Apr 15, 2025
Human Primary Astrocyte (hPA) Culture
- 1Duke University
- Gersbach Lab
Protocol Citation: Samuel Reisman, Charles Gersbach 2025. Human Primary Astrocyte (hPA) Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v97jwzg3e/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: April 09, 2025
Last Modified: April 15, 2025
Protocol Integer ID: 126470
Funders Acknowledgements:
NIH
Grant ID: HG012053
Abstract
This protocols describes methods for culturing human primary astrocytes.
Materials
Ordering information
- Human Astrocytes, SciencCell 1800
- Astrocyte media (complete kit), ScienCell 1801
- Poly-L-Lysine, 10mg/ml, ScienCell 403
- Poly-D-Lysine, 5mg, Sigma P6407
Astrocyte Growth Media
For standard culture use Astrocyte media (ScienCell 1801) supplemented with 10% FBS.
To prepare:
- Remove 60ml basal media from 500mml bottle
- Add thawed AGS, FBS, and P/S supplements (ScienCell 1801)
- Add additional 40ml FBS to bring concentration from 2% to 10%
- Filter
- Media is stable for one month when stored in the dark at 4C
- Media should not be warmed in 37C water bath, but instead should be warmed to RT
Cell counts and size
1. 1 confluent 10cm holds approx. 10 million ScienCell 1800 hPAs
2. Cells range from approx. 10-24um in suspension (via Countess), average size: 15um
Neurogenic media recipe
| A | B | C | D | E | |
| NDM (Guo 2014) | unit | 100 mL | 250 mL | 500 mL | |
| DMEM/F12 (GIBCO) | mL | 97.5 | 243.75 | 487.5 | |
| 0.5% FBS (GIBCO) | mL | 0.5 | 1.25 | 2.5 | |
| 3.5 mM glucose (Sigma)* | mg | 63.056 | 157.64 | 315.28 | |
| penicillin/streptomycin (GIBCO) | mL | 1 | 2.5 | 5 | |
| N2 supplement (GIBCO) | mL | 1 | 2.5 | 5 | |
| Filter sterilize after combining |
Aliquot preparation
PDL aliquot preparation
1. Add 5 ml ddH2O in a 5mg bottle of PDL (Sigma P6407)
2. Filter through 0.45um filter. Make 100ul or 400ul aliquots; store at -20C
- label aliquot size
3. Stocks are 100x, dilute to 1x before coating
- For 100ul aliquots: dilute to 10ml
- For 400ul aliquots: dilute to 40ml
Coating plates (PDL)
Coating plates (PDL)
All plates need to be coated with ply-D-Lysine and rinsed before plating cells
Dilute PDL stock (10x) to 1x with sterile molecular biology grade water
Add to plate (enough volume to cover bottom)
Incubate at 37C for minimum 1hr
Before plating, rinse 2x with water or dPBS
after second rinse, aspirate carefully to remove all liquid
Add cell culture media
Thawing cells
Thawing cells
Prepare plates and prepare a 15ml tube with >=2ml culture media
Remove vial from liquid nitrogen and thaw quickly in water bath until only small ice crystals remain
Carefully pipette cells from seal into 15ml tube
Rinse cryovial once with media
Minimize pipetting, cells are fragile
Add thawed cells directly into TC plate. NO NOT SPIN! Leave DMSO in media
Record the lot #
The next day, change media to remove DMSO
Maintaining cells
Maintaining cells
Change the medium every three days, until the culture is approx. 70% confluent
One the culture riches 70% confluence, change medium every other day until the culture is approx. 90% confluent
Passaging cells
Passaging cells
Passage when culture reaches 90-95% cofluency
Prepare ploy-D-lysine coated plate (see steps 1-6, coating plates)
Warm the following reagents to ROOM TEMPERATURE: media, 0.025% Trypsin/EDTA (note, this is a 1:10 dilution from standard 0.25% Trypsin/EDTA), dPBS, FBS
Rinse cells with dPBS
Add 10ml 0.025% Trypsin/EDTA to cells (10cm dish-scale volume to plate)
While incubating at 37C, add 5ml FBS to 50ml conical tube
Once cells completely round up, transfer Trypsin solution from plate to the 50ml centrifuge tube (a small percentage of cell may detach), and continue to incubate at 37C for another minute (no solution in the dish)
At the end of the incubation, gently tap the side of the dish to dislodge cells from the surface. Check under a microscope to make sure that all cells detach
Add 5ml of media to collect cells and transfer detached cells to the 50ml centrifuge tube.
Rinse the dish with another 5ml of media to collect the residual cells if needed
Check flask to make sure not too many cells were left behind
Centrifuge the cell in the 50ml centrifuge tube at 1000 rpm for 5 minute
Gently resuspend cells in culture media
Plate at desired density
1:6-1:8 for routine split
If transducing for reprogramming: Cells should be 75-90% confluent at transduction