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Lab on aChip
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Page 1 of 28

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DOI: 10.1039/C6LC00638H

Giulia Adriani 1, Dongliang Ma2,3, Andrea Pavesi1, Roger D. Kamm1,4* Eyleen L.K.
Goh2,3,5,6*
1

Singapore-MIT Alliance for Research and Technology, 138602 Singapore

Department of Research, National Neuroscience Institute, Singapore 169856
3
Neuroscience Academic Clinical Programme, Duke-NUS Medical School, Singapore
169857
4
Massachusetts Institute of Technology, Cambridge, MA, 02139 USA
5
Department of Physiology, Yong Loo Lin School of Medicine, National University of
Singapore, Singapore 117597
6
KK Research Center, KK Womens and Childrens Hospital, Singapore 229899, Singapore
2

These

authors contributed equally to the work.

* Corresponding authors

Keywords (4-6): 3D microfluidics, blood-brain-barrier, neurons, astrocytes, endothelial cells,

permeability
Correspondence should be addressed to:
Roger D. Kamm, Ph.D.
Massachusetts Institute of Technology, Cambridge, MA, 02139 USA.
Tel: 617-253-5330; Fax: 617-258-5239; E-mail: rdkamm@mit.edu
Eyleen Goh, Ph.D.
Department of Research, National Neuroscience Institute, 20 College Road, Singapore
169856
Tel: 65-6516-6701; Fax: 65-6557-0729; E-mail: Eyleen_Goh@nni.com.sg

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A 3D neurovascular microfluidic model consisting of neurons, astrocytes and cerebral

endothelial cells as blood-brain barrier

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Abstract
The neurovascular unit is a complex, interdependent system composed of neurons and neural

including endothelial cells, pericytes, and smooth muscle cells. Each cell type in the
neurovascular unit plays an essential role, either in transmitting and processing neural signals
or in maintaining the appropriate microenvironmental conditions for healthy neural function.
In vitro neurovascular models can be useful for understanding the different roles and
functions of the cells composing the neurovascular unit, as well as for assessing the effects on
neural function of therapeutic compounds after crossing the endothelial barrier. Here, we
report a novel three-dimensional neurovascular microfluidic model consisting of primary rat
astrocytes and neurons together with human cerebral microvascular endothelial cells. These
three cell types in our neurovascular chip (NVC) show distinct cell typespecific
morphological characteristics and functional properties. In particular, morphological and
functional analysis of neurons enable quantitative assessment of neuronal responses while
human cerebral endothelial cells form monolayers with size-selective permeability similar to
existing in vitro blood-brain-barrier (BBB) models.

Introduction
The neurovascular unit is composed of neurons, neural supporting cells (astrocytes, microglia,
oligodendrocytes), and vascular cells (cerebral endothelial cells, pericytes, smooth muscle
cells), along with a brain-specific extracellular matrix. This cellular composition and structure
are essential in maintaining homeostasis of the microenvironment in the central nervous
system (CNS) and in the response to neurological disease. 1 Although the widely recognized
role of the cerebral endothelium is to provide a highly selective, protective barrier (the blood
brain barrier or BBB) to control molecular transport into and out of the CNS,
an essential role in regulating neural development
2

2,3

it also plays

through the complex interplay and

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supporting cells such as astrocytes, as well as cells that comprise the vascular system

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signaling between neural and endothelial cells.

Conversely, in the case of Alzheimers

Disease, amyloid-beta accumulation in brain tissues has been found to cause dysfunction of

fully established.

Therefore, a need exists for an in vitro neurovascular model capable of

addressing these questions.

Previous in vitro studies on the neurovascular unit have largely focused on modeling
the endothelial component with the aim of testing strategies to cross the BBB with therapeutic
agents. Two-dimensional (2D) BBB models consist of endothelial cells in monoculture,
co-culture with glial cells or astrocyte-conditioned media

10-12

and neurons.

13,14

8,9

in

Similar

culture conditions have been investigated in 2D models developed within microfluidic

platforms. 11,15-18 But while these existing 2D models may recapitulate some of the important
aspects of neurovascular function, they fail to address the three-dimensional (3D) cellular
organization that is crucial to many cellular processes in vivo. In one 3D study, a multicellular
spheroid system consisting of human primary brain endothelial cells, primary pericytes and
primary astrocytes was shown to spontaneously self-organize into a defined multicellular
BBB-like structure.

19

However, this 3D system does not allow specific manipulations of the

BBB, or allow for real-time monitoring of BBB integrity or function in drug delivery studies.
A few recent studies showed 3D microfluidic BBB models of endothelial cells with astrocytes
and pericytes.

20-22

3D microfluidic platforms offer numerous advantages such as a reduction

in sample volumes and cost, as well as the capabilities to precisely control media flow and
reagent delivery, and to regulate spatiotemporal parameters of the microenvironment.

