Mar 19, 2024

Public workspaceNeuronal co-culture

DOI
dx.doi.org/10.17504/protocols.io.dm6gpze38lzp/v1
  • Nisha Mohd Rafiq1,
  • Pietro De Camilli2,3,4,5
  • 1University of Tuebingen;
  • 2Department of Neuroscience, Yale University School of Medicine, New Haven, Connecticut 06510, USA.;
  • 3Department of Cell biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.;
  • 4Program in Cellular Neuroscience, Neurodegeneration and Repair.;
  • 5Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA.
Berrak Ugur
Open access
DOI: dx.doi.org/10.17504/protocols.io.dm6gpze38lzp/v1
Protocol Citation: Nisha Mohd Rafiq, Pietro De Camilli 2024. Neuronal co-culture. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpze38lzp/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: March 12, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 96913
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000580
Abstract
This protocol describes the co-culturing of iPSC-derived dopaminergic (DA) neurons and iPSC-derived medium spiny neurons (MSNs) in a microfluidic compartmentalization device.
Attachments
Rafiq_co-culture.docx
Rafiq_co-culture.doc...
18KB
Materials
Preparation of device for seeding cells:

Prepare enough amount of NB/B27 medium.

  1. For Amount500 mL

  • NB/B27 medium (see Method section of paper), add:
AB
Neurobasal medium484 mL
B27 supplement without vitamin A10 mL
GlutaMAX 5 mL
Penicillin-Streptomycin1 mL
Storage: NB/B27 medium can be stored for 5 days at Temperature4 °C or for up to one month at Temperature-20 °C .

  • Warm NB/B27 medium at Temperature37 °C .
  • Make NB/B27 complete medium by adding:
AB
BDNF20 ng/ml
Ascorbic acid0.2 mM
GDNF20 ng/ml
db-cAMP0.5 mM
TGFβ31 ng/ml
DAPT10 uM
Y-2763210 uM
Neuronal co-culture device set-up
Neuronal co-culture device set-up

Note
The OMEGA4 device has 2 pairs of interconnected chambers, where each pair of chambers is joined via a series of microfluidic channels.
Coat chambers with Amount200 µL per well with Amount0.1 undetermined Poly-L-Ornithine (PLO) in PBS.
Incubate plates overnight at Temperature37 °C .
Incubation
Overnight
Wash the chambers thrice with PBS.
Wash
Coat chambers with Amount200 µL per well Amount10 undetermined Laminin plus Amount2 undetermined fibronectin, both diluted in PBS.

Incubate plates overnight at Temperature37 °C . Do not store coated plates. Proceed with preparation of plates for seeding cells.

Incubation
Overnight
Preparation of device for seeding cells
Preparation of device for seeding cells
15m
Prepare enough amount of NB/B27 medium.

  1. For Amount500 mL

  • NB/B27 medium (see Method section of paper), add:

AB
Neurobasal medium484 mL
B27 supplement without vitamin A10 mL
GlutaMAX5 mL
Penicillin-Streptomycin1 mL
Storage: NB/B27 medium can be stored for 5 days at Temperature4 °C or for up to one month at Temperature-20 °C .
  • Warm NB/B27 medium at Temperature37 °C .
  • Make NB/B27 complete medium by adding:

AB
BDNF20 ng/ml
Ascorbic acid0.2 mM
GDNF20 ng/ml
db-cAMP0.5 mM
TGFβ31 ng/ml
DAPT10 uM
Y-2763210 uM
Discard coating reagents and add Amount200 µL per well of NB/B27 complete medium.
Keep the plate at Temperature37 °C for Duration00:15:00 before seeding cells.
15m
Replate cultured iPSC-derived dopaminergic neurons (day 30, see Method section of paper) on one side of the two-chamber microfluidic compartmentalization device (OMEGA4, eNuvio) at a cell concentration of 3×105. Only the axons of DA neurons can migrate through the microfluidic channels connected to the adjacent chamber.
Feed neurons with fresh NB/B27 media every 3 days. Add Amount10 undetermined Laminin to NB/B27 media every 10 days before feeding the neurons.

Pipetting
After an additional 25 days in the co-culture device, thaw frozen iPSC-derived medium spiny neurons (MSN) from BrainXell and plate on the other half of the device (where only the axons of DA neurons are present) at a cell concentration of 3x105 cells.
Fix the DA-MSN co-cultures till 7-10 days later for immunofluorescence (see Method section of paper).