Senso s 2012, 12, 1383-1397; doi:10.3390/s120201383
senso s
ISSN 1424-8220
www.mdpi.com/jou nal/senso s
A icle
Mul i-Senso A ays o Online Moni o ing o Cell Dynamics
in in i o S udies wi h Cho oid Plexus Epi helial Cells
Ped o Mes es-Ven u a 1,2,*, And ea Mo gue 1 and Soledad Ga cía Gómez de las He as 2
1 Depa men o Ana omy and Cell Biology, Saa land Uni e si y, Uni e si y Hospi al, Bldg. 61,
66421 Hombu g, Saa , Ge many; E-Mail: wm[email p o ec ed]
2 Depa men o Human His ology and Pa hology, Facul y o Heal h Sciences, Uni e si y Rey Juan
Ca los I, A . de A enas s/n, E-28922 Alco cón, Mad id, Spain; E-Mail: soledad.ga[email p o ec ed]
* Au ho o whom co espondence should be add essed; E-Mail: [email p o ec ed];
Tel.: +34-91-488-8622; Fax: +34-91-488-8955.
Recei ed: 1 Decembe 2011; in e ised o m: 13 Janua y 2012 / Accep ed: 17 Janua y 2012 /
Published: 1 Feb ua y 2012
Abs ac : Senso s and mul i-senso a ays a e he basis o new echnologies o he
non-label moni o ing o cell ac i i y. In his pape we show ha cho oid plexus cells can be
cul u ed on silicon chips and ha senso s egis e in eal ime changes in hei ac i i y,
cons i u ing an in e es ing expe imen al pa adigm o cell biology and medical esea ch.
To alida e he signals eco ded (me abolism = pe i-cellula acidi ica ion, oxygen
consump ion = espi a ion; impedance = adhesion, cell shape and mo ili y) we pe o med
expe imen s wi h compounds ha ac in a well-known way on cells, in luencing hese
pa ame e s. Ou in i o model demons a es he ad an ages o mul i-senso a ays in
assessmen and expe imen al cha ac e iza ion o dynamic cellula e en s—in his case in
cho oid plexus unc ions, howe e wi h applicabili y o o he cell ypes as well.
Keywo ds: senso a ays; pe i-cellula acidosis; cell espi a ion; cell adhesion; impedance;
cell cul u es; elec on mic oscopy; cho oid plexus; chick emb yo
1. In oduc ion
Elec ical and elec ochemical senso s ha e enabled he de elopmen o echnologies, which a e
e y use ul in ob aining da a on cellula ac i i ies wi h minimal ex e nal in e en ion. These
echniques can be applied wi hou any labeling o cells, hus a oiding he possible al e a ion o cells
OPEN ACCESS
Senso s 2012, 12
1384
unde in es iga ion. Fu he mo e, hese echniques ha e pa ed he way o online obse a ion,
enabling con inuous, unin e up ed execu ion o he expe imen . These de elopmen s ep esen a
conside able imp o emen on he mo e adi ional so-called “end-poin ” echniques [1].
Pionee de ices we e equipped wi h senso s de ec ing changes in cell adhesion, cell mo ili y and
cell shape, based on impedance measu emen s [2]. Ano he gene a ion o senso s was he so-called
ligh -add essed po en iome ic senso s (LAPS), able o de ec changes in pe i-cellula acidosis, a
phenomenon which is closely ela ed o he s a e o cell me abolism [3]. Mo eo e , o ensu e he
unc ion and i ali y o he cells, hese de ices we e equipped no only wi h a luidic sys em ha
ensu ed he supply o cul u e medium, bu also wi h sys ems o egula ing empe a u e, humidi y and
gas a mosphe e in he modules whe e he cells we e incuba ed. A common ea u e o he appa a us o
his i s gene a ion was ha hey we e mono-pa ame ic [4]. Du ing he pas 20 yea s hese de ices
ha e ound nume ous applica ions in biology and medicine, o ins ance, in he s udy o ligand- ecep o
in e ac ions, in he analysis o he ac ion o chemicals and d ugs on speci ic cell a ge s, in he
assessmen o he chemosensi i i y o cance cells, and many mo e [5–7].
