The effect of whole blood logistics on neutrophil non-specific activation and kinetics ex vivo.

While the exquisite sensitivity of neutrophils enables their rapid response to infection in vivo; this same sensitivity complicates the ex vivo study of neutrophils. Handling of neutrophils ex vivo is fraught with unwanted heterogeneity and alterations that can diminish the reproducibility of assays and limit what biological conclusions can be drawn. There is a need to better understand the influence of ex vivo procedures on neutrophil behavior to guide improved protocols for ex vivo neutrophil assessment to improve inter/intra-experimental variability. Here, we investigate how whole blood logistics (i.e., the procedure taken from whole blood collection to delivery of the samples to analytical labs and storage before neutrophil interrogation) affects neutrophil non-specific activation (i.e., baseline apoptosis and NETosis) and kinetics (i.e., activation over time). All the experiments (60+ whole blood neutrophil isolations across 36 blood donors) are performed by a single operator with optimized isolation and culture conditions, and automated image analysis, which together increase rigor and consistency. Our results reveal: (i) Short-term storage (< 8 h) of whole blood does not significantly affect neutrophil kinetics in subsequent two-dimensional (2D) cell culture; (ii) Neutrophils from long-term storage (> 24 h) in whole blood show significantly higher stability (i.e., less non-specific activation) compared to the control group with the isolated cells in 2D culture. (iii) Neutrophils have greater non-specific activation and accelerated kinetic profiles when stored in whole blood beyond 48 h.

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.

Liquid-liquid interfaces enable tunable cell confinement to recapitulate surrounding tissue deformations during neutrophil interstitial migration in vivo.

Cell migration is regulated by an interplay between both chemical and mechanical cues. Immune cells navigate through interstitial spaces and generate forces to deform surrounding cells, which in turn exert opposing pressures that regulate cell morphology and motility mechanisms. Current in vitro systems to study confined cell migration largely utilize rigid materials orders of magnitude stiffer than surrounding cells, limiting insights into how these local physical interactions regulate interstitial cell motility. Here, we first characterize mechanical interactions between neutrophils and surrounding cells in larval zebrafish and subsequently engineer in vitro migration channels bound by a deformable liquid-liquid interface that responds to cell generated pressures yielding a gradient of confinement across the length of a single cell. Tuning confining pressure gradients replicates mechanical interactions with surrounding cells during interstitial migration in vivo . We find that neutrophils favor a bleb-based mechanism of force generation to deform a barrier applying cell-scale confining forces. This work introduces a biomimetic material interface that enables new avenues of exploring the influence of mechanical forces on cell migration.

Microphysiological model reveals the promise of memory-like natural killer cell immunotherapy for HIV± cancer.

Numerous studies are exploring the use of cell adoptive therapies to treat hematological malignancies as well as solid tumors. However, there are numerous factors that dampen the immune response, including viruses like human immunodeficiency virus. In this study, we leverage human-derived microphysiological models to reverse-engineer the HIV-immune system interaction and evaluate the potential of memory-like natural killer cells for HIV+ head and neck cancer, one of the most common tumors in patients living with human immunodeficiency virus. Here, we evaluate multiple aspects of the memory-like natural killer cell response in human-derived bioengineered environments, including immune cell extravasation, tumor penetration, tumor killing, T cell dependence, virus suppression, and compatibility with retroviral medication. Overall, these results suggest that memory-like natural killer cells are capable of operating without T cell assistance and could simultaneously destroy head and neck cancer cells as well as reduce viral latency.

The effect of whole blood logistics on neutrophil non-specific activation and kinetics ex vivo.

