マイクロ流体デバイスを用いた細胞のキャプチャ

The devices as we Prepared them, are packaged into kits of 10 or 20, and when they arrive, they're shipped cold in a box and the kit contains all the components that you need to run. The experiment in the kit are, is a packet with all the syringes and buffers, which can remain at room temperature. There's also a copy of the protocol that we're gonna follow in this experiment.

There's also a refrigerated box, which contains the actual devices and all the running buffers which need to be refrigerated. Typically, kits are packaged in groups of 10 or 20. When not in use, the kits should be refrigerated.

Each syringe and each kit component is carefully labeled and the labels correspond to the table in the protocol that we follow. So there's no ambiguity as to which syringe or kit component should be used in the individual steps. After unpacking the, the devices and placing them into a biological safety cabinet, we'll be performing the procedure of isolating the neutrophils and lysing them for downstream genomic applications because we're gonna be, because the life save that we get will be used for isolating or, or we will extract the RNA from the cell lysate.

Because of that, we wanna make sure that our area is clean, not just in the sterile sense, but also clean of any RNAs which are present typically in the environment. So it's very important to wipe down all your area and your devices with the solution which destroys RNAs. So typically we will wipe down all our tools and our work surfaces with RNA SAP or RNAs away, which you can get from a number of manufacturers.

The device has come packaged in a heat seal pouch. Each device is sealed and barcoded this barcode. You should, you should make a note of this barcode if you're working with any clinical samples.

So the device is unsealed, the cover's taken off and it's placed in the pump Holding manifold device. The blood sample we use is a Sample of whole blood. We're gonna flow it through the standard one mil BD syringe, and this is labeled syringe one.

Typically what is done is the, the syringe is removed from the package and the cap is carefully taken off of the blood tube. And what you do is you drop 700 microliters or 0.7 mils of blood and inject half of that or 350 microliters into a 1.5 mil tube.Thanks. That way your, your blood can be passed on for further processing.

Your, your tube of blood can be passed on and yet you still have more blood left over in case of a device failure. So now what we're gonna do is we're gonna load the syringe that has blood into it, onto, onto this needle, which is connected to a piece of tubing with this device. We wanna be very, with, with the microfluidic isolation device.

We wanna be very careful not to introduce any air bubbles into the, into the device or, or the device will malfunction. So typically what we do is we take the syringe and we push down on the plunger and add the liquid to the top surface of the device, make sure it's fully wedded before we've ejected all the liquid. We stop and we carefully remove the syringe body from the needle and basically fill up the needle tip or, or the, the, the lure connector of the needle with the remaining buffer that's in this syringe.

We dispose of that. We then are gonna take our syringe with blood and we're gonna insert it in, into this lure hub. Being careful not to spill the blood and being careful not to introduce any air bubbles.

Find it best to use a kemo holding the body of the syringe and the tubing in case any blood is spilled. Once, once the syringe is connected to the tubing housing or the the needle, we give it a few sharp taps to remove any air bubbles, which may be at the connector. This syringe is then loaded onto the syringe pump and then the, and it's secured with the locking screw.

The syringe pump is turned on and it's already preset to our flow conditions. We're gonna flow this blood through the device at 30 microliters per minute. Once the syringe body is loaded into the syringe pump, we're going to depress the button on the, the movable plunger arm and slide it down until it touches the top of the plunger and starts pushing fluid out through the tip of this.

It's a blunt tip, stainless steel needle. Once we know that the system is primed, we're going to press run. To begin the flow of blood, you press stop to stop it and then you take a clean 1.5 mil centrifuge tube or fendor tube and we're gonna unplug carefully.

Unplug just one, unplug the tubing from one port of the device. Once this tubing is unplugged, we'll insert it into the 1.5 mil fendor tube. We'll press run to begin the blood flow again.

And when we see a drop of blood form, we stop the pump and insert it into the, the inlet that we've just opened up in the device. We'll press run to begin the flow of blood and we'll set a timer for five minutes. We wanna make sure that blood equally fills all the chambers and that there are no obvious air bubbles in the device.

If there are air bubbles that are blocking the flow into any of the chambers, you can take a pair of tweezers and run them along the small channels, which lead up to the capture chambers or the small channels which exit the capture chambers. This device is filling nicely, so after five minutes, you press stop on the syringe pump to stop the flow of blood, you loosen the clamp for the syringe and take it out of the syringe pump holder. Then we're gonna use syringe number three, which is loaded with nuclease free PBS We're gonna use, we're basically gonna, again make sure the surface of the device is wedded to avoid any air bubbles, we'll tap the syringe to make any, make sure any air bubbles are at the top of the syringe body.

Loosen the arm on the syringe pump and insert the three mill syringe into the pump. Tighten it down and then push the plunger down until it contacts the plunger of the syringe. We hit run and we wait for liquid to prime the device and we wait for a to see a droplet at the tip of the stainless steel needle.

