The hCMEC/D3 cell line as a model of the human blood brain barrier
Fluids and Barriers of the CNS
The hCMEC/D3 cell line as a model of the human blood brain barrier
Babette Weksler 2
Ignacio A Romero 1
Pierre-Olivier Couraud 0 3 4
0 Inserm, U1016, Institut Cochin , Paris , France
1 Department of Life, Health and Chemical Sciences, Open University , Milton Keynes, U.K
2 Weill Cornell Medical College , New York, NY , USA
3 Universite Paris Descartes, Sorbonne Paris Cite , Paris , France
4 CNRS, UMR8104 , Paris , France
Since the first attempts in the 1970s to isolate cerebral microvessel endothelial cells (CECs) in order to model the blood-brain barrier (BBB) in vitro, the need for a human BBB model that closely mimics the in vivo phenotype and is reproducible and easy to grow, has been widely recognized by cerebrovascular researchers in both academia and industry. While primary human CECs would ideally be the model of choice, the paucity of available fresh human cerebral tissue makes wide-scale studies impractical. The brain microvascular endothelial cell line hCMEC/D3 represents one such model of the human BBB that can be easily grown and is amenable to cellular and molecular studies on pathological and drug transport mechanisms with relevance to the central nervous system (CNS). Indeed, since the development of this cell line in 2005 over 100 studies on different aspects of cerebral endothelial biology and pharmacology have been published. Here we review the suitability of this cell line as a human BBB model for pathogenic and drug transport studies and we critically consider its advantages and limitations.
Blood-brain barrier; Immortalized cell line; Brain endothelium; In vitro model
Derivation and selection of hCMEC/D3 cells
The hCMEC/D3 cell line was derived from human
temporal lobe microvessels isolated from tissue excised during
surgery for control of epilepsy. The primary isolate was
enriched in CECs. In the first passage, cells were
sequentially immortalized by lentiviral vector transduction with
the catalytic subunit of human telomerase (hTERT) and
SV40 large T antigen, following which CEC were selectively
isolated by limited dilution cloning, and clones were
extensively characterized for brain endothelial phenotype .
The hCMEC/D3 cells form a contact-inhibited
monolayer of elongated cells on collagen type I or type IV.
They do not show adhesion-independent growth in soft
agar but form capillary structures in Matrigel, a
characteristic property of cultured endothelium. They were
reported to have an apparently normal diploid human
karyotype , although a high-resolution multicolor
fluorescence in situ hybridization (FISH) approach
revealed a more complex karyotype at high passages than
initially thought . In addition, they stain positively for
3Inserm, U1016, Institut Cochin, Paris, France
4CNRS, UMR8104, Paris, France
Full list of author information is available at the end of the article
endothelial markers including CD34, CD31, CD40,
CD105, CD144 (VE-cadherin) and von Willebrand
factor, but not for CD36, which is absent from brain
endothelium. They maintain stable growth and endothelial
marker characteristics, at least until the 35th passage.
The physical barrier in hCMEC/D3 cells
Optimal culture conditions are essential for a brain
endothelial phenotype with mature adherens junction
(AJ) and tight junction (TJ) protein expression and a
strong permeability barrier function. Full differentiation
associated with expression of CEC markers like TJ
proteins requires cellular quiescence, achieved either by
removal of key growth factors and/or by exposing cells to
shear stress under flow (see below). Substrates for cell
growth may also contribute to differentiation. For
example, hCMEC/D3 monolayers display five-fold higher
concentrations of TJ proteins on transwell filters than
on plastic coverslips; in the same vein, we shall describe
below the hCMEC/D3 response to activation of Wnt/
- catenin signaling, known to induce BBB formation
during fetal development.
Expression of junctional proteins
In the context of endothelial cell junctions, hCMEC/D3
cells are positive for junction-associated Ig-like proteins
such as PECAM-1 and JAM-A, for AJ and TJ structural
proteins such as VE-cadherin, claudin-3,-5 and occludin
as well as for scaffolding proteins such as beta catenin
and zonula occludens (ZO)-proteins-1 and 2 [1,3]. The
small G-protein Gi2, suggested as a TJ-associated
protein, was indeed identified as a partner of claudin-5 and
its presence was necessary for TJ formation in hCMEC/
D3 cells . Expression of claudins and occludin at
intercellular junctions is best observed when the cells
are confluent, treated with anti-inflammatory steroids
such as hydrocortisone, anti-oxidant agents such as
resveratrol, or the Wnt/-catenin signaling activator,
lithium chloride (LiCl). The Wnt/-catenin pathway acts in
hCMEC/D3 cells to induce/enhance the BBB phenotype
by increasing expression of claudins as demonstrated in
primary mouse CECs . Similarly, all growth factors,
particularly vascular endothelial growth factor (VEGF),
should be removed from the culture medium with the
exception of basic fibroblast growth factor (bFGF) to
enhance expression of junctional proteins. The hCMEC/
D3 cells express other newly identified junctional
proteins such as annexins-1 and -2, which also appear to be
important for maintenance of TJ integrity .
Comparison of transcriptional profiles of hCMEC/D3
cells and primary human CEC with freshly isolated
mouse CEC confirmed the expression by hCMEC/D3
cells of a substantial number of genes expressed by brain
endothelium, but showed lower expression of claudin-5,
occludin, JAM-2, glut-1 and the insulin receptor . The
authors concluded that in order to attain a mature brain
endothelial phenotype, other cell types present in the
neurovascular unit (e.g. astrocytes, pericytes) regulate gene
expression by CEC, suggesting that a more complex
in vitro model might be required to fully mimic the BBB.
