Organoids derived from iPSCs have filled the gap in 3D models for tissues that are inaccessible or for organoids that cannot be developed from adult stem cells. In the last decade, protocols and commercial kits have been developed to differentiate iPSCs into multicellular, neuronal organoids that closely resemble human brain development, including region-specific cellular composition and functional physiology. These models are critical for modeling developmental and neurodegenerative disorders but have somewhat limited utility due to challenges of reproducibility and scalability. Using standard methodologies, bulk iPSCs are seeded into 96 well plates, pipetted into larger-well plates at multiple stages and manually embedded into Matrigel® droplets. In addition to being technically challenging, the standard workflow is limited to generating 96 organoids that cannot be tracked or analyzed throughout differentiation.
We collapsed this complex workflow onto the CellRaft® Array, providing a user-friendly method for growing hundreds of single iPSC-derived neuronal organoids for downstream toxicity screening. Single iPSCs were seeded and differentiated, through embryoid body formation, neural induction and expansion on the CellRaft® Array (Figure 5). On day 10, single cerebral organoids of interest, selected using CellRaft® Cytometry based on diameter (100–300µm), were isolated into maturation media using the CellRaft® AIR System. This process preserves organoid morphology and viability, and organoids were subsequently cultured off-array in cerebral maturation media out to day 42 (Figure 5). Images were captured of each organoid on day 40, after the cerebral organoids were fully mature. By selecting organoids for isolation based on day 10 size, we were able to generate a 96-well plate with single mature organoids that were relatively similar in size for our downstream toxicity screen (Figure 6A). On day 43, the organoids were treated with a six-point dose curve of ethanol (6.25–50mM) for 6 hours to simulate acute alcohol exposure. To evaluate alcohol-induced apoptosis, relative caspase activity was measured using the Caspase-Glo® 3/7 3D Assay. At the highest doses, we observed a dose-dependent activation of caspase activity, indicating alcohol-induced initiation of apoptosis (Figure 6B).
Figure 5. Single iPSCs are differentiated on the 500µm CellRaft® Array to form single neural organoids. iPSCs were seeded on the CellRaft® Array in dilute extracellular matrix (ECM) and imaged daily for a week during embryoid body formation and neural induction for choroid plexus organoid differentiation. On day 10, single organoids were selected based on diameter (100–300µm) and isolated into 96-well plates with maturation media. Cerebral organoids were maintained in maturation media to day 42.
Figure 6. Alcohol-induced apoptosis in neuronal organoids. Images of mature cerebral organoids on day 40, prior to treatment with ethanol, from three of the six replicate wells, demonstrating the ability to generate well-to-well reproducibility in the downstream assay on day 40 by selecting organoids based on size at day 10 (A). On day 43, mature cerebral organoids were exposed to a five-point dose curve of ethanol (6.25–50mM, n = 6 wells per dose) to simulate acute alcohol exposure. After 6 hours of treatment, organoids were evaluated for alcohol-induced apoptosis using the Caspase-Glo® 3/7 3D Assay. We observed a dose-dependent increase in caspase activity, supporting activation of apoptosis at the highest doses of ethanol exposure (B).