Community Research and Development Information Service - CORDIS

FP7

DropTech Report Summary

Project ID: 601865
Funded under: FP7-HEALTH
Country: Germany

Final Report Summary - DROPTECH (Hanging Drop based automated and parallelized cell technology platform for production and testing)

Executive Summary:
The FP7 project DropTech® started in February 2014 with the objective to develop automated handling processes for pluripotent stem cells, like human induced pluripotent stem cells (hiPSC), with integrated readout methods for the use of these cells in high-throughput and high-content assays. Cell-based screenings are used today in all types of clinical developments and safety assessment of new drugs and chemicals. The major change in the last decade is a shift towards more physiologically relevant yet complex and sensitive cell models, like stem cells, and more recently, the shift to hiPSCs. Stem cell technology has the potential to revolutionise drug discovery and safety assessment, and opens up the path for personalized medicine and regenerative medicine approaches.
The DropTech® consortium developed a fully automated platform for the production and handling of stem cell aggregates in hanging drops that allow controlling of cell culture conditions in very small volumes. The platform is hence usable by industry and research for reproducible and standardised high-throughput screening in a variety of applications, ranging from lead compound identification, over toxicity testing, to cell preparation for regenerative medicine.
To validate the fully automated DropTech® platform, the consortium specifically focussed on the Embryonic Stem Cell Test (EST), the only currently available method to assess embryotoxicity in vitro validated by the European Centre for the Validation of Alternative Methods (ECVAM). Until now, the test based on mouse embryonic stem cells (mESC) had to be processed manually following a complex protocol. In the DropTech® project, the complete workflow of the EST, including cell expansion, embryoid body formation, cardiac differentiation and compound addition in hanging drops as well as transfer to 2D conditions has been fully automated using robotic and microfluidic systems and, in a second step, the cell system used has been transferred to a human stem cell model (hiPSC). An integral part of the DropTech® platform are sophisticated integrated cell- and application-specific monitoring solutions to gain operatorless information about the developmental potential of the used cell model. This platform enables standardised, fast and efficient embryotoxicity screenings reducing the need for animal tests.
The DropTech® project was led by Prof. Heiko Zimmermann from Fraunhofer Society (IBMT) and comprised partners from Germany, the United Kingdom, and France. At project start, the DropTech® consortium focussed on analysing the requirements and developing specifications accordingly, and launched the technology development work towards automation of all cell production and cultivation processes in hanging drop suspension. In parallel, standard protocols were established for the different cell workflows including the maintenance, cryopreservation, recovery, differentiation of the iPSC into cardiac cells and shipment of quality-controlled hiPSC lines to the project partners. The complete workflow of the EST, including readout methods, was then adapted to incorporate several refinements to the method.
In the second half of the project, the standard EST using mouse ESC has been translated to a fully automated workflow in the hanging drop system using robotic systems. In the next step, the EST was successfully adapted to a human based cell model. No differences were observed compared to the complex manual procedure. A novel readout device, the so-called “contraction reader”, was developed by Fraunhofer IBMT and implemented in the EST, which allows to readout the contracting cardiac cluster in hanging drops. A cryopreservation method allowing to maintain the contracting cardiomyocyte function upon recovery was furthermore established. To assess the impact on cardiac differentiation and function, assays had been developed to monitor alternative developmental endpoints such as neuronal development.
The developed fully automated DropTech® screening platform thus comprises:
• Automated handling processes for pluripotent cells (e.g. hiPSC)
• Integrated readout methods for the use of pluripotent cells in high-throughput and high-content assays
• Integrated cell- and application-specific sophisticated monitoring solutions to gain operatorless information about the developmental potential of the used cell model
• Automated workflow of the EST including hiPSC expansion, embryoid body formation in hanging drops and transfer to 2D conditions using robotics and microfluidics systems
The advantages of the DropTech® platform are that it enables screening and testing that:
• is reproducible, standardised, fast and efficient
• uses cell models with highest biological relevance (hiPSC) in their native conformation
• enables screening/testing in a small- and medium-scaled budget range affordable for SMEs and academia
• contributes to the 3Rs with regard to animal testing
The DropTech® platform will therefore contribute to the development of new medication and therapies, enable efficient and reliable safety assessment of potentially toxic compounds, and support personalised medicine approaches as well as - in future - regenerative medicine.

Project Context and Objectives:
DropTech® aimed to develop a fully automated platform for the production and handling of stem cell aggregates in hanging drop suspension, with integrated readout methods, for reproducible, standardised, fast and efficient high-throughput screening in a variety of applications, ranging from lead compound identification, over toxicity testing to cell preparation for regenerative medicine.
To achieve the overall project aims, the following detailed objectives had been defined:
• To develop a fully automated workflow for stem cell processing including expansion, differentiation, and cryopreservation both in suspension and anchorage dependent
• To integrate and develop single cell readout systems applicable to 3D aggregates based on optical methods like live-cell imaging for analysis of beating cardiomyocytes and high-resolution multiphoton laser-scanning microscopy for immunophenotyping
• To develop cryopreservation protocols for embryoid bodies based on slow rate and vitrification procedures, ensuring complete functionality (e.g. pluripotency, electrophysiological properties)
• To automate the standard Embryonic Stem Cell Test based on an advanced hanging drop system developed in the HYPERLAB project, allowing all process steps to be performed under fully automated conditions in hanging drops
• To assure robustness, long-term stability and GMP-compliance by developing a standardised quality management system specifically for the DropTech® platform
• To replace the mouse embryonic stem cells traditionally used in the Embryonic Stem Cell Test by human induced pluripotent stem cells
Objectives for the wider application of the DropTech® platform beyond the Embryonic Stem Cell Test were:
• To provide a fully automated platform for miniaturized screenings enabling the development of standardised cell products and services for better and more efficient toxicity and drug tests, able to replace animal testing
• To provide a cell culture system for automated and improved large-scale cultivation of pluripotent stem cells