Alternatively, in line with the aforementioned
differentiating action of the Wnt/-catenin pathway, complementing
the hCMEC/D3 culture medium with astrocyte- and/or
pericyte-derived soluble factors might be sufficient for
further differentiation towards a BBB phenotype.
Restricted permeability to paracellular tracers
Monolayers of hCMEC/D3 show restricted permeability
to lucifer yellow (LY: a low molecular weight paracellular
diffusion marker) and to many hydrophobic and
hydrophilic low molecular weight drugs which correlate with
in vivo permeability coefficients as demonstrated by
Weksler et al  and confirmed by Poller et al . They
also show a restricted permeability to low and high
molecular weight dextrans that is similar to primary CECs
and lower than non-cerebral endothelium (e.g. human
umbilical vein endothelial cells, HUVECs), particularly
under conditions of flow . Indeed, for compounds of
MW>4000, the permeability profile is very similar to
that of bovine and porcine CECs, hitherto the best
characterized in vitro BBB models. As discussed above for
TJ protein expression, the permeability barrier function
is maximized in the presence of LiCl and corticosteroids
(or resveratrol): in these conditions, the permeability
coefficient (Pe) for LY, is: 1.55 +/- 0.16 10-3 cm/min. For
comparison, Pe values for 4 kDa- and 70 kDa-dextrans are:
0.72 +/- 0.07 10-3 cm/min and 0.09 +/- 0.01 10-3 cm/min,
Conversely, stress conditions and extracellular stimuli
have been shown to increase paracellular permeability of
hCMEC/D3 cells via signaling pathways such as JNK, PKC
or NFB. These include mannitol treatment, oxygen and
glucose deprivation (OGD) and pro-inflammatory
cytokines such as TNF and chemokines such as CCL2.
Cowan et al  examined the effects of OGD under
static conditions in hCMEC/D3 cells. They observed a
reversible increase in monolayer permeability to dextran
after 1 h of OGD without cytotoxicity, but permanent
changes in monolayer permeability and marked
cytotoxicity after 12-24 h. The acute permeability changes
involved the generation of nitric oxide and could be
prevented by blocking inducible nitric oxide synthase.
Other studies have demonstrated that
cytokines/chemokines increase the paracellular permeability of hCMEC/D3
cells to dextrans via different mechanisms . With
proinflammatory stimuli, ZO-1, occludin and claudin-5
expression levels are decreased [11,12], whereas JAM-A
translocates away from tight junctions, without any
expression changes . The chemokine CCL2, which is
elevated during CNS inflammation and is associated with
endothelial dysfunction, transiently induces Src-dependent
disruption of hCMEC/D3 AJs, translocation of -catenin
from the AJ to PECAM-1, and increases surface
localization of PECAM-1 .
In brief, these studies illustrate the usefulness of the
hCMEC/D3 model for unraveling the regulatory
mechanisms of junctional integrity and BBB permeability in
pathological conditions (for a review see ).
Transendothelial electrical resistance (TEER)
Although the TEER of human cerebral microvessels has
not been directly determined, it is widely accepted that
mammalian systems such as the rat show high TEER
values well above 1,000 cm2, a characteristic of the
BBB in vivo . However, TEER values above 1,000
cm2 are difficult to achieve in cultured CEC in vitro
and this is particularly true for cell lines compared
to primary cultures. Under static culture conditions,
hCMEC/D3 monolayers develop only a low to
mediumlevel TEER (around 30-50 cm2) in various reports.
Interestingly, higher TEER values close to 300 cm2,
were observed in the presence of hydrocortisone,
probably due to the modulatory activity of corticosteroids on
the expression of TJ proteins such as occludin and
claudin-5 . Another strategy targeted at increasing
TEER values in hCMEC/D3 cells has involved co-culture
with other cell types forming the neurovascular unit,
as suggested above. In a recent paper, co-culture of
hCMEC/D3 cells with astrocytes from different brain
regions evoked a significant TEER increase from 30 to
over 60 cm2 . In both monocultures and
cocultures of hCMEC/D3 with astrocytes, TEER values
increased from baseline over an interval of 5 days,
presumably due to TJ maturation with time. By far the most
promising method to increase hCMEC/D3 cell TEER
values has been exposure to flow-based shear stress.
Indeed, in hCMEC/D3 monolayers subjected to pulsatile
flow after seeding in a capillary cartridge system, the
TEER was reported to rise to 1000-1200 cm2, then to
rapidly drop following flow cessation . Co-culture
with astrocytes did not induce any further increases in
TEER values in this flow-based model suggesting that, at
least in vitro, shear stress may be a more critical factor
in inducing a mature barrier phenotype than interactions
with other cell types.
The transport barrier in hCMEC/D3 cells
Efflux and trans-cellular transport systems expressed by
CECs are key factors for studying and predicting
interactions of drugs at the BBB; an adequate pattern of
transporter expression thus constitutes a prerequisite for
suitable in vitro human BBB models.
