iPSCs and New Approach Methodologies (NAMs) technology journal II

A highly reproducible and precise measurement platform for 3D engineered cardiac muscle tissue contractility

Svetlana Pasteuning1 (svetlana.pasteuning@optics11life.com), Tom Berkers1, Giulia Pilia1, Elizaveta Loseva1, Thorsten Jonas1, Eliano dos Santos2, Katarzyna Kmiotek-Caller2, Ravi A. Kumar2, Anna Zoccarato2

1 Optics11 Life, Amsterdam, The Netherlands,2 School of Cardiovascular & Metabolic Medicine and Science, King’s College London, British Heart Foundation Centre of Excellence, United Kingdom

Abstract

Engineered heart tissues (EHTs) offer more predictive insights into cardiac function than traditional 2D models but are often limited by reproducibility and scalability. Using Cuore, a 3D contractility platform from Optics11 Life, we established a robust workflow for generating consistent 3D EHTs from human Axol’s iPSC-derived ventricular cardiomyocytes and cardiac fibroblasts. The platform enables controlled culture, pacing, and real-time force measurement with nanonewton sensitivity (Figure 1). Tissues show stable maturation and respond sensitively to isoprenaline, confirming reliable detection of functional drug effects. Cuore provides a scalable, high-fidelity solution for human-relevant cardiac drug testing.

Methods

Human EHTs were generated by combining 70% human iPSC-derived ventricular cardiomyocytes from Axol with 30% primary cardiac fibroblasts in a hydrogel matrix to form 3D tissue bundles. Constructs were cast into the 24-well plate Cuore platform (Figure 2), enabling controlled culture conditions and real-time functional assessment. Tissues were maintained for 42 days with daily medium changes and daily longitudinal monitoring. Contractile function of the EHTs was assessed by measuring spontaneous force of contraction, beating frequency, contraction and relaxation kinetics. At the end of the experiment, tissues were challenged with isoprenaline and β1- and β2-adrenergic receptor antagonists, followed by fixation for downstream marker stainings. The tissues were stained with DAPI, Troponin, and Vimentin (Figure 3).

Results

Immunostaining with DAPI, Troponin, and Vimentin revealed well-organized, healthy tissue bundles (Figure 3). No signs of necrosis or tissue damage were observed, supporting the overall robustness and physiological relevance of the model. Engineered heart tissues demonstrated progressive functional maturation over the 42-day culture period, characterized by an increase in force of contraction and spontaneous beating frequency (Figure 4A-B) and faster contraction and relaxation time and velocity (Figure 4C-D). Tissues remained viable and functional throughout the entire duration, indicating long-term stability of the model. Pharmacological stimulation with isoprenaline induced a clear dose-dependent increase in force of contraction under paced (1.5 Hz) and non-paced conditions (Figure 5A). This effect was effectively blocked by commercially available β1 (ICI189)- and β2 (ICI118)-adrenergic receptor antagonists, confirming pathway-specific responsiveness (Figure 5B).

Conclusion

These results demonstrate that the Cuore platform enables the generation of stable, functionally maturing engineered heart tissues with reliable contractile readouts over extended culture periods. Cuore’s non-invasive, real-time force measurement with nanonewton sensitivity, combined with controlled pacing and multi-well scalability, enables standardized, reproducible assay conditions. The tissues exhibit physiologically relevant, dose-dependent pharmacological responses that can be specifically modulated via β-adrenergic pathways. Combined with healthy tissue structure and absence of damage, this supports the robustness and reproducibility of the model. Together, Cuore – a 3D muscle contractility platform and Axol’s hiPSC-derived cardiomyocytes provide a commercially ready, end-to-end solution that aligns with New Approach Methodologies (NAMs) by enabling human-relevant, predictive, and animal-free cardiac efficacy and safety drug testing.

Functional profiling of human iPSC‑derived neuronal networks with 3Brain’s CorePlate™ HD‑MEA technology

Ivan Verduci

3Brain IT SRL | V.le Brigate Partigiane, 14/3-4, 16129, Genova, Italy

Abstract

Functional neuronal networks derived from human iPSC‑based cell types provide a powerful platform for studying synaptic development, network maturation, and pharmacological responses. This study presents the electrophysiological profiling of axoCells™ cortical excitatory neurons co‑cultured with axoCells™ astrocytes, recorded on 3Brain’s CorePlate™ HD‑MEA system. Leveraging high‑resolution sensing and integrated BrainWave6 analysis, we highlight robust, reproducible maturation and clear responses to excitatory and inhibitory modulators. Together, these results demonstrate a sensitive, reproducible, and assay‑ready in vitro model ideally suited for neuroscience research and drug screening.

Methods

Cell Culture

ax0015 Neural Stem Cells were cultured on Poly‑D‑lysine and Axol SureBond‑XF‑coated CorePlate™ devices. At Day 19, ax0665 human iPSC‑derived astrocytes were added at an 8:1 neurons‑to‑astrocytes ratio to support network development.

