Hello and welcome to the laboratory of Dr.Newly John. In a previous video, we demonstrated how we manufacture the neuron device, a platform that allows the researcher to separate the cell bodies of the neurons from a pure axonal fraction. Soft lithography is the technique utilized to make the devices, which consists of casting and curing PDMS onto a silicon wafer containing the device design that was previously manufactured using photolithography.
In this video, we'll demonstrate the preparation of spro dolly E 18 cortical rat neurons, and how to load the neurons into the neuron device. I'd like to introduce you to Kiana Lee. She'll be doing the Demonstrations today.
Before starting Out, it's important to have all necessary media and reagents warm to 37 degrees in the water bath. You can see neuro basal media 0.25%trypsin and DMEM containing 10%FBS. It is important to ensure that everything being placed in the hood to be used for repairing the cells be sterilized and wiped down with 70%ethanol.
First, the tissue culture hood is wiped down with 70%ethanol. Next, the plastic racks used to hold tubes are sprayed with ethanol. Next, the rubber ball used in the tri striation process is cleaning with ethanol.
It's important to do this early on so that it has sufficient enough time to dry. Next, the necessary reagents are wiped down with ethanol and move to the hood. Two Pieces of E 18 fetal rat cortex representing one brain previously dissected are placed in a 15 ml tube containing one ml, ice cold, calcium free, magnesium free dissection buffer, one ml of 0.25%tripsin EDTA is added to the cortex in the dissection buffer, bringing the final volume to two mills and the final tryin concentration to 0.125%The 15 ml tube containing the cortex with the trypsin now added is then placed in a 37 degree water bath for eight minutes, while the fetal cortex is incubating at 37 degrees in trypsin.
Three paste pipettes are fire polished. The tip of the glass pipette is placed just in contact with the flame near the blue part of the flame, and slowly rotated until just the opening turns orange. As it turned orange.
It's important to keep rotating the glass pipette so it does not collapse. Note the openings of the glass pipettes are successively smaller. It is also important to note that you do not wanna make the openings too small as this can cause lysis of the cells after the cortex has been incubated for eight minutes at 37 degrees with the trypsin 10 mls of DMEM containing 10%FBS is added to the cortex to help stop the trypsin reaction.
The 15 ml tube containing the cortex and DMEM 10%FBS is centrifuge at 2, 500 RPM for two minutes. In the biosafety cabinet, vacuum suction attached to a glass pipette is used to remove the supernatant from the cortex. Being careful not to disturb or dislodge the pellet.
One ml of neural basal media containing B 27 and glutamate is added to the cortex pellet and gently pipetted up and down. It is very important to not create air bubbles while pipetting up and down as the air bubbles can damage the cells by oxidation. Next, The fire polished posterior pipette with the widest opening as used to tri the cortex by attaching a sterile rubber bulb to the end of the pipette and pipetting up and down approximately five times.
Being careful not to introduce any air bubbles.Okay. The process has continued with the other two glass pipettes each with the decreased opening size. As previously stated, it is important to ensure that no bubbles are introduced during the tri striation process.
You should also notice that you are no longer seeing any visible chunks. It's important to keep tri rating until you can no longer see any of the chunks of the cortex in the media. After ation, the cells are again centrif fused at 2, 500 RPM for two minutes.
This is about the approximate size of the pellet one would expect from two pieces of cortex. After centrifugation, the sup natin is once again removed from the cell pellet. After super natin is removed, one ml of neuro basal media containing B 27 and glutamate is added to the cortex and used to resus suspend the cortex.
The cell pellet is once again pipetted up and down carefully to avoid adding bubbles and ensuring that the cell pellet is evenly distributed and broken up. One more mil of neuro basal media is added to the cell suspension bringing the final volume to two mils. Next, the resuspended cell solution is filtered through a BD biosciences 40 micron cell strainer next to count the cells and assess viability.
60 microliters of fresh neural basal media is placed into a sterile micro fuge tube. 20 microliters of cells are then added to the 60 microliters of neuro basal media, and finally, 20 microliters of trian blue is added to the cells in the neuro basal media. The trian blue will stain cells which are no longer alive after the cells have been stained with trian blue.
We pipette 20 microliters onto a hemo cytometer and view them under the microscope. Viable cells appear clear and translucent while cells that have their membranes punctured stain blue due to the trian blue. Typically, we obtain about 2.5 million cells and 8 million cells per cortex.
We normally obtain two pieces of the cortex from one brain with the cells resuspended in a final volume of two mils. Thus, the final concentration of cells is typically between 2.5 million cells per mil and 8 million cells per mil. Normally, we load about 20 microliters of cells per device.
This gives us a range of cells per device between 50, 000 cells and 160, 000 cells. Cell densities can be varied depending on the application. However, seeding primary neurons at too low of a density typically leads to cell death Devices Previously prepared containing neural basal media are brought into the biosafety cabinet to maintain sterility.
Excess media from the wells is removed by a vacuum section. However, one must be careful to not remove all the media. There must be some media in the main channel only aspirate media from the reservoirs.
Only 20 microliters of cells is applied to the top left-hand reservoir of the device. The cells flow into the device and attach to the PLL treated surface. Here you can see the cells as they're flowing down through the main channel, and you'll begin to see some of them actually attached to the glass.
After loading, the devices containing the cells are placed back in an incubator for approximately 10 minutes to allow the cells to attach. After 10 minutes in the incubator, the devices are once again brought into the biosafety cabinet, and the reservoirs are filled with media and the devices are then placed back in the incubator. Some important things of note, depending on the incubator and the level of humidity the media need, may need to be changed in the devices every two to three days.
It is good practice to place fresh media in the top wells and allow some fresh media to flow through the device and into the main channels before filling up all of the reservoirs. The Neurons are visualized here with calcium am a cell permeant dye that stains living cells. Here you can see the so compartment or cell side of the device that contains the cell bodies, axons and dendrites.
As we pan across the device, you can see the micro grooves that the axons grow through to reach the other side of the device, the axon side, which contains a pure axonal fraction. For demonstration purposes. These fluorescently labeled cells will be used to illustrate the ax otomy procedure.
One of the properties of the neuron device is that it allows the researcher to perform ex otomy that is cutting the axons from the pure axonal fraction of the device. In order to to conduct research such as studying axonal regeneration. Keano will now demonstrate performing ex otomy in the neuron device, ex otomy is performed by a vacuum.
A sterile glass pipette is placed at the bottom opening of the well on the axonal side, the media is aspirated off by vacuum. All of the media is completely removed from the axonal side of the device, including the media in the main channel. To repeat the process after the media is removed, 150 microliters of media is forced into the main channel by placing the tip of the pipette at the opening of the main channel and depressing the pipette with force.
Next, the vacuum is applied to the opening of the main channel and the top well of the axonal side of the device. Vacuum is once again used to remove all the media from the axonal side, including the main channel. Once again, 150 microliters of media is forced into the main channel by placing the tip of the pipette at the opening of the main channel and depressing the pipette with force.
At this point, the researcher can view the device under a microscope to check the efficiency of the axon cutting. If the axons are not sufficiently cut, then the process can be repeated again as necessary. In This video, we have shown you how to prepare E 18 fetal rack cortical neurons and load them into the neuron device.
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. We hope you have found this video informative and helpful. So that's it from the John Lab.
Thanks for watching.