Expression, function and regulation of ABC transporters
hCMEC/D3 cells express functional efflux transporters
(known as ABC transporters because they contain
ATPbinding cassette(s) for active transport), typical for brain
endothelium, as observed in freshly isolated human
brain microvessels: these include P-glycoprotein (P-gp or
MDR1 or ABCB1), breast cancer resistance protein
(BCRP or ABCG2), and multidrug resistance-associated
proteins (MRP) -4 and -5 (or ABCC4 and 5) . In
addition, hCMEC/D3 cells express MRP-1, as previously
reported with primary human brain endothelial cells in
culture, strongly suggesting that in vitro culturing may
non-physiologically induce the expression of this gene
. Protein expression of P-gp/MDR1, MRP4, BCRP by
hCMEC/D3 cells (grown on collagen-coated dishes) was
further assessed by quantitative proteomic analysis ,
whereas no expression of P-gp/MDR1 was detected in
HUVECs, used as reference non-brain EC. Interestingly,
expression levels of P-gp, BCRP and MRP4 were similar
in hCMEC/D3 cells and in isolated human brain
microvessels . Moreover, these key transporters are
functional in hCMEC/D3 cells as efflux transporter
inhibition studies invariably leads to elevated
intracellular levels of their substrates [1,8,18]. In addition, P-gp
expression is polarized to the apical membrane as
previously demonstrated in situ in human brain microvessels
and appears to be stable for at least 40 passages .
ABC transporter activity and/or expression levels may
be modulated by extracellular stimuli. For example,
Poller et al  noted that P-gp activity was not altered
by TNF- treatment, although P-gp expression levels
increased after treatment. However, it is noteworthy that
apparent increases in P-gp expression have been found
in some cases to be due to selection of high
Pgpexpressing hCMEC/D3 cells, for example, after exposure
to potentially cytotoxic agents, and so may not reflect
real increases in P-gp expression . Substrates for
P-gp can increase its level of expression and activity as
demonstrated in hCMEC/D3 cells exposed to the HIV-1
protease inhibitors ritonavir and atazanavir, both
substrates for P-gp. Inhibition of P-gp (but not of MRP-1)
increased transport of these protease inhibitors. These
drugs bind the xenobiotic receptor PXR which likely acts
as a transcription factor for P-gp . Concern about
P-gp up-regulation during long-term administration of
antiretroviral therapy, thus possibly blocking brain entry
of these protease inhibitors (as well as of other
therapeutic drugs) suggests that the hCMEC/D3 model may
prove useful in designing newer antiretroviral therapies
that use other means of crossing the BBB. Of interest,
HIV-1 Tat can also lead to up-regulation of P-gp
expression and hence contribute to decreased entry of
antiretroviral therapy into the CNS .
In contrast to P-gp, BCRP expression and activity are
decreased by inflammatory cytokines, in particular IL-1
and TNF . Conversely, agonists of the peroxisome
proliferator-activated receptor alpha (PPAR)
upregulate BCRP in hCMEC/D3 cells, and can significantly
decrease accumulation of drugs that are BCRP
substrates (e.g. mitoxantrone). PPAR antagonists
downregulate BCRP in these CECs  suggesting new
targeting strategies for either improving drug brain
bioavailability or increasing neuroprotection. Along the
same lines, BCRP was shown, using hCMEC/D3 cells,
to mediate the transport of nifurtimox, an
antitrypanosomal drug . These observations indicate that
BCRP inhibitors potentially could improve the activity of
anti-trypanosomal drugs and confirm that the hCMEC/
D3 model is appropriate to test novel drugs.
Influx transporters of the solute carrier family and
Brain endothelium is known to express a large number of
membrane receptors and transporters that specifically
control the blood-to-brain transport of nutrients, including
insulin, transferrin and LDL receptors as well as glucose,
amino-acids and organic ion transporters, all members of
the solute carrier family (SLC) of transporters. Accordingly,
hCMEC/D3 cells were tested for expression of these
receptors and transporters by immunochemical analysis,
RTPCR and/or quantitative proteomic analysis. First, they
were shown to express at a high level the glucose
transporter Glut-1 and the transferrin receptor. Indeed, Glut-1
expression was found by quantitative proteomic analysis to
be 15-fold higher in hCMEC/D3 cells than in HUVECs
and similar to that of human brain microvessels .
Influx transporters such as the cation transporter OCT-1,
and to a lesser extent OCT-2 and -3 are expressed and
functional in hCMEC/D3 cells. OCT-1 is responsible for
CEC uptake of the antiepileptic drug lamotrigine, a process
blocked by the selective inhibitor prazosin . Also,
hCMEC/D3 cells express the neutral and cationic amino
acid transporter (ATB0,+), which may be involved in the
brain uptake of the anti-influenza compounds amantadine
and rimantadine . In addition, Carl et al  reported
the expression by hCMEC/D3 cells of the
monocarboxylate transporters SLC16A1 and SLC16A3 (MCT1 and
MCT3), while little or no expression of SLC16A2 (MCT2)
was noted. In agreement with these data, a high level of
SLC16A1 expression at the protein level was detected by
quantitative proteomic analysis of hCMEC/D3 cell extracts
. Regarding the proton-coupled oligopeptide
transporter superfamily (POT, SLC15A) transporters, Carl et al
also reported that hCMEC/D3 cells express both hPHT1
and hPHT2, while little to no expression of either hPepT1
or hPepT2 was observed, in line with previous data in the
human BBB in vivo .
