Jun 17, 2024
iPSC Midbrain differentiation in 96 well plates
- 1University College of London;
- 2Icahn School of Medicine at Mount Sinai
Protocol Citation: gustavo.parfitt Parfitt, Elena Coccia 2024. iPSC Midbrain differentiation in 96 well plates. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l62dj5gqe/v1
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: June 17, 2024
Last Modified: June 17, 2024
Protocol Integer ID: 101924
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 the first passage
StemFlex medium + 10 μM Y-27632 + 1:100 Pen/Strep
Media composition for differentiation
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 plating for differentiation
iPSCs plating for differentiation
8m
Passage 1X106 iPSC into Geltrex coated 10-cm dish with Accutase and rock inhibitor (refer to material section).
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.
Wait until iPSC reaches 60% confluence to start differentiation.
Midbrain Differentiation
Midbrain Differentiation
1w 5d
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 5 mL of Accutase for 00:05:00 .
5m
Add 5 mL of Day 12 media with rock inhibitor, 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 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
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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 |