Name: A Versatile Method of Patterning Proteins and Cells (Video) | JoVE Journal
Uploaded: 2017-02-26T15:00:00.000+00:00
Duration: 9 min 57 s
Description: Learn to pattern cells and substrates in microfluidic channels using low pressure vacuum and simple benchtop tools. This versatile method enables studies of cell-to-cell and cell-to-protein interactions in biological research.

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5 minutes

Funding for this research was provided by the New Jersey Commission on Spinal Cord Research (NJCSCR) (to FHK), grant CSCR14IRG005 (to BLF), NIH grant R15NS087501 (to CHC), and the F.M. Kirby Foundation (to ETA).

NOTE: Please consult all relevant material safety data sheets (MSDS) before use. Some of the chemicals used in this protocol are toxic and carcinogenic. Please use all appropriate safety practices (fume hood, glovebox) and personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes) when using toxic or acid/base materials.
1. Fabrication of Master Molds for Soft Lithography using Photolithography
<ol>
	<li>Draw the layout of the microchannel using a computer-aided design (CAD) drawing tool.</li>
	<li>Print the layout on a blank mask plate using laser mask writer.</li>
	<li>Rinse a 4 inch silicon wafer with acetone followed by isopropanol and dry with a nitrogen air gun to ensure that no solvent is left on the wafer.</li>
	<li>Dehydrate the wafer by baking it on a hot plate at 200 &#176;C for 5 min.</li>
	<li>Select a negative photoresist designed for the desired height of the microchannels. For example, use negative photoresist SU-8 50 to obtain microchannels with a height of 50 &#181;m.</li>
	<li>Allow the wafer to cool to room temperature, and then align the wafer on the chuck of a spin coater.</li>
	<li>Dispense 4 mL (1 mL/inch diameter of the wafer) of negative photoresist onto the center of the wafer.</li>
	<li>Spin coat the wafer with a two-step spin cycle using a spinner. First, apply a spread cycle of 500 rpm with an acceleration of 100 rpm/s for 10 s. Then apply a spin cycle of 2,000 rpm with an acceleration of 300 rpm/s2 for 30 s to obtain a 50 &#181;m coating of photoresist on a wafer.</li>
	<li>Soft bake the wafer in two steps on a hot plate according to the manufacturer&#39;s directions. For a 50 &#181;m coating of photoresist, first pre-bake the wafer at 65 &#176;C for 6 min and then immediately ramp up the hot plate temperature to post-bake the wafer at 95 &#176;C for 20 min.</li>
	<li>Allow the wafer to cool to room temperature and then load the wafer onto the lithography stage.</li>
	<li>Align the mask on the wafer using mask aligner and expose the wafer to UV light (as per the manufacturer&#39;s instructions) at an intensity of 1.5 mW/cm2 for 146 s to apply the total UV dose of 220 mJ/cm2 required for a 50 &#181;m thick layer of photoresist.</li>
	<li>Apply post exposure bake to the wafer in two steps on a hot plate. For a 50-&#181;m coating of photoresist, first pre-bake the wafer at 65 &#176;C for 1 min and immediately ramp up the hot plate temperature to post-bake it at 95 &#176;C for 5 min.</li>
	<li>Develop the wafer in by submerging the wafer in SU-8 developer and gently shaking until the features become clear on the wafer. Develop the wafer for ~6 min for the 50 &#181;m thick photoresist.</li>
	<li>Rinse off excess developer with isopropanol and ethanol, then dry the wafer with a nitrogen air gun.</li>
	<li>Hard bake the silicon wafer on a hot plate at 150 &#176;C for 15 min.</li>
	<li>Place the wafer in a desiccator with 25 &#181;L of the silanizing agent tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane. 
	NOTE: The silanizing agent is corrosive, thus should be used in an acid/base fume hood, with appropriate personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes).</li>
	<li>Apply the vacuum for 5 min and expose the wafer to the vapors of silane for 30 min without releasing the vacuum.</li>
	<li>Transfer the wafer into a suitably sized Petri dish.</li>
</ol>
2. Fabrication of Master Molds for Soft Lithography Using Adhesive Tape
<ol>
	<li>Draw the layout of the microchannel using a CAD drawing tool and print the drawing on white paper.</li>
	<li>Clean a glass slide large enough to accommodate the design with isopropanol and then dry the slide with an air gun.</li>
	<li>Attach adhesive tape onto the cleaned glass slide. Be careful not to trap any air bubbles.</li>
	<li>Tape the sides of the glass slide onto the paper with the design, with the tape side up.</li>
	<li>Use a scalpel to cut the tape on the glass slide using the paper design as a reference and then peel off tape from unwanted areas of the glass slide.</li>
	<li>Rinse the glass slide with isopropanol and then dry with an air gun. Bake the glass slide in an oven at 65 &#176;C for 30 min.</li>
	<li>Gently roll over the tape with a rubber roller to remove air bubbles and allow the slide to cool to room temperature.</li>
