Fabrication of fluid-walled dumbbells and generation of the human corticostriatal pathway
Richard Wade-Martins, Quyen Do, Federico Nebuloni
Abstract
This protocol described the generation of fluid-walled dumbbells for culturing of induced pluripotent stem cell-derived neurons with directional connectivity so as to recapitulate the human corticostriatal connectivity. The fluid-walled microfluidic technology was first reported by Walsh et al., 2017 et al. , 2017 and subsequently shown to be adaptable for cell culture.
Steps
Fabrication of Fluid-Walled Dumbbells
Preparation of petri dishes (Day -7)
Pre-coat 6 cm petri dishes with 7 mL of Poly-D-lysine overnight.
Remove Poly-D-lysine and wash with PBS twice.
Remove PBS and add a thin layer of neurobasal supplemented with B27.
Leave to incubate for at least 15 mins.
Remove neurobasal and add a thin layer of fresh FC40.
Jet an array of 37 dumbbells (33 cm) using an in-house fluid printer.
Coat each dumbbell with 2 µl of 0.46 mg/ml of Geltrex overnight.
Establishment of Human Corticostriatal Pathway
Day -05: Plating Cortical Neurons (CNs)
Preparing media & spinning tubes for CNs plating
2.1.1. On the day of, before starting, add Rock inhibitor (ROCKi) (1:1000) to cMM and pre-warm this media in the water bath.
2.1.2. Add 9 mL of Neurobasal to a 15 mL falcon (spinning tube) for each vial to be thawed and prewarm.
Replating of adherent D32 post-puromycin selection CNs
2.2.1. Aspirate media from CNs wells and rinse with 1 mL PBS.
2.2.2. Aspirate PBS and add 1 mL of room temperature Accutase per well of 6-well plate.
2.2.3. Incubate at 37ºC for ~5 minutes, until cells detach.
2.2.4. Collect all of the well contents and add to the pre-warmed spinning falcon.
2.2.5. Spin at 350g for 5 minutes.
2.2.6. Resuspend cells in the pre-warmed cMM supplemented with ROCKi (1:1000) and count the cell number using either the cell counter or a haemocytometer.
2.2.7. Calculate the appropriate dilution such that CNs are to be deposited into the cortical chambers of all dumbbells at the replating density of 13,000 cells/dumbbell in 1 µl of cell suspension.
2.2.8. Add 3 µl of cMM into the striatal chamber.
Day -03: Transduction of CNs with LV-Ngn2-GFP
Transduce CNs in the cortical chamber with LV-Ngn2-GFP, 50 µl of viral stock/2 µl.
Add 2 µl of fresh cortical media (without lentiviruses) into the striatal chamber.
Day 0: Plating of Day 16 MSN progenitors
Prepare sMM supplemented with ROCKi (1:1000) and pre-warm this media in the water bath.
Add 3 µl of cMM into the cortical chamber.
Add 9 mL of Neurobasal to a 15 mL falcon (spinning tube) for each vial to be thawed and prewarm.
Remove media from both chambers.
Thaw cryovial containing MSN progenitors in water bath until only a small component remains frozen.
Carefully transfer contents of cryovial to pre-warmed spinning tubes.
Centrifuge at 350g for 5 min.
Aspirate media from cell pellet in spinning falcon and replace with 1 mL sMM1 + Rocki, slowly and gently resuspending the pellet.
Count the cell number using either the cell counter or a haemocytometer.
Calculate the appropriate dilution such that MSN progenitors are to be deposited into the striatal chamber of all dumbbells at the replating density of 13,000 cells/dumbbell in 1 µl of cell suspension.
Day 2 – media change
Remove 2 µL (i.e. half) of media from each chamber.
Add 2 µL (i.e. half) of fresh cMM and sMM to the cortical and striatal chamber, respectively.
Day 4, 6 and 8 – media change
Perform similar media change at these days.
