Aug 13, 2023

Public workspaceGenerating regionally specified astrocytes

DOI
dx.doi.org/10.17504/protocols.io.261ged3qov47/v1
  • Tyra Fraser1,
  • Lachlan Thompson1,2
  • 1Florey Institute of Neuroscience and Mental Health;
  • 2University of Sydney
courtney.wright Wright
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DOI: dx.doi.org/10.17504/protocols.io.261ged3qov47/v1
Protocol Citation: Tyra Fraser, Lachlan Thompson 2023. Generating regionally specified astrocytes. protocols.io https://dx.doi.org/10.17504/protocols.io.261ged3qov47/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: August 11, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86378
Keywords: ASAPCRN, astrocytes, stem cells, regional astrocytes, diseased astrocytes
Funders Acknowledgement:
Michael J Fox Foundation
Grant ID: ASAP-000497
Abstract
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
Materials
Material input

  • PPMI stem cells
  • D13 VMDA neurons
  • D13 cortical neurons


Key materials
  • ReagentAdvanced DMEM/F-12Thermo FisherCatalog #12634010
  • ReagentGlutamax (100x)Gibco - Thermo FischerCatalog #35050-061
  • ReagentGibco™ N-2 Supplement (100X)Thermo Fisher ScientificCatalog #17502048
  • ReagentAntibiotic-Antimycotic 9100x0 [Anti-Anti]Thermo Fisher ScientificCatalog #15240062
  • ReagentB-27™ Plus Supplement (50X)Thermo FisherCatalog #A3582801
  • ReagentBasic FGF (FGF2), HumanGold BiotechnologyCatalog #1140-02
  • ReagentEGF, Epidermal Growth Factor, humanBio Basic Inc.Catalog #RC216-15.SIZE.100ug
  • ReagentPDS Kit Papain VialWorthington Biochemical CorporationCatalog #LK003176
  • ReagentDNase I, RNase freeThermo Fisher ScientificCatalog #EN0525









Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet)
Preparation
Preparation
Prepare astrocyte base media by combining;
  • Amount484.5 mL Advanced DMEM/F12
  • Amount5 mL GlutaMAX
  • Amount5 mL N2
  • Amount5 mL ANTI-ANTI
  • Amount500 µL B-27+VITA


Sphere formation
Sphere formation
  1. Disassociate D13 VMDA diff or D19 Cortex diff using Accutase forDuration00:03:00 to Duration00:06:00
  2. Collect cells as small clumps and inhibit using respective diff media + Ri 1:1000
  3. Spin down cells at Centrifigation1300 rpm for Duration00:03:00
  4. Aspirate media and resuspend in respective diff media (with factors required for diff) + Ri 1:1000.
  5. Seed 20 000 cells/ well in a 96-well round bottom plate
  6. Spin plate atCentrifigation1300 rpm for Duration00:03:00
  7. After Duration24:00:00 , 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)
1d 0h 15m
Maintenance
Maintenance
  • 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.
Neurosphere slicing
Neurosphere slicing
  1. When spheres become 0.5cm or larger it is time to chop them to axonotomise the neurons
  2. 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°
  3. Add Amount5 mL of PBS and move sliced spheres into a 15ml Falcon tube
  4. Let sphere settle and aspirate PBS
  5. Resuspend in Amount0.5 mL DNase (1mL in large amount of starting material) and incubate at Temperature37 °C for Duration00:05:00 . Shake tube every few minutes to distribute DNase through fragments
  6. To DNase/cell mixture add Amount9 mL Astro base supplemented w/FGF2 and EGF and re-plate in a new low attachment 10cm plates
  7. After Duration24:00:00 , media change spheres with Amount10 mL Astro base supplemented w/FGF2 and EGF to wash out DNase
1d 0h 5m
Plating down using Papain
Plating down using Papain
53m
53m
  1. 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
  2. Coat plates using 1:80 MG
  3. Use the Worthington kit manual to reconstitute the Papain powder and make up all other reagents
  4. Transfer spheres to a 15ml falcon tube and wash with PBS-/-. Aspirate PBS -/- once spheres have settled
  5. Add Papain DNase mixture to the spheres (Usually double the amount of Papain to volume of spheres) and incubate at Temperature37 °C for Duration00:15:00 to Duration00:30:00 . 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
  6. 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)
  7. Add supernatant to new tube and inhibit with base media
  8. 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).
  9. Spin cells that were inhibited at Centrifigation1000 rpm for Duration00:04:00
  10. Aspirate supernatant and resuspend in Amount300 µL ovomucoid inhibitor +DNase (see Worthington manual)
  11. In a separate tube, add Amount1 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
  12. Transfer the top layer of cells to a fresh tube
  13. Centrifuge at Centrifigation1000 rpm for Duration00:04:00
  14. Resuspend in EGF + FGF2 (DO NOT ADD Ri)

53m
Critical
Freezing astrocytes
Freezing astrocytes
To freeze astrocytes at the precursor stage, perform prep as normal however resuspend in Astrocyte base media + 10% DMSO
M
M
2w
2w
To mature the astrocytes, maintain in CNTF 1:1000 + base media for 2 weeks