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ARTICLE IN PRESSG ModelBIOTEC 6977 110Journal of Biotechnology
xxx (2015) xxxxxx
Contents lists available at ScienceDirect
Journal of Biotechnology
j ourna l ho me page: www.elsev ier .com/ locate / jb io tec
A modular segmented ow-platform for 3D cell c
Karen Lemkea, Tobias Frstera, Robert Rmera, Mandy QuadeQ1Andreas
Grodriana, Gunter Gastrocka,
a Department o niquesGermanyb Faculty of Me sue ReGermany
a r t i c l
Article history:Received 25 AReceived in re21 November Accepted
28 NAvailable onlin
Keywords:DropletsHigh(er) throughputQ2Embryoid body
formationLong-term 3D cultivationAutomated modular
platformPersonalized medicine
to eatrix iasedater-ined u
modld-ba
storage, and an analysis module for monitoring cell aggregation
and proliferation basing on microscopy orphotometry. In this
report, the self-assembly of murine embryonic stem cells (mESCs) to
uniformly sizedembryoid bodies (EBs), the cell proliferation, the
cell viability as well as the inuence on the cell differ-entiation
to cardiomyocytes are described. The integration of a dosage module
for medium exchange oragent addition will enable pbb as 3D
long-term cultivation system for studying stem-cell
differentiation,
1. Introdu
MicrouQ3the 1980s systems tectotal-analysinuence oaffection
ofcell differenmicrouidicmagnitudeschemical faof the technforms are
so
CorresponE-mail add
(K. Lemke), ToRobert.Roeme(M. Quade), St(S.
WiedemeieGunter.Gastro
http://dx.doi.o0168-1656/
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38e this article in press as: Lemke, K., et al., A modular
segmented ow-platform for 3D cell cultivation. J. Biotechnol.
(2015),doi.org/10.1016/j.jbiotec.2014.11.040
e.g. cardiac myogenesis or for diagnostic and therapeutic
testing in personalized medicine. 2015 Published by Elsevier
B.V.
ction
idic technology was developed at the beginning ofas a functional
extension of micro-electromechanicalhnology. Nowadays,
lab-on-a-chip (LOC) or micro-is-systems (TAS) are used to
investigate thef biological, physical, and chemical factors for
any
cells (Wu et al., 2011). For the investigation of stem-tiation
and proliferation or for cancer cell research,
technologies offer the ability to precisely control the and
concentrations of these biological, physical, andctors affecting
the cells (Csete, 2010). However, mostiques used for the
manufacturing of microuidic plat-phisticated but often
inexible.
ding author. Tel.: +49 3606 671 400; fax: +49 3606 671
200.resses:
[email protected]@iba-heiligenstadt.de
(T. Frster),[email protected] (R. Rmer),
[email protected]@iba-heiligenstadt.der),
[email protected] (A.
Grodrian),[email protected] (G. Gastrock).
Due to the preference of 3D to 2D cell culture models in,
e.g.stem cell or cancer research, exible cell cultivation systems
arerequired. Such systems should enable reproducible 3D
long-termcell cultivation in high throughput by precise denition
and controlof the cell microenvironment at any time, which means
the biolog-ical, physical, and chemical parameters. Furthermore, it
would bea great advantage to have either a scaffold-free or a
scaffold-basedcultivation to adjust even different models (Froeling
et al., 2010;Rimann and Graf-Hausner, 2012). Overall, it has to be
easy to use,rapid, and cost-effective. Nowadays, several
microwell-associatedsystems are already on the market, which
initiate the generationof 3D cell structures by gravity-enforced
self-assembly in hangingdroplets, by enabling anchorage-free
culture conditions by chem-ical or nanostructural modication of the
cultivation surface or byproviding degradable and non-degradable
scaffolds. A represent-ing overview of these commercially available
3D culture systems ispresented by Rimann and Graf-Hausner
(2012).
Cell cultivation systems associated to microwells (Tung et
al.,2011; Messner et al., 2013) offer a high degree of
standardization.The possibility to add several cell types
(co-cultivation) or drugs tothe hanging droplets at any time
guarantees a high exibility to runa broad range of SOPs for cell
cultivation. However, these are notclosed systems and there will be
evaporation of the cell medium.
rg/10.1016/j.jbiotec.2014.11.0402015 Published by Elsevier
B.V.
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61f Bioprocess Engineering, Institute for Bioprocessing and
Analytical Measurement Tech
dicine, Technische Universitt Dresden, Centre for Translational
Bone, Joint and Soft Tis
e i n f o
ugust 2014vised form2014ovember 2014e xxx
a b s t r a c t
In vitro 3D cell cultivation is promisedcorresponding to
cellcell and cellmdeveloped. This platform, called pipe-baqueous
droplets are embedded in a wto 20 L and are used as bioreactors
lplatform basically consists of severalgeneration for self-assembly
or scaffoultivationa,b, Stefan Wiedemeiera,
e.V., Rosenhof, D-37308 Heilbad Heiligenstadt,
search, Fetscherstr. 74, D-01307 Dresden,
quate tissue in vivo more realistically than 2D cell
cultivationnteractions. Therefore, a scalable 3D cultivation
platform was
bioreactors (pbb), is based on the segmented ow
technology:immiscible carrier uid. The droplet volumes range from
60 nLp in a tubing-like pearls on a string. The modular
automatedules like a uid management for a high-throughput
dropletsed 3D cell cultivation, a storage module for incubation
and
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ARTICLE IN PRESSG ModelBIOTEC 6977 1102 K. Lemke et al. /
Journal of Biotechnology xxx (2015) xxxxxx
Furthermore, because of the fragility of the hanging droplets,
theirmixing and microscopic detection as well as the automation
ofsuch process steps are complex tasks. Cultivation systems,
whichenable 3D cell culture by chemical or nanostructural
modicationof the cultiture condititherefore hgenerate mformation
iFor stem-ceof the embrferentiationabove-menthe potentiprimary
celthe individu
A segmeapproach. Dserve as bto
microwebioreactorshydrophobirealized for(2007) cultiinner
diamobserved ovoped a droof cells and660 nL drop
Here, wbioreactorscessing and04 226) whcultures. Asalso
asepticand scaffoldparallelizedthe additionculture mebe
monitoretroscopy.
