Sep 09, 2022

Public workspaceAstrocyte extraction from brain organoids V.2

DOI
dx.doi.org/10.17504/protocols.io.261ge364wl47/v2
  • 1Icahn School of Medicine
  • Ahfeldt Lab
  • Organoid and Assembloid
gustavo.parfitt Parfitt
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DOI: dx.doi.org/10.17504/protocols.io.261ge364wl47/v2
Protocol Citation: gustavo.parfitt Parfitt 2022. Astrocyte extraction from brain organoids. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge364wl47/v2Version created by gustavo.parfitt Parfitt
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 09, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69781
Keywords: ASAPCRN
Abstract
Protocol for astrocyte extraction from brain organoids.
Materials
Mature Astrocyte media
Advanced DMEM/F12 (Thermo, 12634-010)250ml
Neurobasal (Life Tech, 21103049)250 ml
1% NEAA (Thermo,11140-050) 5 ml
1% Glutamax (Thermo, 35050-061)5ml
1% N2 (Life Tech, 17502048)5 ml
2% B27 (Thermo, 17504044)10 ml
10ng/ml CNTF (ProSpec, CYT-272)250ul
Protocol materials
ReagentGeltrex LDEV Free hESC Quality 5 mlThermo Fisher ScientificCatalog #A1413302
Step 1
ReagentTrypLE™ Select Enzyme (1X), no phenol redThermo FisherCatalog #12563011
Step 5
Coat a 6 well plate coat with gelatin 0.1% or ReagentGeltrex LDEV Free hESC Quality 5 mlThermo Fisher ScientificCatalog #A1413302 1:100

Collect 10-20 spheres and place in a 6 well plate (day 40+ spheres)

Wash in PBS twice

Aspirate the PBS
Add Amount1 mL of ReagentTrypLE™ Select Enzyme (1X), no phenol redThermo FisherCatalog #12563011 for Duration00:05:00

5m
Triturate using glass a pipette (2-3 up and down)

Transfer the cells and tissue (even the chunks, the trituration is more effective if you have chunks of tissue) to the coated 6 well plate
Aspirate the extra TrypLe
Add Amount3 mL of astrocyte media (https://www.sciencellonline.com/astrocyte-medium.html)

Half media change media every 3 days or until it turns orange.
3d
Keep changing media every 3d until you observe good amount of cell attached to the plate (may vary according to the patterning)
Passage the astrocytes to a gelatin or geltrex coated 15 cm plate in Astrocyte media once they reach 80% confluency
For the experimental protocols after passage cell will be incubated in maturation media (TBD)