Time-resolved monitoring of metabolic activities in vitro (microphysiometry) is necessary to study the dynamic regulations of core biochemical pathways in cellular disease models. Following a brief review of recent developments in the field, this contribution presents the most important branches of current sensor-based methods of microphysiometry. The primary parameters assessed by microphysiometry are extracellular pH changes and the concentration of dissolved oxygen to conclude on extracellular acidification (EAR) and oxygen uptake rates (OUR) as quantitative measures for cell metabolism. Direct sensing of selected small molecule metabolites or metabolic heat are alternative assay strategies. The major physical transduction principles encompass electrochemical and optochemical sensing complemented by less popular approaches for highly specific applications. All microphysiometric devices include tissue culture maintenance systems that have to guarantee physiological conditions and that must be functionally aligned with the sensing component. The interplay of cell metabolic activity and sensors in microscaled reaction volumes can be simulated with appropriate numerical models describing the physical processes of reaction and diffusion. While aspects of automation and throughput belong more to the engineering side of microphysiometry, both form the basis for the inevitable statistical data acquisition and analysis. This paper concludes with a description of two selected applications of microphysiometry, one in toxicology and the other one in clinical cancer research.
cellasys will not have a booth at this years analytica in Munich …
However, you are welcome to visit our microphysiometric systems in our facilities in Munich – Schwabing during analytica 2018.
Please arrange your visit via email@example.com.
You are welcome to visit our presentation at LOPEC 2018:
Low-cost biosensors with printed interdigitated graphene electrodes
Thomas Velten1, Thorsten Knoll1, Axel Brenner1 Anke Schultz1, Joachim Wiest2, Renate Warmers3, Gerald Jenke3, Anna Zumbülte3, Andreas Urban4, Kris Seunarine5
1Fraunhofer-Institute for Biomedical Engineering, 2cellasys GmbH, 3Saueressig GmbH + Co. KG, 4AiCuris Anti-infective Cures GmbH, 5Haydale Limited
Cell-based assays are of enormous importance in anti-viral drug discovery and research on vaccines against viral infections. Therefore, the inhibition of the virus-induced cytopathic effect due to neutralizing antibodies or antiviral agents comprise to the commonly utilized methods. The impedance spectroscopy with commercial platforms demonstrated the principal applicability of this technique for the mentioned applications in well plate format. But costs of disposable components of these systems are high due to the used fabrication technology. We aim at printing low-cost sensor systems for applications in antiviral drug testing and toxicity testing.
Within the 10th World Congress on Alternatives and Animal Use in the Life Sciences in Seattle, we (Annemarie Lang, Frank Schulze) organized and held the You-WC10 as a workshop aiming at young and early career scientists that already work or plan to work in the field of the 3Rs. We wanted to encourage the dialogue of young scientist among themselves and with older peers that have been working in the field for a long time, thereby creating the opportunity for establishing new professional networks.
The You-WC10 included three sections:
1) Follow the 3R Career paths, where experienced researchers talked about their personal career paths that lead them to the 3R’s
2) Meet the Professor, a round table session where young scientist were able to meet with experienced Scientists to discuss and network in an open atmosphere
3) Enjoy Seattle together, a social event were young scientist had the opportunity to enjoy a guided pub crawl through Seattle, get to know each other and form new independent networks
By providing these three segments, we successfully created a free and open atmosphere for the exchange of scientific ideas and career experiences alike and also had a lot of fun!