The DropTech® project has been structured around 6 work packages (WP) which is shown in Figure 1.

Project Results:
As outlined in the previous section, the overall objectives of the DropTech® project are to achieve automation of the Embryonic Stem Cell Test, and to transfer this test currently performed on mouse embryonic stem cells to hiPSC. Progress made in each of the Work-Packages (WPs) towards this goal and the results obtained are described in the following sections.

WP1: Technology requirements and specifications

In the first workpackage, WP1, technical requirements and specifications for setting up an automated cell-based platform for testing purposes have been identified. In order to test the proposed DropTech® platform, the validated Embryonic Stem Cell Test (EST; Seiler and Spielmann, Nature Protocols 2011) was considered as the cell-based reference workflow, since it consists of a variety of different cell manipulation steps. The purpose of the EST is to serve the 3R-principle, namely to reduce, refine and replace animal testing by using cellular model systems to assess cytotoxicity and embryotoxicity (i.e. toxic effects on a cell or on an embryo). The scope of DropTech® was threefold:
1. Automation of the EST
2. “Humanisation” of the EST: The standard EST uses murine embryonic stem cells. The translation of the EST to human cell lines (human induced pluripotent stem cells, hiPSCs) leads to a more precise and relevant method.
3. Translation from a non-physiological, two-dimensional (2D) cultivation to a (3D) three-dimensional cultivation using the hanging drop technique, where cells are forced by gravity to aggregate in a drop hanging from a substrate. Hereby each hanging drop acts as a single micro bioreactor with defined environmental conditions for the cells.
Therefore, in WP1 suitable cultivation and manipulation workflows for relevant cell types, like human induced pluripotent stem cells, were designed and the corresponding parameters, like flow rates, volumes, specific growth surfaces, and factor addition have been defined. In detail, different workflow regimes, e.g. adherent cultivation in 2D cell culture dishes or 3D hanging drops, have been theoretically examined and adapted to automatable routines. Results of WP1 have been delivered to WP2 and WP3 that translated the strategies to laboratory routines. WP2 and WP3 reported back to WP1 in case a workflow had to be adjusted to new or unforeseeable results.
First, a customer needs analysis has been performed by Fraunhofer IME to assess the requirements that the DropTech® platform would have to fulfil. The consortium has interrogated a specified segment of potential users of this technology, in particular in the pharmaceutical and biotech industries worldwide. A key finding with regard to throughput was the necessity to profile approx. 20 compounds (i.e. toxic substances) per week during pharmaceutical hit-to-lead development programmes.
In parallel Fraunhofer IBMT and Censo Biotechnologies (former Roslin Cellab) started with the definition of the necessary functional modules and basic microfluidic setup that would be necessary to adapt the validated EST to an automated routine (Table 1). Two pipet robots have already been in place at Fraunhofer IBMT that were considered as suitable for the tasks:
1. Nanoplotter 2.1, GeSiM mbH, Großerkmannsdorf, Germany
2. Freedom Evo 200, TECAN, Maennedorf, Switzerland.
Their key parameters, like flow rates and volume range of different applicable pipet types, and their specifications, like movements and accuracies in X-, Y-, Z-levels have been compiled. In a second step the pipet robots were prepared for an automated EST as close as possible to the original validated EST (nomenclature: “automated EST”). Here, cells are mainly cultivated adherent on a 2D surface. The plate layouts on the robot’s worktables have been designed and the workflows were simulated with the corresponding software (Nanoplotter software NPC16 V2.15, Tecan software EVOware Standard). The final task in WP1 for DropTech® was the translation of the validated EST to a 3D workflow by introducing the hanging drop routine (nomenclature: “advanced EST”). Therefore, an adaption of the validated EST workflows was crucial, e.g. regarding medium exchanges for a sufficient supply with specific differentiation factors growths or pipet robot movements.
After the customer need analysis, the system simulator setup has been done. Here, the eligibility of the pipet and cell culture robots already available at partner’s sites (Fraunhofer IBMT and IME) has been evaluated. According to the requirements of the EST protocol that was chosen to be used as validation workflow, the system design has been done. The system design included the implementation of the different operations necessary for the two main workflows of the EST.
The first workflow is the one with the cytotoxicity endpoint (see Figure 2). Here, in the validated EST, a defined number of murine fibroblasts are seeded manually in a 96-well in presence of potentially toxic compounds in different concentrations. This validated routine could immediately be adapted through the application of pipet robots since no derivations in the workflow had to be considered, neither for the automated EST workflow nor for the advanced EST workflow completely in hanging drops (Figure 2). The same pipetting steps as well as the schedule apply for the modified cytotoxicity workflow and the media consumption can be reduced by up to 75%.
The second workflow focused on the differentiation endpoint (see Figure 3), i.e. the capacity of the iPSC to develop into different cell types of a human body. In the validated EST, a defined number of stem cells is manually seeded in hanging drops to form EBs and differentiate into cardiomyocytes in presence of potentially toxic compounds. Manually seeded, hanging drops are located on the inner lid side of a Petri dish, what is impossible to automate. Especially medium exchanges are critical (first critical step regarding automation in the validated protocol, marked with purple triangles in Figure 3). Therefore, the formed EBs are rinsed from HDs in the validated protocol on day 3 and are kept in suspension for a sufficient nutrient and differentiation factor supply. Next to a relatively large amount of necessary media; having several EBs floating in one suspension culture leads to the second critical step in the validated protocol when it comes to automation: Free-floating EBs can aggregate and form larger EBs what affects their differentiation. Additionally an automated detection and transfer of single EBs into a 24 well plate at day 5 is unconvertable. The third critical step in the validated EST differentiation endpoint is the adherent cultivation of the EBs from day 5 on. A subsequent relocation of the cell aggregate afterwards is not possible without enzymatic or mechanical detachment. In order to achieve the DropTech® goal of an automation of the EST as close as possible to the original protocol (“automated EST”), the first two critical steps in the validated protocol are circumvented by introducing hanging drop plates in 96 well format. Each hanging drop comprises 20 to 40 µl volume and acts as single micro bioreactor. These perforated hanging drop plates allow the access from above and thus enable medium exchanges to guarantee a sufficient medium supply for the first 5 days in hanging drops. Rinsing the formed EBs at day 3 is no longer necessary. That avoids potential aggregation of two or more EBs, resulting in heterogeneous size distribution affecting the differentiation. Next step for the automated EST is then the transfer of the EBs on day 5 to an adherent cultivation in a flat bottom 96 well plate. By automated addition of 80 µl medium per drop from above, the EBs are rinsed into the well underneath. Hereby, the location of each drop is kept (position A1 from the hanging drop plate is position A1 in the flat bottom target plate). The critical step of a subsequent cultivation of the cell cluster from day 5 onwards is still present in this “automated EST” approach. The extended use of the hanging drop plate throughout the complete differentiation workflow circumvents this critical step as well; however, an additional medium exchange on day 7 has to be performed in order to ensure a sufficient medium supply for the cell aggregates in the micro volume hanging drops (40 µl).
The application of micro carriers in the “advanced EST with carrier” such as alginate micro beads that allow cell-specific coating enables the option of adherent growth. Such carriers allow a highly flexible adaption of the DropTech® platform for various cell types and maintain the option of simple sample transfer at any time throughout the cultivation duration.
Implementing these standard operations in hanging drops in an automated manner, the DropTech® platform even allows the reproducible, homogeneous manipulation of sensitive cell types, like human induced pluripotent stem cells.

