Generating regionally specified astrocytes

Prepare astrocyte base media by combining;

• 484.5mL Advanced DMEM/F12
• 5mLGlutaMAX
• 5mL N2
• 5mL ANTI-ANTI
• 500µL B-27+VITA

Disassociate D13 VMDA diff or D19 Cortex diff using Accutase for0h 3m 0s to 0h 6m 0s 1. Collect cells as small clumps and inhibit using respective diff media + Ri 1:1000

1. Spin down cells at 1300rpm,0h 0m 0s for 0h 3m 0s
2. Aspirate media and resuspend in respective diff media (with factors required for diff) + Ri 1:1000.
3. Seed 20 000 cells/ well in a 96-well round bottom plate
4. Spin plate at1300rpm,0h 0m 0s for 0h 3m 0s
5. After 24h 0m 0s, change the media with Astro base + EGF (20ug/ml stock; 20 ng/ml final 1:1000 dilution) and hLIF (10ug/ml stock; 20ng/ml final 1:500 dilution)

When spheres have been in hLIF and EGF for 4 weeks change the media to EGF (20ug/ml stock; 20 ng/ml final 1:1000 dilution) + FGF2 (100ug/ml stock; 20ng/ml final 1:5000 dilution)* EGF and FGF2 is maintained for a minimum of 2 months.

When spheres become 0.5cm or larger it is time to chop them to axonotomise the neurons1. Aspirate media and slice spheres using an autoclaved blade or two needles. Slice spheres in one direction then rotate plate 90° and slice again. Continue to rotate until you’ve reached 360°

1. Add 5mL of PBS and move sliced spheres into a 15ml Falcon tube
2. Let sphere settle and aspirate PBS
3. Resuspend in 0.5mL DNase (1mL in large amount of starting material) and incubate at 37°C for 0h 5m 0s. Shake tube every few minutes to distribute DNase through fragments
4. To DNase/cell mixture add 9mLAstro base supplemented w/FGF2 and EGF and re-plate in a new low attachment 10cm plates
5. After 24h 0m 0s, media change spheres with 10mL Astro base supplemented w/FGF2 and EGF to wash out DNase

When spheres have been in EGF+FGF2 for 2 months and have been sliced a minimum of 3 times they can be plated down in 2D format1. Coat plates using 1:80 MG

1. Use the Worthington kit manual to reconstitute the Papain powder and make up all other reagents
2. Transfer spheres to a 15ml falcon tube and wash with PBS-/-. Aspirate PBS -/- once spheres have settled
3. Add Papain DNase mixture to the spheres (Usually double the amount of Papain to volume of spheres) and incubate at 37°C for 0h 15m 0s to 0h 30m 0s. Perform triturates at every 5-minute interval **NOTE: If your spheres are healthy and quite large you might need to manually pull them apart before adding Papain like what we do for cutting
4. At the end of 15 minutes, triturate again and let large clumps settle to the bottom and take off all supernatant (this contains a lot of single cells)
5. Add supernatant to new tube and inhibit with base media
6. Add new Papain, and repeat step 3-5 until clumps are disassociated (you will not be able to dissociate the whole thing so some clumps will be left, no longer than 45 minutes).
7. Spin cells that were inhibited at 1000rpm,0h 0m 0s for 0h 4m 0s
8. Aspirate supernatant and resuspend in 300µL ovomucoid inhibitor +DNase (see Worthington manual)
9. In a separate tube, add 1mL of inhibitor albumin. Slowly transfer the cell solution onto the top of the protein gradient and leave to settle. **Note: the Worthington manual says to spin down, this is not necessary
10. Transfer the top layer of cells to a fresh tube
11. Centrifuge at 1000rpm,0h 0m 0s for 0h 4m 0s
12. Resuspend in EGF + FGF2 (DO NOT ADD Ri)

To freeze astrocytes at the precursor stage, perform prep as normal however resuspend in Astrocyte base media + 10% DMSO

To mature the astrocytes, maintain in CNTF 1:1000 + base media for 2 weeks