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

Summary

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.

Abstract

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.

Introduction

Neutrophils, the most abundant white blood cell in the bloodstream¹, are gaining attention as potential diagnostic and therapeutic targets^2,3 because they may be involved in a variety of medical conditions including gout⁴, sepsis³, trauma^5,6, cancer^1,7,8, and various autoimmune diseases^5,9. Neutrophil swarming is a multistage, tightly regulated process with a complexity that makes it a particularly interesting focus of study^5,10,11. During swarming, neutrophils isolate a site of inflammation from the surrounding healthy tissue^5,10,11. Proper regulation of neutrophil swarming is essential to promote wound healing and ultimately inflammation resolution^5,12. Neutrophil swarming has primarily been studied in vivo in rodent^12,13,14,15 and zebrafish^10,11,12,15 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.

Protocol

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

1. Using standard photolithography procedures, generate the master silicon wafer.
   1. Generate a proof of the desired design using a computer-aided design (CAD) software, then send to a photomask manufacturer to produce a chrome photomask. The design used here is 4 mm x 4 mm rectangular arrays of 30 µm diameter filled in circles with a 150 µm center-to-center spacing. This design can be modified as desired for different applications.
   2. Spincoat a 40 µm thick layer of a negative photoresist onto a silicon wafer. Bake wafer at 65 °C for 5 min and 95 °C for 10 min.
   3. Expose wafer to UV light through a chrome photomask with 150–160 mJ/cm² (Figure 1A).
   4. Bake wafer at 65 °C for 5 min and 95 °C for 10 min. Submerge wafer in photoresist developer for 10 min and rinse with isopropyl alcohol. At this stage, the pattern should be visible on the wafer (Figure 1B).
2. Thoroughly mix a 10:1 ratio of polydimethylsiloxane prepolymer and its curing agent (i.e., 20 g of prepolymer and 2 g of curing agent) and pour the uncured polydimethylsiloxane (PDMS) mixture over the master wafer in a Petri dish (Figure 1C).
3. Vacuum treat the uncured PDMS mixture until no air bubbles are present over the master wafer. Cure at 65 °C overnight.
4. Use a scalpel to cut around the exterior of the patterned section of the wafer, and slowly remove the cured PDMS slab. Place the PDMS slab on a clean cutting board with the patterned side facing up (Figure 1D).
5. Punch out individual stamps from the PDMS slab with an 8 mm biopsy punch (Figure 1E). For one glass slide, eight stamps will be needed.
6. Place each stamp face down on the adhesive tape to remove any debris.
7. In advance, prepare a 1.6 mg/mL solution of CP in water.
   1. Add the proper amount of the CP powder to water (e.g., 0.8 g to 500 mL).
   2. Mix on a stir plate at room temperature overnight, or until all the solid is dissolved into the water. The CP solution can be stored at room temperature for 6 months.
   3. If desired, make the CP solution fluorescent by adding poly-L-lysine labelled with fluorescein isothiocyanate (PLL-FITC).
      1. Aliquot about 10 mL of the CP solution. Add a small amount of PLL-FITC (0.05 mg) to the aliquoted volume. The amount can be altered to adjust the brightness of fluorescence as desired.
      2. Vortex the CP solution labeled with FITC for 20 s, or until the solution is a uniform, pale yellow color. Protect from light and store at 4 °C for up to 1 month.
8. With the stamps face-up, prime each stamp with 100 µL of 1.6 mg/mL solution of CP, ensuring no air bubbles form between the CP solution and the stamp (Figure 1F).
9. Invert the stamps onto a layer of CP solution (Figure 1G).
10. Remove the stamps from the CP solution after 1 h.
11. Dab each wet stamp face down onto a clean glass slide 6–8x to remove excess liquid.
12. Vacuum treat the stamps for 1–2 min.
13. Adhere an eight well, 9 mm diameter imaging spacer on the top of a clean glass slide as a guide for stamp placement. With this spacer, each glass slide can have eight microarrays.
14. Place a stamp face down on the glass slide in the center of each well of the imaging spacer (i.e., use eight stamps total).
15. Place a 5.6 ± 0.1 g balanced weight on top of each stamp and allow 10 min for stamping (Figure 1H). A balanced weight is required to ensure the stamp is pressed evenly onto the glass slide and promote even transfer of the CP to the glass slide.
16. Remove the weights and stamps from the glass slide (Figure 1I). Allow the CP layer to dry at room temperature for 24 h before adding the bioparticles, as described in step 1.17. If the CP is tagged with FITC, the effectiveness of the stamping can be checked at this point with a fluorescent microscope at 488 nm before proceeding to step 1.17 (Figure 1M).
17. Cut a blank PDMS slab to the size of the imaging spacer and use the 8 mm biopsy punch to create wells in the PDMS that align with the wells of an imaging spacer. Adhere the PDMS slab to the glass slide with the imaging spacer (Figure 1J).
18. Thaw a solution of bioparticles (e.g., E. coli or zymosan) and dilute to 500 μg/mL in water for injection (WFI).
    NOTE: The bioparticles do not need to be opsonized. Neutrophil surface receptors directly recognize molecules on these bioparticles^19,20,21.
19. Add 100 μL of bioparticle solution to each PDMS well on the glass slide (Figure 1K).
20. Rock the glass slide for 30 min.
21. Rinse the wells thoroughly with water. The bioparticle microarray can be stored in a dust-free environment at 4 °C for up to 3 months. At this point, the pattern should be checked with a fluorescent microscope at 594 nm before proceeding to step 2.1 (Figure 1N).

