A modular segmented-flow platform for 3D cell cultivation

Journal of Biotechnology 205 (2015) 59–69

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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

A modular segmented-flow platform for 3D cell cultivation Karen Lemke a , Tobias Förster a , Robert Römer a , Mandy Quade a,b , Stefan Wiedemeier a , Andreas Grodrian a , Gunter Gastrock a,∗ a Department of Bioprocess Engineering, Institute for Bioprocessing and Analytical Measurement Techniques e.V., Rosenhof, D-37308 Heilbad Heiligenstadt, Germany b Faculty of Medicine, Technische Universität Dresden, Centre for Translational Bone, Joint and Soft Tissue Research, Fetscherstr. 74, D-01307 Dresden, Germany

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Article history: Received 25 August 2014 Received in revised form 21 November 2014 Accepted 28 November 2014 Available online 3 January 2015 Keywords: Droplets High(er) throughput Embryoid body formation Long-term 3D cell cultivation Automated modular platform

a b s t r a c t In vitro 3D cell cultivation is promised to equate tissue in vivo more realistically than 2D cell cultivation corresponding to cell–cell and cell–matrix interactions. Therefore, a scalable 3D cultivation platform was developed. This platform, called pipe-based bioreactors (pbb), is based on the segmented-flow technology: aqueous droplets are embedded in a water-immiscible carrier fluid. The droplet volumes range from 60 nL to 20 ␮L and are used as bioreactors lined up in a tubing like pearls on a string. The modular automated platform basically consists of several modules like a fluid management for a high throughput droplet generation for self-assembly or scaffold-based 3D cell cultivation, a storage module for incubation and storage, and an analysis module for monitoring cell aggregation and proliferation basing on microscopy or photometry. In this report, the self-assembly of murine embryonic stem cells (mESCs) to uniformly sized embryoid bodies (EBs), the cell proliferation, the cell viability as well as the influence on the cell differentiation to cardiomyocytes are described. The integration of a dosage module for medium exchange or agent addition will enable pbb as long-term 3D cell cultivation system for studying stem cell differentiation, e.g. cardiac myogenesis or for diagnostic and therapeutic testing in personalized medicine. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Microfluidic technology was developed at the beginning of the 1980s as a functional extension of micro-electromechanical systems technology. Nowadays, “lab-on-a-chip” (LOC) or “micrototal-analysis-systems” (␮TAS) are used to investigate the influence of biological, physical, and chemical factors for any affection of cells (Wu et al., 2011). For the investigation of stem cell differentiation and proliferation or for cancer cell research, microfluidic technologies offer the ability to precisely control the magnitudes and concentrations of these biological, physical, and chemical factors affecting the cells (Csete, 2010). However, most of the techniques used for the manufacturing of microfluidic platforms are sophisticated but often inflexible.

∗ Corresponding author. Tel.: +49 3606 671 400; fax: +49 3606 671 200. E-mail addresses: [email protected] (K. Lemke), [email protected] (T. Förster), [email protected] (R. Römer), [email protected] (M. Quade), [email protected] (S. Wiedemeier), [email protected] (A. Grodrian), [email protected] (G. Gastrock). http://dx.doi.org/10.1016/j.jbiotec.2014.11.040 0168-1656/© 2015 Elsevier B.V. All rights reserved.

Due to the preference of 3D to 2D cell culture models in, e.g. stem cell or cancer research, flexible cell cultivation systems are required. Such systems should enable reproducible 3D long-term cell cultivation in high throughput by precise definition and control of the cell microenvironment at any time, which means the biological, physical, and chemical parameters. Furthermore, it would be a great advantage to have either a scaffold-free or a scaffold-based cultivation 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-associated systems are already on the market, which initiate the generation of 3D cell structures by gravity-enforced self-assembly in hanging droplets, by enabling anchorage-free culture conditions by chemical or nanostructural modification of the cultivation surface or by providing degradable and non-degradable scaffolds. A representing overview of these commercially available 3D cell culture systems is presented 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 to the hanging droplets at any time guarantees a high flexibility to run a broad range of SOPs for cell cultivation. However, these are not

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closed systems and there will be evaporation of the cell medium. Furthermore, because of the fragility of the hanging droplets, their mixing and microscopic detection as well as the automation of such process steps are complex tasks. Cultivation systems, which enable 3D cell culture by chemical or nanostructural modification of the cultivation surface in order to realize anchorage-free culture conditions, work usually with a bigger cultivation volume and therefore have fewer problems with evaporation. As they often generate more than one 3D cell aggregate per used volume, the formation is not as uniform as by using the hanging drop method. For stem cell research, it was shown that the uniform, precise size of the embryoid bodies (EBs) is essential for the efficiency of cell differentiation (Bauwens et al., 2008; Xu et al., 2011). For the second above-mentioned topic, the cancer research, it is necessary to offer the potential for further parallelization of 3D cell culture based on primary cells of the patient in order to optimize the diagnostics and the individual therapy (Thoma et al., 2014). A segmented-flow-based technique represents an alternative approach. Droplets generated by the segmented-flow technique serve as bioreactors offering essential advantages compared to microwell-based cultivation systems. Segmented-flow-based bioreactors are embedded in a capillary system separated by a hydrophobic oil as separation and transport fluid. They have been realized for several biological models. For example, Funfak et al. (2007) cultivated zebrafish embryos inside a tubing having 1.2 mm inner diameter, and the development of the embryos could be observed over an 80 h period. Clausell-Tormos et al. (2008) developed a droplet-based platform for encapsulation and cultivation of cells and multicellular organisms like Caenorhabditis elegans in 660 nL droplets. Here, we present a segmented-flow-based pipe-based bioreactors-platform (pbb, registered mark of Institute for Bioprocessing and Analytical Measurement Techniques e.V., Reg. no. 305 04 226), which serves as long-term cultivation system for 3D cell cultures. As a closed system avoiding evaporation, it guarantees also aseptic conditions and can be applied for both, scaffold-free and scaffold-based cultivation protocols. The pbb-platform can be parallelized to increase the throughput. Functional modules allow the addition of drugs for screening procedures or the exchange of culture medium for long-term cultivations. Cell proliferation can be monitored using microfluidic modules for microscopy and spectroscopy.

