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

<ol>
	<li>Christian, 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=Control+of+cell+adhesion+using+hydrogel+patterning+techniques+for+applications+in+traction+force+microscopy.">Control of cell adhesion using hydrogel patterning techniques for applications in traction force microscopy.</a> J Vis Exp. 179, e63121 (2022).</li><li>Abbas, Y., Turco, M. Y., Burton, G. 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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levitational+3D+bioassembly+and+density-based+spatial+coding+of+levitoids.">Levitational 3D bioassembly and density-based spatial coding of levitoids.</a> Adv Funct Mater. 32 (50), 2204092 (2022).</li><li>Parfenov, V. A., 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+levitational+bioassembly+of+3D+tissue+construct+in+space.">Magnetic levitational bioassembly of 3D tissue construct in space.</a> Sci Adv. 6 (29), eaba4174 (2020).</li><li>Dell, A. C., Wagner, G., Own, J., Geibel, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+using+hydrogels:+Cell+inks+and+tissue+engineering+applications.">3D bioprinting using hydrogels: Cell inks and tissue engineering applications.</a> Pharmaceutics. 14 (12), 2596 (2022).</li><li>Ino, K., Ito, A., Honda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+patterning+using+magnetite+nanoparticles+and+magnetic+force.">Cell patterning using magnetite nanoparticles and magnetic force.</a> Biotechnol Bioeng. 97 (5), 1309-1317 (2007).</li><li>Okochi, M., Matsumura, T., Honda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+force-based+cell+patterning+for+evaluation+of+the+effect+of+stromal+fibroblasts+on+invasive+capacity+in+3d+cultures.">Magnetic force-based cell patterning for evaluation of the effect of stromal fibroblasts on invasive capacity in 3d cultures.</a> Biosens Bioelectron. 42, 300-307 (2013).</li><li>Mishriki, 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=Rapid+magnetic+3D+printing+of+cellular+structures+with+mcf-7+cell+inks.">Rapid magnetic 3D printing of cellular structures with mcf-7 cell inks.</a> Research (Wash D C). 2019, 9854593 (2019).</li><li>Ozturk-Oncel, M. O., Leal-Martinez, B. H., Monteiro, R. F., Gomes, M. E., Domingues, R. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dive+into+the+bath:+Embedded+3D+bioprinting+of+freeform+in+vitro+models.">A dive into the bath: Embedded 3D bioprinting of freeform in vitro models.</a> Biomater Sci. 11, 5462-5473 (2023).</li><li>Sahni, G., Yuan, J., Toh, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stencil+micropatterning+of+human+pluripotent+stem+cells+for+probing+spatial+organization+of+differentiation+fates.">Stencil micropatterning of human pluripotent stem cells for probing spatial organization of differentiation fates.</a> J Vis Exp. 112, e54097 (2016).</li><li>Joddar, B., 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=Engineering+approaches+for+cardiac+organoid+formation+and+their+characterization.">Engineering approaches for cardiac organoid formation and their characterization.</a> Transl Res. 250, 46-67 (2022).</li></ol>

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.

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

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.

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 ...

cell-patterning-using-magnetic-archimedes-strategy

Research

JoVE Journal

Bioengineering

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.

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

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

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 ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one particular implementation of the MFP, the hierarchical hydrodynamic flow confinement (hHFC), multiple liquids are simultaneously brought in contact with a substrate. Local chemical action and liquid shaping using the hHFC, is exploited to create cell patterns by locally lysing and removing cells. By utilizing the scanning ability of the MFP, user-defined patterns of cell monolayers are created. This protocol enables rapid, real-time and spatially controlled cell patterning, which can allow selective cell-cell and cell-matrix interaction studies.

We present a protocol to perform subtractive patterning of live cell monolayers on a surface. This is achieved by local and selective lysis of adherent cells using a microfluidic probe (MFP). The cell lysate retrieved from local regions can be used for downstream analysis, enabling molecular profiling studies.

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

A Microfluidic System with Surface Patterning for Investigating Cavitation Bubble(s)&#8211;Cell Interaction and the Resultant Bioeffects at the Single-cell Level

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass substrate, that precisely controls the generation of tandem bubbles and individual cells patterned nearby with well-defined locations and shapes. We then demonstrate the generation of tandem bubbles by using two pulsed lasers illuminating a pair of gold dots with a few-microsecond time delay. We visualize the bubble-bubble interaction and jet formation by high-speed imaging and characterize the resultant flow field using particle image velocimetry (PIV). Finally, we present some applications of this technique for single cell analysis, including cell membrane poration with macromolecule uptake, localized membrane deformation determined by the displacements of attached integrin-binding beads, and intracellular calcium response from ratiometric imaging. Our results show that a fast and directional jetting flow is produced by the tandem bubble interaction, which can impose a highly localized shear stress on the surface of a cell grown in close proximity. Furthermore, different bioeffects can be induced by altering the strength of the jetting flow by adjusting the standoff distance from the cell to the tandem bubbles.

A microfluidic chip was fabricated to produce pairs of gold dots for tandem bubble generation and fibronectin-coated islands for single-cell patterning nearby. The resultant flow field was characterized by particle image velocimetry and was employed to study various bioeffects, including cell membrane poration, membrane deformation, and intracellular calcium response.

In this manuscript, we first describe the fabrication protocol of a microfluidic chip, with gold dots and fibronectin-coated regions on the same glass ...

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 ...