iMPS dissociation
Preparation of a Single Cell Suspension of iMPS
*Note: To estimate the number of starting cells that will be required, expected yields of iMPS
cultures from a confluent T25 flask culture are approximately 6-8 x 106 viable cells.
Day 1:
1. iMPS shipped in flask overnight in Hibernate media.
2. Collect and transfer iMPS spheres (between p2-p7) from the flask (T12.5 to T175) in 15 mL
conical tube. 5-10 mL serological pipet.
3. Let iMPS spheres settle by gravity and carefully aspirate out the media without disturbing
the spheres at the bottom.
4. Rinse the spheres once with 2 mL of HBSS/PBS. Let spheres settle by gravity or quick
centrifugation (200 x g for 30 seconds) again.
5. Remove and discard HBSS/PBS.
6. Add 1 mL of accutase per conical.
7. Incubate at 37°C in water bath for 5 minutes with gentle shaking intermittently every 1.5
minutes.
8. Gently pipette the cell suspension up and down in the conical (3-4x) with P1000 to
dissociate the spheres into a single cell suspension.
9. Strain the single cell suspension (still containing some un-dissociated spheres) by passing
the cell suspension through 45 μm Cell Strainer.
10. Collect any un-dissociated spheres from strainer net with P200.
11. Use 1 mL accutase and transfer to a new micro-centrifuge tube. This will ensure any
remaining spheres are dissociated a single cell suspension.
12. Incubate un-dissociated iMPS spheres at 37°C in water bath for additional 3 minutes.
13. Manually gently pipette with P200 (5-8x) up and down to break resistant spheres.
14. Strain this cell suspension by passing again through the same Cell Strainer.
15. Rinse the strainer with 1 mL media.
16. Combine with previously collected single cell suspension filtrate in 15 mL conical.
17. Add 8 mL of PBS containing the cell suspension.
18. Centrifuge the cells at 200 x g for 5 minutes at room temperature (15 - 25°C).
19. Aspirate the supernatant. Resuspend and wash again in 5 mL MNMM stage 1.
20. Count viable cells using Trypan Blue. Dilute a sample of the resuspended cells 1:10 in
Trypan blue and mix gently. Count viable, unstained cells using a hemacytometer.
21. Dilute cells to 3.75x105 cells/ml [75,000 cells/well of 96-well plate, 200ul/well] and
resuspend the appropriate amount of matrigel with cell-media solution (1:100 final dilution)
Day 2-7:
1. Change media every other day (200 ul/well) with MNMM Stage 1 media with DAPT (2.5
uM).
Day 8+:
1. Switch to MNMM Stage 2 media with AraC (0.1 uM) to block residual glial cell proliferation
2. Change media every other day
Day 12: Transfect in Hb9-GFP and Syn-dsRed reporters for live imaging
1. Prepare DNA-Optimem mixture: 600 ng DNA of each reporter with 25 ul/well of Optimem.
Incubate for 5 minutes
2. Prepare Lipofectamine 2000-Optimem mixture: 1.5 ul of lipofectamine in 25 ul of Optimem
per well. Incbuate for 5 minutes.
3. Mix DNA-Optimem and Lipofectamine-Optimem together. Quickly vortex and incubate at
RT for 20 minutes.
4. Remove and save media from iMN plate. Filter and label “Conditioned iMN media.”
5. Wash plate with 200 ul of Neurobasal media twice.
6. Add 100 ul of Neurobasal-KY to each well, followed by 50 ul of the DNA-Lipofectamine-
Optimem mixture.
7. Incubate at 37C for 4 hours.
8. Remove all media and wash twice with 200 ul of Neurobasal.
9. Add 200 ul of a 1:1 mixture of conditioned iMN media with fress MNMM Stage 2 media with
AraC.
10. Assess fluorescence on Day 15 and begin longitudinal imaging on Day 16 if cells are
labeled. Continue to change media every other day.
For 10x KY solution, gradually add small amounts of kynurenic acid to water containing phenol red and use the color of the phenol
red to titrate the pH of the solution back up to about 7.4 as the acid dissolves. Filter the mixture of ingredients below and store at 4C.