WP2: Central functional module development and system design

Main objective of WP2 was the establishment of the central functional modules for the DropTech® platform described in WP1. Therefore diverse hardware like pipet robots and readout devices have been evaluated and linked together to guarantee optimal environmental and culture parameters for murine embryonic stem cells (mESCs) as well as human induced pluripotent stem cells (hiPSCs). For the testing and validation of the platform, the established Embryonic Stem cell Test (EST), published by Seiler and Spielmann in 2011, served as model workflow since it contains numerous cell manipulation steps and long-term cultivations. The EST that has been validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM) as alternative for animal testing consists of two different endpoints:
• Cytotoxicity endpoint: Murine fibroblasts are cultivated in presence of a potentially (cyto-)toxic compound in various concentrations. After 7 days cell’s viability is measured by a metabolic assay (MTT assay)
• Differentiation endpoint: Murine embryonic stem cells are differentiated into autonomously contracting cardiac clusters in presence of a potentially (embryo-)toxic compound in various concentrations. After 14 days their grade of differentiation is evaluated by either the visible inspection of contracting (yes/no-decision) or by an immunofluorescence analysis for cardiac markers (e.g. alpha-actinin)
In a first step, the chosen hardware has been setup to completely automate the standard EST using murine cells with microfluidic and robotic systems according to the system design in WP1. Second, WP2 took care of the humanization of the EST by substituting the murine cell systems with human fibroblasts and induced pluripotent stem cells to increase the significance of the results. Third, the complete workflow of the EST was adapted to a physiological 3D cultivation system instead of using the state-of-the-art 2D plastic lab ware that poorly mimic native environments for cells. Hereby, sophisticated adjustable micro carriers fabricated with biomaterials that offer specific growth surfaces were applied to enable the manipulation of anchorage-dependent cell systems like hiPSCs in their pluripotent state. To achieve this goal, WP 2 also dealt with the implementation and optimisation of available readout systems to the process as well as with the development of an optical detection system for monitoring and fast detection of cell morphology dynamics. These tasks in WP2 finally led to an advanced EST fully integrated in the hanging drop system that enabled an upscaling of the EST for high-throughput screenings. It also comprises the knowledge transfer of the developed DropTech® platform and protocols for evaluation and screening in key laboratories.
The automation of the validated EST has been done according to the system design developed in WP1. The implementation of the routines using the two different types of pipet/cell culture robots has been done in the device-specific scripting language:
• Nanoplotter 2.1, GeSiM mbH
• Freedom Evo 200,TECAN
Standard cell cultivation in sterile conditions has been done in clean benches and standard incubators. The applied cell types were:
• human and murine fibroblast lines (HFF1, 3T3)
• mESC line (ESD3)
• hiPSC lines (ER1918, RCi50, RCi51, RCi53, RCi55, RCi67, RCi68 and RCi69)
In order to adapt the EST to human cells, the following commercially available kits for cardiac differentiation have been evaluated:
• PSC Cardiomyocyte Differentiation Kit, Gibco
• Pluricyte® Cardiomyocyte Differentiation Kit, Pluriomics
Due to their shortcomings for the DropTech® approach, a customised cardiac differentiation protocol, optimized for the differentiation in hanging drops, has been established for mESCs as well as for hiPSCs. Therefore, also different types of micro carriers have been evaluated. After examination of possible lab ware in WP1, the hanging drop workflows have been done in either:
• Perfecta3D, 3D Biomatrix
• GravityPLUS, InSphero
• Customised HD plate, own production
Additionally, cryopreservation approaches for the different cell types have been examined. For quality control of the cells, metabolic assays (MTT, CelltiterGlo), viability staining and cell number determination (Nucleo Counter), as well as immunofluorescence analysis, and quantitative real time polymerase chain reaction for cardiac and pluripotency markers have been done.
To find an optimal on-line readout device, several off-the-shelf devices have been evaluated:
• Cytation 3, Biotek
• FDSS/µCell, Hamamatsu
• ImageXpress, Molecular Devices
• SI8000, Sony
Since all of these systems showed shortcomings, especially when it comes to non-invasiveness and applicability in hangin drops, a functional model for a novel readout device based on laser diffraction has been designed and developed by Fraunhofer IBMT.
