(482a) Characterization of the Permeability of the Brain Endothelium Due to Electroporation Using a Dynamic Microengineered Model

Abstract

The microvascular endothelial cells, which cover the interior of
the brain microvascular network, play a significant role in controlling the
transport of different molecules into the central nervous system (CNS).
Although crucial to human health by maintaining the neuroparenchymal
microenvironment and protecting the neural tissue from toxins, the blood-brain
barrier (BBB) also limits the success rate of new therapies. One significant obstacle
to drug development for targeting the CNS has been the high resistivity of the
BBB in allowing large molecules to penetrate into the CNS. Electroporation, which
is the permeabilization of the cell membrane using pulsed electric fields, is a
non-viral physical method for drug delivery [1]. We fabricated a
microengineered BBB model and evaluated its permeability to different chemicals
via delivery of pulsed electric fields by using a novel laminated microdevice
with embedded electrodes which accommodates the BBB mimic. Our results support
the hypothesis that electroporation is able to increase the permeability of the
BBB without compromising cell viability in an in vitro model.

Introduction

Organs on a chip, which are a class of microdevices that feature
a physiologically relevant function, are a method of in vitro tissue analysis.
The BBB has a significant role in screening certain molecules from the CNS and
is a major obstacle to drug development for many CNS diseases. Hence, many
researches are focused on studying the physiology of this barrier and how to permeate
through it. Booth et al. [2] developed a microfluidic BBB model
incorporating a co-culture of endothelial and astrocytes and monitored the
formation of tight junctions using transendothelial electrical resistance. In
another study Prabhakarpandian et al. [3] modeled a micro BBB using
brain endothelial cells in astrocyte conditioned medium and investigated the
effect of astrocytes on the formation of the tight junctions and the transport
of certain chemicals across the barrier. Flow induced shear force and chemical
factors are also deployed to modulate the permeability of the BBB [4]. In this
study, we stimulated the microengineered BBB model by short pulsed electric
fields (BTX ECM 830, Harvard Apparatus)) to induced cell electroporation and
disrupt the blood-brain barrier. The transport of chemicals across the BBB was
quantified as a function of molecular weight, electric field and flow rate.

Materials and Methods

The microfluidic device was fabricated using photolithography
and replication molding. A polycarbonate membrane was embedded into the device
separating an upper and lower chamber (100µm deep and 300 µm wide). Rat brain
endothelial cells (RBE4) were cultured on the membrane until the formation of a
confluent monolayer and the expression of the tight junctions. The microdevice
features a pair of microelectrodes on one side of the barrier. By applying
electric pulses of different amplitudes, the endothelial cells were reversibly
electroporated and the permeability of the barrier was changed. We injected
Dextran (Sigma) of different molecular weights into one chamber and measured
the diffused amount into the other chamber following electroporation
treatments.

Results and Discussion

Brain endothelial cells were cultured on the porous membrane
inside the microenvironment until a confluent monolayer of cells was formed. We
repeated the experiment with the presence of astrocytes on the opposite side of
the membrane. Astrocytes are star shaped glial cells in the brain and spinal
cord which support the endothelial cells and contribute to the formation of the
tight junctions. To have a better representation of the in vivo conditions,
media was perfused over the endothelial cells in the top chamber. Figure 1
shows a schematic of the experimental setup. By applying electric pulses between
the electrodes, the permeability of the barrier was temporarily increased,
allowing certain molecules of high molecular weight to penetrate through the
BBB. The permeability analysis after the electroporation and live/dead assay
confirms the reversibility of the process without compromising cell viability.

Figure 1. (a) Test setup including the BBB mimic, electrical,
and flow circuits. Two halves of the microfluidics device fabricated in glass
and PDMS are bonded together, encapsulating the membrane and forming separate
luminal and abluminal channels  in both sides of the membrane. (b) a monolayer
of RBE4 cells cultured in the microchannel of the device.

Conclusions:

We modeled the BBB inside the microfluidic device and stimulated
it by electroporation pulses to increase its permeability to certain chemicals
without affecting cell viability. Using this method we are able to quantify the
required electric field for delivery of certain drugs in a dynamic environment.

References:

[1] Garcia, P. et al, PLoS ONE, 2012, 7(11), e50482

[2] Booth, R. et al, Lab Chip, 2012, 12, 1784.

[3] Prabhak, B. et al, Lab Chip, 2013, 13, 1093.

[4] Griep, L. Biomed. Microdevices, 2013, 15, 145-150.