TITLE: Generating regionally specified astrocytes
AUTHORS: Tyra Fraser, Lachlan Thompson

[DESCRIPTION]
This protocol outlines the differentiation of stem cells into regionally specific ventral midbrain and cortical astrocytes.
The protocol is a 4–5-month procedure and requires media changes 3 times a week. 

[GUIDELINES]
Seeding density reference and expansion

3 spheres are plated into a 48 well plate > 24 well plate >12 well plate > 6-well plate > T-25 > T75 > T175
P0 is when you first seed them into 2D format
At P10 VMDA astrocytes > MAX Passage 
At P12 Cortex astrocytes > MAX Passage
At P10 Spinal cord astrocytes > MAX Passage

[STEPS]
SECTION: Preparation
1. Prepare astrocyte base media by combining;
484.5 mL Advanced DMEM/F12
5 mLGlutaMAX
5 mL N2
5 mL ANTI-ANTI
500 µL B-27+VITA


SECTION: Sphere formation
2. Disassociate D13 VMDA diff or D19 Cortex diff using Accutase for3 min to 6 min 
Collect cells as small clumps and inhibit using respective diff media + Ri 1:1000
Spin down cells at 1300 rpm for 3 min
Aspirate media and resuspend in respective diff media (with factors required for diff) + Ri 1:1000. 
Seed 20 000 cells/ well in a 96-well round bottom plate
Spin plate at1300 rpm for 3 min 
After 1440 min, 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)
SECTION: Maintenance
3. 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. 
SECTION: Neurosphere slicing
4. When spheres become 0.5cm or larger it is time to chop them to axonotomise the neurons
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°
Add 5 mL of PBS and move sliced spheres into a 15ml Falcon tube
Let sphere settle and aspirate PBS
Resuspend in 0.5 mL DNase (1mL in large amount of starting material) and incubate at 37 °C for 5 min. Shake tube every few minutes to distribute DNase through fragments
To DNase/cell mixture add 9 mLAstro base supplemented w/FGF2 and EGF and re-plate in a new low attachment 10cm plates
After 1440 min, media change spheres with 10 mL Astro base supplemented w/FGF2 and EGF to wash out DNase
SECTION: Plating down using Papain
5. 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 format
Coat plates using 1:80 MG
Use the Worthington kit manual to reconstitute the Papain powder and make up all other reagents
Transfer spheres to a 15ml falcon tube and wash with PBS-/-. Aspirate PBS -/- once spheres have settled
Add Papain DNase mixture to the spheres (Usually double the amount of Papain to volume of spheres) and incubate at 37 °C for 15 min  to 30 min. 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
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)
Add supernatant to new tube and inhibit with base media
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).
Spin cells that were inhibited at 1000 rpm for 4 min
Aspirate supernatant and resuspend in 300 µL ovomucoid inhibitor +DNase (see Worthington manual)
In a separate tube, add 1 mL 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
Transfer the top layer of cells to a fresh tube
Centrifuge at 1000 rpm for 4 min
Resuspend in EGF + FGF2 (DO NOT ADD Ri) 

SECTION: Freezing astrocytes
6. To freeze astrocytes at the precursor stage, perform prep as normal however resuspend in Astrocyte base media + 10% DMSO
SECTION: M
7. To mature the astrocytes, maintain  in CNTF 1:1000 + base media for 2 weeks