23,24

Therefore, a functional 3D neurovascular microfluidic system will be useful for understanding

cellular interactions in normal and disease-specific conditions in a 3D controlled in vitro
microenvironment overcoming many of the limitations of existing in vitro and in vivo models.
Among these 3D microfluidic BBB models recently reported, only one of them introduced
neurons in their microfluidic chambers, but did not examine the growth and function of those
3

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the BBB. 6 However, the precise role of each cellular component in these processes is not yet

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neurons. 21
Although much progress has been made, there is currently no in vitro 3D

that cross the BBB and quantitatively assessing their influence on neural cell growth and
functionality. To address this need, we developed a microfluidic device consisting of two
central 3D hydrogel regions containing neurons and astrocytes mimicking neural tissues,
flanked by two media channels, one of them hosting cerebral endothelial cells mimicking the
blood vessel wall.25 Each channel communicates with the adjacent ones offering the
possibility for the cells to interact and exchange molecular cues. We demonstrate the
capabilities of this 3D neurovascular model, termed the neurovascular chip (NVC), showing
time-dependent growth and development of each cell type by morphological and quantitative
assessments as well as their functionality by calcium imaging and permeability tests. We
believe the NVC may be useful for studies associated with neurovasculature development and
function as well as for comparative therapeutic studies with the opportunity to assess drug
effects on neural function.

Materials and Methods

Isolation and culturing of cortical neurons and astrocytes
All animal procedures and applicable regulations of animal welfare were in accordance with
the Institutional Animal Care and Use Committee (IACUC) guidelines and approved by
SingHealth IACUC, Singapore.
Primary cultures of cortical neurons were prepared from embryonic day 18 (E18) Long-Evans
rats as described previously.

26,27

Briefly, embryos were decapitated and the cerebral cortex

was carefully extracted from the brain and collected in buffer (127 mM NaCl, 5 mM KCl, 170
M Na2HPO4, 205 M KH2PO4, 5 mM Glucose, 59 mM Sucrose, 100 U/ml
Penicillin/Streptomycin, pH 7.4). Tissues were dissociated using 25 mg/ml papain. After
4

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neurovascular microfluidic system capable of concurrently screening drugs and compounds

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collection in Dulbeccos Modified Eagles Medium with GlutaMax (DMEM, Life

Technologies, Carlsbad, CA) containing 1 M HEPES, 10% foetal bovine serum (FBS) (Life

strainer to remove remaining tissue clumps. The tissue solution was aliquoted into 15 ml
centrifuge tubes. 800 l of 7.5% BSA/PBS were added to the bottom of each tube. Two layers
were formed with the tissue solution on top and BSA at the bottom. After centrifugation at
200 x g for 5 min, primary rat neurons were resuspended in Minimum Essential Medium
(MEM, Life Technologies) supplemented with glucose (0.6% wt/vol), penicillin (100 U/ml),
streptomycin (100 mg/ml), and 10% (vol/vol) FBS before seeding in the device.
For preparation of astrocytes, cortices were dissected from the brains of pups between
postnatal day 0 and 2 (P0-P2) and processed as above. After passing through cell strainer,
astrocytes were washed once, resuspended and cultured in MEM. Astrocytes were maintained
in a humidified CO2 incubator at 37 C and 5% CO2 replacing the medium every 2 days and
detached before seeding in the device using 0.05% trypsin/ethylenediaminetetraacetic acid
(EDTA) (Life Technologies). Although the optimum stages/ages for isolating astrocytes and
neurons from rodents are different, 28 these primary astrocytes (P0-2) and neurons (E18) have
been well established and routinely used in co-culture studies. 26
Culturing of endothelial cells
Experiments were performed with two types of endothelial cells: Human Umbilical Vein
Endothelial Cells (HUVEC, Angio-Proteomie, Boston, MA, USA) expressing green
fluorescence protein (GFP), and Human Cerebral Microvascular Endothelial Cells
(hCMEC/D3, Cedarlane, Ontario, Canada). Both types were cultured on collagen coated (150
g/ml) cell culture dishes with Endothelial Cell Growth Medium (EGM-2, Lonza, Basel,
Switzerland) and maintained in a humidified CO2 incubator at 37 C and 5% CO2, replacing
the medium every 2 days. Cells were detached before seeding using 0.025% trypsin/EDTA
5

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Technologies), and 100 U/ml Penicillin/Streptomycin, cells were filtered through a 70 m cell

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(Lonza). The experiments were performed with HUVEC at passage numbers P6-P7 and

Fabrication of microfluidic device

The microfluidic device is a single layer device made of poly(dimethylsiloxane) (PDMS,
Sylgard 184 Silicone elastomer kit, Dow Corning, Midland, MI, USA) by soft lithography
from a patterned SU-8 silicon wafer. Silicone elastomer and curing agent mixed at a weight
ratio of 10:1 were degassed, poured onto the photolithographically patterned SU-8 structures
and cured in the oven at 80 C for 2 h. I/O ports were created with biopsy punches and the
devices were sterilized by autoclave. Glass coverslips were plasma bonded to the PDMS layer
to create channels of about 190 m in height. Each device consists of four channels connected
for 4.36 mm in length, two for 3D hydrogels and two for culture media (Fig. 1B and 2A). 29,30
The two hydrogel channels are 580 m wide while the two fluidic channels are 920 m wide.
Each hydrogel channel is defined by 9 trapezoidal structures that have bases of 290 m and
120 m, and a height of 140 m (Fig. 2A).
Seeding of endothelial cells, neurons and astrocytes into the NVC
The microchannels were coated with 1 mg/ml Poly-D-lysine (PDL) solution (Sigma-Aldrich,
St. Louis, MO) to prevent the detachment of hydrogels from the channel walls.

