Preparation of human iPSC-derived cortical neuronal progenitors for transplantation into the rodent brain

Prepare a Falcon tube with 2mLbase cortical media plus Rock inhibitor (Ri, 1:1000 dilution)1. Wash cells with 300µL PBS -/- (gently run PBS down the side of each well)

1. Incubate cells in 300µL Accutase (per well) at 37°C to lift cells off wells in small clumps - Monitor the Accutase incubation after 0h 10m 0s; tap the plate to dislodge cells and look for cells lifting in a sheet before proceeding

    - Triturate 5 times to break the cells into clumps
   
    - Incubate clumps for a further `0h 5m 0s` at `37°C`
   
   
   
    - Triturate 10 times to break the cells into single cells and small clumps 
   

2. Transfer cells from the wells into the 15ml Falcon tube containing media + Ri (Step 1)

3. Rinse with plate with a further 300µL of Accutase to ensure all cells are collected

4. Spin cells at 4°C, 1300rpm,0h 0m 0s for 0h 4m 0s

5. Discard supernatant

6. Resuspend cell pellet in 1-2ml base cortical media and add Ri (1:1000)

7. Count cells

   - Take 2 x `10µL`aliquot of cells in two separate tubes
   

Dilute cells 1:1 using 10µL of trypan blue

   - Count cells in a `10µL` aliquot from each tube using a haemocytometer 



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

10. Calculate the total volume needed to resuspend cells to a final density of 100 000/uL

11. Spin cells at 4°C, 1300rpm,0h 0m 0s for 0h 4m 0s

12. Whilst cells are spinning, make up 1mL media + Ri which will be used to resuspend the

cells for implantation surgery

13. At end of the spin, collect tube and discard supernatant

14. Using a P20, add 5µL and resuspend pellet in base cortical media + Ri

15. Transfer resuspended pellet to PCR tube and precisely measure the volume

16. Add the remaining volume needed for cells to reach a density of 100 000 cells/ul as

calculated in step 10.

17. Label the PCR tube containing the cells with details of the cell line and concentration

18. Store cells on ice and transport to animal surgical room.

Athymic mice undergo stereotactic surgery to receive a unilateral graft of 100,000 cortical progenitors in 1µL volume. The surgical coordinates from Bregma are AP: +1, ML: +/-1.5, DV: -1.5

For full details of procedures for implantation of cell suspension can be found athttps://www.protocols.io/view/transplantation-of-fetal-midbrain-dopamine-progeni-261ge4jq7v47/v11