Aug 02, 2023

Public workspaceFabrication of fluid-walled dumbbells and generation of the human corticostriatal pathway

DOI
dx.doi.org/10.17504/protocols.io.kqdg39zb1g25/v1
  • 1Oxford Parkinson's Disease Centre and Department of Physiology, Anatomy and Genetics, University of Oxford, South Park Road, Oxford OX1 3QU, United Kingdom;
  • 2Kavli Institute for Neuroscience Discovery, University of Oxford, Dorothy Crowfoot Hodgkin Building, South Park Road, Oxford OX1 3QU, United Kingdom;
  • 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA;
  • 4Osney Thermofluids Institute, Department of Engineering Science, University of Oxford, Osney Mead, Oxford OX2 0ES, United Kingdom;
  • 5The Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.
Cláudia C. Mendes
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.kqdg39zb1g25/v1
Protocol Citation: Quyen Do, Federico Nebuloni, Richard Wade-Martins 2023. Fabrication of fluid-walled dumbbells and generation of the human corticostriatal pathway. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg39zb1g25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 24, 2023
Last Modified: August 02, 2023
Protocol Integer ID: 80968
Keywords: Corticostriatal pathway, axotomy, fluid-walled microfluidics
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020370
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 and subsequently shown to be adaptable for cell culture.
Materials
Reagents:

Equipment:
  • In-house Fluid Printer (Iota Science Ltd.)

Preparing cMM solution:

Preparing striatal Neuronal Induction base medium (sNIM):
  • DMEM/F12 basal medium
  • 1% MEM Non-Essential Amino Acids (NEAA)
  • 1% Glutamax
  • 1x B27 without vitamin A
  • 1% penicillin/Streptomycin (P/S)
  • 0.05% β-mercaptoethanol
Fabrication of Fluid-Walled Dumbbells
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 3*7 dumbbells (3*3 cm) using an in-house fluid printer.
Note
G-Code to jet-print the microfluid-walled dumbbells can be found here.
Coat each dumbbell with 2 µl of 0.46 mg/ml of Geltrex overnight.
Note
Coat dumbbells as soon as possible to avoid evaporation of FC40 which might cause collapse/merging of fluid walls. If working with many dishes, perform steps 1.6 and 1.7. in smaller batches.

Establishment of Human Corticostriatal Pathway
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.
Note
The volume difference on step 2.2.8. creates a positive fluid pressure gradient toward the cortical chamber, restricting movement of CN cell bodies away from the 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 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.
Note
Swirl the vial to thaw the MSN progenitors and check frequently to ensure the vial do not get completely thawed while in the water bath.

Be very careful to wipe down the vial with ethanol after removing from the water bath, and try to not place lid underwater to remove the risk of contamination.

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.
Note
For different culturing condition, medium from the striatal chamber is removed and replaced with either 1 µl of either MSN cell suspension (13,000 cells/dumbbell) or standard cortical media (i.e. cMM) or cortical media supplemented with 10-fold BDNF (i.e. cMM with 100 ng/ml BDNF).

Add 3 µl of cMM into the cortical chamber.
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.