Oct 27, 2022
Midbrain organoid astrocyte seeding
- 1Icahn School of Medicine
Protocol Citation: gustavo.parfitt Parfitt 2022. Midbrain organoid astrocyte seeding . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbyp4vx1/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: 71926
Abstract
Midbrain organoid astrocyte seeding protocol
Materials
Midbrain organoid media
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
Protocol materials
Geltrex LDEV Free hESC Quality 5 mlThermo Fisher ScientificCatalog #A1413302
Step 12
Astrocyte MediumScienCellCatalog # #1801
Step 7
Refer to the publish protocols to obtain the cell materials required.
From a 90% confluent plate of astrocytes.
Wash with PBS.
Add 1 mL of TrypLE™ Select Enzyme (1X), no phenol red Thermo Fisher Catalog #12563011.
5m
Transfer the cells to 15 ml falcon tubes and add 5 ml of media.
300 rcf, 25°C, 00:03:00
3m
Resuspend the cell pellet in 1ml ofAstrocyte MediumScienCellCatalog # #1801
Count the cells using
Equipment
Countess II
NAME
Life Technologies
BRAND
AMQAX1000
SKU
Individually add day 40 midbrain organoids to a Corning 96 well V bottom shape plate.
Add 50K astrocyte to the organoids.
Add 100 ul of organoid media.
After a week transfer the organoids to a 96 well plate coated with Geltrex LDEV Free hESC Quality 5 mlThermo Fisher ScientificCatalog #A1413302 1/100 .