The YOU-WC10 started at Sunday the 20th of August 4.30 pm with the “Follow the 3R Career Paths”-Session counting about 20 participants. We were delighted about 4 really interesting and inspiring speakers that accepted our invitation and not only gave insights into their career development but also useful advices for career building in general. Maurice Whelan works at the European Commission Joint Research Centre (JRC) where he is head of the Chemical Safety and Alternative Methods Unit and the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). He started out in the engineering field and his early research focused on design of orthopaedic knee prostheses. After joining the JRC his work in optical sensing and imaging brought him into the area of in vitro methods, high throughput screening and alternatives to animal testing. He encouraged young scientists to strive for interdisciplinarity in their research and to consciously reflect if targeted career goals are in accordance with personal life goals. According to him, there is no formula for a successful and fulfilling career since everyone has to find their own path. However it is important to embrace opportunities to learn and grow professionally by taking on challenges that push you out of your comfort zone. Katrin Schütte (EU-Commission) took a more conventional career path based on the study of Nutrition Sciences and a PhD in Neurobiology, followed by an excursion to the industry where she was trained as a human safety toxicologist. As a policy officer at the Directorate-General for Environment (DG ENV) of the EU-Commission, she is now responsible for the implementation of the Directive 2010/63/EU on protection of animals used for scientific purposes which has the final goal of replacing animal testing, in addition to responsibilities for the chemicals regulation REACH. Thomas Hartung the director of the Center for Alternatives to Animal Testing (CAAT) – Johns Hopkins University inspired the young scientists by showing the variety of topics that need to be addressed in the future. Furthermore, he was telling success stories in the field of alternatives to animal testing from the last decades, emphasized the advantages to be a generalist with a wide range of interests and stressed out that an unerring instinct for hot topics is needed for successful career building. In addition, he took us with him through a fascinating professional path including professorships in Germany, the leadership at ECVAM and finally the head of CAAT. Nicole Kleinstreuer from the National Toxicology Programs’ Interagency Center for the Evaluation of Alternative Toxicological Methods (NTP NICEATM) completed the session with an impressive career path starting with a passion for mathematics and physics. She pointed out that sometimes personal goals can very well match professional ones as exemplified by her one year travel in New Zealand that she nicely combined with an internship followed by a PhD-thesis. Additionally, it is beneficial to be more honest about personal deadlines and be self-confident about our work. To summarize, it was a great opportunity for all the young scientists and ourselves to have a look behind the curtain, to get inspired and to gain knowledge about the variety of career opportunities within the field of alternatives to animal testing. Therefore, we like to thank the speakers for their enthusiasm that was present in every talk and for the experience as a whole!
On Tuesday the 22nd of September the “Meet the Professor”-Session took place in the afternoon. About 25 participants used the possibility to get in direct contact with experts in the field and to discuss and exchange ideas in an open atmosphere. Four roundtables were formed with the first one hosted by Joanne Zurlo (Johns Hopkins University) and Christa ThöneReineke (Free University – Berlin, BB3R) on Refinement and Animal Welfare. The second roundtable centered on organ-on-a-chip technology and was hosted by Terry Kavanagh and his colleague Dave Eaton (both University of Washington), discussing the advantages of using primary cells, their experiences with microfluidic renal models and the rationale of sending the latter ones into space. The third roundtable focused on Neurotoxicity and was hosted by Ellen Fritsche (IUF – Leibniz Research Institute for Environmental Medicine, Düsseldorf) and Helena Hogberg (Johns Hopkins University & CAAT) who addressed current hot topics in the field and career opportunities within academic research. The last roundtable was hosted by Jan van der Valk (3Rs-Centre Utrecht Life Sciences (ULS), Utrecht) discussing the established 3R databases (humane endpoints, similarity of animal models) and the critical topic of using animal-derived Fetal Calf Serum (FCS) as a supplement in cell culture. The round table sessions took place in a relaxed and joyful atmosphere as indicated by regular laughing. A general good mood was supported by snacks and soft drinks provided by the WC10 organizing committee. The participants were so adsorbed in the discussion that everyone agreed that the session was over far too soon! We like to thank the experts for their willingness to share their opinions with the young scientists as well as the WC10 organizers for their support!
