3-D

Please cithttp://dx.

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

Please cit ow-http://dx.

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

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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

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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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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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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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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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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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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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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).



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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(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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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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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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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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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

References

Amann, A., Zwierzina, M., Gamerith, G., Bitsche, M., Huber, J.M., Vogel Georg, F.,Blumer, M., Koeck, S., Pechriggl, E.J., Kelm, J.M., Hilbe, W., Zwierzina, H., 2014.Development of an innovative 3D cell culture system to study tumourstroma

ractions, C.L.,Zandsegate300Gmb

kstofferst, Hdhors-termal, A.M

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W., xic m

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-C., Hsughpulyst 13eier, mermotech173.., Lin

01.ridharyoniation

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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

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