31

Two

hydrogel solutions containing collagen type I (BD Biosciences, Franklin Lakes, NJ) were
prepared with collagen concentrations of 7 mg/ml (for astrocytes) and 2.5 mg/ml (for
neurons) at pH ~7.4. Primary rat astrocytes or neurons were mixed in each of their specific
hydrogel solutions at the cell densities of 0.6 106 cells/ml or 5 106 cells/ml, respectively.
The astrocyte hydrogel solution was injected into the device and allowed to polymerize in a
CO2 incubator (37 C, 5% CO2) for 30 min, followed by injection of the neuron hydrogel
solution and addition of supplemented-MEM into the lateral fluidic channel close to astrocyte
hydrogel. After collagen polymerization in the CO2 incubator (37 C, 5% CO2) for 30 min,
6

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hCMEC/D3 at P31-P33.

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supplemented-MEM was injected into the lateral fluidic channel adjacent to the neuron
hydrogel. Medium was changed to neurobasal medium (NB) supplemented with 1x

neuron hydrogel. The two media were refreshed every 24 h for 7 days before seeding of
endothelial cells. After 7 days, the fluidic channels were incubated with collagen (100 g/ml)
in PBS for 45 min in a CO2 incubator (37 C, 5% CO2) to promote endothelial cell adhesion.
After coating, HUVEC (5 106 cells/ml) or hCMEC/D3 (8 106 cells/ml) in EGM-2 were
seeded in the fluidic channel adjacent to the astrocyte hydrogel. Non-adherent cells were
removed 2 h after seeding by washing the devices three times with 100 l of cell culture
media. Both supplemented neurobasal media and EGM-2 were refreshed every 24 h in the
devices for 4 (HUVEC) or 7 days (hCMEC/D3).
Immunocytochemistry and confocal imaging
Primary antibodies against doublecortin (DCX, Santa Cruz Biotechnology, Dallas, TX, USA),
glial fibrillary acidic proteins (GFAP, Merck Millipore, Billerica, MA, USA), an intercellular
junction protein, VE-cadherin (ENZO Life Sciences, Farmingdale, NY, USA), and a tight
junction protein, zonula occuldens-1 (ZO-1, Life Technologies), were used as markers to
identify neurons, glial and endothelial cells, respectively. Antibody against an immediate
early gene, c-Fos (Santa Cruz Biotechnology, Dallas, TX, USA) was used as a marker for
neuronal activity. Cells were fixed with 4% paraformaldehyde (Sigma) for 15 min at room
temperature, permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked with 3%
BSA/5% Donkey serum in PBS for 2 h. Cells were then incubated with primary antibody
DCX (1:500), GFAP (1:2000), c-Fos (1:500), VE-cadherin (1:100) or ZO-1 (1:100) in PBS
overnight at 4 C. The devices were rinsed five times with PBS and incubated in secondary
antibodies (1:500) and NucBlue (Life Technologies) in 0.5% BSA/0.5% donkey serum in
PBS for 3 h at room temperature. For endothelial cells, actin filaments (F-actin) were
7

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GlutaMAX-I and 1x B27 (Life Technologies) after 24 h only in the fluidic channel adjacent to

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identified by incubation with rhodamine phalloidin (1:100) (Life technologies) for 1 h at room
temperature. After incubation, the devices were washed five times with PBS and imaged with

contrast and confocal imaging were used for morphological observations.

Calcium imaging
Cortical primary neurons were washed twice with loading buffer containing (in mM): 118
NaCl, 4.69 KCl, 4.2 NaHCO3, 1.18 KH2PO4, 0.8 MgCl2, 2.0 CaCl2, 20 HEPES, and 30
glucose, with pH 7.4 and incubated with a final concentration of 1 M X-rhod-1 (Molecular
Probes/Invitrogen, Carlsbad, CA) for 30 min at 37 C. Excess dye was removed by washing
twice with loading buffer followed by an additional 20 min incubation to equilibrate
intracellular dye concentration and allow de-esterification. For functional testing of neurons
growing in the 3D hydrogel, 1.0 M KCL was added to the cells for 90 s before washing out
with loading buffer. For endothelial cell barrier functional testing, six devices were seeded
with endothelial cells and six devices without endothelial cells. 1.0 mM monosodium
glutamate (Sigma-Aldrich) was added to the fluidic channel adjacent to the astrocyte hydrogel
after 1 min of baseline recording, for at least 10 min. Time-lapse image sequences of 312
frames were acquired with a sampling rate of 0.64 Hz with a region of 512 x 512 pixels, with
a 534 nm filter on a LSM710 inverted confocal fluorescence microscope. Images were
acquired over a time period of 10 min with ZEN software (Carl Zeiss Pte. Ltd.). c-Fos
immunostaining was done 30 min after applying monosodium glutamate.
Permeability assay
10 kDa Oregon green 488 dextran (Life technologies) and 70 kDa Texas red dextran (Sigma)
were used as fluorescent tracers to assess the permeability of the endothelial barrier. After 4
or 7 days of endothelial cell culture in the device, the cell culture media was removed from
the I/O reservoirs and dextran in EGM-2 was injected through the fluidic channel containing
8

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Zeiss LSM710 or LSM780 confocal microscopes (Carl Zeiss, Pte. Ltd., Singapore). Phase