On Tuesday, directly after the “Meet-the-Professor”-Session we left together for our last segment: “Enjoy Seattle together”, a guided Pub Crawl through microbreweries and bars. Along the way, additional participants of the conference joined the group increasing our group size to 35-40 people. The Pub crawl was conducted by our tour-guide Scott (many thanks to him!) who awaited us with pizza in the “Optimism brewing company” close to Seattle’s Broadway, an optimal spot due to a lot of space and a great selection of beer. In order to further induce networking between the participating scientists, we introduced a game with the promise of winning awards after successful finalization. Therefore, we had prepared several stickers with either an OECD guideline title and number (match pairs) or adverse outcome (AO) and their accompanying molecular initiating event (MIE). The game was a modified version of the so-called “AOP game” introduced to us at the JRC ECVAM Summer School that took place in this year’s May in Ispra, Italy. The aim of the game was to find the matching partner, e.g. for a OECD guideline number the matching title of for a MIE the matching AO. It was a great fun for everyone involved and indeed facilitated an increased exchange among the participants. The German Federal Institute for Risk Assessment (BfR) kindly provided our awards in the form of bottle openers, bags and egg timers. The evening really helped to form new contacts and to exchange opinions and was enjoyed by everyone involved. Therefore, we would like to say a big THANK YOU to the sponsors of the Pub Crawl enabling us to provide pizza and free drinks – these were namely: Cellasys GmbH, Cellbricks GmbH, TissUse GmbH and AniMatch UG.
To conclude, the YOU-WC10 was a complete success and should be continued!
Finally, the organization and implementation would not have been possible without the support of Elaine Faustman, Joanne Zurlo, Robert Kavlock and Ellen Fritsche – big thanks! The events were also supported by the Institute for Risk Analysis & Risk Communication (School of Public Health, University of Washington).
In addition, we like to thank Michele McDermott and Bridget Van Egmond from FASEB as well as the speakers, roundtable-hosts and participants. The rollup, flyers, awards and travel costs for Frank Schulze were kindly provided by the German Federal Institute for Risk Assessment/German Center for the Protection of Laboratory Animals (BfR/Bf3R). We want to express our gratitude for this support and also thank Prof. Gilbert Schönfelder for constructive discussions regarding the YOU-WC10 preparation. Last but not least, we like to thank our helpers: Katharina Hohlbaum, Alexandra Damerau and Moritz Pfeiffenberger.
During the different events we collected a mailing list of the participants that will be used to build a network of young scientists within the field of alternatives to animal testing. If you are interested in the network and want to join, please send us your e-mail.
We hope that you also enjoyed the YOU-WC10 as we did and are looking forward seeing you @ YOU-WC11 in Maastricht 2020
Annemarie Lang & Frank Schulze
Dr. med. vet. Annemarie Lang AG Buttgereit – Bioenergetics, Glucocorticoids & 3R-Research Department of Rheumatology and Clinical Immunology Charité – Universitätsmedizin Berlin Charitéplatz 1, 10117 Berlin, Germany E-Mail: Annemarie.firstname.lastname@example.org Phone: +49 30 450 513384/-64
Dr. Ing. Frank Schulze Federal Institute for Risk Assessment Department Experimental Toxicology and ZEBET Group 92, alternative methods for animal testing Max-Dohrn-Straße 8–10, 10589 Berlin, Germany E-Mail: Frank.Schulze@bfr.bund.de Phone +49 30 18412-3702
53rd Congress of the European Societies of Toxicology (EUROTOX 2017), September 10 – 13, 2017, Bratislava, Slovak National Theatre
Sebastian Eggert, Joachim Wiest: Extension of the cytosensor microphysiometer test method toward cell impedance measurement
The cytosensor microphysiometer test method was validated by the European Commission for Validation of Alternative Methods to identify severe and not classified eye irritating chemicals. The corresponding INVITTOX # 130 protocol was adapted to the IMOLA-IVD technology. This method avoids the use of fetal bovine serum and is fully automated. Recent developments expanded the method toward a wider dilution series and the additional measurement of changes in cellular morphology using impedance measurement with inter-digitated impedance structures (IDES). The combination of extracellular acidification measurement as a metabolic parameter and impedance measurement as a morphological parameter yields better prediction capability to determine the eye irritation potential of new chemicals. First multi-parametric measurements show comparable MRD50 (metabolic rate decrement by 50%) values of sodium dodecyl sulfate (SDS). However, an effect of SDS toward the cellular impedance can only be detected at a higher concentration. The expanded protocol will be used to create a new prediction model and it will be evaluated if it is possible to address the whole range of classification in the field of eye irritation.
cellasys supports “Follow the 3R career paths” with Maurice Wheelan (EURL-ECVAM), Katrin Schutte (European Commission), Thomas Hartung (Johns Hopkins University & CAAT), Nicole Kleinstreuer (NTP NICEATM), “Meet the professor” with Joanne Zurlo (Johns Hopkins University), Christa Thöne-Reineke (FU-Berlin, BB3R), Ellen Fritsche (IUF Düsseldorf), Ed Kelly, Terry Kavanagh (University of Washington), Helena Hogberg (Johns Hopkins University & CAAT), Jan van der Valk (3Rs-Centre ULS, Utrecht) and the “Seattle pub crawl” with YOU.
