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German lab magazine 'Laborjournal' was so kind to present us and our technology in their recent edition. Special thanks to Karin Hollricher for the...

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Venneos was invited by TechCrunch to pitch the CAN-Q on stage at CES2017 in LasVegas / USA. Watch the video of our short presentation on YouTube.

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Cytotoxicity & Viability

Viability (quantifying the number of viable cells) and cytotoxicity (quantifying the number of dead cells) cell-based assays are used to determine the health of a cell. Therefore, they play a major role in the field of human risk assessment and in the drug developmental process during preclinical drug evaluation. Traditionally, toxicity assessment is based on high dose animal studies. However, they are very expensive, time consuming and use a lot of animals which allows only very low throughput, without yielding any information of the mode of toxicity. Since this approach is impractical to evaluate tens of thousands of compounds a rethinking has been fulfilled in the field of toxicology: Turning away from animal studies to in vitro assays. 

The commonly used viability and cytotoxicity assays belong to the group of label-based cellular assays and focus on alteration in membrane integrity and thereby enable the uptake of a specific dye into the cell or the release of a specific dye into the supernatant. However, these assays have the decisive drawback, that for instance, some labels are toxic and can only be used at one point in time – thus the behavior of the cells over time is invisible. In contrast impedance spectroscopy offers the advantage of providing a highly time-resolved information on the viability of the cell, but has the limitation that several cellular alterations lead to the same change in the signal, making results unspecific and less interpretable. Additionally, the outcoming result is only an averaged signal over all cells and heterogenic effects are neglected. 

CAN-Spectroscopy provides an appropriate answer to these downsides by combining the upside of microscopy and impedance spectroscopy.

Performing CAN-Spectroscopy overcomes these limitations due to a label-free electrical readout. It is sensitive for a broad spectrum of cellular behavior but can distinguish between different effects on single cell level. Thus, CAN-Spectroscopy is a suitable tool to investigate and analyze – with an automated and quantitative single cell approach - different mechanism of cell death e.g. discrimination of apoptosis and necrosis, as well as different forms of tissue-specific toxicity. 

Discrimination of apoptosis vs. necrosis

Due to morphological changes, three types of mammalian cell death are known: Apoptosis, autophagy and necrosis. 

Apoptosis also known as programmed cell death is characterized by morphological changes like rounding-up of the cell, retraction of pseudopodes, reduction of cellular volume (pyknosis), condensation of the chromatin, fragmentation of the nucleus (karyorhexis), little or no ultrastructural modification of cytoplasmic organelles, plasma membrane blebbing, and maintenance of an intact plasma membrane until late stages of the process [1]. It plays a major role in tissue homeostasis during development and adultness, wound healing and immune regulation. Especially, tissue homeostasis is of main interest, since a deregulation of apoptosis results in severe malformations and is characteristic for e.g. cancer, AIDS, liver dieses or neurodegenerative disorders [2].

In contrast, morphological hallmarks of necrosis are cytoplasmic swelling, irreversible plasma membrane damage, and organelle breakdown. Although, necrosis was for a long time defined as an accidental uncontrolled type of cell death, more and more evidence has shown that necrosis is a well-defined and regulated process. It is a consequence of a crosstalk between several events on biochemical, molecular and cellular level. 

As well as apoptosis, necrosis occurs under physiological conditions and in disorders such as ischemic injury, neurodegenerative diseases and viral infections [3, 4] 

For the sake of completeness Autophagy cell death is also to be mentioned. It is defined as a type without chromatin condensation but with autophagic vacuolization of the cytoplasm that contains the degenerated cytoplasmic organelles or cytosol [1].

CAN-Spectroscopy is a suitable tool to analyze the two best known types of cell death: Apoptosis and necrosis. A mandatory condition – the discrimination between apoptosis and necrosis is fulfilled by CAN-Spectroscopy as it is possible to determine the membrane integrity on single cell level. Moreover, CAN-Spectroscopy determine the membrane integrity not only at a defined time point than rather facilitates a time resolved investigation of the complete cell death process and thereby yielding further information about the cell death mechanism. Thus, CAN-Spectroscopy offers an easy, unbiased, automated and quantitative analysis tool, contributing to a better understanding and finding answers to still unknown issues regarding apoptosis and necrosis. Thereby gaining knowledge about the mode of action of several neurodegenerative and immunological disease, which further contribute to a more improved and efficient drug discovery process.

