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Microfluidic devices are defined by the application of fluid flow to micron-scale features. Inherent to most experiments involving microfluidic devices is the need to precisely and reproducibly control fluid flow at the microliter scale, often through multiple inlet ports on a single device. While the number of fluid channels per device varies, perfusing multiple inputs requires either the use of multiple flow controllers (often syringe or peristaltic pumps) or the ability to evenly divide fluid across outlets. Towards the latter approach, while a handful of commercial systems exist for splitting fluid flow, these set-ups require significant financial investment, multiple flow control and sensing components, and restrict the user to a predetermined perfusion control system. Simple in-line splitting devices, such a manifolds or T junctions, fail to achieve flow splitting at low flow rates often used in microfluidic systems. To increase capabilities for flow-controlled experiments, we performed experimental analyses of the physical considerations governing even flow splitting under low flow, leading to the design of a microdevice (µ-Split) that can be directly inserted into existing microfluidic set-ups. The µ-Split allows for reproducible, even flow splitting from 10 uL/min to > 2.5 mL/min. By testing multiple device geometries in combination with multiphysics simulations, we identified the design and fabrication features underlying the splitting precision achieved by the µ-Split. Overall, this work provides a useful tool to simplify microfluidic experiments that require evenly divided flow streams, while minimizing the overall device footprint. 

In this work, we present a cost effective and open-source modular cone-and-plate (MoCAP) device that incorporates shear stress in the popular Transwell^®insert system. This system acts as a lid that incorporates flow into 24-well Transwell^®inserts while preserving the ability to conduct molecular profiling assays. Moreover, the MoCAP device can be rapidly reconfigured to test multiple shear stress profiles within a single device. To demonstrate the utility of the MoCAP, we conducted select assays on several different brain microvascular endothelial cell (BMEC) lines that comprise models of the blood-brain barrier (BBB), since shear stress can play an important role in BBB function. Our results characterize how shear stress modulates passive barrier function and GLUT1 expression across the different BMEC lines. Overall, we anticipate this low cost mechanofluidic device will be useful to the mechanobiology community. 

INTRODUCTION: The morphological and molecular changes associated with the degeneration of arterioles in cerebral amyloid angiopathy (CAA) are incompletely understood. METHODS: Post mortem brains from 26 patients with CAA or neurological controls were analyzed using light-sheet microscopy, and morphological features of microvascular degeneration were quantified using surface volume rendering. Vascular stiffness was analyzed using atomic force microscopy. RESULT: Vascular smooth muscle cells (VSMCs) volume was reduced by ≈ 55% inCAA. This loss of VSMC volume correlated with increased arteriolar diameter, variability in diameter, and the volume of amyloid beta (Aβ) deposition in the vessel. Vessels with CAA were > 300% stiffer than controls. The volume of extracellular matrix cross-linking enzyme lysyl oxidase (LOX) correlated closely with vascular degenerative features. DISCUSSION: Our findings provide valuable insights into the connections among LOX, Aβ deposition, and vascular stiffness in CAA. Restoration of physiologic extracellular matrix properties in penetrating arteries may yield a novel therapeutic strategy for CAA. 

Human neural organoid models have become an important tool for studying neurobiology. However, improving the representativeness of neural cell populations in such organoids remains a major effort. In this work, we compared Matrigel, a commercially available matrix, to a neural cadherin (N-cadherin) peptide-functionalized gelatin methacryloyl hydrogel (termed GelMA-Cad) for culturing cortical neural organoids. We determined that peptide presentation can tune cell fate and diversity in gelatin-based matrices during differentiation. Of particular note, cortical organoids cultured in GelMA-Cad hydrogels mapped more closely to human fetal populations and produced neurons with more spontaneous excitatory postsynaptic currents relative to Matrigel. These results provide compelling evidence that matrix-tethered signaling peptides can influence neural organoid differentiation, opening an avenue to control stem cell fate. Moreover, outcomes from this work showcase the technical utility of GelMA-Cad as a simple and defined hydrogel alternative to Matrigel for neural organoid culture. 

Abstract It is increasingly recognized that brain microvascular endothelial cells (BMECs), the principal component of the blood‐brain barrier (BBB), are highly sensitive to soluble cues from both the bloodstream and the brain. This concept extends in vitro, where the extracellular milieu can also influence BBB properties in cultured cells. However, the extent to which baseline culture conditions can affect BBB properties in vitro remains unclear, which has implications for model variability and reproducibility, as well as downstream assessments of molecular transport and disease phenotypes. Here, we explore this concept by examining BBB properties within human‐induced pluripotent stem cell (iPSC)‐derived BMEC‐like cells cultured under serum‐free conditions in DMEM/F12 and Neurobasal media, which have fully defined compositions. We demonstrate notable differences in both passive and active BBB properties as a function of basal media composition. Further, RNA sequencing and phosphoproteome analyses revealed alterations to various signaling pathways in response to basal media differences. Overall, our results demonstrate that baseline culture conditions can have a profound influence on the performance of in vitro BBB models, and these effects should be considered when designing experiments that utilize such models for basic research and preclinical assays. image 

Fabrication of microfluidic devices by photolithography generally requires specialized training and access to a cleanroom. As an alternative, 3D printing enables cost-effective fabrication of microdevices with complex features that would be suitable for many biomedical applications. However, commonly used resins are cytotoxic and unsuitable for devices involving cells. Furthermore, 3D prints are generally refractory to elastomer polymerization such that they cannot be used as master molds for fabricating devices from polymers ( e.g. polydimethylsiloxane, or PDMS). Different post-print treatment strategies, such as heat curing, ultraviolet light exposure, and coating with silanes, have been explored to overcome these obstacles, but none have proven universally effective. Here, we show that deposition of a thin layer of parylene, a polymer commonly used for medical device applications, renders 3D prints biocompatible and allows them to be used as master molds for elastomeric device fabrication. When placed in culture dishes containing human neurons, regardless of resin type, uncoated 3D prints leached toxic material to yield complete cell death within 48 hours, whereas cells exhibited uniform viability and healthy morphology out to 21 days if the prints were coated with parylene. Diverse PDMS devices of different shapes and sizes were easily cast from parylene-coated 3D printed molds without any visible defects. As a proof-of-concept, we rapid prototyped and tested different types of PDMS devices, including triple chamber perfusion chips, droplet generators, and microwells. Overall, we suggest that the simplicity and reproducibility of this technique will make it attractive for fabricating traditional microdevices and rapid prototyping new designs. In particular, by minimizing user intervention on the fabrication and post-print treatment steps, our strategy could help make microfluidics more accessible to the biomedical research community.