Fabricage van een microfluïdische apparaat voor de compartimentering van Neuron Soma en axonen

Welcome to the laboratory of Dr.Newly John. The John Lab specializes in engineering microfluidic platforms for biological applications. In this video, we will demonstrate the production and utilization of a microfluidic device designed for compartmentalizing and the culturing of primary neurons.

The neuron device was first published in Nature Methods in August of 2005. Since its introduction, the neuron device has been used widely by collaborators all over the world. The neuron device consists of a piece of PDMS that has been cast on a silicon wafer containing an SUA pattern of the device, which was previously created by photolithography.

The device has three main features, four reservoirs, eight millimeters in diameter, two main channels each 1.5 millimeter wide, seven millimeters long and a hundred microns tall and micro grooves, which are 10 micrometers wide and three micrometers in height that join the two main channels. The micro grooves can vary in length from 150 microns to 900 microns, though the standard length of choice is 450 microns. Observing the device so that the main channels and the micro grooves are vertical, we can see that the reservoirs on the left of the grooves are connected via the main channels, as are the reservoirs on the right.

This feature allows liquid to flow through the main channel, which is an important feature, making it possible to load cells and change media. The neuron device has three major advantages over traditional tissue culture platforms. One, it allows for the com compartmentalization of cell bodies or soma and the axons With the neuron device, it is possible to obtain pure axonal fractions.

The size and length of the micro grooves allow axons to pass through the other main channel while preventing the saw bodies from crossing over. Number two, due to the design of the device and taking advantage of hydrostatic pressure, it is possible to subject either the cell side and or the axon side of the device to separate treatments simply by maintaining the volume and the reservoirs higher on one side of the device versus the other. And number three, the pure axonal fraction of the device can be cut by vacuum suction, allowing the researcher to study and observe axonal regeneration.

Researcher Hina Lee will now demonstrate preparing the microfluidic neuron Device. A three Ang silicon wafer with a pattern made out of SU eight by a photolithography is used to cast the poly dimethyl Sloane PDMS microfluidic mold. We refer to the SUA pattern silicon wafer as master molds or simply masters.

PDMS is mixed with curing agent at a weight for weight ratio of 10 to one. That is, for example, 15 grams of PDMS per 1.5 grams of curing agent. The PDMS is then mixed well and poured onto the silicon wafer.

The silicon wafer is contained within a 10 centimeter polystyrene Petri dish. It takes approximately 12 grams to 15 grams of PDMS to cover the MAs with a thickness of approximately four millimeter. The master with the PDMS is then placed in a vacuum desiccate with the lid off the Petri dish and vacuum is applied.

This is done to help remove air bubbles from the PDMS that were introduced during the stirring of the curing agent. It takes approximately 15 minutes for the air bubbles to be removed. The master with PDMS is removed from the desiccate after 15 minutes in the vacuum desiccate.

There are still a few remaining bubbles on the PDMS master. An air gun with a 0.45 micron filter is used to remove any remaining bubbles. The master is then placed on an 80 degree hot plate for one hour to cure.

If a vacuum desiccate is not available, the master can be left at room temperature for several hours. The air bubbles will eventually dissipate on their own. If no hot plate is available, the PDMS will cure at room temperature after 24 hours.

It's important to make sure that the master is level During the curing process. Once the PDMS is cured on The master, it is taken to a clean hood using a surgical blade. A circle cut around the perimeter of the devices is made.

It is important that when one inserts the blade into the PDMS to make contact with the silicon wafer, but not to put too much pressure on the wafer or else the master will crack with a circle cut all the way around the devices. There are four devices per each three inch silicon master. The PDMS is carefully lifted off the master.

This can be started by gently twisting the surgical blade as it is inserted into the prior cut. It's really thin with the PDMS devices on the mold. Face up reservoirs are punched into the device using an eight millimeter tissue biopsy punch.

The debris left in the puncher is pushed out with a rod. The devices are then quartered and excess PDMS trimmed away so that the device will fit neatly onto a Corning cover. Glass number one size 24 millimeter by 40 millimeter nitrogen air is used to blow away any excess debris.

Stubborn debris can also be removed using 3M Scotch Brand 4 7 1 vinyl tape. The clean devices are now ready for plasma bonding. A herrick plasma cleaner is used to Bond the neuron devices to the cover slips briefly.

The devices and the cover slips are placed on a tray, and the tray will then be placed into the plasma cleaner and a vacuum pump used to evacuate air from the chamber. It is important to note that the PDMS devices are placed face up. That is with the design face up so that they will be exposed to the plasma gas.

After the vacuum pressure has reached 300, millitorr power is turned onto electric coil and set to high. All is cracked on the door to let a little bit of air into the chamber, which will help generate the plasma. The devices and the cover glass are plasma treated for two minutes.

Notice the purple color in the chamber. That's the actual plasma. The electric coil creates plasma, which is a partially ionized gas consisting of electrons, ions, and neutral atos or molecules.

The plasma creates reactive species on the surface of the glass and the device, which when placed together will form a permanent bond. The plasma also turns the surface's hydrophilic, which helps facilitate the addition of liquids. The plasma cleaner also sterilizes the devices.

After plasma treating for two minutes, the power in the vacuum is turned off and air is led into the chamber. The plasma treated services of the glass and the microfluidic device are assembled in contact with each other in a clean bench. The devices are then placed in a sterile 60 millimeter dishes.

The plasma treated surfaces of the glass and the device form a tight irreversible bond. Within 10 minutes of plasma bonding, the device is to the glass polyol lysine is added to the device. After 10 minutes, the hydrophilicity of the device will decrease.

Making it difficult for the PLL to enter the device. It is important to check for air bubbles in the device after the addition of PLL. An easy method to remove air bubbles present is to use a P 200 pipette filled with 100 microliters of liquid.

In this case, PLL place the tip directly at the opening of the main channel, then depress the P 200 forcefully. This may need to be repeated several times, but eventually the force from the pipette will drive the bubbles out. The devices containing PLL are allowed to incubate overnight and a standard tissue culture incubator at 37 degrees with 5%CO2.

After 24 hours of incubation with PLL, the devices are rinsed twice quickly with autoclave deionized water. During this process, liquid is never entirely removed from the main channels, only from the reservoirs. Removing liquid from the main channels will introduce bubbles.

After two quick rinses, the devices are filled with autoclave deionized water once again and placed back in the incubator for a minimum of three hours or overnight. The next day, after the devices have been incubated with water overnight or for a minimum of three hours, they're once again brought into the biosafety cabinet and rinse twice with deionized water. Then neural basal media containing the necessary supplements, B 27 and glutamate for culturing primary rat neurons is added to the devices.

The devices are then placed back in the incubator for future use. The devices are now ready for seating neurons. In this video, We have demonstrated the technique of soft lithography, which we use to manufacture our neuron devices.

These devices can be used to culture neurons and other cell types of interest. The primary advantage of these devices is the ability to separate and compartmentalize the culturing of the neuron cell bodies on one side of the device from a pure axonal fraction on the other side of the device. In the next video, we will show you how we prepare E 18 fetal rack cortical neurons and how we load the cells into the device.

So that's it from the John Lab. Thanks for watching.