The two workflows of the DropTech® approach, each consisting of two different endpoint analyses (cytotoxicity endpoint and differentiation endpoint), that have been proposed and designed in WP1, were implemented on both pipet robots. The first DropTech® workflow automated the EST as closely as possible to the validated EST according to the protocol published by Seiler and Spielmann in 2011 (workflow “automated EST”). Here, the cytotoxicity endpoint of this automated EST was implemented completely in two-dimensional lab wares (96 well plate format). For the differentiation endpoint of the “automated EST” workflow, hanging drop plates were used for the first five days to generate embryoid bodies (EBs), the starting point of cardiac differentiation. The subsequent cultivation until the endpoint analysis at day 14 has also been implemented in 96-well plates. Through this deviation regarding the validated EST the aggregation of several EBs to a single large cluster that might cause adverse and unforeseeable effects for the differentiation process, can successfully be prevented. The second DropTech® workflow adapted the “automated EST” workflow completely to hanging drop cultivation to maintain the physiological 3D structure and enable permanent access to each sample for transferring (workflow “advanced EST”). Here, the positive effect of separation could be maintained by keeping the 96-well plate format. Further the application of micro carriers as optional growth surface can be added any time into the hanging drops and enable the manipulation of anchorage-dependant cells, like hiPSCs. Since a full medium exchange is obviously not possible in hanging drops, the differentiation protocols had to be adapted in close collaboration with WP3, regarding the correct concentration of factors that are relevant for the process. Especially for hiPSCs this had been a major task, because commercially available media did not perform to a sufficient amount in the DropTech® routines and do not come with a detailed specification of factors. As soon as a stable differentiation protocol for the hiPSCs had been established, the “advanced EST” workflow, completely in hanging drops, has been compared to the manual procedure. As Figure 4 shows, both methods gave similar ID50 values and showed minimal standard deviations, meaning that the automation of the process is as gentle and significant as the manual one. Thus DropTech® enabled the “humanisation” of the EST to predict cytotoxicity and embryotoxicity by introducing the hiPSCs that are more significant for biomedical research.
Since the evaluated off-the-shelf readout devices showed shortcomings regarding their applicability for on-line measurements and for measurements in hanging drops, a functional module for a fast and non-invasive detection of cardiac contractions has been developed. The method is based on the differences in the diffraction pattern of a laser beam applied on potentially contracting cardiac clusters and is depicted in Figure 5. Shown is the one channel functional model that has the potential to be parallelized to achieve higher throughput.
The establishment of the functional model of the contraction reader enabled the DropTech® platform to automate the “advanced EST” workflow including on-line monitoring of cardiac contraction in the differentiation process. Figure 6 schematically shows the differentiation endpoint in the “advanced EST” workflow for hiPSCs, ending with an immunofluorescence staining for the cardiac marker alpha-actinin. The contraction readout has been done using the contraction reader. Applying a threshold and a peak detection on the resulting graph, the contraction frequency could be resolved.
Another main task in WP2 was the establishment of suitable cryopreservation protocols for the various cell types and applications. In case of the hiPSCs for example, the cells have been expanded after their reception, frozen in a computer-controlled freezer in barcoded vials, and have been stored in the automated Askion cryobanking system as a DropTech® mirror bank. Exemplarily for the established application-driven cryopreservation protocol, the developed Cryo-hanging-drops workflow is shown in Figure 7. Hereby, droplets of cell suspension dedicated for cytotoxicity analysis are frozen via controlled-rate freezing to the customised hanging-drop-plate. In the frozen state, hanging drops can be stored for long periods and enable stock keeping. Upon application, the Cryo-hanging-drop plate can immediately be thawed in a target plate and assessed by a metabolic assay like CelltiterGlo (Promega). Thus time- and material consuming cultivation- and washing steps before and after standard cryopreservation procedures in cryo-vials can be avoided. The Cryo-hanging-drops technique provides ready-to-use frozen biological samples for screening purposes in 96 well plate format.