</ol>
3. Soft Lithography Fabrication of the PDMS Devices
<ol>
	<li>Mix PDMS elastomer and its curing agent in a ratio of 10:1 (w:w), stir the mixture vigorously, and then degas the mixture by placing it into a vacuum chamber until no air bubbles appear in the mixture.</li>
	<li>Pour the mixture onto a silanized silicon mold or adhesive tape mold in a petri dish to obtain a ~2 mm thick layer of PDMS and degas until all air bubbles disappear.</li>
	<li>Cure the PDMS in an oven at 65 &#176;C for 2 h.</li>
	<li>Use a scalpel to cut the PDMS layer at least 5 mm away from the features and then peel the cured PDMS off the mold using tweezer.</li>
	<li>Punch a single inlet hole with a 1 mm biopsy punch anywhere on the microchannel, preferably at the end of a network of microchannels as seen in Figure 2a and 4b.</li>
	<li>Clean the PDMS cast with adhesive tape to remove dust particles adsorbed onto the device. Sterilize the PDMS microchannel device (PDMS cast) by rinsing with a solution of 70% ethanol followed by sterile deionized (DI) water, and expose to UV for 30 min.</li>
	<li>Keep the sterile PDMS device in a sterile petri dish until use.</li>
</ol>
4. Substrate Patterning
<ol>
	<li>Place the sterile PDMS cast using tweezers onto a sterile glass coverslip or petri dish and apply gentle pressure using the tip of the tweezers. 
	NOTE: The PDMS cast makes a conformal but reversible seal with the glass coverslip or petri dish forming microchannels without use of any adhesive.</li>
	<li>Place a droplet (20 - 40 &#181;L) of the substrate solution on the inlet completely covering the inlet hole. Pattern Poly-D-Lysine (PDL) by placing a 20 &#181;L droplet of PDL in sodium tetraborate buffer on the inlet of the microchannels.</li>
	<li>Put the petri dish in a vacuum of ~254 mmHgA (equivalent to house vacuum in biological laboratories) for 10 min, and observe air coming out of the channel in the form of bubbles.</li>
	<li>Release the vacuum and observe the substrate solution flowing into the microchannel.</li>
	<li>Incubate the dish at the appropriate conditions for substrate adherence. See Tables 1 and 2 for incubation conditions (these vary depending on the substrate). For PDL patterning, incubate for 1 h at 37 &#176;C.</li>
	<li>Carefully peel off the PDMS stamp using sterile tweezers and wash the pattern on glass coverslip thrice with DI water.</li>
	<li>Add a 1% bovine serum albumin (BSA) solution to the petri dish to cover the dish or the glass coverslip and incubate overnight at 37 &#176;C. 
	NOTE: This will reduce non-specific adherence in uncoated regions.</li>
	<li>Aspirate off the 1% BSA solution, and the patterned substrate is now ready to use.</li>
</ol>
5. Indirect Patterning of Cells
<ol>
	<li>Prepare the cell suspension in serum-free culture medium. The cell type and cell density are listed in Table 1.</li>
	<li>Add the cell suspension to the patterned Petri dish and completely submerge the patterned region in cell suspension.</li>
	<li>Incubate the petri dish at 37 &#176;C in an incubator for 15 min to allow cells to attach to the patterned substrate.</li>
	<li>Aspirate the excess cell suspension from the Petri dish and wash the patterned cells three times with phosphate buffered saline (PBS) while gently shaking for 10 s to remove unattached cells.</li>
	<li>Add the appropriate culture medium to the patterned cell cultures and incubate the patterned cells at 37 &#176;C in a CO2 incubator.</li>
</ol>
6. Direct Patterning of Cells
NOTE: This technique is an alternative to the indirect cell patterning described in step 5. However, unlike in step 5, in this technique cells are patterned on tissue culture surfaces with or without substrate coating.
<ol>
	<li>Sterilize the microfluidic device by rinsing with a solution of 70% of ethanol followed by DI water.</li>
	<li>Soak the device overnight in a solution of 1% BSA to prevent the cell adhesion to the PDMS.</li>
	<li>Dry the device at room temperature and attach it to the bottom of the tissue culture-treated petri dish.</li>
	<li>Prepare the cell suspension in serum-free culture media. The cell type and cell density are listed in Table 2.</li>
	<li>Place a droplet (4 - 8 &#181;L) of the cell suspension, enough to fill the microchannels, on the inlet completely covering the inlet hole.</li>
	<li>Put the petri dish in a vacuum for 10 min and observe air coming out of the channel in the form of bubbles. Release the vacuum and observe the cell suspension flowing into the microchannel.</li>
	<li>Incubate the Petri dish in an incubator to promote cell adhesion as per the incubation conditions (depend on type of cell patterned) mentioned in Table 2. Add PBS to the petri dish submerging the PDMS device.</li>
	<li>Peel the PDMS carefully off the petri dish with tweezers and wash the pattern with PBS. Add cell culture media to the Petri dish and place the cell culture in the incubator.</li>
</ol>

