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.

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