2. Materia
2.1. Convenundifferenti
Human DSMZ-GermACC-305) w(DMEM, Sifetal bovin(Sigma-Aldand
antibimycin, Sigapproximat12 104 cplasmid pEorescent prwere
alway(G418-disu
Undifferwere kindlof the Acta(Potta et al
translation initiation site of the Acta2 gene was isolated by
BACrecombineering method and then subcloned into the ESC
reporterconstruct pPuroIRES2-EGFP. This linearized construct was
elec-troporated into CGR8 ESCs, in order to generate after
neomycin
on ths weial mminerthered p
conh celing
vitroyoc
Actsionardiomscripence,04 cedium) sup
acidated
the was ationltice
dishere
cultu untidiumd ESce of
iffere-bas3 in
threoid bis puizedDTA 93 c, T4g a sionm co0 orta2
ES ce0 nL)sing -lysinmetotal differb-pl
d-frehe fu
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127e this article in press as: Lemke, K., et al., A modular
segmented doi.org/10.1016/j.jbiotec.2014.11.040
vation surface in order to realize anchorage-free cul-ons, work
usually with a bigger cultivation volume andave fewer problems with
evaporation. As they oftenore than one 3D cell aggregate per used
volume, thes not as uniform as by using the hanging drop method.ll
research, it was shown that the uniform, precise sizeyoid bodies
(EBs) is essential for the efciency of cell dif-
(Bauwens et al., 2008; Xu et al., 2011). For the secondtioned
topic, the cancer research, it is necessary to offeral for further
parallelization of 3D cell culture based onls of the patient in
order to optimize the diagnostics andal therapy (Thoma et al.,
2014).nted-ow-based technique represents an alternativeroplets
generated by the segmented-ow techniqueioreactors offering
essential advantages comparedll-based cultivation systems.
Segmented-ow-based
are embedded in a capillary system separated by ac oil as
separation and transport liquid. They have been
several biological models. For example, Funfak et al.vated
zebrash embryos inside a tubing having 1.2 mmeter, and the
development of the embryos could beer an 80 h period.
Clausell-Tormos et al. (2008) devel-
plet-based platform for encapsulation and cultivation
multicellular organisms like Caenorhabditis elegans inlets.e
present a segmented-ow based pipe-based
-platform (pbb, registered mark of Institute for Biopro-
Analytical Measurement Techniques e.V., Reg. no. 305ich serves as
long-term cultivation system for 3D cell
a closed system avoiding evaporation, it guarantees conditions
and can be applied for both, scaffold-free-based cultivation
protocols. The pbb-platform can be
to increase the throughput. Functional modules allow of drugs
for screening procedures or the exchange of
dium for long-term cultivations. Cell proliferation cand using
microuidic modules for microscopy and spec-
ls and methods
tional cell culture of EFG-expressing HEK 293 andated Acta 2
murine embryonic stem cells (mESCs)
embryonal kidney (HEK) 293 cells (Leibniz Institutean Collection
of Microorganisms and Cell Cultures;ere expanded in Dulbeccos
modied Eagles mediumgma-Aldrich D5523) supplemented with 10% (v/v)e
serum (FBS, Biochrom, S0115), 2 mM l-glutaminerich, R8758), 4.5 g/l
glucose (Sigma-Aldrich, G7021)otics (100 units/mL penicillin/100
g/mL strepto-ma -Aldrich, P0781). Conuent cultures were splitely
two times a week and seeded out at aboutells/cm2. The HEK 293 cells
were stably transfected withGFP-C1 expressing high-intensity
enhanced green u-oteins. Starting from 2 days after transfection
the cellss maintained in culture containing 750 g/mL
geneticinlfate, Applichem, A2167).entiated Acta2 murine embryonic
stem cells (mESCs)y provided by Prof. Dr. A. Sachinidis. The
generation2 ESC line has been described in detail previously.,
2009). Briey, the promoter region upstream of the
selectiES cellessentl-glutaLIF, fudescribwhen
Botcontain
2.2. Incardiom
Thedimeninto caous deconu2.5 1EB me(IMDMaminoaggregsion
onwhichevaporthe mulogical7. EBs w7 and day 10EB
meentiatepresen
2.3. DscaffoldHEK 29
TheembryFor thtrypsinwith EHEK 2Aldrichby usinsuspenmediu200,
30500 AcActa 2per 40exprespoly-lage dia(1 mL tThese ular
pbscaffol3.3). Tplatform for 3D cell cultivation. J. Biotechnol.
(2015),
e stable Acta2 ESC line. These undifferentiated Acta2re cultured
without feeder cells in Glasgow minimumedium (GMEM) supplemented
with 10% (v/v) FBS, 2 mM, 50 M -mercaptoethanol (-ME), and 100
units/mL
called ES medium, in 0.2% gelatin-coated asks asreviously (Potta
et al., 2010). The cells were passeduence reached upon 70%.l types
were cultivated at 37 C in a humidied 5% CO2-atmosphere.
Acta2 ESC differentiation of embryoid bodies intoytes using the
hanging drop method
a2 ESCs were differentiated in the form of three-l multicellular
aggregates called embryoid bodies (EBs)yocytes using the hanging
drop method as per previ-
tion by Potta et al. (2010). Briey, upon reaching 70% ES cells
were trypsinized and a single-cell suspension oflls/mL was prepared
in differentiation medium, called, consisting of Iscoves modied
Dulbeccos mediumplemented with 20% (v/v) FBS, 1% (v/v)
non-essential
s, 2 mM l-glutamine, and 100 M -ME. ES cells were in hanging
droplets containing 500 cells/20 L suspen-
inner surface of the lid of a 10-cm bacteriological dish,lled
with 5 mL of sterile PBS in order to prevent the
of the droplets, and placed in an incubator. After 2 days,llular
aggregates were transferred into a new bacterio-
using EB medium and cultured in suspension until day transferred
to 0.2% (w/v) gelatine-coated dishes on dayred for further 8 days
as adherent EBs. Starting froml day 15, EBs were cultured in the
same differentiation
but with 5 g/mL puromycin for selecting the differ-Cs. The
enriched cardiomyocytes were indicated by the
EGFP-expressing beating areas within a treated EB.
nt cell seeding densities for scaffold-free and/ored 3D cell
culture of Acta2 ESCs and EGFP-expressingthe pipe based bioreactors
(pbb)
e-dimensional multicellular spheroids as well as theodies (EBs)
were generated by self-assembly in pbb.rpose, cells cultured as
monolayers in asks were
in the case of Acta2 ES cells with 0.05% (w/v) trypsin(Gibco,
25200-056) and in the case of EGFP-expressingells with 0.25% (w/v)
trypsin-EDTA solution (Sigma-049) in order to determine the
single-cell suspensionNeubauer cell counting chamber. Different
single-cell
concentrations were prepared in the cell type specicrresponding
to the seeding density per droplet (100,
500 Acta2 ES cells per 800 nL ES medium, 200, 300 orES cells per
800 nL EB medium, 300, 500, 750 or 1000lls per 20 L EB medium, 50
EGFP-expressing HEK 293. In the case of a scaffold-based
cultivation of EGFP-HEK 293 cells 150 L of a 10 mg/mL stock
solution ofe (PLL)-coated glass beads (Section 2.4) with an aver-er
of about 100 m were added to the cell suspensionvolume) in order to
generate a single bead per droplet.ent solutions were used in the
basic setup of the mod-atform (Section 3.1) in order to generate
droplets fore or scaffold-based 3D cell culture (Sections 3.2
andndamental process steps of the generation of droplets
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ARTICLE IN PRESSG ModelBIOTEC 6977 110K. Lemke et al. / Journal
of Biotechnology xxx (2015) xxxxxx 3
Fig. 1. Photog e-basediameter of 1 m system660970 nL; (C two
by means omodied tw
2.4. Poly-l-
The PLL-performed G4519, 5.96The beads (RT) in the the PLL
HEPstep, the glto enable anphosphate-
2.5. Modula
The pbbtem based ouid peruwhich was the transpoThe dropletsion
as wellThere was order to guaprotocols. Ioxygen (DOwith air
enrerated and was equippand reproduhave to havrequiremenwhich
wereand incubatone hand, aumes rangivolumes ranally
connecAdditionallthe gas exch37 C in a htion of micrscalability
ocultivation droplet genanalysis en(SOPs) (Seccells could b
yger was0 (Wduleinat
nerabased
the goduleere c
pum drop. The
b-plang the, a cmmeted tee tion 2008d its 2008sensL. T
ry w locatric
tatin00 rrier .3 mmted.therpillaThe
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230raphic images of the different droplet generation and storage
modules of the pipm as storage and incubation module with about 800
nL droplets; (B) a uid micro) the two-capillary probe for droplet
generation of 800 nL to 2 L; (D) the modied
f a uidic microsystem, the two-capillary probe or theo-capillary
probe are described in Section 2.6 in detail.