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</ol>

The authors declare no competing financial interests.

While conventional photolithography is a well-established technique for the creation of molds for soft lithography, the equipment, materials, and skills necessary to use conventional photolithography are not readily available to most laboratories. For laboratories without access to these resources, we have presented adhesive tape fabrication as a method of creating molds with relatively simple features for microfluidic devices. This method allows any laboratory to create and utilize microfluidic devices for research purp...

In tissue engineering and biosensing, the ability to control the spatial organization of proteins and cells on a &#181;m scale, has become increasingly important over the last four decades1,2,3. Precise spatial organization of proteins and cells has allowed researchers to examine the interaction between cells and substrates containing similar or different types of cells, to guide cell growth, and to immobilize biomolecules for the fabrication of biosensors4,5,6,7,8,9.
Current methods of patterning proteins include photopatterning and microcontact printing. Photopatterning utilizes light sensitive material which is crosslinked upon exposure to ultra violet (UV) light. UV light directed at a photomask (consisting of transparent areas with darker regions to prevent UV light transmission) causes crosslinking in specific regions which can then be used for subsequent attachment of biomaterials or cells10,11. While this scheme is very accurate and allows for precise control of the topography of the culture surface, it is limited to UV-sensitive biomolecules that can be patterned by UV radiation12. Microcontact printing is another popular method of patterning specific proteins13,14. In this method, a poly-dimethyl siloxane (PDMS) stamp is treated with a variety of surface modification reagents before being soaked in a solution of the chosen biomolecular substrate. It is then gently pressed onto a glass coverslip or other surface thus &#34;stamping&#34; the biomolecule onto the culture surface. However, stamping is limited to the type of material that can be transferred as well as the wettability of biomolecules to the surface of the PDMS stamp15.
Direct patterning of cells can be more difficult and relies on complex methods such as switchable substrates, stencil based methods, or patterning with specific cell adhesion molecules16,17. These methods are limited in their ability to pattern cells due to the lack of compatible cell adhesion substrates, incompatibility of the process to work with sensitive biological cells and constraints, inconsistency in reproducing the patterning, and complexity of the procedure. For example, with switchable substrates, custom substrates need to be designed for every cell type, to switch their adherence to specific cell types without degradation upon exposure to the UV light and heat used in process17,18,19,20. Stencil based patterning methods are versatile in their ability to pattern cells; however, it is difficult to manufacture PDMS stencils at the appropriate thicknesses for use16,21. Direct injection of cells into PDMS microfluidic channels have some advantages such as: 1) ease in fabrication of microfluidic channels and 2) suitability for many different cells and substrates. However, the prevalent issue of air bubble capture during the injection process due to the hydrophobicity of PDMS without the use of plasma cleaning, or other methods to decrease air bubbles, makes it difficult to consistently create patterned cells on glass or plastic surfaces21.
This work expands upon capillary micromolding22,23,24,25,26 and reports a method to inject protein and cell suspensions into microchannels. The method used here demonstrates the patterning of substrates and both direct and indirect patterning of specific cell types. This technique overcomes the high hydrophobicity of PDMS and eliminates the presence of bubbles during injection of either substrates or cells by taking advantage of the gas permeability of PDMS27. This paper demonstrates the use of the technique with several different substrates and cell types. The article also highlights the fabrication of molds for soft lithography using conventional photolithography as well as a simple and low-cost adhesive tape method useful in resource limited settings28,29.

