Sep 19, 2022
Midbrain organoid differentiation in spinner flasks.
This protocol is a draft, published without a DOI.
- 1Icahn School of Medicine
Protocol Citation: gustavo.parfitt 2022. Midbrain organoid differentiation in spinner flasks.. protocols.io https://protocols.io/view/midbrain-organoid-differentiation-in-spinner-flask-cgretv3e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 16, 2022
Last Modified: December 26, 2022
Protocol Integer ID: 70150
Keywords: ASAPCRN
Funders Acknowledgement:
Lily Sarrafha
Tim Ahfeldt
Joel Blanchard
Abstract
Midbrain differentiation protocol using spinner flasks.
Materials
Media for iPSC
StemFlex medium
Media for spheres making
StemFlex medium + 10 μM Y-27632 + 1:100 Pen/Strep
Media composition for diferenciation
D0-1 - DMEM/F-12+Glutamax + 13 B-27 minus vitamin A + 13 N-2 + 100 nM LDN193189 + 10 μM SB431542
D2-3 - DMEM/F-12+Glutamax + 13 B-27 minus vitamin A + 13 N-2 + 100 nM LDN193189 + 10 μM SB431542+ 2 μM Purmorphamine + 1 μM SAG
D4-7 - DMEM/F-12+Glutamax + 13 B-27 minus vitamin A + 13 N-2 + 100 nM LDN193189 + 10 μM SB431542
+ 2 μM Purmorphamine + 1 μM SAG + 3 μM CHIR99021
D8-11 - DMEM/F-12+Glutamax + 13 B-27 minus vitamin A + 13 N-2 + 100 nM LDN193189 + 3 μM CHIR99021
D12-21 (Terminal Media) DMEM/F-12 + Glutamax + 13 B-27 minus vitamin A + 13 N-2 + 20 ng/mL BDNF + 20 ng/mL GDNF + 0.2 mM Ascorbic Acid + 10 μM DAPT + 0.1 μM dcAMP
D22-34 (Terminal Media ) DMEM/F-12+Glutamax + 13 B-27 minus vitamin A + 13 N-2 + 10 ng/mL BDNF + 10 ng/mL GDNF + 0.2 μM Ascorbic Acid + 10 μM DAPT + 0.1 mM dcAMP
D35+ (Long-Term Media) DMEM/F-12+Glutamax + 13 B-27 minus vitamin A + 13 N-2 + 10 ng/mL BDNF + 10 ng/mL GDNF + 0.2 μM Ascorbic Acid
iPSCs aggregation in spinner flasks
iPSCs aggregation in spinner flasks
8m
Passage iPSC lines to four 10-cm dishes to generate 40X106 cells.
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 40X106 cells into a spinner flask.
For the passaging add 3 mL of Accutase for 00:05:00 .
5m
Add 5 mL of StemFlex , gently mix and transfer to a 15 ml conical tube.
300 x g, 25°C, 00:03:00
3m
Resuspend the cells in 1 mL of StemFlex with rock inhibitor and Count the cells.
Put the cells in the correct cell concentration and add to 5 mL 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 60 mL of media using StemFlex until the spheres reach 300 μM.
Midbrain Differentiation
Midbrain Differentiation
1w 5d
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
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| 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 |