lysine (PLL)-coating of glass beads
coating of the glass beads (P2636, Sigma-Aldrich) waswith a 10%
(w/v) PLL HEPES/NaCl buffer, pH 7.4 (PLL,
g/l HEPES, H4035, 8 g/l NaCl, S5886, Sigma-Aldrich).rstly were
swollen for 30 min at room temperatureHEPES/NaCl buffer without PLL
and then incubated inES/NaCl buffer for further 20 min. During this
coating
ass beads were periodically slightly dispersed in order even
coating. Afterwards, the beads were washed withbuffered saline.
r pbb-platform characterization
-platform was developed as a closed microuidic sys-n aqueous
droplets embedded in the water-immiscibleorodecalin (PFD, A18288,
Alfa Aesar GmbH & Co. KG),proofed to be biocompatible and is
commonly used forrt of xenografts (Brandhorst et al., 2008; Lowe,
1999).s were used as bioreactors for cell culture in suspen-
as for scaffold-free and scaffold-based 3D cell culture.no
addition of surfactants for droplet stabilization inrantee the
compatibility to established cell cultivation
n order to maintain a sufcient supply with dissolved) during
long-term cultivation, the PFD was aeratediched with 5% CO2 before
usage. The droplets were gen-transported by a syringe pump (cetoni
GmbH), whiched with glass syringes (ILS GmbH). To guarantee
stablecible uid manipulation conditions, the microchannelse
hydrophobic surfaces. A simple way to realize thesets is to use
PTFE-tubings (Jasco Deutschland GmbH),
applied with typical lengths of up to 3 m as storageion modules
for highly parallelized approaches, on thend with different
diameters, e.g. 1.0 mm for droplet vol-ng from 800 nL to 2 L (Fig.
1a) and 1.6 mm for droplet
(Fritz Gof 6 bater S-1the mocontam
2.6. Geprobe-
Fortion mThey wsyringeeratedstoragethe pb
Usimoduland cointegraguarangeneraet al., tion anet al.
(stress-of 1.9 mcapillatricallyan electhe rosion (1the ca(i =
0genera
Anotwo-caother. e this article in press as: Lemke, K., et al., A
modular segmented ow-doi.org/10.1016/j.jbiotec.2014.11.040
ging from 2 to 20 L, respectively. They were occasion-ted and
disconnected to the uid management module.y, the gas permeability
of the tubing wall guaranteedange maintaining the pH and DO during
incubation at
umidied 5% CO2-containing atmosphere. The applica-ochannels with
different inner diameters enabled thef the droplets and thereby the
scalability of the cellprocesses. The integration of functional
modules foreration (Fig. 1bd) and manipulation and
non-invasiveabled, e.g. automated standard operation procedurestion
3.1). Furthermore, the microenvironment of thee precisely composed
by using a microvalve SMLD 300
tem GmbHoccasionallprobe was While the IQ, Qinstruwas
pumpesuspensionillary (180 embedded with a lengtof this coil
wincubated ad bioreactor (pbb)-platform: (A) the PTFE-tubing coil
with an inner with milled hydrochannels microchannels for droplet
generation of
capillary probe for droplet generation of 220 L (scale bars 20
mm).
r AG, Switzerland) for agent addition. Here, a pressure applied,
which was controlled by a pressure transmit-IKA Alexander Wiegand
SE & Co. KG). All materials ofs could be sterilized by an
autoclave and neither cross-ion nor evaporation was observed.
tion of droplets by means of chip-based or droplet generation
modules of the pbb-platform
eneration of droplets, different kinds of droplet genera-s were
developed regarding to its usage and scalability.onnected to a uid
management module consisting of ap with two independently working
syringes. The gen-lets were pumped into a PTFE-tubing for
incubation orse three kinds of modules represent the basic set-up
oftform (see details in Section 3.1).e uidic microsystem (Fig. 1b)
as droplet generationell mixing module, developed as minispinner by
ibarcially available by cetoni GmbH, Germany, had to bedirectly in
front of the uidic microsystem in order toa homogeneous single-cell
suspension and thereby aof homogeneous cell suspension droplets
(Schumacher; Schemberg et al., 2009). All details of the
construc-functionality were published previously by Schumacher).
Briey, the minispinner is used for mixing of shearitive cell
suspensions and consists of a working volumehe agitator is attached
to the top and is made of a PTFEith an attached permanent magnet at
its end. An eccen-ted permanent magnet on a turning plate driven
bymotor enables the rotation of the agitator because ofg magnetic
eld. By pumping the mixed cell suspen-L/min) into the main
microchannel (i = 1.0 mm) anduid PFD (500 L/min) into the other
microchannel) of a T-junction chip droplets of about 830 nL
were
kind of droplet generation module is probe-based. Thery probe
consists of two capillaries, which sticks in eachprobe was mounted
on a 10 mL vessel (Schrfe Sys-
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266platform for 3D cell cultivation. J. Biotechnol. (2015),
) lled with 12 mL of a single-cell suspension (andy the
PLL-coated glass beads, Fig. 1c). The setup of thedescribed
previously in detail (Schemberg et al., 2009).cell suspension was
mixed with a shaker (BioShakements GmbH) by 300 rpm at RT, the
carrier uid PFDd into the outer capillary (148 L/min) and the
cell
together with the PFD were drawn into the inner cap-L/min).
Thereby about 850 nL cell suspension droplets
in PFD were generated and drawn into a PTFE tubing coilh of 2 m
and a 1.0 mm inner diameter (Fig. 1a). Both endsere sealed with
prepared cannulas or ttings and weret 37 C in a humidied 5%
CO2-containing atmosphere.
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ARTICLE IN PRESSG ModelBIOTEC 6977 1104 K. Lemke et al. /
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In order to generate droplets with a volume of 2 L up to 20 L,
asecond droplet generation system based on the principle of the
two-capillary probe, which is called the modied two-capillary
probe,was developed (Fig. 1d). For this purpose, a PTFE-tubing with
aninner diameter of 1.6 mm and a sucking ow rate of 600 L/minwas
used while the pumping ow rate of PFD was 600 L/min,which
wasgenerate drcell suspencapillary pr450 rpm.