<table><tbody><tr><td>CorelDRAW X4 CAD Drawing Tools</td><td>Corel Corporation, Canada</td><td>X4 Version 14.0.0.701</td><td>CAD tool used to draw the layout of the microfluidic device</td></tr><tr><td>Laser Printer HP</td><td>Hewlett Packard, CA</td><td>1739629</td><td>Used to print the layout of microfluidic device for adhesive tape technique</td></tr><tr><td>Bel-Art Dessicator</td><td>Fisher Scientific, MA</td><td>08-594-16B</td><td>Used to degass the PDMS mixture</td></tr><tr><td>Adhesive Scotch Tape</td><td>3M Product, MN</td><td>Tape 600</td><td>Used to fabricate adhesive tape Master</td></tr><tr><td>PDMS Sylgard 184</td><td>Dow Corning, MI</td><td>1064291</td><td>Casting polymer</td></tr><tr><td>Petri Dish</td><td>Fisher Scientific, MA</td><td>08-772-23</td><td>Used to keep the mold to cast with PDMS</td></tr><tr><td>Stainless steel Scalpel (#3) with blade (# 11)</td><td>Feather Safety Razor Co. Ltd. Japan</td><td>2976#11</td><td>Used to cut the PDMS</td></tr><tr><td>Tweezers</td><td>Ted Pella, CA</td><td>5627-07</td><td>Used to handle the PDMS cast during peeling</td></tr><tr><td>Glass slides</td><td>Fisher Scientific, MA</td><td>12-546-2</td><td>Used as surface to pattern the Substrate</td></tr><tr><td>Glass slides</td><td>Fisher Scientific, MA</td><td>12-544-4</td><td>Used as surface to pattern the Substrate</td></tr><tr><td>Rubber Roller</td><td>Dick Blick Art Materials, IL</td><td>40104-1004</td><td>Used to attach adhesive tape on glass without trapping air bubbles</td></tr><tr><td>Laser Mask Writer</td><td>Heidelberg Instruments, Germany</td><td>DWL66fs</td><td>Used to fabricate quartz mask used in photolithography fabrication process</td></tr><tr><td>EVG Mask Aligner (Photolithography UV exposure tool)</td><td>EV Group, Germany</td><td>EVG 620T(B)</td><td>Used to expose the photoresist to UV light</td></tr><tr><td>Spin Coater Headway</td><td>Headway Research Inc, TX</td><td>PWM32-PS-CB15PL</td><td>Used to spin coat the photoresist on silicon wafer</td></tr><tr><td>Photoresists SU-8 50</td><td>MicroChem, MA</td><td>Y131269</td><td>Negative photoresist used for mold fabrication</td></tr><tr><td>SU-8 Devloper</td><td>MicroChem, MA</td><td>Y020100</td><td>Photoresist developer</td></tr><tr><td>Tridecafluoro-1,1,2,2-Tetrahydrooctyl-1-Trichlorosilane</td><td>UCT Specialties, PA</td><td>T2492-KG</td><td>Coat mold to avoid PDMS adhesion</td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich, MO</td><td>190764</td><td>Cleaning Solvent</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich, MO</td><td>24102</td><td>Sterilization Solvent</td></tr><tr><td>Poly-D-Lysine hydrobromide (PDL)</td><td>Sigma-Aldrich, MO</td><td>P0899-10MG</td><td>PDL solution is made at 0.1 mg/mL in Sodium Tetraborate Buffer</td></tr><tr><td>Laminin</td><td>Sigma-Aldrich, MO</td><td>L2020</td><td>Laminin aliquoted into 10 &amp;micro;L aliquots and diluted to 20 &amp;micro;g/&amp;micro;L in PBS prior to use</td></tr><tr><td>BSA</td><td>Fisher Scientific, MA</td><td>BP1605100</td><td>Cell culture</td></tr><tr><td>C2C12 Myoblast cell lline</td><td>ATCC, VA</td><td>CRL-1722</td><td>Used to demonstrate C2C12 patterning</td></tr><tr><td>PC12 Cell Line</td><td>ATCC, VA</td><td>CRL-1721</td><td>Used to demonstrate PC12 patterning</td></tr><tr><td>Collagen type 1, rat tail</td><td>BD Biosciences</td><td>40236</td><td>Cell culture</td></tr><tr><td>DMEM</td><td>GIBCO, MA</td><td>11965-084</td><td>Cell culture</td></tr><tr><td>Horse Serum, heat inactivated</td><td>Fisher Scientific, MA</td><td>26050-070</td><td>Cell culture</td></tr><tr><td>Phalloidin-tetramethylrhodamine B isothiocyanate (TRITC)</td><td>Sigma-Aldrich, MO</td><td>P1951</td><td>To label cells</td></tr><tr><td>Calcein-AM live dead cell Assay kit</td><td>Invitrogen, MA</td><td>L-3224</td><td>Cell viability Assay</td></tr><tr><td>Biopsy Hole Punch</td><td>Ted Pella, CA</td><td>15110-10</td><td>Punched hole in PDMS</td></tr></tbody></table>

