Co-Culturing Glioblastoma Cells on Patterned Neurons: A Technique to Monitor the Migration of Glioblastoma Cells on Rat Hippocampal Neurons In Vitro

Abstract

Source: Guyon, J. et al. Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions. J. Vis. Exp. (2021)

This video describes a co-culture assay of glioblastoma cells on micropatterned neurons, which mimic the myelinated neuronal tracts invasion by the glioblastoma stem-like cells as they migrate on neurons in the peritumoral spaces. This assay can help analyze the effects of pharmacological drugs that inhibit glioblastoma cells' migration on neurons in a glioma.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation of the Patterned Slides Substrate Preparation for Micropatterning Treat 18 mm circular glass coverslips by air/plasma activation for 5 min. Place the coverslips in a closed chamber with 100 μL of (3-aminopropyl) triethoxysilane in a desiccator for 1 h. Incubate wi…

Materials

(3-aminopropyl) triethoxysilane	Sigma	440140-100ML	The amino group is useful for the bioconjugation of mPEG-SVA
96-well round-bottom plate	Sarstedt	2582624	Used to prepare spheroids
Accutase	Gibco	A11105-01	Stored at -20 °C (long-term) or 4°C (short-term), sphere dissociation enzyme
B27	Gibco	12587	Stored at -20 °C, defrost before use
Basic Fibroblast Growth Factor	Peprotech	100-18B	Stored at -20 °C, defrost before use
Countess Cell Counting Chamber Slides	Invitrogen	C10283	Used to cell counting
Coverslips	Marienfeld	111580	Cell culture substrate
Dessicator cartridges	Sigma	Z363456-6EA	Used to reduce moisture during (3-aminopropyl) triethoxysilane treatment
DPBS 10x	Pan Biotech	P04-53-500	Stored at 4 °C
Flask 75 cm²	Falcon	10497302
HBSS	Sigma	H8264-500ML
Heparin sodium	Sigma	H3149-100KU	Stored at 4 °C
Laminin		114956-81-9	Promotes neuronal adhesion
Flask 75 cm²	Falcon	10497302
Heparin sodium	Sigma	H3149-100KU	Stored at 4 °C
Leonardo software			Loading of envisioned micropatterns
MetaMorph Software	Molecular Devices LLC	NA	Microscopy automation software
Methylcellulose	Sigma	M0512	Diluted in NBM for a 2% final concentration
Neurobasal medium	Gibco	21103-049	Stored at 4 °C
Nikon TiE (S Fluor, 20x/0.75 NA)			Inverted microscope equipped with a motorized stage
Penicillin – Streptomycin	Gibco	15140-122	Stored at 4 °C
PLPP	Alveole		Photoinitiator used to degrade the PEG brush
Poly(ethylene glycol)-Succinimidyl Valerate (mPEG-SVA)	Laysan Bio	VA-PEG-VA-5000-5g	Used as an anti-fouling coating
Trypsin-EDTA	Sigma	T4049-100ML	Used to detach adherent cells