2.7. Live/de
The celmetabolic awith enzympropidiumidescribed (Eof
dropletswith the c48-well plaaqueous sothe life/deaSigma-Aldrcent
images(Olympus microscopethe sedimenImage J softmeasuremecolor
channmanually mwashing stestructures, Therefore, twere
subtraculation of tof the red aviability in pa spread sh
2.8. In situ
Bright-spheroids wthe digital cto the IX50land GmbHthe
cross-seusing the xland GmbHshaped likedepend onerror
associncreasing respectively
In orderEGFP-exprean invertedMT20 (Olymarc burner
photodiodeguaranteed
2.9. Online photometric measurements
Based on a photoelectric barrier consisting of a
light-emittingdiode (520 nm) and a photodiode (3801100 nm, Srel(520
nm) = 48%)(i) the exacthe EB size f
dete size.sitio
ults
esignlatfo
piped owter-
ctors2007ed foor thd madepedrope (Figding
waspilla
uidinnen dro
2.6 mme of tplet ere de ogate
scaly mge onera
to 2lized.
gener diath an
of thulatients
coultails ct mpletine mh thestagee ogion
in SeFig. 2modtion
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340e this article in press as: Lemke, K., et al., A modular
segmented doi.org/10.1016/j.jbiotec.2014.11.040
stopped automatically every 4 s for 2.2 s in order tooplets. In
each of these intervals, a 20 L droplet ofsion was pulled into the
tubing. The modied two-obe containing the cell suspension was
rocked with
ad cell staining
l viability based on the membrane integrity andctivity was
assessed using a double uorescent staininge substrate uorescein
diacetate (FDA) and DNA-dye
odide (PI) instead of ethidium bromide as previouslyhrhart et
al., 2009). For this purpose, a dened number
containing 3D cell structures were pumped togetherarrier uid out
of the PTFE tubing into a well of ate, where the carrier uid was
separated from thelution by aspiration. The droplets were mixed 1:1
withd staining solution containing 8 g/mL FDA (F2756,ich) and 20
g/mL PI (P4170, Sigma-Aldrich). Fluores-
were obtained using the triple band lter U-N61000v2Deutschland
GmbH) and the inverted uorescence
IX50 (Olympus Deutschland GmbH). These images ofted 3D cell
structures were analyzed on one z-level by
ware (NIH, Bethesda, MD, USA). Therefore, only relativents were
performed. The images were split into threeels: red, green and
blue. The 3D cell structures werearked in the green and the red
channel. As no furtherp was performed in order to avoid damage of
3D cell
the background of the images were not perfectly black.he mean of
red and green intensities of cell-free areacted from those of the
3D cell structures. Then the cal-he gray values between the green
channel and the sumnd green channel were made. Based on this
result, theercent was calculated, and this was commonly done in
eet program.
imaging and microscopic measurements
eld or uorescent images of the embryoid bodies orere obtained by
imaging through the tubing wall withamera XC10 (Olympus Deutschland
GmbH) connected
inverted uorescence microscope (Olympus Deutsch-). The spheroid
size was determined by measuringctional area (in m2) on one z-level
of each spheroidcellence image analysis software (Olympus
Deutsch-). As the embryoid bodies or spheroids are not
perfectly
a sphere, the measured areas of their cross-sections their
orientation inside the droplet. However, theiated with these
different orientations decreases withnumber of measured embryoid
bodies and spheroids,.
to determine the cell proliferation of the transfectedssing HEK
293 during 8 days by uorescent intensity
microscope equipped with the illumination systempus Deutschland
GmbH) was used. The MT20 xenon
was controlled by an electronic feedback loop and a, so that a
light intensity with minimal uctuations was.
ricallythe EBThe po
3. Res
3.1. D(pbb)-p
Thementein a wabioreaet al., was ustion. F(i) uitwo inbased
modulDepenerationtwo-ca
TheminisppensioSection0.11.6the sizfor droture wand
thinvesti
Theactor bthe usaone gefrom 2are reaand d)
Thean innand wilengthmanipadd agwhichsee deA correthe dro
Onlthrougscope and ththe redetailsform (
Its applicaplatform for 3D cell cultivation. J. Biotechnol.
(2015),
t position of a droplet for droplet manipulation and (ii)or
monitoring the cell proliferation could be photomet-cted. The
strength of the electric signal correlated with
Both diodes were arranged gapless at the PTFE tubing.n of this
analysis module was exible.
and discussion
and scalability of the modular pipe based bioreactorsrm
-based bioreactors (pbb)-platform is based on the seg-
technology. Using this technology, droplets embedded
immiscible carrier uid can be applied as micro-scaled for
different applications in life sciences (e.g. Funfak;
Clausell-Tormos et al., 2008). Here the pbb-platformr the
scaffold-based and scaffold-free 3D cell cultiva-ese applications,
the basic setup of pbb consisted of anagement module including a
syringe pump drivingndently working syringes, (ii) a chip-based or
probe-let generation module and (iii) a storage and incubation. 2a,
b and e; Gastrock et al., 2009; Lemke et al., 2008).on the cell
culture and the application, the droplet gen-
performed by means of the uidic microsystem or thery probe (Fig.
1bd, see details in Sections 3.2 and 3.3).ic microsystem was always
used in connection with ther to guarantee the generation of
homogenous cell sus-plets (Schumacher et al., 2008; Schemberg et
al., 2009;). It is based on milled hydrophobic microchannels of
inner diameter. Regarding to microchannel diameter,he droplets
varies. The simplest channel congurationgeneration is the
T-junction. Construction and manufac-one by iba. The inuence of the
channel constructionw rate on the accuracy of the droplet
generation wasd in detail and will be published separately.ability
of the droplets and therefore the size of the biore-eans of
probe-based generation of droplets depend onf the two-capillary
probe or the modied one. The rsttes droplets from 800 nL to 2 L and
the second one0 L. The homogeneity of the cell suspension
droplets
in both cases by mounting the probe on a shaker (Fig. 1c
erated droplets were drawn into a PTFE tubing coil withmeter of
1.0 mm for droplets of 800 nL to 2 L (Fig. 1a)
inner diameter of 1.6 mm for droplets of 220 L. Thee tubing did
not exceed 5 m in order to enable easy
on of each droplet, which is necessary if one want toor medium
to a droplet by means of a dosage module,d be easily integrated
into the pbb-platform (Fig. 2d;in Section 3.4 and supplementary
material Section 2.2).anipulation could be enabled by the
determination ofs position based on a photoelectric
barrier.onitoring could be performed easily by microscope
tubing wall while the tubing was xed at the micro- (Fig. 2c).
