Long-term culture and electrophysiological characterization of human brain organoids

Abstract

Here we describe a modified version of the culturing protocol first described by Lancaster et al.,1 that supports prolonged development of self?organizing 3D human brain organoids for over 9 months. Prolonged culture allows maturation of organoids that acquire a high degree of cellular diversity and neuronal maturation, including formation of dendritic spines and spontaneously active neuronal networks. In this model, photoreceptor?like cells mature substantially and become responsive to non? invasive, light?based sensory stimulation that appears capable of affecting neuronal activity. In Quadrato et al.2 we have used large?scale, single cell RNA sequencing to provide a first measure of the diversity of cells generated across organoids grown in different bioreactors. Here, we describe in detail the protocol for dissociation and cell isolation in brain organoids for large?scale single?cell droplet sequencing3. We also describe an electrophysiological rig specialized for performing extracellular recordings in whole organoids with controlled light stimulation, and methods for recording spontaneously active neuronal networks in organoids. Subject