These senso s we e ollowed by a new gene a ion o senso s cha ac e ized by he p esence on he
silicon chip o se e al senso ypes, he so-called mul i-senso a ays, wi h which many pa ame e s can
be measu ed simul aneously: impedance (adhesion, cell shape), acidi ica ion o pe i-cellula acidosis
(me abolism) and oxygen consump ion ( espi a ion) [8–10]. F om he e y beginning, hese new sys ems
we e de eloped wi h a iew o hei possible applica ions in esea ch a eas such as he chemosensi i i y
o umo cells and d ug sc eening. Mo e ecen ly, examples o success ul applica ions in oxicology,
pha macology, and e en in he ield o ecology can be ound in ele an li e a u e [11–14].
Wi h he applica ion o mul i-senso echnology a ose he necessi y o he de elopmen o me hods
o p epa a ion o cells and issues sui able o he in es iga ion o such specimens in hese new de ices.
A i s , he use o pe manen cell lines was impe a i e, because hey a e easie o handle in he senso
de ices. Bu in he mean ime, conside able expe ience has been acqui ed wi h p ima y and o gano ypic
cul u es, as well as issue slices [15].
This pape desc ibes he p epa a ion and managemen o cho oid plexus epi helial cells (CPC) on
mul i-senso a ays as an in i o cell model and p esen s esul s o how hese cells eac when ea ed
wi h compounds in luencing hei me abolism and anspo unc ions. The signi icance o cho oid
plexus lies in he ac ha i is a majo sou ce o ce eb ospinal luid, which ci cula es h ough he b ain
en icles and he suba achnoid space [16]. Because o he complex ne wo k o in e ac ions in he
whole body, app op ia e cellula models unde axenic condi ions a e equi ed o unde s and he
molecula basis o speci ic mechanisms o he cho oid plexus epi helial cells, since hey a e he main
cell elemen o his o gan. These equi emen s we e aken in o accoun in he expe imen al app oach
desc ibed he e wi h a esul ing high ele ance o cell biology in gene al and medical esea ch in
pa icula (neu ology, neu osu ge y, pha macology, e c.).
2. Expe imen al Sec ion
2.1. Cell Cul u es
Cho oid plexus o chicken emb yo 14 ED emb yos (20–30 emb yos p o assay) we e dissec ed and
collec ed in cul u e medium (DMEM/F12 Gibco, supplemen ed wi h penicillin and s ep omycin,
Senso s 2012, 12
1385
L-glu amine, insulin, EGF, hyd oco isone, NEA, ans e in, 10% FCS). A e dissec ion, he cho oid
plexus we e dissocia ed wi h ypsin + EDTA (Gibco) in a Pe i dish and using a magne ic s i e a
37 °C in a CO2 incuba o (He aeus). Subsequen ly, he cell suspension was passed h ough a cell s aine
(BD) and insed wi h medium wi h 10% FCS ( o inac i a e ypsin). The esul ing cell suspension was
cen i uged a 2,400 pm o 4 min (cy o-cen i uge Sa o ius), he supe na an disca ded and he pelle
e-suspended in esh medium and he cells coun ed wi h T ypan Blue (Sigma-Ald ich). Subsequen ly,
he chips we e inocula ed wi h 1.5 o 3 × 105 cells each. Pa allel o his, well pla es wi h The manox
slides (Nunc) we e also seeded wi h a simila cell suspension (2 × 106 p o well).
Al hough he chips a e made o silicon, a ma e ial sui able o cell cul u es, we coa ed hei su aces
wi h collagen I, which conside ably imp o es a achmen condi ions o he cho oid plexus cells [17].
A solu ion o collagen I (20 µg/mL in 0.02 N ace ic acid) was applied o he su aces o he chips,
which we e hen incuba ed a 37 °C o 1 h. Subsequen ly, hey we e washed wi h cul u e medium and
used immedia ely o e ige a o -s o ed. The manox slides we e coa ed ollowing same p o ocol.
The cells we e i s cul u ed wi hou cys-a abinoside (Sigma-Ald ich) on bo h chips and The manox
slides. A e 24 h he medium was eplaced by esh medium con aining cys-a abinoside, a subs ance
able o supp ess he g ow h o ib oblas s and endo helial cells, bu which does no a ec he epi helial
cells o he cho oid plexus. A e a cul u e pe iod o 24 h o longe (un il 70–80% o he chip su ace
was co e ed wi h cho oid plexus cells) he chips we e ans e ed o he senso de ice. The
measu emen s in he senso de ice we e conduc ed wi h a medium (low bu e ing capaci y), which did
no con ain cys-a abinoside. The measu emen imes in he senso uni anged om 24 o 72 h, o e en
longe acco ding o expe imen equi emen s. The g ow h o cells on The manox pla es was egula ly
moni o ed using an in e ed mic oscope (Olympus) wi h phase con as . As, in his case, he chips
we e opaque, he examina ion was pe o med by e lec ed ligh wi h a s e eomic oscope (Wild,
Hee b ugg, Swi ze land).
