Joachim Wiest¹, Frank Schulze²

11th World Congress on Alternatives and Animal Use in the Life Science

Corresponding author’s e-mail: wiest@cellasys.com

1 cellasys GmbH
2 BfR – Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments

Organ-on-chip (OoC) systems are a promising alternative to the still common animal experimentation. However, when compared to OoC systems found in literature such as lung, liver or kidney, bone is underrepresented as indicated by the rather low number of
proposed systems. One reason is that the development of bones in-vitro is a time-consuming process that can take up to several weeks. Currently, the process of matrix mineralization can only be investigated by end-point assays that require the destruction of the organoid. Therefore, a sensor-based, reliable and robust method for a non-invasive and continuous determination of in vitro matrix mineralization would be of great advantage, especially when 3D constructs are cultured in microphysiological bioreactors. In the presented work, we show data from impedance measurement on a collagen scaffold-dummy with osteogenic medium. Measurements were performed in a bioreactor (ID = 13 mm, height = 3mm) which was sandwiched by two impedance foils (Bio24, well B3 with passivated feed line). For impedance measurement a VersaSTAT 3 was used. Impedance spectroscopy from 1 Hz to 1MHz with an RMS of 30 mV was performed. The setup was mounted in a Faradic cage which was heated to 37°C. With the performed measurement we were able to distinguish pure osteogenic medium from a medium soaked collagen scaffold by approximately 60 Ω in the real part. During the performed proof-of-principle measurement no clear difference in the imaginary part of the impedance was visible. Further measurements with mineralized bone tissue are planned to further investigate the possibilities of the technology. The results indicate that the proposed geometry can be used to perform impedance measurement at bone scaffolds. Furthermore, a fixed frequency of 10kHz and 30mV seems to be suitable for the measurement.

cellasys was nominated by Bayern Innovativ for the Innovation Leben Award 2020.

Christian Schmidt^1,2 and Joachim Wiest^1,2*

11th World Congress on Alternatives and Animal Use in the Life Science, Abstract overview, 2020, 53

1 cellasys GmbH, Kronburg / Germany
2 Technical University of Munich, Heinz-Nixdorf-Chair of Biomedical Electronics, Department of Electrical and Computer Engineering, TranslaTUM, Munich / Germany

Corresponding author: wiest@cellasys.com

Finding a chemically-defined cell culture medium as a replacement for FBS is a timeconsuming and costly process. In this work, we present a three-stage approach for evaluating the chemically-defined DMEM/F12+ITS medium (SOP-G200-005_DME_F12+ITS) for the Caco-2 cell line. The three stages are increasingly lengthy and identify adhesion, proliferation and metabolism changes of the cell culture. The first stage utilizes the microphysiometric system intelligent mobile lab for in-vitro diagnostic (IMOLA-IVD) to reveal any short-term effects caused by the chemically-defined medium. The IMOLA-IVD device enables an automated cell analysis by label-free measurements of the acidification rate and impedance. Additionally, the device provides fresh nutrients to the Caco-2 cells regularly by a pre-programmable protocol. Herein, the used protocol cellasys #8 is defined as follows: 6 h DMEM + 5% FBS, 6 h DMEM/F12+ITS, 4 h DMEM + 5% FBS, 4 h DMEM/F12+ITS, 4h positive control with 2% Sodium dodecyl sulphate (SDS). In the experimental results no short-term effects and cellular stress responses are visible. This suggests that all major nutrients are present in the chemically-defined medium. The second stage is a differentiation cultivation in a T25 flask for 40 days while the third stage is a long-term cultivation for 100 days. The qualitative results of these stages obtained by a light microscope show that the proliferation and adhesion is reduced compared to DMEM + 5% FBS cultivated cells but is constant during the long-term cultivation suggesting that there are no missing nutrients. With the IMOLA-IVD system, the screening time for finding minimal, chemically-defined media can be reduced. The real-time measurement of the device shows any cell stress caused by missing nutrients in a chemically-defined medium formulation. This enables a rapid first stage screening with a duration of 24 hours.