a versatile method of patterning proteins and cells

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 method allows the patterning of proteins and indirect patterning of cells using dead-end microfluidic channels with dimensions as small as 10 µm and equipment available in almost all biological laboratories once the master mold is made. This technique can be utilized with PDMS microfluidic channels created using traditional soft photolithography, or with PDMS microfluidic channels created with adhesive tape fabrication (Figure 1)28,...

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 ...

The overall goal of this procedure is to pattern cells or substrates by distributing them into complex long and closed microchannels using simple bench top tools available in most biological laboratories. This method can help answer key questions in the field of biology regarding cell-to-cell and cell-to-protein interactions. The main advantage of this technique is that it is easy to perform and it only requires tools that are readily available in most biological laboratories.Generally, individuals new to this method will struggle because it requires optimization of the incubation conditions for the cells and substrates chosen by the researcher. Visual demonstration of this method is critical, as the proper placement of the PDMS stamp and solution significantly improves the reproducibility of the experiment. Start by drawing out the layout of the microchannel using a CAD drawing tool.Then, print the drawing onto white paper. Next, use isopropanol to clean a glass slide that is large enough to accommodate the design. And then dry the slide with an air gun.Attach adhesive tape to the cleaned glass slide, taking care not to trap any air bubbles. Then, tape the sides of the glass slide onto the paper with the design, so that the tape side is facing up. Use a scalpel to cut the tape on the glass slide, using the paper design as a reference.Once cut, peel off the tape from the unwanted areas of the glass slide. Next, rinse the glass slide with isopropanol to remove any residual adhesive. And then use an air gun to dry the surface.Once patterned and cleaned, bake the glass slide in an oven at 65 degrees Celsius for 30 minutes. Then, gently roll over the tape with a rubber roller, to remove air bubbles and allow the slide to cool to room temperature in a petri dish. Begin by mixing 10 parts of a PDMS elastomer with 1 part of its curing agent and stir the mixture vigorously.Then, place the mixture into a vacuum chamber to de-gas the mixture until no air bubbles remain. Pour a 2mm thick layer of the mixture onto the adhesive tape mold, and de-gas the mold until all air bubbles disappear. Once cooled, use a scalpel to cut the PDMS layer at least 5mm away from the features.And then, peel the cured PDMS off the mold using a pair of tweezers. Punch a single inlet hole with a 1mm biopsy punch, preferably at the end of a network of microchannels. Then, clean the PDMS cast with adhesive tape, to remove dust particles from the device.Sterilize the PDMS microchannel device by rinsing it with a solution of 70%ehtanol, followed by sterile deionized water, and expose it to UV light for 30 minutes. Use tweezers to place the sterile PDMS cast onto a sterile glass cover slip, and apply gentle pressure using the tip of the tweezers. Next, place a droplet of poly d lysine in sodium tetraborate buffer on the inlet of the microchannels.Place the petri dish under vacuum for 10 minutes. During this time, observe air coming out of the channel in the form of bubbles. Release the vacuum and observe the substrate solution flowing into the microchannel Incubate the dish at 37 degrees Celsius for one hour.Then, carefully peel off the PDMS stamp using sterile tweezers, and wash the pattern three times with deionized water. Add a 1%BSA solution to the glass cover slip, to reduce non-specific adhesion, and incubate the sample overnight at 37 degrees Celsius. The next day, aspirate the BSA solution.To start the indirect patterning of cells, prepare a cell suspension in serum free culture medium as described in the accompanying text protocol. Add the cell suspension to the patterned petri dish, completely