2.2. Senso De ice
The Bionas 2500® analyzing sys em (Bionas GmbH, Ros ock, Ge many) was used in hese s udies.
A de ailed desc ip ion o his sys em has been published p e iously [13]. B ie ly, he de ice is
composed o six modules, which a e connec ed wi h a pump and con ol uni s. The cul u e media, wi h
o wi hou d ugs, i s low o and hen h ough each module con aining he senso chips and a e hen
di ec ed o a was e collec o . The e e ence elec ode is placed in he luidic pa hway behind he senso
modules. The senso chip (me abolic chip Bionas Disco e y™ SC1000) used he e, con ains a senso
a ay composed o : (a) wo Cla k- ype senso s, (b) i e ISFETs (ionic-sensi i e ield-e ec ansis o s)
and (c) one IDES (in e -digi ized elec ode senso s). Such senso s allow simul aneous de e mina ion o
cellula oxygen consump ion (a), pe i-cellula acidi ica ion (b) and cell impedance (c) in on-line
egime and wi hou any labeling.
Be o e he senso analyze is used, he ubes o he luidic sys em ha e o be disin ec ed by
pe using hem wi h 70% e hanol, ollowed by PBS and a inal washing wi h cul u e medium, in ou
case DMEM/F12 (see abo e). The senso chips should also be disin ec ed acco ding o a simila
p o ocol as abo e. The senso chips wi h CPC cells (see abo e) we e placed in o he modules and
main ained in he Bionas de ice a 37 °C.
Senso s 2012, 12
1386
The ollowing ope a ional pa ame e s o he Bionas Analyzing Sys em 2500® we e selec ed. The
low a e was 56 µL/min wi h an al e na e cycle o “go” and “s op” phases o 4 min each. Du ing he
“go” phase cho oid plexus cells a ached o he chip ecei ed esh medium wi h o wi hou
compounds (see below). Du ing he “s op” phase oxygen consump ion and ex acellula acidi ica ion
we e con inuously measu ed in he supe na an . Changes in he a es o hese pa ame e s we e de ec ed
o each single s op phase, ep esen ing he me abolic ac i i y o he cells a ha ime and a e gi en as
single da a poin s in he diag ams.
The p esence o cells in con ac wi h IDES was de ec ed and exp essed in e ms o cell impedance.
These measu emen s p o ided in o ma ion on he ex en o cell adhesion o he subs a e p ecisely a
he IDES a eas and we e independen o pump ac i i y. Howe e , in conside a ion o he in luence o
in e e ences, he da a poin alues du ing he “s op” phase we e a e aged. All da a ob ained ha e been
p esen ed no malized and we e p ocessed wi h O igin 8.0.
2.3. Compounds
To induce in e p e able changes in me abolism and beha io o cho oid plexus cells he ollowing
compounds we e selec ed:
(a) Sodium luo ide (NaF, Me ck) ha dose-dependen ly inhibi s glycolysis and anae obic
me abolism; in addi ion, di ec e ec s o NaF on mi ochond ia ha e been desc ibed (see below)
(b) Po assium cyanide (KCN, Riedel-de-Haen) ha inhibi s cellula espi a o y p ocesses,
especially a he le el o mi ochond ia,
(c) Fo skolin (gene ously p o ided by P o . D . M. Diene , Uni e si y o Giessen, Ge many), an
agonis o adenyla e cyclase, an enzyme ha s imula es and egula es he syn hesis o cAMP,
(d) Ace azolamide (Sigma-Ald ich) ha inhibi s ca bonic anhyd ase, a key enzyme in wa e
anspo p ocesses in he cho oid plexus.
These compounds we e implemen ed in he ollowing concen a ions: NaF 20 mM, KCN 5 mM and
10 mM, o skolin 5, 10 and 25 µM, ace azolamide 10 µM, 100 µM, 1 mM and 2 mM. The compounds
we e applied du ing pe iods o ime anging om 1 o 24 h, ollowed by a a iable pe iod, du ing
which he cells ecei ed cul u e medium con aining no compound, wi h he aim o de ec ing possible
la e e ec s and es ima ing he deg ee o cell eco e y a e a ce ain ea men .
