TITLE: iPSC Midbrain differentiation in 96 well plates
AUTHORS: gustavo.parfitt Parfitt, Elena Coccia

[DESCRIPTION]
Midbrain differentiation protocol using spinner flasks.

[STEPS]
SECTION: iPSCs plating for differentiation
1. Passage 1X106 iPSC into Geltrex coated 10-cm dish with Accutase and rock inhibitor (refer to material section).

SECTION: iPSCs plating for differentiation
2. For the passaging add 3 mL of Accutase for 5 min .

SECTION: iPSCs plating for differentiation
3. Add 5 mL  of StemFlex , gently mix and transfer to a 15 ml conical tube.

SECTION: iPSCs plating for differentiation
4. 300 x g, 3 min, 25 °C 

SECTION: iPSCs plating for differentiation
5. Resuspend the cells in 1 mL  of StemFlex with rock inhibitor and Count the cells.

SECTION: Midbrain Differentiation
7. Starting on day D0, Aspirate all the media, wash the plate with PBS, and add  D0-D1 media (refer to the materials). 
SECTION: Midbrain Differentiation
8. D1 change half the media with D0-1 medium.

SECTION: Midbrain Differentiation
9. 
D2 change half the media with D2-3 medium.

SECTION: Midbrain Differentiation
10. 
D3 Change half the media with D2-3 medium.

SECTION: Midbrain Differentiation
11. D4 change half the media with D4-7 medium.
SECTION: Midbrain Differentiation
12. D5 change half the media with D4-7 medium.
SECTION: Midbrain Differentiation
13. D6 change half the media with D4-7 medium.
SECTION: Midbrain Differentiation
14. D7 change half the media with D4-7 medium.
SECTION: Midbrain Differentiation
15. D8 change all the media with D8-11 medium.
SECTION: Midbrain Differentiation
16. D9 change half the media with D8-11 medium.
SECTION: Midbrain Differentiation
17. D10 change half the media with D8-11 medium.
SECTION: Midbrain Differentiation
24. On D12 change to D12 terminal media for final differentiation, change half media every 48h.
SECTION: Midbrain Differentiation
25. On D35 change to long-term maintaining media.
SECTION: Midbrain Differentiation
26. Protocol Media schedule
<!--td {border: 1px solid #cccccc;}br {mso-data-placement:same-cell;}-->
 
  Day x--1StemFlexDays 0-1DMEM/F12+Glutamax + B27 + N2 + SB + LDNDays 2-3DMEM/F12+Glutamax + B27 + N2 + SB + LDN + Purmorphamine + SAGDays 4-7DMEM/F12+Glutamax + B27 + N2 + SB + LDN + Purmorphamine + SAG + CHIRDays 8-11DMEM/F12+Glutamax + B27 + N2 + LDN + CHIRDays 12-21DMEM/F12+Glutamax + B27 + N2 + BDNF + GDNF + DAPT+ Ascorbic Acid + dCAMP Days 22-35DMEM/F12+Glutamax + B27 + N2 + 0.5(BDNF + GDNF) + DAPT + Ascorbic Acid + dCAMPDays 35-endDMEM/F12+Glutamax + B27 + N2  + 0.5(BDNF + GDNF) + Ascorbic Acid 
 
SECTION: iPSCs plating for differentiation
6. Wait until iPSC reaches 60% confluence to start differentiation.
SECTION: Midbrain Differentiation
18. On the D12 passage, plate 10000 neural progenitors cells into a Geltrex-coated 96-well plate.
SECTION: Midbrain Differentiation
20. For the passaging add 5 mL of Accutase for 5 min .
SECTION: Midbrain Differentiation
22. 300 x g, 3 min, 25 °C 
SECTION: Midbrain Differentiation
23. Resuspend the cells in 1 mL of Day 12 media with rock inhibitor and Count the cells.
SECTION: Midbrain Differentiation
19. Wash the cells with PBS.
SECTION: Midbrain Differentiation
21. Add 5 mL of Day 12 media with rock inhibitor, gently mix and transfer to a 15 ml conical tube.