3Rs IN ACTION

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 [1]. A remaining issue is that many laboratories use fetal bovine serum (FBS) in standard cell culture. Beside the questionable way of manufacturing of FBS [2], 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 [2]. 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 [3] and in microphysiometric experiments to determine the eye irritation potential of chemicals.

 

[1]     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

[2]     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

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

IEEE EMBC 2017

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.

ICBA 2017

2nd int’l Conference on  Biomaterials and Applications (ICBA 2017)

 

Oral presentation: Automated microphysiometer for assessment of cytotoxicity

Cytotoxic properties of new chemicals or drugs have to be assessed before they enter into the market. Here we present an automated method to investigate the interaction of soluble substances with living cells. Living cells are cultivated on a BioChip which measures their cellular respiration, extracellular acidification and changes in impedance online. With an automated fluidic system different concentrations of the substance under investigation can be transported to the cells. This makes it possible to determine the cytotoxic potential of the substance. By removing the substance it is possible to monitor the recovery of the cells and to distinguish between a true toxic effect and an inhibitory effect. Examples from fields as environmental monitoring, eye irritation testing and repeated dose toxicology are presented. An outlook toward the development of organ on chip systems is given.

Sebastian Eggert receives VDI award

Sebastian Eggert was awarded with the 2016 VDI award for his master thesis “Development and evaluation of a 3D hepatocyte model for metabolic monitoring”. The work was supervised by Prof. Dr. Dirk Weuster-Botz (TU München) and Dr. Frank A. Alexander (cellasys) and integrated human liver spheroids and the IMOLA-IVD technology.

http://www.verein-der-ingenieure.de/bezirksverein-muenchen/was-wir-tun/berichte-von-veranstaltungen/vdi-preis-2016/index.html

ARTE docu

“Tierversuche – es geht nicht ohne” (animal experiments – we cannot yet replace all of them) / “Tests animaliers, ne pourrait-on pas s’en passer?”


The joint French-Germany cultural TV channel ARTE broadcasted “Tierversuche – es geht nicht ohne” (animal experiments – we cannot yet replace all of them) / “Tests animaliers, ne pourrait-on pas s’en passer?” on 07.02.2016 with participation of cellasys.

 

… please send an eMail to info@cellasys.com to receive a download link of the broadcasting …

ROCHE SERVICE DAY 2016

Meet Bavarian Biotech – innovative technology solution


cellasys attends the Roche Service Day 2016.

(Download brochure here)

EUROTOX 2016

Meet us in Seville at the 52nd European Congress of the European Society of Toxicology.


S. Eggert, F. A. Alexander Jr., J. Wiest: Real-time Spheroid Monitoring via an Automated Microphysiometer

Currently, in vitro toxicity studies rely heavily on chemical labelling and end-point assays to characterize the response of the body to newly developed compounds. While satisfactory for simple toxicity screening using planar cultures, chemical labels may affect the basal metabolic function of certain cell types. More physiologically relevant 3D cultures, such as Organs-on-Chips, are emerging as a advantageous tool for more accurate toxicology studies. However, conversion of these models into a high-throughput format is complicated by the requirement of significantly larger numbers of cells per model. Additionally, long-term studies require multiple samples for each time-point investigated, further increasing the necessary cell stock. For this reason, real-time monitoring techniques, like cellular microphysiometry, can provide a beneficial platform for studying 3D tissue cultures by screening multiple time-points using a single sample. Herein, we have developed a label-free technique for the culture and monitoring of 3D spheroids on a BioChip, which is able to measure extracellular acidification rate and oxygen consumption rate in real-time. 3D-printed microwell arrays were incorporated with porous layers into Intelligent Mobile Lab for In Vitro Diagnostics (IMOLA-IVD) BioChips to maintain spheroids. HepG2 liver cells were seeded to form liver spheroids at 1000 cells per spheroid over the course of four days. Mature liver spheroids measuring 622 microns in diameter were transferred onto newly designed chips and perfused for 36 hours prior to lysis with sodium dodecyl sulfate. The presented work serves as a candidate study for extended long-term monitoring of spheroids on the IMOLA-IVD.

F.A. Alexander J., J. Wiest: Skin-On-A-Biochip: Leveraging Cellular Microphysiometry for IMOLA-based Organ-on-a-Chip Studies

Currently, protocols that predict organ toxicity using animal testing are inaccurate at predicting toxicity in vivo and present an ethical dilemma in regards to animal welfare. The advent of more accurate 3D in vitro microphysiological systems, so-called organs-on-a-chip (OOCs), has improved our ability to probe the potential effects of drug candidates in vivo. Cellular microphysiometry systems like the IMOLA-IVD (cellasys GmbH), a microsensor array-based assaying technique, offer a solution to these issues with the ability to noninvasively monitor biological changes in real-time. A candidate 3D in vitro culture, the reconstructed human epidermis (RHE) artificial skin, was integrated with a prototype IMOLA-IVD biochip designed for online monitoring of commercially manufactured artificial skin models. Automated monitoring of an RHE in real-time can reveal time-resolved data on the toxic effect new compounds have on the epidermis. For this reason, we developed a protocol for an automated skin corrosion/irritation assay that monitors transepithelial electrical resistance (TEER) and extracellular acidification (ECAR). Metabolic signals were recorded in real-time in an incubator via a modified IMOLA-IVD system. EpiDerm RHE’s from MatTek In Vitro Life Sciences Laboratories were perfused automatically with medium via a peristaltic pump, IMOLA fluidic modules and the DALiA control software. Future work will involve developing this method into more assays that monitor other in vivo-like 3D cultures (i.e. liver spheroids) to develop a plug and play suite of IMOLA organ models for in vitro toxicity testing.

