Application Note
Functional analysis of spontaneous calcium oscillations of iPSC-derived 3D neural organoids and evaluation of responses to neuroactive compounds
- High-throughput screening – Rapid, screening of large numbers of compounds, accelerated and short-time studies.
- Real-time, functional readouts of Physiologically relevant models - have used organoids that are physiologically relevant and provide a more accurate representation of drug effects.
- Automated monitoring and feeding – ensures optimal health of organoids and maintains them with minimal manual intervention.
Krishna Macha, Oksana Sirenko, Jeffrey Tang | Molecular Devices, LLC
Leon Chew | STEMCELL Technologies Inc.
Introduction
Neural organoids, derived from human induced pluripotent stem cells (iPSCs), are a rapidly evolving technology with great potential to understand brain development, disease, and disorders within the context of diverse genetic backgrounds. In this study, we focus on the functional characterization of the spontaneous activity of neural organoids measured by calcium oscillations. Neural organoids were formed using standardized reagents and protocols from STEMCELL™ Technologies and shipped to Molecular Devices before conducting assays to measure spontaneous activity.
Morphological characterization of 3D organoids was performed by brightfield imaging. Organoid’s diameters ranged from 1800–2000 µm. Expression of neural markers including MAP2 and GAFP was detected by RT-qPCR analysis. Functional characterization of neural activity was done via calcium oscillation assay and was recorded on the FLIPR® Penta High-Throughput Cellular Screening System that measured fast kinetic changes in calcium signal. Calcium oscillations were visualized and analyzed by ScreenWorks® Peak Pro 2 software. In addition, kinetic imaging was recorded on the ImageXpress® Micro Confocal system. Importantly, the calcium-sensitive dye used contains a background fluorescence masking technology that enables sensitive detection of calcium oscillation without the need to wash calcium dye. The calcium oscillation patterns were analyzed for multiple parameters including peak count, and amplitude. The majority of organoids demonstrated spontaneous calcium oscillation activity with a consistent rate of oscillations. Some organoids did not show synchronous activity but such activity was induced by stimulation with 4-aminopyridine or AMPA.
Several compounds were used for pharmacological characterization to show the appropriate functional responses. AMPA and 4-AP addition resulted in a dosedependent increase in the frequency of calcium oscillations, while GABA caused a decrease in oscillation amplitude. Taken together, this biological system of iPSC-derived 3D neural organoids paired with high-content imaging and detailed analysis of calcium oscillations demonstrates a promising tool for compound testing.
Methods
H-iPSCs maintained in mTeSR™ Plus were dissociated and seeded at a density of 3 x 106 cells/well in Seeding Medium (Formation Medium + 10 M rho-kinase inhibitor (ROCKi)) in AggreWell™ 800 plates. Cultures were fed daily with Formation Medium. After 6 days, organoids were transferred to a 6-well plate in Expansion Medium and maintained on an orbital shaker at 70 RPM (INFORS HT Celltron). Organoids were fed every 2–3 days with an Expansion Medium until day 25, at which point organoids were fed with a Differentiation Medium until day 43. From day 43 onward, organoids were fed every 2–3 days with Maintenance Medium. Neural organoids were shipped at day 60 and fed with Maintenance media with feeds every 2–3 days.
On the day of assay, cell spheroids were loaded with 2X conc. of FLIPR Calcium 6 dye indicator (Molecular Devices) and incubated for 2h. We used a high-speed EMCCD camera on the FLIPR Penta instrument (Molecular Devices) to measure the patterns and frequencies of spontaneous calcium waveforms from 3D neural organoids. Baseline recordings were acquired for ≥10 min, and then plates were dosed with drugs for 30–90 min. Peak analysis was accomplished with ScreenWorks Peak Pro 2 software (Molecular Devices), allowing the characterization of both primary and secondary peaks, as well as complex calcium oscillation patterns. High-content imaging was done on the ImageXpress Micro Confocal System (Molecular Devices) and was used to capture 3D structures of the spheroids and for viability evaluation.
Automated feeding and monitoring using CellXpress.ai
Figure 1. Schematic diagram of the process workflow. (1–4) Organoids were formed and differentiated from human pluripotent stem cells using STEMCELL™ Technologies products i.e., seeding, formation, expansion medium, and differentiation medium. (5) Maintenance of the organoids was done using a maintenance medium at Molecular Devices. (6) Assay and imaging were performed using the FLIPR Penta instrument (Molecular Devices) and the ImageXpress micro confocal system (Molecular Devices).
Results
Formation and characterization of 3D neural organoids
Figure 2. The STEMdiff™ Dorsal Forebrain Organoid Kit (STEMCELL™ Technologies Catalog # 08620) and STEMdiff™ Midbrain Organoid Differentiation Kit (STEMCELL Technologies Catalog #100-1096) were used to generate dorsal forebrain organoids or midbrain organoids, respectively, according to manufacturer protocols using the human pluripotent stem cell (hPSCs) line SCTi003-A (Catalog # 200-0511). hPSCs maintained in mTeSR™ Plus were dissociated and seeded at a density of 3 x 106 cells/well in Seeding Medium (Formation Medium + 10 M rho-kinase inhibitor (ROCKi)) in AggreWell™800 plates. Cultures were fed daily with Formation Medium. After 6 days, organoids were transferred to a 6-well plate in Expansion Medium and maintained on an orbital shaker at 70 RPM (INFORS HT Celltron). Organoids were fed every 2–3 days with an Expansion Medium until day 25, at which point organoids were fed with a Differentiation Medium until day 43. From day 43 onward, organoids were fed every 2–3 days with Maintenance Medium. Neural Organoids were shipped at day 60 and fed with Maintenance media with feeds every 2–3 days.
