Recovery and preparation for transplantation of cryopreserved vmDA progenitors for transplantation.

Prepare NBB27 base media according to the materials table.

Place5mL of NBB27 media + All + Ri 1:1000 in a 15mL falcon tube. 1. Warm cryopreserved cells in hand or in a water bath until a small chunk of ice is remains

1. Remove thawed portion into the tube containing the 5mL (prepared in as per Step 1)
2. Take 500µL of media and use it to thaw the remaining chunk of ice.
3. Remove all media from cryopreserved tube and place into the falcon tube with media.
4. Spin cells(300x g,4°C).

Aspirate supernatant. Flick pellet twice.1. Resuspend in 1mLof NBB27 + All + Ri 1:1000.

1. Pipette 10µL into a small Eppendorf tube for cell counting. Repeat for a second Eppendorf tube.
2. Take the 2 tubes and add 10µL trypan blue to cells in each tube.
3. Place 10µL of mixed cells in haemocytometer.
4. Count cells in each quadrant.
5. Calculate total number of cells. Repeat this for tube 2 to ensure an accurate cell count.

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.

9. Spin cells(300x g,4°C)

Label small Eppendorf tube with specific details about the transplant.1. Aspirate supernatant

1. Add half the required media on top of the cells gently (i.e. if you have 2 million cells total final volume is 20µL to achieve 100 000 cells/uL, so add 10µL of media (NBB27 + All + Ri 1:1000) to pellet.
2. 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.
3. Once mixed, take up the suspension once or twice and transfer to a small Eppendorf tube.
4. Measure the volume of cell suspension.
5. Add the appropriate volume of NBB27 + All + Ri 1:1000 required to reach the final volume.
6. Place cells on ice ready for use