J. Wiest: Automated INVITTOX protocol # 130

The IMOLA-IVD technology monitors the extracellular acidification, cellular respiration and changes in impedance of living cells. In combination with a standard peristaltic pump, proprietary fluidic modules and control software it was possible to set up different cell based assays or organ-on-chip models. In this work we set up a configuration to transfer the cytosensor microphysiometer test method for identification of eye irritation which was validated by the European commission for Validation of Alternative Methods (ECVAM). It measures the extracellular acidification rate (EAR) of L929 mouse fibroblasts and the influence of e.g. the detergent sodium dodecyl sulfat (SDS) toward their EAR. The results show that the determination of the metabolic rate decrement by 50% (MRD50) value can be automated with the proposed set-up. Furthermore it was possible to further develop the INVITTOX protocol toward a fetal bovine serum (FBS) free assay. This simplifies the assay (no difference between seeding and low-buffered treatment medium) and excludes ethical issues related to FBS. Furthermore an increase in reproducibility is expected since inter-laboratory differences due to the problem of different personnel and different lots of FBS are overcome. The necessary adaptations of the INVITTOX protocol # 130 are described and measurements using FBS-free L929 fibroblasts are presented.

J. Wiest: Automated long-term monitoring of extracellular acidification and changes in impedance of living cells

Label-free monitoring of living cells is useful to determine e.g. delayed effects of chemicals which can not be addressed by conventional endpoint-assays. In the presented work a technology to dynamically monitor the energy metabolism of living cells is described. The IMOLA-IVD technology is introduced which monitors extracellular acidification and changes in bioimpedance of living cells. To allow long-term experiments, a fluidic system is included to supply the cells with fresh cell culture media and to add or remove the chemical compound under investigation. A short historical review on microphysiometry is given. Examples from application fields as oncology, toxicology, regenerative medicine and environemental monitoring are presented and further challenges such as data processing are highlighted. The presented technology is able to perform label-free long-term experiments on living cells and to reveal delayed effects of drugs or chemicals.

BIOGRAPHY

Press release: Printed sensors for research and development on antiviral drugs


Printed sensors for research and development on antiviral drugs

The efficacy of novel antivirals is increasingly studied with modern measuring methods such as real-time monitoring of live human cells. This helps to minimize the use of animal testing and increases the significance of the test for its relevance to patients. An interdisciplinary team of researchers including cellasys GmbH, AiCuris GmbH + Co. KG, SAUERESSIG GmbH + Co. KG and Haydale Limited, led by the Fraunhofer Institute for Biomedical Engineering IBMT, have now reached a milestone in the BMBF funded project BIOGRAPHY. For the first time, living cells have been examined with the help of printed graphene sensors.

Within this project, cellasys is responsible for the specification and characterization of the sensors. Currently, the growth behavior of living cells can be monitored electronically on the graphene structures using the IMOLA-Technology. To achieve this goal the scientists have developed a new graphene ink and have optimized the printing process. Stability and biocompatibility of the microstructures were both confirmed during the initial phase of the project starting in 2014.

cellasys’ BioChips have been used primarily in medical research. With these new advances in printing technology, they can now be manufactured at considerably lower costs. Thus, they are now suitable for industrial applications such as pharmaceutical research and development of drugs to combat viral infections. Additionally, applications in the field of comprehensive water monitoring are now conceivable. The projected completion date of this collaborative research project is in 2017, ending with a parallelization of the measurement setup and a final testing.

Further information: www.graphene-biosensors.eu

This research and development project is partially funded by the Innovate UK and the German Federal Ministry of Education and Research (BMBF) within the Framework Concept “Research for Tomorrow’s Production” (funding number 02PN2240) and managed by the Project Management Agency Karlsruhe (PTKA).

The company

cellasys GmbH offers system solutions for microphysiometry. These include services such as contract research, research & development, and production & maintenance. Furthermore cellasys works as consultants for development of applications, data analysis and data interpretation. The microphysiometric systems monitor different parameters directly from living cells. These parameters include extracellular acidification (pH), cellular respiration (pO2) and morphology (impedance). The measurements are label-free, parallel, continuous and in real-time. With the BioChip technology you can e.g. determine the efficiency of a drug outside of humans (or animals) prior to the start of the therapy.

Further information: www.cellasys.com

Top: Printed graphene on a transparent foil. Bottom: Encapsulated sensor for use in cellasys’ microphysiometric systems.

Top: Printed graphene on a transparent foil. Bottom: Encapsulated sensor for use in cellasys’ microphysiometric systems.

EMBC 2016

Visit our presentation “Automated Transepithelial Electrical Resistance Measurements of the EpiDerm Reconstructed Human Epidermis Model” at the 38th Annual International Conference of the IEEE Engineering in Medicine and Biology Society.


Automated Transepithelial Electrical Resistance Measurements of the EpiDerm Reconstructed Human Epidermis Model

Understanding the effect of exogenous substances on human skin is critical for toxicology assessment. To address this, numerous artificial models of the topmost layer of human skin, so-called reconstructed human epidermis (RhE), have been created in an attempt to produce a clear analogue for testing. Unfortunately, current testing modalities still rely on endpoint assays and are not capable of monitoring time-resolved changes in barrier function without using numerous redundant samples. In this work, a novel, time-resolved approach is realized by monitoring the transepithelial electrical resistance (TEER) of MatTek EpiDerm® reconstructed human epidermis model, utilizing an automated protocol with the Intelligent Mobile Lab for in vitro diagnostics (IMOLA-IVD).