WP3: Cell sources and cell based workflows

The third work-package, WP3, was responsible for identifying cell lines and defining optimal culture conditions for the automated production of human induced pluripotent stem cells. The main objectives of WP3 was to develop protocols and quality control procedures for the cultivation of human cell models in the hanging drop and ultimately, to adapt the mouse-based standard EST to human induced pluripotent stem cells (hiPSC). WP3 also had the role to validate frozen cardiac clusters derived from hiPSC produced in the DropTech® platform.
Censo Biotechnologies Ltd (former Roslin Cellab Ltd) has maintained and expanded twelve hiPSC lines of unaffected (RCi30, RCi50, RCi51, RCi53, RCi55, RCi68, GIBCO, UKBi005-A, UKKi011-A, UKKi012-A) and affected (UKKi007-A, UKKi008-A) status. All twelve cell lines have been expanded at Censo Biotechnologies Ltd and banks created for each cell line. Protocols for the maintenance, cryopreservation and recovery of hiPSCs were established. For the maintenance of all hiPSC lines, a xeno-free and feeder free culture system was utilised for a culture period of >50 passages, maintaining the ability to differentiate towards cardiomyocytes (the lineage of preference for DropTech). The cryopreservation of hiPSC lines was achieved using an animal component-free, defined cryopreservation medium with 10% DMSO (CryoStor® CS10) and storage performed at -150°C for an extended period of >2 years with successful recovery rates and without any issues identified.
EB generation is extensively used as the preliminary step in various differentiation protocols, as cells within the EBs can be directed towards specific cell lineages through exposure to differentiation factors. For the project, Censo Biotechnologies Ltd tested culture in hanging drops and methods using a U- or V-bottomed 96-well plates for the formation of EBs, from pre-determined numbers of hiPSCs. EBs from hiPSC lines were generated using both the Perfecta3D® Hanging Drop plates and petri dishes. EBs were grown for 24 and/or 48 hrs prior commencing cardiomyocyte differentiation. All twelve cell lines were used to generate EBs with success rates >80% (i.e. EB formation) and subsequent generation of spheroids where >95% contained contracting cardiomyocytes. The protocol for the formation of EBs was transferred to an automated platform and collaborative experiments were performed between Censo Biotechnologies Ltd and IBMT. The automated approach was successful and matched the manual handling approach in terms of EB formation and subsequent media exchange.
Several studies have evaluated murine and human ESCs as a model for embyotoxicity. A number of compounds whose effects are well documented in vivo and in vitro such as busulfan, retinoic acid, indomethacin, caffeine, saccharin, 5-fluorouracil, penicillin G and Hydroxyurea, have been classified and evaluated as strongly, weakly and non-embryotoxic (Mehta, et al., 2008) using human ESCs. Censo Biotechnologies Ltd has completed the work on the validation of the EST-like hiPSC assay using two hiPSC lines (RCi50 and RCi53) which were selected based on their potential to generate contracting cardiomyocytes, and on one HDF (Human Dermal Fibroblast) cell line. Ten compounds were tested using the concentration ranges for the compounds, as identified by Fraunhofer IME-ScreeningPort. Those compounds were: Ascorbic Acid, Saccharin, Penicillin G, Isoniazid, Caffeine, Aspirin, Indomethacin, 5-Fluorouracil, Hydroxyurea and Retinoic Acid. The culture assay format used was 2D in 96-well plates. Following a culture period of seven days, an Alamar Blue cell viability assay was performed on, both hiPSC and HDF lines resulting in the IC50 values for all compounds being identified.
Finally, Censo Biotechnologies Ltd established protocols for the successful cryopreservation and recovery of contracting hiPSC-derived cardiomyocytes using a 3D culture format. Contracting hiPSC-derived cardiomyocytes from nine hiPSC lines (RCi50, RCi53, RCi55, GIBCO, UKBi005-A, UKKi007-A, UKKi008-A, UKKi011-A, UKKi012-A) were cryopreserved for 1, 4 and 8 weeks at -150°C followed by successful recovery of between 85% and 95% in cell viability and contractibility. The cryopreservation of contracting cardiomyocytes was achieved using CryoStor® CS10. The protocol for the cryopreservation of contracting cardiomyocyte spheroids has been transferred to Fraunhofer IBMT partners and experiments have been replicated successfully in two laboratories.
Censo Biotechnologies Ltd extensively tested its established protocols for the maintenance, cryopreservation, and recovery using, as mentioned above, twelve hiPSC lines of unaffected and affected status. These lines were generated from different reprogramming methods. Successful protocols for the generation of contracting cardiomyocytes, again using the twelve different hiPSC lines, have been established using 3D (spheroids) formation, cell aggregate formation and 2D monolayer cultures. In addition, six different matrices (Geltrex, Matrigel, Vitronectin, Fibronectin, Laminin and Tropoelastin), were successfully evaluated for cardiomyocyte differentiation potential in a 2D monolayer culture format for five of the hiPSC lines (GIBCO, UKBi005-A, UKKi007-A, UKKi008-A and UKKi011-A). Cryopreservation protocols, for contracting hiPSC-derived cardiomyocytes, using a 3D and cell aggregate culture format were also established. Contracting hiPSC-derived cardiomyocytes from nine of the hiPSC lines (RCi50, RCi53, RCi55, GIBCO, UKBi005-A, UKKi007-A, UKKi008-A, UKKi011-A, UKKi012-A) have been cryopreserved for 1, 4 and 8 weeks at -150°C followed by successful recovery of greater than 85% and up to 95% in cell viability and contractibility.
In summary, examining a total of twelve different iPSC lines in three different culture formats, Censo Biotechnologies Ltd has established robust and reproducible protocols for the differentiation of hiPSCs into cardiomyocytes and a successful cryopreservation method retaining contracting cardiomyocyte function upon recovery.