(Potta et al., 2009). Briefly, the promoter region upstream of the translation initiation site of the Acta2 gene was isolated by BAC recombineering method and then subcloned into the ESC reporter construct pPuroIRES2-EGFP. This linearized construct was electroporated into CGR8 mESCs, in order to generate after neomycin selection the stable Acta2 mESC line. These undifferentiated Acta2 mES cells were cultured without feeder cells in Glasgow’s minimum essential medium (GMEM) supplemented with 10% (v/v) FBS, 2 mM l-glutamine, 50 ␮M ␤-mercaptoethanol (␤-ME), and 100 units/mL LIF, further called ES medium, in 0.2% gelatin-coated flasks as described previously (Potta et al., 2010). The cells were passed when confluence reached upon 70%. Both cell types were cultivated at 37 ◦ C in a humidified 5% CO2 containing atmosphere.

2. Materials and methods

The three-dimensional multicellular spheroids as well as the embryoid bodies (EBs) were generated by self-assembly in pbb. For this purpose, cells cultured as monolayers in flasks were trypsinized in the case of Acta2 mES cells with 0.05% (w/v) trypsin with EDTA (Gibco, 25200-056) and in the case of EGFP-expressing HEK 293 cells with 0.25% (w/v) trypsin-EDTA solution (Sigma-Aldrich, T4049) in order to determine the single-cell suspension by using a Neubauer cell counting chamber. Different single-cell suspension concentrations were prepared in the cell type specific medium corresponding to the seeding density per droplet (100, 200, 300 or 500 Acta2 mES cells per 800 nL ES medium, 200, 300 or 500 Acta2 mES cells per 800 nL EB medium, 300, 500, 750 or 1000 Acta 2 mES cells per 20 ␮L EB medium, 50 EGFP-expressing HEK 293 per 400 nL). In the case of a scaffold-based cultivation of EGFPexpressing HEK 293 cells 150 ␮L of a 10 mg/mL stock solution of poly-l-lysine (PLL)-coated glass beads (Section 2.4) with an average diameter of about 100 ␮m were added to the cell suspension (1 mL total volume) in order to generate a single bead per droplet. These different solutions were used in the basic setup of the modular pbb-platform (Section 3.1) in order to generate droplets for scaffold-free or scaffold-based 3D cell culture (Sections 3.2 and 3.3). The fundamental process steps of the generation of droplets

2.1. Conventional cell culture of EGFP-expressing HEK 293 and undifferentiated Acta 2 murine embryonic stem cells (mESCs) Human embryonal kidney (HEK) 293 cells (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures; ACC-305) were expanded in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich D5523) supplemented with 10% (v/v) fetal bovine serum (FBS, Biochrom, S0115), 2 mM l-glutamine (Sigma-Aldrich, R8758), 4.5 g/L glucose (Sigma-Aldrich, G7021) and antibiotics (100 units/mL penicillin/100 ␮g/mL streptomycin, Sigma-Aldrich, P0781). Confluent cultures were split approximately two times a week and seeded out at about 1–2 × 104 cells/cm2 . The HEK 293 cells were stably transfected with plasmid pEGFP-C1 expressing high-intensity enhanced green fluorescent proteins. Starting from 2 days after transfection the cells were always maintained in culture containing 750 ␮g/mL geneticin (G418-disulfate, Applichem, A2167). Undifferentiated Acta2 murine embryonic stem cells (mESCs) were kindly provided by Prof. Dr. A. Sachinidis. The generation of the Acta2 mESC line has been described in detail previously

2.2. In vitro Acta2 mESC differentiation of embryoid bodies into cardiomyocytes using the hanging drop method The Acta2 mESCs were differentiated in the form of threedimensional multicellular aggregates called embryoid bodies (EBs) into cardiomyocytes using the hanging drop method as per previous description by Potta et al. (2010). Briefly, upon reaching ∼70% confluence, mES cells were trypsinized and a single-cell suspension of 2.5 × 104 cells/mL was prepared in differentiation medium, called EB medium, consisting of Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 20% (v/v) FBS, 1% (v/v) non-essential amino acids, 2 mM l-glutamine, and 100 ␮M ␤-ME. ES cells were aggregated in hanging drop containing 500 cells/20 ␮L suspension on the inner surface of the lid of a 10-cm bacteriological dish, which was filled with 5 mL of sterile PBS in order to prevent the evaporation of the droplets, and placed in an incubator. After 2 days, the multicellular aggregates were transferred into a new bacteriological dish using EB medium and cultured in suspension until day 7. EBs were transferred to 0.2% (w/v) gelatin-coated dishes on day 7 and cultured for further 8 days as adherent EBs. Starting from day 10 until day 15, EBs were cultured in the same differentiation EB medium but with 5 ␮g/mL puromycin for selecting the differentiated mESCs. The enriched cardiomyocytes were indicated by the presence of EGFP-expressing beating areas within a treated EB. 2.3. Different cell seeding densities for scaffold-free and/or scaffold-based 3D cell culture of Acta2 mESCs and EGFP-expressing HEK 293 in the pipe-based bioreactors (pbb)

