A 3D bioelec ical in e ace o assess colo ec al cance p og ession
in i o
J. Saez
a
,
b
,
c
,
*
, A. Dominguez-Al a o
d
, C. Ba be io
a
, A.M. Wi he s
a
, D. Mece eyes
c
,
d
,
R.M. Owens
a
,
**
a
Depa men o Chemical Enginee ing and Bio echnology, Uni e si y o Camb idge, Philippa Fawce D i e, Camb idge, CB3 0AS UK
b
Mic ofluidics Clus e UPV/EHU, BIOMICs Mic ofluidics G oup, Lasca ay Resea ch Cen e , Uni e si y o he Basque Coun y UPV/EHU, A enida Miguel de
Unamuno, 3, 01006, Vi o ia-Gas eiz, Spain
c
Ike basque, Basque Founda ion o Science, E-48011 Bilbao, Spain
d
POLYMAT Uni e si y o he Basque Coun y UPV/EHU, Joxe Ma i Ko a Cen e , A da. Tolosa 72, 20018 Donos ia-san Sebas ian, Spain
a icle in o
A icle his o y:
Recei ed 8 Janua y 2022
Recei ed in e ised o m
11 May 2022
Accep ed 13 May 2022
A ailable online 17 June 2022
Keywo ds:
PEDOT
Hyalu onic acid
Collagen
Conduc i e sca olds
Elec ochemical impedance spec oscopy
Colon cance
Me as asis
abs ac
Conduc ing polyme s such as PEDOT ha e a ac ed conside able a en ion in he issue enginee ing field
o add an ac i e elec ical ead-ou o 3D cell cul u es. Howe e , PEDOT is no mally copolyme ized wi h
PSS
ha possibly deg ades acidic by-p oduc s in he long- e m. Gi en his d awback, i is p e e able o
ailo PEDOT:polyelec oly e dispe sions ha be e mee he mo phological and physiological mic o-
en i onmen o he human issues. He ein, a no el bioelec ical in e ace in he shape o a 3D po ous
sca old made o he conduc ing PEDOT/hyalu onic acid (HA) and collagen (COL) is p esen ed. Fo his
pu pose, fi s , he oxida i e chemical polyme iza ion o 3,4-e hylenedioxy hiophene (EDOT) was ca ied
ou in he p esence o he biopolyme s. Then, po ous sca olds we e cons uc ed by eeze-d ying he
dispe sions which allows good con ol o po e size and mo phology, showing unique mechanical
p ope ies. In e es ingly, hese biocompa ible, conduc ing sca olds success ully suppo g ow h o 3D
cell cul u es o sw480 colon adenoca cinoma cance cells, achie ing good cell a achmen and p oli -
e a ion. When in eg a ed wi h elec odes, hey u he allow eal- ime elec ical moni o ing o cell
g ow h and p oli e a ion. Upon he addi ion o he fla onoid mo in, cell apop osis and dea h we e
moni o ed by elec ochemical impedance spec oscopy and op ical immunos aining., demons a ing he
p omise o hese sca olds o cance cell p og ession modeling. We belie e ha ou findings ha e
demons a ed he g ea p omise o combining PEDOT wi h biopolyme s o cance cell p og ession
modeling bu also will be o in e es in b oade applica ions in he fields o biomedicine, wea able
elec onics, and p ospec i ely applied o clinic.
©2022 The Au ho (s). Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND
license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
1. In oduc ion
Resea ch in cance undamen als is c i ical o unde s anding
and ea ing di e en ypes o cance . Among a ious ypes o
cance , colo ec al cance is he hi d mos commonly diagnosed
cance a e lung and b eas cance [1]. In he Eu opean Union
(EU) alone, he annual numbe o dea hs due o colo ec al cance
is p ojec ed o each almos 89,000 in 2021 [2]. Mos o hese
dea hs occu wi h me as asis o o he o gans when cance cells
mig a e om a p ima y umo o he blood essels and lym-
pha ics. Recen s udies ha e shown ha he ex acellula ma ix
(ECM) plays a key ole in me as a ic cance p og ession, being
c i ical in p omo ing esis ance and ecu ence in he umo
mic oen i onmen [3]. These findings ha e inspi ed he de elop-
men o 3D issue-enginee ed cons uc s esembling he in i o
en i onmen and enable he disco e y o e ficien a ge ing he -
apy d ugs o co e he g owing needs o he wo ldwide popula-
ion [4]. O e all, cance cells g own in 3D configu a ion exp ess
di e en mo phology, mo ili y, and p oli e a ion capabili ies, and
exhibi highe esis ance o an icance d ugs compa ed o 2D
adi ional models [5].
3D cell cul u es in i o can be sus ained in sca olds ha
imp o e cell a achmen and guide cells o specific si es o issue
*Co esponding au ho .
** Co esponding au ho .
E-mail add esses: [email p o ec ed] (J. Saez), mo3[email p o ec ed] (R.M. Owens).
Con en s lis s a ailable a ScienceDi ec
Ma e ials Today Chemis y
jou nal homepage: www.jou nals.else ie .com/ma e ials- oday-chemis y/
h ps://doi.o g/10.1016/j.m chem.2022.100990
2468-5194/©2022 The Au ho (s). Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/
).
Ma e ials Today Chemis y 24 (2022) 100990
de elopmen [6]. These sca olds a e made o polyme s wi h
physiological and biochemical ele ance o he issue o in e es
allowing he mo phological design o s uc u es wi h dedica ed
mechanical and composi ional p ope ies. Mo phologically
speaking, na i e cell en i onmen s like he na u al ECM a e
mimicked by adap ing he mechanical aspec o he sca old.
P ope ies such as po osi y and s i ness a e c ucial o ensu e
in e connec ed po e ne wo ks ha aid di usion o nu ien s and
oxygen and p omo e cell a achmen wi h subsequen sca old
coloniza ion [7]. Physiologically speaking, he use o na u al poly-
me s such as collagen (COL) and glycosaminoglycans (GAGs) in he
composi ion o he sca old could be he way o enhance biocom-
pa ibili y gene a e empla es ha suppo cell g ow h in he long-
e m. Among GAGs, hyalu onic acid (HA) is a nega i ely cha ged
polysaccha ide wi h ca boxylic and sul a e unc ional g oups,
shown o influence cells h ough bo h biochemical ( ecep o -
media ed) and biophysical (s uc u al and mechanical) mecha-
nisms [8]. Biochemically, HA ecep o s and in acellula -binding
p o eins ha e been iden ified and linked o in acellula signaling
a ec ing cell adhesion, mig a ion, and p oli e a ion. In he umo
mic oen i onmen , HA is known o p omo e umo cell mig a ion
o enhance mig a ion ac i i y in i o [9]. As an example, a colon
adenoca cinoma cell line (sw480) showed a highe spon aneous
mig a ion in he p esence o HA [10]. Collec i ely, hese findings
sugges ha HA con ibu es o ele an changes in he ECM physical
p ope ies which may se e as biophysical cues con ibu ing o cell
esponse and cell mig a ion.
O e he pas decade, conduc ing polyme s (CPs) such as
poly(3,4-e hylenedioxy hiophene) (PEDOT) [11,12], polypy ole
(PPy) [13], and polyaniline (PANI) [14], ha e eme ged in he issue
enginee ing field o add an elec oac i e capabili y o 3D cell cul-
u e moni o ing. Sca olds made o he semiconduc o PEDOT a e
pa icula ly in e es ing o p omo e cell g ow h by elec ical s im-
ula ion in bone, muscle, b ain [15]. Many g oups ha e exploi ed he
polyme poly(3,4-e hylenedioxy hiophene) doped wi h poly-
s y ene sul ona e (PSS), as a suppo o cell g ow h and di e en-
ia ion o cells, ega dless o he possible elease o acidic PSS
deg ada ion by-p oduc s in he long- e m [16]. Gi en his d aw-
back, i is p e e able o ailo PEDOT:polyelec oly e dispe sions
ha be e mee he mo phological and physiological mic oen i-
onmen o he human issues [17]. A a ie y o nega i ely cha ged
biomolecules ha e been s udied as polyelec oly es such as chon-
d oi in sul a e [18], dex an sul a e [19], xan ham gum [20], hepa in
[21], algina e [22], and HA [23] showing imp o ed cell g ow h and
di e en ia ion compa ing o he comme cial PEDOT:PSS.
3D po ous sca olds can be ab ica ed by he eeze-d ying
echnique [24]. This echnique consis s o a fi s s ep whe e dis-
pe sions a e ozen a a specific cooling a io, ollowed by a second
s ep whe e ice c ys als om he ozen dispe sions a e sublimed a
a ce ain acuum p essu e o se e al hou s. In his way, 3D po ous
sca olds a e ob ained wi h mo phology and mechanical p ope ies
akin o human body issues [25]. This ab ica ion me hod has a ely
been used in he case o PEDOT conduc ing polyme s.
In his wo k we p esen a 3D in e ace o assess cance p o-
g ession based on elec oac i e sca olds made o PEDOT:HA and
COL ha ecapi ula e he na u al ECM in i o. The po ous sca olds
we e cons uc ed by he eeze-d ying echnique and we e used o
elec ically moni o cance cell p og ession by Elec ochemical
Impedance Spec oscopy (EIS). sw480 cells we e used as a cance
cell model o s udy he biochemical and biophysical (mig a ion,
p oli e a ion) cell-ECM in e ac ion in he ECM mic oen i onmen .
The phy ochemical compound mo in [26] was used as an an i-
p oli e a i e d ug o induce he apop osis o cells and alida e he
sys em.
2. Resul s and DISCUSSION
2.1. Syn hesis and cha ac e iza ion o PEDOT:Hyalu onic acid and
PEDOT:Hyalu onic acid/collagen aqueous dispe sions
The syn he ic ou e o dispe sions con aining he conduc ing
polyme PEDOT and polysaccha ide has been epo ed p e iously
[23]. In his wo k, a simila syn he ic ou e was ollowed o he
syn hesis o PEDOT:HA and PEDOT:HA/COL aqueous dispe sions.
The chemical oxida i e polyme iza ion p ocess ollowed o ob ain
he PEDOT:HA and PEDOT:HA/COL dispe sions employed di e en
o mula ions o he ini ial componen s. Thus, h ee di e en w %
a ios we e ini ially p epa ed: 15:85, 50:50, and 85:15 o each
dispe sion. The dispe sions we e p epa ed by polyme izing EDOT
monome in he p esence o he polysaccha ide sodium hyalu o-
na e dissol ed in wa e a oom empe a u e. The dispe sions
con aining 1, 3, and 5 mL o COL (3 mg/mL) (PEDOT:HA/COL1,
PEDOT:HA/COL3, and PEDOT:HA/COL5, espec i ely) we e syn he-
sized by adding COL wi h he sodium hyalu ona e, Fig. 1A.
Ammonium pe sul a e ((NH
4
)
2
S
2
O
8
) was used as an oxidan in he
p esence o a ca aly ic amoun o i on (III) chlo ide (FeCl
3
)(Fig. S1).
The eac ion akes place slowly and he ini ial yellowish iscous
solu ion u ns o a g eyish less iscous solu ion wi hin he fi s 6 h.
This phenomenon was obse ed p e iously wi h o he PEDOT:po-
lysaccha ide dispe sions and i is asc ibed o he PEDOT chains
b eaking he s ong in amolecula hyd ogen bonds o he poly-
saccha ide [20]. The polyme iza ion o EDOT is comple ed in 72 h
acqui ing a cha ac e is ically da k blue colo and a iscosi y simila
o ha o wa e .
While 50:50 and 15:85 w % a ios p esen highly con inuous
d op-cas ed film o ma ion, 85:15 w % films p esen highe
discon inui y wi hin he film s uc u e as shown in he scanning
elec on mic oscopy (SEM) pic u es (Fig. S2). As a esul , jus he
15:85 w % was ound o be sui able o u he in es iga ion as he
one showing be e aspec - a io p ope ies. To simpli y, he wo k-
ing aqueous dispe sions unde s udy we e PEDOT:HA, PEDOT:HA/
COL1, PEDOT:HA/COL3, and PEDOT:HA/COL5 in w % 15:85 a io.
UVe iseNIR spec oscopy was pe o med o obse e he
PEDOT oxida ion s a e ob ained in he p esence o wo insula o s
addi i es, sodium hyalu ona e, and COL. The dispe sions showed
wo cha ac e is ic PEDOT abso p ion bands (Fig. 1B). The b oad
abso p ion band o PEDOT
p
-
p
*is cen e ed a ⁓800 nm and he
bipola on band is cen e ed a 1200 nm [20]. These wo abso p ion
bands p esen in he UVe iseNIR spec a indica e he p esence o
doped PEDOT chains as ca ion and di-ca ion species and a e ela ed
o he conduc i i y o he ma e ial, confi ming ha PEDOT:HA
dispe sions a e conduc ing. The p esence o hese bands confi ms
he highe p esence o doped chains in his PEDOT:HA dispe sion, a
sign o high conduc i i y. Howe e , he dispe sions con aining COL
display a deple ion o he bipola on band, indica ing ha COL may
ac as an insula o , educing conduc i i y.
This obse a ion is in line wi h he elec ical conduc i i y
measu ed on d op-cas ed films p epa ed on o silicon molds and
analyzed by a ou -poin p obe (see Fig. S3). PEDOT:HA films
possess a conduc i i y abo e 10
2
S/cm as epo ed p e iously [23],
which is simila o PEDOT:PSS films o med wi hou addi i es like
COL. The addi ion o COL shows a dec ease in he conduc i i y in
ag eemen wi h he p esence o insula ing species. The addi ion o
1 mL o COL (PEDOT:HA/COL1) d ops he conduc i i y alues o
8$10
3
S/cm and o 2$10
3
S/cm when COL is used o concen a-
ions highe han 3 mL eaching a pla eau in conduc i i y (PEDO-
T:HA/COL3 and PEDOT:HA/COL5). Al hough PEDOT:HA dispe sions
con aining COL we e ound o be less conduc ing, hey we e kep
unde s udy due o hei p omising mechanical and biocompa ible
J. Saez, A. Dominguez-Al a o, C. Ba be io e al. Ma e ials Today Chemis y 24 (2022) 100990
2
p ope ies in ecapi ula ing he physiological mic oen i onmen o
he ECM.
The mo phology o he PEDOT:HA and PEDOT:HA/COL dispe -
sions was in es iga ed by TEM, Fig. 1CeF. As obse ed in simila
sys ems [27], he PEDOT:HA dispe sions consis o sphe ical PEDOT
pa icles co e ed by HA o an a e age size o 200 nm. In con as ,
PEDOT:HA/COL dispe sions display a ne wo k o in e connec ed
pa icles wi h diame e s be ween 600 nm and 1
m
m. In e es ingly,
he addi ion o COL o he mix u e c ea es a la ge COL ne wo k
co e ing he PEDOT:HA pa icles, hus, a ec ing he colloidal
mo phology (Fig. S4).
Fu he in o ma ion on he dispe sion p ope ies was ob ained
ia SEM (Fig.1GeJ). Op ical analysis was pe o med on SEM pic u es
and i was ound ha PEDOT:HA sphe ical pa icles a e o med as a
esul o PEDOT chains being d op-cas ed, making he sphe ical
pa icles agglome a e oge he in he o m o a film. PEDOT:HA films
p esen a flowe -like s uc u e o med by discon inuous clus e s. In
con as , films wi h inc easing concen a ions o COL showed a
con inued and smoo he s uc u e, p esen ing elonga ed c ys als
and, as discussed be o e o TEM, a simila spide web-like s uc u e
o med by elonga ed c ys als in he PEDOT:HA/COL3 film.
2.2. P epa a ion and cha ac e iza ion o PEDOT:HA and PEDOT:HA/
COL sca olds
To c ea e a 3D elec oac i e ECM, po ous sca olds we e p e-
pa ed by he eeze-d ying me hod [25]. The eeze-d ying me hod
consis s o wo pa s, Fig. 2A. Fi s , dispe sions a e ozen down a a
se empe a u e and cooling a e, and second, he ice is sublimed a
a se p essu e, ob aining po ous sca olds and elec odes o he
EIS cha ac e iza ion. In his p ocess, di e en pa ame e s such as
solid con en o he dispe sion, p esence o a c osslinke ,
and eeze-d ying condi ions will a ec he mo phology and me-
chanical p ope ies o he sca old. A e eeze-d ying he di e en
dispe sions, he ou ypes o sca old (Fig. 2B) we e analyzed
chemically by Fou ie ans o m in a ed (FTIR) spec oscopy
(Fig. S5) and he mog a ime ic analysis (TGA) (Fig. S6). Also, he
mo phology o he sca olds was cha ac e ized by SEM (Fig. 2CeF).
O e all, he sca olds p esen an iso opic s uc u e as de e mined
by he ice c ys al g ow h and he solid con en . The sca olds con-
aining COL show a mo e compac s uc u e due o he highe solid
con en and wo phases sepa a ed as he concen a ion o COL in-
c eases ( op en iched wi h COL). We hypo hesize ha as he ice
c ys als g ow, he polyme chains a e pushed aside, concen a ing
a ound he g owing c ys als. Once he ice is emo ed by sublima-
ion, only a po ous skele on is le whe e he ice s uc u e was.
PEDOT:HA and PEDOT:HA/COL sca olds show an in e connec ed
po ous ne wo k ha will enhance wa e and nu ien s e en ion,
and cell mig a ion and p oli e a ion. In con as o PEDOT:HA (po e
size 32 ±6
m
m), sca olds con aining COL p esen a mo e poly-
dispe sed and compac s uc u e ea u ing sligh ly smalle po e
sizes anging om PEDOT:HA/COL1 31 ±2 o PEDOT:HA/COL5
26 ±5
m
m(Fig. S7). In pa icula , PEDOT:HA/COL3 sca olds show a
spide -web s uc u e (Fig. 2E) ha co ela es well wi h he spide -
web obse ed in 2D (Fig. 1I). We en isioned ha his po ous
s uc u e ha mimics he ECM would acili a e cell nu ien
Fig. 1. A) PEDOT:HA and PEDOT:HA/COL syn he ic scheme. Inse : ca oon o PEDOT:HA eac ion ial a 72 h. B) UVe iseNIR spec a o PEDOT:HA and PEDOT:HA/COL dispe sions
showing he highe conduc i i y o PEDOT:HA in he absence o collagen. CeF) T ansmission Scanning Mic oscopy (TEM) pic u es o he dispe sions highligh ing he collagen
ne wo ks c ea ed a ound PEDOT:HA pa icles (in o de PEDOT:HA, PEDOT:HA/COL1, PEDOT:HA/COL3, PEDOT:HA/COL5). G-J) Scanning Elec on Mic oscopy (SEM) pic u es o he
films ea u ing di e en su ace displays, gene ally, and inc emen o collagen c ea es a smoo he su ace (in o de PEDOT:HA, PEDOT:HA/COL1, PEDOT:HA/COL3, PEDOT:HA/COL5).
J. Saez, A. Dominguez-Al a o, C. Ba be io e al. Ma e ials Today Chemis y 24 (2022) 100990
3
pe cola ion, cell a achmen , and coloniza ion [28]. This demon-
s a es ha he addi ion o inc easing concen a ions o COL can
a y he po osi y o he sca old and i s mechanical s i ness.
I is well known ha he s i ness o he sca old a ec s cell
a achmen and p oli e a ion [29] and load ans e o he a ached
cells when he sca old de o ms unde physiological condi ions.
PEDOT:HA and PEDOT:HA/COL mechanical p ope ies we e hus
in es iga ed. Di e ences among he mechanical p ope ies o he
di e en sca olds we e e alua ed by comp ession es ing. The
comp ession es was pe o med in d y sca olds showing Young's
modulus alues be ween PEDOT:HA/COL5 20 ±7 kPa and PEDO-
T:HA 156 ±12 kPa (Fig. 3A). As a gene al end, he highe he COL
con en , he highe he Young's modulus o he sca olds. While i is
di ficul o compa e he sca olds since hey ha e di e en po e
sizes and con en s, he measu emen s sugges ha he sca olds
con aining inc easing amoun s o COL p esen less s i ness
compa ing o PEDOT:HA. While inc easing he COL con en p e-
sumably educes he s i ness o he cell wall ma e ial, he inc e-
men in PEDOT:HA/COL5 sca olds no iced in he Young's modulus
is p obably o se by he wo phase sepa a ed displayed o he
PEDOT:HA/COL5 sca olds. I is impo an o men ion ha he
sca olds we e obse ed o main ain good s uc u al in eg i y
h oughou he 7 days in cell cul u e media, confi ming sca old
s abili y unde physiological condi ions.
The swelling capaci y o sca olds is linked o po osi y and hy-
d ophilici y, being he las key o ans e ing cell nu ien s and
cellula me aboli es [30]. As explained abo e, he sca olds con ain-
ing COL p esen a mo e compac and less po ous s uc u e compa ed
o PEDOT:HA sca olds, likely due o he g ea e solid con en . The
sca olds unde s udy p esen ed a highe swelling capaci y in he
absence o COL a e 24 h in PBS (pH ¼7.4) as shown in Fig. 3B. In
con as , he PEDOT:HA/COL sca olds we e obse ed o be mo e
hyd ophilic when inc easing he COL con en ( isual obse a ion)
showing a as e speed o we ing in PBS. PEDOT:HA/COL3 sca olds
exhibi ed bo h a decen swelling a io (SR) and good hyd ophilici y.
EIS measu emen s we e un o analyze he e ec o he
inc easing concen a ion o COL on he o e all elec ochemical
p ope ies o he sca old. As an icipa ed, he inclusion o COL is
ound o be associa ed wi h an inc ease in he ma e ial impedance
o e he mid-low equency ange, as shown in he Bode plo (|Z| s
F equency) in Fig. 3C. This e ec is mo e significan in PEDOT:HA/
COL5. To be highligh ed is he exhibi ion o he ohmic fla cu e o
he sca olds in he absence o p esence o COL [24,25]. The
appa en changes we e accompanied by he p esence o a b oad
peak in he phase spec um and an inc ease in he phase angle
maximum wi h COL con en ( om 34
o 38
, PEDOT:HA and
PEDOT:HA/COL5, espec i ely), Fig. 3D. O e all, he COL-con aining
sca olds we e ound o keep hei conduc ing cha ac e is ics,
holding a g ea p omise o using hese s uc u es in cell mig a ion
and p oli e a ion elec ical moni o ing.
The p esence o HA wi hin he sca olds was assessed by im-
munos aining using a bio inyla ed HA-binding p o ein (HABP)
Fig. 2. A) Gene al scheme o manu ac u e sca olds. B) Image o he s and-alone sca olds PEDOT:HA, PEDOT:HA/COL1, PEDOT:HA/COL3, PEDOT:HA/COL5 (le o igh ). CeF) SEM
mic og aphs showing he po es and s uc u e o he sca olds. Inse : zoom image o he po es o he sca old C) PEDOT:HA, D) PEDOT:HA/COL1, E) PEDOT:HA/COL3; black a ows
indica e he spide -web s uc u e, F) PEDOT:HA/COL5.
J. Saez, A. Dominguez-Al a o, C. Ba be io e al. Ma e ials Today Chemis y 24 (2022) 100990
4
me hod. Bio in was non-co alen ly bonded o s ep a idin-FITC
conjuga e wi h high a fini y o de ec HA in he s uc u e o he
sca old (ATTO488). Mo eo e , he p esence o COL was de ec ed by
using a COL ype I an ibody ha binds specifically, using Alexa Fluo
647 as he seconda y an ibody. As shown in Fig. 3EeF, HA (g een)
was localized wi hin he whole s uc u e o he PEDOT:HA and
PEDOT:HA/COL sca olds. In con as , COL p esence in PEDOT:HA/
COL sca olds was ound o be mo e localized in he o m o clus e s
as depic ed in Fig. 3F and Fig. S8. Non-con aining HA and COL
PEDOT:PSS sca olds we e used as con ols wi hou showing any
simila ace o clus e wi hin he s uc u e (Fig. 3G).
2.3. PEDOT:HA and PEDOT:HA/COL sca olds p omo e cell
p oli e a ion
Gi en ha bo h HA and COL a e ECM-de i ed cons i uen s, an
enhanced cy ocompa ibili y o e o he simila ma e ials such as
PEDOT:PSS is expec ed. To e alua e cell p oli e a ion on he
di e en sca olds, an XTT cell iabili y assay was ca ied ou o
quan i y me abolic ac i i y o sw480 cance cells seeded in sca old
slices o 500
m
m hickness o 7 days (Fig. 4A). In e es ingly,
PEDOT:HA showed simila me abolic ac i i y and indi ec ly cy o-
compa ibili y o he con ol (p is ine PEDOT:PSS). In con as ,
Fig. 3. A) Young's modulus o d y sca olds (n ¼3). PEDOT:HA/COL5 p esen s a highe Young's modulus due o he wo dis inc egions o he sca old ( op pa collagen
concen a ed and bo om pa PEDOT:HA concen a ed) ANOVA *p0.05. B) Swelling a io (%) o PEDOT:HA and PEDOT:HA/COL sca olds (n ¼3) ANOVA *p0.05. C-D)
Compa a i e EIS measu emen s o he di e en sca olds showing he C) Bode plo o PEDOT:HA and PEDOT:HA/COL sca olds, and. D) phase angle s equency o PEDOT:HA and
PEDOT:HA/COL sca olds. E) HA (g een) immunos aining o PEDOT:HA sca old, F) HA (g een) and collagen (o ange, as indica ed by whi e a ow heads) immunos aining o
PEDOT:HA/COL3 sca old and G) B igh field image o PEDOT:PSS sca old.
J. Saez, A. Dominguez-Al a o, C. Ba be io e al. Ma e ials Today Chemis y 24 (2022) 100990
5
sca olds con aining COL showed imp o ed cell su i al and p o-
li e a ion compa ed o bo h PEDOT:HA and PEDOT:PSS sca olds. O
no e is he highe cell iabili y and cell g ow h obse ed o
PEDOT:HA/COL3 sca olds (Fig. 4B). F om his poin , jus PEDOT:HA/
COL3 sca olds we e conside ed o de eloping he elec oac i e
ECM bioin e ace.
A e 7 days o cul u e, sw480s cells s ained o nuclei wi h DAPI
and cy oskele on (F-ac in) wi h Alexa Fluo 594 Phalloidin, showed
high locomo ion and mig a ion o he bo om pa s o he sca old
p esumably enhance by he p esence o HA and COL and he in e -
connec ed po ous ne wo k (Fig. 4CeF). Fig. 4G shows an o ho iew
whe e sw480s cells ha e in aded he bo om pa s o he sca old,
displaying high cell a achmen and co e age. The Video S1 shows a
s ack o he immunos ained cells seeded on o a 500
m
m slice.
Supplemen a y ideo ela ed o his a icle can be ound a
h ps://doi.o g/10.1016/j.m chem.2022.100990
2.4. 3D elec ical and op ical moni o ing o sw480s p og ession
As demons a ed by o he s be o e [31], conduc ing sca olds a e
capable o showing changes in hei elec ochemical p ope ies when
cells in e ac , g ow, and colonized hesca old. In his way, conduc ing
sca olds can be used o ace cell g ow h and dea h employing EIS.
Only PEDOT:HA/COL3 sca old-based elec odes we e selec ed o
unde go his s udy as i was conside ed o be he op imal condi ion
showing he bes biochemical and biophysical p ope ies, as
explained abo e. sw480 cells we e seeded on a s e ilized sca old a
2.5 10
5
cells and kep unde incuba ion o 7 days (Fig. 5A). F esh
media changes we e made a day 3 and 6. A e 7 days o cul u e,
sca olds main ained good s uc u al in eg i y and s i ness. A wo-
elec ode configu a ion was used o ake he measu emen s, using a
pla inum (P ) mesh as he coun e elec ode and he connec ions
being, WeS (sca old-based elec ode), and CE-RE (P mesh). F esh
media was used as he elec oly e solu ion. The impedance da a
showed in he Bode plo (|Z| s F equency), e ealed a significan in-
c ease in he impedance alue o a hal o de o magni ude ( om |Z|⁓
49 k
U
o |Z
cell
|⁓100 k
U
a 10
1
Hz) in he case o sca olds con aining
cells, in he low- equency ange (10
1
-10
2
Hz) (Fig. 5B). In his e-
quency ange, ionic anspo domina es, hus indica ing ha cells
ha e g own and filled he po ous o he sca old ac ing as a ba ie o
ions o flow. In he mid o high- equency ange (10
2
e10
5
Hz), a
disc e e inc emen o he impedance alues indica es he p esence o
lining cells on he walls o he sca old, hinde ing ion anspo . The
Nyquis plo u he confi ms he e ec s o cells lining he po es o he
sca old in he high- equency ange (Fig. 5C). The phase da a sugges s
he addi ion o capaci i e elemen s in he ci cui as wellas an inc ease
o he phase angle om 4⁓65
o 4
cells
⁓71
, which co esponds o
he g ow h o cells in he po es o he sca old (Fig. 5D).
A e es ablishing he sw480 g ow h bioelec onic model,
apop osis o cells was induced by he fla onoid compound mo in.
Fig. 4. A) Scheme o a 500
m
m sca old slice seeded wi h sw480s. B) Viabili y o sw480 cells measu ed by XTT assay (n ¼3). Abso bance a 450 nm o he 4 samples compa ed o
PEDOT:PSS con ol. Abso bance alues ob ained a e 7 days o cell cul u e wi h an ini ial seeding cell densi y o 4 10
4
ANOVA *p0.05. C) Top iew, D) Bo om iew and E) o ho
iew o con ocal mic oscopy images showing he nuclei s ained wi h DAPI (blue) and F-ac in cy oskele on s ained wi h Alexa Fluo 594. The o ho iew shows he in asi eness o
he sw480s h ough he sca old (270
m
m dep h).
J. Saez, A. Dominguez-Al a o, C. Ba be io e al. Ma e ials Today Chemis y 24 (2022) 100990
6
Fig. 5. A) Schema ic o a sca old-based elec ode wi h sw480 cells p oli e a ing. B-G) Impedance s udy o he adhesion and p oli e a ion o sw480s on he PEDOT:HA/COL sca old-
based elec odes. B-G) Rep esen a i e B) Bode impedance magni ude C) Nyquis D) Phase plo showing he ini ial (black line, day 1 be o e seeding), final ( ed line, day 7 a e
seeding), and mo in esponse (blue line, day 8 a e mo in mo phology dis up ion) o he cul u ed cells on he elec oac i e sca old. Inse in Bode plo shows he e olu ion o he
complex impedance du ing he issue o ma ion, om day 1 o day 8 a e mo in ea men . E-F) Ba plo s showing E) he ela i e change in he esis ance(
D
R/R
0
), and F)
capaci ance (
D
C/C
0
) o he po ous elec odes (n ¼3) a e each s age o seeding. G-H) sw480s mic oscopy pic u es displaying he change o mo phology a e mo in ea men in 2D.
I-J) SEM images illus a ing cell co e age o he sca olds be o e and a e mo in ea men . K-L) T ea ed and non- ea ed sca olds seeded wi h sw480s showing DNA damage and
ch oma in condensa ion a e Hoechs immunos aining. M N) Con ocal mic oscopy pic u es o sw480 cells s ained wi h Annexin-V (FITC) and PI a day 8 wi h and wi hou mo in
ea men . Whi e dashes indica e wo po es o he sca old comple e filled wi h cells.
J. Saez, A. Dominguez-Al a o, C. Ba be io e al. Ma e ials Today Chemis y 24 (2022) 100990
7
Mo in has been p e iously epo ed o show an icance ac i i y and
induce apop osis causing mo phology dis up ion in sw480s cells
[32]. 2D expe imen s o sw480s cells showed a d as ic change in he
mo phology o he cell on mo in ea men a e 24 h. While un-
ea ed con ol cells showed a smoo h su ace (Fig. 5H), ea men
wi h mo in esul ed in se e e damage o cells wi h os ensible
de o ma ion, sh unken o abno mal ound ype and cell numbe was
significan ly dec eased (Fig. 5I). Elec ically, ea men wi h mo in
shows a dec ease o he impedance alues in he low- equency
ange o |Z
mo in
|⁓36 k
U
a 10
1
Hz ( om |Z
cell
|⁓100 k
U
) indi-
ca ing cells p og ession owa ds dea h (Fig. 5B). Also, in he high-
equency ange (see Bode and Nyquis plo s), a dec ease o he
impedance ini ial alues sugges s ha cells ha e de ached om he
sca old. This was also confi med by SEM images o he 3D con-
duc ing sca olds aken be o e and a e mo in ea men (Fig. 5IeJ).
The phase angle also is educed o 4
mo in
⁓65
( om 4
cells
⁓71
)
showing a hal ing phase alue a ibu ed o he p esence o a dis-
u bing elemen in he cell cul u e medium (mo in) (Fig. 5D).
Ionic anspo opposi ion due o he p esence o g owing/
apop o ic cells con ibu es o he inc emen /dec ease o he Rela-
i e Resis ance (
D
R/R
0
) and Rela i e Capaci ance (
D
C/C
0
)
(Fig. 5FeG) calcula ed wi h he co esponding EIS fi ing model
(Fig. S9)[33]. The esul ing EIS model allows moni o ing changes in
he esis ance ( om
D
R/R
0
0.281 ±0.039 o 0.007 ±0.026) and
capaci ance ( om
D
C/C
0
2.590 ±0.235 o 0.844 ±0.062) as a
esul o cell mig a ion and p oli e a ion h ough he bulk o he
sca old, as well as cell dea h and loss o connec i i y be ween cells.
The an ip oli e a i e ac i i y shown by mo in could be due o
he induc ion o apop osis and i can be obse ed by s aining he
cells wi h a fluo escen DNA-binding dye. Nuclea al e a ions like
ch oma in condensa ion and DNA agmen a ion which a e he
hallma k o apop osis we e de e mined by Hoechs 33342 s aining.
As shown in Fig. 5L, un ea ed con ol cells did no show much blue
fluo escence, indica ing ha cells a e ali e and Hoechs does no
easily pe mea e in o he cell nuclei. The fluo escence ligh was
dense and b igh e in cells ea ed wi h mo in along wi h
ema kable nuclea changes o apop osis such as he o ma ion o
apop o ic bodies, condensa ion o ch oma in, and nuclea ag-
men a ion (Fig. 5M). Fu he confi ma ion o apop osis in cells was
ca ied ou by s aining un ea ed (Fig. 5N) and mo in- ea ed
sw480s (Fig. 5O) wi h Annexin-V/FITC and p opidium iodide (PI).
Vi al cells a e nega i e o fluo escence-conjuga ed Annexin-V
binding and PI, while ea ly apop o ic cells a e posi i e o Annexin-
V bu nega i e o PI, whe eas nec o ic cells a e Annexin-V nega i e
and PI posi i e. A significan inc ease in he numbe o s ained
apop o ic cells was obse ed a e mo in ea men and a sligh
inc ease in he numbe o dead cells (s ained o PI), in con as o
un ea ed cells.
In con as , con ols do no show significan changes in
impedance alues in he whole equency ange (Fig. S10). The
abo emen ioned findings a e consis en wi h p e ious s udies on
simila 3D conduc ing cell cul u e sys ems. A mo e in-dep h s udy
o he elec ochemical mig a ion o cells could be done by modi-
ying he sca old se up and in eg a ing an ex a gold elec ode in a
di e en pa o he sca old.
3. Conclusions
In his s udy, no el PEDOT:HA and COL sca olds wi h he abili y
o hos and moni o cells a e p esen ed. Conduc ing sca olds we e
ob ained by syn hesizing and eeze-d ying PEDOT:HA and PEDO-
T:HA/COL dispe sions h ough oxida i e polyme iza ion. The me-
chanical p ope ies and po osi y o he conduc ing sca olds can be
uned wi h he concen a ion o he COL and he a io in be ween
he conduc ing polyme and polysaccha ide, gi ing he possibili y
o op imize po e size and s i ness acco ding o he a ge cell
cul u e. Among all o he sca olds, PEDOT:HA/COL3 p o ed i s
po en ial o ac as a bioelec ical in e ace o hos and moni o
sw480 colon adenoca cinoma cance cells’g ow h and p og ession
all o e he sca old. By inducing cell apop osis wi h mo in, op ical
immunos aining p o ed ha cells lose hei mo phology and he
elec ical impedance eco e s o ini ial alues. Significan ly, his
wo k p esen s a conduc ing and biocompa ible unc ional ma e ial
ha p o ides a powe ul ool o be e mimic na i e issues as well
as a pla o m o moni o elec ically small changes p oduced in
cance cell p og ession.
4. Expe imen al sec ion
4.1. Syn hesis o PEDOT:HA and PEDOT:HA/COL dispe sions
PEDOT:HA and PEDOT:HA/COL dispe sion wi h 2% solid con en
and a 15:85 a io we e syn hesized by oxida i e polyme iza ion ac-
co ding o he p e ious p ocedu e [20]. In a 10 ml ial, 170 mg o so-
dium hyalu ona e was dissol ed in 10 mL 9, 7, and 5 mL o Milli-Q
wa e o PEDOT:HA, PEDOT:HA/COL1, PEDOT:HA/COL3, and PEDO-
T:HA/COL5, espec i ely. The iscous solu ion was s i ed o 20 min
un il he HA comple ely dissol es. Righ a e ,1 mL, 3 mL, and 5 mL o
COL ype I om a ail (3 mg/mL, Gibco) was added espec i ely
(PEDOT:HA/COL1, PEDOT:HA/COL3, and PEDOT:HA/COL5). A e wa d,
a 1.5 equi o ammonium pe sul a e ((NH4)
2
S
2
O
8
, Sigma Ald ich)
espec o monome oge he wi h a ca aly ic amoun (10 mg) o i on
(III)chlo ide (FeCl
3
, Sigma Ald ich) was added o he p e ious aqueous
solu ion. Once he mix u e is dissol ed, 30 mg o EDOT monome was
added. The eac ion was kep a oom empe a u e o 72 h un il an
in ense deep blue colo was ob ained.
Films o he di e en dispe sions we e p epa ed by d op-cas ing
200
m
L o dispe sion in sphe ical silicon molds o 1 cm diame e .
The dispe sions we e le a oom empe a u e o d y o e nigh o
c ea e a film.
4.1.1. Dispe sion cha ac e iza ion
4.1.1.1. UVeVis cha ac e iza ion. UVe iseNIR measu emen s we e
aken wi h a UV/ is/NIR Pe kin-Elme lambda 950 spec ome e
using qua z cu e es. 5 / % o PEDOT:HA o PEDOT:HA:col so-
lu ion we e dissol e in Milli-Q wa e and he UVe iseNIR da a was
acqui ed. Spec a we e no malized o pe o m a ai compa ison.
4.1.1.2. TEM cha ac e iza ion. TEM images we e collec ed using a
JEOL JEM-2100F model EM-20014 (JEOL L d, Japan), which ea u es
a 200 kV ield emission gun (Scho ky) “FEG”and an ul a-high-
esolu ion pole piece “UHR.”Fi s , 1
m
L o he s udied sample was
solubilized in 1 mL o deionized wa e o ob ain a clea solu ion.
Finally, 5
m
L o he solu ion was deposi ed on o a coppe film TEM
g id and e apo a ed a oom empe a u e.
4.1.1.3. Fou -poin p obe. A ou -poin p obe sys em (Ossila) was
used o e alua e he conduc i i y o he solid films. PEDOT:HA o
PEDOT:HA/COL solu ions we e d op-cas ed in sphe ical glass slides
o 12 mm diame e . The films we e d ied a oom empe a u e.
4.2. Sca old p epa a ion by eeze-d ying me hod
10 w% PEDOT:PSS (He aeous PH 1000), 3 w % o (3-
glycidyloxyp opyl) ime hoxysilane (GOPS), and 0.5 w % o dode-
cylbenzosul onic acid (DBSA) pe mL o dispe sion we e added o
bo h PEDOT:HA and PEDOT:HA/COL dispe sions. Sca olds we e
o med by he eeze-d ying me hod wi h a Vi is Ad an age 2.0
Bench op (SP Scien ific). Dispe sions we e dispe sed wi h 150
m
Lo
each solu ion in a 96 mul i-well pla e. The eeze-d ying p ocess was
J. Saez, A. Dominguez-Al a o, C. Ba be io e al. Ma e ials Today Chemis y 24 (2022) 100990
8
adap ed om [24] by changing he empe a u e o 35
Cwi ha
eezing a e o 0.19 C/min. Fo he p epa a ion o sca old-based
elec odes, 1.5 cm leng h, 3 mm wid h gold-co e ed Kap on shee s
we e imme sed in o each well be o e eeze-d ying. Gold-Kap on
was co e ed wi h a PDMS solu ion (1:10 PDMS, cu ing agen ) o
p e en seconda y sho -ci cui ing. A e eeze-d ying, he sca olds
we e hea ed up o 49
C o 4 h o s a he c osslinking p ocess.
Sca olds ob ained we e ⁓5mmdiame e and⁓3 mm heigh .
Sca olds no con aining gold-Kap on we e sliced wi h a Vib a-
ome (Leica Biosys ems) and 500
m
m hickness slices we e ob ained.
4.2.1. Sca old cha ac e iza ion
4.2.1.1. FTIR and TGA cha ac e iza ion. In a ed spec a we e eco -
ded a oom empe a u e wi h a The mo scien ific model Nicole
is20 FT-IR, The mo Scien ific Inc., USA. The measu e was pe o med
on 1 mm slice o he desi ed sca old, applying 32 scans in ans-
mission mode.
TGA was pe o med unde ai a mosphe e (25 mL/min flow a e)
using a TGA Disco e y (TA Ins umen s). A piece o 1 mm o he
desi ed sca old was placedin he P pans andinse ed in he chambe .
Fi s , an equilib a ion s ep was se a 100
C o 20 min. Then, he
sample was hea ed om 100 o 800
Ca a a eo 10
C/min.
4.2.1.2. SEM cha ac e iza ion. SEM was pe o med on JEOL JSM-
6490LV. PEDOT:HA and PEDOT:HA:col wi h di e en COL compo-
si ions we e sliced in 2.5 mm hickness and moun ed on aluminum
holde wi h double-sided ca bon ape. The ou e and inne shell was
e alua ed a di e en magnifica ions using poin -by-poin mode.
4.2.1.3. Mechanical cha ac e iza ion. Sca old po e size was calcu-
la ed by Image J (Ja a) analysis. 30 po es o 3 pic u es o each
sca old we e measu ed wi h a ule and he media was calcula ed.
Sca olds we e cha ac e ized mechanically using Tinius Olsen
5ST (Tinius Olsen L d, UK) 1e50 kN. Wi h a Tinius Olsen appa a us,
he comp essi e moduli o he d y sca olds we e calcula ed. The
Tinius Olsen had a load cell o 25 N and he comp ession speed was
se a 1 mm/min. Young's modulus alues we e calcula ed om he
slope o he linea pa o he s ain s ess cu e. Sca old densi y
was calcula ed acco ding o hei olume and weigh .
