200810 blog image special announcement nanion and friends week 2020

Presentation: 3D Bioprinting of Pathophysiological Human Heart Models using Vascularized Cardiac Spheroids as Building Blocks

Speakers:
Dr. Carmine Gentile | University of Technology Sydney

Abstract:
Current animal and 2D in vitro models fail to accurately recapitulate the complex human heart microenvironment. This leads to a poor translation of findings in the field of cardiovascular research from bench to the bedside. 3D bioprinting technology has emerged in the past 15 years as a tool for the bioengineering of complex tissues and organs with defined geometries. In this approach, tissue-tailored bioinks (composed of cells and other biomaterials) are deposited within permissive hydrogels to 3D bioprint viable and functional tissues. We developed cardiac bioinks by co-culturing cardiac myocytes, endothelial cells and fibroblasts in vascularized cardiac spheroids (or VCSs) that are embedded in alginate/gelatin-hydrogels. 3D bioprinted heart tissues (3DHTs) contain VCSs that are viable for at least 30 days in culture, are highly vascularized, act as building blocks by fusing within the hydrogel over time and contract synchronously when electrically paced. Currently, 3DHTs are used for in vitro drug discovery and toxicity studies, in disease modeling of myocardial damage (heart attack in a Petri dish) and for cardiac regeneration purposes in vivo. Given their unique features in better recapitulating the microenvironment typical of the human heart, 3DHTs have the potential to be used to both prevent and treat cardiovascular disease in patients.

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(due to a pending manuscript submission - this reproduced copy is delayed)* 


Presentation: ICH S7B best practices considerations - New Q&As

Speaker:
Dr. Sonja Stoelzle-Feix, Director Scientific Affairs Nanion, CiPA committee member Ion Channel Working Group

Abstract:
The ICH E14/S7B Implementation Working Group released a draft version on August 28th 2020 on “Clinical and Nonclinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential Questions and Answers”. This document is open for public consultation and comprises proposed revisions for some sections of the current Q&A´s for ICH E14. Furthermore, new Q&A for ICH S7B are included.

This talk will focus on best practice outlines as depicted in the draft version, specifically on in vitro cardiac ion channel assays. The ultimate goal is to provide a more robust and reproducible evaluation of potency of drug block of cardiac ion channel current using patch clamp techniques and heterologous expression systems.


Presentation: Benchmarking best practices and calibration standards for HTS hERG recordings for improved proarrhythmic assessment

Speaker:
Dr. Alison Obergrussberger | Scientific Communications Manager (Nanion Technologies)

Abstract:
The use of automated patch clamp (APC) electrophysiology in cardiac safety screening has increased over the years, and APC is now an established and accepted technique in most, if not all, safety testing laboratories. Since the introduction of the ICH S7B non-clinical guidance in November 2005 which requires all new drugs to be tested for activity on the IKr current carried by hERG expressed in recombinant cell lines using the patch-clamp technique, very few drugs have been withdrawn from the market due to pro-arrhythmic complications. APC has become the major workhorse in safety testing laboratories and is now considered to be the gold standard. Furthermore, with the introduction of the comprehensive in vitro pro-arrhythmia assay (CiPA) which recommends expanding electrophysiological recordings to include other cardiac ion channels, APC will continue to play a major role in cardiac safety testing. Recently, a large study comparing the results of a set of standard compounds tested on different instruments at different sites has been published[1] which highlights the need for standardized protocols for reliable results, for example, for hERG recordings. 

We have undertaken a study to identify key parameters that can affect IC50 values of compounds acting on hERG using the medium and high throughput APC systems, Patchliner, SyncroPatch 384PE and SyncroPatch 384i. Effects of experimental parameters such as voltage protocol, incubation time, labware, compound storage time and replicate number on IC50 values of a set of CiPA compounds will be presented and recommendations for best practices for hERG measurements using APC is provided. Furthermore, as outlined in the 2020 Best Practice Consideration for In vitro Studies, ‘The concentration of compound to which the cells were exposed should be verified by applying a validated analytical method to the solution collected from the cell chamber[2] in patch clamp studies. Nanion has implemented a new procedure that enables sample collection from used wells from the NPC-384 chips and this will be described.

  1. Kramer J, Himmel HM, Lindqvist A, et al. (2020) Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells. Nat Sci Reports 10:5627. doi: 10.1038/s41598-020-62344-w
  2. (2020) ICH E14 / S7B Implementation Working Group: Clinical and Nonclinical Evaluation of QT / QTc Interval Prolongation and Proarrhythmic Potential Questions and Answers. https://database.ich.org/sites/default/files/ICH_E14-S7B_QAs_Step2_2020_0827_0.pdf. Accessed 22 Sep 2020

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Presentation: Taking the FLEXcyte 96 to a new dimension: Integration of mechanical stimulation into a high-throughput cardiac contractility assay

Speakers:
Dr. Matthias Gossmann | co-founder and CEO (innoVitro GmbH)

Abstract:
In pre-clinical drug development, cardiac contraction analysis of potential drug candidates is one of the crucial steps to ensure a successful and reliable outcome for clinical studies. Safety, toxicology, and efficacy departments worldwide are seeking a modern contractility assay that combines the reliability of traditional in/ex vivo techniques with a high throughput and predictive human cell models.