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Fig. 1. Photographic images of the different droplet generation and storage modules of the pipe-based bioreactors (pbb)-platform: (A) the PTFE-tubing coil with an inner diameter of 1 mm as storage and incubation module with about 800 nL droplets, (B) a fluid microsystem with milled hydrophobic microchannels for droplet generation of 660–970 nL, (C) the two-capillary probe for droplet generation of 800 nL to 2 ␮L, (D) the modified two-capillary probe for droplet generation of 2–20 ␮L (scale bars 20 mm).

by means of a fluidic microsystem, the two-capillary probe or the modified two-capillary probe are described in Section 2.6 in detail. 2.4. Poly-l-lysine (PLL)-coating of glass beads The PLL-coating of the glass beads (P2636, Sigma-Aldrich) was performed with a 10% (w/v) PLL HEPES/NaCl buffer, pH 7.4 (PLL, G4519, 5.96 g/L HEPES, H4035, 8 g/L NaCl, S5886, Sigma-Aldrich). The beads firstly were swollen for 30 min at room temperature (RT) in the HEPES/NaCl buffer without PLL and then incubated in the PLL HEPES/NaCl buffer for further 20 min. During this coating step, the glass beads were periodically slightly dispersed in order to enable an even coating. Afterwards, the beads were washed with phosphate-buffered saline. 2.5. Modular pbb-platform characterization The pbb-platform was developed as a closed microfluidic system based on aqueous droplets embedded in the water-immiscible fluid perfluorodecalin (PFD, A18288, Alfa Aesar GmbH & Co. KG), which was proofed to be biocompatible and is commonly used for the transport of xenografts (Brandhorst et al., 2008; Lowe, 1999). The droplets were used as bioreactors for cell culture in suspension as well as for scaffold-free and scaffold-based 3D cell culture. There was no addition of surfactants for droplet stabilization in order to guarantee the compatibility to established cell cultivation protocols. In order to maintain a sufficient supply with dissolved oxygen (DO) during long-term cultivation, the PFD was aerated with air enriched with 5% CO2 before usage. The droplets were generated and transported by a syringe pump (cetoni GmbH), which was equipped with glass syringes (ILS GmbH). To guarantee stable and reproducible fluid manipulation conditions, the microchannels have to have hydrophobic surfaces. A simple way to realize these requirements is to use PTFE-tubings (Jasco Deutschland GmbH), which were applied with typical lengths of up to 3 m as storage and incubation modules for highly parallelized approaches and with different inner diameters, e.g. 1.0 mm for droplet volumes ranging from 800 nL to 2 ␮L (Fig. 1A) and 1.6 mm for droplet volumes ranging from 2 to 20 ␮L, respectively. They were occasionally connected and disconnected to the fluid management module. Additionally, the gas permeability of the tubing wall guaranteed the gas exchange maintaining the pH and DO during incubation at 37 ◦ C in a humidified 5% CO2 -containing atmosphere. The application of microchannels with different inner diameters enabled the scalability of the droplets and thereby the scalability of the cell cultivation processes. The integration of functional modules for droplet generation (Fig. 1B–D) and manipulation and non-invasive analysis enabled, e.g. automated standard operation procedures (SOPs) (Section 3.1). Furthermore, the microenvironment of the

cells could be precisely composed by using a microvalve SMLD 300 (Fritz Gyger AG) for agent addition. Here, a pressure of 6 bar was applied, which was controlled by a pressure transmitter S10 (WIKA Alexander Wiegand SE & Co. KG). All materials of the modules could be sterilized by an autoclave and neither crosscontamination nor evaporation was observed. 2.6. Generation of droplets by means of chip-based or probe-based droplet generation modules of the pbb-platform For the generation of droplets, different kinds of droplet generation modules were developed regarding to their usage and scalability. They were connected to a fluid management module consisting of a syringe pump with two independently working syringes. The generated droplets were pumped into a PTFE-tubing for incubation or storage. These three kinds of modules represent the basic set-up of the pbb-platform (see details in Section 3.1). Using the fluidic microsystem (Fig. 1B) as droplet generation module, a cell mixing module, developed as “minispinner” by iba and commercially available by cetoni GmbH had to be integrated directly in front of the fluidic microsystem in order to guarantee a homogeneous single-cell suspension and thereby a generation of homogeneous cell suspension droplets (Schumacher et al., 2008; Schemberg et al., 2009). All details of the construction and its functionality were published previously by Schumacher et al. (2008). Briefly, the minispinner is used for mixing of shear stress-sensitive cell suspensions and consists of a working volume of 1.9 mL. The agitator is attached to the top and is made of a PTFE capillary with an attached permanent magnet at its end. An eccentrically located permanent magnet on a turning plate driven by an electric motor enables the rotation of the agitator because of the rotating magnetic field. By pumping the mixed cell suspension (100 ␮L/min) into the main microchannel (∅i = 1.0 mm) and the carrier fluid PFD (500 ␮L/min) into the other microchannel (∅i = 0.3 mm) of a Tjunction chip droplets of about 830 nL were generated. Another kind of droplet generation module is probe-based. The two-capillary probe consists of two capillaries, which stick in each other. The probe was mounted on a 10 mL vessel (Schärfe System GmbH) filled with 1–2 mL of a single-cell suspension (and occasionally the PLL-coated glass beads, Fig. 1C). The setup of the probe was described previously in detail (Schemberg et al., 2009). While the cell suspension was mixed with a shaker (BioShake IQ, Qinstruments GmbH) by 300 rpm at RT, the carrier fluid PFD was pumped into the outer capillary (148 ␮L/min) and the cell suspension together with the PFD were drawn into the inner capillary (180 ␮L/min). Thereby about 850 nL cell suspension droplets embedded in PFD were generated and drawn into a PTFE tubing coil with a length of 2 m and a 1.0 mm inner diameter (Fig. 1A). Both ends of this coil were sealed with prepared cannulas or fittings

