In Vitro Blood-Brain Barrier Models-An Overview of Established Models and New Microfluidic Approaches

REVIEW

In Vitro Blood–Brain Barrier Models—An Overview of EstablishedModels and New Microfluidic Approaches

ANETTE WOLFF,1 MARIA ANTFOLK,1 BIRGER BRODIN,2 MARIA TENJE1,3

1Lund University, Department of Biomedical Engineering, Lund, Sweden2University of Copenhagen, Department of Pharmacy, Copenhagen, Denmark3Uppsala University, Department of Engineering Sciences, Uppsala, Sweden

Received 14 October 2014; revised 5 December 2014; accepted 5 December 2014

Published online 28 January 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24329

ABSTRACT: The societal need for new central nervous system (CNS) medicines is substantial, because of the global increase in lifeexpectancy and the accompanying increase in age-related CNS diseases. Low blood–brain barrier (BBB) permeability has been one of themajor causes of failure for new CNS drug candidates. There has therefore been a great interest in cell models, which mimic BBB permeationproperties. In this review, we present an overview of the performance of monocultured, cocultured, and triple-cultured primary cells andimmortalized cell lines, including key parameters such as transendothelial electrical resistance values, permeabilities of paracellular fluxmarkers, and expression of BBB-specific marker proteins. Microfluidic systems are gaining ground as a new automated technical platformfor cell culture and systematic analysis. The performance of these systems was compared with current state-of-the-art models and it wasnoted that, although they show great promise, these systems have not yet reached beyond the proof-of-concept stage. In general, it wasfound that there were large variations in experimental protocols, BBB phenotype markers, and paracellular flux markers used. It is theauthor’s opinion that the field may benefit greatly from developing standardized methodologies and initiating collaborative efforts onoptimizing culture protocols. C© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:2727–2746,2015Keywords: BBB; in vitro models; membranes; blood–brain barrier; BBB on a chip; cell culture; CNS

INTRODUCTION

The pharmaceutical industry is faced with one of the greatestchallenges of the modern age—developing medicines that canreach the brain. One of the incentives is the fact that the totalcost of the neurovascular health care system in Europe wasestimated in 2010 at 800 billion €PPP in direct and indirectcosts,1 with an estimated 85% increase in costs for dementiacare by 2030, according to Alzheimers Disease International.2

These numbers can only be expected to increase with the age-ing population. The challenge lies in the existence of the highlyeffective blood–brain barrier (BBB) that strictly controls whatmolecules are allowed to pass and reach the brain. Drug screen-ing is very expensive and time-consuming in the current drugdevelopment process, where in vivo screening is applied at thepreclinical test stage. Much research effort is therefore directedtoward the development of functional BBB in vitro models,which allows the speed by which new drugs are made avail-able to increase.

Already in 1885, Paul Ehrlich found that dyes injected intothe circulatory system stained all organs in the mammalian

Abbreviations used: BAEC, bovine aortic endothelial cells; BCEC, brain capil-lary endothelial cells; BBEC, bovine brain endothelial cells; PBEC, porcine brainendothelial cells; HBEC, human brain endothelial cells; HBMECs, human brainmicrovascular endothelial cells; HBVEC, human brain vascular endothelial cells;hCMEC, human cardiac microvascular endothelial cells; MBEC, murine brainendothelial cells; NPC, neural progenitor cells; RBCECs, rat brain capillaryendothelial cells; RBECs, rat brain endothelial cells; RBMECs, rat brain mi-crovascular endothelial cells; TEER, transendothelial electrical resistance; TJ,tight junction.

Correspondence to: Anette Wolff (Telephone: +46-709948974; Fax: +46-462224527; E-mail: [email protected])

Journal of Pharmaceutical Sciences, Vol. 104, 2727–2746 (2015)C© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association

body, except for the brain and the spinal cord.3 On the basis ofthis work, in 1900, the term BBB or really bluthirnshranke, wascoined by Berlin-based Max Lewandowsky.4 One of Ehrlich’sstudents went on, in 1913, to show that the effect could be seenin the reverse situation as well, by injecting trypan blue into thecerebrospinal fluid and observing that it did not spread outsideof the central nervous system (CNS).5 It then became apparentthat the brain is protected by a very effective barrier shownlater to be the result of tight connections between the cerebralendothelial cells.6

The BBB can be characterized as a highly effective and se-lective barrier in the interface between the blood of the brainmicrovasculature and the brain tissue and is crucial for achiev-ing a normal function of the CNS. Small lipophilic gases, suchas O2 and CO2 may diffuse freely across the BBB, but tight junc-tions (TJs) restrict paracellular fluxes of hydrophilic molecules.Various nutrients and large molecules are actively transportedacross the cellular membranes through transporter proteins orreceptor-mediated endocytosis.

Recent excellent reviews in the field has dealt with only mi-crofluidic systems,7 had a more biological approach,8–11 or afocus on drug permeability and applications.12,13 This reviewintends to compare the various different models designed toimitate the BBB. Papers have been selected based on influenceon the field, as well as model development, and little focus hasbeen given to application-oriented studies. Current state-of-theart BBB in vitro research models are presented both with re-spect to the cell cultures used and the engineering of the cellculture setup and its integration. This is, to our knowledge, thefirst time an all-encompassing view is taken on the researchwhere equal efforts have been made to analyze the biologicalas well as the technical aspects of the complete in vitro systems.

Wolff et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2727–2746, 2015 2727

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This work intends to contribute to a stronger interdisciplinaryapproach to BBB research.

The Neurovascular Unit

The neurovascular unit is the five cell types that constitutethe BBB: endothelial cells, astrocytes, pericytes, neurons, andmicroglia (Fig. 1).

Although the different cell types of the BBB have beenidentified,14,15 their interdependence and individual roles arestill not fully understood. Here, we list the current level of un-derstanding of the roles of the different cell types: the endothe-lial cells in the brain are specialized BBB cells that form themicrovessels permeating the brain. They differ from regularendothelial cells in four aspects:

1. An absence of fenestrae.2. A continuous basement membrane that is shared with

pericytes, as have been discovered in recent years.16–18

3. A high complexity of TJs.4. Limited pinocytic vesicular transport.

The barrier is tight for polar compounds because of theirlow lipid permeability, and the presence of extremely tight TJs,leaving only very small polar molecules crossing the barrier.19

The transport pathways available for larger molecules are theparacellular and transcellular pathways.20 The paracellularpathways are passive, thus relying solely on the concentrationgradient and the permeability, which is very low for macro-molecules. The transcellular pathways are energy dependentand substance specific.

Tight junctions are structures assisting the endothelialcells in accomplishing the tightness of the BBB. They com-prise at least three different types of transmembrane pro-teins; claudins, occludins, and junctional adhesion molecules.21

Claudin and occludin have both been shown to be paramount tothe selective paracellular permeability.22,23 Three specific trans-

membrane proteins are primarily present in cells with effectivebarrier functions, such as the BBB endothelial cells. They are:ZO-1, claudin-5, and occludin, where ZO-1 is needed as an an-chor point for the claudin and occludin proteins24 and is nec-essary for TJ formation.25 Because of the specificity of thesetransmembrane proteins to the BBB, they are often used asmarkers for successful BBB formation.