While the exquisite sensitivity of neutrophils enables their rapid response to infection in vivo; this same sensitivity complicates the ex vivo study of neutrophils. Handling of neutrophils ex vivo is fraught with unwanted heterogeneity and alterations that can diminish the reproducibility of assays and limit what biological conclusions can be drawn. There is a need to better understand the influence of ex vivo procedures on neutrophil behavior to guide improved protocols for ex vivo neutrophil assessment to improve inter/intra-experimental variability. Here, we investigate how whole blood logistics (i.e., the procedure taken from whole blood collection to delivery of the samples to analytical labs and storage before neutrophil interrogation) affects neutrophil non-specific activation (i.e., baseline apoptosis and NETosis) and kinetics (i.e., activation over time). All the experiments (60+ whole blood neutrophil isolations across 36 blood donors) are performed by a single operator with optimized isolation and culture conditions, and automated image analysis, which together increase rigor and consistency. Our results reveal: i) Short-term storage (<8 h) of whole blood does not significantly affect neutrophil kinetics in subsequent two-dimensional (2D) cell culture; ii) Neutrophils from long-term storage (>24 h) in whole blood show significantly higher stability (i.e., less non-specific activation) compared to the control group with the isolated cells in 2D culture. iii) Neutrophils have greater non-specific activation and accelerated kinetic profiles when stored in whole blood beyond 48 h.

Microfluidic Model to Evaluate Astrocyte Activation in Penumbral Region following Ischemic Stroke.

Stroke is one of the main causes of death in the US and post-stroke treatment options remain limited. Ischemic stroke is caused by a blood clot that compromises blood supply to the brain, rapidly leading to tissue death at the core of the infarcted area surrounded by a hypoxic and nutrient-starved region known as the penumbra. Recent evidence suggests that astrocytes in the penumbral region play a dual role in stroke response, promoting further neural and tissue damage or improving tissue repair depending on the microenvironment. Thus, astrocyte response in the hypoxic penumbra could promote tissue repair after stroke, salvaging neurons in the affected area and contributing to cognitive recovery. However, the complex microenvironment of ischemic stroke, characterized by gradients of hypoxia and nutrients, poses a unique challenge for traditional in vitro models, which in turn hinders the development of novel therapies. To address this challenge, we have developed a novel, polystyrene-based microfluidic device to model the necrotic and penumbral region induced by an ischemic stroke. We demonstrated that when subjected to hypoxia, and nutrient starvation, astrocytes within the penumbral region generated in the microdevice exhibited long-lasting, significantly altered signaling capacity including calcium signaling impairment.

Microfluidic Tumor-on-a-Chip Model to Study Tumor Metabolic Vulnerability.

Tumor-specific metabolic adaptations offer an interesting therapeutic opportunity to selectively destroy cancer cells. However, solid tumors also present gradients of nutrients and waste products across the tumor mass, forcing tumor cells to adapt their metabolism depending on nutrient availability in the surrounding microenvironment. Thus, solid tumors display a heterogenous metabolic phenotype across the tumor mass, which complicates the design of effective therapies that target all the tumor populations present. In this work, we used a microfluidic device to study tumor metabolic vulnerability to several metabolic inhibitors. The microdevice included a central chamber to culture tumor cells in a three-dimensional (3D) matrix, and a lumen in one of the chamber flanks. This design created an asymmetric nutrient distribution across the central chamber, generating gradients of cell viability. The results revealed that tumor cells located in a nutrient-enriched environment showed low to no sensitivity to metabolic inhibitors targeting glycolysis, fatty acid oxidation, or oxidative phosphorylation. Conversely, when cell density inside of the model was increased, compromising nutrient supply, the addition of these metabolic inhibitors disrupted cellular redox balance and led to tumor cell death.

Development of a Microfluidic Array to Study Drug Response in Breast Cancer.

Luminal geometries are common structures in biology, which are challenging to mimic using conventional in vitro techniques based on the use of Petri dishes. In this context, microfluidic systems can mimic the lumen geometry, enabling a large variety of studies. However, most microfluidic models still rely on polydimethylsiloxane (PDMS), a material that is not amenable for high-throughput fabrication and presents some limitations compared with other materials such as polystyrene. Thus, we have developed a microfluidic device array to generate multiple bio-relevant luminal structures utilizing polystyrene and micro-milling. This platform offers a scalable alternative to conventional microfluidic devices designed in PDMS. Additionally, the use of polystyrene has well described advantages, such as lower permeability to hydrophobic molecules compared with PDMS, while maintaining excellent viability and optical properties. Breast cancer cells cultured in the devices exhibited high cell viability similar to PDMS-based microdevices. Further, co-culture experiments with different breast cell types showed the potential of the model to study breast cancer invasion. Finally, we demonstrated the potential of the microfluidic array for drug screening, testing chemotherapy drugs and photodynamic therapy agents for breast cancer.