Perform every step under sterile conditions (e.g. laminar flow cabinet).
This protocol is standardised to human PSC culture in T25 flasks and generation of BENOs with 90.000 cells per organoid (3,000 cells/µL).
Day -4 PSC culture
1. Coat T25 flask with 1:120 (in DPBS) growth factor reduced Matrigel™ (100 µL/cm²) and incubate for 30 min in ambient air incubator with 5.0% CO2 and 37 °C.
2. Plate cells (optimal cell density will have to be established experimentally for every individual PSC line and clone) with 4 mL of iPS-Brew XF media and 5 mmol/L ROCKi.
3. Place PSC in ambient air incubator with 5.0% CO2 and 37 °C.
4. Change medium daily (4 mL) until cells reach confluence of 70-80%.
Day -1 BENO generation
1. Aspirate cell culture media in T25 flask
2. Wash with 3 mL 1x DPBS two times, aspirate DPBS after each washing step
3. Add 3 mL of 0.5 mmol/L EDTA to PSCs and incubate for 2-3 min (some lines may require different incubation times and/or higher EDTA concentrations).
4. Carefully aspirate EDTA solution and tap the side of the flask until cell detachment.
5. Resuspend iPSC in 10 mL Brew supplemented with 10 µmol/L ROCKi.
6. Determine cell count (e.g. Neubauer chamber with 1:5 0.4 % Trypan blue or by an automated cytometer) and viability.
7. For pluripotency confirmation via flow cytometry, fix 2x106 cells with 4 % Histofix at a 1:1 ratio and incubate for 15 min. Centrifuge at 300 g for 10 min. Wash two times in 1x DPBS. Store in DPBS at 4 °C for further analysis (up to 1 week).
8. Centrifuge remaining cell resuspension at 100 g for 3 min.
9. Resuspend cell pellet in Brew supplemented with 20 ng/mL FGF2 and 10 µmol/L ROCKi to achieve cell concentration of 4500 cells/µL.
All steps are performed on ice:
10. To generate BENOs, add a 1:1 ratio of Collagen and 2x DMEM to a pre-cooled vessel.
11. Neutralised by 0.1 mol/L NaOH.
12. Add an appropriate amount of cell resuspension to achieve a concentration of 3,000 cells/µL in the final BENO reconstitution mixture.
13. Pipette 30 µL of the BENO reconstitution mixture into a single well of a 96-well plate (U-bottom, low attachement); fill as many wells as experimentally desired.
14. Incubate for 30 min in ambient air incubator with 5.0% CO2 and 37 °C.
15. Add 300 µL of Brew supplemented with 10 ng/mL FGF2 and 10 µmol/L ROCKi.
16. Incubate overnight in ambient air incubator with 5.0% CO2 and 37 °C.
Differentiation Day 0 to 3: Neuronal commitment phase in 96 well plate
1. Prepare sufficient amounts of neuronal commitment medium (NCM):
Add 10 µmol/L SB 431542, 50 ng/ml LDN and 1 µmol/L RA to Basal Medium (Neurobasal-A containing 2 mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 2% B27, 1% N2 supplement, 200 µmol/L ascorbic acid).
2. Carefully remove 200 µL of medium per well and add 200 µL of pre-warmed NCM. Change medium daily.
3. Tap plate to make sure that BENOS sediment to the bottom of the wells. Incubate BENOs in ambient air incubator with 5.0% CO2 and 37 °C.
4. On day 3, transfer BENOs into 6-well plates (10 BENOs / well) with 5 mL NCM per well.
Differentiation Day 6 and 8: Neuronal commitment phase in 6 well plate
1. Carefully aspirate 4 mL of NCM per well.
2. Add 4 mL of fresh, pre-warmed NCM to each well.
Differentiation Day 10 and 13: Neuronal progenitor growth phase
1. Prepare sufficient amounts of neural progenitor expansion medium (NPEM):
Basal Medium complemented with 10 ng/mL FGF2 and 5 ng/mL TGFB1.
2. Carefully aspirate 4 mL of NPEM per well.
3. Add 4 mL of fresh, pre-warmed NPEM to each well.
Differentiation Day 15, 17, 20, 22, 24, and 27: Neuronal differentiation phase
1. Prepare sufficient amounts of neural progenitor differentiation medium (NDM):
Basal medium complemented with 5 ng/ml TGFB1 and 2.5 µM DAPT.
2. Carefully aspirate 4 mL of NDM per well.
3. Add 4 mL of fresh, pre-warmed NDM to each well.
Differentiation Day 29 onwards: Neuronal maturation phase
1. Assess BENOs on day 28 (e.g. by immunofluorescence or gene expression analysis).
2. Carefully aspirate 4 mL of Basal Medium per well.
3. Add 4 mL of fresh, pre-warmed Basal Medium to each well.
4. Change medium every second day.
BENOs can be cultured according to your experimental needs. Neuronal network maturation occurs at day 40 onwards. Palpable gliogenesis can be observed from day 60, with first notable astrocyte formation followed by the appearance of myelinating oligodendrocytes from day 90 onwards. Substantial myelination can be observed from culture day 150.