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and were incubated at 37 ◦ C in a humidified 5% CO2 -containing atmosphere. In order to generate droplets with a volume of 2 ␮L up to 20 ␮L, a second droplet generation system based on the principle of the twocapillary probe, which is called the modified two-capillary probe, was developed (Fig. 1D). For this purpose, a PTFE-tubing with an inner diameter of 1.6 mm and a sucking flow rate of 600 ␮L/min was used while the pumping flow rate of PFD was 600 ␮L/min, which was stopped automatically every 4 s for 2.2 s in order to generate droplets. In each of these intervals, a 20 ␮L droplet of cell suspension was pulled into the tubing. The modified two-capillary probe containing the cell suspension was rocked with 450 rpm. 2.7. Live/dead cell staining The cell viability based on the membrane integrity and metabolic activity was assessed using a double fluorescent staining with enzyme substrate fluorescein diacetate (FDA) and DNA-dye propidiumiodide (PI) instead of ethidium bromide as previously described (Ehrhart et al., 2009). For this purpose, a defined number of droplets containing 3D cell structures were pumped together with the carrier fluid out of the PTFE tubing into a well of a 48-well plate, where the carrier fluid was separated from the aqueous solution by aspiration. The droplets were mixed 1:1 with the life/dead staining solution containing 8 ␮g/mL FDA (F2756, Sigma-Aldrich) and 20 ␮g/mL PI (P4170, Sigma-Aldrich). Fluorescent images were obtained using the triple band filter U-N61000v2 (Olympus Deutschland GmbH) and the inverted fluorescence microscope IX50 (Olympus Deutschland GmbH). These images of the sedimented 3D cell structures were analyzed on one z-level by Image J software (NIH, Bethesda, MD, USA). Therefore, only relative measurements were performed. The images were split into three color channels: red, green and blue. The 3D cell structures were manually marked in the green and the red channel. As no further washing step was performed in order to avoid damage of 3D cell structures, the background of the images were not perfectly black. Therefore, the mean of red and green intensities of cell-free area were subtracted from those of the 3D cell structures. Then the calculation of the gray values between the green channel and the sum of the red and green channel were made. Based on this result, the viability in percent was calculated, and this was commonly done in a spread sheet program. 2.8. In situ imaging and microscopic measurements Bright-field or fluorescent images of the embryoid bodies or spheroids were obtained by imaging through the tubing wall with the digital camera XC10 (Olympus Deutschland GmbH) connected to the IX50 inverted fluorescence microscope (Olympus Deutschland GmbH). The spheroid size was determined by measuring the cross-sectional area (in ␮m2 ) on one z-level of each spheroid using the xcellence image analysis software (Olympus Deutschland GmbH). As the embryoid bodies or spheroids are not perfectly shaped like a sphere, the measured areas of their cross-sections depend on their orientation inside the droplet. However, the error associated with these different orientations decreases with increasing number of measured embryoid bodies and spheroids, respectively. In order to determine the cell proliferation of the transfected EGFP-expressing HEK 293 during 8 days by fluorescent intensity an inverted microscope equipped with the illumination system MT20 (Olympus Deutschland GmbH) was used. The MT20 xenon arc burner was controlled by an electronic feedback loop and a photodiode, so that a light intensity with minimal fluctuations was guaranteed.

2.9. Online photometric measurements Based on a photoelectric barrier consisting of a light-emitting diode (520 nm) and a photodiode (380–1100 nm, Srel(520 nm) = 48%) (i) the exact position of a droplet for droplet manipulation and (ii) the EB size for monitoring the cell proliferation could be photometrically detected. The strength of the electric signal correlated with the EB size. Both diodes were arranged gapless at the PTFE tubing. The position of this analysis module was flexible.

3. Results and discussion 3.1. Design and scalability of the modular pipe based bioreactors (pbb)-platform The pipe-based bioreactors (pbb)-platform is based on the segmented-flow technology. Using this technology, droplets embedded in a water-immiscible carrier fluid can be applied as micro-scaled bioreactors for different applications in life sciences (e.g. Funfak et al., 2007; Clausell-Tormos et al., 2008). Here, the pbbplatform was used for the scaffold-based and scaffold-free 3D cell cultivation. For these applications, the basic setup of pbb consisted of a (i) fluid management module including a syringe pump driving two independently working syringes, (ii) a chip-based or probebased droplet generation module and (iii) a storage and incubation module (Fig. 2A, B and E; Gastrock et al., 2009; Lemke et al., 2008). Depending on the cell culture and the application, the droplet generation was performed by means of the fluidic microsystem or the two-capillary probe (Fig. 1B–D, see details in Sections 3.2 and 3.3). The fluidic microsystem was always used in connection with the minispinner to guarantee the generation of homogenous cell suspension droplets (Schumacher et al., 2008; Schemberg et al., 2009; Section 2.6). It is based on milled hydrophobic microchannels of 0.1–1.6 mm inner diameter. Regarding to microchannel diameter, the size of the droplets varies. The simplest channel configuration for droplet generation is the T-junction. Construction and manufacture were done by iba. The influence of the channel construction and the flow rate on the accuracy of the droplet generation was investigated in detail and will be published separately. The scalability of the droplets and therefore the size of the bioreactor by means of probe-based generation of droplets depend on the usage of the two-capillary probe or the modified one. The first one generates droplets from 800 nL to 2 ␮L and the second one from 2 to 20 ␮L. The homogeneity of the cell suspension droplets are realized in both cases by mounting the probe on a shaker (Fig. 1C and D). The generated droplets were drawn into a PTFE tubing coil with an inner diameter of 1.0 mm for droplets of 800 nL to 2 ␮L (Fig. 1A) and with an inner diameter of 1.6 mm for droplets of 2–20 ␮L. The length of the tubing did not exceed 5 m in order to enable easy manipulation of each droplet, which is necessary if one want to add agents or medium to a droplet by means of a dosage module, which could be easily integrated into the pbb-platform (Fig. 2D; see details in Section 3.4 and supplementary material Section 2.2). A correct manipulation could be enabled by the determination of the droplet’s position based on a photoelectric barrier. Online monitoring could be performed easily by microscope through the tubing wall while the tubing was fixed at the microscope stage (Fig. 2C). The droplets were guided through the tubing and the flow was periodically stopped when one droplet reached the region of observation. Further online analysis modules (see details in Section 3.5) can be occasionally integrated into the platform (Fig. 2). Its modularity allows the platform to be adapted to specific applications and SOPs, respectively. The fluidic interfaces are