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endothelial cells (lumen side) simultaneously with blank media in the other fluidic channel to
maintain equal hydrostatic pressures in the device. Fluorescence images were captured before

with an IX81 inverted fluorescence microscope (Olympus, Tokyo, Japan) using a 4X

objective.
Quantification of permeability coefficient
The permeability of the endothelial barrier was quantified by measuring the rate of dextran
diffusion across the barrier immediately after dextran injection into the lumen side of the
device. Considering the principle of mass conservation, the rate at which dextran crosses the
endothelial barrier (J) from the lumen side equals the rate at which it accumulates in the
hydrogel, as reflected in the equation:

J = Ab PC =

d
dt

C dV

(1)

where Ab is the surface area of the endothelial barrier at the interface between the gel region
and the endothelial channel that fluorescent molecules have to pass to reach the hydrogel
(Ab=lbh, where lb is the length of the barrier and h is the height of the channel), P is the
permeability coefficient, C is the concentration change across the barrier, and the integral is
taken over the volume (V) of the gel region.
Considering the dextran concentration to be proportional to the fluorescence intensity:

J = Ab PI =

d
dt

I dV = dt (I

V)

ave

(2)

where is the proportionality constant defined by the equation C = I, I is intensity change

between the two sides of the barrier during the permeability measurement, Iave is the volumeaveraged intensity within the gel region, and V is the hydrogel volume (V=Agh, where Ag is
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the fluorescent solution injection (background) and every min after the injection for 20 min

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the surface area in the hydrogel region). Simplifying the above algebraically, we compute the

d
d
d
I
I
I
V dt ave Ag h dt ave Ag dt ave
P=
=
=
lb h I
Ab I
lb I

(3)

Where Ag is 158676 m2 and lb, calculated as distance between two trapezoidal posts, is 195
m. The fluorescent intensity values were computed from the fluorescence images with
ImageJ (NIH).
Neurite analysis
Neurite growth was analysed with the IMARIS software (Bitplane, Zurich, Switzerland).
Specifically, the imaging software defines a neurite segment as the portion of a neurite
between two branch points and the branch level as a number that increases moving outward.
The branch level is determined by the relative diameter of the individual segments. The initial
branch level of each neurite was set to 1. At each branch point, the branch level of the
segment with smaller relative mean diameter increases by one, while the segment with the
greater mean diameter maintains the same branch level. Five regions of interest for each
experimental group were imaged for the analysis.
Statistical Analysis
All data were plotted as mean SEM and a student t-test or one-way ANOVA were used to
determine if two (t-test) or more (ANOVA) datasets were statistically different from each
other. At least three devices ( 3 regions per device) for each condition were used for the
imaging and data analysis unless otherwise stated.

Results and Discussion

Co-culture of endothelial cells, astrocytes and neurons in microfluidic device
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permeability coefficient P as:

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We established a neurovascular in vitro model using astrocytes and neurons to mimic the
brain tissues with endothelial cells to form the blood vessel. This neurovascular in vitro model

accommodate the different cells separately in a single platform (Fig. 1B), we fabricated a
microfluidic device with a patterned PDMS surface having four channels after being plasmabonded to a glass coverslip (Fig. 2A). Each channel communicated directly with the adjacent
ones allowing cell contact and signaling. Two channels in the middle of each microfluidic
device were designated for 3D hydrogels while the two flanking fluidic channels contained
different culture media. Each channel was optimized for the needs of each cell type, in terms
of the coatings, hydrogels and media used.
One optimization consists of the concentration of collagen in the hydrogel for the
growth of each cell type. A collagen type I hydrogel with a concentration of 2.5 mg/ml was
initially used for seeding both astrocytes and neurons in their respective channels, following
previous works on cell-cell interactions and migration in collagen.

21,32-36

Under these

conditions, however, we observed significant gel contraction in the region containing

astrocytes (Fig. S1A). To limit gel contraction, the collagen concentration was increased to 7
mg/ml (Fig. S1B,C). The collagen concentration of 2.5mg/ml was found acceptable for
neuron growth and kept unchanged. Therefore, seeding neurons and astrocytes in different
compartments allows each to be manipulated and studied independently and also makes it
possible to determine if the effect of any specific compound is direct or acts indirectly,
mediated by the other cell type. Moreover, it is well-established that different culture
conditions are required for optimum growth and development of neurons and astrocytes.

28

Culturing neurons and astrocytes in different chambers could also limit, at least partially, the
flow and diffusion of media and factors between adjacent compartments. This NVU could
have the flexibility to specifically examine interactions between astrocytes and neurons, if
desired, by limiting the physical interaction area between both cell types similarly to what has
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was designed based on the organization of the in vivo neurovascular unit (Fig. 1A). To

