Griddient: a microfluidic array to generate reconfigurable gradients on-demand for spatial biology applications.

Biological tissues are highly organized structures where spatial-temporal gradients (e.g., nutrients, hypoxia, cytokines) modulate multiple physiological and pathological processes including inflammation, tissue regeneration, embryogenesis, and cancer progression. Current in vitro technologies struggle to capture the complexity of these transient microenvironmental gradients, do not provide dynamic control over the gradient profile, are complex and poorly suited for high throughput applications. Therefore, we have designed Griddent, a user-friendly platform with the capability of generating controllable and reversible gradients in a 3D microenvironment. Our platform consists of an array of 32 microfluidic chambers connected to a 384 well-array through a diffusion port at the bottom of each reservoir well. The diffusion ports are optimized to ensure gradient stability and facilitate manual micropipette loading. This platform is compatible with molecular and functional spatial biology as well as optical and fluorescence microscopy. In this work, we have used this platform to study cancer progression.

Microfluidic device with reconfigurable spatial temporal gradients reveals plastic astrocyte response to stroke and reperfusion.

As a leading cause of mortality and morbidity, stroke constitutes a significant global health burden. Ischemic stroke accounts for 80% of cases and occurs due to an arterial thrombus, which impedes cerebral blood flow and rapidly leads to cell death. As the most abundant cell type within the central nervous system, astrocytes play a critical role within the injured brain. We developed a novel microphysiological platform that permits the induction of spatiotemporally controlled nutrient gradients, allowing us to study astrocytic response during and after transient nutrient deprivation. Within 24 h of inducing starvation in the platform, nutrient deprivation led to multiple changes in astrocyte response, from metabolic perturbations to gene expression changes, and cell viability. Furthermore, we observed that nutrient restoration did not reverse the functional changes in astrocyte metabolism, which mirrors reperfusion injury observed in vivo. We also identified alterations in numerous glucose metabolism-associated genes, many of which remained upregulated or downregulated even after restoration of the nutrient supply. Together, these findings suggest that astrocyte activation during and after nutrient starvation induces plastic changes that may underpin persistent stroke-induced functional impairment. Overall, our innovative device presents interesting potential to be used in the development of new therapies to improve tissue repair and even cognitive recovery after stroke.

Astrocyte innate immune activation and injury amplification following experimental focal cerebral ischemia.

Astrocytes are a distinct population of glial cells responsible for many homeostatic functions in normal neural architecture. In the healthy brain, astrocyte functions range from maintenance of the blood brain barrier to modulation of synaptic transmission and neuronal plasticity to glial scar formation post-ischemic injury. In humans, this group of cells exhibits far greater heterogeneity than previously thought-with distinct subpopulations that likely carry out specialized functions. Following ischemic injury, astrocytes take on a distinct phenotype-known as the reactive astrocyte. This phenotype is responsible for both the propagation and amelioration of neuronal injury during ischemia. Following ischemia, astrocytes undergo temporal and spatial-dependent changes in morphology, gene expression, hypertrophy and hyperplasia as a result of signaling within the local microenvironment of the penumbra compared to the core infarct. This elicits a cascade of downstream effects, including inflammation and activation of the innate immune system, which both propagates and ameliorates local injury within the brain parenchyma. This review will focus upon the double-edged sword-that are astrocytes and the innate immune system. We will discuss the role that astrocytes and the innate immune system play in amplifying secondary brain injury, as well as attenuating ischemic damage. Specifically, we will focus on molecular signaling and processes that could be targeted as potential therapeutic interventions.