Oct 27, 2022
Midbrain astrocyte and co-culture
- 1Icahn School of Medicine
Protocol Citation: gustavo.parfitt Parfitt 2022. Midbrain astrocyte and co-culture. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8j9yrg2w/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: October 27, 2022
Last Modified: October 27, 2022
Protocol Integer ID: 71928
Abstract
Midbrain astrocyte and co-culture for mix-genetic experiments.
Materials
Media for iPSC
StemFlex medium
Media for iPSC passaging
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
D12-24 (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
D25+ (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 + 10 ng/mL CNTF.
Protocol materials
ACCUTASE™ 100 mL
STEMCELL Technologies Inc.Catalog #7920
Step 4
Geltrex LDEV Free hESC Quality 5 mlThermo Fisher ScientificCatalog #A1413302
In 2 steps
STEM-CELLBANKER - GMP GradeamsbioCatalog #11890
Step 20
Astrocyte MediumScienCellCatalog # #1801
Step 29
Intro
Intro
Midbrain NPC were generated following the publish protocol https://www.protocols.io/view/midbrain-organoid-differentiation-in-spinner-flask-rm7vzbnr4vx1/v1 with modifications listed bellow.
NPC generation
NPC generation
8m
8m
From a 80% confluent plate of iPSCs in a 10 cm plate.
Wash with 5ml of PBS
For the passaging add 3 mL of ACCUTASE™ 100 mL
Stemcell TechnologiesCatalog #7920
5m
Add 5 mL of StemFlex , gently mix and transfer to a 15 ml conical tube.
300 rcf, 25°C, 00:03:00
3m
Resuspend the cells in 1 mL of StemFlex with rock inhibitor and Count the cells using
Equipment
Countess II
NAME
Life Technologies
BRAND
AMQAX1000
SKU
Plate 2 million cells in a 1/100 Geltrex LDEV Free hESC Quality 5 mlThermo Fisher ScientificCatalog #A1413302 coated plate in Stemflex with 10 μM Y-27632.
D0 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.
Freeze the NPCs in STEM-CELLBANKER - GMP GradeamsbioCatalog #11890
NPC Plating
NPC Plating
Thaw the NPCs and transfer to a 15 ml conical tube.
Add 5 ml of D12 terminal media with 10 μM Y-27632
300 rcf, 25°C, 00:03:00
3m
Resuspend the cells in 1 mL D12 terminal media with 10 μM Y-27632 and count the cells using
Equipment
Countess II
NAME
Life Technologies
BRAND
AMQAX1000
SKU
Astrocyte co-culture
Astrocyte co-culture
3w 4d 0h 3m
3w 4d 0h 3m
In a 96 well plate coated with Geltrex LDEV Free hESC Quality 5 mlThermo Fisher ScientificCatalog #A1413302 1/100 plate 50k NPCs
Change half media every day until day 25.
3w 4d
Obtain astrocytes following protocol https://www.protocols.io/view/astrocyte-extraction-from-brain-organoids-261ge364wl47/v2
Thaw the Astrocytes and transfer to a 15 ml conical tube.
Add 5 ml of AAstrocyte MediumScienCellCatalog # #1801 with 10 μM Y-27632.
300 rcf, 25°C, 00:03:00
3m
Resuspend the cells in 1 mL D12 terminal media with 10 μM Y-27632 and count the cells using
Equipment
Countess II
NAME
Life Technologies
BRAND
AMQAX1000
SKU
Plate 10k on the neurons.
Mature the co-cultures until day 50 changing media every other day with day 25 media.