Aug 02, 2023

Public workspaceLocalised axotomy of human Cortical Neurons (CNs) from induced pluripotent stem cells (iPSCs)

DOI
dx.doi.org/10.17504/protocols.io.rm7vzbp14vx1/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.rm7vzbp14vx1/v1
Protocol Citation: Quyen Do, Federico Nebuloni, Richard Wade-Martins 2023. Localised axotomy of human Cortical Neurons (CNs) from induced pluripotent stem cells (iPSCs). protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbp14vx1/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: 80974
Keywords: Laplace pressure, automatic flow, dumbbell circuits, fluid-walled microfluidics
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020370
Abstract
This protocol describes the process followed to perform localised axotomy of iPSC-derived human cortical axons cultured within fluid-walled dumbbells in 6 cm TCT-treated Petri dishes. A similar protocol was first described by Soitu et al., 2020 to perform wounds assays in monolayer cell cultures.
Materials
Reagents:

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

Preparation of coating-prep medium:
  • Neurobasal
  • 1x B-27 supplement

Localised axotomy assay
Localised axotomy assay
On Day 20, remove the FC40 overlay from the Petri dish where human Cortical Neurons (CNs) were cultured inside fluid-walled dumbbells.
Gently add ~2 ml of coating-prep medium (see Materials) to destroy every fluid wall without peeling cells off the substrate.
Remove medium and wash with PBS twice. Be careful with cells peeling.
After last wash, add ~5 ml of fresh medium (Neurobasal supplemented with B27).
Note
Cells can now be stored in incubator for several minutes if needed.

Equip the fluid printer with a 1 mL glass syringe filled with medium.
Place dish into the fluid printer.
Fluid printer automatically perform axotomy by means of a submerged medium jet. Such jet is held at a fixed height above cells and it is moved around the dish by the printer traverse to cross perpendicularly across axon bundles at the midpoint.

Key parameters:
  • jet height =300 μm
  • jet flow rate = 480 μl/min
  • traverse speed = 960 mm/min
Note
Parameters must be finely tuned, depending on cells maturation and concentration.

Fluid printer is controlled by scripts written in G-code.

Remove medium and overlay fresh FC40.
New fluid-walled dumbbells can be fabricated around axotomized cultures following steps 1.5 and 1.6 in Protocol: Fabrication of fluid-walled dumbbells and generation of the human corticostriatal pathway
Live-imaging of axonal regeneration
Live-imaging of axonal regeneration
On DIV 0, fluorescent live images of all dumbbell conduits were taken just prior to CNs replating to serve as initial normalising timepoint.

Images were taken on a digital SLR camera (Nikon D7100 DSLR) connected to an epi-fluorescence microscope (Olympus IX53; 1.25×, 4×, 10×, 25× objectives) equipped with a translation stage and an overhead illuminator (Olympus IX3 with filters).
Medium was changed every other day from now on for the next 20 days.
Similar live images were taken again on day 20 prior to axotomy, and subsequently 36, 60, and 84 hours post-axotomy.