Modeling and optimization of parallelized immunomagnetic nanopore sorting for surface marker specific isolation of extracellular vesicles from complex media.

The isolation of specific subpopulations of extracellular vesicles (EVs) based on their expression of surface markers poses a significant challenge due to their nanoscale size (< 800 nm), their heterogeneous surface marker expression, and the vast number of background EVs present in clinical specimens (1010-1012 EVs/mL in blood). Highly parallelized nanomagnetic sorting using track etched magnetic nanopore (TENPO) chips has achieved precise immunospecific sorting with high throughput and resilience to clogging. However, there has not yet been a systematic study of the design parameters that control the trade-offs in throughput, target EV recovery, and ability to discard background EVs in this approach. We combine finite-element simulation and experimental characterization of TENPO chips to elucidate design rules to isolate EV subpopulations from blood. We demonstrate the utility of this approach by reducing device background > 10× relative to prior published designs without sacrificing recovery of the target EVs by selecting pore diameter, number of membranes placed in series, and flow rate. We compare TENPO-isolated EVs to those of gold-standard methods of EV isolation and demonstrate its utility for wide application and modularity by targeting subpopulations of EVs from multiple models of disease including lung cancer, pancreatic cancer, and liver cancer.

Parallelized immunomagnetic nanopore sorting: modeling, scaling, and optimization of surface marker specific isolation of extracellular vesicles from complex media.

The isolation of specific subpopulations of extracellular vesicles (EVs) based on their expression of surface markers poses a significant challenge due to their nanoscale size (< 800 nm), their heterogeneous surface marker expression, and the vast number of background EVs present in clinical specimens (10 10 -10 12 EVs/mL in blood). Highly parallelized nanomagnetic sorting using track etched magnetic nanopore (TENPO) chips has achieved precise immunospecific sorting with high throughput and resilience to clogging. However, there has not yet been a systematic study of the design parameters that control the trade-offs in throughput, target EV recovery, and specificity in this approach. We combine finite-element simulation and experimental characterization of TENPO chips to elucidate design rules to isolate EV subpopulations from blood. We demonstrate the utility of this approach by increasing specificity > 10x relative to prior published designs without sacrificing recovery of the target EVs by selecting pore diameter, number of membranes placed in series, and flow rate. We compare TENPO-isolated EVs to those of gold-standard methods of EV isolation and demonstrate its utility for wide application and modularity by targeting subpopulations of EVs from multiple models of disease including lung cancer, pancreatic cancer, and liver cancer.

Endothelial barrier function is co-regulated at vessel bifurcations by fluid forces and sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid mediator of endothelial barrier function. Prior studies have implicated mechanical stimulation due to intravascular laminar shear stress in co-regulating S1P signaling in endothelial cells (ECs). Yet, vascular networks in vivo consist of vessel bifurcations, and this geometry generates hemodynamic forces at the bifurcation point distinct from laminar shear stress. However, the role of these forces at vessel bifurcations in regulating S1P-dependent endothelial barrier function is not known. In this study, we implemented a microfluidic platform that recapitulates the flow dynamics of vessel bifurcations with in situ quantification of the permeability of microvessel analogues. Co-application of S1P with impinging bifurcated fluid flow, which is characterized by approximately zero shear stress and 38 dyn•cm-2 stagnation pressure at the vessel bifurcation point, promotes vessel stabilization. Similarly, co-treatment of S1P with 3 dyn•cm-2 laminar shear stress is also protective of endothelial barrier function. Moreover, it is shown that vessel stabilization due to bifurcated fluid flow and laminar shear stress is dependent on S1P receptor 1 or 2 signaling. Collectively, these findings demonstrate the endothelium-protective function of fluid forces at vessel bifurcations and their involvement in coordinating S1P-dependent regulation of vessel permeability.

Competing Fluid Forces Control Endothelial Sprouting in a 3-D Microfluidic Vessel Bifurcation Model.

Sprouting angiogenesis-the infiltration and extension of endothelial cells from pre-existing blood vessels-helps orchestrate vascular growth and remodeling. It is now agreed that fluid forces, such as laminar shear stress due to unidirectional flow in straight vessel segments, are important regulators of angiogenesis. However, regulation of angiogenesis by the different flow dynamics that arise due to vessel branching, such as impinging flow stagnation at the base of a bifurcating vessel, are not well understood. Here we used a recently developed 3-D microfluidic model to investigate the role of the flow conditions that occur due to vessel bifurcations on endothelial sprouting. We observed that bifurcating fluid flow located at the vessel bifurcation point suppresses the formation of angiogenic sprouts. Similarly, laminar shear stress at a magnitude of ~3 dyn/cm2 applied in the branched vessels downstream of the bifurcation point, inhibited the formation of angiogenic sprouts. In contrast, co-application of ~1 µm/s average transvascular flow across the endothelial monolayer with laminar shear stress induced the formation of angiogenic sprouts. These results suggest that transvascular flow imparts a competing effect against bifurcating fluid flow and laminar shear stress in regulating endothelial sprouting. To our knowledge, these findings are the first report on the stabilizing role of bifurcating fluid flow on endothelial sprouting. These results also demonstrate the importance of local flow dynamics due to branched vessel geometry in determining the location of sprouting angiogenesis.