Astrocytes are characteristically star-shaped glial cellsfound only in the CNS and they perform different tasks in thebrain, one of them being to give biochemical aid to the endothe-lial cells that form the BBB.26 It has been found that astrocytessecrete factors are needed for BBB function27,28 and assist inregulating transport across the capillaries.29

Pericytes cover 22%–33% of a capillary,30 varying betweenmicrovessel type,31 seemingly correlating with the degree oftightness of the endothelial junctions,32 and pericyte deficiencyis known to increase permeability of the BBB.33 Although it hasbeen suggested that pericytes affect the BBB phenotype,34 it isbelieved that they instead inhibit the expression of moleculesthat increase vascular permeability35 and induce polarity to theastrocyte end feet that surrounds the microvessels,31 resultingin a tightening of the barrier. Pericytes also synthesize elementsnecessary for the differentiation of the BBB36 and are involvedin transport across capillary walls.31

Neurons are the main functional constituents of the brain.They need to be protected against the fluctuations inherentin the mammalian system, for example, temperature fluctua-tions, variations of O2 or CO2 levels, or chemical concentrationvariations, which is the reason for the existence of the BBB.Although the exact mechanism is unknown, Minami37 has re-cently shown that the presence of neurons increases the barriereffects, suggesting that neurons, or communication to the bar-rier, are also a crucial part of the BBB.

The final group of cells that form the neurovascular unitare microglia, which are responsible for clearing debris andhandling apoptotic cells in the brain. They are found inthe perivascular space but will not be discussed further, as

Figure 1. The overview of the neurovascular unit, with the basal lamina denoted as BL. Reproduced from Abbott et al.14 with permission fromElsevier B.V.

Wolff et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2727–2746, 2015 DOI 10.1002/jps.24329

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activated macrophages are one of the hallmarks of an infectedbrain17 and have been shown to drastically reduce barrier ef-fectiveness when activated.38

BBB Models

The oldest and best-established method to study the transportof molecules across the BBB is to perform in vivo experiments.The main advantage of in vivo experiments is that it allowsfor studies of the brain in its natural environment. As it isunethical to perform these studies on the human brain, it ne-cessitates the use of laboratory animals. The versatility of theresults is however questionable and it has been shown that ap-proximately 50% of results obtained from animal models cannotbe translated into correct human responses.39 To overcome theethical issues, in vitro models based on cell cultures are applied.The most commonly used methods to determine the quality ofthe in vitro models, is to measure the transendothelial electri-cal resistance (TEER), and to measure the permeability of spe-cific marker substances. Additionally, cell-type-specific mark-ers, such as ZO-1, Claudin-5, Occludin, and von Willebrandsfactor, are targeted for immunostaining to ensure the presenceof the correct cell phenotypes. The different analysis techniquesare described briefly below.

Transendothelial electrical resistance is a quantitative mea-surement of the resistance over the cell layer and the cell cul-ture membrane. Several factors affect the resistance, such ascell origin and the level of confluency of the cells. The measure-ment is performed by connecting electrodes to either side of thecell culture membrane and measuring the momentary resis-tance upon an applied potential (Fig. 2). The in vivo reportedvalues of the resistance across the BBB40–42 is 1500–8000 �cm2,and a value of 150–200 �cm2 is considered the lowest functionallimit for in vitro models.43

The method of measuring TEER differs between groups, be-cause of the various instruments available. “Chopstick” elec-trodes, Endohm cup electrodes, and CellZscope culture cham-bers may give readings that differ. Furthermore, althoughTEER is a relatively simple way of measuring barrier tightness,it is not enough to judge the selectivity of the barrier. Compar-isons of barrier tightness between in vitro models cultured in

Figure 2. The original in vitro Transwell setup. Endothelial cellsgrown on a Transwell membrane. A 4-electrode system is used to mea-sure the resistance over the cells and the membrane. The resistanceover an empty membrane is then subtracted from the cell culture re-sistance.

different laboratories should therefore ideally be accompaniedby comparisons of size selectivity of passively diffusing com-pounds and TJ protein profiles, as the composition of TJ pro-teins will be crucial for the permeation process (for references,see Haseloff et al.44).

Sucrose and mannitol are the most commonly used com-pounds for permeability studies, where the in vivo permeabil-ity has been noted to be approximately 3 × 10−8 and 1.4 ×10−7 cm/s, respectively.45 In vitro models are generally consid-ered useful if the permeabilities are within a factor 100 of the invivo values. Permeability studies often do not reflect the pres-ence of transporter proteins, but the authors have chosen to notgo into detail regarding active transport and efflux proteins.Investigations of this character are highly specific for each sub-stance evaluated, and are beyond the scope of this work. For anin-depth discussion on active transporter and efflux proteins,see the review article by Cardoso et al.10

The three methods to investigate the BBB (to measureTEER, to analyze permeabilities, and to immunostain forknown markers) may thus provide enough information thatconfluence uniformity and tightness of the model barrier canbe estimated.

In Vitro Models

In 1974, the first documented successful culture of bovine cere-bral cortex endothelial cells on nylon sieves was published.46

The most common platform of in vitro models used todayis created using Transwells (Corning Inc., Corning, NY) andmost publications are reported using 10 :m thick polystyreneor polycarbonate membranes with 400 nm pores with a108 pores/mm2 pore density.28,47–54

The models can be developed using one, two, or more celltypes, as described below. The endothelial cells are usuallygrown in the upper (luminal) compartment of the Transwellin cell-specific growth medium. Additional cells, such as astro-cytes or pericytes, are normally cultured on the lower (ablumi-nal) side of the membrane (Fig. 3).

The major challenge with the development of successful invitro models is to identify and culture pure cell types that re-sult in high TEER values and low permeabilities. Althoughthere is some use for nonbrain-derived endothelial cell culturesto establish a new model system, one should strive for usingbrain-derived endothelial cells in a live setup. Brain-derivedendothelial cells will retain some of their BBB phenotypic ex-pressions, whereas those derived from the kidney or gut willgive a tight barrier but will inherently differ widely in trans-port and efflux protein expression.

Preferably, human cells should be used to minimize species-specific responses, though they are not often used, as immor-talized human cell lines do not create a sufficiently tight bar-rier. In addition, primary cells of human origin often stemfrom biopsies of diagnosed patients giving rise to the ques-tion whether the cells are suitable when extracted from pos-sibly diseased tissue.55–57 Shusta and coworkers48,58–60 circum-vent this issue by instead using human pluripotent stem cells.As stem cell-derived brain endothelial cells are difficult toobtain, immortalized human cell lines such as human car-diac microvascular endothelial cells (hCMEC)/D3 are normallychosen for a human model. The cell line human brain mi-crovascular endothelial cells (HBMECs) has been shown to

DOI 10.1002/jps.24329 Wolff et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2727–2746, 2015

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Endothelial cellsAstrocytesNeurons/Pericytes

d

a

b c

Figure 3. The different kinds of in vitro models. (a) A monocultureof endothelial cells. (b) A contact coculture of endothelial cells andastrocytes or pericytes. (c) A noncontact coculture of endothelial cellsand astrocytes or pericytes. (d) A triple culture of endothelial cells withastrocytes in contact and pericytes or neurons in a noncontact position.

give the best barrier properties for permeability studies usingTranswells.61

Immortalized animal-derived cell lines are most commonlyused, although most of these models do not reach TEER valuesabove 300–500 �cm2. Barrier properties are commonly betterin rat than mouse models, but are outperformed by porcineand bovine models.50,62–68 Franke et al.69 have however shownthat it is possible to increase the TEER to near-in vivo lev-els by adding hydrocortisone to the culture, which is now acommon approach. Their maximum reported TEER value was1800 �cm2 with a low permeability for sucrose of 0.2–1.8×10−6

cm/s. There are definite advantages to using immortalized ani-mal cell lines—number one being the relative ease with whichthey are cultured.