The metabolic barrier in hCMEC/D3 cells
The activity of drug-metabolizing enzymes, especially
phase 1 cytochromes P450 (CYPs), might also indirectly
control the cerebral uptake of compounds from the blood
. The aryl hydrocarbon nuclear receptor (AhR) was
detected in hCMEC/D3 cells and dioxin (a ligand of AhR)
treatment increased cytochromes P450 CYP1A1 and
CYP1B1 over 20-fold . Interestingly, CYP1B1 was
previously identified as the major CYP in freshly isolated
human brain microvessels , suggesting that the
hCMEC/D3 model may be well adapted for further studies
regarding the regulatory mechanisms of CYP1B1
expression by brain endothelium.
Drug vectorization and trans-cellular transport
Numerous studies of liposomes and nanoparticles as
vehicles for crossing the BBB while avoiding efflux
transporters have utilized hCMEC/D3 cells. For example,
Chattopadhyay et al  showed that solid lipid
nanoparticles encapsulating atazanavir can circumvent P-gp
efflux activity that usually limits uptake of the drug.
Markoutsa et al  tested immunoliposomes bearing
both a monoclonal antibody to the transferrin receptor
(OX-28) and another isotype-matched monoclonal
antibody linked to the lipid particles via a biotin-streptavidin
technique, and showed that these structures were well
taken up and transcytosed. These authors concluded
that the hCMEC/D3 model was useful for particle
transport studies. More recently, a combination of LDL
receptor-targeted liposome-encapsulated doxorubicin
and statins, known to increase LDL receptor expression,
was shown to increase the drug delivery across hCMEC/
D3 monolayers , suggesting a new concept of drug
delivery to the brain. The toxicity of gold nanoparticles
was evaluated in hCMEC/D3 compared to epithelial
cells . Sodium citrate on the particle surface but not
particle size contributed to impaired viability and
proliferation of endothelial cells, which internalized fewer
nanoparticles than epithelial cells.
Single chain camelid antibody VHH fragments with
anti-glial fibrillary protein (GFP) activity as well as
fusion protein VHH- GFP were able to cross hCMEC/D3
monolayers as fluobodies . Indeed, the same VHH
crossed the BBB in vivo in mice and localized to
astrocytes, showing for the first time that an antibody was
efficiently able to penetrate the BBB and target resident
cells in the brain.
Interactions of immune cells with hCMEC/D3 cells
Although the CNS was originally considered an
immuneprivileged site because of the presence of the BBB and the
apparent absence of lymphatic drainage, it is now well
recognized that activated lymphocytes and monocytes
do infiltrate the CNS by crossing the BBB and that
neuroimmune diseases such as multiple sclerosis are
characterized by massive perivascular infiltrates around brain
microvessels. The hCMEC/D3 cell line provides a useful
model for deciphering the modes of interactions between
human brain endothelium and activated immune cells.
Response of hCMEC/D3 cells to inflammatory mediators
The hCMEC/D3 cells respond to inflammatory stimuli
by increasing paracellular permeability to tracers (see
previous section) and are able to support adhesion and
migration of leukocytes by increased expression of
adhesion proteins like ICAM-1 and VCAM-1 . They
express functional cytokine and chemokine receptors such
as TNFR1 and 2, IFNGR1 and CXCR1-5 and CCR3-6
[1,37]. Indeed, Fasler-Kan et al  demonstrated TNF
activation of NFB signaling, whereas interferon gamma
(IFN induced activation of JAK/STAT signaling
pathways, and upregulated MHC Class I. In addition,
secretion of chemokines by CECs may be an additional
mechanism for modulating leukocyte extravasation.
Furthermore, hCMEC/D3 cells secrete chemokines in a
similar fashion to primary human brain endothelium
both under basal conditions (CCL2 and CXCL8)
or following stimulation by cytokines (CCL5, CXCL10,
CX3CL1 or fractalkine) [39,40].
Leukocyte adhesion to and transmigration across hCMEC/
Monocytes adhere to activated hCMEC/D3 cells and
migrate across the monolayer. The interaction between
human monocytes and hCMEC/D3 cells involves the
generation of reactive oxygen species (ROS), release of
tissue-plasminogen activator (tPA) from the endothelial
cells and a subsequent increase in permeability of the
endothelial monolayer to large molecules (>150 kDa).
Degradation of occludin appears to mediate the opening
of endothelial-endothelial TJs . Blocking the ERK1/2
pathway can partly reverse the monocyte-induced
opening of monolayer TJs and impede occludin degradation.
The same mechanism as demonstrated in the hCMEC/
D3 model underlies brain changes in experimental
autoimmune encephalomyelitis in the rat, a model of
multiple sclerosis, as well as in rat monocytes and rat brain
endothelial cells in vitro, suggesting that it is a
generalized mechanism and may be pertinent in multiple
sclerosis pathology. The same authors recently reported that
a modulator of the sphingosine-1-phosphate (S1P)
receptor, known to reduce inflammatory lesions in
multiple sclerosis (FTY720P or GilenyaW), actually maintains
hCMEC/D3 cells in a state of immune quiescence
associated with decreased transmigration of monocytes .
This result further validates the hCMEC/D3 model for
investigating the regulatory mechanisms of inflammation
at the BBB.
Monocyte adhesion to hCMEC/D3 cells is enhanced
by endothelial treatment with TNF or IFN and can be
inhibited by antibodies to the integrin VLA-4. A role for
the junction-associated prion protein PrPC in monocyte
transmigration through brain endothelial cells was
demonstrated with hCMEC/D3 cells, using either the U937
monocytic cell line or fresh primary blood monocytes:
antibodies to the prion protein inhibited monocyte
transmigration across the endothelial layer, whereas
anti-PECAM 1 antibodies had no effect . This
inhibition was also observed with mouse primary brain EC
and with a rat brain endothelial cell line, suggesting, as
above, a mechanism common to brain endothelium
from several species.
