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><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 &amp;times; 50 mm</td></tr><tr><td>DiD</td><td>MedChemExpress (MCE)&amp;nbsp;</td><td>HY-D1028</td><td>For labeling cells with red fluorescence (Ex: 640 nm)</td></tr><tr><td>DiI</td><td>MedChemExpress (MCE)&amp;nbsp;</td><td>HY-D0083&amp;nbsp;</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&amp;nbsp;</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 &amp;times; 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...

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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

Universidad de Cartagena UDEC

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 

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

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

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

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

Biology

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

Chemistry

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 ...

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro when stem cells are spatially confined onto micropatterns, thus setting up experimental models which offer unique opportunities to identify, in vitro, the fundamental principles of biological organisation.
Here we describe our own implementation of the methodology. We adapted a photo-patterning technique to reduce the need for specialized equipment to make it easier to establish the method in a standard cell biology laboratory. We also developed a free, open-source and easy to install image analysis framework in order to precisely measure the preferential positioning of sub-populations of cells within colonies of standard shapes and sizes. This method makes it possible to reveal the existence of patterning events even in seemingly disorganized populations of cells. The technique provides quantitative insights and can be used to decouple influences of the environment (e.g., physical cues or endogenous signaling), on a given patterning process.

The method presented here uses micropatterning together with quantitative imaging to reveal spatial organization within mammalian cultures. The technique is easy to establish in a standard cell biology laboratory and offers a tractable system to study patterning in vitro.

A fundamental goal in biology is to understand how patterns emerge during development. Several groups have shown that patterning can be achieved in vitro ...

Developmental Biology

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, including studies of microbial physiology and interactions. At the simplest level, accurate spatial patterning of microorganisms would enable reliable, long-term imaging of large numbers of individual cells and transform the ability to quantitatively study distance-dependent microbe-microbe interactions. More uniquely, coupling accurate spatial patterning and full control over environmental conditions, as offered by microfluidic technology, would provide a powerful and versatile platform for single-cell studies in microbial ecology. 
This paper presents a microfluidic platform to produce versatile and user-defined patterns of microorganisms within a microfluidic channel, allowing complete optical access for long-term, high-throughput monitoring. This new microfluidic technology is based on capillarity-assisted particle assembly and exploits the capillary forces arising from the controlled motion of an evaporating suspension inside a microfluidic channel to deposit individual microsized objects in an array of traps microfabricated onto a polydimethylsiloxane (PDMS) substrate. Sequential depositions generate the desired spatial layout of single or multiple types of micro-sized objects, dictated solely by the geometry of the traps and the filling sequence. 
The platform has been calibrated using colloidal particles of different dimensions and materials: it has proven to be a powerful tool to generate diverse colloidal patterns and perform surface functionalization of trapped particles. Furthermore, the platform was tested on microbial cells, using Escherichia coli cells as a model bacterium. Thousands of individual cells were patterned on the surface, and their growth was monitored over time. In this platform, the coupling of single-cell deposition and microfluidic technology allows both geometric patterning of microorganisms and precise control of environmental conditions. It thus opens a window into the physiology of single microbes and the ecology of microbe-microbe interactions, as shown by preliminary experiments.

We present a technology that uses capillarity-assisted assembly in a microfluidic platform to pattern micro-sized objects suspended in a liquid, such as bacteria and colloids, into prescribed arrays on a polydimethylsiloxane substrate.

Controlled patterning of microorganisms into defined spatial arrangements offers unique possibilities for a broad range of biological applications, ...

Engineering

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 ...

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons

The ability to direct neurons into organized neural networks has great implications for regenerative medicine, tissue engineering, and bio-interfacing. Many studies have aimed at directing neurons using chemical and topographical cues. However, reports of organizational control on a micron-scale over large areas are scarce. Here, an effective method has been described for placing neurons in preset sites and guiding neuronal outgrowth with micron-scale resolution, using magnetic platforms embedded with micro-patterned, magnetic elements. It has been demonstrated that loading neurons with magnetic nanoparticles (MNPs) converts them into sensitive magnetic units that can be influenced by magnetic gradients. Following this approach, a unique magnetic platform has been fabricated on which PC12 cells, a common neuron-like model, were plated and loaded with superparamagnetic nanoparticles. Thin films of ferromagnetic (FM) multilayers with stable perpendicular magnetization were deposited to provide effective attraction forces toward the magnetic patterns. These MNP-loaded PC12 cells, plated and differentiated atop the magnetic platforms, were preferentially attached to the magnetic patterns, and the neurite outgrowth was well aligned with the pattern shape, forming oriented networks. Quantitative characterization methods of the magnetic properties, cellular MNP uptake, cell viability, and statistical analysis of the results are presented. This approach enables the control of neural network formation and improves neuron-to-electrode interface through the manipulation of magnetic forces, which can be an effective tool for in vitro studies of networks and may offer novel therapeutic biointerfacing directions.

This work presents a bottom-up approach to the engineering of local magnetic forces for control of neuronal organization. Neuron-like cells loaded with magnetic nanoparticles (MNPs) are plated atop and controlled by a micro-patterned platform with perpendicular magnetization. Also described are magnetic characterization, MNP cellular uptake, cell viability, and statistical analysis.

The ability to direct neurons into organized neural networks has great implications for regenerative medicine, tissue engineering, and bio-interfacing. ...

Pattern Generation for Micropattern Traction Microscopy

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

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

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

Stimulation of Patterned Neuronal Cultures by a Magnetic Field

Source: Stern, S., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54357">External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures.</a> J. Vis. Exp. (2017)
This video demonstrates a method for stimulating neuronal cultures grown in a patterned circular ring on a coverslip, using a time-varying magnetic field.

1. Preparation of coverslips for 1D cultures.

	Clean glass coverslips by immersion in a base piranha solution consisting of 75 mL DDW, 25 mL 25% ammonia ...

Cell Patterning Using Magnetic-Archimedes Strategy

Summary

Explore More Videos

Chapters in this video

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

A Versatile Method of Patterning Proteins and Cells

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons

Pattern Generation for Micropattern Traction Microscopy

Stimulation of Patterned Neuronal Cultures by a Magnetic Field

Cell Patterning Using Magnetic-Archimedes Strategy

Summary

Explore More Videos

Chapters in this video

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates

Mapping the Emergent Spatial Organization of Mammalian Cells using Micropatterns and Quantitative Imaging

Patterning of Microorganisms and Microparticles through Sequential Capillarity-assisted Assembly

A Versatile Method of Patterning Proteins and Cells

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons

Pattern Generation for Micropattern Traction Microscopy

Stimulation of Patterned Neuronal Cultures by a Magnetic Field

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates