Ci a ion: E xebe ia, L.; Messelmani,
T.; Badiola, J.H.; Llobe a, A.;
Fe nandez, L.; Vilas-Vilela, J.L.;
Lecle c, E.; Legallais, C.; Jellali, R.;
Zaldua, A.M. Valida ion o
HepG2/C3A Cell Cul u es in Cyclic
Ole in Copolyme Based Mic o luidic
Bio eac o s. Polyme s 2022,14, 4478.
h ps://doi.o g/10.3390/polym14214478
Academic Edi o : Yiqiang Fan
Recei ed: 26 Sep embe 2022
Accep ed: 19 Oc obe 2022
Published: 22 Oc obe 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
polyme s
A icle
Valida ion o HepG2/C3A Cell Cul u es in Cyclic Ole in
Copolyme Based Mic o luidic Bio eac o s
Lei e E xebe ia 1,2,3,* , Taha Messelmani 4, Jon Hai z Badiola 1, And eu Llobe a 2, Luis Fe nandez 2,
JoséLuis Vilas-Vilela 3,5, E ic Lecle c 4,6, Cécile Legallais 4, Rachid Jellali 4,*,† and Ane Mi en Zaldua 1,†
1Lea ike S. Coop., Xemein E o bidea 12, 48270 Ma kina-Xemein, Spain
2mic oLIQUID S.L, Goi u 9, 20500 A asa e-Mond agon, Spain
3Mac omolecula Chemis y Resea ch G oup (labquimac), Depa men o Physical Chemis y,
Facul y o Science and Technology, Uni e si y o he Basque Coun y (UPV/EHU), 48940 Leioa, Spain
4CNRS, Biomechanics and Bioenginee ing, Cen e de Reche che Royallieu-CS 60319,
Uni e si éde Technologie de Compiègne, 60203 Compiègne, F ance
5BC Ma e ials, Basque Cen e o Ma e ials, Applica ions and Nanos uc u es, UPV/EHU Science Pa k,
48940 Leioa, Spain
6CNRS IRL 2820, Labo a o y o In eg a ed Mic o Mecha onic Sys ems, Ins i u e o Indus ial Science,
Uni e si y o Tokyo, 4-6-1 Komaba, Tokyo 153-8505, Japan
*Co espondence: le xebe ia@lea ike .com (L.E.); [email p o ec ed] (R.J.)
† These au ho s con ibu ed equally o his wo k.
Abs ac :
O gan-on-chip (OoC) echnology is one o he mos p omising
in i o
ools o eplace he
adi ional animal expe imen -based pa adigms o isk assessmen . Howe e , he use o OoC in d ug
disco e y and oxici y s udies emain s ill limi ed by he low capaci y o high- h oughpu p oduc ion
and he incompa ibili y wi h s anda d labo a o y equipmen . Mo eo e , polydime hylsiloxanes, he
ma e ial o choice o OoC, has se e al d awbacks, pa icula ly he high abso p ion o d ugs and
chemicals. In his wo k, we epo he de elopmen o a mic o luidic de ice, using a p ocess adap ed
o mass p oduc ion, o cul u e li e cell line in dynamic condi ions. The de ice, made o cyclic
ole in copolyme s, was manu ac u ed by injec ion moulding and in eg a es Lue lock connec o s
compa ible wi h s anda d medical and labo a o y ins umen s. Then, he COC de ice was used o
cul u ing HepG2/C3a cells. The unc ionali y and beha iou o cul u es we e assessed by albumin
sec e ion, cell p oli e a ion, iabili y and ac in cy oskele on de elopmen . The cells in COC de ice
p oli e a ed well and emained unc ional o 9 days o cul u e. Fu he mo e, HepG2/C3a cells in
he COC biochips showed simila beha iou o cells in PDMS biochips. The p esen s udy p o ides
a p oo -o -concep o he use o COC biochip in li e cells cul u e and illus a e hei po en ial o
de elop OoC.
Keywo ds: cyclic ole in copolyme ; injec ion moulding; O gan-on-Chip; li e ; cell g ow h
1. In oduc ion
The li e pe o ms a g ea numbe o asks o suppo o he o gans and impac s all
physiological sys em. I is in ol ed in se e al essen ial unc ions including de oxi ica ion,
p o ein syn hesis and me abolism, glycogen s o age, bile sec e ion, as well as he emo al
o pa hogens and exogenous an igens o m he sys emic ci cula ion [
1
,
2
]. As he majo si e
o xenobio ic me abolism, he li e is exposed o a wide ange o exogenous subs ances
and is o en he subjec o chemical-induced oxici y [
2
]. The e o e, he in es iga ion o
d ugs/chemicals hepa o oxici y is c i ical o hei success ul use.
Mos o he s anda d oxicological app oaches o e alua ing chemical oxici y in ol e
complex
in i o
animal s udies, which a e bo h ime consuming and cos ly. Mo eo e ,
he da a p o ided by animal models canno always be ex apola ed o human me abolic
si ua ion due o he complexi y o he me abolism eac ions in he human body [
3
–
5
]. Due
Polyme s 2022,14, 4478. h ps://doi.o g/10.3390/polym14214478 h ps://www.mdpi.com/jou nal/polyme s
Polyme s 2022,14, 4478 2 o 15
o he conce n o animal wel a e and cos o he expe imen s,
in i o
cul u e sys ems
become highly in e es ing o oxici y s udies. Howe e , he con en ional
in i o
me hods
a e mainly based on s a ic 2D cul u es in Pe i dishes, which a e poo ly ep esen a i e o
human in i o physiology, me abolism and oxici y [6].
Among he new echnologies, O gan-on-a-Chip (OoC) de ices based in mic o lu-
idics a e ex emely a ac i e o
in i o
pha maceu ical and oxicological s udies. The
p og ess made in mic o echnology allows o imp o e
in i o
cul u es and cons uc ele-
an
in i o
models wi h complex geome ies [
7
–
12
]. The dynamic OoC cul u es allows
o ep oduce se e al cha ac e is ics o
in i o
en i onmen such as physiological shea
s ess, h ee-dimensional o ganiza ion, dynamic low, zona ion and homogenous anspo
o molecules such as ho mones, d ugs and me abolic was e [
13
]. Thus, se e al ad anced
wo ks o bioa i ical
in i o
Li e -on-a-Chip (LoC) de ices has been published in he las
yea s [14–19].
One o he main unc ions o a LoC de ices is o con ol he g ow h and he cell
beha iou in he mic oen i onmen , so he selec ed ma e ial o he manu ac u ing on he
de ice is c ucial o he co ec de elopmen o he analysis [
20
]. Polyme based mic o lu-
idic de ices a e o g ea in e es due o hei ligh weigh , low cos , op ical anspa ency
and chemical esis ance [
21
,
22
]. Due o i s p ope ies and he acili y o p o o yping manu-
ac u ing, polydime hylsiloxane (PDMS) is he mos used ma e ial o mic o luidic de ices
due o i s biocompa ibili y and ease o ans o ma ion [
23
–
25
]. Howe e , di e en s ud-
ies showed se e al disad an ages associa ed wi h he use o he PDMS in mic o luidic
de ices as wa e abso p ion, non-speci ic adso p ion o molecules and po osi y among
o he s [
26
,
27
]. To da e, he moplas ic polyme s, such as Polyca bona e (PC), Polyme hyl-
me hac yla e (PMMA), Polyes y ene (PS) and Cyclic Ole in Polyme (COP), in pa icula
Cyclic Ole in Copolyme s (COC), ha e eme ged as he mos p omising ma e ials o he
mass manu ac u ing o low-cos mic o luidic de ices [28,29].
COCs a e enginee ing he moplas ics p oduced by copolyme iza ion o cyclic monome s
like no bo nene wi h e hylene. I has a ully ca bon-based main chain and no double bonds
as shown in Figu e S1 (Supplemen a y File). COCs show a unique combina ion o p ope ies
such as glass-like op ical anspa ency, low wa e abso p ion, excellen wa e - apou ba ie
p ope ies, low dielec ic loss, high hea esis ance, biocompa ibili y and low-cos . In e ms
o chemical p ope ies, COCs show esis ance o ino ganic acids and bases as well as
pola o ganic sol en s [
30
,
31
]. Fu he mo e, scaling up he p oduc ion o o gan-on-a-chip
de ices is one impo an issues o o e come as mos de ices analysed in li e a u e is based
on labo a o y based ma e ials, such as PDMS, and ab ica ion me hods [
32
,
33
]. Injec ion
moulding o he moplas ics, such as COC, is epo ed o be cheap, as and p omising
app oach o a massi e ab ica ion o O gan-on-chip de ices [34–36].
In his wo k, he manu ac u ing o a COC biochip was pe o med a e adap ing i
o mass p oduc ion by injec ion moulding. The biochip is based on p e ious design, ha
showed good pe o mances as LoC manu ac u ed wi h PDMS and Pe luo opolye he s,
being bo h o hem biocompa ible [
37
–
42
]. The alida ion o he COC biochip o a Li e -
on-a-chip bio eac o was pe o med. The g ow h, iabili y, beha iou and me abolism
o HepG2/C3A cells, which a e commonly used as li e cell model, we e s udied and
compa ed wi h esul s ob ained using PDMS biochips.
2. Ma e ials and Me hods
2.1. Ma e ials
The selec ed ma e ials ha e been polydime hylsiloxane (PDMS) and Cyclic Ole in
Copolyme (COC). The used PDMS was Sylga d 184 ki (PDMS polyme + cu ing agen ;
10:1 mixing a io) pu chased om Dow Co ning (Midland, TX, USA). COC polyme was
pu chased om TOPAS Ad anced Polyme s (Raunheim, Ge many).
Polyme s 2022,14, 4478 3 o 15
2.2. Design and Manu ac u ing o he Bio eac o
The biochip consis s o 2 pa s: he mic os uc u ed bo om laye con ains cell cul u e
chambe s and mic ochannels (high o 100
µ
m), and he op laye , wi h a ese oi
100 µm
in dep h, includes an inle and ou le o cul u e medium pe usion. The aim o he design
is o allow a uni o m low wi hin he mic os uc u es. The mic os uc u e design and
ab ica ion o he bio eac o has been epo ed in p e ious wo ks (Figu e 1A) [
37
–
40
]. The
PDMS laye s was manu ac u ed by eplica moulding p ocess using SU-8 (epoxy-based
nega i e pho o esis ) mould ab ica ed by pho oli hog aphy p ocess. Then, he wo laye s
we e sealed oge he a e su aces ac i a ion wi h eac i e ai plasma (1 min; Ha ick
Scien i ic, Pleasan ille, NY, USA).
Polyme s 2022, 14, x FOR PEER REVIEW 3 o 17
2. Ma e ials and Me hods
2.1. Ma e ials
The selec ed ma e ials ha e been polydime hylsiloxane (PDMS) and Cyclic Ole in
Copolyme (COC). The used PDMS was Sylga d 184 ki (PDMS polyme + cu ing agen ;
10:1 mixing a io) pu chased om Dow Co ning (Midland, TX, USA). COC polyme was
pu chased om TOPAS Ad anced Polyme s (Raunheim, Ge many).
2.2. Design and Manu ac u ing o he Bio eac o
The biochip consis s o 2 pa s: he mic os uc u ed bo om laye con ains cell cul u e
chambe s and mic ochannels (high o 100 µm), and he op laye , wi h a ese oi 100 µm
in dep h, includes an inle and ou le o cul u e medium pe usion. The aim o he design
is o allow a uni o m low wi hin he mic os uc u es. The mic os uc u e design and ab-
ica ion o he bio eac o has been epo ed in p e ious wo ks (Figu e 1A) [37–40]. The
PDMS laye s was manu ac u ed by eplica moulding p ocess using SU-8 (epoxy-based
nega i e pho o esis ) mould ab ica ed by pho oli hog aphy p ocess. Then, he wo laye s
we e sealed oge he a e su aces ac i a ion wi h eac i e ai plasma (1 min; Ha ick Sci-
en i ic, Pleasan ille, NY, USA).
Figu e 1. (A) Design o he biochip laye s, (B) CAD design o COC bio eac o : (1) comple e Biochip,
(2) and (3) Lue lock connec o s.
Figu e 1.
(
A
) Design o he biochip laye s, (
B
) CAD design o COC bio eac o : (1) comple e Biochip,
(2) and (3) Lue lock connec o s.
Being he aim o his wo k o alida e COC o he mass manu ac u ing o LoC de ices,
he design o he mic os uc u es and mic ochambe s has been upda ed o adap ed o mass
manu ac u ing p ocesses. Rounded e ches, ypically ob ained a e a machining p ocess o
injec ion moulding ools, ha e been in oduced in he biochips. The modi ied geome ies
o he bo om laye o COC de ices a e shown in Figu e 1B. In he case o he biochip op
laye , he adap a ion o scaling pu pose has been pe o med adding Lue lock connec o s,
one o he gold s anda ds o medical and labo a o y ins umen s, such as needles, sy inges,
Polyme s 2022,14, 4478 4 o 15
and cannulas due o i s easy handling and compa ibili y [
43
,
44
]. The design o he co e
con aining Lue lock connec o s based on ISO 80369-7 [45] a e shown in Figu e 1B.
The injec ion moulding p ocess has been ca ied ou in A bu g 270 injec ion machine.
The injec ion mould was designed using he so wa e CREO Pa ame ics (6.0.5.1) and he
machining and manu ac u ing o he inse s and mould was ca ied ou in Ruimoldes 2012
S.L. The clamping o ce o he machine is 25 T, sc ew o 20 mm diame e and 80 cm
3
o
maximum injec ion olume. The selec ed injec ion pa ame e s we e; a mel empe a u e o
270
◦
C, mould empe a u e o 30
◦
C and injec ion speed o 13 mm/s. The bonding o he
biochip was pe o med using a doble-sided p essu e sensi i e adhesi e.
2.3. Scanning Elec on Mic oscopy
The PDMS and COC mic os uc u ed laye s we e cha ac e ized by scanning elec on
mic oscopy (SEM) using a Quan a 250 FEG mic oscope (The mo ische , Eindho en, The
Ne he land). The samples we e eco e ed by a hin palladium laye p io o analysis and
he images we e acqui ed wi h a 20 kV accele a ing ol age using he hi ac mode.
2.4. Flow and P essu e Measu emen s
Flow and p essu e measu emen s ha e been pe o med o analyse he hyd aulic
esis ance o he biochips. The luidic ci cui o he analysis consis s o a p essu e con olle
(MFCS-EX, Fluigen ) ha was connec ed he biochip and o a low senso (Flow Uni ype
M, Fluigen ). PEEK ubes connec ed all he ci cui o allow he cul u e medium o low
in o he biochip. A eedback loop adjus ed he p essu e applied in he ese oi o ensu e
he main enance o he desi ed low a e. The schema ic ep esen a ion o he mic o luidic
ci cui is shown in Figu e S2 (Supplemen a y File).
The p essu e di e en ial is gene a ed by p essu izing he inle and he ou le , and he
esul an low a e is measu ed o each biochip. The hyd aulic esis ance o he ci cui is
calcula ed ollowing he Equa ion (1):
RH=
∆P
Q(1)
whe e
∆P
is he p essu e di e en ial be ween inle and ou le and
Q
is he low a e. The
hyd aulic esis ance o PEEK ubing wi hin he ci cui is de e mined by Equa ion (2):
h=
8·µ·l
π·R4(2)
whe e
µ
is he iscosi y o he luid, lis he leng h o he ube, and Ris he in e nal adio o
ube. The hyd aulic esis ance o he biochips is calcula ed by sub ac ing he hyd aulic
esis ance o PEEK ubes o he hyd aulic esis ance o he whole ci cui .
2.5. Cells
HepG2/C3a hepa oca cinoma cells we e p o ided by he Ame ican Type Cul u e
Collec ion (ATCC, CRL-10741). The used cul u e media con ains Minimal Essen ial Medium
(MEM, Gibco, Wal ham, MA, USA), 2 mM L-Glu amine (Gibco), 0.1 mM non-essen ial
amino acids (Gibco), 1 mM sodium py u a e (Gibco), 10% ( / ) e al bo ine se um (Gibco)
and penicillin-s ep omycin (100 uni s/mL–100
µ
g/mL, Pan Bio ech, Aidenbach, Ge many).
The ba ch cul u es we e pe o med in T75 lasks (Falcon, Me k Eu olab, S asbou g, F ance)
using 15 mL o cul u e medium and he cells we e main ained a 37
◦
C in a humidi ied
a mosphe e supplied wi h 5% o CO
2
. The cells we e passaged weekly a a con luence o
80–90% and he cul u e medium was enewed e e y wo days.
2.6. Dynamic Cul u e in Biochip
Two dynamic cul u e expe imen s o di e en du a ions ha e been pe o med, bo h
including wo phases: he adhesion phase (24 h) and he pe usion phase (3 and 8 days).
Figu e 2A shows he iming and s eps o bo h sho and long- ime expe imen s. The expe i-
Polyme s 2022,14, 4478 5 o 15
men al se up used o dynamic cells cul u e was composed o a pe usion loop, including
he cul u e medium ank (bubble ap), he pe is al ic pump and one biochip. They we e
in e connec ed using 0.65 mm in e io diame e silicone/Te lon ubing (Figu e 2B). Be o e
each expe imen , he ubing and bubble ap we e s e ilized by au ocla ing, while he
biochips (PDMS and COC) we e s e ilized using e hanol (70%).
Polyme s 2022, 14, x FOR PEER REVIEW 5 o 17
Ge many). The ba ch cul u es we e pe o med in T75 lasks (Falcon, Me k Eu olab, S as-
bou g, F ance) using 15 mL o cul u e medium and he cells we e main ained a 37 °C in
a humidi ied a mosphe e supplied wi h 5% o CO
2
. The cells we e passaged weekly a a
con luence o 80–90% and he cul u e medium was enewed e e y wo days.
2.6. Dynamic Cul u e in Biochip
Two dynamic cul u e expe imen s o di e en du a ions ha e been pe o med, bo h
including wo phases: he adhesion phase (24 h) and he pe usion phase (3 and 8 days).
Figu e 2A shows he iming and s eps o bo h sho and long- ime expe imen s. The ex-
pe imen al se up used o dynamic cells cul u e was composed o a pe usion loop, in-
cluding he cul u e medium ank (bubble ap), he pe is al ic pump and one biochip. They
we e in e connec ed using 0.65 mm in e io diame e silicone/Te lon ubing (Figu e 2B). Be-
o e each expe imen , he ubing and bubble ap we e s e ilized by au ocla ing, while he
biochips (PDMS and COC) we e s e ilized using e hanol (70%).
Figu e 2. Design o he expe imen s: (A) Timing and s eps o sho and long- ime expe imen s in-
cluding he adhesion and pe usion phases, (B, C) schema ic ep esen a ion ad image o dynamic
low se up espec i ely.
To enhance he cell adhesion, be o e he seeding o he cells, he bio eac o s we e
coa ed wi h a ail ype 1 collagen (Co ning, NY, USA; 300 µg/mL in bu e saline solu-
ion: PBS Gibco) and incuba ed a 37 °C in an a mosphe e supplied wi h 5% CO
2
. A e 1h,
he cleaning o he collagen was pe o med wi h cul u e medium and 0.2 ± 0.03 × 10
5
cells
we e inocula ed inside o each biochip. The cells we e incuba ed in s a ic condi ions o
adhesion du ing 24 h in a 5% CO
2
incuba o a 37 °C. To keep he cul u e medium inside
he cul u e chambe , he biochip inle po s we e closed using wo sy inges (con aining
500 µL o cul u e medium).
A e 24 h o adhesion, 3 biochips we e chosen o ini ial cell coun ing (adhe en cell) and
he es we e p epa ed o he pe usion phase. The biochips we e hen connec ed o he pe -
usion loop, and 2 mL o cul u e medium we e added in each bubble ap (Figu e 2A,B). The
en i e se up was incuba ed a 37 °C in a 5% CO
2
supplied incuba o and he pe is al ic
Figu e 2.
Design o he expe imen s: (
A
) Timing and s eps o sho and long- ime expe imen s
including he adhesion and pe usion phases, (
B
,
C
) schema ic ep esen a ion ad image o dynamic
low se up espec i ely.
To enhance he cell adhesion, be o e he seeding o he cells, he bio eac o s we e
coa ed wi h a ail ype 1 collagen (Co ning, NY, USA; 300
µ
g/mL in bu e saline solu ion:
PBS Gibco) and incuba ed a 37
◦
C in an a mosphe e supplied wi h 5% CO
2
. A e 1 h, he
cleaning o he collagen was pe o med wi h cul u e medium and 0.2
±
0.03
×
10
5
cells
we e inocula ed inside o each biochip. The cells we e incuba ed in s a ic condi ions o
adhesion du ing 24 h in a 5% CO
2
incuba o a 37
◦
C. To keep he cul u e medium inside
he cul u e chambe , he biochip inle po s we e closed using wo sy inges (con aining
500 µL o cul u e medium).
A e 24 h o adhesion, 3 biochips we e chosen o ini ial cell coun ing (adhe en cell)
and he es we e p epa ed o he pe usion phase. The biochips we e hen connec ed o he
pe usion loop, and 2 mL o cul u e medium we e added in each bubble ap (Figu e 2A,B).
The en i e se up was incuba ed a 37
◦
C in a 5% CO
2
supplied incuba o and he pe is al ic
pump was s a ed a low a e o 25
µ
L/min. The medium was collec ed ( o subsequen
analysis) and enewed e e y day.
2.7. Cell Coun ing and Viabili y
Cell coun ing was made by de achmen wi h ypsin-EDTA (0.25%, Gibco) and coun -
ing using a g adua ed Malassez cells. The p oli e a ion a e was calcula ed by di iding he
cell numbe coun ed a he end by he numbe o seeded cells. T ypan blue s aining was
used o iabili y analysis.
Polyme s 2022,14, 4478 6 o 15
Each analysis was epea ed h ee imes in iplica e o each ma e ial and h ee biochips
we e coun ed o each expe imen (3 expe imen s
×
3 biochips = 9 eplica es). Da a is
plo ed as mean ±SD.
2.8. Albumin Measu emen s
The albumin p oduced by cells and eleased in cul u e medium measu emen s we e
measu ed using ELISA sandwich es in a 96-well pla e. The assays we e pe o med using
a human albumin ELISA Quan i a ion Se (E80-129, Be hyl Labo a o ies, Mon gome y, TX,
USA), ollowing he manu ac u e ins uc ions. The pla e was ead wi h an abso bance
wa eleng h o 490 nm, using a Spec a luo Plus mic opla e eade (TECAN, Männedo ,
Swi ze land). The analyses we e pe o med wi h cul u e medium collec ed om h ee
biochips and epea ed h ee ime in eplica e, leading o n= 9 (3 expe imen s
×
3 biochips
= 9 eplica es). Da a is plo ed as mean ±SD.
2.9. Immunos aining and Con ocal Mic oscopy
A e adhesion and pe usion phases (end o he expe imen ), he biochips we e
washed wi h PBS (Gibco), ixed in pa a o maldehyde 4% (PFA, MP biomedicals, Illki ch-
G a ens aden, F ance) o 30 min a oom empe a u e and washed and s o ed in PBS un il
s aining. Cell nuclei we e s ained wi h DAPI a 10
µ
g/mL (4
0
,6-diamidino-2-phenylindole,
D1306, In i ogen) and phalloidin (Alexa Fluo 488 Phalloidin, The mo Fishe (Wal ham,
MA, USA)) s aining was used o F-ac in isualiza ion. The samples we e incuba ed, in
da k and a oom empe a u e, wi h phalloidin o 3 h and DAPI o 30 min. A he end o
he incuba ion, he samples we e washed wi h PBS.
The obse a ions o he s ained samples we e made wi h a lase scanning con ocal
mic oscope (SM 710, Zeiss (Obe kochen, Alemania)) a 647 and 488 nm, espec i ely.
3. Resul s
3.1. Biochips Cha ac e iza ion
The pic u es o COC (injec ion moulding) and PDMS ( eplica moulding) biochips
a e p esen ed in Figu e 3A. The injec ion moulding o COC allows o p oduce e y hin
biochip wi h good op ical anspa ency. The design o he biochip acili a es he connec ion
wi h s anda d Lue lock connec o s ( ypically used o medical de ices), and hus, he
wo ld- o-chip in e connec ion o cell inocula ion and dynamic cell cul u e. In he case
o PDMS biochips, he used Lue lock connec o s a e comme cial and a e added o he
biochips a e he manu ac u ing o he comple e biochips whe eas in he case o COC
biochips, and due o he adap a ion o he design o mass manu ac u ing pu poses, Lue
lock connec o s a e pa o he injec ion moulded co e as can be obse ed in Figu e 3A.
Polyme s 2022, 14, x FOR PEER REVIEW 7 o 17
This con i ms an op imum moulding condi ion ega dless he ma e ial (COC o PDMS)
and echnology used (injec ion moulding o eplica moulding).
Figu e 3. COC and PDMS biochips cha ac e iza ion: (A) biochips pic u es, (B,C) phase con as mi-
c oscopy and MEB images, espec i ely.
3.2. Flow and P essu e Measu emen s
To e alua e he biochips sealing and he low ci cula ion inside he bio eac o s, we
pe o med a dynamic es in which se e al low a es and p essu e we e es ed (expe i-
men al de ail in Sec ion 2.4). Rega ding he maximum ou le p essu e ha he biochips
can wi hs and, COC biochips s a ed leaking a 400 mba whe eas PDMS biochips can
wi hs and 500 mba ou le p essu es. Ne e heless, o COC biochip, no leakage was ob-
se ed in con inuous pe usion a a p essu e o 300 mba .
Using he equa ions 1 and 2 (see Sec ion 2.4), we calcula ed he hyd aulic esis ance
o COC and PDMS biochips. The esul s ob ained du ing he low and p essu e measu e-
men s a e p esen ed in Figu e 4. No signi ican di e ence was obse ed be ween he wo
ypes o biochips. The hyd aulic esis ances we e app oxima ely o 2.2·1012 ± 0.35·1012 and
2.75·1012± 1.3·1012Pa·s·m−3 o COC and PDMS biochips, espec i ely. The hyd aulic e-
sis ance alues o COC biochips show less s anda d a ia ion han PDMS biochipswhich
can be due o he manual manu ac u ing s eps ypically used o PDMS biochip ab ica-
ion.
Figu e 3.
COC and PDMS biochips cha ac e iza ion: (
A
) biochips pic u es, (
B
,
C
) phase con as
mic oscopy and MEB images, espec i ely.
Polyme s 2022,14, 4478 7 o 15
Figu e 3B shows he phase con as mic oscopy obse a ions o he bo om laye o
he biochips manu ac u ed wi h PDMS and COC ma e ials. The lexibili y o he PDMS
makes he demoulding easie o PDMS han o COC. Ne e heless, he eplicas ob ained
in COC a e p ecise and main ain s uc u es dimensions. The mic os uc u ed bo om laye s
(PDMS and COC) we e also analysed using SEM. The images a e p esen ed in Figu e 3C.
They show an accu a e eplica ion o he mic os uc u es in bo h ma e ials, good accu acy
in he dimensions, excellen su ace quali ies and no de o ma ions in he mic os uc u es.
This con i ms an op imum moulding condi ion ega dless he ma e ial (COC o PDMS)
and echnology used (injec ion moulding o eplica moulding).
3.2. Flow and P essu e Measu emen s
To e alua e he biochips sealing and he low ci cula ion inside he bio eac o s, we
pe o med a dynamic es in which se e al low a es and p essu e we e es ed (expe i-
men al de ail in Sec ion 2.4). Rega ding he maximum ou le p essu e ha he biochips
can wi hs and, COC biochips s a ed leaking a 400 mba whe eas PDMS biochips can
wi hs and 500 mba ou le p essu es. Ne e heless, o COC biochip, no leakage was
obse ed in con inuous pe usion a a p essu e o 300 mba .
Using he equa ions 1 and 2 (see Sec ion 2.4), we calcula ed he hyd aulic esis ance o
COC and PDMS biochips. The esul s ob ained du ing he low and p essu e measu emen s
a e p esen ed in Figu e 4. No signi ican di e ence was obse ed be ween he wo ypes
o biochips. The hyd aulic esis ances we e app oxima ely o 2.2
·
10
12 ±
0.35
·
10
12
and
2.75
·
10
12 ±
1.3
·
10
12
Pa
·
s
·
m
−3
o COC and PDMS biochips, espec i ely. The hyd aulic
esis ance alues o COC biochips show less s anda d a ia ion han PDMS biochipswhich
can be due o he manual manu ac u ing s eps ypically used o PDMS biochip ab ica ion.
Polyme s 2022, 14, x FOR PEER REVIEW 8 o 17
Figu e 4. Hyd aulic esis ance o PDMS and COC Biochips.
3.3. HepG2/C3a Cell Adhesion on COC and PDMS Biochips
Cells adhesion on he bo om mic os uc u ed laye o biochip is c ucial be o e s a -
ing he dynamic pe usion. Fo his, a i s se ie o expe imen s was pe o med in o de o
in es iga e he beha iou o HepG2/C3a cells in con ac wi h COC subs a e. The COC
and PDMS (con ol) biochips we e p elimina ily coa ed wi h collagen. The cells mo phol-
ogy a e seeding a e p esen ed in Figu e 5A. In bo h COC and PDMS biochips, he cells
p esen ounded shape and we e in suspension in he cul u e chambe . Fu hemo e, he
HepG2/C3a cells we e homogeneously dispe sed h oughou he mic ochambe s and mi-
c ochannels o he bio eac o s.
Figu e 4. Hyd aulic esis ance o PDMS and COC Biochips.
3.3. HepG2/C3a Cell Adhesion on COC and PDMS Biochips
Cells adhesion on he bo om mic os uc u ed laye o biochip is c ucial be o e s a ing
he dynamic pe usion. Fo his, a i s se ie o expe imen s was pe o med in o de o
in es iga e he beha iou o HepG2/C3a cells in con ac wi h COC subs a e. The COC and
PDMS (con ol) biochips we e p elimina ily coa ed wi h collagen. The cells mo phology
a e seeding a e p esen ed in Figu e 5A. In bo h COC and PDMS biochips, he cells
p esen ounded shape and we e in suspension in he cul u e chambe . Fu hemo e, he
Polyme s 2022,14, 4478 8 o 15
HepG2/C3a cells we e homogeneously dispe sed h oughou he mic ochambe s and
mic ochannels o he bio eac o s.
Polyme s 2022, 14, x FOR PEER REVIEW 8 o 17
Figu e 4. Hyd aulic esis ance o PDMS and COC Biochips.
3.3. HepG2/C3a Cell Adhesion on COC and PDMS Biochips
Cells adhesion on he bo om mic os uc u ed laye o biochip is c ucial be o e s a -
ing he dynamic pe usion. Fo his, a i s se ie o expe imen s was pe o med in o de o
in es iga e he beha iou o HepG2/C3a cells in con ac wi h COC subs a e. The COC
and PDMS (con ol) biochips we e p elimina ily coa ed wi h collagen. The cells mo phol-
ogy a e seeding a e p esen ed in Figu e 5A. In bo h COC and PDMS biochips, he cells
p esen ounded shape and we e in suspension in he cul u e chambe . Fu hemo e, he
HepG2/C3a cells we e homogeneously dispe sed h oughou he mic ochambe s and mi-
c ochannels o he bio eac o s.
Figu e 5.
Cha ac e iza ion o HepG2/C3a cells adhesion on COC and PDMS biochips: (
A
) cell
mo phology a e seeding and a e 24 h o adhesion (scale ba 150
µ
m), (
B
) Phalloidin and DAPI
s ainings o cells a e adhesion phase: DAPI (nuclei, blue) and phalloidin (F-ac in, g een); scale ba
100 µm.
A e 24 h o adhesion phase in s a ic condi ions, he cul u e medium was changed o
emo e non-adhe en cells and he biochips we e obse ed using phase con as mic oscopy.
Figu e 5A p esen s he cell mo phology inside he biochips. The adhesion o HepG2/C3a
o he bo om su aces o he mic o luidic bio eac o s was success ul o bo h ma e ials
used (COC and PDMS). We did no de ec any signi ican di e ence be ween COC and
PDMS. The cells exhibi ed an elonga ed shape and homogeneously occupy he su ace o
he cul u e chambe s. The cell coun ing pe o med pos -adhesion (a e cell de achmen
wi h ypsin) e ealed 0.185
±
0.02
×
10
5
and 0.15
±
0.007
×
10
5
a ached cells in COC and
PDMS biochips, espec i ely. In compa ison wi h he ini ial seeded cells, he pe cen age
o adhe ed cells was o 90
±
5% o COC biochip and 75
±
8% o PDMS biochip. Finally,
he cy oskele on o ganiza ion was in es iga ed by ac in s aining. As shown in Figu e 5B
and Figu e S3 (Supplemen a y File), he ac in cy oskele on o he cells can be seen clea ly
(in ense g een luo escence signal) in he whole cul u e chambe . The ac in ilamen s appea
o be concen a ed benea h he cell memb ane (a ound nuclei).
3.4. Sho Time Dynamic Cul u e o HepG2/C3a in Biochips
A e adhesion alida ion and o e alua e he po en ial o COC biochips o dynamic
cell cul u e, we pe o med a se ies o dynamic cul u es o sho ime. These cul u es we e
pe o med o 4 days: 24 h o adhesion and 72 h o dynamic pe usion. Fo compa ison, he
expe imen s we e ealized wi h COC and PDMS biochips
The mo phology o HepG2/C3a cells a e adhesion phase and a he end o he
expe imen (24 h o adhesion + 72 h o dynamic cul u e) is p esen ed in Figu e 6A. As
obse ed in he p e ious Sec ion 3.3, he cells ha e success ully adhe ed o he biochips.