Corresponding demo data including detailed experimental set up, CAN-Spectroscopy performance and data analysis are coming soon.

Organ-specific toxicity has become of major concern, since drugs and chemicals have been withdrawn from the market due to severe side effects. Especially, cardiotoxicity and neurotoxicity contribute to drug attrition [10]. Since the cardiovascular system and nervous system are very complex, highly regulated and specialized, even small interferences can result in severe malfunctions. To minimize toxicity, human- and cell-type specific assays with the presence of a relevant endpoint, which can be potentially disrupted by the drug / compound are needed.

Neurotoxicity

Important processes and functions of the (developing) nervous system that are targeted by potential neurotoxic compounds are proliferation, differentiation, migration, myelination, synapse formation and excitability as well as formation and function of neurons and glia cells.

Beside analyzing the influence of a specific compound on general cytotoxicity, CAN-Spectroscopy facilitates the simultaneous investigation of nervous system relevant endpoints. For instance, alterations in excitability can be determined. 

The nervous system is very complex, comprising of a heterogeneous population of billions of cells. Each cell type is characterized by a unique protein and ion channel composition which leads to a unique electrical fingerprint. Performing CAN-Spectroscopy allows the identification of electrical fingerprints of each cell within the population. Based on that, several questions concerning neurotoxicity can be investigated. For instance, whether a chemical rather equally affects all neural cell types within the population or shows cell-type specific toxicity. In addition, during development neuronal cells undergo several developmental stages with exclusive molecular and cellular characteristics that may contribute to changes in the sensitivity to certain chemicals. Thus, CAN-Spectroscopy is a suitable tool to identify the susceptible time window of specific neurotoxic compounds. 

CAN-Spectroscopy enables a multi-parametric screening and subsequent correlation of these phenotypic changes. 

Cardiotoxicity

The electrical pathways, which are responsible for the heart's rhythmic contraction is a highly complex and well coordinated process. Under physiological conditions, ions (Ca2+, K+, Na+) pass in and out of the cell resulting in cardiomyocyte contraction. Any interferences with that process result for example in cardiac arrhythmia, which is one of the most common causes of death [10]. Therefore, testing for such interferences is in the field of human risk assessment and drug discovery process of high demand.

 

Performing CAN-Spectroscopy with e.g. induced pluripotent stem cell derived cardiomyocytes is a suitable tool to monitor and quantify in real-time the beating signal of the cell population on single cell level. Moreover, the frequency and amplitude of the beating signal can be determined over a defined period of time, which enables the identification of the susceptible time window of specific cardiotoxic compounds. 

Sources

1. Kroemer G, El-Deiry WS, Golstein P, et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death. Cell death and differentiation. 2005;12 Suppl 2:1463-1467.

2. Elmore S. Apoptosis: a review of programmed cell death. Toxicologic pathology. 2007;35:495-516.

3. Festjens N, Vanden Berghe T, Vandenabeele P. Necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response. Biochimica et biophysica acta. 2006;1757:1371-1387.

4. Vandenabeele P, Galluzzi L, Vanden Berghe T, et al. Molecular mechanisms of necroptosis: an ordered cellular explosion. Nature reviews. Molecular cell biology. 2010;11:700-714.

5. Vaccarino FM, Ganat Y, Zhang Y, et al. Stem cells in neurodevelopment and plasticity. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 2001;25:805-815.

6. Du C, Xie X. G protein-coupled receptors as therapeutic targets for multiple sclerosis. Cell research. 2012;22:1108-1128.

7. Scott CW, Peters MF. Label-free whole-cell assays: expanding the scope of GPCR screening. Drug discovery today. 2010;15:704-716.

8. Zhang R, Xie X. Tools for GPCR drug discovery. Acta pharmacologica Sinica. 2012;33:372-384.

9. NRC. Toxicity Testing in the 21st Century. A Vision and a Strategy. The National Research Council Committee on Toxicity Testing and Assessment of Environmental Agents: The National Academies Press; 2007:p. 196.

10. Braam SR, Tertoolen L, van de Stolpe A, et al. Prediction of drug-induced cardiotoxicity using human embryonic stem cell-derived cardiomyocytes. Stem cell research. 2010;4:107-116.