Bahbouhi et al  used hCMEC/D3 cells as a BBB
model to compare adhesion and transmigration across
CEC by peripheral blood mononuclear cells (PMBC) or
purified T cells from multiple sclerosis patients versus
PBMC or T cells from healthy individuals. They
observed that PBMC migration is dependent on PSGL-1
and LFA-1 present on the PBMC. Both CD4+ and CD8+
T cells utilize these ligands to adhere to brain
endothelium via P-selectin and VLA-4, respectively, and
adherence can be blocked by anti-ligand antibodies. In
multiple sclerosis, the frequency of CD4+ T cells that
are PSGL-1+ is significantly greater than in healthy
individuals; CD8+ cell populations were similar in both MS
patients and controls. Transmigration of PBMC from
multiple sclerosis individuals was enhanced across both
resting and TNF-activated hCMEC/D3 cells. The
absolute transmigration was much greater across
TNF-activated hCMEC/D3 cells. Interestingly, PMBC from
individuals treated with IFN a widely used first-line
treatment of multiple sclerosis) had lower rates of
transmigration and demonstrated lower LFA-1 levels.
Whether human neutrophils induce permeability
changes in brain endothelium was studied by Joice et al
 using hCMEC/D3 monolayers. This study was
undertaken to understand whether neutrophil
accumulation contributes to vasogenic edema in stroke. Untreated
neutrophils applied to the hCMEC/D3 monolayers for
30 min actually decreased baseline permeability to low
molecular weight (4 kDa) dextran by 53%, whereas
neutrophils preactivated with TNF, LTB4 or PMA
(treatments that induced marked release of ROS) had no
effect on baseline permeability. The authors then
showed, in rats injected intracerebrally with human
neutrophils, that very similar changes in brain vascular
edema were seen. The authors concluded that the
hCMEC/D3 model was useful in evaluating potential
contributions to vasogenic edema.
The hCMEC/D3 model for investigating
The hCMEC/D3 cell line has been widely used to model
brain endothelium for investigating the molecular
mechanisms of its interaction with and response to multiple
human pathogens (viruses, fungi, bacteria and parasites)
known to affect the CNS. Below are mentioned some of
the most exciting results reported in this field.
Studies related to retroviral infection have concerned
two pathogens, HTLV-1 and HIV-1. HTLV-1 infects
hCMEC/D3 cells via their receptors for viral entry,
Glut-1 and neuropilin-1, an observation that has been
confirmed in situ in necropsy material from patients
with TSP/HAM (tropical spastic paraparesis/human
T-lymphotropic virus type-I-associated myelopathy) .
CEC infection leads to increases in paracellular
permeability and TJ disorganization, probably via expression of
the viral protein Tax. An additional mechanism leading
to BBB disruption is via secretion of TNF and IL1 by
HTLV-1 infected T cells .
In the context of HIV-1, studies on hCMEC/D3 cells
have focused on 1) mechanistic studies on
HIV-1induced BBB breakdown or 2) a model to investigate
effects of anti-HIV therapeutics, particularly protease
inhibitors, on BBB function (see previous section). For
mechanistic studies, it has been demonstrated that
HIV1 and/or Tat protein induces disruption of claudin-5 and
increases permeability of hCMEC/D3 cells in a similar
fashion to effects on primary rodent BECs .
Tatinduced delocalization of ZO-1 from the membrane into
the nucleus is mediated by Rho signaling and CREB .
In addition, Tat induces hCMEC/D3 cells into an
activated inflammatory state by inducing increased
expression of IL-1, E-selectin, CCL-2, and IL-6 , an effect
that is attenuated by PPAR and PPAR agonists 
via matrix metalloproteases . As a result,
HIV-1infected monocytes, or Tat protein itself, have been
shown to increase ICAM-1 expression and to favor
transmigration of the infected monocytes across
hCMEC/D3 cells by a mechanism that involved
NFBinduced release of MMP-9 .
HIV Tat also induces amyloid beta (A) peptide
accumulation in hCMEC/D3 cells which may contribute to
its effect on BBB function . A accumulation and
Tat-induced barrier dysfunction are lipid raft- and
caveolae-dependent and involve caveolae-associated Ras
signaling [54,55]. As mentioned above, Tat can also lead
to up-regulation of P-gp expression and hence
contribute to decreased entry of antiretroviral therapy into the
Adhesion to and penetration across a monolayer of
hCMEC/D3 cells by the fungal pathogen Cryptococcus
neoformans was demonstrated by Vu et al , who
found that a large polysaccharide capsule on the fungus
plus CD44, the hyaluronic acid receptor present on the
hCMEC/D3 cells, were both important for the
adherence of fungal particles to endothelial cells. Upon
adherence of Cryptococci, the endothelial cells developed
microvilli that attached to the fungi and appeared to aid
in their transcytosis. Conversely, removal of hyaluronic
acid or use of non-encapsulated organisms blocked
adherence. The authors pointed out that although the
TEER of the monolayers was lowabout half that of
primary brain endothelial cellsit was not further lowered
by the adherence of Cryptoccocci and appeared to
constitute a genuine barrier.
Although meninogocci (Neisseria meningitidis) are
commonly carried in the nasal and oral mucosa of humans,
direct meningococcal infection of the brain, a
devastating illness, is fortunately rare. How meningococci enter
the brain has long been poorly understood, but hCMEC/
D3 cells used as a model of the BBB have importantly
contributed to the elucidation of this mechanism.