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Fig. 2. Scheme of the modular pbb-platform: (A) fluid management module, (B) storage module, (C) analysis module, (D) dosage module, (E) droplet generation module.

unified regarding their geometry (diameter, seal tightness) and their surface properties (surface energy). After processing steps, e.g. the storage module, can be easily disconnected and sealed at the tubings’ ends before it can be transported to the incubator without danger of contamination. Among others, the modularity makes the pbb-platform appropriate for high(er) throughput applications without need of pipette robots. 3.2. Scaffold-based 3D cell culture in high(er) throughput Scaffold-based 3D cell culture systems, which are already available on the market, can be divided into two different technologies: (i) cell-seeding on an acellular 3D matrix, which is commonly based on the multi-well plate system, and (ii) dispersion of cells in a liquid hydrogel, followed by polymerization (Rimann and Graf-Hausner, 2012). Additionally, there are further custom-made approaches in individually shaped chips, which partially have the advantage to be performed in perfusion (Wu et al., 2011). The pbb cultivation system can be performed using the dispersion in a hydrogel (Wiedemeier et al., 2011) as well as using acellular matrices like, e.g. microcarriers, whose density should be mostly equal to the density of water in order to prevent rapid sedimentation. Here, the droplet generation with PLL-coated glass beads was exemplarily performed. In order to enable a simplified monitoring and a homogenous cell proliferation droplets having one microcarrier inside were intended to generate. When approximately 2750 PLL-coated glass beads/mL were used for the droplet generation (Section 2.3), 40.5% of the droplets contained a single bead, 46.1% no beads, 8.9% two beads, and 4.5% three beads (Fig. S13A, see details in the supplementary material Section 1). The mean of the distribution of the beads per droplet was determined with 0.7, although a mean of 1.1 was preliminarily calculated. Obviously, the beads influenced the fluid flow, which is usually generated at the probe head of the two-capillary probe during

the droplet generation step, and causes the lower bead distribution. The same PLL-coated glass beads were here exemplarily used for scaffold-based cell cultivation in pbb generating 400 nL droplets of EGFP-expressing HEK 293 cells together with one PLL-coated glass bead in a PTFE tubing with an inner diameter of 0.75 mm, using a pumping flow rate of 50 ␮L/min, and a drawing flow rate of 60 ␮L/min. The EGFP-expressing HEK 293 cells attached to the PLL-coated glass beads within 1 day like they usually do, when they were mixed together in a batch approach for further usage in a bigger bioreactor (Fig. S13B, see details in the supplementary material Section 1). 3.3. Scaffold-free 3D cell cultivation in high(er) throughput: uniform EB formation and cell proliferation One important 3D cell culture model system is the embryoid body formation of ESCs in order to differentiate into specific cell types. The ESC differentiation is influenced by many physical and chemical parameters including the extracellular microenvironment (Bratt-Leal et al., 2009). One first essential step is the controlled uniformly sized EB formation. Using 400 Acta2 mESCs in 800 nL droplets of pbb one EB per droplet was formed by selfassembly within one day (Fig. 3). In order to generate uniformly sized EBs, the influence of the cell seeding density, the medium composition and/or the droplet volume were investigated. For this purpose, different cell seeding densities in ES and EB medium were tested within 800 nL droplets with a PTFE-tubing of an inner diameter of 1.0 mm (Fig. 4). Independent of the used medium as well as of the cell seeding density, small standard deviations of the EB sizes were determined. Regarding the cell proliferation behavior, the optimized cell seeding density in ES medium seems to be between 100 and 200 mESCs per 800 nL droplets, while in the EB medium the optimized cell seeding density

Fig. 3. EB formation of Acta2 mESCs by self-assembly in an 800 nL droplet of pbb within 1 day: 400 Acta2 mESCs per EB medium droplet at the beginning, after 6 h of incubation and the already formed EB after 24 h (from left to right, scale bars 500 ␮m).

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Fig. 4. Influence of the cell seeding density and the medium composition on EB formation and cell proliferation in 800 nL droplets of pbb: the EB diameter of cell seeding densities of 100 mESCs, 200 mESCs, 300 mESCs and 500 mESCs in ES medium, and 200 mESCs, 400 mESCs and 500 mESCs in EB medium was determined. For each data point, 10 droplets were monitored.

