TITLE: Midbrain organoid differentiation in spinner flasks.
AUTHORS: gustavo.parfitt

[DESCRIPTION]
Midbrain differentiation protocol using spinner flasks.

[STEPS]
SECTION: iPSCs aggregation in spinner flasks
1. Passage iPSC lines to four 10-cm dishes to generate 40X106 cells.

SECTION: iPSCs aggregation in spinner flasks
4. Wait for the cells to reach 80% confluence and seed iPSC 40X106 cells into a spinner flask.

SECTION: iPSCs aggregation in spinner flasks
2. Prepare 120 mL of StemFlex  per flask with rock inhibitor (refer to material section) and add 115 to the spinner flask.
SECTION: iPSCs aggregation in spinner flasks
3. Place the spinner flask on the stir plate and set to 65 rpm. 
SECTION: iPSCs aggregation in spinner flasks
5. For the passaging add 3 mL of Accutase for 5 min .

SECTION: iPSCs aggregation in spinner flasks
6. Add 5 mL  of StemFlex , gently mix and transfer to a 15 ml conical tube.

SECTION: iPSCs aggregation in spinner flasks
7. 300 x g, 3 min, 25 °C 

SECTION: iPSCs aggregation in spinner flasks
8. Resuspend the cells in 1 mL  of StemFlex with rock inhibitor and Count the cells.

SECTION: iPSCs aggregation in spinner flasks
9. Put the cells in the correct cell concentration and add to 5 mL  of StemFlex with rock inhibitor. 

SECTION: iPSCs aggregation in spinner flasks
10. From the main opening of the spinner flask add the cells and plate back on the stir plate.
SECTION: iPSCs aggregation in spinner flasks
11. Every other day change 60 mL of media using StemFlex until the spheres reach 300 μM.

SECTION: Midbrain Differentiation
12. On the starting day D0, filter the spheres in a 300 μM to 500μM range.
SECTION: Midbrain Differentiation
13. 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. 
SECTION: Midbrain Differentiation
14. D1 change half the media with D0-1 medium.

SECTION: Midbrain Differentiation
15. 
D2 change half the media with D2-3 medium.

SECTION: Midbrain Differentiation
16. 
D3 Change half the media with D2-3 medium.

SECTION: Midbrain Differentiation
17. D4 change half the media with D4-7 medium.
SECTION: Midbrain Differentiation
18. D5 change half the media with D4-7 medium.
SECTION: Midbrain Differentiation
19. D6 change half the media with D4-7 medium.
SECTION: Midbrain Differentiation
20. D7 change half the media with D4-7 medium.
SECTION: Midbrain Differentiation
21. D8 change all the media with D8-11 medium.
SECTION: Midbrain Differentiation
22. D9 change half the media with D8-11 medium.
SECTION: Midbrain Differentiation
23. D10 change half the media with D8-11 medium.
SECTION: Midbrain Differentiation
24. D11 change half the media with D8-11 medium.
SECTION: Midbrain Differentiation
25. 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.
SECTION: Midbrain Differentiation
26. On D35 change to long-term maintaining media.
SECTION: Midbrain Differentiation
27. 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