This work was supported by funding from the William G. Lowrie Department of Chemical and Biomolecular Engineering and the Comprehensive Cancer Center at The Ohio State University. Data presented in this report came from images processed using Imaris x64 (ver. 9.3.0 Bitplane) available at the Campus Microscopy and Imaging Facility, The Ohio State University. This facility is supported in part by grant P30 CA016058, National Cancer Institute, Bethesda, MD.

The authors acknowledge the healthy volunteers who kindly donated their blood. Blood specimens were obtained after informed volunteer consent according to institutional review board (IRB) protocol #2018H0268 reviewed by the Biomedical Sciences Committee at The Ohio State University.
1. Microfabrication of bioparticle microarray
<ol>
	<li>Using standard photolithography procedures, generate the master silicon wafer.
	<ol>
		<li>Generate a proof of the desired design using a computer-aided des...

<ol>
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The authors declare no conflicts of interest.

We developed a microstamping platform to generate uniform arrays of bioparticles to stimulate in vitro neutrophil swarming. The in vitro nature of our platform allows us to circumvent the complications that arise with in vivo swarming experiments, namely the poor ability to analyze mediators released by swarming neutrophils5. Additionally, in vivo models are typically performed in rodents11,12,13,</s...

Neutrophils, the most abundant white blood cell in the bloodstream1, are gaining attention as potential diagnostic and therapeutic targets2,3 because they may be involved in a variety of medical conditions including gout4, sepsis3, trauma5,6, cancer1,7,8, and various autoimmune diseases5,9. Neutrophil swarming is a multistage, tightly regulated process with a complexity that makes it a particularly interesting focus of study5,10,11. During swarming, neutrophils isolate a site of inflammation from the surrounding healthy tissue5,10,11. Proper regulation of neutrophil swarming is essential to promote wound healing and ultimately inflammation resolution5,12. Neutrophil swarming has primarily been studied in vivo in rodent12,13,14,15 and zebrafish10,11,12,15 models. However, the nature of these in vivo animal models gives rise to limitations5. For example, the mediators released by neutrophils during swarming are not easily accessible for analysis5. Additionally, there are many potential sources for a given mediator in vivo, so an in vivo experiment must introduce a genetic deficiency to inhibit cellular production and/or interaction in order to investigate the role of that mediator in a given process13. An in vitro experiment circumvents this complication by enabling neutrophil observation without the context of additional cells. Additionally, research describing human neutrophil coordinated migration is limited16. On an in vitro swarming platform, human neutrophils can be directly analyzed. An in vitro swarming platform could expand upon the knowledge gained from in vivo studies by providing opportunities to fill the gaps left by the limitations of in vivo studies.
To address the need for an in vitro platform that mimics in vivo neutrophil swarming, we developed a microstamping platform that enables us to pattern bioparticle microarrays that stimulate neutrophil swarming in a spatially controlled manner. We generate bioparticle microarrays on glass slides in a two-step process. First, we use microstamping to generate a microarray of cationic polyelectrolyte (CP) spots. Second, we add a solution of bioparticles that adhere to the CP spots via electrostatic interaction. By first patterning the CP layer, we can selectively pattern negatively charged bioparticles to generate the desired neutrophil swarming pattern. The positively charged layer holds the negatively charged bioparticles through the vigorous washing step that removes the bioparticles from the areas on the glass slide that do not have the CP. Additionally, the CP used here, a copolymer of acrylamide and quaternized cationic monomer, is biocompatible, so it does not induce a response from the neutrophils. It has a very high surface charge that immobilizes the micron-sized bioparticles to the glass slide, thus inhibiting neutrophils from removing the particles from the patterned position on the glass slide. This results in bioparticle clusters arranged in a microarray. When we added neutrophils to the microarray, they formed stable swarms around the bioparticle clusters. Through tracking neutrophil migration, we found that swarming neutrophils actively migrate toward the bioparticle clusters. Furthermore, we used this platform to analyze certain mediators that neutrophils release during swarming. We found 16 mediators that are differentially expressed during swarming. Their concentrations follow three general trends over time: increase, decrease, or spike. Our in vitro neutrophil swarming platform facilitates the analysis of spatially controlled human neutrophil swarming, as well as the collection and analysis of mediators released by neutrophil swarming. In a previous publication, we demonstrated that patients with certain medical conditions (trauma, autoimmune disease, and sepsis), had neutrophils that functioned differently than those from healthy donors5. In future research studies, our platform could be used to analyze neutrophil function among a variety of patient populations. This platform can quantitatively analyze the complex coordination involved in neutrophil swarming. Additional studies can be done to provide insight on the neutrophil function of a specific patient population or neutrophil response to a pathogen of interest.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#34;The Big Easy&#34; EasySep Magnet</td>
