The interactions between the malignant glioma cells and their surrounding neurons are gaining interest. Here, we show the development of an in vitro model co-culturing derived cortical glutamatergic neurons and tumor cells. The microfludic devices used for co-cultures coupled to multielectrode arrays allow the study of different cell types and perform electrical activity recordings at different time points of the cultures.
This method is particularly helpful for functional and mechanistic studies and for analyzing the effects of pharmacological agents that block pediatric high-grade glioma cell migration and interaction with neurons. 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 four 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 three to four 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 UW479 and BT35 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 experiment. Observe each cell line to reach 80%confluency.
On day 21 post-culture of glutamatergic neurons, start co-culture by trypsinizing UW479 and BT35 cells. Seed the trypsinized UW479 and BT35 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. Place the MFD in the recording device and use a commercially available software to perform the electrophysiological recording of glutamatergic neurons on the 21st day. Before seeding pediatric high-grade glioma cells, perform a second electrophysiological recording of the differentiated glutamatergic neurons cultured as control parallel to the co-culture.
Perform another electrophysiological recording on the 23rd day after two days of the co-culture. Human IPS-derived cortical glutamatergic neurons were characterized and assessed using nestin, SOX2, metabotropic glutamate receptors II, and VGLUT1 immunostaining. Nestin is an intermediate filament protein and it was expressed in undifferentiated CNS cells.
SOX2 was also expressed in undifferentiated cells of the neural epithelium in CNS. Nestin and SOX2-positive cells percentage decreased from the fourth day to the 21st day, confirming the differentiated state of glutamatergic neurons. The microscopic images revealed a progressive distribution of glutamatergic neurons across microfluidic devices.
When BT35 and UW479 were added to the culture, it was observed that the floating cells were present after glioma cell seeding on the 21st day, which progressively disappeared until the 23rd day and became adherent in the device. The percentages of viable cells for BT35 and UW479 were comparable and implied that patient-derived cell lines could be used appropriately. Spike detection and raster plot on the 23rd day of culture showed increased electrical activity after adding pediatric high-grade glioma cells.
To monitor the synchronicity of the neural network activity, the instantaneous firing rate in BT35 or glutamatergic neurons was recorded. The difference in electrical activity between the individually cultured and co-cultured glutamatergic neurons was significant on the 23rd day. Further methodological development may include the functional assessment of the network with burst analysis and special temporal cross-correlations of the network.
Now, researchers have a promising tool to explore interactions and pharmacological targeting of pediatric high-grade glioma tumor lines and high PSC neurons. Several tumor lines and several neuron types might be used.