Adhesion of meningococci on hCMEC/D3 monolayers
induces translocation of multiple endothelial membrane
proteins, including ezrin, moesin, and actin to form
honeycomb cortical plaques beneath the meningococcal
colonies. Coureuil et al  observed that type IV pili
present on pathogenic meninogocci recruited to the site
of bacterial colonies the Par3/Par6/PKC endothelial
polarity complex. This complex normally plays a pivotal
role in the establishment of eukaryotic cell polarity and
governs the formation of intercellular junctions; its
translocation to these cortical plaques led to the
formation of ectopic intercellular junctional domains at the
sites of bacteria-endothelial cell interactions and
depleted junctional proteins at endothelial cell-cell
interfaces. This response of hCMEC/D3 cells resulted in the
opening of intercellular junctions, thus permitting
paracellular bacterial infiltration across the endothelial
barrier. Coureuil et al further  explored the hCMEC/D3
model to ascertain the signaling pathway that recruits
the cortical plaques to meningococcal colony sites. They
elegantly demonstrated that meningococci hijack
another endothelial physiological pathway through
activation of -adrenergic receptors by their Type IV pili,
followed by activation of the scaffolding protein
arrestin and the tyrosine kinase Src. Activation of this
pathway favors endocytosis of phosphorylated
VEcadherin, a normal component of TJs, further opening
up endothelial TJs. Of note, these authors recently
reported that this pathway is also used by non-brain
microvascular endothelial cells, but is clearly distinct
from that used by epithelial cells .
Cerebral malaria, a common complication of
Plasmodium falciparum infection particularly in children, is
one of the most severe and often lethal manifestations of
this common tropical disease. Induction of cerebral
edema during cerebral malaria is among the most feared
complications of this disease, yet the mechanisms are
not well understood. The hCMEC/D3 cell line has
provided an excellent in vitro model for studying the
detailed interactions between P. falciparum parasites and
brain endothelium. Jambou et al  evaluated the
mechanism of P. falciparum-parasitized erythrocyte
adhesion to hCMEC/D3 cells and showed for the first time
that this process involved trogocytosis, the transfer of
membrane material from one cell (malarial antigens on
parasitized erythrocyte) to another cell (endothelial cell),
followed by ingestion of the entire parasitized
erythrocyte. These authors compared the hCMEC/D3 cell line
with the HBEC-5i cell line and showed that the
HBEC5i line displayed a more activated phenotype when
unstimulated, expressing much higher levels of ICAM-1,
an important receptor in the interaction between
parasitized erythrocytes and brain endothelial cells .
Blocking ICAM-1 or TNF activation of endothelial
cells prevented cytoadhesion of parasitized erythrocytes
and their ingestion. More recently, hCMEC/D3 cells
were used by Zougbede et al  to demonstrate that
P. falciparum-parasitized red blood cells could alter BBB
integrity also by a mechanism independent of
cytoadhesion, namely, by induction of metabolic acidosis,
which also resulted in opening TJs in the hCMEC/D3
monolayer, a process which also would favor
development of cerebral edema.
The hCMEC/D3 model for investigating
It is now well recognized that brain endothelium
dysfunction likely contributes to the progression of several
neurodegenerative diseases, initially considered as purely due to
neuronal alterations, like Alzheimers or Parkinsons
diseases. The hCMEC/D3 model has been widely used to
study the toxic effects of A peptides on brain
microvasculature in the context of Alzheimers disease. A 1-40,
the most abundant toxic A peptide around blood vessels,
was shown to increase hCMEC/D3 monolayer
permeability, in the absence of cytotoxic effects, via the
downregulation of the TJ protein occludin, without changing
levels of claudin-5 or ZO-1 . The A 1-40 effect on
permeability could be prevented by inhibiting JNK or
p38MAPK, suggesting that these signaling pathways
represented a possible therapeutic target in the treatment of
A peptides have been shown to decrease the activity
of efflux transporters in hCMEC/D3 cells . Indeed,
when hCMEC/D3 cells were exposed to A peptides,
P-gp mRNA and protein levels decreased through
down-modulation of the Wnt/-catenin signaling
pathway (by decreasing -catenin level and increasing
DKK1, an endogenous Wnt signaling inhibitor). These
changes were reversed by administration of Wnt3a. The
Table 1 Published immortalized human brain EC lines
Cell lines Immortalization procedure
effect was specific for P-gp, as MDR4 and BRCP were
not affected in these studies.
The hCMEC/D3 cell line was used to study cerebral
amyloid angiopathy (CAA), an age-associated
hemorrhagic condition commonly found in sporadic as well as
some familial types of Alzheimers disease. Fossati et al
 observed that A peptides induce caspase-mediated
mitochondrial dysfunction, then apoptosis in hCMEC/D3
cells; A peptides bearing familial CAA mutations were
more toxic to CEC than wild type A peptides. Apoptosis
of hCMEC/D3 cells was associated more with oligomeric
peptide forms than with amyloid fibrils, a finding
consistent with increasing evidence that oligomers of A rather
than the precipitating fibers are the most neurotoxic form.
