Cryopreservation of stem cell derived ventral midbrain neural progenitors

Take Mr. Frosty^TM out of -80°Cfreezer and keep at Room temperature .1. Label the cryogenic vials with a label machine to include the following information: cell line, clone number, day of differentiation, freezing date and initials.

1. Wash cells with PBS -/-
2. Put 484µL of Accutase per cm² of cells.
3. Incubate cells at 37°C``0h 4m 0s
4. Whilst cells are incubating place 5mL of PBS -/- into a 15mL Falcon tube.
5. Remove cells from incubator and pipette up and down twice. If cells come off in large clumps place them back into the incubator for 0h 2m 0s. 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.
6. Place cells in the falcon tube prepared earlier (Step 6) with Ri 1:1000.
7. Spin cells300x g,4°C

Aspirate supernatant and flick pellet twice. 1. Resuspend in1mLof NBB27+ Ri (1:1000).

1. Put 10µLof cell mix into a small Eppendorf tube for cell counting.
2. Add 10µL trypan blue to cells and mix well by pipetting up and down.
3. Transfer 10µL of cells + Trypan blue mix in haemocytometer.
4. Count cells in each quadrant.
5. 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 cells`300x g,4°C`
   

1. Aspirate supernatant and flick pellet twice.

2. Resuspend in desired amount of Cryostor^® CS10 for each cryovial (Calculated in Step 8 above).

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

3. Quickly aliquot1mLof cells + Cryostor ^® CS10 into cryovials.

4. Put the vials in Mr.Frosty and place in the -80°C freezer as fast as possible.

5. 24h 0m 0s after freezing, transport the cells on dry ice and store in Liquid Nitrogen for

long-term storage.

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