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 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.
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.
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
2. Sample Preparation
3. Running the assay and image analysis
4. Supernatant collection and protein detection
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 in the cluster. Thus, it is not possible to accurately track a neutrophil within the cluster using this method. Zymosan and E. coli bioparticle clusters both result in the generation of neutrophil swarms. For our results, Figure 2B uses data from neutrophil swarms generated by E. coli particles. Figure 3 and the other panels of Figure 2 use neutrophil swarms generated by zymosan particles. The results obtained demonstrate that bioparticle clusters stimulates neutrophil activation when a neutrophil contacted the cluster, which ultimately led to the formation of stable neutrophil swarms around each cluster after 30–60 min (Figure 2A, top). In contrast, neutrophils did not show collective migration in the absence of bioparticle clusters (Figure 2A, bottom, and Video S2). Using the fluorescent intensity of stained neutrophil nuclei at 405 nm, the average neutrophil swarm size around 30 µm diameter E. coli bioparticle clusters was found to be 1,490 ± 680 µm2 (mean ± SD, Figure 2B, top). The fluorescence intensity of a given region of interest where no bioparticle clusters are present was approximately constant over time, which confirmed the absence of collective migration in this setting (Figure 2B, bottom). Tracks of neutrophil migration show that neutrophils converged on a bioparticle cluster when one was present (Figure 2C, top). Conversely, no convergence was observed in the control system (Figure 2C, bottom). The speed (distance travelled/time) of swarming and nonactivated neutrophils was measured and a statistically significant difference in the speed distributions was found (ANOVA, p < 0.0001), as shown in Figure 2D. The average speed for swarming neutrophils was 20.6 ± 13.0 µm/min (mean ± SD), and the average speed for control neutrophils was 2.0 ± 2.2 µm/min.
Additionally, the concentration of 16 proteins that neutrophils released during swarming were analyzed (Figure 3). The normalized concentration of each protein at different time points in the swarming process (t = 0, 0.5, 1, and 3 h) were calculated, where the normalized concentration is (C – Cmin) / (Cmax – Cmin). 10 proteins increased in concentration throughout swarming. These proteins were adipsin, galectin-3, GROa, IL-6R, MIP-1a, MMP-8, MMP-9, Nidogen-1, TIMP-1, and TLR2. Two proteins (pentraxin 3 and RANK) decreased in concentration throughout swarming. The remaining four proteins (clusterin, PF4, RANTES, and Trappin-2) increased during the first hour of swarming but decreased thereafter. In other words, the concentrations of those proteins "spiked" during swarming. Of the 16 proteins identified, 12 proteins were identified in our previous publication5. Adipsin, galectin-3, nidogen-1, pentraxin 3, TIMP-1, and TLR2 were shown to be swarming-specific, while clusterin, IL-6R, MMP-8, and MMP-9, RANK, and trappin-2 were not5.
Figure 1: Production of bioparticle microarray. A silicon wafer coated with negative photoresist is exposed to UV light through a chrome photomask (A). After the silicon wafer is baked and developed, a photoresist pattern remains on the surface of the silicon wafer. This is the master wafer (B). In a Petri dish, a PDMS mixture is added to the top of the master wafer. The PDMS is cured overnight at 65 °C to form the PDMS mold (C). The PDMS mold is cut from the master wafer and a biopsy punch is used to punch out individual PDMS stamps (D). A PDMS stamp (E) is coated with CP solution (F). The stamp is inverted onto a thin layer of CP solution (G). After incubating in the CP solution for 1 h, the stamp is blotted onto a glass slide to remove excess CP. Eight stamps are then pressed onto a clean glass slide with a 5.6 ± 0.1 g weight, aligned with an imaging spacer (H). When the stamp is removed, a CP pattern remains (I). A PDMS slab with precut wells is adhered to the glass slide (J). Then, a bioparticle solution is added over the CP pattern (K). The negatively charged bioparticles bind to the positively charged polyelectrolyte via electrostatic interaction. The excess bioparticle solution is washed away, leaving bioparticles patterned on top of the CP pattern (L). (M) Fluorescent image of the CP layer labeled with FITC (Scale bars = 50 µm, large image; Scale bar = 25 µm, inset). (N) Fluorescent image of patterned zymosan bioparticles conjugated with Texas Red (Scale bar = 100 µm, large image; Scale bar = 50 µm, inset). Please click here to view a larger version of this figure.
Figure 2: Neutrophil swarm growth around bioparticle clusters. In the presence of bioparticle clusters, neutrophils undergo collective migration toward the bioparticle clusters (bottom, control neutrophils). When no bioparticles are present, the neutrophils do not perform collective migration. (A) In the presence of zymosan bioparticle clusters, neutrophils form swarms in 30 min. Without bioparticle clusters, no swarms form (Scale bars = 50 µm). (B) Neutrophil swarms grow to an average size of 1,490 ± 680 µm2 (mean ± SD) around 30 µm diameter E. coli bioparticle clusters. Control neutrophils exhibit a constant density that corresponds to no swarm growth (ANOVA, p < 0.0001, n = 32 neutrophil swarms, N = 1 donor, error bars = standard deviation). (C) Tracks of swarming neutrophils converge on the zymosan bioparticle clusters, while control neutrophils show no converging point (Scale bars = 50 µm). (D) Neutrophils swarming toward a zymosan target have a mean speed of 20.6 ± 13.0 µm/min (mean ± SD), while control neutrophils have a mean speed of 2.0 ± 2.2 µm/min. Each count on the heat map represents a neutrophil with the given instantaneous speed at the given time point. These heat maps are representative of one experiment (n = 1 swarm; N = 1 donor; ANOVA, 6,114 = swarming neutrophils, 32,116 = control neutrophils, p < 0.0001). Please click here to view a larger version of this figure.