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been done in Gao et al. 37

The co-culture in this NVC was established by seeding primary astrocytes and neurons

two central gel channels labeled in blue and orange, respectively (Fig. 2A and B). Both
astrocytes and neurons were cultured in the device with supplemented-MEM for 24 h. From
day 1 onwards, supplemented-MEM or supplemented-NB were injected into the designated
medium channels (Fig. 2B). At day 7, endothelial cells were seeded in the medium channel
(shown in green in Fig. 2A and B) adjacent to the hydrogel containing astrocytes. The growth
and development of endothelial cells, primary astrocytes and primary neurons in their
respective microfluidic channels were monitored over time. Phase contrast images showed
that the three different cell types were able to grow in their designated channels optimized for
their growth and development (Fig. 2C, Fig. S2).
Although it would be preferable to use cells isolated from subjects of the same species
and same age, it is not feasible to isolate or purchase human primary neurons and astrocytes
for routine experimental needs. The more affordable and readily accessible primary cells
isolated from rodents have frequently been used to recapitulate cellular phenomena or
processes in experimental models of the brain vasculature and the BBB in co-culture with
human cells12,38. Thus, we chose to use primary neurons and astrocytes isolated from rats for
our studies to establish a 3D neurovascular microfluidic model consisting of rat neurons and
astrocytes together with human cerebral endothelial cells. As the ultimate aim of this work is
to develop this NVC for drug screening, future work includes differentiating neural cells and
endothelial cells from the same patient-derived induced pluripotent stem (iPS) cells
reprogrammed from healthy controls or subjects with neurological disorders.
Growth and characterization of multiple cell types in the NVC
Astrocytes are critical components of the neurovascular unit and their interactions with
cerebral endothelial cells have been shown to modulate endothelial barrier function.
12

20,32

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suspended in their specific hydrogel solutions, one after another on day 0 (Fig. 2B) into the

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Specifically, astrocytes cultured with endothelial cells in 3D matrices have been shown to
increase BBB permeability in response to TGF-1 stimulation which is known to increase

monoculture. 39 The presence of astrocytes and endothelial cells also supports the growth and
maturation of neurons.

4,40,41

Although neurons are not considered as cellular component of

the BBB, they are important component of the neurovascular unit.

42-44

To characterize the

multiple cell types in the 3D microfluidic devices, immunocytochemistry was used to stain
the different cell types. Doublecortin (DCX) is a microtubule-associated protein specific for
immature neurons, and the antibody against DCX allows a clear visualization of the cell body
and neurites. Astrocytes were identified with an antibody against glial fibrillary acidic protein
(GFAP). The side and top views of the three types of cells in the 3D microfluidic devices
showed neurons identified by DCX, astrocytes positive for GFAP while HUVEC were GFP
labeled (Fig. 3A). Neurons and astrocytes in 3D hydrogels showed specific cell morphologies
that are signatures of both cell types: neurons showed neurite outgrowth (Fig. 3B,C) while
astrocytes exhibited characteristic star-shaped morphology (Fig. 3C). Endothelial cells formed
a monolayer in the channel and expressed ZO-1 (Fig. 3C). All three cell types were able to
grow, express cell-specific markers and display morphological characteristics specific for
each individual cell type.
Human endothelial cells in microfluidic channels show morphological and functional
characteristics of an endothelial barrier
We assessed the integrity of the in vitro monolayer formed by endothelial cells. Two human
endothelial cell lines were compared in our microfluidic device, namely HUVEC isolated
from umbilical veins and hCMEC/D3 isolated from cerebral microvasculature. The
hCMEC/D3 cell line is known to exhibit typical BBB characteristics, such as expression of
endothelial-specific junction proteins, expression of efflux transporters such as P-glycoprotein
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vascular permeability while no effect was observed in 2D co-culture or endothelial

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and influx transporters such as glucose transporter, and importantly has the ability to exclude
drugs. 45 Moreover, both cell lines have been used in previous BBB models for a variety of

cells

48,49

stimuli.

50

as well as for investigating the cerebral endothelium response to inflammatory

Indeed, both HUVEC and hCMEC/D3 in our NVC expressed F-actin, the

intercellular junction protein VE-cadherin (Fig. 4A,B) and the tight junction protein ZO-1
(Fig. 3C and S3), and were able to form monolayers along the walls of the media channels by
4 and 7 days, respectively. Representative 3D reconstructions (Fig. 4C,D) and cross sections
(Fig. 4E,F) of the endothelial monolayers showed the integrity of the wall on the side of the
channel at the interface between the medium channel and the astrocyte-containing hydrogel.
Although one microfluidic-based study has shown increase ZO-1 expression in brain
endothelial cells in the presence of astrocyte-conditioned medium (ACM),
either showed no significant differences upon the addition of ACM

21

11

other studies

or did not make any

direct comparison of ZO-1 expression between endothelial cells in monoculture and coculture conditions.

17,22

Our data show a trend towards increased ZO-1 expression in

endothelial cells cells co-cultured with astrocytes as compared to those in monoculture,

although this increase was not statistically significant (Fig. S3). These data are in agreement
with a previous study using primary human brain-derived microvascular endothelial cells
(hBMVEC) and primary human astrocytes

21

that showed an increase trend but not

statistically significant change in ZO-1 expression in the ACM-treated endothelial cells as

compared to the untreated endothelial cells.
To investigate if this endothelial monolayer alongside the channel constituted a
functional barrier, fluorescent dextrans with different molecular weights were used as tracers
to assess their permeability across the endothelial wall. Another methods to assess tight
junction formation is to measure TEER, but this method was not used here due to the
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studies such as nanoparticle uptake by the BBB 46,47 and interactions of BBB with immune

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difficulty in controlling the variability in the measurements with factors such as temperature,
culture media, media volume and hydrogels with or without cells. Instead, we relied on the

in combination with immunofluorescent labeling of the junctional proteins.