Additionally, there is a growing interest in using primaryanimal cells to increase the fidelity of the in vitro models, al-though this is a time-consuming and sensitive process.70–76 Themajor drawback of this approach is that primary cells culturedex vivo lose some of their phenotypic expressions with each pas-sage as a result of the lack of exposure to physiological factors.This results in a leaky barrier with low TEER values and highpermeability of marker substances. The negative effects can beminimized by using primary cells within the first couple of pas-sages, preferably using the first passage, and all reports studiedin this review article are performed on cells prior to passage 10.

In the following sections, we will review the current workusing immortalized cell lines and primary cell cultures, andcompare the BBB model system qualities (as defined by re-ported TEER, permeability, and cell type purity) when mono-,

co-, and triple-cultures are applied. For the interested reader,we refer you to the reviews of Gumbleton and Audus,77 Deliet al.,13 and Abbott et al.8 for in-depth discussions about pri-mary cells and cell lines in in vitro studies.

Monoculture

One of the most widely used BBB models is developed by cul-turing endothelial cells alone in a Transwell (Fig. 3a). Hawkinset al.78 established that the microvascular endothelial cellsform the basis of the BBB by showing that frogs have a function-ing BBB, even without astrocyte assistance. Thus, the monocul-tures of endothelial cells have been a reasonable model despiteits simplicity and have been published recently,76,79,80 some-times assisted by the use of astrocyte-conditioned medium, asthis has been shown to increase barrier function.27 Another ap-proach to increase the tightness of a monoculture of endothelialcells is to culture the endothelial cells on a glia-conditioned ex-tracellular matrix, as presented by Hartmann et al.81 However,as established by Patabendige et al.,82 sometimes a monocul-ture can nonetheless be an informative choice when one is in-terested in a robust model with which to perform permeabilitystudies.

Monocultures have seen a revival upon the introduction ofmicrofluidic cell culture systems, which are often limited in thenumber of different cell types they can manage. By integratingthe cells into a microfluidic network, it is possible to intro-duce shear stresses to the cells through the fluid flow. Dane-man et al.27 showed that shear stress, together with astrocyte-conditioned medium can increase the TEER of primaryHBMECs by a factor of 3, from 500 to 1500 �cm2. Similarwork has been presented by Griep et al.83 where they used thehuman cerebral microvascular endothelial cell line hCMEC/D3and showed a 300% increase in TEER when shear stresses wereintroduced (Fig. 4). Cucullo et al.84 cultured hCMEC/D3 undera pulsating flow and could report on an increase in TEER val-ues of close to 20 times compared with static cultures, furtherindicating the importance fluid flow and shear stresses play onthe permeability of the BBB.

Prabhakarpandian et al.85 created a microfluidic chip with amicrocirculation two-compartment chamber in an effort to in-troduce shear stress, whereas at the same time keeping chipmanufacturing simple. Rat brain endothelial cell (RBEC) lineRBE4 was cultured with astrocyte-conditioned medium for theexperiments.85 Low permeability for dextran was presented,

Figure 4. The microsystem developed by Griep et al.83 to mimic the invivo situation of shear stress that affects the microvessels made from aPDMS chip with a Transwell membrane insert. In (a), numbers denote(I) top part, (II) Transwell membrane, (III) bottom part, and (IV) thePt electrodes. Image (b) gives the assembled device and (c) a photo ofthe BBB chip. Reproduced from Griep et al.83 with permission fromSpringer US.


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but unfortunately no TEER values were reported. Dextran isavailable as quite large molecules at 3–5 kDa, so it is not im-mediately clear to see the effectiveness of the barrier in thiswork. This may be attributed to a higher focus of the articleon chip manufacture, and their proof-of-concept approach. Thefield of integrated systems is still young and more publishedwork where the main focus is on the microsystem engineeringcan be expected, though results from monocultures should gen-erally be regarded with caution because of the lack of astrocyteinduction.

Cocultures

In most common coculture models, endothelial cells are grownon the luminal side of a Transwell membrane, whereas astro-cytes, pericytes, or neurons are grown either on the bottomof the well (noncontact) or directly on the abluminal side ofthe membrane (contact) (Figs. 3b and 3d). The contact modelallows the nonendothelial cells to affect the endothelial cellssolely by close-range association, such as by processes throughthe membrane.86 The noncontact model ensures that any ef-fect the nonendothelial cells have on the endothelial cells isstrictly through chemical gradients. Thus, separate and im-portant information on the fundamental understanding of theBBB is provided by the two different methods. A more detaileddescription follows, where additional references are found inTable 1 below.

Endothelial Cells and Astrocytes

In 1980, it was recognized that many BBB features could beat least partly reestablished by coculturing endothelial cells to-gether with astrocytes.124 This work has been extremely influ-ential in the research field and astrocytes are the most commoncell type that endothelial cells are cocultured with.125 Typically,TEER values are increased up to a factor 2 in astrocyte cocul-tures compared with monocultures of endothelial cells.68 If thisis modified to a contact model, the improvement can give asmuch as nine times higher TEER values than the monolayer inprimary cultures.72,87 In addition, astrocytes have been shownto be central to the vasodilation of microvessels, which plays amajor part of the BBB.126

Harris and Shuler127 investigated the effect of the thick-ness of the cell culture membrane between the endothelialcells and the astrocytes. They developed a 1-:m thin silicon ni-tride membrane with 2-:m-wide pores where they cocultured ahuman cerebromicrovascular endothelial cell line (SV-HCEC)with astrocytes.127 The performance of the cell culture scaf-fold was evaluated by observing the cell density on the novelmembrane in relation to an identical culture on traditionalTranswell membranes. The number of cells was higher on thetreated silicon nitride membranes, but unfortunately no TEERvalues, or other quantifiable factors were reported, so it is dif-ficult to judge the barrier performance.

Stanness et al.128 created the first microfluidic system usingthe morphological structure of capillaries to coculture a bovineaortic endothelial cells (BAEC) with a rat astrocyte glioma cellline (C6) under shear stress (DIV-BBB). Hollow fibers with en-dothelial cells cultured on the luminal side, and astrocytes onthe abluminal side, is suspended in a chamber (Fig. 5), withmedium pumped into the chamber. Although this system givesrelevant resistances and permeability results, it is limited infunction and application, as evaluated by Pooja and Cucullo.129

The DIV-BBB is the only system using hollow fibers, whereasthe following microfluidic systems use planar structures, whichis the conventional fabrication method in microengineering. Ifone uses planar structures, you do however risk loosing somefeatures inherent in the BBB by removing a physiological as-pect of the barrier.