2. Sample Preparation

1. Collect at least 2 mL of fresh blood in K2-EDTA tubes from the desired donor. The expected yield of neutrophils is 1–2 x 10⁶ cells/1 mL whole blood. The imaging assay requires approximately 1.5 x 10⁵ neutrophils, and the analysis of the supernatant requires 1 x 10⁶ neutrophils. Use the blood within 4 h.
2. Separate red blood cells (RBCs) by adding an erythrocyte aggregation agent in a 1:5 ratio to the whole blood. Wait 45 min for a translucent layer (buffy coat) to separate from the layer of RBCs.
3. Remove the buffy coat and wash with phosphate buffered saline (PBS) using 1 mL buffy coat: 9 mL PBS.
4. Centrifuge for 5 min at 190 x g and 20 °C.
5. Aspirate the supernatant and resuspend the pellet at 5 x 10⁷ cells/mL.
6. Use a negative immunomagnetic selection kit to isolate neutrophils.
   1. Add 50 μL of antibody cocktail/1 mL cell suspension. Wait 10 min.
   2. Add 100 μL of magnetic beads/1 mL cell suspension. Wait 10 min.
   3. Add cell suspension to a round-bottom tube and place in a cylindrical magnet. Wait 10 min.
7. Pour supernatant into a centrifuge tube. Add up to 10 mL of PBS. Centrifuge for 5 min at 190 x g and 20 °C.
8. Aspirate supernatant. Resuspend white pellet in IMDM with 0.4% human serum albumin.
9. Stain nuclei with 20 µg/mL Hoechst 33342 for 10 min at 37 °C.
10. Add 5 mL of IMDM with 0.4% human serum albumin to rinse. Centrifuge for 5 min at 190 x g and 20 °C.
11. Resuspend cells at 7.5 x 10⁵ cells/mL in IMDM with 0.4% human serum albumin.
12. Add 100 µL of the cell suspension to a PDMS well containing a bioparticle microarray. Ensure the cell suspension is convex over the top of the PDMS well and does not contain any bubbles.
13. Seal with a 12 mm diameter coverslip. Cover the opening of the PDMS well with a 12 mm diameter coverslip. Press down gently onto the coverslip with tweezers so the excess cell suspension escapes to the edge of the well. Use a tissue to remove the excess cell suspension.

3. Running the assay and image analysis

1. Load microparticle array with cells on the live cell imaging station with a microscope equipped with a cage incubator set to 37 °C, 5% CO₂, and 90% relative humidity.
2. Use time-lapse fluorescent and brightfield microscopy to record images at 10x magnification every 10 s at 405 nm, 594 nm, and brightfield. In a typical experiment, images are collected up to 2 h.
3. Use an automated cell tracking software to track the migration of individual neutrophils toward the bioparticle cluster.
   1. Use the autoregression mode of a spot detection cell tracking software. Set the spot radius to 5 µm (the approximate size of a neutrophil nucleus). Set the minimum track length to 120 s and a maximum gap size of one frame.
   2. From the data generated by the cell tracking software, extract the files that contain the neutrophil position and speed. These files can be used with a graphing software to generate neutrophil migration tracks (Figure 2C) and a heat map of speed vs. time (Figure 2D), respectively.
4. Use the 405 nm fluorescent images to track swarm size over time on an image analysis software of your choice.
   1. Define regions of interest (ROIs) around each bioparticle cluster where neutrophils will swarm. Keep the same size ROI to analyze each bioparticle cluster.
   2. Analyze the mean fluorescent intensity of the 405 nm images within each ROI over time.
   3. Generate a calibration curve of mean fluorescent intensity to swarm size by taking manual measurements at various swarm sizes from 0 µm² to the maximum swarm size. Use this calibration to calculate the swarm size over time.

