Hi, I'm Kevin won. I'm a master's of engineering student at the Biofluids Lab at Cornell University. And today I'm gonna talk about using a an agros based microfluidic device to study the effect of EGF epidermal growth factor on Maureen neuro stem cells.
The device basically consists of three microfluidic channels, each 150 microns wide. The EGF is pumped into one of the side channels and the buffer is pumped into the other side channel. It's basically sets up static linear chemical gradient across the center channel.
Since the EGF diffuse easily diffuses through the agro gel, the neuron stem cells are seeded in the center channel and the progress of their migration is monitored through a microscope. So the devices basically comprised of plexiglass top layer that has all the inlets and outlets for connecting to the tubing. And underneath that is a agro gel membrane, which has four pattern devices in it.
This is surrounded by PDMS spacer, which guarantees that we have constant thickness throughout the overall device. The PDMS and the gel assemble on a glass slide, which is coated with ect. This helps the cells to adhere to the glass slide.
In addition, the stainless steel plate is used to mount the entire device and we Sam sandwich the device together with screws. The wild type cell line has been transfected with a retrovirus vector to over express the wild type EGF receptor and the delta cell line has been transfected with a mutated version of the EGF receptor that enables it to be constituently active without the EGF ligand. Okay, so I'm going To begin by explaining the assembly process of our micro fluidic device.
So I first placed the PDMS spacer around the relief features of the silicon micro channels in our masters. So now I'm going to weigh out 0.3 grams of agros. So now I'm gonna bring up 10 mils of CO2 independent media and add that to the agros.
And I'm gonna pipet up and down to mix up the aros powder with the CO2 independent media. Now we're ready for the microwave. As you can see now, the agros is now liquified, but there are a few granules still remaining.
I will try to swirl this around to dissolve the granule and get rid of the air bubbles. So I'm pouring the molten agros onto the relief features of the silicon master and I'll now take a sterile glass slide and place it over the PDMS spacer. And I put it at an angle to try to force most of the air bubbles in the agro gel out.
And I applied pressure and I will continue applying pressure until the agros shell solidifies. So after one to two minutes it has solidified. Now I will remove the excess agros from the slide.
The next step is to gently slide off the glass slide and this glass slide can mount be disposed. Now I am going to try to gently transfer the pattern aros to a glass slide that I've previously coated with fibronectin. I basically use this plastic sterile sheet to help me lift off the pattern agros with the PDMM MS spacer and I will transfer the pattern agros with the channels down towards the slide.
Now that this is firmly on the glass slide, I will punch holes into the individual reservoirs so that the plastic manifold has access to the channels. I'll do this using a small metal hole puncher. Now we've finished punching holes in the device.
I will add 500 microliters of CO2 independent media to prevent desiccation of the micro channels and I'll now let it sit for one to two minutes. Okay, after one to two minutes, I will try to drain the excess media and now I'll align the holes that punched in into the pattern agros with the inlet and outlet holes of the plexiglass manifold. And once the holes have been aligned, I press firmly to form a nice seal.
Now I place the plexiglass manifold over the stainless steel mounts and now I will gently sandwich the device together using screws. I am very careful not to overtighten the screws because this will squish the channels and I place the screws in in a clockwise manner to prevent the inside a pattern Agros gel from slotting. I'm gonna draw one mil of media and I will use this syringe to test the device.
And as you can see, I'm injecting fluid into each microchannel and looking for fluid to come out of each outlet. So Right now I've placed the assembled device into the environmental chamber of the microscope. This is maintained at 37 degrees Celsius, so this will warm up the device and get it ready for when we need to seed the cells.
And as you can see, I've already threaded the two, the parasol pump tubing through the setup. And I am currently flushing the tubing with PBS in preparation for our Experiments. So now we're gonna Set up the tubing.
I'm gonna thread the parasol pump tubing through the caps with holes already punched into them. And each tubing has a, has a specific number of notches on it to make that I can identify both the inlet and the outlet of the tubing. So the tubing with four notches on it will receive the 10 nanogram per mil.
EGF solution will now attach the tubing to the device. So I'll attach it to each device according to how many notches it has. So it corresponds to the solution that it's taking up.
And now I attach the outlet tubing. We use a clear tigon tubing to ensure that the media is indeed flowing through the outlet tubing and there are no blockages in the channel. And now I will start the parasol pumps.
So now I'll begin the procedure for seeding the cells into the center channel. I'll start by taking out the excess media in the sensor channel. So now I will see the cells.
The cells are already in suspension in CO2 independent media. And I was sell, I was seated 60 in the inlet of the center channel and I was seated 20 microliters in the outlet. And hopefully the cells will be seated by gravity driven flow.
And I'll repeat this procedure for the other two sensitive channels and we'll now observe this under the microscope to see if cell seating was successful. So now the cells have adhered to the glass slide and in a few minutes they will begin to spread and attain their unique morphology. So today we covered the assembly of the actual device, the preparation of the reagents and the microscope techniques that I use to image the cell migration within the device.
In conclusion, we hope that this device will have much broader abdication, specifically in the clinical setting. So figure one horizontally. It shows the trajectories of the C 17.2 cells and increasing EGF concentrations vertically.
It shows the trajectories of the various strains of the C 17.2 cells that we've used in our experiments with the same EGF concentration. The parental cell data shows that there's a peak in motility at the 10 nanogram per mil EGF concentration gradient, indicating that the receptors have become saturated with ligand at a higher EGF concentration. And wild type data shows that there's an increase in motility at the a hundred nanogram per mil concentration or EGF.
This indicates that the receptors do not become saturated at higher EGF concentrations. This makes sense since they've been designed to overexpress the EGF receptor. Finally, the delta cell line data indicates that the motility of the cells is roughly independent of EGF concentration.
This is also expected since the the mutated EGF receptors were designed to be activated independent of the presence of EGF.
