This protocol establishes a novel neuronal axon and Astroglia co-culture platform to analyze neuron to ggl interactions using high resolution imaging. First, assemble the microfluidic culture chamber, prepare neuronal cultures and induce the neuronal axons to enter into the other side of the MCP. Next, add cultured astrocytes to establish a compartmentalized co-culture system results, including time-lapse imaging with confocal immunofluorescence microscopy can help understand the interactions between axons, astroglia, and neuronal axons.
We first had the idea for this method when trying to separate the effect of neuronal process versus neuronal secretion to the GT one regulation. Typically, individuals new to the method will struggle with inducing axon into other part of the cellular compartment. It requires a high density of healthy neurons plated on the neuronal side with the neuron plated near the central channels.
The MCP open chambers are designed for compartmented cultures of different types of cells, polyurethane and laminin coatings are necessary for plating neurons and astrocytes in the co-culture. First, apply one milligram per milliliter polyurethane solution to the sterile glass bottom dishes and incubate at 37 degrees Celsius overnight. Wash the coated glass bottom dishes three times with double distilled water and then place under a sterile fume hood to dry.
Next, coat the dish with one milligram per milliliter, laminin and dry under UV light for one hour. The assembly of MCP on the glass bottom dish should be freshly prepared before use. Position the microfluidic culture platform with the central connecting channels on its bottom side and form a tight seal with the coated glass.
Bottom dishes. Add regular neuron or astrocyte culture mediums on both sides of the assembled MCP. Incubate the chamber for two to four hours at 37 degrees Celsius and verify that there is no leakage between the MCP and the glass.
Bottom dish. Cortico neuronal cell cultures are freshly prepared from embryonic 14 to 16 day old mouse brains. Following dissection of the mouse brain, remove the meninges from the cortex under the dissecting microscope.
Use a razor blade to mince the tissue into small tissue blocks and place the mince tissue blocks into a tube after centrifuging. Discard the media above the pellet and add 0.05%Try in for 10 minutes and a 37 degrees Celsius water bath. Dissociate the cells by gentle tation with a fire polished pasta pipette.
Pass the sample through a 70 micron strainer to collect a clear neuronal cell suspension using neuron plating. Medium supplemented with 5%FBS seed. 150 microliters of neurons on the right side of the assembled MCP plate.
A high density of neurons as only the neurons that attach in the cell retention area are able to grow axons through the central channels into the other side of the assembled MCP. On the following day. Carefully aspirate the medium from the cell plating area of the chamber and replace the neuron plating medium with regular neuron culture.
Medium, add 20 nanograms per milliliter, glial derived neurotrophic factor or GDNF to the astrocyte side of the chamber. This creates a chemical gradient that will help induce axon outgrowth from the neuronal side across the central channels of the assembled MCP. Two days later monitor for axons that enter into the central channel.
Once the axons enter into the central channel, they usually enter the other side within one day. Axons are normally intact for at least one week after entering the other side. I after dissociating the brain from P one to three mouse puffs seed astrocytes into polyurethane coated 10 centimeter dishes.
Culture, the cells overnight replenish the astrocyte culture medium daily for the next two days, and then once every three days after seven days when the astrocytes have formed a 90%confluent monolayer harvest cells using trypsin About five days after the neurons were plated aspirate off the GDNF. Then seed 150 microliters of the resuspended astrocytes into the left side of the assembled MCP. When axons are about to enter or have just entered after the replating of the astrocytes axons that entered the left side of the assembled MCP directly interact with the astrocytes either by direct axonal contact or release of soluble factors.
Monitor the astrocytes for attachment to the cell retention area. Immunostain the cells in the compartmentalized co-culture system to visualize specific proteins like those involved in the interaction between the axons and the astrocytes. The compartmented neuron and astrocyte co-culture system allows only the neuronal processes, especially the axons to selectively interact with astrocytes.
Here we describe axon induced transcriptional activation of astroglia glutamate transporter. These studies use transgenic mice, which harbor an expression of an EGF fluorescence reporter under the control of the GT one genomic promoter. A high density of neurons is cultured in the cell retention area of the assembled MCPA magnified view shows the axons crossing the central channels and entering into the other side with beta three tubulin staining revealing the axon growth cone.
The MCP system permits monitoring of the axon bundles as they grow through the central channel and enter the other side. Immunos staining for GT one identifies astrocytes in the glial side of the MCP system emerged. Image displays contact between axons and GLT one positive astrocytes here.
The expression of the EGFP reporter correlates with endogenous GT one promoter activation, protein expression, and functional activity. Assessment of the GT one promoter activity in single astrocytes in C two show a significantly increased GLT one immunoreactivity in compartmentalized co-culture system. Importantly, the MCP system allows time-lapse images to monitor dynamic changes.
For example, these data show axon induced GT one promoter activation with back GT one EGFP astrocytes and wild type neurons a day after plating of the astrocytes. Very minimum, EGFP fluorescence is observed. The EGFP intensity increases significantly 48 hours after co-culture when axons are well into the astrocytes side and interact directly contacting with astrocytes here.
Kinine treatment on the neuronal side of the assembled MCP induced neuronal cell death. Interestingly, it also stimulated axon retraction and degeneration as well as the decrease in axonal EGFP fluorescence. These dynamic changes of EGFP fluorescence intensity in response to the axons clearly demonstrated that Astroglia GT one promoter activity is modulated by the axon interaction.
After watching this video, you should have a good understanding of how to establish the core culture system using the microfluidic culture platform once mastered, this technique can be performed properly in a few days.