A he end o each expe imen , he chips we e examined and pho og aphed unde a s e eoscopic
mic oscope, and hen ixed chemically o elec on mic oscopy. The Bionas de ice has six modules
ha we e dis ibu ed as ollows: wo modules we e des ined o he con ols, wo o compound 1 and
he emaining wo o compound 2. This p ocedu e acili a es compa a i e analysis. A leas ou chips
pe compound and concen a ion we e measu ed and e alua ed. Chips which c ea ed dis u bances
du ing expe imen s we e excluded. The expe imen s we e pe o med (pe compound and concen a ion)
on a leas wo di e en days.
2.4. Elec on Mic oscopy
Chips and The manox slides we e ixed in 3% glu a aldehyde (Polysciences Inc.) in PBS. Fo
scanning elec on mic oscopy in a FEI 30XL ESEMTM a 10 kV, specimens we e pos - ixed wi h
Senso s 2012, 12
1387
osmium e oxide, dehyd a ed in a se ies o alcohols, c i ical poin d ied and spu e ed wi h
pla inum [18,19]. T ansmission elec on mic oscopy ollowed a p o ocol desc ibed elsewhe e [20,21].
B ie ly, ixa ion was pe o med wi h 3% glu a aldehyde in PBS, pos - ixa ion wi h osmium e oxide,
dehyd a ion in a se ies o alcohols and embedding in Embed 812 (Elec on Mic oscopy Sciences,
USA). Epoxy esin mix u e was pou ed on he cells and polyme ized a 60 °C. The polyme ized esin
laye wi h he cells can be easily emo ed om he silicon chip. Ul a hin sec ions (80 nm hick) we e
s ained wi h u anyl ace a e (Me ck) and lead ci a e (EMS, USA) and examined in a FEI TecnaiTM
Bio win 120 ansmission elec on mic oscope a 60 kV.
3. Resul s and Discussion
3.1. Cul u es o Cho oid Plexus Epi helial Cells (CPC)
The p epa a ion o he cho oid plexus o he chick emb yo was pe o med—wi h sligh
modi ica ions—acco ding o a p o ocol al eady desc ibed [22]. Following his p o ocol, we ha e
achie ed he o ma ion o cell monolaye s composed almos exclusi ely o cho oid plexus epi helial
cell (Figu e 1). This was made possible by he addi ion o cys-a abinoside, an inhibi o o DNA
syn hesis, o he cul u e medium, [22]. The CPCs ha e a nucleo ide anspo sys em unable o up ake
cys-a abinoside; he e o e g ow h and mo phology o hese cells emain unchanged. In con as , he
o he cells o plexus ( ib oblas s, endo helia) ake up he cy os a ic, which p e en s hei p oli e a ion
and g ow h. The esul is pu e cul u es o CPC.
Figu e 1. Phase con as o cho oid plexus cells g own on The manox o 4 days. The cells
o m a monolaye displaying a polygonal shape. The cul u e was p e iously ea ed wi h
cys-a abinoside o elimina e cell con amina ion. 20X objec i e.
When he cell suspension was applied o ei he chips o The manox slides, he cells a ached a an
ea ly s age du ing he i s 24 h, showing a a iable shape, consis en wi h he p ocess o a achmen o
subs a e. A e 2 days cul i a ion he cells began o agg ega e wi h hei congene s, cons i u ing he
monolaye 2 days la e (Figu e 1). Compa a i e s udies wi h o he coa ings (laminin, ib onec in and
collagen IV) ha e shown ha CPCs a ach as e on collagen I-coa ed su aces, a ci cums ance
bene icial o ou s udies wi h he senso s, as we can s a he measu emen s ea lie .

Senso s 2012, 12
1388
Elec on mic oscopy e ealed he su ace ul as uc u e o hese monolaye s and con i med hei
speci ic cell composi ion (Figu e 2). The cell bo de s we e ma ked by nume ous hin mic o illi, gi ing
he image a mosaic-like appea ance. In he cen e o he cells, apical pole mic o illi and cilia, ypical
a ibu es o hese cells los du ing enzyma ic dissocia ion, eappea ed. T ansmission elec on
mic oscopy con i med hese diagnoses, and also enabled de e mina ion o he pa icipa ion o
cy oskele on and cell memb ane, o ming in e cellula con ac s and ancho ages o subs a e (no
shown). Cells g own on silicon chips o The manox displayed exac ly he same pa e ns.
