Midbrain organoid differentiation in spinner flasks.
gustavo.parfitt Parfitt
Abstract
Midbrain differentiation protocol using spinner flasks.
Steps
iPSCs aggregation in spinner flasks
Passage iPSC lines to four 10-cm dishes to generate 40X106cells.
Prepare 120 mL of StemFlex per flask with rock inhibitor (refer to material section) and add 115 to the spinner flask.
Place the spinner flask on the stir plate and set to 65 rpm.
Wait for the cells to reach 80% confluence and seed iPSC 40X106cells into a spinner flask.
For the passaging add 3mL of Accutase for 0h 5m 0s .
Add 5mL of StemFlex , gently mix and transfer to a 15 ml conical tube.
300x g,25°C
Resuspend the cells in 1mL of StemFlex with rock inhibitor and Count the cells.
Put the cells in the correct cell concentration and add to 5mL of StemFlex with rock inhibitor.
From the main opening of the spinner flask add the cells and plate back on the stir plate.
Every other day change 60mL of media using StemFlex until the spheres reach 300 μM.
Midbrain Differentiation
On the starting day D0, filter the spheres in a 300 μM to 500μM range.
Aspirate all the media in the flask and replace by D0-D1 media (refer to materials). Add the filtered spheres and place the spinner flask back on the stir plate.
D1 change half the media with D0-1 medium.
D2 change half the media with D2-3 medium.
D3 Change half the media with D2-3 medium.
D4 change half the media with D4-7 medium.
D5 change half the media with D4-7 medium.
D6 change half the media with D4-7 medium.
D7 change half the media with D4-7 medium.
D8 change all the media with D8-11 medium.
D9 change half the media with D8-11 medium.
D10 change half the media with D8-11 medium.
D11 change half the media with D8-11 medium.
On D12 change to D12 terminal media for final differentiation. From this point on the organoids can be transfer to low attachment plates and place on shaker.
On D35 change to long-term maintaining media.
Protocol Media schedule
| A | B |
|---|---|
| Day x--1 | StemFlex |
| Days 0-1 | DMEM/F12+Glutamax + B27 + N2 + SB + LDN |
| Days 2-3 | DMEM/F12+Glutamax + B27 + N2 + SB + LDN + Purmorphamine + SAG |
| Days 4-7 | DMEM/F12+Glutamax + B27 + N2 + SB + LDN + Purmorphamine + SAG + CHIR |
| Days 8-11 | DMEM/F12+Glutamax + B27 + N2 + LDN + CHIR |
| Days 12-21 | DMEM/F12+Glutamax + B27 + N2 + BDNF + GDNF + DAPT+ Ascorbic Acid + dCAMP |
| Days 22-35 | DMEM/F12+Glutamax + B27 + N2 + 0.5(BDNF + GDNF) + DAPT + Ascorbic Acid + dCAMP |
| Days 35-end | DMEM/F12+Glutamax + B27 + N2 + 0.5(BDNF + GDNF) + Ascorbic Acid |