Permeability, P, for 10 kDa and 70 kDa dextrans was first computed for the
monoculture of endothelial cells. The permeability time points chosen for hCMEC/D3 and
HUVEC were 7 days in vitro (DIV) and 4 DIV, respectively. The different time points
represent the observed days in which the respective endothelial monolayers were fully formed
and had not yet begun to detach from the gel. Permeability at 7 days in vitro (DIV) for
hCMEC/D3 for 10 kDa and 70 kDa dextrans were 1.23 10-5 cm/s and 3.3 0.03 10-6 cm/s,
respectively (Fig. 4G). Permeability at 4 DIV for HUVEC for 10 kDa dextrans and 70 kDa
were 6.58 10-5 cm/s and 4.23 10-5 cm/s, respectively (Fig. 4G, Fig. S4A). These
permeability values for 10 kDa and 70 kDa dextrans showed that both barriers formed by
HUVEC and hCMEC/D3 were selective according to size; barriers presented statistically
significant higher permeability to tracers of lower molecular weight. Notably, the monolayer
walls formed by hCMEC/D3 showed significantly lower permeability and retained their
barrier integrity longer (7 DIV) as compared to those formed by HUVEC (4 DIV). Our data
for different time points indicated that the culture period in vitro is an important factor for the
permeability of endothelial cells. Moreover, to obtain a functional barrier we optimized the
cell seeding density to 5 106 cells/ml for HUVEC and 8 106 cells/ml for hCMEC-D3.
Lower concentrations did not form an endothelial barrier (data not shown).
We then determined if these permeability values change when HUVEC and
hCMEC/D3 cells are cultured in the NVC containing astrocytes and neurons (Fig. S4B and
S5B). In the co-culture systems, permeabilities for hCMEC/D3 of HUVEC were in the range
of ~10-5 - 10-6 cm/s (Fig. 4H) and both HUVEC and hCMEC/D3 barriers remained selectively
permeable in co-culture conditions. Although HUVEC barriers in co-culture show lower
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permeability coefficients for molecular fluorescent dextrans in the different culture conditions

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permeability coefficients for both 10 and 70 KDa dextrans as compared to HUVEC

monoculture (Fig. 4G), hCMEC barriers in co-culture exhibited somewhat higher

may be due to several factors. For example, as the cells in the co-culture experiment are from
different species, this may have an adverse effect on the vascular permeability in the coculture condition. However, to confirm whether the species difference plays a role,
comparative studies using the same conditions and methodology would be necessary.
Although still far from the in vivo permeability values for 10 and 70 KDa dextrans
(~10-7 cm/s) measured from microvessels in rats,

51

our permeability values fall within the

previously reported range of values for 3D monolayers in microfluidic devices (~10-5 - 10-6
cm/s using 3, 10 and 70 KDa Dextrans).

17,22,52,53

These data suggest that human endothelial

cell lines can be used for studies on neurovasculature function, for comparative large
molecule screening, and also have the advantage of being readily available and highly
reproducible.

54

Other possible modifications to the present NVC to reduce permeability

include the addition of pericytes and flow-mediated shear stress on the endothelial layer that
have been implicated to influence endothelial barrier integrity in in vitro BBB models. 10,20 In
addition, it would be beneficial to include the trans-endothelial electrical resistance (TEER)
measurement as another assessment of permeability and BBB integrity.

Primary neurons show neurite outgrowth and exhibit neuronal activity in 3D

Neuronal development within the 3D matrices was examined by morphological and functional
analysis. For morphological analysis, we examined the growth of these neurons in the 3D
hydrogel within the NVC over time (Fig. 5). 3D reconstructed images (Fig. 5A) show that the
neurons in co-culture with astrocytes and HUVEC were growing progressively in 3D within
the hydrogel over the three time points examined (3, 7 and 11 DIV). This progressive growth
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permeability coefficients as compared to monoculture. This is a limitation of the system that

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was documented by analysing the extent of neurite branching of each individual neuron (Fig.
5B), total neurite length per neuron (Fig. 5C) and the complexity of neurite outgrowth (Fig.

Only 1.21% of neurons fall in the 700 - 1400 m range and 0% neuron has a total length of
1400 - 2100 m. At 7 DIV and 11 DIV, the percentages of neurons within 0 - 700 m, 700 1400 m and 1400 - 2100 m are 92%, 7.4%, 0.57% and 88.5%, 10.6%, 0.92%, respectively
(Fig. 5C). This increase in total neurite length over time in culture can be attributed to
increased complexity of neurite outgrowth as demonstrated by the increased number of
neurite segments for each branch level over time (number of neurite segments was normalized
with the total number of neurons in the ROI) (Fig. 5D). Furthermore, the presence of
astrocytes was found to play an important role in neurite outgrowth as shown by the increased
number of neurite segments for each branch level (Fig. 5E) and by the higher number of
neurite segments per neuron (Fig. 5F) when neurons were in co-culture with astrocytes or in
triple co-culture with astrocytes and endothelial cells compared to the neurons in monoculture.
To further verify if these neurons growing in the 3D matrix were functional, we
measured neuronal activity using calcium imaging. Calcium oscillations upon chemical
stimulation such as potassium chloride (KCl) are indicators of neuronal activity. This type of
calcium oscillations is typically detected in primary neurons cultured on 2D after 10 days in
culture. Thus, we performed calcium imaging at 11 DIV. A fluorescent calcium indicator, XRhod-1 was added to the neurons in the NVC and the fluorescence intensity of randomly
selected neurons were tracked with confocal microscopy before and after stimulation with
KCl (Fig. S6A). We found that KCl stimulations resulted in a significant increase in calcium
concentrations in the neurons as demonstrated by the fluorescence intensities of a
representative neuron (Fig. S6B) and the normalized fluorescence intensities of multiple
neurons (n=5) (Fig. S6C).