The FLEXcyte 96 technology promises to remedy this situation by using human iPSC-derived cardiomyocytes in a 96 well approach to analyze cardiac contractility in vitro. Compared to standard cell culture substrates for cell-based assays, the physiological environment of the flexible membranes significantly improves the maturation status of the cells and enhances the validity of pharmacological tests. By application of static and dynamic mechanical stimuli, the FLEXcyte 96 mimics the environment cardiomyocytes would experience in a beating human heart during systolic and diastolic stress as well as during acute and chronic hypertension.

We conclude that the FLEXcyte 96 is the ideal tool for time and cost-effective cardiac contractility research and the only in vitro assay that supports human iPSC derived cardiomyocyte maturation via a physiological environment and mechanical stimulation.


Presentation: Turning Cells into Reagents [3PM CET / 9AM EDT]

Speakers:
Dr. Oliver Wehmeier and Dr. Tim Strassmaier | acCELLerate GmbH & Nanion Technologies

Abstract:
Assay Ready Cells represent a significant advantage over the traditional use of cells from a continuous culture. The cells can be used in the assay instantly after thawing, which eliminates the need for a complex cell culture. Assay Ready Cells are produced in large homogeneous batches of up to 1000 vials and can be stored for years in liquid nitrogen without loss of quality. Because individual vials of frozen Assay Ready Cells derive from a single batch the reproducibility can be significantly improved. Moreover, with Assay Ready Cells a user gains additional flexibility because the cells are always available from the frozen stock in a constant and pre-validated quality.

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Presentation: Evaluation of possible proarrhythmic potency: variability of IC50 values of drugs under different conditions and in different platforms

Speakers:
Dr. Péter Orvos | University of Szeged

Abstract:
The conventional microelectrode technique and the manual patch clamp method offer direct, information-rich, and real-time in vitro technologies to study proarrhythmic effect of drugs and drug candidate compounds. Although providing excellent data quality, these tests are complicated, time consuming and expensive for the large numbers of compounds. Automated patch-clamp platforms are mainly used with stably expressing cell lines and suitable for rapid and high-quality pharmacological investigation of drug candidates. The Comprehensive in Vitro Proarrhythmia Assay (CiPA) was initiated to further improve these preclinical drug safety paradigms. However, some evidence indicates that the different proarrhythmic pharmacological assays result in contradictory outcomes raising serious questions regarding their predictability for in vivo situations including clinical settings. IC50 values may varied between platforms, therefore, aim of our study was to compare the effect of proarrhythmic compounds on hERG and IKr currents and on cardiac action potential. The hERG current was measured by using both automated and manual patch clamp methods on HEK293 cells. The native ion current (IKr) were recorded from rabbit ventricular myocytes by manual patch clamp technique.

Dofetilide, cisapride, sotalol, terfenadine and verapamil were tested in hERG assay at both room temperature and 37°C with Patchliner. All these compounds were more potent at physiological temperature and therefore, it is a desirable option to study hERG currents at physiological temperature. To evaluate the prognostic value of hERG assay these agents were subjected for further investigations. The IKr current blocking capability of the compounds was tested on rabbit ventricular myocytes with manual patch clamp method at 37°C. The corresponding IC50 values of dofetilide, cisapride and verapamil were in good agreement with IC50 values obtained with Patchliner in hERG assays. As sotalol and terfenadine have stronger effect on IKr measured by manual patch clamp method compared with hERG automated patch clamp experiments, the effects of these drugs on hERG current using manual patch clamp technique were also investigated to study how the potency of these drugs are influenced by the experimental techniques themselves. In contrast with the hERG automated patch clamp assays, the effects of sotalol and terfenadine on hERG current were stronger measured by the manual patch-clamp technique.

In conclusion, results obtained with automated patch-clamp equipment in HEK-hERG cells usually show a reasonable conformity with outcomes of IKr current experiments. The Patchliner system used in our study is well suited to perform safety pharmacological studies. Variability of IC50 values of drugs in different platforms observed in certain cases, which could have been caused by the lack of continuous flow of compound-containing solutions. 

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Presentation: Heart slice culture system reliably demonstrates clinical drug-related cardiotoxicity

Speakers:
Dr. Tamer M. Mohamed | University of Louisville

Abstract:
The limited availability of human heart tissue and its complex cell composition are major limiting factors for the reliable testing of drug efficacy and toxicity. Recently, we developed functional human and pig heart slice biomimetic culture systems that preserve the viability and functionality of 300 μm heart slices for up to 6 days. Here, we tested the reliability of this culture system for testing the cardiotoxicity of anti-cancer drugs. We tested three anti-cancer drugs (doxorubicin, trastuzumab, and sunitinib) with known different mechanisms of cardiotoxicity at three concentrations and assessed the effect of these drugs on heart slice viability, structure, function and gene expression. Slices incubated with any of these drugs for 48 h showed diminished in viability as well as loss of cardiomyocyte structure and function. Mechanistically, RNA sequencing of doxorubicin-treated tissues demonstrated a significant downregulation of cardiac genes and upregulation of oxidative stress responses. Trastuzumab treatment downregulated cardiac muscle contraction-related genes consistent with its clinically known effect on cardiomyocytes. Interestingly, sunitinib treatment resulted in significant downregulation of angiogenesis-related genes, in line with its mechanism of action. Similar to hiPS-derived-cardiomyocytes, heart slices recapitulated the expected toxicity of doxorubicin and trastuzumab, however, slices were superior in detecting sunitinib cardiotoxicity and mechanism in the clinically relevant concentration range of 0.1–1 μM. These results indicate that heart slice culture models have the potential to become a reliable platform for testing and elucidating mechanisms of drug cardiotoxicity.

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