Booth and Kim104 also approached the issue of the lack ofa combined shear stress, reported to play a role in the BBBphysiology,100 and dual cell layer setup. They used cut-outs fromTranswell membranes with luminally cultured mouse braincapillary endothelial cell (BCEC) line bEnd.3 and astrocytes incontact abluminally in a microfluidic chip of their own design.A dynamic coculture was shown to be the most effective forestablishing improved barrier functions, outperforming staticcoculture, and monoculture as well as a dynamic monolayer(Fig. 6).104 However, they report unexpectedly low TEER val-ues (never reaching more than 25 �cm2) for their monocul-ture Transwell setup where a TEER for bEnd.3 cells reaches100 �cm2, normally.54 This makes it difficult to comment on thequality of the in vitro model, although the effect of a dynamicenvironment is clearly demonstrated.

Endothelial Cells and Pericytes

In recent years, it has become clear that not only astrocytesplay a major role in the dynamic regulation of the BBB phe-notype in vivo and in vitro, but that also pericytes are veryinfluential. In vivo, it has been shown that pericytes play arole in angiogenesis.35 The effects of pericytes and astrocytesin vitro seem, however, to be strongly linked to cell type origin.Dohgu et al.130 presented a study on the pericyte productionof transforming growth factor-$ type I where they showed asignificant decrease in permeability for sodium fluorescein fora coculture of mouse BCEC and rat brain pericytes. Anotherstudy, using rat brain capillary endothelial cells (RBCECs) re-ports on a 100% increase in TEER to a final value of 200 �cm2

for cocultures with rat brain pericytes.111

The opposite is however reported by Hatherell et al.102 us-ing hCMEC/D3 cocultured with either astrocytes or pericytes ofhuman origin. Here, astrocyte contact resulted in an increasein TEER from 39 to 60 �cm2, whereas cocultures with pericytesresulted in an increase from 39 to 46 �cm2. Although these re-sistances are in line with other studies made with hCMEC/D3,the lack of reported permeabilities makes the study hard tocompare with others presenting higher TEER values.

Another feature that differs the actions of the astrocytes andthe pericytes is that astrocytes can affect the BBB also from adistance, whereas contact seems crucial for pericyte contribu-tion to barrier property.113 Surprisingly, for short-term hypoxicevents, pericytes seem to have a detrimental effect on barrierproperties, significantly lowering the TEER compared with amonoculture of RBE4 cells.49 However, under prolonged O2 de-privation astrocytes proved less efficient at preserving barrierfunction than did pericytes.49 This indicates a bearing for in-cluding pericytes when establishing a BBB model intended forischemic studies.

Triple-Culture

Although it is largely accepted that the endothelial cells formthe basis for the BBB structure, a higher level of resemblancebetween the in vitro model systems and in vivo can be achieved


2732 REVIEW

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m-f

ree

med

ium

wit

hh

ydro

cort

ison

eO

mid

iet

al.53

Pro

pan

olol

:18.

4±

0.7

Con

tin

ued


REVIEW 2733

Tab

le1.

Con

tin

ued

Res

ult

sO

verv

iew

ofB

BB

Mod

els

Mod

elS

peci

esP

rim

ary

Cel

lTyp

eC

ellT

ype

inC

onta

ctC

ellT

ype

inN

onco

nta

ctT

EE

R(�

cm2)

Per

mea

bili

ty(P

app;1

0−6

cm/s

)O

ther

Info

Ref

eren

ce

Hu

man

hB

ME

CA

stro

cyte

s55

–297

N/A

Kas

aet

al.97

hB

MV

EC

Ast

rocy

tes

260

±13

0S

ucr

ose:

16.7

±2.

5P

erm

eabi

liti

esre

port

edas

cm/m

inM

egar

det

al.98

Caf

fein

e:50

±15

Sta

vudi

ne:

83.3

±8.

3A

nti

pyri

ne:

100

±13

.3P

ropa

nol

ol:2

00±

16.7

HB

ME

CA

stro

cyte

s60

N/A

Zys

kan

dS

chn

eide

r-W

ald63

Hu

man

brai

nva

scu

lar

endo

thel

ialc

ell

Ast

rocy

tes

700

Su

cros

e:3.

16D

IV-B

BB

wit

hfl

owC

ucu

llo

etal

.99

Hu

man

brai

nen

doth

elia

lcel

l(H

BE

C)

Ast

rocy

tes

700

Su

cros

e:0.

005

DIV

-BB

Bw

ith

flow

.P

erm

eabi

liti

esre

port

edas

cm/m

in

Cu

cull

oet

al.10

0

Man

nit

ol:0

.016

D-G

luco

se:0

.3D

ilan

tin

:0.2

5L

-asp

:0.3

Mor

phin

e:0.

009

D-a

sp:0

.008

HB

EC

Ast

rocy

tes

100

Su

cros

e:0.

33D

IV-B

BB

no

flow

.P

erm

eabi

liti

esre

port

edas

cm/m

in

Cu

cull

oet

al.10

0

Man

nit

ol:3

3.3

D-G

luco

se:1

.2D

ilan

tin

:0.2

2L

-asp

:8.3

Mor

phin

e:1.

7D

-asp

:13.

3h

BM

EC

SV

G-A

22–2

6N

aFl:

7.22

±0.

45ce

llZ

scop

e,n

ogr

owth

fact

ors

Eig

enm

ann

etal

.61

EC

V30

4A

stro

cyto

ma

1321

N1

<80

N/A

Tan

etal

.101

EaH

y929

Ast

rocy

tom

a13

21N

1<

20N

/AT

anet

al.10

1

hC

ME

C/D

3A

stro

cyte

sC

C-2

565/

SC

1810

60±

1N

/AH

ath

erel

let

al.10

2

BB

19S

VG

-A/H

BP

CT

<20

N/A

cell

Zsc

ope

Eig

enm

ann

etal

.61

TY

10S

VG

-A/H

BP

CT

<20

N/A

cell

Zsc

ope

Eig

enm

ann

etal

.61

hC

ME

C/D

3S

VG

-A/H

BP

CT

<20

NaF

l:6.

88±

0.52

cell

Zsc

ope,

no

grow

thfa

ctor

sE

igen

man

net

al.61

Con

tin

ued


2734 REVIEW

Tab

le1.

Con

tin

ued

Res

ult

sO

verv

iew

ofB

BB

Mod

els

Mod

elS

peci

esP

rim

ary

Cel

lTyp

eC

ellT

ype

inC

onta

ctC

ellT

ype

inN

onco

nta

ctT

EE

R(�

cm2)

Per

mea

bili

ty(P

app;1

0−6

cm/s

)O

ther

Info

Ref

eren

ce

Mu

rin

eM

uri

ne

brai

nen

doth

elia

lcel

l(M

BE

C)

Ast

rocy

tes

N/A

NaF

l:25

±0.