Figure 3: Free mediators released by swarming neutrophils. Neutrophil swarming affects the production of various proteins over time. Protein concentration was measured at 0, 0.5, 1, and 3 h. The data points were fitted with smoothing splines (λ = 0.05). These proteins tend to follow one of three characteristic trends: decreasing over time, increasing over time, or spiking around 1 h and then decreasing (Error bars = standard deviation; n = 3 replicates; N = 1 donor). Please click here to view a larger version of this figure.
Video S1: Neutrophil swarming toward zymosan bioparticle clusters. Neutrophil nuclei are shown in blue. Zymosan targets are marked with red circles (Scale bar = 50 µm; original acquisition time = 60 min). Please click here to download this video.
Video S2: Nonactivated neutrophil random migration. Neutrophil nuclei are shown in blue (Scale bar = 50 µm; original acquisition time = 60 min). Please click here to download this video.
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 rodents11,12,13,15,22,23, or zebrafish11,12,15,23. Our platform uses human neutrophils, which enables us to more directly interpret our results in the context of human disease, though certain similarities between mouse and human neutrophils have been observed5,11. Additionally, we maintain a spatially-controlled swarming environment that distinguishes our platform from in vivo models by providing a high level of reproducibility that facilitates the analysis of human neutrophil collective migration as well as the collection and analysis of mediators released by swarming neutrophils.
During the development of our microstamping protocol, several challenges arose that required careful troubleshooting. First, the CP used for the microstamping is highly hydrophilic, and the PDMS stamps are hydrophobic. Because the CP does not have a high affinity for PDMS, our procedure was carefully designed to avoid the formation of bubbles and promote wetting. By first priming the stamp with CP while face-up (step 1.8), we minimize the formation of bubbles between the CP and the stamp. The stamp is then inverted onto a layer of CP and incubated for 1 h. This long incubation time ensures that every section of the stamp is wetted. Second, the process of removing excess CP before stamping on a clean glass slide (step 1.11) can be inconsistent. While performing step 1.11, the stamp must be carefully examined. When the pattern begins to become visible, the stamp is ready for step 1.12. Additionally, the required vacuum time to dry the stamps (step 1.12) can vary. This is primarily dependent on the weather. On a warm, humid day, 2 min of vacuum time is required. On a cool, dry day, 1 min of vacuum time is sufficient.
We have shown that neutrophils from healthy donors form stable swarms around bioparticle clusters. With time-lapse fluorescent microscopy, we can quantify swarm size and track neutrophil migration, which enables us to analyze neutrophil chemotaxis quantitatively5. For example, we have previously shown that this platform can be used to calculate the chemotactic index (CI, the cosine of the angle between the neutrophil velocity vector and the position vector between the neutrophil and the nearest bioparticle cluster), speed (distance the neutrophil travels divided by time), radial velocity (the speed multiplied by CI), and the total distance traveled (the difference between initial and final neutrophil position) of individual migrating neutrophils5. Unlike most in vitro studies24,25,26, our platform does not have an artificial chemotactic gradient, so neutrophil intercellular communication is the sole driving force of neutrophil migration. Additionally, the supernatant generated by swarming neutrophils is easily accessible. We can collect and analyze the supernatant for intercellular mediators released by neutrophils without interference from other cell types that are present in vivo. 16 proteins were observed whose expression during swarming could be described as one of three trends: increase (10 proteins), decrease (two proteins), and spike (four proteins). Six of these proteins confirmed previously reported trends during swarming over time5. Some of the identified proteins were previously shown to be swarming-specific, while others were expressed differentially by activated non-swarming neutrophils5. Proteins that increase in concentration over time are likely associated with the pro-inflammation response. Some of the proteins that increase in concentration over time are already known to be involved in the pro-inflammatory response (e.g., galectin-3 and MMP-9)27,28. The relationship between other proteins and inflammation is less well understood. The proteins that spike or decrease during swarming may be involved in the regulation of inflammation. However, further research is necessary to understand the role in inflammation of many of the proteins that are differentially expressed during swarming. Analyzing released mediators along with neutrophil migration can help us better understand the complex picture of inflammation and how neutrophils impact the surrounding tissue during swarming.
In future studies, our platform can be used to thoroughly study the ability of unhealthy neutrophils to generate stable swarms around patterned bioparticle clusters. Various medical conditions have been related to neutrophils, including sepsis3, trauma6, and cancer1,8,17,18, which suggests neutrophils in these patients may have altered function. This platform can be used to examine differences between the intercellular mediators released by healthy and unhealthy neutrophils. Additionally, this platform could be modified to pattern live microbes and used to analyze neutrophil response to live microbes in vitro.
In conclusion, we have developed a novel platform for analyzing neutrophil swarming in vitro. The highly controlled nature of our platform allows us to mitigate issues that arise during in vivo neutrophil swarming experiments. Neutrophil swarming on a bioparticle microarray is easily quantifiable via time-lapse fluorescent microscopy. Additionally, we can collect the mediators released by the neutrophils without the interference of other tissues that are present in vivo. This platform can be used in future research to quantify differences between the migration behaviors of neutrophils from healthy and unhealthy donors.
The authors have nothing to disclose.
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 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 |