Similarly, hCMEC/D3 cells were used to evaluate the
contribution of metalloproteases to the pathogenesis of CAA
. When hCMEC/D3 were exposed to A peptides, the
cells increased both production and enzymatic activity of
MMP2 which in turn degraded A peptides to A 1-16
C-terminal fragments resulting in decreased CEC
apoptosis. Conversely, silencing MMP-2 led to further A 40/
42-induced mitochondrial dysfunction and increased
apoptosis of hCMEC/D3 cells. Thus, MMP2 may represent a
potential vasoprotective and neuroprotective response of
the brain vasculature.
Finally, the hCMEC/D3 cell line has also been used to
investigate A clearance mechanisms from the CNS to
prevent both neurotoxic and vasculotoxic effects.
Indeed, a first report on hCMEC/D3 cells showing that A
is selectively effluxed when present on the luminal, but
not the abluminal side  has been also confirmed in
primary bovine CEC models  suggesting that P-gp
may act as a protective mechanism against plasma A
but not participate in the clearance of brain A although
its relevance in vivo remains to be determined.
Advantages and limitations of hCMEC/D3 cells
In summary, results from various laboratories worldwide
indicate that hCMEC/D3 cells retain the expression of
TEER LY/sucrose permeability Year
(.cm2) coefficient (10-3 cm/min) (first pub)
No permeability characterization
No expression of CD31
Low level of functional P-gp
Promising preliminary characterization nd
most transporters and receptors expressed in vivo at the
human BBB, including MDR1, BCRP, MRP4, transferrin
receptor, insulin receptor, Glut-1; they also express
metabolizing enzymes and TJ proteins, as expected.
Relatively few alternative models of the human BBB
have been proposed, either as primary human CEC or
cell lines. The following table (Table 1) summarizes
other human CEC lines that have been used within the
last decade. In contrast to the hCMEC/D3 cell line, most
of them have been only minimally characterized. This
strengthens the conclusion that the hCMEC/D3 cell line
constitutes a unique model for investigating the biology
of human brain endothelium.
However, a recent publication elegantly described the
preparation of human BBB ECs from induced
pluripotent stem (iPS) cells or embryonic stem (ES) cells .
Indeed, pure brain EC populations were isolated
following serial incubation of human iPS or ES cells first with
medium favoring neural differentiation and later with
medium favoring endothelial differentiation. These stem
cell-derived CECs grew as pure cultures, exhibited brain
TJ molecules and transporters and developed a high
TEER, significantly higher than hCMEC/D3 cells.
Although the reproducibility of this sophisticated approach
remains to be confirmed, these results demonstrate that
understanding the molecular mechanisms of BBB
development and regulation permits efficient modeling of the
human BBB in vitro. This new model displays excellent
barrier characteristics and may, in the future, constitute
for the pharmaceutical industry a key tool for
investigating BBB permeability to candidate drugs.
To date, the main advantage of the hCMEC/D3 cell line
is that it represents a stable, easily grown and
transferable population of human microvascular CEC that stably
maintains a normal BBB phenotype. As illustrated above,
it appears particularly well adapted for drug uptake and
active transport studies, as well as for understanding the
brain endothelium response to various human pathogens
and inflammatory stimuli. Optimizing the TJ tightness of
hCMEC/D3 cell monolayers still remains a major
challenge in order to provide an in vitro model that might
recapitulate all the characteristics of human BBB,
encompassing permeability restriction with appropriate
molecular exclusion and functional efflux and influx
transport systems. As suggested above, culture under
flow together with treatment with recently identified
BBB modulators may greatly help design strategies for
hCMEC/D3 optimization. The large network of
laboratories currently working with this model worldwide
actually constitutes a major asset for achieving this
ABC-transporters: ATP-binding cassette transporters; AJ: Adherens Junction;
BBB: Bloodbrain barrier; BCRP: Breast cancer resistance protein;
CYP: Cytochrome P-450; CECs: Cerebral endothelial cells; CNS: Central
nervous system; LiCl: Lithium chloride; LY: Lucifer yellow; MDR-1: Multidrug
resistance protein-1; MRPs: Multidrug resistance-associated proteins;
OGD: Oxygen and glucose deprivation; P-gp: P-glycoprotein; PPAR
alpha: Peroxisome proliferator-activated receptor alpha;
SLCtransporters: Solute carrier transporters; TEER: Transendothelial electrical
resistance; hTERT: Catalytic subunit of human telomerase; TJ: Tight junction.
BW, IAR and POC jointly analysed literature and wrote the review. All authors
have read and approved the final version of the manuscript.
The authors thank F. Glacial, K. Ganeshamoorthy and C. Artus (Institut Cochin, Paris)
for providing unpublished observations on hCMEC/D3 cells.
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13. Haarmann A , Deiss A , Prochaska J , Foerch C , Weksler BB , Romero IA , Couraud PO , Stoll G , Rieckmann P , Buttmann M : Evaluation of soluble junctional adhesion molecule-A as a biomarker of human brain endothelial barrier breakdown . PLoS One 2010 , 5 : e13568 .
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20. Tai LM , Reddy PS , Lopez-Ramirez MA , Davies HA , Male DK , Loughlin AJ , Romero IA : Polarized P-glycoprotein expression by the immortalized human brain endothelial cell line, hCMEC/D3, restricts apical-to-basal permeability to rhodamine 123 . Brain Res 2009 , 1292 : 14 - 24 .
21. Poller B , Drewe J , Krahlenbuhl S , Huwyler J , Guttman H : Regulation of BCRP (ABCG2) and P-glycoprotein (ABCB1) by cytokines in a model of the human blood-brain barrier . Cell Mol Neurobiol 2010 , 30 : 63 - 70 .
