Mar 19, 2026
Astrocyte-Conditioned Media (ACM) cultures
- Elena Coccia1
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. Astrocyte-Conditioned Media (ACM) cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv55m1nv1b/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: February 12, 2026
Last Modified: March 19, 2026
Protocol Integer ID: 243129
Keywords: astrocyte-conditioned media, ACM, midbrain organoids, neuronal cultures, neuronal culture, 2d neuronal culture, involving d25 midbrain organoid, d25 midbrain organoid, astrocyte, conditioned media, acm, culture, use in experiment
Abstract
To generate astrocyte-conditioned media (ACM) for use in experiments involving D25 midbrain organoids or 2D neuronal cultures.
Materials
Mature Astrocyte Media (Complete medium for astrocyte culture)
15 mL centrifuge tubes (Sterile, disposable)
50 mL centrifuge tubes (Sterile, disposable)
37°C water bath (For incubation)
Centrifuge (Capable of 2000 x g)
-80°C freezer (For storage of conditioned media)
Reagents:
Cell Culture Medium Mature Astrocyte Media - Medium for maintaining astrocyte cultures
Reagent 0.25% Trypsin-EDTA - Enzyme for cell dissociation
Reagent DNase I - Enzyme to prevent DNA clumping
Troubleshooting
Problem
Low astrocyte yield
Solution
Ensure proper seeding density and media quality.
Problem
Contamination
Solution
Maintain sterile techniques throughout the procedure.
Safety warnings
Follow standard laboratory safety protocols. Use gloves and goggles when handling reagents and biological materials.
Astrocyte Culture Preparation
Astrocytes will be cultured in Mature Astrocyte Media for ACM generation.
Cell Seeding
1. Prepare 500 mL of Mature Astrocyte Media. 2. Seed astrocytes at a density of 80,000 cells/mL in 100 mm plates. 3. Incubate at 37°C with 5% CO2 until confluent. Notes: Monitor for confluence and replace media every 4 days.
ACM Harvesting
Every 4 days, ACM will be harvested.
Media Collection
1. Remove the existing media from the flasks. 2. Add fresh Mature Astrocyte Media to each flask. 3. Incubate for an additional 4 days. - Duration: 4 days - Notes: Ensure that the media is not disturbed during incubation.
Centrifugation
1. After 4 days, collect the conditioned media from each flask into sterile 15 mL centrifuge tubes. 2. Centrifuge at 2000 x g for 5 minutes to remove cellular debris.
Storage
1. Transfer the supernatant to new sterile tubes and store at -80°C.
Application of ACM
ACM can be used to maintain D25 midbrain organoids or 2D neuronal cultures.
Media Exchange
1. Maintain organoids or neuronal cultures in ACM for 30 days. 2. Perform 50% media exchanges every 3 days. - Duration: 30 days