WP4: Platform development and validation

In WP4 the system components identified in WP1, 2 and 3 were integrated into a workflow to assess compound characteristics with regard to their potential to induce developmental and reproductive toxicity (DART) with the DropTech® platform. In particular, the specifications for cellular reagents, microplate types, liquid handling and media exchange, compound addition, detection systems for cardiac contraction and assay readouts for cytotoxicity and differentiation capacity have been amalgamated into a workflow and their combined operational performance has been assessed in an industrial-type working environment. To this end, a panel of reference compounds with known physico-chemical properties, solubility, toxicity profile, molecular mechanism-of-action and validated quantitative reference data (provided for instance by ECVAM) have been assessed with the DropTech® platform. The primary endpoints defined by Seiler and Spielmann (Nature Protocols, 2011) have been quantified in murine and human cells, i.e. the cytotoxicity in pluripotent stem cells, the cytotoxicity in somatic fibroblast cells and the impact of the respective compound on the differentiation capacity of stem cells to form cardiac tissues. Parts of the automated workflow for cytotoxicity assessment were carried out in 96-well format the lab automation platform Fluent (Tecan) (Figure 8) and protocols and scripts were harmonized between DropTech® partners Fraunhofer IBMT and Fraunhofer IME-ScreeningPort (former European ScreeningPort).
Compound toxicity was evaluated in parallel either on the murine cell lines ES-D3/NIH3T3 or hiPSC (RCi50, RCi53)/HFF1. The assay was set up for testing of up to 18 different compounds in 7-point dose response per week, thereby meeting the customer requirements as specified in WP1. For the automatic workflow a customised script was developed (Figure 9).
The following reference compounds were tested using the DropTech® workflow in both murine and human assay formats: Saccharin, Penicillin G, Caffeine, Dexamethasone, Isoniazid, Ascorbic acid, Indomethacin, 5-Fluorouracil, Hydroxyurea, Retinoic acid, Thalidomide, Paraquat, Glyphosate, Fumonisin, Miltefosine, nanoparticles with various coating chemistries and proprietary small molecule compounds from in-house Fraunhofer IME-SP drug discovery projects. This variety of chemical space analysed enabled the project partners to fine-tune the platform components during the course of the project in order to achieve optimal performance by termination of the project in the end of January 2107. Figure 10 shows the representative cytotoxic performance of the platform with regard to 5-Fluorouracil and Penicillin-G in the murine assay format.
With regard to the differentiation endpoint also significant enhancements of the classical EST protocol have been achieved: Protocols for embryoid body (EB) formation (Figure 12) and differentiation into beating spheres containing cardiomyocytes were established and transferred to the Fluent lab automation platform (Figure 11).
The newly developed workflow enables the production of standardised EBs, robust differentiation into beating cardiac structures and the subsequent analysis of molecular marker proteins. For quantification of endogenous marker expression in fixed cells Fraunhofer IME-ScreeningPort has validated antibodies for marker protein detection for pluripotency (e.g. Oct4, Sox2), for (neuro-)ectodermal tissue (e.g. beta-III-Tubulin, Nestin), for mesodermal tissue (e.g. alpha-Actinin, Myosin Heavy Chain) and for endodermal tissue (e.g. Sox17, FoxA2, Albumin). These antibodies have then been used to quantify the amount of marker protein expression either using A) flow cytometry analytics or B) high content imaging technologies. Furthermore, representative compounds have been tested for their ability to impact on the process of differentiation. Exemplary data using flow cytometry are summarised in Figure 13 which shows the impact of Saccharin, Fumonisin and Hydroxyurea. These data are in accordance with previously published results, confirming the validity of the DropTech® technology.
Alternative to the flow cytometric approach high content imaging based protocols for marker detection have been developed (Figure 14) and validated using standard compounds. This allows for a higher compound throughput compared to laborious hands-on processing and sample preparation for FACS.
Apart from cardiac tissues also alternative tissue types have been generated and appropriate differentiation protocols have been established accompanied by evaluation routines to quantify marker expression. They could provide a basis for extending the DropTech® platform to other relevant in vitro endpoints which are of high biological and pharmaceutical relevance. In particular, cells of neuronal or hepatocyte lineages have been created and respective analysis routines put in place.
In summary, in WP4 an industry-compliant platform was created which was instructed by WP1, WP3 and WP3 and was found suitable for use by pharmaceutical industry clients in the Hit-to-Candidate selection regimen for compound stratification and ranking as a prelude to in vivo compound stratification. Here, a 3R compliant platform (enabling the reduction, refinement and replacement of animal testing) was created that could – upon thorough validation by regulatory bodies – provide an alternative to existing workflows in pharmaceutical development programmes.