			<td>STEMCELL Technologies</td>
			<td>18001</td>
			<td>Magnet to use with neutrophil isolation kit</td>
		</tr>
		<tr>
			<td>Cell Incubator</td>
			<td>Okolab</td>
			<td>777057437 / 77057343</td>
			<td>Okolab cage incubator for temperature and CO2 control</td>
		</tr>
		<tr>
			<td>EasySep Human Neutrophil Isolation Kit</td>
			<td>STEMCELL Technologies</td>
			<td>17957</td>
			<td>Kit for immunomagnetic negative selection of human neutrophils</td>
		</tr>
		<tr>
			<td>Eclipse Ti2</td>
			<td>Nikon Instruments</td>
			<td>MEA54010 / MEF55037</td>
			<td>Inverted research microscope</td>
		</tr>
		<tr>
			<td>Escherichia coli (K-12 strain) BioParticles Texas Red conjugate</td>
			<td>Invitrogen</td>
			<td>E2863</td>
			<td>Bioparticle powder, dissolve in water prior to addition to Zetag&#174; array</td>
		</tr>
		<tr>
			<td>Harris Uni-Core 8-mm biopsy punch</td>
			<td>Sigma Aldrich</td>
			<td>Z708925</td>
			<td>To cut PDMS stamps</td>
		</tr>
		<tr>
			<td>HetaSep</td>
			<td>STEMCELL Technologies</td>
			<td>7906</td>
			<td>Erythrocyte aggregation agent for separating buffy coat from red blood cells in fresh human blood</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
			<td>Nucleus fluorescent stain</td>
		</tr>
		<tr>
			<td>Human L1000 Array</td>
			<td>Raybiotech Inc.</td>
			<td>AAH-BLG-1000-4</td>
			<td>High density array to detect 1000 human proteins</td>
		</tr>
		<tr>
			<td>Human Serum Albumin (HSA)</td>
			<td>Sigma Aldrich</td>
			<td>A5843</td>
			<td>Low endotoxin HSA, to prepare 2 % solutions in IMDM for isolated neutrophils</td>
		</tr>
		<tr>
			<td>Iscove&#39;s Modified Dulbeccos&#39; Medium (IMDM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12440053</td>
			<td>To resuspend isolated neutrophils</td>
		</tr>
		<tr>
			<td>K2-EDTA tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-657-32</td>
			<td>Tubes for blood collection</td>
		</tr>
		<tr>
			<td>Low Reflective Chrome Photomask</td>
			<td>Front Range Photomask</td>
			<td>N/A</td>
			<td>Dimensions 5&#34; x 5&#34; x 0.09&#34; (L x W x D)</td>
		</tr>
		<tr>
			<td>Microarray Scanner</td>
			<td>Perkin Elmer</td>
			<td>ASCNGX00</td>
			<td>Fluorescence reader of protein patterned microdomains</td>
		</tr>
		<tr>
			<td>Microscopy Image Analsysis Software - Imaris</td>
			<td>Bitplane</td>
			<td>9.3.0</td>
			<td>Software for automatic cell tracking analysis</td>
		</tr>
		<tr>
			<td>NiS Elements Advanced Research Software Package</td>
			<td>Nikon Instruments</td>
			<td>MQS31100</td>
			<td>Software for automatic live cell imaging and swarm size calculation</td>
		</tr>
		<tr>
			<td>Poly-L-lysine fluorescein isothiocyanate (PLL-FITC)</td>
			<td>Sigma Aldrich</td>
			<td>P3069-10MG</td>
			<td>30,000 - 70,000 MW PLL labeled with FITC, used to fluorescently label CP solution</td>
		</tr>
		<tr>
			<td>SecureSeal 8-well Imaging Spacer</td>
			<td>Grace Bio-Labs</td>
			<td>654008</td>
			<td>8-well, 9-mm diameter, adhesive imaging spacer</td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>University Wafer</td>
			<td>590</td>
			<td>Silicon 100 mm N/P (100) 0- 100 ohm-cm 500 &#956;m SSP test</td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>Laurell</td>
			<td>WS-650MZ-23NPPB</td>
			<td>Used to spincoat a 40-&#181;m layer of photoresist onto silicon wafer</td>
		</tr>
		<tr>
			<td>SU-8 2025</td>
			<td>MicroChem</td>
			<td>2025</td>
			<td>Negative photoresist to make silicon master wafer</td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>MicroChem</td>
			<td>Y020100</td>
			<td>Photoresist developer. Remove non-crosslinked SU-8 2025 from silicon wafer</td>
		</tr>
		<tr>
			<td>Sylgard 184 (polydimethylsiloxane, PDMS)</td>
			<td>Dow</td>
			<td>1673921</td>
			<td>2-part silicone elastomer kit for making microstamps and PDMS wells</td>
		</tr>
		<tr>
			<td>UV Exposure Masking System</td>
			<td>Klo&#233;</td>
			<td>UV-KUB 2</td>
			<td>Used to crosslink photoresist on silicon wafer through chrome mask with UV light</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1287303</td>
			<td>High quality water to dilute bioparticles</td>
		</tr>
		<tr>
			<td>Zetag 8185</td>
			<td>BASF</td>
			<td>8185</td>
			<td>Cationic polyelectrolyte (CP), powder, Copolymer of acrylamide and quaternized cationic monomer, forms &#34;inking solution&#34; for microstamping when dissolved in water</td>
		</tr>
		<tr>
			<td>Zymosan A S. cerevisiae BioParticles Texas Red conjugate</td>
			<td>Invitrogen</td>
			<td>Z2843</td>
			<td>Bioparticle powder, dissolve in water prior to addition to Zetag array</td>
		</tr>
	</tbody>
</table>