The wa e - e aining capaci y o he sca olds was ob ained by
pe o ming a swelling analysis. Sca olds we e weighed be o e and
a e soaking o 24 h in PBS. The SR o he sca olds was analyzed
acco ding o Equa ion 1.
Equa ion 1 L¼
W
W
W
D
W
D
x100 whe e.
whe e, W
W
is he weigh o he sca old a e soaking wi h PBS
and W
D
is he weigh o he d y sca old.
4.2.1.4. Elec ical cha ac e iza ion. Sca olds we e cha ac e ized by
EIS using an Au olab po en ios a (PGSTAT128 N, Me ohm, UK) in
he equency ange o 10
1
o 10
5
Hz. A wo-elec ode configu a-
ion was se up o he EIS measu emen s using a comme cial
pla inum mesh as he coun e elec ode (WeS and RE-CE). The
dis ance be ween he elec odes was se o ⁓3 mm. The applied AC
ol age was 0.01 V and measu emen s we e ca ied ou a 0 V DC
po en ial s open ci cui po en ial.
4.2.1.5. Sca old immunofluo escence s aining. COL ype I and HA
immunos aining was pe o med sequen ially. Fi s ly, 500
m
m
sca old slices we e blocked wi h 1% Bo ine Se um Albumin (BSA,
Fishe BioReagen s) in 0.01% Tween-20 o 2 h a oom empe a-
u e. A e washing 3 imes in PBS, slices we e incuba ed wi h COL
ype I p ima y an ibody (1:50) (In i ogen) in 1% PBS/BSA o e nigh
a 4
C. The nex day, slices we e washed 3 imes wi h PBS o ge id
o he non-bonded molecules and incuba ed wi h 1:200 and an i-
abbi Alexa Fluo 647 (The moFishe ) in PBS o 2 h a 4
C. The
slices we e hen washed wi h PBS, and p epa ed o he HA im-
munos aining. Fo his, he slices we e again blocked wi h 1% BSA in
0.01% Tween-20 o 2 h a oom empe a u e and washed wi h PBS,
ho oughly. Then, slices we e incuba ed wi h HABP, bio inyla ed
(1:100, Sigma-Ald ich) in 1% PBS/BSA o e nigh a 4
C. The
ollowing day, slices we e washed 3 imes in PBS and incuba ed
wi h 1:200 FITC-S ep a idin (The moFishe ) in PBS o 2 h a oom
empe a u e. The final sample obse a ion was pe o med wi h he
con ocal mic oscope (Axio Obse e Z1 LSM 800, Zeiss).
4.3. Cell expe imen s
4.3.1. Cell cul u e main enance
Human sw480s colon adenoca cinoma cells we e cul u ed in
DMEM (Gibco, Sigma-Ald ich) supplemen ed wi h 10% Fe al Bo ine
Se um (FBS, Sigma Ald ich), 1% Glu amax (Gibco, Li e echnologies),
and 1% Pens ip (Gibco, Li e echnologies). 20.000 cells/cm
2
cells
we e cul u ed in T-75 flasks, main ained a 37
, 5% CO2 humidified
a mosphe e, and ha es ed wi h 0.25% ypsin be o e seeding o
passaging.
4.3.2. Seeding p ocess
Cell seeding was pe o med in bo h slices and sca old-based
elec odes o elec ical measu emen s and hey we e ully hy-
d a ed o 2 h and s e ilized wi h 70% e hanol o an hou . Then,
hey we e ho oughly washed wi h UHP wa e and PBS and le in
cell media o 2 h a 37
C, o allow o p o ein adhesion. Slices we e
hen seeded wi h 10
m
Lo 410
4
cell suspension and sca old-
based elec odes we e seeded wi h 20
m
L o 2.5 10
5
cell sus-
pension on op o each sample and le incuba ing o 2 h a 37
C.
Then, 400
m
L and 800
m
L o media we e added o slices and sca old-
based elec odes, espec i ely, and kep incuba ing a 37
C, 5% CO
2
.
The media was changed e e y 3 days.
To alida e he sys em, he an i-p oli e a i e d ug mo in was
added a e 7 days o incuba ion. 1
m
L o mo in was added o cell
cul u e media a day 7 a a concen a ion o 250
m
M and le o ac ua e
o a day o induce apop osis o cells. Con ols we e ea ed equally.
4.3.3. XTT assay
Assessmen o cell iabili y and p oli e a ion o sw480 cells was
pe o med using he XTT Cell Viabili y Assay Ki (Cell Signalling
Technology Inc.) which consis s o an indi ec quan i a i e colo i-
me ic assay de ec ing he cellula me abolic ac i i ies. In b ie ,
410
4
sw480s seeded on slices we e cul u ed o 7 days. Cul u e
media was eplaced wi h 500
m
L esh media and 100
m
l yellow
e azolium sal XTT solu ion was added o each well. This eagen
unde goes educ ion in o a highly colo ed o mazan dye by dehy-
d ogenase enzymes in me abolically ac i e cells, enabling he
es ima ion o li e cells wi hin a gi en cell cul u e. A e 3.5 h in-
cuba ion a 37
C, 100
m
l sample was collec ed om each well
con aining sca old slices and ans e ed o a 96-well pla e, ol-
lowed by abso bance eading a 450 nm using a mic opla e eade
TECAN SPARK®.
4.3.4. sw480s immunos aining
Sw480s we e fixed in 4% pa a o maldehyde (PFA, The mofishe
Scien ific) o 20 min, a oom empe a u e. All samples we e
ho oughly washed wi h PBS, pe meabilized in 0.1% T i on x-100
o 15 min, and hen blocked o nonspecific binding wi h 1% BSA
and 0.1% Tween-20 o 1 h a oom empe a u e. To label, he
samples o nuclei and F-ac in cy oskele on, DAPI and Alexa Fluo
647 Phalloidin we e used, espec i ely. Fluo escence images o cells
wi hin he sca old we e aken using an epifluo escence/con ocal
mic oscope (Axio Obse e Z1 LSM 800, Zeiss).
J. Saez, A. Dominguez-Al a o, C. Ba be io e al. Ma e ials Today Chemis y 24 (2022) 100990
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