Abstract
Background & Purpose
Drug development often faces challenges in accurately predicting neurotoxicity and therapeutic efficacy. Traditional methods such as ATP-based viability assays are destructive, while non-destructive membrane-based assays are costly and time-consuming. To address this, 28bio developed a non-invasive, image-based cytotoxicity assay for human iPSC-derived brain organoids. This model allows simultaneous evaluation of drug safety and efficacy with improved scalability and reproducibility.
Methods
- Organoid Culture & Model Development:
- iPSC-derived cortical organoids from both CDKL5 deficiency disorder (CDD) patient cells and healthy control (CTL) lines were cultured in 384-well plate formats for high-throughput drug screening.
- CDD and CTL organoids grew to similar sizes and showed appropriate cellular composition (neuron to astrocyte ratio), however, functional characterization revealed a striking difference in the spontaneous firing pattern between these organoids making them ideal for functional phenotypic screening.
- Creating an Image-Based Toxicity Assay:
- Brightfield images of CTL and CDD organoids following a 4-day treatment with a panel of known neurotoxic compounds were processed using ImageJ to extract features like intensity, texture, and morphology.
- A neural network trained in JMP software predicted Cell Titer Glo (CTG) luminescence values, serving as a proxy for cell viability.
- High Throughput Screening for Novel Targets Protocol:
- CDD and CTL organoids were treated with following a chronic, 3-week dosing paradigm to identify disease-modifying therapeutics rather than acute, symptomatic treatments
- Prior to performing FLIPR functional assay, brightfield images of organoids were captured for image-based toxicity assessment.
Results
- The image-based model accurately predicted IC50 values for neurotoxic agents across multiple cell lines with a high correlation to actual CTG values (R² = 0.92 – 0.93).
- High throughput phenotypic screening in combination with image-based toxicity assessment generated both toxicity scores (safety) and disease selectivity (efficacy) data for novel CDD therapeutics.
- The image-based toxicity assay combined with FLIPR successfully distinguished compounds with:
- Selective rescue effects – improving CDD organoid activity without harming healthy organoids.
- Non-selective rescue effects – affecting both CDD and control organoids.
- Compounds with toxic profiles were identified early, enabling improved screening efficiency.
- Key Therapeutic Hits Identified:
- From an initial screen of over 5000 compounds, 22 top hits (across 15 unique biological targets) were identified as both safe and efficacious in rescuing CDD hyperactivity phenotype
Conclusion
28bio’s microBrain™ image-based cytotoxicity assay is a non-destructive, scalable, and cost-effective alternative to traditional toxicity endpoints. This technology enables combined safety and efficacy testing, improving the identification of promising drug candidates for neurodevelopmental disorders such as CDKL5 deficiency disorder (CDD).