Fig. 5. Influence of the cell seeding density on EB formation and cell proliferation in 20 ␮L droplets of pbb: the EB diameter of cell seeding densities of 300 mESCs, 500 mESCs and 1000 mESCs in EB medium was determined. Independent of the cell seeding density, one major and up to a few minor EBs per droplet were formed during the first 2 days. Therefore, here the EB diameter of the major EB was integrated. 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 cells in ES medium in comparison to 500 cells in EB medium after 1 day could be a hint that this cell density together with the medium composition maybe resulted in a spatial high content of growth factors which further stimulates the proliferation immediately after the EB formation. But as the EB diameter determination only estimates the cell proliferation this needs further investigation. Additionally, at that time, no analyses of nutrients or toxic metabolites like lactate were performed as these parameters should not be the limiting ones at the time of EB formation. Therefore, in all cases, the EB formation in 800 nL droplets was followed by a cell proliferation monitored by the increase of the EB diameter as well as their crosssectional area (Section 2.8). Monitoring the EB formation and proliferation of mESCs in 20 ␮L droplets in a PTFE-tubing of 1.6 mm inner diameter the first detection was that independent of the tested cell seeding densities from

300 to 1000 cells per 20 ␮L during the first initial days one major and up to a few 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 the EB formation. The advantage of the 20 ␮L droplet cell cultivation was the determined ongoing proliferation till day 8. But as analyses of nutrients and metabolites were not performed, no precise prediction regarding the end of proliferation could be made. As expected, only few dead cells could be detected by determining the cell viability using live/dead staining (Section 2.7) even after 8 days of proliferation (Fig. 6). To further characterize the pbb-platform, the EB formation and the cell proliferation were compared with the hanging drop method using representatively for the pbb-platform 500 mESCs in 800 nL EB medium droplets and 500 mESCs in 20 ␮L EB medium droplets performed by the hanging drop method. After one day, both techniques formed EBs with equal size corresponding to the EB diameter as

Fig. 6. Live/dead staining of EBs generated by means of the modified two-capillary probe: (A) using cell seeding density of 300 Acta2 mESCs per 20 ␮L droplet after 1 day, (B) using cell seeding density of 300 Acta2 mESCs per 20 ␮L droplet after 8 days, (C) using cell seeding density of 1000 Acta2 mESCs per 20 ␮L droplet after 1 day, (D) using cell seeding density of 1000 Acta2 mESCs per 20 ␮L droplet after 8 days (upper panels: FDA staining images, lower panels: PI staining images, scale bars 200 ␮m).

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Fig. 7. Comparison of EB formation and cell proliferation in 800 nL droplets (pbb) with 20 ␮L droplets (hanging drop method) with 500 mESCs regarding to EB diameter (on the right-hand side) and cross-sectional area of the EB (on the left-hand side). For each data point, 10 EBs were monitored.

well as to the cross-sectional area of the EB. However, during the following day, the EBs in the pbb-platform proliferated much less than the EBs in the hanging drops (Fig. 7). The reasons could be an insufficient supply of nutrients, oxygen or carbon dioxide for pH maintenance or an accumulation of toxic metabolites. The ability to cultivate both, scaffold-free and scaffold-based cell cultures, opens up a broad range of applications. The described examples showed that cells proliferate inside the pipe-based bioreactors. However, there are parameters, e.g. thickness of the tubing wall, that have to be optimized in order to improve the gas exchange between the bioreactors and the environment. Furthermore, the exchange of metabolized culture medium is necessary for longterm cultivations (see detail in Section 3.4). 3.4. Influences on cell proliferation in pbb There is a range of parameters that influence the cell proliferation in pbb. During the development and investigation of the pbb-platform, the most important ones were identified. Compared to the cell proliferation in hanging drops having a volume of 20 ␮L cell culture, the cell proliferation in 800 nL droplets of the pbbplatform seemed to be slower. To investigate the reasons for this discrepancy, the DO and/or the nutrient supply in droplets of the pbb-platform were estimated. As the cultivation of pbb was performed in an incubator at 37 ◦ C in a humidified 5% CO2 -containing atmosphere, the DO and the pH should be maintained by the gas permeability of the PTFE-tubing wall (BOLA GmbH, 2014). The oxygen permeability of the tubing wall should be high enough to guarantee a sufficient DO supply for cell proliferation. This was proofed by determining the DO of 20 ␮L droplets containing 500 Acta2 mESCs (five droplets each day) and the carrier fluid in between the droplets by means of DO microsensor (PreSens GmbH) over a time period of 6 days. The measurements are described in detail together with Fig. S14 in the supplemental material Section 2.1. The DO content of the droplets decreased after one day from 96 to 89% as a result of cell proliferation. In the same time the DO content of the carrier fluid did not change. After 3 days of cell proliferation, the DO content of the droplets and the carrier fluid was determined about 91%. Even till the sixth day, the DO content of the droplets and the carrier fluid did not change significantly. Therefore, no DO limitation could be detected. As the DO value was not the reason for slow cell proliferation, different kinds of dosage modules for nutrient supply were developed (Fig. 8), in order to enable a comparable proliferation in the 800 nL droplets as in the 20 ␮L droplets. Using the T-junction module, each 20 ␮L medium droplet was fused with

Fig. 8. Examples of dosage module, here a schematic view: (A) the droplet fusion module for the dosage of shear stress-sensitive cell suspensions, (B) detailed view of the fusion process, (C) the medium exchange module, (D) detailed view of the medium exchange process.

