Oct 27, 2022

Midbrain organoid astrocyte seeding

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzbyp4vx1/v1
  • 1Icahn School of Medicine
  • Ahfeldt Lab
  • Organoid and Assembloid
gustavo.parfitt Parfitt
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzbyp4vx1/v1
Protocol Citation: gustavo.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
Keywords: astrocyte seeding, midbrain organoid
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
Astrocyte MediumScienCellCatalog # #1801
Geltrex LDEV Free hESC Quality 5 mlThermo Fisher ScientificCatalog #A1413302
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 .