A Technique to Generate Brain Organoids from Human Embryonic Stem Cells

Abstract

Source: Boisvert, E. M., et al. A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells. J. Vis. Exp  (2020).

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…

Materials

B27 Supplement	Gibco, Waltham, MA, USA	17504-044
bFGF	Life Technologies, Carlsbad, CA, USA	PHG0263
BSA	Sigma-Aldrich, St. Louis, MO, USA	A9647
Dispase	STEMCELL Technologies, Vancouver, Canada	7913
DMEM/F12	Thermo Fisher Scientific, Waltham, MA, USA	11330-032
DPBS	Gibco, Waltham, MA, USA	10010023
Growth Factor Reduced Matrigel (Matrix)	Corning, Corning, NY, USA	356231
H9 hESCs	WiCell, Madison, WI, USA	WA09
Heparin	Sigma-Aldrich, St. Louis, MO, USA	9041-08-01
L-glutamine	Gibco, Waltham, MA, USA	25030-081
Monothioglycerol	Sigma-Aldrich, St. Louis, MO, USA	M6145
mTESR media	STEMCELL Technologies, Vancouver, Canada	85850
N2 NeuroPlex	Gemini Bio Products, West Sacramento, CA, USA	400-163
NEAA	Gibco, Waltham, MA, USA	11140-050
Triton X-100	Sigma-Aldrich, St. Louis, MO, USA	T8787-100ML
Ultra-low attachment T75 flasks	Corning, Corning, NY, USA	3814