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23. Zastre JA , Chan GN , Ronaldson PT , Ramaswamy M , Couraud PO , Romero IA , Weksler B , Bendayan M , Bendayan R : Up-regulation of P-glycoprotein by HIV protease inhibitors in a human brain microvessel endothelial cell line . J Neurosci Res 2009 , 87 : 1023 - 1036 .
24. Zhong Y , Hennig B , Toborek M : Intact lipid rafts regulate HIV-1 Tat proteininduced activation of the Rho signaling and upregulation of P-glycoprotein in brain endothelial cells . J Cereb Blood Flow Metab 2010 , 30 : 522 - 533 .
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26. Watson CP , Dogruel M , Mihoreanu L , Begley DJ , Weksler BB , Couraud PO , Romero IA , Thomas SA : The transport of nifurtimox, an anti-trypanosomal drug, in an in vitro model of the human blood-brain barrier: evidence for involvement of breast cancer resistance protein . Brain Res 2012 , 1436 : 111 - 121 .
27. Dickens D , Owen A , Alfirevic A , Giannoudis A , Davies A , Weksler B , Romero IA , Couraud PO , Pimohamed M : Lamotrigine is a substrate for OCT-1 in brain endothelial cells . Biochem Pharm 2012 , 83 : 805 - 814 .
28. Kooijmans SA , Senyschyn D , Mezhiselvam MM , Morizzi J , Charman SA , Weksler B , Romero IA , Couraud PO , Nicolazzo JA : The involvement of a Na+ and Cl -dependent transporter in the brain uptake of amantadine and rimantadine . Mol Pharm 2012 , 9 : 883 - 893 .
29. Carl SM , Lindley DJ , Couraud PO , Weksler BB , Romero I , Mowery SA , Knipp GT : ABC and SLC Transporter expression and Proton Oligopeptide Transporter (POT) mediated permeation across the human blood-brain barrier cell line, hCMEC/D3 . Mol Pharm 2010 , 7 : 1057 - 1068 .
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32. Chattopadhyay N , Zastre J , Wong HL , Wu XY , Bendayan R : Solid lipid nanoparticles enhance the delivery of the HIV protease inhibitor, atanazavir, by a human brain endothelial cell line . Pharm Res 2008 , 25 : 2262 - 2271 .
33. Markoutsa E , Pampalakis G , Niarakis A , Romero IA , Weksler BB , Couraud PO , Antimisiaris SG : Uptake and permeability studies of BBB-targeting immunoliposomes using the hCMEC/D3 cell line . Europ J Pharmaceut Biopharmaceut 2011 , 77 : 263 - 274 .
34. Pinzn-Daza M , Garzn R , Couraud PO , Romero IA , Weksler BB , Ghigo D , Bosia A , Riganti C : The association of statins plus LDL receptor-targeted liposome-encapsulated doxorubicin increases in vitro drug delivery across blood-brain barrier cells . Br J Pharmacol 2012 , 167 : 1431 - 1447 .
35. Freese C , Uboldi C , Gibson MI , Unger RE , Weksler BB , Romero IA , Couraud PO , Kirkpatrick CJ : Uptake and cytotoxicity of citrate-coated gold nanospheres: comparative studies on human endothelial and epithelial cells . Part Fibre Toxicol 2012 , 3 : 9 - 23 .
36. Li T , Bourgeois JP , Celli S , Glacial F , LeSourd AM , Mecheri S , Weksler BB , Romero IA , Couraud PO , Rougeon F , Lafaye P : Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging . FASEB J 2012 , 26 : 3969 - 3979 .
37. Lopez-Ramirez MA , Fischer R , Torres-Badillo CC , Davies HA , Logan K , Pfizenmaier K , Male DK , Sharrack B , Romero IA : Role of caspases in cytokine-induced barrier breakdown in human brain endothelial cells . J Immunol 2012 , 189 : 3130 - 3139 .
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40. Hurst LA , Bunning RA , Couraud PO , Romero IA , Weksler BB , Sharrack B , Woodroofe MN : Expression of ADAM-17, TIMP-3 and fractalkine in the human adult brain endothelial cell line, hCMEC/D3, following proinflammatory cytokine treatment . J Neuroimmunol 2009 , 210 : 108 - 112 .
41. Reijerkerk A , Kooij G , van der Pol SM , Leyen T , van Het Hof B , Couraud PO , Vivien D , Dijkstra CD , de Vries HE : Tissue-type plasminogen activator is a regulator of monocyte diapedesis through the brain endothelial barrier . J Immunol 2008 , 181 : 3567 - 3574 .
42. van Doorn R , Lopes Pinheiro MA , Kooij G , Lakeman K , van Het Hof B , van der Pol SM , Geerts D , van Horssen J , van der Valk P , van der Kam E , Ronken E , Reijerkerk A , de Vries HE : Sphingosine 1-phosphate receptor 5 mediates the immune quiescence of the human brain endothelial barrier . J Neuroinflammation 2012 , 9 : 133 .
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44. Bahbouhi B , Berthelot L , Pettre S , Michel L , Wiertlewski S , Weksler BB , Romero IA , Miller F , Couraud PO , Brouard S , Laplaud DA , Soulillou JP : Peripheral blood CD4+ T lymphocytes from multiple sclerosis patients are characterized by higher PSGL-1 expression and transmigration capacity across a human blood-brain barrier-derived endothelial cell line . J Leukocyte Biol 2008 , 86 : 1049 - 1059 .
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