Differentiation of cerebral orgnaoid#

Reagent#

  • Cell

  • Knockout Serum Replacement (KOSR)

  • DMEM /F12

  • Neurobasal medium

  • Penicillin/Streptomycin (P/S)

  • N2 supplement

  • B27 (+ Vitamin A) plus supplement

  • Human insulin

  • Ascorbic acid

  • GlutaMAX

  • dibutyryl-cAMP

  • NEAA

  • LDN-193189

  • SB431542

  • XAV939

  • BDNF

Preparation#

Neural induction medim#

Reagent Volume
Knock out DMEM/F12 75 ml
KSOR 15 ml
GlutaMAX 1 %
NEAA 1 %
LDN-193189 100 nM
SB431542 10 μM
XAV939 10 μM
Total 100 ml

Differentiaion medium#

Reagent Volume
Knock out DMEM/F12 50 ml
Neurobasal medium 50 ml
N2 supplement 0.5 %
B27 (+ Vitamin A) plus supplement 1 %
GlutaMAX 1 %
MEM-NEAA 1 %
Human insulin 0.25 mg/ml (v/v)
Total 100 ml

Maturation medium#

Reagent Volume
Neurobasal medium 100 ml
N2 supplement 1 %
B27 (+ Vitamin A) plus supplement 2 %
GlutaMAX 1 %
MEM-NEAA 1 %
Human insulin 0.25 mg/ml (v/v)
Ascorbic acid 200 mM
BDNF 20 ng/ul
dibutyryl-cAMP 1 mM
Total 100 ml

Maintanance medium#

Reagent Volume
Brainphys 100 ml
B27 (+ Vitamin A) plus supplement 2%
GlutaMAX 1 %
P/S 1 %
BDNF 20 ng/ml
GDNF 20 ng/ml
Total 100 ml

Ca2+ imaging recording medium#

Reagent Volume
Brainphys Imaging optimization medium 100 ml
B27 (+ Vitamin A) plus supplement 2%
GlutaMAX 1 %
P/S 1 %
BDNF 20 ng/ml
GDNF 20 ng/ml
Total 100 ml

Protocols#

  1. iPS cells on 6 well plate were dissociated into single cells with TrypLE express.

  2. Plated at 30,000 cell per each well of U-bottom ultra-low attachment 96 well plate (Corning) with mTeSR plus supplemented with 20 μM of Y-23632.

  3. After 24 h, culture medium was replaced with neural induction medium and changed every other day.

  4. After 10 days of culture, the culture medium was replaced with neuronal differentiation medium and the medium was changed every 2 days until 18 days.

  5. After 18 days of culture, organoid were transferred to flat bottom ultra-low attachment 24 well plate (Corning) on the rocking shaker (120 rpm) with switching to maturation medium.

  6. After 30 days from the differentiation, the culture medium was changed two-time per week with maintanance medium until imaging session.