iPSC Midbrain differentiation in 96 well plates
gustavo.parfitt Parfitt, Elena Coccia
Abstract
Midbrain differentiation protocol using spinner flasks.
Steps
iPSCs plating for differentiation
Passage 1X106 iPSC into Geltrex coated 10-cm dish with Accutase and rock inhibitor (refer to material section).
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.
Wait until iPSC reaches 60% confluence to start differentiation.
Midbrain Differentiation
Starting on day D0, Aspirate all the media, wash the plate with PBS, and add D0-D1 media (refer to the materials).
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.
On the D12 passage, plate 10000 neural progenitors cells into a Geltrex-coated 96-well plate.
Wash the cells with PBS.
For the passaging add 5mL of Accutase for 0h 5m 0s .
Add 5mL of Day 12 media with rock inhibitor, gently mix and transfer to a 15 ml conical tube.
300x g,25°C
Resuspend the cells in 1mL of Day 12 media with rock inhibitor and Count the cells.
On D12 change to D12 terminal media for final differentiation, change half media every 48h.
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 |