The droplets were guided through the tubingw was periodically
stopped when one droplet reachedof observation. Further online
analysis modules (seection 3.5) can be occasionally integrated into
the plat-).ularity allows the platform to be adapted to specics and
SOPs, respectively. The uidic interfaces are
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ARTICLE IN PRESSG ModelBIOTEC 6977 110K. Lemke et al. / Journal
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Fig. 2. Scheme (C) an
unied regtheir surface.g. the storthe tubingsout danger the
pbb-plawithout nee
3.2. Scaffold
Scaffold-able on the (i) cell-seedon the multhydrogel, fo2012).
Addin individuto be perfotion system(Wiedemeie.g. microcdensity of
the dropletplarily perfa homogenrier inside wPLL-coated (Section
2.3beads, 9.1%in the suppbution of tha mean of 1inuenced
f theuses
samd-baP-exead
pumL/mated ixedreac
1).
affol EB
impdy foes. Temicnt (Blled
nL dly wrderedinge wees inPTFE
Fig. 3. EB formincubation and
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429 of the modular pbb-platform: (A) uid management module, (B)
storage module,
arding their geometry (diameter, seal tightness) ande properties
(surface energy). After processing steps,age module, can be easily
disconnected and sealed at ends before it can be transported to the
incubator with-of contamination. Among others, the modularity
makestform appropriate for high(er)-throughput applicationsd of
pipette robots.
-based 3D cell culture in high(er) throughput
based 3D cell culture systems, which are already avail-market,
can be divided into two different technologies:ing on an acellular
3D matrix, which is commonly basedi-well plate system, and (ii)
dispersion of cells in a liquidllowed by polymerization (Rimann and
Graf-Hausner,itionally, there are further custom-made
approachesally shaped chips, which partially have the advantagermed
in perfusion (Wu et al., 2011). The pbb cultiva-
can be performed using the dispersion in a hydrogeler et al.,
2011) as well as using acellular matrices like,arriers, whose
density should be mostly equal to thewater in order to prevent
rapid sedimentation. Here
generation with PLL-coated glass beads was exem-ormed. In order
to enable a simplied monitoring andous cell proliferation droplets
having one microcar-ere intended to generate. When approximately
2750
glass beads/mL were used for the droplet generation), 41.3% of
the droplets contained a single bead, 47.0% no
head oand ca
Thescaffolof EGFglass busing aof 60 PLL-cowere mger
bioSection
3.3. Scuniform
Oneoid bocell typand chronmecontroin 800assemb
In ocell sevolumdensitiwith a e this article in press as: Lemke,
K., et al., A modular segmented
ow-doi.org/10.1016/j.jbiotec.2014.11.040
two beads, and 4.6% three beads (Fig. S13A, see detailslementary
material Section 1). The mean of the distri-e beads per droplet was
determined with 0.7, although.1 was preliminarily calculated.
Obviously, the beads
the uid ow, which is usually generated at the probe
dent of the ustandard dthe cell proin ES mediudroplets, w
ation of Acta2 mESCs by self-assembly in an about 800 nL droplet
of pbb within 1 day: 4 the already formed EB after 24 h (from left
to right, scale bars 500 m).alysis module, (D) dosage module and
(E) droplet generation module.
two-capillary probe during the droplet generation step,the lower
bead distribution.e PLL-coated glass beads were here exemplarily
used forsed cell cultivation in pbb generating 400 nL
dropletspressing HEK 293 cells together with one PLL-coatedin a
PTFE tubing with an inner diameter of 0.75 mm,ping ow rate of 50
L/min, and a drawing ow rate
in. The EGFP-expressing HEK 293 cells attached to theglass beads
within 1 day like they usually do, when they
together in a batch approach for further usage in a big-tor
(Fig. S13B, see details in the supplementary material
d-free 3D cell cultivation in high(er) throughput:formation and
cell proliferation
ortant 3D cell culture model system is the embry-rmation of ESCs
in order to differentiate into speciche ESC differentiation is
inuenced by many physicalal parameters including the extracellular
microenvi-ratt-Leal et al., 2009). One rst essential step is
theuniformly sized EB formation. Using 400 Acta2 ESCsroplets of pbb
one EB per droplet was formed by self-ithin one day (Fig. 3).
to generate uniformly sized EBs, the inuence of the density, the
medium composition and/or the dropletre investigated. For this
purpose, different cell seeding
ES and EB medium were tested within 800 nL droplets-tubing of an
inner diameter of 1.0 mm (Fig. 4). Indepen-
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456platform for 3D cell cultivation. J. Biotechnol. (2015),
sed medium as well as of the cell seeding density,
smalleviations of the EB sizes were determined. Regardingliferation
behavior, the optimized cell seeding densitym seems to be between
100 and 200 mESCs per 800 nLhile in the EB medium the optimized
cell seeding density
00 Acta2 mESCs per EB medium droplet at the beginning, after 6 h
of
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ARTICLE IN PRESSG ModelBIOTEC 6977 1106 K. Lemke et al. /
Journal of Biotechnology xxx (2015) xxxxxx
Fig. 4. Inuence of the cell seeding density and the medium
composition on EBformation and cell proliferation in 800 nL
droplets of pbb: the EB diameter of cellseeding densities of 100
ESCs, 200 ESCs, 300 ESCs and 500 ESCs in ES medium, and200 ESCs,
400 ESCs and 500 ESCs in EB medium was determined. For each data
point,10 droplets were monitored.
was estimated between 200 and 300 mESCs per 800 nL
droplets.However, the big difference of estimated EB diameter of
500 cellsin ES medium in comparison to 500 cells in EB medium after
1 daycould be a hint that this cell density together with the
medium com-position maybe resulted in a spatial high content of
growth factorswhich further stimulates the proliferation
immediately after the EBformation. But as the EB diameter
determination only estimates thecell proliferthat time, nwere
perfoones at theformation imonitored bsectional ar
Monitordroplets inindependen
Fig. 5. Inuence of the cell seeding density on EB formation and
cell proliferationin 20 L droplets of pbb: the EB diameter of cell
seeding densities of 300 ESCs, 500ESCs and 1000 ESCs in EB medium
was determined. Independent of the cell seedingdensity, one major
and up to a few minor EBs per droplet were formed during therst 2
days. Therefore, here the EB diameter of the major EB was
integrated. For eachdata point, 10 droplets were monitored.
cells per 20 L during the rst initial days one major and up to
afew minor EBs were formed. These EBs fused to one EB on day
3,which further proliferated till day 8, the end of the monitoring
time(Fig. 5). Therefore, the 800 nL droplet volume seems to support
theEB formation. The advantage of the 20 L droplet cell
cultivationwas the determined ongoing proliferation till day 8. But
as analy-ses of nutrients and metabolites were not performed, no
precise
tion regarding the end of proliferation could be made. Ased, ol
viabf prourthl proleprem dr
by t EBs
Fig. 6. Live/deusing cell seedcell seeding de
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486ation this needs further investigation. Additionally, ato
analyses of nutrients or toxic metabolites like lactatermed as
these parameters should not be the limiting
time of EB formation. Therefore, in all cases, the EBn 800 nL
droplets was followed by a cell proliferationy the increase of the
EB diameter as well as their cross-ea (Section 2.8).ing the EB
formation and proliferation of ESCs in 20 L
a PTFE-tubing of 1.6 mm the rst detection was thatt of the
tested cell seeding densities from 300 to 1000
predicexpectthe celdays o
To fthe celusing rmediuformedformede this article in press as:
Lemke, K., et al., A modular segmented
ow-doi.org/10.1016/j.jbiotec.2014.11.040
ad staining of EBs generated by means of the modied
two-capillary probe: (A) using celling density of 300 Acta2 mESCs
per 20 L droplet after 8 days, (C) using cell seeding dennsity of
1000 Acta2 mESCs per 20 L droplet after 8 days (upper panels: FDA
staining imnly few dead cells could be detected by determiningility
using live/dead staining (Section 2.7) even after 8
liferation (Fig. 6).er characterize the pbb-platform, the EB
formation andiferation were compared with the hanging drop
methodsentatively for the pbb-platform 500 ESCs in 800 nL EBoplets
and 500 ESCs in 20 L EB medium droplets per-he hanging drop method.
