(77b) Microfluidic Assay for Neutrophil Chemotaxis towards Chemoattractant Gradients

Chemoattractants represent an essential group of molecular guidance signals that regulate the transit of leukocytes out of the mainstream of blood and into tissues at sites of inflammation. Neutrophils are uniquely sensitive to a vast array of chemoattractants including complement fragments (C5a), lipid mediators (Leukotriene B4 (LTB4)), a multitude of chemokines (Interleukin 8 (IL-8)), as well as exogenous mediators produced by pathogens (Formyl-Methionyl-Leucyl-Phenylalanine (fMLP)). Cell adhesion molecules, including fibronectin, E-selectin, and P-selectin, are also known to play an important roll in neutrophil capture from the bloodstream and subsequent transmigration into the target tissue during inflammation. The goal of this project is to validate a robust biomimetic assay for neutrophil chemotaxis towards gradients of chemoattractants and cell adhesion molecules using enclosed microfluidic channels previously developed by our group. These devices consist of non-planar, enclosed microenvironments of increasing complexity, from straight channels, to channels with posts and simple mazes. The cross-section of the channels (3 × 6 μm) is smaller than the human neutrophils, so that cells are mechanically confined and allowed to move only along the axis of the channels toward a gradient of chemoattractactant and cell adhesion molecules.

For the study, neutrophils were isolated from the whole blood of at least six healthy donors and cells were loaded into an array of twelve microfluidic devices allowing a combinatorial analysis of four chemoattractants and three cell adhesion molecules simultaneously. Cell motility was recorded on a Nikon TiE inverted microscope with a heated tissue incubating chamber set at 37 °C. Individual frames recorded at 2 min intervals and the speed of at least fifty cells per condition was measured using ImageJ. In straight channels, neutrophils undergo sustained, unidirectional motion towards a chemoattractant source. In more complex maze-like geometries, neutrophils are able to select the most direct route over 78% of the time for all chemoattractants tested.

LTB4 [100 nM] was shown to be the most potent chemoattractant and neutrophil migration speed was increased two-fold compared to fMLP [100 nM] on fibronectin [25 nM] (41.1 µm/min vs. 19.2 µm/min). The device was also used to create dose-response curves with LTB4. Interestingly, neutrophil migration speed was shown to increase in a dose-dependent manner to LTB4 concentration. P-selectin [50 ng/mL] was also shown to have a significant negative affect on neutrophil migration decreasing speed to both fMLP [100 nM] (19.2 µm/min vs. 10.7 µm/min) and LTB4 [100 nM] (41.1 µm/min vs. 11.7 µm/min). In this study, we have validated a robust and easy to use microfluidic device for the analysis of neutrophil chemotaxis. In the future, this device will be used to investigate impaired neutrophil migration in burn and transplant patients.