This video demonstrates a technique for generating cerebral organoids from human embryonic stem cells (hESCs). The adherent culture of hESC colonies is treated with enzymes to detach and break them into clusters. These clusters form three-dimensional spheres in suspension, which are then induced to differentiate into organoids that mimic the brain cortex with layers of neurons and glial cells.
Protocol
1. Stem Cell Maintenance Maintain H9 hESCs on a layer of growth factor reduced basement membrane matrix (see the Table of Materials, henceforth simply referred to as matrix) according to the manufacturer's instructions. To coat one 6-well plate or one 10 cm dish, combine 100 µL of matrix with 5.9 mL of ice-cold Dulbecco's modified Eagle medium (DMEM)/F12 media. Wrap plates in paraffin film and store overnight at 4 °C. Use them on the next day for…