2S

hay

anet

al.50

b.E

nd5

Ast

rocy

tom

a13

21N

1<

20N

/AT

anet

al.10

1

b.E

nd3

C6

glio

ma

130

Su

cros

e:19

.4±

2.6

Om

idie

tal

.53

Pro

pan

olol

:23.

4±

1.9

b.E

nd3

C6

glio

ma

<15

0S

ucr

ose:

16.4

±5.

2cA

mp

addi

tion

Om

idie

tal

.53

Pro

pan

olol

:23.

4±

5.9

b.E

nd3

Ast

rocy

tes

N/A

TA

MR

A:5

.9±

1.9

Lie

tal

.103

Dex

tran

10k:

0.78

±0.

24D

extr

an70

k:0.

16±

0.09

b.E

nd3

Ast

rocy

tes,

C8D

1A25

0N

/A:

BB

BB

ooth

and

Kim

104

b.E

nd3

Ast

rocy

tes,

C8D

1A25

N/A

Tra

nsw

ells

Boo

than

dK

im10

4

b.E

nd3

Ast

rocy

tes,

C8D

1A34

5N

/A:

BB

B,s

hea

rst

ress

opti

mal

at1.

2dy

n/c

m2

Boo

than

dK

im10

5

Rat

RB

EC

Ast

rocy

tes

N/A

Su

cros

e:2

DIV

-BB

Bpe

rmea

bili

ties

esti

mat

edfr

omgr

aph

sS

tan

nes

set

al.10

6

D-A

sp:2

L-A

sp:9

.3R

CE

CA

stro

cyte

sN

/AN

aFl:

4.7

Per

mea

bili

ties

repo

rted

ascm

/min

Kis

etal

.64

Eva

ns

blu

eal

bum

in:�

0.75

RB

EC

Ast

rocy

tes

N/A

Su

cros

e:7.

5±

1P

erm

eabi

liti

esre

port

edas

cm/m

inB

lasi

get

al.10

7

NaF

l:11

±1.

3D

ich

lork

ynu

ren

icac

id:1

1±

1.5

RB

EC

Ast

rocy

tes

130

±7

N/A

EC

GF

+20

%P

DS.

Per

mea

bili

ties

repo

rted

ascm

/min

Dem

euse

etal

.108

RB

ME

CA

stro

cyte

s40

0–45

0N

/AK

riza

nac

-Ben

gez

etal

.109

RB

ME

CA

stro

cyte

sN

/AS

ucr

ose:

9.9

Kri

zan

ac-B

enge

zet

al.11

0

Con

tin

ued


REVIEW 2735T

able

1.C

onti

nu

ed

Res

ult

sO

verv

iew

ofB

BB

Mod

els

Mod

elS

peci

esP

rim

ary

Cel

lTyp

eC

ellT

ype

inC

onta

ctC

ellT

ype

inN

onco

nta

ctT

EE

R(�

cm2)

Per

mea

bili

ty(P

app;1

0−6

cm/s

)O

ther

Info

Ref

eren

ce

RB

EC

Ast

rocy

tes

<12

5N

aFl:

�4

App

roxi

mat

ion

sof

Pe

valu

esfr

omta

bles

Nak

agaw

aet

al.11

1

RB

EC

Ast

rocy

tes

<16

0N

aFl:

�4

Nak

agaw

aet

al.68

RB

EC

Ast

rocy

tom

a,13

21N

1<

20N

/AT

anet

al.10

1

Bov

ine

Bov

ine

len

sen

doth

elia

lcel

lP

eric

ytes

150

±17

0L

uci

fer

yell

ow:1

0.1

±2.

5E

ndo

thel

ialc

ells

deri

ved

from

hem

atop

oiet

icst

emce

lls.

Per

mea

bili

ties

repo

rted

ascm

/min

Cec

chel

liet

al.11

2

Hu

man

hB

ME

CP

eric

ytes

26±

0.16

N/A

cell

Zsc

ope

Eig

enm

ann

etal

.61

hC

ME

C/D

3H

BV

P46

±1.

1N

/AH

ath

erel

let

al.10

2

Rat

RB

EC

Per

icyt

es43

8±

75N

/A1:

mpo

res

Dem

euse

etal

.108

RB

EC

Per

icyt

es13

0±

7N

/A0.

45:

mpo

res

Dem

euse

etal

.108

RB

EC

Per

icyt

es97

.5±

8.5

N/A

Nor

mal

ized

valu

esfo

rpe

rmea

bili

tyH

ayas

hie

tal

.113

RB

EC

Per

icyt

es<

200

NaF

l:�

3.4

App

roxi

mat

ion

sof

Pe

valu

esfr

omta

bles

Nak

agaw

aet

al.11

1

RB

EC

Per

icyt

es<

230

NaF

l:�

4.1

Nak

agaw

aet

al.68

RB

E4

Per

icyt

es<

200

Su

cros

e:<

25P

erm

eabi

liti

esre

port

edas

cm/m

inA

lAh

mad

etal

.49

Dex

tran

:<3

Non

-con

tact

cocu

ltu

reB

ovin

eB

BE

CA

stro

cyte

sN

/AS

ucr

ose:

8.5

±0.

7P

erm

eabi

liti

esre

port

edas

cm/m

inH

amm

etal

.114

Inu

lin

:2.8

±0.

3B

BE

CA

stro

cyte

s66

1±

48N

/AD

ehou

cket

al.62

BB

EC

Ast

rocy

tes

500–

800

Su

cros

e:3.

8G

arbe

rget

al.11

5

Ala

nin

e:15

.2A

nti

pyri

ne:

54.0

AZ

T:5

.7C

affe

ine:

60.2

Cim

etid

ine:

4.0

Cyc

losp

orin

e:4.

6D

iaze

pam

:38.

0D

igox

in:3

.1L-D

opa:

16.3

Gly

cero

l:6.

8In

uli

n:0

.67

Lac

tic

acid

:9.3

Leu

cin

e:13

.0M

orph

ine:

22.0

Nic

otin

e:66

.0P

hen

ytoi

n:5

0.3

Ure

a:26

.3V

erap

amil

:23.

4V

inbl

asti

ne:

11.6

Vin

cris

tin

e:2.

3W

arfa

rin

:17.

9

Con

tin

ued


2736 REVIEW

Tab

le1.

Con

tin

ued

Res

ult

sO

verv

iew

ofB

BB

Mod

els

Mod

elS

peci

esP

rim

ary

Cel

lTyp

eC

ellT

ype

inC

onta

ctC

ellT

ype

inN

onco

nta

ctT

EE

R(�

cm2)

Per

mea

bili

ty(P

app;1

0−6

cm/s

)O

ther

Info

Ref

eren

ce

BA

EC

C6

736

±0.

38S

ucr

ose:

0.08

8D

IV-B

BB

hol

low

fibe

rS

tan

nes

set

al.11

6

Th

eoph

ylli

ne:

1.88

Mor

phin

e:0.

