THREE-STAGE APPROACH FOR EVALUATION OF A CHEMICALLY DEFINED CELL CULTURE MEDIUM FOR THE CACO-2 CELL LINE: SHORT-TERM EFFECTS, DIFFERENTIATION POTENTIAL AND LONG-TERM CULTIVATION
Christian Schmidt1,2 and Joachim Wiest1,2*
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.
Replacing Fetal Bovine Serum
The Academy for Animal Welfare and cellasys host a Symposium about Replacing Fetal Bovine Serum. It will take place online on November 3rd, 2020.
Tissue-on-a-Chip: Microphysiometry With Human 3D Models on Transwell Inserts
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
cellasys #8: A microphysiometric assay to asses enhanced cell culture media
Christian Klenk1,2 and Joachim Wiest1,2*
11th World Congress on Alternatives and Animal Use in the Life Science, Abstract overview, 2020, 33
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
A new protocol based on a microphyiometric system (McConnell et al., 1992, Hartung et al., 2010, Wiest et al., 2016, Alexander et al., 2018, Brischwein and Wiest, 2019) to analyze cell culture medium (CCM) is described. With the presented cellasys #8 protocol, significant data can be gained in 24 h compared to conventional weaning experiments which need several weeks to perform. First, L929 cells are supplied for 6 hours with DMEM + FBS reference medium to gain initial data. In a second step, 6 hours of the investigated test medium is supplied to see if there are any changes in cellular vitality or morphology. Then again 4 hours DMEM + FBS and 4 hours of test medium to monitor if the effect of the CCM to the cells is reversible. The experiment ends with 4 hours of test medium + 0.2% SDS to induce cell death. In the presented work, two chemically defined CCM and a common serum-containing medium DMEM + FBS were tested on the L929 cell line. Compared to the reference medium, cells in the DMEM/Ham’s F12 + ITS medium pursue a loss in adherence, but no decrease in extracellular acidification rate. This was substantiated by the observation that the acidification rate remained constant and the impedance recovered after changing back to the reference medium. Cells in NCTC 135 retained their impedance values but lost vitality. It seems reasonable to suppose that cells in NCTC 135 medium are slowly suffering as indicated by a slow decrease in impedance and highly fluctuating acidification rates. Further experiments for the presented method could be the improvement of the DMEM/Ham’s F12 + ITS medium. With the new method electrical cell-substrate impedance and extracellular acidification responds of the cells can be measured immediately and consequently the quality of new CCM can be quantified.
Alexander, F., Eggert, S. and Wiest, J. (2018) ‘A novel lab-on-a-chip platform for spheroid metabolism monitoring’, Cytotechnology. Springer Netherlands, 70(1), pp. 375–386. doi: 10.1007/s10616-017-0152-x.
Brischwein, M. and Wiest, J. (2019) ‘Microphysiometry’, in Wegener, J. (ed.) Label-Free Monitoring of Cells in vitro. Cham: Springer International Publishing, pp. 163–188. doi: 10.1007/11663_2018_2.
Hartung, T. et al. (2010) ‘First alternative method validated by a retrospective weight-of-evidence approach to replace the Draize eye test for the identification of non-irritant substances for a defined applicability domain.’, Altex, 27(1), pp. 43–51. doi: 10.14573/altex.2010.1.43.
McConnell, H. M. et al. (1992) ‘The cytosensor microphysiometer: Biological applications of silicon technology’, Science, 257(5078), pp. 1906–1912. doi: 10.1126/science.1329199.