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5D-F). At 3 DIV, almost all neurons (98.8%) have total neurite length of 700 m and below.

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Application of the NVC for compound testing

To assess the possibility of using our NVC for compound testing, we designed a functional

barrier by assessing neuronal activity. Glutamate is a potent neurotransmitter that plays a

critical role in neural function. Thus, the concentration of glutamate in the brain is tightly
regulated as compared to other tissues, largely through the presence and specific function of
the BBB. Therefore, if the endothelial cell barrier in our NVC is functional, it will prevent the
monosodium glutamate added into the endothelial cell channel to pass through the barrier and
activate the neurons. For this purpose, we measured neuronal activity by calcium imaging and
c-Fos expression. c-Fos is an immediate early gene that indicates neuronal activation.
Monosodium glutamate injected into the endothelial cell channel resulted in a significant
increase of calcium concentration in the neurons within the devices without endothelial
barrier compared to the devices with endothelial cells (Fig. 6A). This suggests that the
presence of the endothelial barrier restricted the passage of glutamate and the consequent
increase of calcium in neurons. Biochemically, we observed a significant decrease in the
density of c-Fos-immunopositive neurons in devices with endothelial barrier as compared to
devices without endothelial cells (Fig. 6B,C).
Notably, the response time and magnitude of increase in the fluorescence intensity of
these neurons in the 3D hydrogel are considerably lower compared to other studies that added
monosodium glutamate directly onto the cells in media. Our data show a 30% increase upon
addition of glutamate in fluorescence intensity in the NVU without endothelial cells as
compared to that with endothelial cells (Fig. 6A). KCl treatment shows a 15% increase in
fluorescence intensity in the neurons (Fig. S6C). These magnitudes of change are
considerably lower compared to other studies that added monosodium glutamate or KCL
directly onto the cells growing in media probably because the compounds in our system have
to diffuse through the hydrogel in order to reach the cells. The difference caused by the
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assay with monosodium glutamate to determine the functionality of the endothelial cell

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compounds having to diffuse through a hydrogel in order to reach the cells may be especially
obvious for readouts that exhibit a quick response as in calcium imaging. On the other hand,

of all DCX positive neurons that are c-Fos positive as compared to the ~20 % in untreated
control. These data are consistent and comparable with previous publications using similar
compound and readout assays. 55,56
Our results indicate that both primary neurons in the 3D hydrogel and the endothelial
cell barrier were functional within the NVC. Therefore, neurons in the devices could
potentially be used to test compounds that are known to influence survival, growth or function
of neurons after they have crossed the endothelial barrier. Moreover, it will be possible in the
future to examine diseased neurons or patient-derived neurons morphologically and
functionally for their responses to drugs or compounds passing through the BBB.

Conclusions
We have developed a NVC containing neurons and astrocytes mimicking the brain tissues and
cerebral endothelial cells mimicking the blood vessel wall. We demonstrate functionality of
this 3D microfluidic model showing that each cell type in the co-culture system exhibits celltype specific morphology and express characteristic cellular markers. Moreover, quantitative
measurements of neurite outgrowth suggest the possibility to compare the effect of a variety
of compounds and factors on neural growth and maturation. Endothelial cell functionality is
confirmed by permeability tests that demonstrate the selectivity of the endothelial monolayer
while neural functionality is demonstrated by calcium imaging. Finally, the present platform
supports the development of a more sophisticated and complex 3D in vitro neurovascular
model, including the addition of other cell types present in the neurovasculature such as
pericytes and microglia. Thus, we believe that the NVC will be a useful platform for

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c-Fos expression examined 30 min after the addition of monosodium glutamate shows ~ 80 %

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neurovascular studies such as comparative therapeutic tests with the opportunity to assess

Acknowledgements
This work was supported by Competitive Research Program (CRP) funds from National
Research Foundation (NRF-CRP 002-082), Singapore, the GlaxoSmithKline (GSK)
Academic Center of Excellence (ACE) Award, Abbott Nutrition - Cognition Center of
Excellence, Singapore R&D, the National Research Medical Council (NMRC) - Collaborative
Research Programme Grant (CBRG) - (NMRC/CBRG/0094/2015) and the Ministry of
Education (MOE) TiER 2 (MOE2015-T2-1-022) to E.L.G. and by the National Research
Foundation (NRF), Prime Ministers Office, Singapore, under its CREATE
programme, Singapore-MIT Alliance for Research and Technology (SMART) BioSystems
and Micromechanics (BioSyM) IRG.

Author contributions
G.A., D.M., A.P. and E.L.G. designed the NVC. G.A. fabricated the devices, optimised 3D
gel components, performed endothelial barrier characterisation, analysed data for the
experiments and performed the neurite outgrowth analysis. D.M. isolated and cultured
primary astrocytes and neurons, performed neurite outgrowth assays, calcium imaging and
analysed data for the experiments. A.P. assisted with the experimental work. G.A., D.M. and
E.L.G. wrote the manuscript. R.D.K. co-supervised the project and provided critical inputs to
the experimental design and writing of the manuscript. E.L.G. initiated and directed the entire
study, designed experiments and analysed data.