an EB consisting 800 nL droplet for nutrient supply (Fig. 8A). This kind of dosage module can be used for the dosage of any shear stress-sensitive solution. To further elongate the cell proliferation in 20 ␮L droplets, a new developed medium exchange module was tested by using Acta2 EBs (Fig. 8B). The aspiration of metabolized medium and the addition of fresh medium did not affect the EBs inside the droplets. During this automated process step of aspiration, the gravity force acting on the EB exceeded the force caused by the velocity of the medium (data are presented in supplementary material Section 2.2, Fig. S15). The transport of the droplets was performed with 400 ␮L/min, whereas the 12 ␮L of the metabolized medium was aspirated with 300 ␮L/min. The integration of a dosage module for medium exchange prolonged the cell cultivation and guaranteed no limitation by nutrient supply or inhibition by waste products like lactate. As no DO limitation was determined and a further nutrient supply is possible, the pbb-platform has the potential for a long-term cultivation system. In contrast to a medium exchange, the dosage of drugs has to enable little dosages following by a fast mixing, in order to avoid hot spots within one droplet, which could be toxic for the cells. Mixing efficiency depends on the magnitude of the dosage momentum and the position of the droplet toward the by-channel during the dosage momentum (Fig. 9), respectively. As shown in Fig. 9D, 520 ms after the application of the dosage momentum, the droplet mixing was still incomplete. The mixing was finished after the droplet had moved about 40 mm inside the microchannel. The application of the medium exchange module and the dosage module enables long-term cultivation of cell cultures in 20 ␮L droplets, on the one hand, and drug screening processes, on the other hand. The automated procedure allows a high(er) throughput, which is important for scaling up by fusion of 800 nL droplets with 20 ␮L droplets and for numbering up for statistical evaluation. 3.5. Online monitoring in pbb Although pbb works in high(er) throughput and therefore the usage of 10 or 20 droplets for an endpoint detection analysis will not necessarily quit the experiment as there will still remain many droplets for further determinations, an online monitoring as a

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Fig. 9. Dosage module for drug screening: dosing of bromophenol blue solution (2%, w/v, in NaOH 0.1 mol/L) into a DMEM droplet, running with a flow rate of 250 ␮L/min, (A) 0 ms, droplet enters the module, (B) 240 ms, droplet reaches dosing spot, (C) 280 ms, dosage procedure, (D) 800 ms, passive mixing of the droplet is still incomplete (scale bars 2 mm). Lower panels show the enlarged framed sections of the corresponding images A–D.

Fig. 10. Photometric analysis module: (A) schematic view of the module, (B, C) the electric signal identifies the size of the EB, after (B) 45 h and (C) 141 h cultivation time, and the size of the droplet.

usually non-invasive and a fast analysis method is the much more sophisticated variant of analysis. Therefore, both, a photometric and a microscopic analysis, were enabled. The photometric analysis module is based on a photoelectric barrier (Fig. 10). Using this module, either the position of a droplet, e.g. for its further manipulation like droplet fusion or the EB size, which correlated to the strength of the signal, were detected. This module is cost-effective as the wavelength could be easily adapted to the application by changing the LED and the photo diode. Additionally, the position of the module at the tubing is flexible. The microscopic detection is easily performed through the tubing wall. In order to enable measuring of fluorescent intensity of the 3D cell structure, the utilized inverted microscope has to be equipped with the illumination system MT20 (Section 2.8). As the light intensity of the MT20 system was controlled in order to minimize the fluctuation, the cell proliferation of EGFP-expressing HEK 293 cells as spheroids or as adherent growing cells on a microcarrier could be measured (Fig. 11). Focalizing was realized manually in such a way to detect simultaneously both, the maximal crosssection area and the periphery of the spheroid or the adherent growing cells on a microcarrier as high-contrast as possible. The increase of the fluorescent intensity could be correlated to the increase of the cross-sectional area. The error associated with different orientations of spheroids or the adherent growing cells on microcarriers decreased with increasing number of measurements.

Fig. 11. Measurement of cell proliferation of EGFP-expressing HEK 293 cells as spheroids and as adherent growing cells on PLL-coated glass beads by microscopic software via continuous increase in fluorescent intensity and fluorescent area. 400 nL droplets were generated by means of a two-capillary probe from a cell suspension of 1.25 × 105 cells/mL corresponding to a cell seeding density of 50 cells per 400 nL droplet. In the case of scaffold-based cultivation 1.5 mg PLL-coated glass beads were added to 1 mL cell suspension (Section 2.3).

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Fig. 12. Scheme of the successful integration of the pbb-based EB formation step into the mESC differentiation: successful Acta2 mESCs differentiation into cardiomyocytes was indicated by the presence of EGFP-expressing beating cell clusters within a treated EB.

3.6. Integration of the pbb-platform into the mESC differentiation into cardiomyocytes The mESCs differentiation into cardiomyocytes starting with the EB formation by self-assembly using hanging droplets is well described (Hescheler et al., 1997; Potta et al., 2010). As the EB formation of 500 Acta2 mESCs in 800 nL droplets was yielded in uniformly sized EBs this process step was integrated into the conventional procedure of mESC differentiation into cardiomyocytes. The further process steps like transfer into a rocked Petri dish for 5–6 days performing EB cultivation in suspension, the EB outgrowth for 3 days after the transfer to gelatinized cell flask (beating and fluorescent cell cluster), and at last the enrichment of the differentiated cardiomyocytes by the addition of puromycin for further 5 days were performed in the conventional way. The successful mESC differentiation into cardiomyocytes was indicated and detected by the presence of EGFP-expressing beating areas within treated EBs (Section 2.2). As shown in the scheme in Fig. 12, the mESC Acta2 differentiation into cardiomyocytes was successful when the EB formation was performed in 800 nL droplets in pbb with a cell seeding density of 500 mESCs. Even without the conventional EB cultivation in suspension, the mESCs were differentiated into cardiomyocytes after an EB formation step for 2 days (Fig. 12). Neither the elongation of the EB formation step in pbb nor the variation of the cell seeding density yielded into a successful mESCs differentiation. The failure in differentiation after 8 days cultivation in pbb indicated that the cells lost their differentiation capacity due to the non-optimal conditions. A high DO content of the 20 ␮L droplets was determined during 6 days of cultivation in pbb. So far DO measurements in the 800 nL droplets were not performed as a measurement with the microsensor based on an optical fiber was not possible. As it is known that the low-oxygen tension (hypoxic) is one of the regulatory signals for maintenance, proliferation and differentiation of several stem cells, e.g. hematopoietic stem cells