Wiest, J. et al. (2016) ‘Data Processing in Cellular Microphysiometry’, IEEE Transactions on Biomedical Engineering, 63(11), pp. 2368–2375. doi: 10.1109/TBME.2016.2533868
Chemically defined cell culture media – a contribution to address the reproducibility crisis in biomedical sciences
Tilo Weber1, Kristina Wagner1, Joachim Wiest2*
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
Chemically defined formation of spheroids loaded with superparamagnetic iron oxide nanoparticles
J. Wiest1*, E. Stetter2, M. Koch3
11th World Congress on Alternatives and Animal Use in the Life Science, Abstract overview, 2020, 60
1 cellasys GmbH, Kronburg / Germany,
2 Stetter Elektronik, Seeheim-Jugenheim / Germany,
3 Feldkraft Ltd., Copenhagen / Denmark
Corresponding author: J. Wiest, wiest@cellasys.com
To increase physiological relevance and to develop new therapies 3D spheroids are of increasing interest. The addition of superparamagnetic iron oxide nanoparticles (SPIONs) into the spheroid allows for investigation of new therapeutic strategies (Zhang et al., 2014) without the use of animals (Lei and Schaffer, 2013; Whatley et al., 2014; Marx et al., 2016). L929 fibroblasts were maintained in 25 mL Greiner Bio-One AdvancedTC© cell culture flasks in chemically defined DME/F12+ITS cell culture medium. Cells received fresh medium twice a week and were passaged at about 80% confluency once a week. Spheroids were created similar to a previously developed method (Alexander, Eggert and Wiest, 2018). The 20 nm SPIONs beads were obtained from micromod Partikeltechnologie GmbH (Rostock / Germany) and a stock solution with 100 µg/mL was prepared. Spheroids were prepared in a 96 well-plate with cell-repellent surface (Greiner Bio-One GmbH, #650970). Each well was filled with 190 µL DME/F12+ITS containing 10.000 L929. For the SPION loaded spheroids (SPION-LS), 10 µL of magnetic beads stock solution was added, whereas for the non-magnetic control spheroids (NM-CS) 10 µL of cell culture medium was added. Then the 96well plate was centrifuged at 1000 g for 5 min and finally incubated at 37°C with 5% CO2. 100 µL of medium in each well was replace by fresh DME/F12+ITS (preheated to 37°C) daily. Spheroids were used for the experiments on day 5. For manipulation of the spheroids a neodym magnet (length 25 mm, diameter 25 mm) was used in a distance of approximately 30mm. The calculated field force acting on the spheroids was approximately 30mT. To investigate if the SPIONs are incorporated into the spheroids one SPION-LC was transferred to a well with one NM-CS. The movement of the SPION-LC due to the applied magnetic field can be seen at https://youtu.be/ev9bZnKyvGA .
Alexander, F., Eggert, S. and Wiest, J. (2018) ‘A novel lab-on-a-chip platform for spheroid metabolism monitoring’, Cytotechnology. Springer Netherlands, 70(1), pp. 375–386. doi: 10.1007/s10616-017-0152-x.
Lei, Y. and Schaffer, D. V. (2013) ‘A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation’, Proceedings of the National Academy of Sciences of the United States of America, 110(52). doi: 10.1073/pnas.1309408110.
Marx, U. et al. (2016) ‘Biology-inspired microphysiological system approaches to solve the prediction dilemma of substance testing’, Altex, 33(3), pp. 272–321. doi: 10.145/3/aitex.1603161.
Whatley, B. R. et al. (2014) ‘Magnetic-directed patterning of cell spheroids’, Journal of Biomedical Materials Research – Part A, 102(5), pp. 1537–1547. doi: 10.1002/jbm.a.34797.
Zhang, E. et al. (2014) ‘Dynamic Magnetic Fields Remote-Control Apoptosis via Nanoparticle Rotation’, ACS Nano. American Chemical Society, 8(4), pp. 3192–3201. doi: 10.1021/nn406302j.
Cell line authentication
For biocompatibility assays and the cellasys #8 protocol we use the L929 cell line. As a prerequisition for reliability of data, our cell line was recently analysed by the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH.
Please find the species identification in our quality management section.
EUSAAT 2019
cellasys, Technische Universität München and MatTek In Vitro Life Science Laboratories present recent updates about our “Tissue-on-a-Chip” at the 22nd European Congress on Alternatives to Animal Testing:
Tissue-on-a-Chip
In this study we demonstrate a microphysiometric system based on IMOLA‑IVD device that will allow real-time measurement of multiple parameters in human small intestine tissue model (EpiIntestinalTM). The parameters are measured non-invasively, while the system provides fresh nutrients and allows programmable repeated dosing of test articles. In the presented set of experiments we have concentrated on trans-epithelial electric resistance (TEER) as this parameter was previously established to reflect an integrity of EpiIntestinal tissue model. The performance of the system was verified and baselines were established by long term incubation of EpiIntestinal tissue in IMOLA-IVD, followed by single application of 2% SDS.