Jun 14, 2024

Public workspaceCryopreservation of stem cell derived ventral midbrain neural progenitors

DOI
dx.doi.org/10.17504/protocols.io.14egn6696l5d/v1
  • Tyra Fraser1,
  • Lachlan Thompson1,2
  • 1Florey Institute of Neuroscience and Mental Health;
  • 2The University of Sydney
courtney.wright Wright
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DOI: dx.doi.org/10.17504/protocols.io.14egn6696l5d/v1
Protocol Citation: Tyra Fraser, Lachlan Thompson 2024. Cryopreservation of stem cell derived ventral midbrain neural progenitors. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6696l5d/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 14, 2024
Protocol Integer ID: 100999
Keywords: ASAPCRN, Cryopreservation, Neural progenitors, ventral midbrain, freezing, iPSC
Funders Acknowledgements:
Michael J Fox Foundation
Grant ID: ASAP-000497
Abstract
This protocol outlines the cryopreservation procedure for stem cell derived neural progenitors. It can be used for the cryopreservation and long-term storage of ventral midbrain dopamine neuron progenitors at Day 13 or Day 17 of differentiation in liquid nitrogen tanks. 
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
  • Mr.Frosty‱ (isopropanol-based cell freezing container)Cryovials
  • Labels
  • P1000 pipette
  • 15 mL falcon tubes
  • Accutase
  • Cryostor‱ CS10
  • Rock inhibitor (Ri)
  • NBB27
NBB27 Base media (For Terminal vmDA) 100ml
DMEM/F12 47ml
NBM 47ml
B27 + VitA 2ml
N2 1ml
ITS-A 1ml
NEAA 1ml
GMAX 500ul
Pen strep 500ul
NBB27 base media for terminal vMDA
Safety warnings
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet) for each of the raw materials used
Before start
Day 17 (D17) VmDA and day 13 (D13) VmDA progenitors are used in this protocol
Experimental procedure
Experimental procedure
1d 0h 12m
1d 0h 12m
  1. Take Mr. Frosty‱ out of Temperature-80 °C freezer and keep at TemperatureRoom temperature .
  2. Label the cryogenic vials with a label machine to include the following information: cell line, clone number, day of differentiation, freezing date and initials.
  3. Wash cells with PBS -/-
  4. Put Amount484 µL of Accutase per cm2 of cells.
  5. Incubate cells at Temperature37 °C Duration00:04:00
  6. Whilst cells are incubating place Amount5 mL of PBS -/- into a Amount15 mL Falcon tube.
  7. Remove cells from incubator and pipette up and down twice. If cells come off in large clumps place them back into the incubator for Duration00:02:00 . Repeat this step until cells form small clumps (~5cells).  If cells form small clumps after the first 4-minute incubation move straight to step 8.
  8. Place cells in the falcon tube prepared earlier (Step 6) with Ri 1:1000.
  9. Spin cellsCentrifigation300 x g, 4°C, 00:03:00
9m
  1. Aspirate supernatant and flick pellet twice.
  2. Resuspend inAmount1 mL of NBB27+ Ri (1:1000).
  3. Put Amount10 µL of cell mix into a small Eppendorf tube for cell counting.
  4. Add Amount10 µL trypan blue to cells and mix well by pipetting up and down.
  5. Transfer Amount10 µL of cells + Trypan blue mix in haemocytometer.
  6. Count cells in each quadrant.
  7. Calculate total number of cells.

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

8. Calculate volume required for desired freezing concentration. We recommend freezing no less than 1x106 cells per vial.
9. Spin cellsCentrifigation300 x g, 4°C, 00:03:00
3m
1. Aspirate supernatant and flick pellet twice.
2. Resuspend in desired amount of Cryostor‱ CS10 for each cryovial (Calculated in Step 8 above).

IMPORTANT: Be fast to avoid cell death due to toxic Cryostor ‱ CS10.

3. Quickly aliquotAmount1 mL of cells + Cryostor ‱ CS10 into cryovials.
4. Put the vials in Mr.Frosty and place in the -80°C freezer as fast as possible.
5. Duration24:00:00 after freezing, transport the cells on dry ice and store in Liquid Nitrogen for long-term storage.

IMPORTANT: Do not store cells in the -80 freezer for longer than 1 week.
1d
Critical