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

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

1. Substrate Preparation for Micropatterning
   1. 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.
   2. Incubate with 100 mg/mL of poly (ethylene glycol)-succinimidyl valerate (molecular weight 5,000 (Peg- SVA)) in 10 mM carbonate buffer, pH > 8, for 1 h. Rinse extensively with ultrapure water and dry under a chemical hood.
      NOTE: At this stage, the sample can be stored at 4°C in the dark for further use.
   3. Add the photoinitiator, 4-benzoyl benzoyl trimethylammonium chloride (PLPP), at 14.7 mg/mL in phosphate-buffered saline (PBS).
      NOTE: A concentrated form of PLPP, a PLPP gel, can also be used. It results in a shorter ultraviolet (UV) illumination time required to degrade the PEG brush (100 mJ/mm²).
2. Photoinitiator Gel Deposition
   1. Prepare a mixture of 3 μL of PLPP gel and 50 μL of absolute ethanol to deposit in the center of the slide. Place the sample under a chemical hood until complete evaporation of the absolute ethanol.
      NOTE: At this stage, the sample can be stored at 4°C in the dark for further use.
3. Glass Slide Micropatterning
   1. Mount the coverslip in a Ludin chamber and place it on the motorized stage of a microscope equipped with an auto-focus system.
   2. Load images corresponding to the envisioned micropatterns into the software. Apply these parameters: replication 4 x 4 times, spacing of 200μm, UV dose of 1,000 mJ/mm². After the automatic UV-illumination sequence, rinse the PLPP away with multiple PBS washes.
      NOTE: If PLPP gel was used, remove it by extensive washes with deionized water, dry in a stream of N₂, and store at 4 °C.
   3. Incubate with laminin (50 μg/mL in PBS) for 30 min. Wash extensively with PBS.
      ​NOTE: A fluorescent solution of purified green fluorescent protein (GFP, 10 μg/mL in PBS) can be mixed with laminin to visualize the micropatterns by fluorescence microscopy.

2. Preparation of Neurons and GBM Cells for Co-culture

1. Culture of Embryonic Rat Hippocampal Neurons
   1. Dissect the hippocampus of embryonic (E18) rats and transfer the tissue into a Hank's balanced salt solution (HBSS)/1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)/penicillin-streptomycin solution in a 15 mL tube. Remove excess solution without drying the hippocampus.
   2. Add 5 mL of trypsin-ethylenediamine tetra acetic acid (EDTA) supplemented with penicillin (10,000 units/mL)/streptomycin (10,000 μg/mL) and 1 mM HEPES, and incubate for 15 min at 37 °C. Wash 2x with the HBSS/HEPES/penicillin-streptomycin solution, and let the tissue remain in this solution for 2–3 min.
   3. Dissociate the tissue using two flame-polished Pasteur pipettes, by pipetting up and down 10x with each tissue, taking care to minimize foaming. Count the cells and evaluate the viability of the cell suspension. Plate the neurons on micropatterned coverslips as indicated below.
      NOTE: Cell viability rate is 85–90% after extraction.
2. Cell culture for Neurons on Micropatterned Coverslips
   1. Rehydrate micropatterned glass slides with PBS and incubate complete neuronal cell culture medium.
   2. Seed the hippocampal neurons obtained from E18 Sprague-Dawley rats directly over the micropatterned glass coverslip at a density of 50,000 cells per cm² in neurobasal medium (NBM) enriched with 3% horse serum. Place the micropatterned neurons in the incubator (37 °C, 5% CO₂) for 48 h.
      NOTE: After ~6 h, primary hippocampal neurons can be seen adhering to the laminin micropatterns.
3. Co-culture of Human GBM Stem-like Cells on Neurons
   1. As the spheroid-shaped cells grow in suspension, centrifuge the suspension for 5 min at 200 × g. Wash the spheroids with 5 mL of PBS, and incubate the cells with 0.5 mL of the cell dissociation reagent (see Table of Materials) for 5 min at 37 °C.
   2. Add 4.5 mL of complete NBM (complemented with B27 supplement, heparin, fibroblast growth factor 2, penicillin, and streptomycin, as described previously), and count the cells using an automatic counting technique.
   3. Seed 1,000 GBM cells over the micropatterned neuronal culture in NBM enriched with 3% horse serum. Incubate the plate at 37 °C, 5% CO₂, and 95% humidity.