WP5: Dissemination and Innovation management

Based on a specific DropTech® dissemination plan, all elementary tools for a successful dissemination of future results were implemented in the beginning of the project: A clear project “corporate” identity with a powerful logo was designed and a flyer (Figure 15 and Figure 16) as well as public website were created in collaboration with a professional graphic design agency. A Knowledge portfolio was established and internal discussions concerning the exploitation of project results were regularly taking place.
Based on the first project achievements, an important number of high-impact dissemination activities were carried out in the second half of the project to spread information on and promote the DropTech® platform towards a wide group of stakeholders (e.g. chemical, pharmaceutical and cosmetics industry, biotechnology companies, regulatory authorities, academic organisations etc.). The DropTech® stakeholder workshop on 22-24 November 2016 at Fraunhofer IME-ScreeningPort enabled potential future users to work with elements of the DropTech® platform for the time of the hands-on training. Project results were presented at several important conferences such as the ISSCR Annual Meeting 2016 in San Francisco, the EUSAAT conference 2016 in Linz while the DropTech® booth at the EC scientific conference on ‘Non-Animal Approaches’ provided opportunity for further face-to-face discussions.
The project video prepared towards the end of the project gives a comprehensive summary of the achievements made in DropTech® and demonstrates not only the added value of the DropTech® platform for the different stakeholders but also the impact that the novel technology may have on the European citizen.

WP6: Project management

Right from the project launch, the Project Office represented by the management company ARTTIC had put into place different tools for partners such as mailing lists, monitoring tools and a project internal webspace to support the exchange of information, to facilitate remote communication among partners, to ensure a close follow-up of the project progress and to provide a project document repository.
A management structure and management procedures adapted to the size of the project ensured smooth collaborations toward the overall project goals. The General Assembly (GA) met on a bi-annual basis (Figure 18) and regular teleconferences of the GA and between WPs were held on an as-needed basis. Documentation related to meetings such as an agenda, logistics packets and slide templates were circulated to all partners ahead of time and minutes reporting on the project progress, key actions and decisions taken were circulated following the meetings.
Contractual and financial issues were handled by the Coordinator Fraunhofer IMBT with support from the Project Office. An amendment to the Grant Agreement was submitted to the EC and approved in the first period which was related to the partial transfer of rights and obligations from European ScreeningPort to Fraunhofer IME-ScreeningPort. Later into the project, Roslin Cellab Ltd. changed its name to Censo Biotechnology Ltd. The preparation of the contractual reporting to the EC was handled by the Project Office with contributions from all partners. EC payments were distributed by the Coordinator to all partners according to the agreed timeline and breakdown and in line with the Grant and the Consortium Agreements rules.

Potential Impact:
Socio-economic impact and the wider societal implications of the project

The final result of the DropTech® project is a fully automated DropTech® profiling platform for small molecule or biological substrates. The platform consists of “hardware” (automation, liquid handling, microfluidics, microplates) as well as “software” (cells, cell-based workflows, algorithms to quantify cellular contraction). It includes automated handling processes for pluripotent (e.g. human induced pluripotent cells, hiPSCs) and differentiated cells, integrated readout methods for the use of pluripotent cells and cells derived thereof in high-throughput and high-content assays, sophisticated integrated cell- and application-specific monitoring solutions to gain information on the developmental potential of the cell model applied. The project partners have established and validated an automated workflow for the Embryonic Stem Cell Test in a “humanised” form, based on hiPSC, including hiPSC expansion, embryoid body formation in hanging drops and transfer to 2D conditions using robotic and microfluidics systems. The platform is configured to reliably test up to 20 compounds per week.
The screening/profiling platform will be exploitable in an industrial setting in a variety of biotechnological sectors, including drug discovery, toxicity testing and regenerative medicine.
The novel DropTech® platform thus enables screening and profiling that
• is reproducible, standardised, fast and efficient
• uses cell models with highest biological relevance (hiPSCs and hiPSC-derivated tissue-specific cells, in particular without transgene expression)
• enables screening/profiling in a small- and medium-scaled budget range affordable for SMEs and academia
• contributes to the 3R’s with regard to animal testing (reduction, refinement, replacement)
• can be configured to hiPSCs from i) healthy donors or ii) patients burdened with monogenic or complex diseases, thereby enabling rational and personalised therapeutics development
• can be extended to alternative tissues (in addition to cardiac cells) that are also of key interest for toxicological and pharmacological studies (e.g. neuronal cells or hepatocytes) and
• can be used for studies on small molecules or biologicals.
The DropTech® platform will therefore contribute to the development of new medication and therapies, enable efficient and reliable safety assessment of potentially toxic compounds, and support personalised medicine approaches as well as - in future - regenerative medicine.