Electrophysiological Recordings

Electrophysiological activity was recorded using the 3Brain HyperCAM Alpha platform with CorePlate 6W 38/60, featuring 13,824 simultaneously active electrodes (2,304 per well) arranged within a 2.9 × 2.9 mm² active area (21 × 21 µm electrodes, 60 µm pitch). Signals were sampled at 10 kHz with a 100 Hz high‑pass filter and acquired longitudinally from Day 19 to track network maturation.

Pharmacological Assay

5 µM AMPA + 5 µM NMDA or 10 µM NBQX + 50 µM AP5 were used to modulate electrical activity.

Analysis Software

Data were analyzed in BrainWave6, extracting spikes and network burst metrics, raster plots, connectivity maps, and CAT analysis within a unified, fully integrated workflow.

Results

The findings from this study highlight the complementary strengths of Axol’s human iPSC‑derived neuronal biology and 3Brain’s CorePlate‑enabled HD‑MEA technology in resolving the functional properties of cortical excitatory networks. axoCells™ Cortical Excitatory Neurons co‑cultured with axoCells™ Astrocytes showed clear and reproducible maturation signatures, developing stable, synchronized activity and predictable responses to excitatory modulation, demonstrating their suitability for mechanistic studies and pharmacological evaluation.

Conclusion

The findings from this study highlight the complementary strengths of Axol’s human iPSC‑derived neuronal biology and 3Brain’s CorePlate‑enabled HD‑MEA technology in resolving the functional properties of cortical excitatory networks. axoCells™ Cortical Excitatory Neurons co‑cultured with axoCells™ Astrocytes showed clear and reproducible maturation signatures, developing stable, synchronized activity and predictable responses to excitatory modulation, demonstrating their suitability for mechanistic studies and pharmacological evaluation.

By leveraging 3Brain’s CMOS‑based HD‑MEA platform, 13,824 simultaneously recording electrodes across the six‑well format, we achieved high‑resolution mapping of network behavior, capturing both large‑scale population dynamics and subtle changes arising during maturation or after pharmacological intervention. This spatial resolution supported detailed assessment of connectivity, network propagation, and activity distribution.

The integration with BrainWave 6, a unified environment for acquisition, visualization, and advanced analysis, ensured a coherent and reproducible workflow, enabling complex metrics and spatiotemporal insights without external tools.

Together, the Axol-3Brain system provides a sensitive, high‑resolution, and efficient platform for neuropharmacology, toxicity testing, and the study of human neuronal circuit function.

Profiling ALS variants and ion channel expression changes with HT automated patch clamp

Nina Brinkwirth, Alison Obergrussberger, Nadine Becker, Elena Dragicevic, Niels Fertig

Nanion Technologies GmbH, Ganghoferstr 70A, Munich, Germany

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease resulting in motor neuron loss, muscle weakness, paralysis, and death. While most cases are sporadic, over 40 genes are implicated, and mechanisms remain unclear, highlighting the need for relevant New Approach Methodologies (NAMs) and human models. Here, we functionally characterized ion channel properties of axoCells™ human iPSC-derived motor neurons from unaffected donors and C9orf72 ALS patients using the SyncroPatch 384. We observed clear differences in potassium and sodium channel properties, current densities, and neurotransmitter responses, demonstrating the value of combining iPSC-derived motor neurons with automated patch clamp for high-throughput ALS phenotyping.

Methods

axoCells™ human iPSC-derived motor neurons from an unaffected donor and a C9orf72 ALS patient (Axol Bioscience) were used. Cells were generated via Sendai virus reprogramming and cultured in axoCells™ maintenance media with supplements. After thawing, cells were maintained under standard conditions, becoming assay-ready ~10 days post-thaw, with maturation up to 120 days. Identity was confirmed by immunocytochemistry (TUJ-1, HB9, ChAT, Isl-1). Functional electrophysiology recordings were performed using the high-throughput automated patch clamp (APC) system, the SyncroPatch 384 (Nanion Technologies). Cells were recorded in whole-cell mode, enabling parallel analysis of up to 384 cells. Sodium and potassium currents were recorded, and neurotransmitter responses were assessed, with both cell types measured on the same chip for direct comparison.

Results

axoCells™ iPSC-derived motor neurons from an unaffected donor (Male) and a C9orf72 ALS patient (ALS) were characterized using the SyncroPatch 384. At DIV10, brightfield imaging revealed clear morphological differences (Fig. 1A). Motor neuron identity was confirmed by high expression of TUJ-1, HB9, ChAT, and Isl-1 (Fig. 1B). High-quality whole-cell recordings with consistently high seal resistances were achieved across both cell types, enabling robust and reproducible comparisons. C9orf72 neurons exhibited significantly reduced Na⁺ current amplitudes compared to controls, while voltage-dependent activation and inactivation parameters remained unchanged, indicating preserved channel gating but altered channel expression or membrane properties (Fig. 2A,B,C). K⁺ current analysis revealed pronounced functional remodeling. Unaffected motor neurons exhibited fast activation with pronounced inactivation, characteristic of transient outward K+ currents (IA). In contrast, C9orf72 motor neurons displayed sustained currents with little or no inactivation, consistent with delayed rectifier K+ currents (IK) (Figure 3A,B). These changes were accompanied by significantly reduced current amplitudes and current density, as well as a shift in waveform distribution, indicating altered channel composition and repolarization dynamics (Fig. 3A,C). Neurotransmitter profiling further highlighted disease-relevant alterations. While glutamate-evoked responses showed no differences in amplitude or EC50 (Fig. 4A,B), GABA-evoked currents were significantly reduced in C9orf72 neurons without changes in EC50 values, suggesting impaired inhibitory signaling without altered receptor sensitivity (Fig. 4C,D).