Flow dynamics control endothelial permeability in a microfluidic vessel bifurcation model.

Endothelial barrier function is known to be regulated by a number of molecular mechanisms; however, the role of biomechanical signals associated with blood flow is comparatively less explored. Biomimetic microfluidic models comprised of vessel analogues that are lined with endothelial cells (ECs) have been developed to help answer several fundamental questions in endothelial mechanobiology. However, previously described microfluidic models have been primarily restricted to single straight or two parallel vessel analogues, which do not model the bifurcating vessel networks typically present in physiology. Therefore, the effects of hemodynamic stresses that arise due to bifurcating vessel geometries on ECs are not well understood. Here, we introduce and characterize a microfluidic model that mimics both the flow conditions and the endothelial/extracellular matrix (ECM) architecture of bifurcating blood vessels to systematically monitor changes in endothelial permeability mediated by the local flow dynamics at specific locations along the bifurcating vessel structure. We show that bifurcated fluid flow (BFF) that arises only at the base of a vessel bifurcation and is characterized by stagnation pressure of ∼38 dyn cm-2 and approximately zero shear stress induces significant decrease in EC permeability compared to the static control condition in a nitric oxide (NO)-dependent manner. Similarly, intravascular laminar shear stress (LSS) (3 dyn cm-2) oriented tangential to ECs located downstream of the vessel bifurcation also causes a significant decrease in permeability compared to the static control condition via the NO pathway. In contrast, co-application of transvascular flow (TVF) (∼1 µm s-1) with BFF and LSS rescues vessel permeability to the level of the static control condition, which suggests that TVF has a competing role against the stabilization effects of BFF and LSS. These findings introduce BFF at the base of vessel bifurcations as an important regulator of vessel permeability and suggest a mechanism by which local flow dynamics control vascular function in vivo.

Writing Wrinkles on Poly(dimethylsiloxane) (PDMS) by Surface Oxidation with a CO2 Laser Engraver.

Surface wrinkles formed by the buckling of a strained stiff layer attached to a soft elastomer foundation have been widely used in a variety of applications. Micropatterning of wrinkled topographies is, however, limited by process/system complexities. In this article, we report an approach to write surface wrinkles with desired pattern geometries on poly(dimethylsiloxane) (PDMS) elastomers using a commercial infrared laser engraver with a spot size of 127 µm. Wrinkled micropatterns with wavelength from <50 to >300 µm were obtained in minutes without using special facilities or atmospheres. The minimal achievable pattern sizes of one-dimensional and two-dimensional patterns and the change of the minimal achievable pattern size with wrinkle orientation were investigated under a given set of operating parameters. Sub-spot size patterning was also demonstrated. To reduce surface cracking, a typical problem in large-area wrinkle patterning, a patterning scheme that separates neighboring laser exposure areas by nonexposure gaps was developed. In addition, micropatterns with gradient wrinkles were created on the surface. This is the first report that patterns microscale surface wrinkles on elastomer surfaces using infrared laser irradiation. The simple and versatile approach is expected to provide a fast yet controllable way to create wrinkled micropatterns at low cost to facilitate a broad array of studies in surface engineering, cellular biomechanics, and optics.

Microfluidic approaches to the study of angiogenesis and the microcirculation.

Microfluidic systems have emerged as a new class of perfusable in vitro culture models that have helped advance and refine our understanding of microvascular function. Cutting-edge microfluidic models have successfully integrated principles from quantitative analysis of vascular function, in vitro flow chambers, microfabrication techniques, and 3D tissue scaffolds. Here, we review the evolution of microfluidic systems, namely their progression from 2D planar microchannel arrays to 3D microtissue analogs, and highlight their recent contributions in elucidating the role of biomolecular transport and fluid mechanical stimuli in controlling angiogenesis. Further advancement of microfluidic systems in recapitulating tissue-level phenomena in vitro, controlling important physiochemical and biological parameters, and integrating cellular and molecular analysis will help further enhance their application within the microcirculation research community.