After one day, both techniques
with equal size corresponding to the EB diameter as
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495platform for 3D cell cultivation. J. Biotechnol. (2015),
seeding density of 300 Acta2 mESCs per 20 L droplet after 1 day,
(B)sity of 1000 Acta2 mESCs per 20 L droplet after 1 day and (D)
usingages, lower panels: PI staining images, scale bars 200 m).
-
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ARTICLE IN PRESSG ModelBIOTEC 6977 110K. Lemke et al. / Journal
of Biotechnology xxx (2015) xxxxxx 7
Fig. 7. Compawith 20 L dro(on the right-hFor each data p
well as to tfollowing dthan the EBinsufcientmaintenanc
The abilcell cultureexamples shactors. Howwall, that habetween
thexchange ocultivations
3.4. Inuen
There iseration in ppbb-platforto the cell pcell cultureplatform
sediscrepancypbb-platforformed in aatmospherepermeabilitgen
permeguarantee aproofed by Acta2 mESCbetween thover a timedetail
toget2.1. The DO96 to 89% acontent of tproliferatiouid was dcontent
of signicantly
As the Dtion, differedeveloped (the 800 nL
xampfor thsion p
excha
e, ea 800
modolutts, a g Acn of
thison thm (d. S1/min00 ge p
by .o DOossition ontr
little dosages following by a fast mixing, in order to avoid
hotithin one droplet, which could be toxic for the cells. Mixingcy
depends on the magnitude of the dosage momentum andition of the
droplet toward the by-channel during the dosage
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562rison of EB formation and cell proliferation in 800 nL
droplets (pbb)plets (hanging drop method) with 500 ESCs regarding
to EB diameterand side) and cross-sectional area of the EB (on the
left-hand side).oint, 10 EBs were monitored.
he cross-sectional area of the EB. However, during theay, the
EBs in the pbb-platform proliferated much lesss in the hanging
drops (Fig. 7). The reasons could be an
supply of nutrients, oxygen or carbon dioxide for pHe or an
accumulation of toxic metabolites.ity to cultivate both,
scaffold-free and scaffold-baseds, opens up a broad range of
applications. The describedowed that cells proliferate inside the
pipe-based biore-ever, there are parameters, e.g. thickness of the
tubingve to be optimized in order to improve the gas exchangee
bioreactors and the environment. Furthermore, thef consumed culture
medium is necessary for long-term
(see detail in Section 3.4).
ces on cell proliferation in pbb
a range of parameters that inuence the cell prolif-bb. During
the development and investigation of them, the most important ones
were identied. Comparedroliferation in hanging drops having a
volume of 20 L, the cell proliferation in 800 nL droplets of the
pbb-emed to be slower. To investigate the reasons for this, the DO
and/or the nutrient supply in droplets of them were estimated. As
the cultivation of pbb was per-n incubator at 37 C in a humidied 5%
CO2-containing, the DO and the pH should be maintained by the gasy
of the PTFE-tubing wall (BOLA GmbH, 2014). The oxy-ability of the
tubing wall should be high enough to
Fig. 8. Emodule of the fumedium
modulsistingdosagesitive sdropleby usinadditioDuringacting
mediu2.2, Fig400 Lwith 3exchanitationlactate
As nply is pcultiva
In cenablespots wefcienthe pose this article in press as: Lemke,
K., et al., A modular segmented
ow-doi.org/10.1016/j.jbiotec.2014.11.040
sufcient DO supply for cell proliferation. This wasdetermining
the DO of 20 L droplets containing 500s (ve droplets each day) and
the separation uid in
e droplets by means of DO microsensor (PreSens GmbH) period of 6
days. The measurements are described inher with Fig. S14 in the
supplemental material Section
content of the droplets decreased after one day froms a result
of cell proliferation. In the same time the DOhe separation uid did
not change. After 3 days of celln, the DO content of the droplets
and the separationetermined about 91%. Even till the sixth day, the
DOthe droplets and the separation uid did not change. Therefore, no
DO limitation could be detected.O value was not the reason for slow
cell prolifera-nt kinds of dosage modules for nutrient supply
wereFig. 8), in order to enable a comparable proliferation
indroplets as in the 20 L droplets. Using the T-junction
momentumthe applicastill incommoved abo
The applmodule endroplets, onother handput, which with 20 L
3.5. Online
Althougusage of 10not necessadroplets foles of dosage module,
here a schematic view: (A) the droplet fusione dosage of shear
stress-sensitive cell suspensions, (B) detailed viewrocess, (C) the
medium exchange module, (D) detailed view of thenge process.
ch 20 L medium droplet was fused with an EB con- nL droplet for
nutrient supply (Fig. 8a). This kind ofule can be used for the
dosage of any shear stress sen-
ion. To further elongate the cell proliferation in 20 Lnew
developed medium exchange module was testedta2 EBs (Fig. 8b). The
aspiration of used medium and thefresh medium did not affect the EB
inside the droplets.
automated process step of aspiration, the gravity forcee EB
exceeded the force caused by the velocity of theata are presented
in supplementary material Section5). The transport of the droplets
was performed with, whereas the 12 L of the used medium was
aspiratedL/min. The integration of a dosage module for
mediumrolonged the cell cultivation and guaranteed no lim-nutrient
supply or inhibition by waste products like
limitation was determined and a further nutrient sup-ble, the
pbb-platform has the potential for a long-termsystem.ast to a
medium exchange, the dosage of drugs has to
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558platform for 3D cell cultivation. J. Biotechnol. (2015),
(Fig. 9), respectively. As shown in Fig. 9D, 520 ms aftertion of
the dosage momentum, the droplet mixing wasplete. The mixing was
nished after the droplet hadut 40 mm inside the
microchannel.ication of the medium exchange module and the
dosageables long-term cultivation of cell cultures in 20 L
the one hand, and drug screening processes, on the. The
automated procedure allows a high(er) through-is important for
scaling up by fusion of 800 nL dropletsdroplets and for numbering
up for statistical evaluation.
monitoring in pbb
h pbb works in high(er) throughput and therefore the or 20
droplets for an endpoint detection analysis willrily quit the
experiment as there will still remain manyr further determinations,
an online monitoring as a
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ARTICLE IN PRESSG ModelBIOTEC 6977 1108 K. Lemke et al. /
Journal of Biotechnology xxx (2015) xxxxxx
Fig. 9. Dosage H 0.1 (A) 0 ms, drop ocedubars 2 mm). Lo
Fig. 10. Photoand the size of
usually nonsophisticateand a microysis modulemodule, eitulation
likestrength of as the wavchanging ththe module
The micring wall. Inof the 3D cbe equippethe light inminimize
thHEK 293 cmicrocarriemanually inimal cross-adherent grThe
increasthe increasdifferent oron microcaments.