Main dissemination activities and exploitation of results

A large range of high-impact dissemination activities have been carried out during the project’s lifetime to reach out to a wide group of DropTech® stakeholders with the overall objectives:
• To spread knowledge generated by the project
• To make the project visible by providing to all identified audiences appropriate targeted information about the DropTech® technology for in vitro toxicity testing and drug screening, and hence to facilitate its future commercialisation
• To inform the public about ongoing research activities and their possible impact on society.
The broad dissemination of project results comprised the following key dissemination activities towards stakeholders:
• A poster presentation of DropTech® in the ISSCR Annual Meetings 2015 in Stockholm / Sweden in June 2015
• The participation in the Annual Meetings 2015 and 2016 of the German Stem Cell Network including the Partnering Form in 2016 where DropTech® was presented to a small group of industry
• The organisation of a DropTech® Stakeholder Meeting in Cambridge/UK in October 2015 to discuss the potential implementation of the DropTech® technology in hiPSC generating projects (e.g. ForIPS, EBiSC)
• An oral DropTech® presentation given by Dr. Ina Meiser from Fraunhofer IBMT at the ISSCR Annual Meeting 2016 in San Francisco/USA on 22 June 2016 (Figure 19a) - the recording of the talk has been made available via the DropTech® public website (www.droptech.eu)
• The participation in the 17th EUSAAT 2016 3R congress (European Society for Alternatives to Animal Testing) in August 2016 in Linz/Austria where DropTech® was presented in three different ways: a poster, a talk given by Dr. Ole Pless who also co-chaired the session on “Stem Cells & Reproductive Toxicity”
• The organisation of a DropTech® Stakeholder Workshop at Fraunhofer IME Screening-Port in November 2016 in Hamburg/Germany where representatives of pharma and research companies could test elements of the DropTech® technology
• The preparation of a DropTech® video prepared by ARTTIC which provides a comprehensive summary of the project objectives, achievements and furthermore provides first user feedback on the DropTech® added value from participants of the DropTech® Stakeholder Workshop. The DropTech® video has been made available on YouTube (and via the DropTech® public website) and is thus easily accessible to the public (https://www.youtube.com/watch?v=ArGm6xFzzLM&feature=youtu.be)
• The organisation of a DropTech® booth at Scientific Conference on Non-Animal Approaches at the EC in Brussels in December 2017 (Figure 19b) gave opportunity for face-to-face discussions with different stakeholders and the public in general.
• Finally, the DropTech® public website was regularly updated and additional dissemination material have been prepared such as a project flyer for distribution at conferences and two roll-up banners which were used at the DropTech® booth in Brussels as well as for the workshop and video shooting.
The DropTech® dissemination activities have been successful in raising large interest in the DropTech® technology. As a result, DropTech® has been invited to give a talk at the 14th Cryogenics 2017 IIR International Conference in May 2017 in Dresden. The participation to the next ISSCR 2017 Annual Meeting is furthermore planned.
All key dissemination activities carried out have successfully supported the project exploitation goal: To get in contact with potential future DropTech® users in the drug discovery and toxicity testing market as well as the scientific community, to launch negotiations for commercialising the DropTech® platform and the services offered and thus to exploit the project results.
Towards the end of the project, the consortium partners discussed the possible future enhancement of the DropTech® platform and identified a list of five key exploitable results (including the Contraction Reader developed by Fraunhofer IBMT, the human based Embryonic Stem Cell Test and automated formation of embryoid body). Beta customer sites and potential industrial commercialisation partners have been identified and discussions are in process at Fraunhofer IBMT and Fraunhofer IME-SP.

List of Websites:
DropTech® consortium

The DropTech® project is coordinated by Prof. Dr. Heiko Zimmermann from the Fraunhofer Institute for Biomedical Engineering (IBMT). The consortium is composed of the following partner organisations:

Fraunhofer Gesellschaft zur Förderung der Angewandten Forschung e.V., involved through the following two institutes:
• Fraunhofer Institute for Biomedical Engineering IBMT, Sulzbach, Germany
• Fraunhofer Institute for Molecular Biology and Applied Ecology IME-SP, Hamburg, Germany
Censo Biotechnology Ltd. (former Roslin Cellab Ltd.), Roslin Midlothian, United Kingdom
ARTTIC, Paris, France

Contact

Project Coordinator
Prof. Dr. Heiko Zimmermann
Head of Institute
Fraunhofer Institute for Biomedical Engineering (IBMT)
Josef-von-Fraunhofer-Weg 1
D-66280 Sulzbach – GERMANY
heiko.zimmermann@ibmt.fraunhofer.de

Project Administration Office
Beate Kreisel / Annette Ringwald
ARTTIC
58a rue du Dessous des Berges
75013 Paris - FRANCE
droptech@eurtd.com

Project website: www.droptech.eu

Related information

Reported by

FRAUNHOFER-GESELLSCHAFT ZUR FOERDERUNG DER ANGEWANDTEN FORSCHUNG E.V
Germany

Subjects

Life Sciences
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