Microfluidic assay for precise measurements of mouse, rat, and human neutrophil chemotaxis in whole-blood droplets

Abstract

Animal models of human disease differ in innate immune responses to stress, pathogens, or injury. Precise neutrophil phenotype measurements could facilitate interspecies comparisons. However, such phenotype comparisons could not be performed accurately with the use of current assays, as they require the separation of neutrophils from blood using species-specific protocols, and they introduce distinct artifacts. Here, we report a microfluidic technology that enables robust characterization of neutrophil migratory phenotypes in a manner independent of the donor species and performed directly in a droplet of whole blood. The assay relies on the particular ability of neutrophils to deform actively during chemotaxis through microscale channels that block the advance of other blood cells. Neutrophil migration is measured directly in blood, in the presence of other blood cells and serum factors. Our measurements reveal important differences among migration counts, velocity, and directionality among neutrophils from 2 common mouse strains, rats, and humans.

Keywords: inbred lines; migration; neutrophil.

Figure 1

Microfluidic platform for nonlethal measurements of murine neutrophil migration patterns. (A) For mouse models, a 50 µl droplet of capillary whole blood is taken via facial venipuncture procedure without anesthesia. Heparin anticoagulant and Hoechst stain are added to sample, and 2 µl blood is added to each microfluidic neutrophil chemotaxis assay. Each neutrophil chemotaxis assay device includes 1 WBLC surrounded by 16 FCCs primed with chemoattractant. Each device is 5 mm in diameter, and 12 assays can be run in parallel in 12‐well, glass‐bottom plates. (B) Neutrophils migrate out of the WBLC along the chemoattractant gradient(green) in the migration channel. The RBC filter that includes a 3 µm pinch and right‐angle geometries traps RBCs but allows actively migrating neutrophils to pass. A bifurcation before each FCC facilitates quantification of directional neutrophils following the chemoattractant gradient from randomly migrating cells that exit the device. Neutrophil (blue) counts accumulating in the FCC are obtained with time‐lapse imaging.

Figure 2

Microfluidic platform to measure mouse, rat, and human neutrophil migratory function from a droplet of whole blood. (A) After injury and microbial infection (green grains), damaged tissue and bacteria produce gradients of chemoattractants that act as a compass for neutrophil chemotaxis. (B) To prevent the granular flow of RBCs through the migration channels, we implemented mechanical filters that selectively block the advancement of human and murine RBCs. The RBC filter that includes a 3 µm constriction and right‐angle geometries traps RBCs but allows actively migrating neutrophils (blue) to pass. (C) The constriction just upstream of the neutrophil migration channel significantly reduces the number of mouse RBCs in the migration channel (P < 0.05). This prevents clogging of the migration channel and allows mouse neutrophils to migrate actively without obstruction. (D) RBC filtration comb. High‐resolution (40×), bright‐field images of the RBC filtration comb. A 3 µm pinch in the RBC filtration comb size excludes mouse RBCs from obstructing the upstream migration channel, whereas actively migrating neutrophils (blue‐stained nucleus—Hoechst dye) are deformable and can migrate through this bottleneck without slowing in velocity.

Figure 3

Characterization of mouse, rat, and human neutrophil migration from whole blood. (A) Neutrophil accumulation profiles in response to LTB₄ were consistent over a 3 wk time period for the same mice (Sv129S6). This suggests that a neutrophil migration baseline is feasible and could provide a reliable reference in models of disease that evolve over time. (B) Neutrophil migration from a droplet of whole blood from human healthy donors (black bars), Sv129S6 mice (129; blue bars), C57BL/6 mice (B6; red bars), and Wistar rat (gray bars) toward 2 standard chemoattractants (fMLP and LTB₄) was compared. Human cells that migrated to all chemoattractants and mouse cells (C57BL/6 and Sv129S6) only directionally migrated toward LTB₄. Rat neutrophils migrated to fMLP in lower numbers (6.9‐fold less; P < 0.05) than human neutrophils. (C) Human, mouse, and rat neutrophil migration velocities toward LTB₄ were compared. Human (22 µm/min), Sv129S6 (25 µm/min), and rat (20 µm/min) neutrophils migrated with comparable velocities, whereas C57BL/6 mouse neutrophil velocity was significantly lower (12 µm/min; P < 0.05). (D) Mouse and human neutrophil directional index toward LTB₄ was compared. Human (0.85) and Sv129S6 (0.9) neutrophils migrated with comparable directionality, whereas C57BL/6 neutrophil directional index was significantly lower (0.6; P < 0.05). Experiments presented in B–D were repeated 3 times.

Figure 4

Directional versus nondirectional cell migration toward C5a in human and Sv129S6 mouse neutrophils. (A) The percentage of neutrophils migrating toward C5a (1 µM), from a droplet of whole blood is different for humans and Sv129S6 mice (n = 3; P < 0.05). (B) The percentage of neutrophils activated, based on migration phenotype inside of the whole‐blood compartment over distances larger than 1 cell length, reveals differences between human and Sv129S6 mouse neutrophils (n = 3, P < 0.05). (C) C5a causes less‐persistent directional migration in humans and nondirectional cell activation in mouse (Sv129S6) neutrophils compared with migration toward LTB₄ (total length of axis is 1 mm distance inside of the device).