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603 module for drug screening: dosing of bromophenol blue
solution (2%, w/v, in NAOlet enters the module, (B) 240 ms, droplet
reaches dosing spot, (C) 280 ms, dosage prwer panels show the
enlarged framed sections of the corresponding images AD.e this
article in press as: Lemke, K., et al., A modular segmented
ow-doi.org/10.1016/j.jbiotec.2014.11.040
metric analysis module: (A) schematic view of the module, (B, C)
the electric signal iden the droplet.
-invasive and a fast analysis method is the much mored variant
of analysis. Therefore, both, a photometricscopic analysis, were
enabled. The photometric anal-
is based on a photoelectric barrier (Fig. 10). Using thisher the
position of a droplet, e.g. for its further manip-
droplet fusion or the EB size, which correlated to thethe
signal, were detected. This module is cost-effectiveelength could
be easily adapted to the application bye LED and the photo diode.
Additionally, the position of
at the tubing is exible.oscopic detection is easily performed
through the tub-
order to enable measuring of uorescent intensityell structure,
the utilized inverted microscope has tod with the illumination
system MT20 (Section 2.8). Astensity of the MT20 system was
controlled in order toe uctuation, the cell proliferation of
EGFP-expressing
ells as spheroids or as adherent growing cells on ar could be
measured (Fig. 11). Focalizing was realized
such a way to detect simultaneously both, the max-section area
and the periphery of the spheroid or theowing cells on a
microcarrier as contrast as possible.e of the uorescent intensity
could be correlated toe of the cross-sectional area. The error
associated withientations of spheroids or the adherent growing
cellsrriers decreased with increasing number of measure-
3.6. Integrainto cardiom
The ESCEB formatidescribed (formation o
Fig. 11. Measspheroids andsoftware via c400 nL dropletpension of
1.2per 400 nL drobeads were admol/L) into a DMEM droplet, running
with a ow rate of 250 L/min,re, (D) 800 ms, passive mixing of the
droplet is still incomplete (scaleplatform for 3D cell cultivation.
J. Biotechnol. (2015),
ties the size of the EB, after (B) 45 h and (C) 141 h
cultivation time,
tion of the pbb-platform into the mESC
differentiationyocytes
s differentiation into cardiomyocytes starting with theon by
self-assembly using hanging droplets is wellHescheler et al., 1997;
Potta et al., 2010). As the EBf 500 Acta2 mESCs in 800 nL droplets
was yielded in
urement of cell proliferation of EGFP-expressing HEK 293 cells
as as adherent growing cells on PLL-coated glass beads by
microscopicontinuous increase in uorescence intensity and uorescent
area.s were generated by means of a two-capillary probe from a cell
sus-5 105 cells/mL corresponding to a cell seeding density of 50
cellsplet. In the case of scaffold-based cultivation 1.5 mg,
PLL-coated glassded to 1 mL cell suspension (Section 2.3).
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ARTICLE IN PRESSG ModelBIOTEC 6977 110K. Lemke et al. / Journal
of Biotechnology xxx (2015) xxxxxx 9
Fig. 12. Schem ifferewas indicated
uniformly sventional pThe further56 days pefor 3 days
auorescenttiated cardidays were pdifferentiatthe presenc(Section
2.2differentiatformation wseeding dencultivation diomyocytethe
elongatof the cell ferentiationin pbb indidue to the ndroplets waDO
measurmeasuremenot possibleis one of thdifferentiat(Kimura ancould
be anthe 800 nL sized EB fo800 nL dropdroplets, suinto cardiom
This exaple for the rcell differen2010, Sectiodroplets
(Sesuspensionby means oif it will be
4), (ieadsectioally lls bharacn 3.4
clus
mad wiws: (lifercell crdiomcell-dinueler on of
formroug
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651e of the successful integration of the pbb-based EB formation
step into the mESC dby the presence of EGFP-expressing beating cell
clusters within a treated EB.
ized EBs this process step was integrated into the con-rocedure
of mESC differentiation into cardiomyocytes.
process steps like transfer into a rocked Petri dish forrforming
EB cultivation in suspension, the EB outgrowthfter the transfer to
gelatinized cell ask (beating and
cell cluster), and at last the enrichment of the
differen-omyocytes by the addition of puromycin for further
5erformed in the conventional way. The successful mESCion into
cardiomyocytes was indicated and detected bye of EGFP-expressing
beating areas within treated EBs). As shown in the scheme in Fig.
12, the mESC Acta2ion into cardiomyocytes was successful when the
EBas performed in 800 nL droplets in pbb with a cellsity of 500
ESCs. Even without the conventional EB
in suspension, the mESCs were differentiated into car-s after an
EB formation step for 2 days (Fig. 12). Neitherion of the EB
formation step in pbb nor the variationseeding density yielded into
a successful mESCs dif-. The failure in differentiation after 8
days cultivationcated that the cells lost their differentiation
capacityon-optimal conditions. A high DO content of the 20 Ls
determined during 6 days of cultivation in pbb. So far
and 3.glass btion (Sprincipated cehere c(Sectio
4. Con
Tworealizeas follocell proon 3D into caof ES which
(Heschentiati
Thehigh the this article in press as: Lemke, K., et al., A
modular segmented ow-doi.org/10.1016/j.jbiotec.2014.11.040
ements in the 800 nL droplets were not performed as ant with the
microsensor based on an optical ber was. As it is known that the
low-oxygen tension (hypoxic)
e regulatory signals for maintenance, proliferation andion of
several stem cells, e.g. hematopoietic stem cellsd Sadek, 2012),
the detected failure in differentiation
additional and indirect proof of the high DO content indroplets
over several days. Nevertheless, an uniformlyrmation within one day
by means of self-assembly inlets, which will remain no longer than
2 days in 800 nLpport or at least not inhibit the mESC
differentiationyocytes.
mple proves that the pbb-platform is qualied in
princi-ealization of the individual major process steps of
stemtiation based on the hanging drop method (Potta et al.,n 2.2):
(i) formation of uniformly sized EBs in 800 nLctions 3.3 and 3.6),
(ii) cultivation for further 5 days in
by means of EB transfer into a 20 L medium dropletf a dosage
module combined with a medium exchange
necessary to guarantee cell proliferation (Sections 3.3
proliferatiodays has becells could be reducedlimiting
faccultivation of these twsteps. Thereliferation ststep by trane.g.
