Jun 15, 2024

Public workspacePreparation of ventral midbrain cells for transplantation

DOI
dx.doi.org/10.17504/protocols.io.rm7vzjjkxlx1/v1
  • Tyra Fraser1,
  • Lachlan Thompson1,2
  • 1Florey Institute of Neuroscience and Mental Health;
  • 2The University of Sydney
courtney.wright Wright
Open access
DOI: dx.doi.org/10.17504/protocols.io.rm7vzjjkxlx1/v1
Protocol Citation: Tyra Fraser, Lachlan Thompson 2024. Preparation of ventral midbrain cells for transplantation. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjjkxlx1/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: May 31, 2024
Last Modified: June 15, 2024
Protocol Integer ID: 100998
Keywords: ASAPCRN, transplantation, ventral midbrain, iPSC, cell preparation, neurons
Funders Acknowledgement:
Michael J Fox Foundation
Grant ID: ASAP-000497
Abstract
This protocol outlines the preparation of iPSC derived ventral midbrain progenitors for xenotransplantation.
Guidelines
All work is to be conducted in well sterilised laminar flow hoods designated for human iPSC work where possible to minimise contamination
Materials
Materials
  • PBS -/-
  • Accutase
  • P10, 20, 200, 1000
  • 15ml falcon tube
  • 3 small Eppendorf tubes
  • Trypan blue
  • NBB27 + ALL (add this fresh)
  • Rocki (Ri)


ABC
NBB27 Base media (For Terminal vmDA) 100ml +ALL (added as needed)
DMEM/F12 47ml BDNF (20ng/ml)
NBM 47ml GDNF (20ng/ml)
B27 + VitA 2ml TGFB3 (1ng/ml)
N2 1ml DAPT (10uM)
ITS-A 1ml AA (200uM)
NEAA 1ml dcAMP (0.25mM)
GMAX 500ul
Pen strep 500ul
Components of NBB27 base media
Safety warnings
Attention
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet) for each of the raw materials used
Before start
D19 iPSC derived ventral midbrain progenitors are used in this protocol
Experimental details
Experimental details
15m
  1. Wash wells with 1 x PBS -/-
  2. Incubate cells in Amount484 µL of Accutase per cm2 at Temperature37 °C . Note: D19 typically dissociate between 10-20mins.
  3. Prepare a Amount15 mL falcon tube for cells.
  4. Check dissociation after Duration00:06:00 by using a p1000 to pipette up and down twice onto the cells. Observe if cells dislodge and are present in small clumps of 5-10 cells.
  5. If YES: Collect all cells into a Amount15 mL tube. Wash wells with Accutase to ensure all cells are collected.
  6. If NO: Incubate plate for intervals of Duration00:02:00 Pipette suspension up and down twice targeting the large clumps/sheets at each interval to promote dissociation. Repeat until cells are dissociated into small clumps when you can transfer to a Amount15 mL tube.
8m
Critical
  1. Add Ri (1:1000)
  2. Incubate the tube for Duration00:01:00 intervals until cells are broken into mostly single cells/small clumps (~5cells).
  3. Cancel the reaction with PBS -/- at a 1:1 dilution to Accutase.
  4. Spin cells (Centrifigation300 x g, 4°C, 00:03:00 )
4m
  1. Aspirate supernatant. Flick pellet twice.
  2. Resuspend in NBB27 + All (~2ml per 48well) + Ri 1:1000.
  3. Resuspend suspension and pipette Amount10 µL into a small Eppendorf tube for cell counting. Repeat for a second Eppendorf tube.
  4. Take 2 tubes and add Amount10 µL trypan blue to cells in each tube.
  5. Transfer Amount10 µL of mixed cells in haemocytometer.
  6. Count cells in each quadrant.
  7. Calculate total number of cells. Repeat this for tube 2 to ensure accurate cell counts.

Total cells = Average count of quadrants x Dilution factor x Volume (ml) x 10^4

8. Calculate the total volume needed to resuspend cells to a final density (typically between 100-150K/µl)and the amount of Ri 1:1000 required.
9. Spin cells (Centrifigation300 x g, 4°C, 00:03:00 )
3m
  1. Aspirate supernatant.
  2. Add half the required media on top of the cells gently (i.e. if you have 2 million cells total final volume is Amount20 µL to achieve 100 000 cells/µL so add Amount10 µL of media (NBB27 + All + Ri 1:1000) to pellet.
  3. Using a P20 gently disturb the pellet in a circular motion, taking care not to damage cells by hitting the edge of the tube with the pipette tip.
  4. Once mixed, take up the suspension once or twice and transfer to a small Eppendorf tube.
  5. Measure the volume of cell suspension.
  6. Add the appropriate volume of NBB27 + All + Ri 1:1000 required to reach the final volume.
  7. Place cells on ice and transfer to surgical room immediately for transplantation.


Critical