(Kimura and Sadek, 2012), the detected failure in differentiation could be an additional and indirect proof of the high DO content in the 800 nL droplets over several days. Nevertheless, an uniformly sized EB formation within one day by means of self-assembly in 800 nL droplets, which will remain no longer than 2 days in 800 nL droplets, support or at least not inhibit the mESC differentiation into cardiomyocytes. This example proves that the pbb-platform is qualified in principle for the realization of the individual major process steps of stem cell differentiation based on the hanging drop method (Potta et al., 2010, Section 2.2): (i) formation of uniformly sized EBs in 800 nL droplets (Sections 3.3 and 3.6), (ii) cultivation for further 5 days in suspension by means of EB transfer into a 20 ␮L medium droplet by means of a dosage module combined with a medium exchange if it will be necessary to guarantee cell proliferation (Sections 3.3 and 3.4), (iii) outgrowth of the EBs on scaffolds (e.g. gelatin-coated glass beads) for further 3 days by means of scaffold-based cultivation (Section 3.2)— a dosage of the scaffold to each droplet would be principally possible, too, and (iv) the enrichment of the differentiated cells by pyromycin treatment realizing by the dosage module, here characterized by the dosage of bromophenol blue solution (Section 3.4). 4. Conclusions Two main topics of the 3D cell cultivation, which can be well realized with this new modular cell cultivation pbb-platform, are as follows: (i) mESC differentiation based on EB formation followed by cell proliferation and differentiation, and (ii) disease research based on 3D cell culture model systems. Here, the differentiation of mESCs into cardiomyocytes was exemplarily investigated. The importance of ES cell-derived cardiomyocytes and many of the parameters, which influence their differentiation, are known since the 1990s (Hescheler et al., 1997). However, a robust and

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standardized differentiation of cardiomyocytes in high throughput is still missing. The formation of uniformly sized EBs by self-assembly in pbb in high throughput has already been established. Additionally, the cell proliferation of EBs in 20 ␮L droplets of pbb over a time period of 8 days has been presented. Using the medium exchange module, the cells could be supplied with nutrients and toxic metabolites could be reduced at any time. Therefore, these parameters need not be a limiting factor for cell proliferation in the pbb-platform, and the cell cultivation can be further elongated if necessary. The functionality of these two process steps has been presented here as individual steps. Therefore, a next aim has to be the integration of a cell proliferation step on a matrix comparable to the conventional process step by transferring the EBs to 0.2% gelatin-coated dishes, in pbb, e.g. on gelatin-coated beads. As the handling and the cell proliferation on beads in pbb is presented here in principle, this step seems to be possible, too. Additionally, the dosage module working with a magnetic valve enables the precise addition of different growth factors or antibiotics for the enrichment of differentiated cells. But only the combination and automation of these steps will enable the standardized generation of, e.g. cardiomyocytes in pbb using EB formation as a 3D cell structure as starting point. In addition, the targeted integration of low-oxygen tension in pbb, e.g. mimicking a hypoxic niche for cardiac progenitors, could enable the study of its influence on maintenance, proliferation and differentiation of stem cells in cardiac homeostasis and regeneration (Kimura and Sadek, 2012). One prerequisite to successful investigations is the integration of an online DO probe by means of, e.g. fluorescent particles. Another one would be the technical adaptation of the pbb-platform to the low-oxygen tension as the modules so far seemed to realize normoxic conditions. The application of 3D cell culture models will get more and more importance in different applications like, e.g. cancer research. Therefore, another aim will be the usage of primary cells and the establishment of co-culture in droplets. As pbb is a miniaturized high throughput system, which also integrates technical modules for drug screening, it will be a promising tool for diagnostic and therapeutic tests in the field of personalized medicine. However, nowadays some 3D cell culture systems based on the hanging drop method or on chemical and nanostructural modification are already on the market (Spies et al., 2008; Tung et al., 2011; Amann et al., 2014). They are compatible to the established cell-based assays based on the microwell plate scale and therefore set a benchmark. Although, compared to these systems, pbb has advantages according to evaporation, speed of droplet generation, addition of drugs, droplet mixing (higher throughput) and in situ detection of the cells inside the droplets by, e.g. microscopy. However, to utilize all possible functional features of pbb, technical effort is necessary and time-consuming. Common online analyses have to be adapted. Furthermore, the status quo of the manual handling of microfluidic systems demands skill. Nevertheless, pbb could be a valuable alternative to established systems as soon as the integration of further SOPs for common cell proliferation and cell differentiation assays like, e.g. ATP measurement and immunochemical staining, can be guaranteed. As single process steps are already automated, reaching this aim seems to be possible, so that pbb will have the potential to be an autonomous 3D cell cultivation system. Optional parallelization of pbb can increase the statistical accuracy of studies. The small volume of the droplets reduces the needed amount of medium and other reagents drastically. The possibility of contaminations is low because all the modules of the pbb-platform have closed microchannels. Most of the modules can be realized as disposables and so they are suitable as cost-effective alternative to currently available techniques for applications in personalized medicine or pharmacy.

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