05B

BE

CC

6<

200

Su

cros

e:<

10P

erm

eabi

liti

esre

port

edas

cm/m

inA

bbru

scat

oan

dD

avis

117

BA

EC

C6

<50

0D

IV-B

BB

Cu

cull

oet

al.11

8

BC

EC

C6

<30

00N

/AR

O20

–172

4(p

roin

flam

mat

ory)

Zen

ker

etal

.47

BC

EC

C6

2000

N/A

Zen

ker

etal

.47

BB

EC

C6

100

Su

cros

e:8.

1G

arbe

rget

al.11

5

Ala

nin

e:10

.1In

uli

n:0

.6L

euci

ne:

9.0

Vin

cris

tin

e:3.

7B

AE

CC

665

0±

26.5

Su

cros

e:5.

34±

0.23

DIV

-BB

BS

anta

guid

aet

al.11

9

Ph

enyt

oin

:1.4

6±

0.17

Por

cin

eP

BE

CA

stro

cyte

s21

4±

22N

/AG

iese

etal

.94

PB

EC

Ast

rocy

tes

<78

0M

ann

itol

:<1

CP

T-c

AM

P,R

O20

–172

4an

dh

ydro

cort

ison

etr

eate

dT

ran

swel

ls

Pat

aben

dige

etal

.82

PB

EC

C6

834

±13

6S

ucr

ose:

1.6

±1.

0S

eru

m-f

ree

med

ium

,wit

hsu

pple

men

tof

hyd

roco

rtis

one

Sm

ith

etal

.66

Pro

pan

olol

:18.

4±

0.7

PB

EC

C6

352

±34

N/A

Sm

ith

etal

.66

Hu

man

hB

ME

CA

stro

cyte

s<

500

N/A

Per

cen

tage

su

sed

for

perm

eabi

liti

esS

iddh

arth

anet

al.27

Hu

man

plu

ripo

ten

tst

emce

lls

Ast

rocy

tes

860

±26

0In

uli

n:0

.029

±0.

02H

um

anpl

uri

pote

nt

stem

cell

-der

ived

endo

thel

ial

cell

s

Lip

pman

net

al.59

Su

cros

e:0.

034

±0.

015

Glu

cose

:0.2

2±

0.07

Vin

cris

tin

e:0.

062

±0.

02C

olch

icin

e:0.

092

±0.

1P

razo

sin

:0.2

9±

0.04

Dia

zepa

m:1

.1±

0.5

Hu

man

prim

ary

brai

nen

doth

elia

lce

lls

Ast

rocy

tes

N/A

Gar

berg

etal

.115

Con

tin

ued


REVIEW 2737T

able

1.C

onti

nu

ed

Res

ult

sO

verv

iew

ofB

BB

Mod

els

Mod

elS

peci

esP

rim

ary

Cel

lTyp

eC

ellT

ype

inC

onta

ctC

ellT

ype

inN

onco

nta

ctT

EE

R(�

cm2)

Per

mea

bili

ty(P

app;1

0−6

cm/s

)O

ther

Info

Ref

eren

ce

Su

cros

e:21

.7A

lan

ine:

25.0

An

tipy

rin

e:45

.0C

affe

ine:

24.2

Cim

etid

ine:

31.7

Cyc

losp

orin

e:1.

0D

igox

in:1

.5L-D

opa:

20.0

Gly

cero

l:20

.0In

uli

n:1

1.7

Lac

tic

acid

:40.

0L

euci

ne:

28.3

Nic

otin

e:27

.3P

hen

ytoi

n:5

.8U

rea:

70.0

Ver

apam

il:1

6.0

Vin

blas

tin

e:16

.7V

incr

isti

ne:

2.2

War

fari

n:2

1.7

Mu

rin

eM

BE

CA

stro

cyte

sN

/AN

aFl:

3.5

±0.

1S

hay

anet

al.50

Rat

RB

EC

Ast

rocy

tes

358

±40

Su

cros

e:5

±0.

3P

erm

eabi

liti

esre

port

edas

cm/m

inB

over

iet

al.12

0

Inu

lin

:1.8

±0.

3R

BE

CA

stro

cyte

s<

130

NaF

l:�

5.5

Nak

agaw

aet

al.68

RB

EC

Ast

rocy

tes

252

±38

NaF

l:0.

16±

0.03

Lip

pman

net

al.58

SV

-AR

BE

CA

stro

cyte

s50

–70

Su

cros

e:6.

7G

arbe

rget

al.11

5

Ala

nin

e:9.

4A

nti

pyri

ne:

20.4

AZ

T:1

6.5

Caf

fein

e:11

.2C

imet

idin

e:6

Cyc

losp

orin

e:1.

4D

iaze

pam

:22.

7D

igox

in:2

.6L-D

opa:

15.1

Gly

cero

l:20

.1In

uli

n:5

Lac

tic

acid

:14.

5L

euci

ne:

17.8

Mor

phin

e:18

.9N

icot

ine:

20.1

Ph

enyt

oin

:16.

3U

rea:

32.7

Ver

apam

il:9

Vin

blas

tin

e:2

Vin

cris

tin

e:4.

9W

arfa

rin

:3.2

Con

tin

ued


2738 REVIEW

Tab

le1.

Con

tin

ued

Res

ult

sO

verv

iew

ofB

BB

Mod

els

Mod

elS

peci

esP

rim

ary

Cel

lTyp

eC

ellT

ype

inC

onta

ctC

ellT

ype

inN

onco

nta

ctT

EE

R(�

cm2)

Per

mea

bili

ty(P

app;1

0−6

cm/s

)O

ther

Info

Ref

eren

ce

RB

EC

C6

239

±51

Su

cros

e:11

.5±

3.3

Per

mea

bili

ties

repo

rted

ascm

/min

Bov

erie

tal

.120

Inu

lin

:4.3

±0.

3B

ovin

eB

ME

CP

eric

ytes

4450

±17

0N

/AN

PC

-der

ived

BM

EC

.M

odifi

edE

Cm

ediu

m(n

oex

ogen

ous

bFG

F)

Lip

pman

net

al.60

Rat

RB

EC

Per

icyt

es19

.8±

2.6

N/A

Nor

mal

ized

valu

esfo

rpe

rmea

bili

tyH

ayas

hie

tal

.113

RB

EC

Per

icyt

es<

150

NaF

l:�

4.5

Per

mea

bili

ties

repo

rted

ascm

/min

Nak

agaw

aet

al.68

Bov

ine

BM

EC

NP

Cs

2940

±80

0S

ucr

ose:

0.53

–0.5

7±

0.17

Tra

nsw

ellw

ith

NP

Cs

and

NP

C-d

eriv

edB

ME

CL

ippm

ann

etal

.60

BM

EC

NP

Cs

5160

±32

0N

/AN

PC

-der

ived

BM

EC

.M

odifi

edE

Cm

ediu

m(n

oex

ogen

ous

bFG

F)

Lip

pman

net

al.60

Rat

RB

EC

NP

Cs

110

±5

NaF

l:5.

5±

0.8

Wei

den

fell

eret

al.12

1

RB

EC

NP

Cs

246

±19

NaF

l:0.

170

±0.

05�

20%

fray

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2740 REVIEW

Figure 5. The DIV-BBB system. Image taken fromhttp://www.flocel.com/technology.html.

