Multi compartment microfluidic neuron co-culture device fabrication begins with defining reservoirs on the PMMA block with a milling machine, followed by hot embossing of microchannel arrays that connect the compartments. Once A-P-M-M-A mold has been prepared, A-P-D-M-S mold master is replicated from the PMMA mold and coated with trichlorethylene. The final device is replicated from the PDMS mold master and assembled on A PDL coated culture plate.
Primary E 16 to 18 rat neurons are then loaded into the soma compartment and cultured for two weeks prior to adding P one to two rat oligodendrocyte cells on top of axonal layers inside the axon ggl compartment for co-culture. Hi, I'm J Par from Nano Bio Systems Laboratory in Electrical and computer engineering department at Texas a and m University. Today we will show you a procedure for fabricating multi compartment microfluidic neuron co culture platform.
We use this procedure in our lab to study central nervous system neuron glia interaction in vitro. So let's get started. To begin this procedure, use a micro milling machine to make 3.5 millimeter deep compartments.
In A-P-M-M-A block define one soma compartment and six axon ggl compartments. After preparing the compartments, sonicate and isopropanol for 10 minutes to remove debris, make micro ridge structures on the glass slide using conventional photolithography and wet etching process hot emboss the micro ridge patterns from the glass. Slide onto the PMMA master first, manually align the PMMA mold master and the glass substrate.
Then raise the temperature to 115 degrees Celsius on a hot press. And once the temperature is reached, apply 0.5 tons of weight for five minutes. After five minutes, cool down to 40 degrees Celsius and release the pressure.
Now start preparing the PDMS master. Mix the PDMS pre polymer with its curing agent at a 10 to one ratio by weight, cut the PMMA master into 50 millimeter by 50 millimeter size blocks. Using a bend saw, place it inside a cask and pour the PDMS mixture on top of the PMMA master place the cask in a desiccate connected to a vacuum pump and DGAs for 15 minutes.
To remove bubbles generated during the PDMS mixing process. Once all the bubbles are removed, put the cask in a leveled 85 degrees Celsius oven for one hour for polymerization. Now that the PDMS is fully cured, peel the PDMS master off of the PMMA master vapor coat the PDMS master by placing it inside a desiccate with two to three drops of trichlorethylene and apply a vacuum for 15 minutes.
Coating of the PDMS master facilitates the release of the final PDMS neuron co-culture device from the PDMS master mold After coating, the PDMS master is briefly rinsed with isopropyl alcohol to remove excessive trichlorethylene and and dried with nitrogen gas neuron co-culture device Fabrication from the PDMS polymer is similar to preparing the PDMS master mold described in the previous section. Start by mixing the PDMS pre polymer with obscuring agent at a ratio of 10 to one by weight proceed to pour the PDMS mixture on top of the PDMS master. It is important not to pour too much PDMS so that the 3.5 millimeter high PDMS structure on the master is not fully immersed.
DGAs inside a desiccate connected to a vacuum pump for 15 minutes. When all the bubbles are removed, put the PDMS inside a leveled 85 degrees Celsius oven for one hour for polymerization at the end of one hour, take the PDMS out of the oven and leave it at room temperature to cool down. Then gently peel off the PDMS neuron culture devices from the PDMS master.
Wrap the PDMS devices in paraform to protect them from dust or debris. Finish by cutting the paraform wrapped PDMS into individual pieces using a drill press equipped with a blade, the neuron co-culture PDMS devices are nearly ready for use in cell culture plates. Prior to placing the neuron co-culture PDMS device in a cell culture plate, put the device inside the plasma cleaner and expose to air plasma for 90 seconds to make the surface hydrophilic.
This treatment will facilitate filling of the micro channels with culture media after assembly onto poly de lycine coated plates. In order to sterilize the plasma treated device, immerse it in 70%ethanol for 30 minutes and move the container into the sterile bio hood. After 30 minutes, take out the device and dry with nitrogen gas filtered with a 0.2 micron millipore filter.
Next place the dried devices on top of a poly D lysine coated polystyrene six well culture plate and apply gentle pressure to ensure a tight seal between the surface of the culture plate and the device ice. Now fill the soma compartment with culture media. Wait for a minute or two to ensure that no bubbles are trapped within the micro channels.
Then fill the six axon glia compartments. Finally incubate the devices filled with culture media in a 37 degrees Celsius incubator overnight to remove any possible debris or toxic residue. After the overnight incubation, the culture can be started to prepare for cell loading into the device.
Use a pipette to aspirate the culture media from the Soma and Axon compartments. Then load 40 microliters of primary forebrain neuron cells from embryonic day 16 to 18 rats into the soma compartment around the microchannel inlets at an aerial density of 500 cells per square millimeter. It is important not to touch the PDMS device while loading the cells since it can break the seal between the device and the cell culture plate.
These microscopic images show calcium am stained neuron cells. Inside the soma compartment cells are uniformly loaded inside the compartment and located closely to microfluidic channels. Incubate the devices loaded with the cells for 30 minutes in a 37 degrees Celsius incubator to enhance cell adhesion to the substrate.
After this incubation, fill up all the compartments with culture media. Maintain the cells growing in the device at 37 degrees Celsius in a humidified 5%CO2 incubator change out only half of the culture media every three or four days. A dense axonal network should be formed inside the Axon glia compartments.
After two weeks of culture after 14 days of culturing, the primary neurons in vitro CLIA cells are added prior to loading cells aspirate approximately 60%of the culture media from the compartments oligodendrocytes were dissected from the cerebral hemispheres of Sprague Dolly rats at postnatal. Day one to two add five microliters of oligodendrocyte cells to each axon glia compartment at an aerial density of 1000 cells per square. Millimeter caution must be made not to touch the bottom substrate using the pipette during loading, since it can damage the axonal network formed on the substrate after the addition of the oligodendrocytes, incubate the cells for one hour at 37 degrees Celsius and then fill up the compartments.
With culture media continue to culture the neurons as before the addition of the CLIA change only half of the culture media every three or four days for two more weeks while studying mammalian central nervous system. Axon glia interaction when experiments are carried out properly. Neuron cells loaded to the soma compartment locate close to microchannel inlets and an axonal layer starts to form inside the axon Ggl compartments.
After one week of culture, signs of neuron cell aggregation inside the soma compartment can be an indication that cells are not healthy when loading oligodendrocytes. After two weeks of neuron culture axon ggl compartments should be covered with a dense layer of axons and oligodendrocytes should be loaded on top of the axonal layer. We have just shown you how to fabricate multi compartment neuroco culture platform and how to use it to study CNS Neuroglia interaction in vitro.
When doing this procedure, it is important to remember to keep devices from dust and not to sterilize them in ethanol for too long since they can't deteriorate the bonding. That's it. Thanks for watching and g lucking your experiments.
