Figure 6.

Functional assessment of cerebral organoids by calcium imaging. (A) A representative image of a culture derived from a YOAD cerebral organoid loaded with the ratiometric calcium indicator Fura2-AM. A 9-month-old organoid was seeded onto a glass coverslip for Ca²⁺ imaging. The image shows the overlay of 340 nm and 380 nm channels; each colored circle corresponds to a region of interest (ROI) represented in (B). When Ca²⁺ binds to the indicator, fluorescence at 340 nm increases, while 380 nm fluorescence decreases. The 340/380 ratio is thus used as a measurement of Ca²⁺ responses to drugs or agonists or to assess spontaneous activity. (B) Relative change in fluorescence intensity over time of specific ROIs from (A) using Fura2-AM. Neurons can be stimulated with chemicals that are perfused into the bath chamber and the Ca²⁺ responses recorded. The culture was exposed to the excitatory neurotransmitter glutamate (20 µM; perfusion indicated by the horizontal black bar) to elicit a Ca²⁺ response, followed by high K⁺ (60 mM; perfusion indicated by the horizontal black bar) to mimic membrane depolarization. Responses may be fast or slow transient increases or prolonged increases that do no return to baseline within the timeframe of the experiment.