submerging the patterned region in cell suspension. Then, incubate the petri dish at 37 degrees Celsius in an incubator for 15 minutes to allow cells to attach to the patterned substrate.Following incubation, aspirate the excess cell suspension from the petri dish and wash the patterned cells three times with PBS, while gently shaking for ten seconds to remove the unattached cells. Then, add the appropriate culture medium to the patterned cell cultures, and incubate the patterned cells at 37 degrees Celsius in a carbon dioxide buffered incubator. For the direct patterning of cells, first, sterilize the microfluidic device by rinsing it with a solution of 70%ethanol followed by deionized water.Then, soak the device overnight in a solution of 1%BSA to prevent non-specific cell adhesion to the PDMS. Dry the device at room temperature, and then attach it to the bottom of the tissue culture treated petri dish. Place 4 to 8 microliters of the cell suspension, enough to fill the microchannels, on the inlet, completely covering the hole.Then, put the petri dish in a vacuum chamber for 10 minutes. Release the vacuum and observe the cell suspension flowing into the microchannel. Incubate the petri dish at 37 degrees Celsius in a carbon dioxide buffered incubator, for up to one hour.Then, remove the dish from the incubator and use a pair of tweezers to carefully peel the PDMS from the dish. Wash the pattern with PBS, and then add cell culture media to the petri dish. When finished, place the cells back into the incubator.Shown here are embryonic rat cortical neurons patterned on poly d lysine and laminin surfaces. The location of the cells demonstrate general adherence to the patterned areas and growth along the PDL and laminin stripes. Shown here, at a higher magnification, are C2C12 myoblasts.This cell type also adheres to the patterned areas and demonstrates fusion into multinucleated myotubes. This technique has also been adapted for direct cell patterning, shown here with fibroblasts. This direct cell patterning method has no adverse effect on cell survival.Following this procedure, other methods like fluorescent microscopy can be performed in order to answer additional questions involving protein localization or cell-substrate interactions. While attempting this procedure, it is important to remember that proper placement of the droplet of cells or substrate solution to cover the entire inlay is critical for success. After its development, this technique paved the way for researchers in the field of neuroscience to explore photoreceptor axon guidance in retinal biology.After watching this video, you should have a good understanding of how to pattern cells and substrate with gas permeable PDMS microfluidic devices using negative pressure. Thanks for watching and good luck with your experiment.

a-versatile-method-of-patterning-proteins-and-cells

Research

JoVE Journal

Bioengineering

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.

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces.  We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

Microcontact Printing of Proteins for Cell Biology

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 Co-culture Patterning Using Aqueous Two-phase Systems

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 on Photolithographically Defined 
Parylene-C: SiO2 Substrates

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

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.

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

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

Simple, Affordable, and Modular Patterning of Cells using DNA

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

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.

Pattern Generation for Micropattern Traction Microscopy

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

Learn to pattern cells and substrates in microfluidic channels using low pressure vacuum and simple benchtop tools. This versatile method enables studies of cell-to-cell and cell-to-protein interactions in biological research.

A Versatile Method of Patterning Proteins and Cells

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