Figu e 2. Scanning elec on mic oscopy o CPCs g owing on he senso chip. The cells
ha e nume ous mic o illi a he apical pole and show a ma ked elie in he cen al pa
ha co esponds o he nucleus.
Be o e he silicon chips wi h he cells we e placed in o he senso de ice, e e y chip was examined
unde a s e eomic oscope o es ima e he quali y o he monolaye s and hei opog aphic ela ionship
wi h he senso s (Figu e 3).
Figu e 3. Scanning elec on mic oscope image shows g oups o cells a anged in monolaye
g owing on he chip. The CPCs can be seen as a g ey eil co e ing he majo pa o he
isible chip su ace.
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n
7
). Impeda
n
a
dhesion ha
d
a
pp ox. 60
%
h
ing le els
c
yanide. N
o
h
in a ew h
o
13
9
o
ugh i la
e
a
yed a a h
e
o NaF.
.
n
me abolis
m
n
ide om
h
n
ce emain
e
d
aken pla
c
%
(Figu e
9
close o
h
o
e he
o
u s.
9
0
e
e
m
h
e
e
d
c
e
9
).
h
e
S
g
c
s
h
d
h
o
w
d
S
enso s 201
Fi
g
u
e
as Fi
g
wi h c
Fi
g
u
e
ype a
The me
a
g
lycolysis [
2
c
ell [3,6,15]
s
ess, inclu
d
h
a accom
p
d
e achmen
h
e changes
o
impai m
e
The exp
e
w
i h he bl
o
d
oes no ap
p
om he gl
y
2, 12
e
8. Online
g
u e 7). No
yanide.
e
9. In co
n
s expe ime
n
a
bolic chan
g
2
3], esul i
n
. Mo eo e
d
ing chang
e
p
anied hes
e
o he cell
s
in espi a
o
e
n o he s
a
e
imen s w
i
o
cking o
e
p
ea o be l
e
y
colysis, w
h
moni o in
g
able ac i a
n
as , adhe
s
n
Figu e 7
)
g
es igge e
n
g in a edu
, i has bee
n
e
s in cell p
e
p ocesses
s
[26]. Suc
h
o
y pa ame
a
bili y o
h
i
h KCN a
e
e
spi a ion c
o
e
hal (unde
h
ich appea
g
o me abo
ion o me
s
ion o CP
C
)
.
d
b
y NaF a
c ion in he
n
well-doc
u
o eins, mi
o
diminishe
d
h
e en s a
e s could
b
h
ei memb
a
e
in e es i
n
o
uld ha e
a
he condi
ed s ongl
y
lism o CP
C
abolism (a
c
C
s (imped
a
e explaine
d
ex usion
o
u
men ed h
a
o
chond ia a
d
memb an
e
e consis e
n
b
e due o h
e
a
nes h oug
h
n
g, as he i
n
a
compens
a
ions used
h
y
ac i a ed
i
C
s exposed
c
idi ica ion
)
a
nce) does
n
d
by he in
h
o
p o ons (
p
a
NaF can
i
n
d cell nuc
l
e
luidi y,
w
n
wi h ou
e
e ec o
N
h
lipid pe o
n
c ease in
g
a
o y signi
i
h
e
e) becau
s
i
n hese ex
p
o cyanide
)
obse ed
n
o show a
n
h
ibi ion o
e
p
e i-cellul
a
i
nduce apo
p
l
eus [24,25
]
w
i h he app
e
impedance
N
aF on he
xida ion [2
7
g
lycolysis
o
i
cance. Th
e
s
e he ATP
p
e imen s (
(same exp
e
in all expe
n
y change
s
e
nolase, a k
e
a
acidi ica
i
p
osis h ou
g
]
. The lipid
e
a ance o
b
measu em
e
mi ochond
7
].
o
bse ed si
m
e
blocking
o
needed ca
n
inc ease o
13
9
e
imen
imen s
s
(same
e
y enzyme
i
i
on) om
h
g
h me abol
i
pe oxida i
o
b
lebbing a
n
e
n s. Finall
y
ia as a es
u
m
ul aneous
l
o
espi a i
o
n
be p o id
e
pe i-cellul
a
9
1
i
n
h
e
i
c
o
n
n
d
y
,
u
l
l
y
o
n
e
d
a