Conclusion

iPSC-derived motor neurons are a physiologically relevant ALS model, preserving patient genetics and enabling functional phenotyping. Automated patch clamp was used to robustly characterize C9orf72 neurons, confirming disease-relevant changes. These included altered morphology consistent with cytoskeletal dysfunction. APC revealed remodelled K⁺ currents, reduced Na⁺ currents, and impaired inhibitory (GABA) signalling, while glutamate and glycine responses were unchanged. High data quality and throughput support scalable, disease-relevant drug discovery and ion channel screening, even beyond the ALS phenotype.

A human cell-based atrial fibrillation disease model

Bettina Lickiss, Jan Hunker, Britta Simons, Peter Linder, Matthias Gossmann

innoVitro GmbH, Juelich, Germany

Abstract

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia, significantly impacting morbidity and mortality worldwide. Using commercially available human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes (axoCellsTM, Axol Bioscience Ltd.) (Lickiss & Hunker et al., 2024), we developed an AF-like human phenotype in vitro disease model based on the protocol of Seibertz et al., 2022. The drug screening assay is designed to assess AF-related functional changes in a human-relevant context, with extracellular field potential duration (EFP duration, CardioExcyte 96, similar to MEA) as readout parameter.

Methods

Preculture of atrial hiPSC-CMs on CardioExcyte 96 plates (Figure 1A) for 9 days including medium changes every other day. Begin of tachypacing in CardioExcyte 96 device on day 9 for 24 h -> induction of electrical remodelling (AF phenotype) shown by reduced EFP duration (Figure 2). Compound addition 24 h after begin of tachypacing followed by chronic EFP duration assessment (Figure 1B).

Results

Tachypacing of atrial hiPSC-CMs induced electrical remodeling, evidenced by a significant shortening of EFP duration, corresponding to QT interval shortening (Figure 2). Upon cessation of tachypacing, cells exhibited gradual recovery, with EFP duration increasing from ~55% to ~80% and ~ 90% respectively over 24 hours (Figure 3).

S-bay treatment induced a dose-dependent prolongation of EFP duration in atrial hiPSC-CMs. This effect was fully reversible upon washout (Figure 4), demonstrating the reversible/rescue effects that can be tested using this human-based AF approach.

Conclusion

This human-based disease model presents a significant advancement for AF research, providing a human-relevant platform for identifying novel therapies with greater translational potential and accelerating the development of effective AF treatments.

High-throughput electrophysiological characterization and screening of ventricular axoCellsTM cardiomyocytes, using Sophion’s APC platforms QPatch and Qube 384

Atanaska Velichkova, Kadla R. Rosholm

Sophion, Ballerup, Denmark

Abstract

Automated patch clamp (APC) systems enable high-throughput, precise electrophysiological measurements of cardiac ion channels in human induced pluripotent stem-cell derived cardiomyocytes

(hiPSC-CMs), accelerating screening workflows of these key therapeutic and safety targets.

This study employs Sophion QPatch and Qube 384 APC platforms (figure 1) to biophysically and pharmacologically evaluate cardiac ion channels (Nav1.5 and Cav1.2) and paced action potentials in axoCellsTM ventricular hiPSC-CMs, highlighting the potential of this technology in cardiac safety and drug-discovery.

Biophysical characterization and screening of Nav1.5 on Qube 384

Using customized dissociation and whole-cell protocols we obtained a good-quality single-cardiomyocyte suspension that could be recorded on Qube 384 with up to 80 % success rates (figure 2A). First, we did a biophysical characterization of Nav1.5, with the current-voltage relationship and Vhalf of activation and inactivation in good agreement with literature values (figure 2B-E).

The good experiment success rate and data quality allow us to do a sample Nav1.5 compound concentration-response experiment using the well-known state-dependent Nav blocker, tetracaine (figure 3).

Recording and compound modulation of Cav1.2 and action potentials in physiological solutions on QPatch

We recorded L-type Cav1.2 channel currents and paced action potentials (APs) in about 60 % of the axoCellsTM hiPSC-CMs using physiological solutions on QPatch (figure 4). As expected, the APs were significantly prolonged when the Cav1.2 current was potentiated and shortened when the Cav1.2 current was blocked.