm, as illustrated in Figure 4A.</li>
		<li>Flatten a 0.5 mm thick silicone plate. Cut the silicone plates according to the shape of the fluid channel, which has a rectangular cavity of 15 mm &#215; 20 mm &#215; 0.5 mm with triangular buffer areas on both the inlet and outlet sides, as illustrated in Figure 4A,B.</li>
		<li>Customize 40 mm &#215; 75 mm rectangular glass plates. Clean the glass plates with air plasma for 30 s.</li>
		<li>Immediately after air plasma cleaning, cover the glass plates with 0.5 mL of a commercially available anti-adhesion buffer (see Table of Materials) and allow them to air dry. Wash the glass plates once with ethanol and let them air dry.</li>
		<li>Attach a silicone plate prepared in step 5.1.2 securely to the middle of a glass plate, as illustrated in Figure 4A.</li>
		<li>Prepare polydimethylsiloxane (PDMS) prepolymer according to the manufacturer&#39;s instructions (see Table of Materials). For each PDMS chip, weigh 10 g of the base constituent and 1 g of the firming agent into a 50 mL centrifuge tube.</li>
		<li>Mix the agents thoroughly with a glass stirring rod until small bubbles are uniformly distributed in the colloid. The mixing takes 5-10 min, depending on the colloid volume. Centrifuge the colloid prepolymer for 1 min at 500 x g (at room temperature) to eliminate bubbles.</li>
		<li>Slowly pour ~10 mL of the prepolymer into the cavity to fill the PTFE mold.</li>
		<li>Carefully place the mold into a vacuum reservoir and keep it horizontal to prevent the prepolymer from flowing away.</li>
		<li>Remove residual air bubbles from the prepolymer with a vacuum pump. The vacuuming takes 120-180 min.</li>
		<li>If needed, pour more prepolymer to further fill the mold cavity. Vacuum for another 60 min.</li>
		<li>Carefully cover the PTFE mold with the glass plate, with the silica side facing the cavity, as illustrated in Figure 4Bi.</li>
		<li>Ensure that all bubbles are eliminated. Transfer the mold to a 60 &#176;C drying oven for the prepolymer to solidify overnight.</li>
		<li>Remove the mold from the oven. After cooling, demold the solidified PDMS cover carefully. Create an inlet and an outlet with a &#934;1 mm needle puncher.</li>
		<li>Wash the PDMS cover and 24 mm &#215; 50 mm cover slides according to step 2.1.2. 
		NOTE: Proceed with the following steps under sterile conditions.</li>
		<li>Attach a PDMS cover and a cover slide to create a microfluid chip containing a flow channel of approximately ~500 &#181;m in height. The bottom side should consist of a ~0.15 mm glass cover slide to enable cell patterning with the Mag-Arch-based strategy.</li>
	</ol>
	</li>
	<li>Assemble commercially available sterile adapters into both the inlet and outlet of the microfluid chip8.</li>
	<li>Prepare a 2% (w/v, dissolved in PBS) gelatin coating buffer. Sterilize the buffer by autoclaving.</li>
	<li>Inject the gelatin coating buffer into the chip via the inlet adapter using a 1 mL syringe. If bubbles emerge, flush them out with extra coating buffer.</li>
	<li>Place the chip into a 10 cm cell culture dish. Add 1 mL of PBS around the dish bottom to avoid drying. Transfer the dish to a cell culture incubator. The coating takes 30 min.</li>
	<li>Flush out the coating buffer with 2 mL of Gd-contained medium (30 mM) via the inlet.</li>
	<li>Gently inject 1 mL of cell suspension containing 30 mM Gd-DTPA with a 1 mL syringe.</li>
	<li>Pattern cells as illustrated in steps 2.2-2.3 using miniature cylindrical magnets from step 1.2. 
	NOTE: However, attaching a silicone plate with a hollow that fits the magnet sets to the undersurface of the glass slide is recommended, as shown in Figure 4Ci.</li>
	<li>After patterning, gently refresh the Gd-DTPA-contained medium with 1 mL of complete medium by flushing with a 1 mL syringe. 
	NOTE: The process of cell patterning in microfluidics, from steps 5.7-5.9, takes about 180 min, which is similar to cell patterning on standard glass slides.</li>
	<li>Quantify the patterns under a microscope. If needed, connect the microfluid chip to a circulating culture device for further cultivation. 
	NOTE: In our experience, cells are able to grow for at least 72 h when supported by a simple micropump-based device, which provides the microfluidics with a continuous flow of fresh culture medium in a cell incubator8.</li>
</ol>

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

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The authors have no competing financial interests.

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.

Cell patterning, allowing precise control of cell positioning, presents a unique advantage in the study of cell behavior. In this protocol, a cell ...

cell-patterning-using-magnetic-archimedes-strategy

Research

JoVE Journal

Bioengineering

Assembling Magnet Sets and Conducting Multilayered Cell Patterning Using Magnetic Fields

1.3K Views.  Zhejiang University School of Medicine.  This protocol describes an ink-free, label-free, substrate-independent, high-throughput cell patterning method based on the Magnetic Archimedes effect.

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

Summary