(250g) Nvu-on-a-Chip: Optimizing Brain Endothelial Cell Culture for Microfluidic Modeling of the Nvu | AIChE

(250g) Nvu-on-a-Chip: Optimizing Brain Endothelial Cell Culture for Microfluidic Modeling of the Nvu

Authors 

Roy, S., New Jersey Institute of Technology
Basuray, S., New Jersey Institute of Technology
Organ-on-a-chip devices are an emerging class of in vitro models that combine microfabrication and spectroscopic techniques with cell culture to study organ physiology. We are developing an organ-on-chip model of the neurovascular unit (NVU), NVU-on-a-chip, which analyzes real-time NVU dynamics in a controlled microenvironment. NVU-on-chip incorporates human brain endothelial cells (hBECs), astrocytes and neuronal cell lines to mimic physiological phenomena of the neurovascular unit (NVU). A key component of the NVU is a selectively permeable layer of endothelial tissue, the blood-brain barrier (BBB), which prevents passage of most small particle nanotherapeutics from the bloodstream into the brain. We have optimized brain endothelial cell culture in a monoculture, microfluidic device for future incorporation with NVU tissue. Endothelial cells are seeded into a microfluidic organ-on-a-chip device containing a single microfluidic channel and two electrode interfaces; tissue integrity is analyzed with electrical impedance spectroscopy (EIS). The device contains a pair of (5 nm Titanium, 25 nm Gold) interdigitated electrodes that form the top and bottom layers. It contains a 100-300 μm thin PDMS channel (500 × 18000 μm) between the two electrodes which complete the microfluidic device. The PDMS channel is necessary to facilitate gas exchange to the cells in the microfluidic channels. Cell death is observed if other materials like laser cut PMMA channels are used. The top and bottom electrode/channel pairs sandwich an extracellular matrix (ECM) coated Transwell membrane seeded on one side with rat brain microvascular endothelial cells (RBMEC). The ECM coating needs to be uniform which allows the cells to grow in the microfluidic channel. The uniformity of the ECM coating is validated by using a fluorescent microscope.Cells mature for several days in a 37°C, 5% CO2 humidified incubator post-seeding to allow the formation of barrier properties. The fabricated device is characterized using optical imaging, permeability assays, such as fluorescence microscopy, and EIS. Optical imaging confirms endothelial cell adhesion and confluency. Fluorescence microscopy signifies the presence of ZO-1, an accessory protein indicative of tight junction formation. EIS measures resistance and capacitance across the seeded endothelial membrane. A resistance value of ~1000Ω indicates a functional blood-brain barrier, while lower values implicate a compromised or ‘open’ BBB. EIS measurements are advantageous because they provide real-time capacitance and resistance measurements of transient BBB activities, such as permeability changes. Additionally, EIS capacitance data distinguishes transcellular resistance from paracellular resistance. This novel approach provides insight to transcellular BBB kinetics as well as paracellular (tight junction) kinetics. The device will be incorporated into a more sophisticated NVU-on-a-chip. NVU-on-a-chip will be used to characterize the interaction and mechanistic pathway for drug-loaded nanoparticles.