Figure 3. (A) Representative brightfield morphology of a day 60 dorsal forebrain organoids generated using the STEMdiff™ Dorsal Forebrain Organoid Kit (B)RT-qPCR analysis of day 60 dorsal forebrain organoids. Results show upregulation of forebrain specification marker FOXG1, progenitor marker PAX6, neuronal marker MAP2, and glial marker GFAP. Results displayed as Log10(Fold Change 2-∆∆Ct Method) (Average ± SEM n = 3 organoids) Data is normalized to TBP and compared to undifferentiated hPSC control.
Figure 4. (A) Representative brightfield morphology of a day 60 midbrain organoids generated using the STEMdiff™ Midbrain Organoid Kit (B)RTqPCR analysis of day 60 midbrain organoids. Results show upregulation of midbrain progenitor marker FOXA2, dopaminergic neuron marker TH, neuronal marker MAP2, and glial marker GFAP. Results displayed as Log10(Fold Change 2-ΔΔCt Method) (Average ± SEM n = 3 organoids) Data is normalized to TBP and compared to undifferentiated hPSC control.
Compound testing using organoids
Figure 5. Baseline calcium oscillations vs. drug response of dorsal forebrain organoids. Ca2+ waveforms were recorded by kinetic calcium imaging using the FLIPR Penta instrument and analyzed using Peak Pro 2 software. Organoids had consistent baseline activity in the absence of compound additions (left panel). After the addition of indicated compounds (4 dose concentrations, 0.4μM presented here), the pattern was modified as indicated on the right panel.
Figure 6. Bar graphs represent peak counts and amplitudes in controls and after the addition of indicated concentrations of compounds. We were able to observe expected phenotypic responses to 4-AP and AMPA causing an increase in the frequency of calcium oscillations. Interestingly, GABA and Baclofen were causing some frequency increase, while the amplitude of oscillations decreased.
Automated monitoring and media exchanges
with CellXpress.ai™ Automated Cell Culture System
Since the continuous culture of neural organoids is labor-intensive, we developed protocols for automated imaging and media exchanges using the CellXpress.ai™ Automated Cell Culture System. A subset of the organoids was placed into the CellXpress.ai system and cultured in 96-well format for 10 days with imaging and media exchange every 24h. Images were taken with transmitted light (see below) and media exchanges were done by a liquid handler replacing 2/3 media volumes.
Figure 7. A. Schematic diagram of CellXpress.ai Automated Cell Culture system. B. Images of the subset of 96-well plates with cultured neural organoids
Summary
This study showcases the use of 3D neural organoids derived from human pluripotent stem cells (hPSCs) as an advanced model for evaluating neurotoxicity and the effects of compounds on neural function. The organoids were cultured and analyzed using kinetic calcium imaging, a technique that tracks spontaneous calcium oscillations, which serve as a real-time indicator of neural network activity. By leveraging the FLIPR Penta High-Throughput Screening System, researchers can capture detailed functional data, such as changes in frequency and amplitude of calcium oscillations, providing critical insights into how compounds affect neuronal activity and health. In the FLIPR Penta assay, we observed distinct changes in the peak counts and amplitudes of calcium oscillations following compound treatments. Interestingly, although GABA and Baclofen are typically associated with a decrease in peak count and an increase in amplitude due to their inhibitory effects, we saw an unexpected trend in this study. Instead, these compounds led to a slight increase in peak frequency and a reduction in amplitude, suggesting a more complex modulation of neural activity. In contrast, 4-AP and AMPA caused a dose-dependent increase in peak frequency, indicating heightened neuronal excitability. These unique peak data offer deeper insights into how different compounds impact neural network dynamics and can help identify potential therapeutic or toxic effects on brain function. To address the challenges of scalability and throughput in long-term organoid culture, this study also utilizes the CellXpress.ai™ Automated Cell Culture System. This system automates key processes, including media exchange and daily imaging, in a 96-well plate format, reducing manual labor while maintaining optimal culture conditions. The automation of these processes ensures consistent monitoring of organoid development and activity over extended periods, enabling efficient highthroughput screening of compounds. By combining the FLIPR Penta for functional assessment and CellXpress. ai™ for automated culture management, this integrated approach offers a scalable, reproducible platform for studying drug effects, neurotoxicity, and neural circuits. This workflow accelerates the evaluation of compounds for drug discovery and toxicology, providing a more physiologically relevant model than traditional 2D systems.
Conclusion
- In vitro 3D neural organoids, generated using human pluripotent stem cells, present a useful cell-based assay for assessment of neurotoxicity and neuro-active effects of various neuromodulators.
- This assay platform shows promise for the evaluation of compound effects and early detection of neurotoxicity in vitro.
- Analysis of kinetic calcium imaging demonstrates spontaneous activity of neural organoids and provides read-outs for the functional neural activity and can be used for the evaluation of phenotypic changes and compound effects.
- Organoid culture is amenable to automation which would allow scaling up and increased throughput of the assay.