4. Supernatant collection and protein detection

1. Incubate the neutrophils in the wells containing the bioparticle microarray at 37 °C and 5% CO₂ for 3 h. Take samples at desired time points. Typically, samples will be taken at 0, 0.5, 1, and 3 h. To overcome the limit of detection of the protein array assay, the entire volume of supernatant of a single well (200 µL) was used for each time point. Each time point was analyzed in triplicate.
2. Using a brightfield microscope, verify that swarms are formed on the microarray.
3. Aspirate the supernatant with a 200 µL pipette and load in a 0.45 μm centrifuge filter tube.
4. Centrifuge the supernatant at 190 x g and 20 °C for 5 min and collect the filtrated volume.
5. Store samples at -80 °C until the processing time.
6. Use a microarray kit that detects a range of human proteins to process samples.
   1. Add 200 μL of each sample to a separate dialysis tube provided with the kit.
   2. Place the dialysis tubes in a beaker containing at least 500 mL of PBS (pH = 8.0). Stir gently on a stir plate for at least 3 h at 4 °C. Change the PBS in the beaker and repeat this step.
   3. Transfer each sample to a clean centrifuge tube and centrifuge at 9,000 x g for 5 min to remove any precipitates. Transfer each supernatant to a clean tube.
   4. Biotinylate each sample by adding 36 µL of 1x labeling reagent from the kit per 1 mg of total protein in the dialyzed sample to 180 µL of dialyzed sample. Incubate at 20 °C for 30 min. Mix gently every 5 min.
   5. Add 3 µL of stop solution provided with the kit into each sample tube. Transfer each sample to a fresh dialysis tube and repeat steps 4.6.2–4.6.3. At this stage, the sample can be stored at -20 °C or -80 °C until you are ready to proceed.
   6. The glass slide provided with the kit is stored at -20 °C. Allow it to come to room temperature. Place the assembled glass slide in a laminar flow hood for 1–2 h at room temperature.
   7. Add 400 μL of the blocking buffer provided with the kit into each well of the assembled glass slide. Incubate at room temperature for 30 min.
   8. Centrifuge the prepared samples for 5 min at 9,000 x g to remove precipitates or particulates. Dilute 5x with blocking buffer.
   9. Remove the blocking buffer from each well. Add 400 μL of the diluted samples into the appropriate wells. Incubate for 2 h at room temperature while rocking.
   10. Decant the samples from each well. Wash 3x with 800 μL of the 1x wash buffer I provided with the kit at room temperature for 5 min each while rocking.
   11. In a clean container, submerge the assembled glass slide in 1x wash buffer I. Wash 2x at room temperature for 5 min each while rocking.
   12. Add 400 μL of 1x Cy3-conjugated streptavidin to each sub-array. Cover with plastic adhesive strips. Protect from light for the remainder of the protocol.
   13. Incubate for 2 h at room temperature while rocking.
   14. Decant the solution and disassemble the glass slide from the sample chambers.
   15. In the 30 mL centrifuge tube provided with the kit, carefully add the glass slide and enough 1x wash buffer I to cover the glass slide. Wash 3x for 10 min each at room temperature while rocking.
   16. In the 30 mL centrifuge tube, wash 2x with 1x wash buffer II for 5 min each at room temperature while rocking.
   17. Wash the glass slide with 30 mL of ddH₂O for 5 min. Remove the glass slide from the centrifuge tube and allow to dry for 20 min in a laminar flow hood. The prepared glass slide may be stored at -20 °C until ready to scan.
   18. Scan the glass slide with a microarray scanner at a fluorescence emission of 555 nm.

Results

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...

Discussion

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 rodents^11,12,13,

Materials

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