Figure 6. The effect of shear stress on cell cultures, where a dynamiccoculture is shown to be more than 10 times better than a static mono-layer of endothelial cells. Reproduced from Booth and Kim104 withpermission from RSCPublishing.

by introducing additional cell types from the neurovascularunit.

Generally, data on triple cultures are gathered using theTranswell culture platform. Astrocytes are commonly usedas the first additional cell type, as more than 30 years ago,they were the first cell type shown to be essential for BBBfunction.131 Neurons are traditionally used as the second sup-plementary cell type and are most frequently grown out ofcontact on the abluminal side of the membrane (Fig. 3c). Al-though the role of pericytes is not yet fully understood, theyhave gained some interest in recent years as a supplementarycell type. Accordingly, not many research groups have adoptedthis new model.68,102,113

For many groups studying drug transport or otherapplication-oriented studies, a triple culture may be more com-plicated than needed. If however one is interested in, for in-stance, ischemia, a triple (or quadruple) culture may be neces-sary to reach a sufficiently in vivo-like model.

Endothelial Cells with Astrocytes and Neurons

In a report, Schiera et al.132 investigated the permeability of[3H]sucrose in a triple-culture using RBCECs, astrocytes, andneurons. The endothelial cells were cocultured in contact withthe astrocytes on the membrane, and neurons were cultured onthe bottom of the well. Four different configurations (endothe-lial cells, endothelial cells with astrocytes, endothelial cellswith neurons, and endothelial cells with both astrocytes andneurons) were studied and the most notable significance (p <

0.005) could be seen for the triple-culture.132 Again, no TEERvalues were reported, which makes it difficult to assess the ac-tual tightness of the barrier, although the work clearly shows areduced permeability of the barrier upon the addition of morecell types in accordance with the hypothesis.

Recent work, using the same triple-culture setup with en-dothelial cells, astrocytes, and neurons, have been shown togive an increase in TEER value by 35.9% compared withmonocultures,123 reaching a TEER of 268 �cm2. A significant(p < 0.01) rise in ZO-1 expression was also seen, indicating atighter barrier than for monocultures. Triple-cultures with en-dothelial cells, astrocytes, and neurons also result in a ninefoldincrease in the expression of the enzyme (-glutamyl transpep-tidase (which aids in reducing the net influx of amino acidsto the brain and plays a part in the BBB function78) comparedwith both monocultures of endothelial cells and cocultures withastrocytes.123

Rat brain endothelial cells synthesize occludin faster whencocultured with neurons,92,133 and Schiera et al.134 has shownthat RBE4.B cells manage to synthesize even more occludinwhen cocultured for 5 days with both astrocytes and neurons.With a noticeable increase in TEER value, it is clear that oc-cludin synthesizing has a visible impact on barrier function.It has since been demonstrated that occludin is expendable inTJ construction, as other proteins are upregulated in the eventof occludin shortage,135 making it unclear whether the rise inoccludin synthesis is the sole reason for the increased TEERvalues.

Endothelial Cells with Astrocytes and Pericytes

Nakagawa et al.111 used RBECs cocultured with astrocytes andpericytes to study the effect of cell type combination on the re-sulting barrier properties. By varying the seeding site of theastrocytes and the pericytes in relation to the endothelial cellsand comparing this to a coculture with endothelial cells and ei-ther of the other two cell types, they could show that the TEERincreased almost twofold (from �100 to �200 �cm2) when thepericytes were in contact with the endothelial cells instead ofthe astrocytes. This further confirms that pericytes are most ef-ficient in upholding BBB functionality in the endothelial cellsin contact mode.113 When pericytes and astrocytes were usedtogether and pericytes were cultured in contact, abluminallyon the membrane (named EPA in Fig. 6), TEER increased withnearly a factor 8 (Fig. 7), compared with a monoculture. On thecontrary, permeability studies showed that sodium fluoresceinpermeability did not change depending on which cell was cul-tured in contact,111 suggesting that triple-cultures may not benecessary to study drug permeability.

A number of studies indicate that TEER values aloneare not sufficient to determine the quality of the BBB asTEER values above 150 �cm2 do not necessarily decreasethe permeability of given tracer substances.13,43,111,136,137 To


REVIEW 2741

TE

ER

(cm

2 )

Days

0

2 3 4 5

TE

ER

(cm

2 )

0

5

10

0Days

a, b

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200

300

2 3 4

E00

EP0

EA0

E0P

E0A

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EAP

0

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000

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0A0

5

Figure 7. The effect of different co- and triple-cultures on a BBB model using Transwells. With an inset graph showing that neither astrocytesor pericytes have any relevant resistances. E00, a monoculture of endothelial cells. E0A, noncontact coculture with astrocytes. EA0, contactcoculture with astrocytes. E0P, noncontact coculture with pericytes. EP0, contact coculture with pericytes. EAP, triple-culture with astrocytesin contact and pericytes in noncontact. EPA, triple culture with pericytes in contact and astrocytes in noncontact. Reproduced from Nakagawaet al.111 with permission from Springer US.

Figure 8. Graph of the correlation between permeability coefficientsof drugs in a triple culture in the EPA setup (in vitro), and the apparentpermeability coefficients of the same drugs in animal models (in vivo).Reproduced from Nakagawa et al.68 with permission from Elsevier B.V.

evaluate their previous work, Nakagawa et al.68 comparedthe absorption of 19 compounds with their known in vivovalues using the same seven models as in the 2007 exper-iment in a new study published in 2009. This study, witha larger number of trials, showed the highest TEER for thetriple-culture with the EPA setup, with a reduced perme-ability in regard to a monoculture of endothelial cells. Thegroup also showed that the expression of TJ proteins wasincreased by the presence of astrocytes or pericytes but in-creased most in the EPA model. Permeabilities of low Pe

substances remained low in the EPA model at 0.44–3.51×10−6 cm/s. When compared with in vivo values, a correlation ofR2 = 0.89 was found, (Fig. 8). When evaluated together, the two

studies68,111 give a good overall image of their model with manyof the parameters that have bearing on the tightness of thebarrier.

Hatherell et al.102 repeated Nakagawa’s experiment, usinghCMEC/D3 cocultured with human pericytes and astrocytes.Their obtained TEER values differed noticeably from Naka-gawa’s results with the endothelium/astrocyte culture result-ing in the highest TEER values, even compared with the twocombinations of triple cultures (Fig. 9). This effectively con-firms that even though two cell types may be similar in func-tion, they can still differ widely in application122 and thatspecies-specific parameters have substantial impact on barrierbehavior.39

Using RBE4, Al Ahmad et al.49 analyzed the stability oftriple cultures upon anoxic events. They showed that the pres-ence of astrocytes and pericytes in a triple-culture was theonly means of maintaining the original TEER values even48 h after oxygen deprivation. The triple-culture also gavethe lowest increase in permeability of sucrose (2.81 ± 0.81× 10−3 cm/s as opposed to 10.6 ± 1.28 × 10−3 cm/s for amonoculture) 48 h after the anoxic event. The mechanism bywhich the astrocytes and pericytes promoted the survival of theREB4 cells during anoxia was determined to be by caspase-3inhibition.