bioparticle microarrays for chemotactic and molecular analysis of human neutrophil swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

When neutrophils are added to the bioparticle microarray, neutrophils that contact the bioparticle clusters become activated and initiate the swarming response. The bioparticle microarray was validated using time-lapse fluorescent microscopy to track neutrophil migration toward the bioparticle clusters (Video S1). The migration of individual neutrophil nuclei is tracked as they migrate toward the bioparticle cluster. When neutrophils reach the bioparticle cluster, their nuclei overlap with other nuclei i...

Watch this Scientific Journal Video about Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro at JoVE.com

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

This protocol is significant because it allows for an in vitro analysis of neutrophil swarming, which overcomes many limitations of our present inability to experiment. The main advantage of this technique is that it provides easy access to the secreted cytokines and lipid mediators that neutrophils release during swarming and allows for quantitative imaging analysis. While we applied this technique to neutrophils, it could be extended to analyze the migration of other white blood cells like monocytes and T cells.Begin by preparing a 1.6 milligram per milliliter cationic polyelectrolite, or CP solution, by adding the proper amount of CP powder to water and mixing it on the stir plate overnight or until all solids are dissolved. If desired, the solution can be made fluorescent by adding poly-L-lysine labeled with FITC. Generate the master silicon wafer using standard photolithography procedures.The design used here is a four by four milliliter rectangular array of 30 micrometer diameter filled-in circles, with a 150 micrometer center to center spacing, but it can be modified as desired for different applications. Spin-coat a 40 micrometer thick layer of negative photoresist onto a silicon wafer. Then bake the wafer at 65 degrees Celsius for five minutes.And 95 degrees Celsius for 10 minutes. Expose the wafer to UV light through a chrome photomask with 150 to 160 millijoules per centimeter squared. Bake the wafer again at 65 degrees Celsius for five minutes and 95 degrees Celsius for 10 minutes.Then submerge it in photoresist developer for 10 minutes. And rinse it with isopropyl alcohol. The pattern should now be visible on the wafer.Next, thoroughly mix a 10 to one ratio of polydimethylsiloxane, or PDMS, prepolymer and its curing agent. And pour the uncured mixture over the master wafer in a Petri dish. Vacuum treat the uncured PDMS mixture until no air bubbles are present.Then cure it overnight at 65 degrees Celsius. On the next day, use a scalpel to cut around the exterior of the patterned section of the wafer. And slowly remove the cured PDMS slab, placing it on a clean cutting board with the patterned side facing up.Punch out individual stamps from the PDMS slab with an eight millimeter biopsy punch and place each stamp face-down on the adhesive tape to remove debris. With these stamps facing up, prime each stamp with 100 microliters of the pre-prepared CP solution, ensuring that no air bubbles form between the solution and the stamp. Then invert the stamps onto a layer of CP solution and remove them after one hour.Dab each wet stamp face down onto a clean glass slide six to eight times to remove excess liquid. Then vacuum treat the stamps for one to two minutes. Adhere an eight well, eight millimeter diameter imaging spacer on top of a clean glass slide as a guide for stamp placement.Place a stamp face down on the glass slide in the center of each well of the imaging spacer. Then place a 5.6 gram balanced weight on top of each stamp and allow 10 minutes for stamping. Remove the weights and the stamps from the slide and allow the CP layer to dry at room temperature for 24 hours before adding bioparticles.If the CP is tagged with FITC, check effectiveness of the stamping with a fluorescent microscope. Cut a blank PDMS slab to the size of the imaging spacer and use the eight millimeter biopsy punch to create wells in the PDMS that align with the wells of the spacer. Then adhere the PDMS slab to the glass slide.Thaw a solution of bioparticles and dilute it to 500 micrograms per milliliter in water for injection. Add 100 microliters of bioparticle solution to each PDMS well on the glass slide and