on gelation on beato be possiba magneticfactors or aonly the cothe
standarEB formatiothe targeteding a hypoxntiation: successful Acta2
mESCs differentiation into cardiomyocytes
ii) outgrowth of the EBs on scaffolds (e.g. gelatinized) for
further 3 days by means of scaffold-based cultiva-n 3.2)a dosage of
the scaffold to each droplet would bepossible, too, and (iv) the
enrichment of the differenti-y pyromycin treatment realizing by the
dosage module,terized by the dosage of bromophenol blue
solution).
ions
in topics of the 3D cell cultivation, which can be wellth this
new modular cell cultivation pbb-platform, arei) ESC
differentiation based on EB formation followed byation and
differentiation, and (ii) disease research basedulture model
systems. Here, the differentiation of ESCsyocytes was exemplarily
investigated. The importanceerived cardiomyocytes and many of the
parametersence their differentiation are known since the 1990set
al., 1997). However, a robust and standardized differ-
cardiomyocytes in high throughput is still missing.ation of
uniformly sized EBs by self-assembly in pbb inhput has already been
established. Additionally, the cell
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671platform for 3D cell cultivation. J. Biotechnol. (2015),
n of EBs in 20 L droplets of pbb over a time period of 8en
presented. Using the medium exchange module, thebe supplied with
nutrients and toxic metabolites could
at any time. Therefore, these parameters need not be ator for
cell proliferation in the pbb-platform, and the cellcan be further
elongated if necessary. The functionalityo process steps has been
presented here as individualfore, a next aim has to be the
integration of a cell pro-ep on a matrix comparable to the
conventional processsferring the EBs to 0.2% gelatin-coated dishes,
in pbb,
tin-coated beads. As the handling and the cell prolifera-ds in
pbb is presented here in principle, this step seemsle, too.
Additionally, the dosage module working with
valve enables the precise addition of different growthntibiotics
for the enrichment of differentiated cells. Butmbination and
automation of these steps will enabledized generation of, e.g.
cardiomyocytes in pbb usingn as a 3D cell structure as starting
point. In addition,
integration of low-oxygen tension in pbb, e.g. mimick-ic niche
for cardiac progenitors, could enable the study
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Please cit ow-http://dx.
ARTICLE IN PRESSG ModelBIOTEC 6977 11010 K. Lemke et al. /
Journal of Biotechnology xxx (2015) xxxxxx
of its inuence on maintenance, proliferation and
differentiationof stem cells in cardiac homeostasis and
regeneration (Kimura andSadek, 2012). One condition precedent to
successful investigationsis the integration of an online DO probe
by means of, e.g. uores-cent particlthe pbb-plaseemed to r
The appmore imporTherefore, aestablishmehigh-througfor drug
sctherapeutic
Howevehanging drocation are a2011; Amancell-based afore set a
bhas advantaation, additin situ detecHowever, toeffort is nechave
to be handling of
Neverthsystems as proliferatioment and im
As singleseems to beautonomouof pbb can volume of and other ris
low becamicrochannand so theyavailable tepharmacy.
Acknowled
The authdevelopingInstitute ofe.V.
We are gogy and Patof Cologne)and introdudures.
This woribas basic rQ4LAB, grant aBioChanceP
Appendix A
Supplemfound, in th2014.11.04
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753754755e this article in press as: Lemke, K., et al., A
modular segmented doi.org/10.1016/j.jbiotec.2014.11.040
es. Another one would be the technical adaptation oftform to the
low-oxygen tension as the modules so farealize normoxic
conditions.lication of 3D cell culture models will get more
andtance in different applications like, e.g. cancer
research.nother aim will be the usage of primary cells and thent of
co-culture in droplets. As pbb is a miniaturizedhput system, which
also integrates technical modules
reening, it will be a promising tool for diagnostic and tests in
the eld of personalized medicine.r, nowadays some 3D cell culture
systems based on thep method or on chemical and nanostructural
modi-lready on the market (Spies et al., 2008; Tung et al.,n et
al., 2014). They are compatible to the establishedssays based on
the microwell plate scale and there-enchmark. Although, compared to
these systems, pbbges according to evaporation, speed of droplet
gener-ion of drugs, droplet mixing (higher throughput) andtion of
the cells inside the droplets by, e.g. microscopy.
utilize all possible functional features of pbb, technicalessary
and time-consuming. Common online analysesadapted. Furthermore, the
status quo of the manual
microuidic systems demands skill.eless, pbb could be a valuable
alternative to establishedsoon as the integration of further SOPs
for common celln and cell differentiation assays like, e.g. ATP
measure-munochemical staining, can be guaranteed.
process steps are already automated, reaching this aim possible,
so that pbb will have the potential to be ans 3D cell cultivation
system. Optional parallelizationincrease the statistical accuracy
of studies. The smallthe droplets reduces the needed amount of
mediumeagents drastically. The possibility of contaminationsuse all
the modules of the pbb-platform have closedels. Most of the modules
can be realized as disposables
are suitable as cost-effective alternative to currentlychniques
for applications in personalized medicine or
gements
ors wish to thank Katja Wlfer for her assistance on the
pbb-platform during the HYPERLAB project at the
Bioprocess- and Analytical Measurement Techniques
rateful to Prof. Dr. Agapios Sachinidis (Centre of
Physiol-hophysiology, Institute of Neurophysiology, University
for providing the murine Acta2 embryonic stem cellscing us into
their cultivation and differentiation proce-
k was supported by the State of Thuringia in the frame ofesearch
program, by the European Commission, HYPER-greement number
FP7-223011, and by the BMBF (PTJ)lus program, AirJet, grant no.
0313680.
. Supplementary data
entary data associated with this article can bee online version,
at http://dx.doi.org/10.1016/j.jbiotec.0.
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A modular segmented flow-platform for 3D cell cultivation1
Introduction2 Materials and methods2.1 Conventional cell culture of
EFG-expressing HEK 293 and undifferentiated Acta 2 murine embryonic
stem cells (mESCs)2.2 In vitro Acta2 ESC differentiation of
embryoid bodies into cardiomyocytes using the hanging drop
method2.3 Different cell seeding densities for scaffold-free and/or
scaffold-based 3D cell culture of Acta2 ESCs and EGFP-expres...2.4
Poly-l-lysine (PLL)-coating of glass beads2.5 Modular pbb-platform
characterization2.6 Generation of droplets by means of chip-based
or probe-based droplet generation modules of the pbb-platform2.7
Live/dead cell staining2.8 In situ imaging and microscopic
measurements2.9 Online photometric measurements
3 Results and discussion3.1 Design and scalability of the
modular pipe based bioreactors (pbb)-platform3.2 Scaffold-based 3D
cell culture in high(er) throughput3.3 Scaffold-free 3D cell
cultivation in high(er) throughput: uniform EB formation and cell
proliferation3.4 Influences on cell proliferation in pbb3.5 Online
monitoring in pbb3.6 Integration of the pbb-platform into the mESC
differentiation into cardiomyocytes
4 ConclusionsAcknowledgementsAppendix A Supplementary
dataReferences