DISCUSSION ON CURRENT STATE-OF-THE-ART ANDNEW TRENDS

As mentioned before, endothelial cells from different speciesmay share many BBB attributes at a cellular or molecular levelbut they can still react differently in experimental setups.122

Given this, it is truly difficult to assess a model without testingit for all types of relevant endothelial cells; a model might workwell for one endothelial cell but not for another. Minor modifi-cations and additions to models can result in widely differentresults, as apparent in Table 1. In the author’s opinion, the


2742 REVIEW

30

35

40

45

50

55

60

65

70

10987654321

TE

ER

(Ω

cm

2 )

Time (days)

A

B C

E FG H

D

*

hCMEC/D3 mono-cultivation (A) hCMEC/D3 and CC-2565 co-cultivation (B)

hCMEC/D3 and SC-1810 co-cultivation (C) hCMEC/D3 and HBVP co-cultivation (D)

hCMEC/D3, CC-2565, and HBVP tri-cultivation (E) hCMEC/D3, SC-1810, and HBVP tri-cultivation (F)

hCMEC/D3, HBVP, and CC-2565 tri-cultivation (G) hCMEC/D3, HBVP, and SC-1810 tri-cultivation (H)

Figure 9. The effect of co- and triple-cultures on hCMEC/D3 and twodifferent astrocyte cell lines (CC-2565 and SC1810) and one pericytecell line (HBVP) in three independent experiments. The asterisk marksthe most significant points compared with a monoculture (p < 0.001).Reprinted from Hatherell et al.102 with permission from Elsevier B.V.

field would benefit greatly from commonly accepted standardsfor permeability measurements, TEER, fluorescence, and otherquantitative evaluations.

Another area of the BBB in vitro research field with high po-tential is the development of models based on pluripotent stemcells, or neural progenitor cells (NPC). This approach couldenable human in vitro model systems with a much higher ex-pected prediction rate compared with animal-derived systems.In work from 2011, Lippmann et al.58 differentiated NPC forup to 12 days to reach a ratio of 3:1 of astrocytes and neu-rons (Fig. 10), which were then seeded on the bottom of aTranswell. The membrane cup with rat brain microvessel en-dothelial cells (RBMECs) was added to the well and TEER val-ues were monitored for 5 days. Their optimized NPC-derivedcultures increased membrane resistance by up to 200% com-pared with endothelial cell monoculture,58 raising TEER to avalue of approximately 250 �cm2. This work builds upon previ-ous research, where a controlled differentiation of NPC aimedat in vitro BBB models were presented already in 2007.121

The group has been further developing their work, and in2012 they showed that human pluripotent cells that were cod-ifferentiated with neural cells produced endothelial cells withBBB properties, such as TJ expression and presence of rel-evant nutrient transporters.59 The stem cells also respondedto astrocytes in cocultures, giving relevant TEER values(1450 �cm2) and a permeability of several compounds withinacceptable levels of the in vitro model. In 2014, additional im-provements were presented where TEER values greater than5000 �cm2 were achieved by first enhancing the BBB pheno-type in the HBMECs using retinoic acid.60 This is the highestpublished TEER value to date from any in vitro BBB model,indicating that triple-cultures are superior to mono- and cocul-tures with respect to obtaining high-barrier resistances. It is,

Figure 10. Immunofluorescent images of NPC cultured for 12 dayswith 10% fetal bovine serum (a) astrocytes labeled with GFAP (red) ornestin (green) (b) and neurons labeled for $III-tubulin (red) or nestin(green). Scale bars indicate 50 :m and nestin labels undifferentiatedNPC. Reproduced from Lippmann et al.58 with permission from WileyInc.

however, not completely clear whether triple-cultures are nec-essary to achieve reliable in vitro data because several otherreports are available (Table 1), where high TEER values arereached without using triple cultures. Using NPC instead ofmature astrocytes and neurons is, however, not a simple taskand the protocol for reaching the correct ratio of astrocytesand neurons is in itself a major undertaking. So far, only EricShusta’s group has worked with NPC cultures in this fashion.

For drug delivery studies, permeability is the most inter-esting performance factor to evaluate an in vitro model upon.Although many researchers are active in this field, few studiesuse the same test substances, making it hard to ascertain thetrue permeability ratio between the different models and celltypes. Even so, a BBB model with shear stress75 or a model withNPC-derived astrocytes and neurons100,116 gives the lowest re-ported permeabilities for both mannitol and sucrose. However,too little data exist regarding membrane permeability and pro-tein expression to claim superiority over other models. Lipp-mann et al.127 show a low permeability for sucrose at 0.57 ±0.12 × 10−6 cm/s in their coculture with cells derived from NPCand Cucullo et al.100 show sucrose at 0.29 × 10−6 cm/s with


REVIEW 2743

their DIV-BBB, while also showing low-permeability values forsmaller molecules such as mannitol. In this particular compar-ison, the data clearly demonstrate how low permeabilities canbe found with relatively low TEER values and higher perme-abilities for a system with high TEER values, which makes itimpossible to correlate resistance to permeability. As microflu-idic devices today do not yet measure permeabilities, it is im-possible to evaluate them properly.

Most studies published to date on in vitro BBB research areperformed using the Transwell cell culture systems, but duringthe last 10 years, a major increase of interest in microchip BBBmodels has been seen.84,85,104,127,138,139 The motivation for this isthe fact that the capillaries in the microvasculature are as smallas 7–10 :m in diameter with an average intercapillary distanceof 40 :m140,141 and an average distance to the nearest neuronof only 10–20 :m.142 The standard semipermeable membraneof the Transwells has a thickness of 10 :m, which is more than300 times thicker than the naturally occurring basement mem-brane of the endothelial cells. A system developed in the samescale as the neurovascular unit is expected to more closely re-semble the in vivo situation and in turn result in in vivo-likebarrier properties. Most of the studies published on microfluidicBBB systems have not yet reached beyond the proof-of-conceptstage and the reported TEER values and permeability valuesare often of a lower standard than the Transwell-based sys-tems. It is likely several more articles in this highly interestingresearch area will be published in the coming years, with greatpotential for the BBB research field.

CONCLUSIONS

This review article has presented some of the most influentialand most recent work regarding in vitro BBB models to date.Monocultures, cocultures, and triple-cultures of the major con-stituent cell types of the neurovascular unit have been investi-gated to better understand the important aspects of achieving ahigh-fidelity model of the BBB. Different setups, ranging fromthe classical Transwells to more novel approaches using mi-crofluidics, have also been studied.

This review article further set out to evaluate the perfor-mance of cell-based in vitro systems of the BBB. It was foundto be very difficult to evaluate the performance of the plethoraof different models used because of the lack of coherence inthe research field. The authors therefore call for a more stan-dardized method to evaluate the performance of the differentin vitro systems developed as an effort to jointly bring the re-search field forward. It would also be interesting to see moreresearch groups taking an interdisciplinary approach to the is-sue as both the biological as well as the more technical aspectsof the culture platform are likely to be highly influential on thefinal in vitro performances.

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In Vitro Blood-Brain Barrier Models-An Overview of Established Models and New Microfluidic Approaches

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