Christian Schmidt¹, Jan Markus², Helena Kandarova ^3,4 and Joachim Wiest ^1,5*

Front. Bioeng. Biotechnol., 04 August 2020 | https://doi.org/10.3389/fbioe.2020.00760

¹cellasys GmbH, Kronburg, Germany
²MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
³Centre of Experimental Medicine SAS, Slovak Academy of Sciences, Bratislava, Slovakia
⁴Institute of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Slovakia
⁵Heinz Nixdorf Chair of Biomedical Electronics, Department of Electrical and Computer Engineering, Technical University of Munich, Munich, Germany

Corresponding author: wiest@cellasys.com

Microphysiometry has proved to be a useful tool for monitoring the energy metabolism of living cells and their interactions with other cells. The technique has mainly been used for monitoring two-dimensional (2D) monolayers of cells. Recently, our group showed that it is also possible to monitor the extracellular acidification rate and transepithelial electrical resistance (TEER) of 3D skin constructs in an automated assay maintaining an air–liquid interface (ALI) with a BioChip extended by 3D-printed encapsulation. In this work, we present an optimized multichannel intestine-on-a-chip for monitoring the TEER of the commercially available 3D small intestinal tissue model (EpiIntestinal^TM from MatTek). Experiments are performed for 1 day, during which a 60 min cycle is repeated periodically. Each cycle consists of three parts: (1) maintain ALI; (2) application of the measurement medium or test substance; and (3) the rinse cycle. A cytotoxic and barrier-disrupting benchmark chemical (0.2% sodium dodecyl sulfate) was applied after 8 h of initial equilibration. This caused time-dependent reduction of the TEER, which could not be observed with typical cytotoxicity measurement methods. This work represents a proof-of-principle of multichannel time-resolved TEER monitoring of a 3D intestine model using an automated ALI. Reconstructed human tissue combined with the Intelligent Mobile Lab for In vitro Diagnostic technology represents a promising research tool for use in toxicology, cellular metabolism studies, and drug absorption research.

Tilo Weber¹, Kristina Wagner¹, Joachim Wiest^2*

11th World Congress on Alternatives and Animal Use in the Life Science, Abstract overview, 2020, 114

1 German Animal Welfare Federation
2 cellasys GmbH

Corresponding author: wiest@cellasys.com

The use of fetal bovine serum (FBS) in cell culture media has different drawbacks. The
harvesting of FBS from bovine fetuses after slaughter of the pregnant parent (dam)
raises ethical and legal concerns. From a pure scientific point of view the use of FBS is
inacceptable since regional differences in type and concentration of ingredients exist
(Baker, 2016, van der Valk et al., 2010). Hence, a non-definable quality of FBS
undermines data reliability and decrease or even prevent experimental reproducibility.
However, the use of chemically defined cell culture media is still scarce.
The introduction of chemically defined cell culture media is a contribution to fight the
reproducibility crisis in the biomedical sciences and an approach to address animal
welfare concerns. Usage of chemically defined medium will eliminate some unknowns in
cell culture experiments. A procedure to develop serum free media was introduced by
van der Valk and colleagues (van der Valk et al., 2010). In the presented work, a detailed
recipe to prepare a defined DMEM / Ham’s F12 + ITS cell culture medium is given. This
medium has proven to work in our laboratory for L929 and Caco-2 cell lines in
combination with a certain plastic ware (Greiner Bio-One, Advanced-TC). To prepare the
chemically defined cell culture medium (DME /F12+ITS) mix 50% DMEM (e.g. Sigma
Aldrich, D5648) and 50% Ham’s F12 (e.g. Sigma Aldrich, N6760), add 14.7 mmol/l NaCl,
20.9 mmol/l NaCHO3 and 5 ml/l ITS (Sigma Aldrich, I3146) (cellasys, 2019). The medium
was developed for cell culture in a 5% CO2 incubator. The presented method was
employed to develop a chemically defined cell-based assay for cytotoxicity
determination according to ISO10993-5 (Wiest, 2017), furnishing a first proof of its
applicability as an alternative to cell culture media containing FBS.

Baker, M. (2016). Reproducibility: Respect your cells!. Nature 537, 433–435.
doi:10.1038/537433a;

cellasys (2019). https://www.cellasys.com/wpcontent/
uploads/2019/10/wwwSOP-G200-005_DME-F12-ITS_medium_preparation_V1_3-
1.pdf;

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(4), 1053–1063. doi:10.1016/j.tiv.2010.03.016;

van der Valk, J., Bieback, K., Buta, C. et al. (2018). Fetal Bovine Serum (FBS): Past – Present –
Future. ALTEX 35(1), 99–118. doi:10.14573/altex.1705101;

Wiest, J. (2017). Chemisch definiert – ein zellbasierter Zytotoxizitätsassay ohne fötales Kälberserum. BIOspektrum
23, 61. doi:10.1007/s12268-017-0768-6