Microliter Whole Blood Neutrophil Assay Preserving Physiological Lifespan and Functional Heterogeneity.

For in vitro neutrophil functional assays, neutrophils are typically isolated from whole blood, having the target cells exposed to an artificial microenvironment with altered kinetics. Isolated neutrophils exhibit limited lifespans of only a few hours ex vivo, significantly shorter than the 3-5 day lifespan of neutrophils in vivo. In addition, due to neutrophils' inherently high sensitivity, neutrophils removed from whole blood exhibit stochastic non-specific activation that contributes to assay variability. Here, a method - named "µ-Blood" - is presented that enables functional neutrophil assays using a microliter of unprocessed whole blood. µ-Blood allows multiple phenotypic readouts of neutrophil function (including cell/nucleus morphology, motility, recruitment, and pathogen control). In µ-Blood, neutrophils show sustained migration and limited non-specific activation kinetics (<0.1% non-specific activation) over 3-6 days. In contrast, neutrophils isolated using traditional methods show increased and divergent activation kinetics (10-70% non-specific activation) in only 3 h. Finally, µ-Blood allows the capture and quantitative comparison of distinct neutrophil functional heterogeneity between healthy donors and cancer patients in response to microbial stimuli with the preserved physiological lifespan over 6 days.

In Vitro Neutrophil-Bacteria Assay in Whole Blood Microenvironments with Single-Cell Confinement.

Blood is a common medium through which invasive bacterial infections disseminate in the human body. In vitro neutrophil-bacteria assays allow flexible mechanistic studies and screening of interventional strategies. In standard neutrophil-bacteria assays, both the immune cells and microorganisms are typically interrogated in an exogenous, homogeneous, bulk fluid environment (e.g., culture media or bacterial broth in microtiter plates), lacking the relevant physicochemical factors in the heterogenous blood-tissue microenvironment (e.g., capillary bed) with single-cell confinement. Here we present an in vitro neutrophil-bacteria assay by leveraging an open microfluidic model known as "µ-Blood" that supports sub-microliter liquid microchannels with single-cell confinement. In this study we compare the exogenous and endogenous fluids including neutrophils in RPMI (standard suspension cell culture media) and whole blood in response to Staphylococcus aureus ( S. aureus , a gram-positive, non-motile bacterium) in phosphate buffered saline (PBS), Mueller Hinton Broth (MHB), and human serum. Our results reveal a significant disparity between the exogenous and endogenous fluid microenvironments in the growth kinetics of bacteria, the spontaneous generation of capillary (i.e., Marangoni) flow, and the outcome of neutrophil intervention on the spreading bacteria.

Microliter whole blood neutrophil assay preserving physiological lifespan and functional heterogeneity.

For in vitro neutrophil functional assays, neutrophils are typically isolated from whole blood, having the target cells exposed to an artificial microenvironment with altered kinetics. Isolated neutrophils exhibit limited lifespans of only a few hours ex vivo, significantly shorter than the 3-5 day lifespan of neutrophils in vivo. In addition, due to neutrophil inherently high sensitivity, neutrophils removed from whole blood exhibit stochastic non-specific activation that contributes to assay variability. Here we present a method - named micro-Blood - that enables functional neutrophil assays using a microliter of unprocessed whole blood. micro-Blood allows multiple phenotypic readouts of neutrophil function (including cell/nucleus morphology, motility, recruitment, and pathogen control). In micro-Blood, neutrophils show sustained migration and limited non-specific activation kinetics (<0.1% non-specific activation) over 3-6 days. In contrast, neutrophils isolated using traditional methods show increased and divergent activation kinetics (10-70% non-specific activation) in only 3 h. Finally, micro-Blood allows the capture and quantitative comparison of distinct neutrophil functional heterogeneity between healthy donors and cancer patients in response to microbial stimuli with the preserved physiological lifespan over 6 days.