TENTH WOLRD CONGRESS – ALTERNATIVES AND ANIMAL USE IN THE LIFE SCIENCES, August 20-24, 2017, Seattle, Washington, USA
Roman Kolar, Joachim Wiest, Michael Feil, Tilo Weber: Chemically defined cell culture practice of L929 fibroblasts
Living cells are a promising tool to develop alternatives to animal experiments. To provide them for cell based assays e.g. cell lines are kept in CO2 incubators and split once a week with feeding approximately every third day. The guidelines for good cell culture practice give recommendations how to work in cell culture . A remaining issue is that many laboratories use fetal bovine serum (FBS) in standard cell culture. Beside the questionable way of manufacturing of FBS , it is chemically not defined. The variations in FBS may be a driver for the reproducibility problem in biomedical research. For our L929 cell line, the cell culture media with 10% FBS was replaced by the chemically defined DME/F12 + ITS mixture . With this procedure we maintained the cell line now for more than a year. Although the cells changed their morphology they are used successful in biocompatibility testing  and in microphysiometric experiments to determine the eye irritation potential of chemicals.
 Coecke S, Balls M, Bowe G, Davis J, Gstraunthaler G, Hartung T, Hay R, Merten OW, Price A, Schechtman L, Stacey G, Stokes W; Second ECVAM Task Force on Good Cell Culture Practice (2005) Guidance on good cell culture practice. a report of the second ECVAM task force on good cell culture practice. Altern Lab Anim 33: 261-87
 van der Valk J, Brunner D, De Smet K, et al. (2010) Optimization of chemically defined cell culture media – Replacing fetal bovine serum in mammalian in vitro methods. Toxicology in Vitro 24: 1053-1063. Doi:10.1016/j.tiv.2010.03.016
 Wiest, J.: Chemisch definiert – ein zellbasierter Zytotoxizitätsassay ohne fötales Kälberserum. Biospektrum (2017) 23: 61. doi:10.1007/s12268-017-0768-6
The paper “Biology-inspired microphysiological system approaches to solve the prediction dilemma of substance testing” wins the 2017 ALTEX award for the best ALTEX publication in 2016.
39th Annual International Conference of the IEEE Engineering in Medicine & Biology Society (EMBC’17), JeJu Island, S. Korea, from 11-15 July 2017:
Michael Feil, Joachim Wiest: Evaluation of Printed Microsensors for Microphysiometry
In this work, investigation of the electrical properties of carbon-based printed microsensors by impedance measurements and their suitability for cell-based assays are presented. Special attention was devoted to the most sensitive measurement frequency, which could be determined at 10 kHz for the investigated L929 cell line.
Sebastian Eggert, Frank Alexander, Joachim Wiest: Enabling 3D hepatocyte spheroids for microphysiometry
Advances in the areas of tissue engineering and microfabrication techniques have enabled promising in vitro platforms, known as Organs-on-Chips, with the aim of mimicking complex in vivo conditions for more accurate toxicology studies. To analyze the physiological change induced by chemicals or toxic substances continuously, sensors can be used in order to measure the intracellular and extracellular environment of single cells, cell constructs, or tissue, and therefore the integration of monitoring techniques into 3D tissue culture platforms provides an essential step for the next generation Organ-on-Chip platforms. However, current in vitro platforms are not capable of combining the culture of 3D models with monitoring techniques. To address this, a novel spheroid encapsulation is designed for fluidic contact between 3D models in microwells and Intelligent Mobile Lab for In Vitro Diagnostics (IMOLA-IVD) BioChip sensors while preventing spheroid fusion. In this work, spheroid culturing protocols were developed for optimized spheroid growth and an evaluation of spheroid integrity on different porous layers was performed in order to provide a defined spheroid encapsulation on BioChip sensors.
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