The cell p oli e a ion was e iden be ween he i s 24 h o cul u e (a e adhesion) and he
end o he expe imen (72 h o pe usion). The cells ha e p oli e a ed o e con luence and
s a ed o o m a second laye . As cell densi y was high, i is di icul o dis inguish he
cellula pheno ype. We we e no able o obse e a di e ence be ween he mo phologies o
Polyme s 2022,14, 4478 9 o 15
cells cul u ed in COC biochips and cells cul u ed in PDMS biochips. The cell de achmen
a he end o he expe imen s allowed cells coun ing and iabili y assessmen ( ypan blue
s aining). The p oli e a ion a io was o 2.3
±
0.6 and 2
±
0.3 o COC and PDMS biochips,
espec i ely (Figu e 7A). Mo eo e , he iabili y es s pe o med by ypan blue a e 96 h
o cul u e (24 h s a ic cul u e and 72 h o pe usion) e ealed good iabili y in bo h cul u es’
condi ions ( iabili y abo e 90%). The phalloidin s aining con i med he de elopmen o
ac in cy oskele on in COC and PDMS cul u e (no speci ic di e ence was de ec ed be ween
he wo ma e ials, Figu e 7B and Figu e S3, Supplemen a y File).
Polyme s 2022, 14, x FOR PEER REVIEW 10 o 17
Figu e 6. Cha ac e iza ion o dynamic cul u es in biochip: (A) cell mo phology a e adhesion and
a he end o 72 h o pe usion (scale ba 150 µm), (B) Phalloidin and DAPI s ainings o cells a he
end o pe usion: DAPI (nuclei, blue) and phalloidin (F-ac in, g een); scale ba 100 µm.
To e alua e he unc ionali y o HepG2/C3a cells cul u ed in COC biochips, albumin
p oduc ion was moni o ed h oughou he 72 h o pe usion and compa ed wi h he
esul s ob ained in PDMS biochips. The esul s a e shown in Figu e 7B. he albumin
p oduc ion o cells cul u ed in COC bio eac o s was o 135 ± 27, 123 ± 32 and 87 ± 25
ng/h/106 cells a e 24, 48 and 72 h o pe usion, espec i ely. The quan i y o albumin
p oduced by HepG2/C3a cells in PDMS biochips was simila : 149 ± 41 (24 h), 113 ± 39 (48
h) and 90 ± 5 (72 h) ng/h/106 cells.
Figu e 7. P oli e a ion and unc ionali y o HepG2/C3a cells cul u ed in dynamic COC and PDMS
biochips o 96 h including 24 h o adhesion and 72 h o pe usion: (A) P oli e a ion Ra io a he end
o he expe imen , (B) Albumin p oduc ion a e 24 h, 48 h and 72 h o pe usion.
Figu e 6.
Cha ac e iza ion o dynamic cul u es in biochip: (
A
) cell mo phology a e adhesion and a
he end o 72 h o pe usion (scale ba 150
µ
m), (
B
) Phalloidin and DAPI s ainings o cells a he end
o pe usion: DAPI (nuclei, blue) and phalloidin (F-ac in, g een); scale ba 100 µm.
Polyme s 2022, 14, x FOR PEER REVIEW 10 o 17
Figu e 6. Cha ac e iza ion o dynamic cul u es in biochip: (A) cell mo phology a e adhesion and
a he end o 72 h o pe usion (scale ba 150 µm), (B) Phalloidin and DAPI s ainings o cells a he
end o pe usion: DAPI (nuclei, blue) and phalloidin (F-ac in, g een); scale ba 100 µm.
To e alua e he unc ionali y o HepG2/C3a cells cul u ed in COC biochips, albumin
p oduc ion was moni o ed h oughou he 72 h o pe usion and compa ed wi h he
esul s ob ained in PDMS biochips. The esul s a e shown in Figu e 7B. he albumin
p oduc ion o cells cul u ed in COC bio eac o s was o 135 ± 27, 123 ± 32 and 87 ± 25
ng/h/106 cells a e 24, 48 and 72 h o pe usion, espec i ely. The quan i y o albumin
p oduced by HepG2/C3a cells in PDMS biochips was simila : 149 ± 41 (24 h), 113 ± 39 (48
h) and 90 ± 5 (72 h) ng/h/106 cells.
Figu e 7. P oli e a ion and unc ionali y o HepG2/C3a cells cul u ed in dynamic COC and PDMS
biochips o 96 h including 24 h o adhesion and 72 h o pe usion: (A) P oli e a ion Ra io a he end
o he expe imen , (B) Albumin p oduc ion a e 24 h, 48 h and 72 h o pe usion.
Figu e 7.
P oli e a ion and unc ionali y o HepG2/C3a cells cul u ed in dynamic COC and PDMS
biochips o 96 h including 24 h o adhesion and 72 h o pe usion: (
A
) P oli e a ion Ra io a he end
o he expe imen , (B) Albumin p oduc ion a e 24 h, 48 h and 72 h o pe usion.
