Co-culturing Glutamatergic Neurons and Pediatric High-Grade Glioma Cells

Begin with a polymer-coated microfluidic device comprising reservoir wells connected by channels and filled with culture medium.

Remove the medium from the reservoirs and seed human induced pluripotent stem cells.

The cells enter the channels and adhere to the coated surface.

Now, fill the reservoirs with a differentiation medium.

Growth factors in the medium induce stem cell differentiation into neuronal progenitor cells.

Next, add a medium containing specific nutrients and neurotrophic factors that induce neuronal progenitor maturation into glutamatergic neurons.

These neurons release the excitatory neurotransmitter glutamate from their presynaptic terminals, which bind to postsynaptic neuron receptors, generating excitatory signals.

Now, take pediatric high-grade glioma cells, cancer cells derived from aggressive brain tumors.

Add the cells to the reservoirs and incubate. The glioma cells enter the channels and adhere.

Gradually, these cells form functional synapses with the glutamatergic neurons, facilitating neuronal-glioma cell crosstalk and leading to neuronal hyperactivity.

To begin culturing the commercialized human iPS-derived cortical glutamatergic neurons, empty the inlet and outlet reservoirs of the microfluidic device by pipette aspiration, letting only the device channels be filled with medium.

Seed the human iPS cells by adding 10 microliters of 6.5 million human iPS cells per milliliter suspension in the medium, and let the device be under the hood for 15 minutes to allow cells to attach. After 15 minutes, fill both inlet and outlet reservoirs with 50 microliters of day 4 culture medium. Then transfer the device into the incubator to maintain the cells at 37 degrees Celsius and 5% carbon dioxide for 23 days. Replace the medium every 3 to 4 days as described in the text.

To count the cells and assess viability using a standard phase contrast microscope, take microscopic pictures at 4th day, 21st day, and 23rd day. Culture both UW-479 and BT-35 cell lines in DMEM and F-12 glutamax supplemented with 10% fetal bovine serum in a cell culture flask. Maintain cell culture under a controlled environment at 37 degrees Celsius in normoxic conditions throughout the experiments. Observe each cell line to reach 80% confluency.

On day 21 post-culture of glutamatergic neurons, start co-culture by trypsinizing UW-479 and BT-35 cells. Seed the trypsinized UW-479 and BT-35 cells on top of the mature adherent glutamatergic neurons in each dedicated microfluidic device.

Maintain the co-cultures for two days under a controlled environment at 37 degrees Celsius and 5% carbon dioxide with glutamatergic neuron D11 and onward medium. Count pediatric high-grade glioma cells using the microscopic pictures, analyzed with the image analysis software to assess their viability and calculate the percentage of cells.