Additional Information
Competing financial interests: The authors declare no competing financial interests.

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Figure 1. Neurovascular Unit. (A) Schematic representation showing blood vessel, astrocytes
and neurons within the brain tissue. Insert shows selectivity of the endothelial monolayer. (B)
Scheme of the four channels in the neurovascular chip (NVC) and the location of the three
different cell types. From left to right: medium (pink), neurons (orange), astrocytes (blue), and
endothelial cells (green).

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Figure 2. (A) Photo of the device with gel channels filled with food dye for visualization
purpose (left), schematic layout of the 3D neurovascular chip (NVC) and enlarged view of the
channels: two central hydrogel regions for co-culturing astrocytes (blue) and neurons (orange),
and two side channels for hosting endothelial cells and media (green and red, respectively).
(B) Time line of the experiment. (C) Phase contrast images showing growth of primary
neurons, primary astrocytes and endothelial cells (HUVEC and hCMEC/D3), in their
respective microfluidic channels.

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x
y

200 m

GFAP

GFP

200 m

200 m

C DCX

DAPI GFAP DAPI

50 m

ZO-1
DAPI ZO-1 DAPI GFAP
HUVEC
hCMEC/D3
DAPI

50 m

50 m

50 m

GFP

50 m

Figure 3. Immunocytochemistry of primary neurons, primary astrocytes and endothelial cells

with specific cell type markers. (A) Representative images showing top and side views of the
three cell types in 3D co-culture: neurons (red), astrocytes (white), GFP-labeled HUVEC
(green). (B) 3D view of the neuron gel region. (C) Representative images (left to right)
showing immature neurons identified by DCX, astrocytes characterized by GFAP, HUVEC
and hCMEC/D3 expressing ZO-1 and GFAP positive astrocytes (red) residing in close
proximity with GFP-labeled endothelial cells (green) in the NVC.

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DCX DAPI

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Figure 4. Endothelial barrier characterization. Representative images showing HUVEC

(DIV4, A) and hCMEC/D3 (DIV7, B) monolayers expressing Hoechst, F-actin and VEcadherin. 3D visualization (C,D) and sections (E,F) of the endothelial walls for HUVEC (C,E)
and hCMEC/D3 (D,F). (G) Graph showing calculated permeability coefficients in
monoculture (with ECs only) for 10kDa and 70kDa dextrans comparing HUVEC (DIV4) and
hCMEC/D3 (DIV7). (H) Graph showing calculated permeability coefficients for 10kDa and
70kDa dextrans comparing HUVEC (DIV4) and hCMEC/D3 (DIV7) in the triple co-culture
system. Data show mean values and SEM. Students t-test. *p<0.05, **p<0.01, ***p<0.001,
****p<0.0001.

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Figure 5. Morphological assessment of primary neurons in 3D hydrogel. (A) Representative

3D reconstructed images showing neurons growing in 3D gel at 3, 7 and 11 DIV. (B)
Representative tracings of neurites reconstructed from confocal images and color-coded by
branch level at 3, 7, and 11 DIV. (C) Graph plotting the total neurite lengths of each neuron at
3, 7 and 11 DIV. The filaments in the graph represent the actual neurites for each neuron. (D)
Graph showing the total number of neurite segments for each branch level normalized with
the total number of neuron in the region examined at the 3 different time points as indicated.
(E) Graph showing the total number of neurite segments for each branch level normalized
with the total number of neuron in the region examined at 11 DIV for the 4 different
conditions: only neurons (N); neurons and astrocytes (N+A); neurons, astrocytes and
hCMEC/D3 (N+A+hCMEC/D3); neurons, astrocytes and HUVECs (N+A+HUVECs). (F)
Graph showing the total number of neurite segments in neurons at 11 DIV for the 4 conditions
as indicated. All data in D, E and F show mean values and SEM. Statistical analysis students
t-test. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001. At least 5 ROI for each condition were
used for analysis (C, D, E, F).

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Lab on a Chip Accepted Manuscript

Published on 08 December 2016. Downloaded by National University of Singapore on 09/12/2016 07:07:17.

DOI: 10.1039/C6LC00638H

Lab on a Chip

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Figure 6. Calcium imaging and c-Fos expression show reduced glutamate transport across the
endothelial barrier in the NVC. (A) Graph showing average normalized changes in
fluorescence intensities (F / F0) of X-Rhod-1 (measuring calcium concentrations) of neurons
at 11 DIV from 3-5 NVC units per condition. Data show mean values and SEM. Arrow
indicates the addition of 1 mM monosodium glutamate to the endothelial cell channel after
approximately 1 min of baseline fluorescence recording. (B) Representative
immunofluorescence images showing c-Fos (green), DCX (red) and DAPI (blue) in neurons.
(C) Graph showing percentage of cells immunopositive for both c-Fos and DCX over total
DCX positive cells in NVC unit with or without endothelial cells.

28

Lab on a Chip Accepted Manuscript

Published on 08 December 2016. Downloaded by National University of Singapore on 09/12/2016 07:07:17.

DOI: 10.1039/C6LC00638H