TITLE: Fabrication of fluid-walled dumbbells and generation of the human corticostriatal pathway
AUTHORS: Quyen Do, Federico Nebuloni, Richard Wade-Martins

[DESCRIPTION]
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 and subsequently shown to be adaptable for cell culture.

[STEPS]
SECTION: Fabrication of Fluid-Walled Dumbbells
1. Preparation of petri dishes (Day -7)
SECTION: Fabrication of Fluid-Walled Dumbbells
1.1. Pre-coat 6 cm petri dishes with 7 mL of Poly-D-lysine overnight. 
SECTION: Fabrication of Fluid-Walled Dumbbells
1.2. Remove Poly-D-lysine and wash with PBS twice.
SECTION: Fabrication of Fluid-Walled Dumbbells
1.3. Remove PBS and add a thin layer of neurobasal supplemented with B27.
SECTION: Fabrication of Fluid-Walled Dumbbells
1.4. Leave to incubate for at least 15 mins.
SECTION: Fabrication of Fluid-Walled Dumbbells
1.5. Remove neurobasal and add a thin layer of fresh FC40.
SECTION: Fabrication of Fluid-Walled Dumbbells
1.6. Jet an array of 3*7 dumbbells (3*3 cm) using an in-house fluid printer. 
 
 
SECTION: Fabrication of Fluid-Walled Dumbbells
1.7. Coat each dumbbell with 2 µl of 0.46 mg/ml of Geltrex overnight. 
 

SECTION: Establishment of Human Corticostriatal Pathway
2. Day -05: Plating Cortical Neurons (CNs)
SECTION: Establishment of Human Corticostriatal Pathway
2.1. 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.
SECTION: Establishment of Human Corticostriatal Pathway
3. Day -03: Transduction of CNs with LV-Ngn2-GFP
SECTION: Establishment of Human Corticostriatal Pathway
2.2. 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.
 

SECTION: Establishment of Human Corticostriatal Pathway
3.1. Transduce CNs in the cortical chamber with LV-Ngn2-GFP, 50 µl of viral stock/2 µl.

SECTION: Establishment of Human Corticostriatal Pathway
3.2. Add 2 µl of fresh cortical media (without lentiviruses) into the striatal chamber.
SECTION: Establishment of Human Corticostriatal Pathway
4. Day 0: Plating of Day 16 MSN progenitors
SECTION: Establishment of Human Corticostriatal Pathway
4.1. Prepare sMM supplemented with ROCKi (1:1000) and pre-warm this media in the water bath.
SECTION: Establishment of Human Corticostriatal Pathway
4.2. Add 9 mL of Neurobasal to a 15 mL falcon (spinning tube) for each vial to be thawed and prewarm.
SECTION: Establishment of Human Corticostriatal Pathway
4.3. Remove media from both chambers.
SECTION: Establishment of Human Corticostriatal Pathway
4.4. Thaw cryovial containing MSN progenitors in water bath until only a small component remains frozen.
 

SECTION: Establishment of Human Corticostriatal Pathway
4.5. Carefully transfer contents of cryovial to pre-warmed spinning tubes.
SECTION: Establishment of Human Corticostriatal Pathway
4.6. Centrifuge at 350g for 5 min.
SECTION: Establishment of Human Corticostriatal Pathway
4.7. Aspirate media from cell pellet in spinning falcon and replace with 1 mL sMM1 + Rocki, slowly and gently resuspending the pellet.
SECTION: Establishment of Human Corticostriatal Pathway
4.8. Count the cell number using either the cell counter or a haemocytometer.
SECTION: Establishment of Human Corticostriatal Pathway
4.9. 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.
 

SECTION: Establishment of Human Corticostriatal Pathway
4.10. Add 3 µl of cMM into the cortical chamber.
SECTION: Establishment of Human Corticostriatal Pathway
5. Day 2 – media change
SECTION: Establishment of Human Corticostriatal Pathway
5.1. Remove 2 µL (i.e. half) of media from each chamber.
SECTION: Establishment of Human Corticostriatal Pathway
5.2. Add 2 µL (i.e. half) of fresh cMM and sMM to the cortical and striatal chamber, respectively.
SECTION: Establishment of Human Corticostriatal Pathway
6. Day 4, 6 and 8 – media change
Perform similar media change at these days.