rock the slide for 30 minutes. Rinse the wells thoroughly with water and check the pattern on the slide with a fluorescent microscope.The bioparticle microarray can be stored in a dust-free environment at four degrees Celsius for up to three months. Prepare the cells by resuspending them at 7.5 times 10 to the fifth cells per milliliter in IMDM with 0.4%human serum albumen. Add 100 microliters of the suspension to a PDMS well containing the bioparticle microarray, making sure that the cell suspension is convex over the top of the PDMS well and doesn't contain any bubbles.Seal the well with a 12 millimeter diameter cover slip and press down gently with tweezers so the excess cell suspension escapes to edge of the well, then use a tissue to remove the excess. To image the cells, load the microparticle array with cells on the live cell imaging station of a microscope equipped with a cage incubator set to 37 degrees Celsius, 5%carbon dioxide, and 90%relative humidity. Use time lapse, fluorescent, and bright field microscopy to record images at 10X magnification every 10 seconds at 405 nanometers, 594 nanometers, and bright field.Incubate the neutrophils in the wells with bioparticles at 37 degrees Celsius and 5%carbon dioxide for three hours, taking samples at desired time points. Use a bright field microscope to verify that swarms are formed on the microarray. Collect the entire volume of supernatant of a single well, and load it into a 0.45 micrometer centrifuge filter tube, making sure to analyze each time point in triplicate.Centrifuge the tubes at 190 times G and 20 degrees Celsius for five minutes, and collect the filtered volume. Then store the samples at minus 80 degrees Celsius until processing time. When attempting this protocol, work with photoresist and developer in the fume hood.Have an IRB approved protocol in place before taking blood from human donors. Remember that materials derived from human blood are BSL2. When neutrophils are added to the bioparticle microarray, neutrophils that contact the bioparticle clusters become activated and initiate the swarming response.The bioparticle microarray was validated using time lapse fluorescent microscopy to track neutrophil migrations toward the bioparticle clusters. Stable neutrophil swarms are formed around each cluster after 30 to 60 minutes of exposure. In contrast, neutrophils do not show collective migration in the absence of bioparticle clusters.Fluorescence intensity of stained neutrophil nuclei was used to determine that the average swarm size around the bioparticle clusters was approximately 1490 micrometers squared. In the absence of clusters, fluorescence of a given region of interest remained constant over time. Tracks of neutrophil migration show that neutrophils converged on a bioparticle cluster when one was present, but no convergence was observed in the control system.The speed of swarming and nonactivated neutrophils was measured, and a statistically significant difference in the speed distributions was found. The average speed for swarming neutrophils was 20.6 micrometers per minute, while the average speed for control neutrophils was two micrometers per minute. The concentrations of 16 proteins that neutrophils release during swarming were analyzed over time.10 proteins increased in concentration. Two decreased. And the remaining four increased during the first hour of swarming but decreased thereafter.While performing this procedure, ensure that everything is clean and your stamps are dried properly, as these are essential for successful patterning of the bacterial particles. The development of this technique will enable researchers to explore new questions in the field of neutrophil swarming, including how free mediators and extracellular vesicles are involved in neutrophil intercellular communication.

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Research

JoVE Journal

Bioengineering

5.0K Views.  The Ohio State University. This protocol is significant because it allows for an in vitro analysis of neutrophil swarming, which overcomes many limitations of our present inability to experiment. The main advantage of this technique is that it provides easy access to the secreted cytokines and lipid mediators that neutrophils release during swarming and allows for quantitative imaging analysis. While we applied this technique to neutrophils, it could be extended to analyze the migration of other white blood cells like ...