When you see a drop forming, you stop the pump. We're gonna take out the blood, the the stainless steel tip that has the blood. We're gonna insert the wash buffer and press run.

And we're going to let this run for five minutes. And you should take a clean wipe and just wipe up any small amount of blood, which may be at the inlet or the outlet for After five minutes of running wash buffer through the device, the device should be completely cleared of any blood and you can see that by basically there won't be any red blood left in the Device, the device will look clear. So what we're gonna do is after the five minutes, we're Going to stop the wash buffer from running.

And again, we're gonna take the syringe off of the syringe pump. We're gonna cap the 1.5 mil micro centrifuge tube, right on the flexible outlet tubing. And we're gonna carefully lift up the tube, the micro centrifuge tube, pulling the, the waste tubing out from the device.

We're gonna throw that away in a biohazard container. In the kit is a new outlet tubing, which is actually made out of Teflon. It has again, a stainless steel tip on it.

We're gonna replace the old outlet tubing with this new outlet tubing. We're gonna take that outlet tubing and we're gonna try to bend it around sort of a U or a V shape. And then also with the kit is a kaya shredder column, this shears DNA and basically homogenizes your cell life.

So we're gonna, we're gonna open the purple cap of the shredder column and place the outlet tubing into the Kay Shredder column. We're gonna take out from the kit syringe number two, which says, for RLT we're going to use it. It's a standard BD one mil to syringe with a 22 gauge blunt tip, stainless steel.

We're gonna use that to inject 350 microliters of standard RLT from kyogen into the device. Because the device has a dead volume about 50 microliters, what we wanna do is we want to inject the RLT, but behind the RLT, we want to inject a plug of air to, to release that dead volume of the device into the chi shredder column. So the way we'll do that is we'll take our syringe, we'll pull, draw up 350 microliters of air and then followed by 350 microliters or 0.35 mils of RLT and the RLT is now at the bottom of the syringe.

Resist the temptation to flip the syringe over and e inject the air. The air is there to make sure we get all the RLT into our Kay shredder column. We're gonna pull out the tubing that from the wash buffer.

We're gonna insert our syringe with the RLT and we're gonna slowly inject the RLT into the device over a period of about 30 seconds, making sure that the outlet tubing is ejecting its waste into the the chi shredder column. Once air starts getting injected into the device, you push down on the plumer and wait for all the fluid to be released into the Kay Shredder column. We ca the kay shredder column and we spin it on a microcenter fuge with a balance at 15, 000 RPM or or maximum RPM on a benchtop microcenter fuse for two minutes.

So after spinning the column for two minutes, we're gonna take the sample out of it, which now is in RLT and we're gonna place it into a one of the supplied purple cap cryo vials. If you're working with a clinical sample, sample these cryo vials, you'll label it at the beginning of the experiment. Here is the lysate in the cryo vial.

This cryo vial is immediately placed in a minus 80 degree AC C freezer and stored for shipment or Later RNA extraction. So this is an alternate protocol For isolation of neutrophils. Instead of lysing the cells with RLT buffer, we're going to stain them with a standard histological stain.

A stain. So we take the device sealed device outta the bag and we unwrap it again, we note down the lot number on the device and place the Petri dish. In the Petri dish holder.

We're gonna take the one mil syringe from its bag syringe number one, we're going to load it with 700 microliters of blood and inject half of it 350 microliters into an eend DPH tube. We'll set that aside. Take got syringe four, which has the tubing we'll use to select in, inject the blood into the device.

Gonna hold the base of the needle from the tip of the tubing in a chem way. Take off the syringe body and fill the lure. Adapter would buffer and dispose of that syringe.

We'll insert the blood containing syringe into the needle using the chem wipe to collect any spillover. We'll tap the syringe to remove any air bubbles at the connection between the needle and the syringe body. We'll load the syringe into the syringe pump, we'll prime or the tubing by pushing down on the moving arm of the syringe pump.

We'll disconnect the tubing from one of the outlets of the device, the capture device, and place it into a 1.5 mil einor tube. And then we'll begin flowing blood by pushing run on the syringe pump. We'll wait for a droplet of blood to form at the stainless steel tip.

Once the droplet forms you press stop dab off that blood, insert it into the device and press run. Now we set a timer for five minutes. Okay, after flowing through the device for five minutes, press stop on the syringe pump and release the syringe.

And we're gonna replace that syringe with the three mil syringe containing our wash buffer, which is just one XPBS. Again, we wanna make sure the top of the device is wedded and then we're going to remove the needle tip containing blood or plus run. Begin priming the device.

We're gonna droplet forms, we insert it into the device and press run dab off any blood at the inlet or the exit tubing and we'll let that flow for five minutes after the washing step. Again, we'll stop the syringe pump and remove the syringe from the pump holder. To do standard GIM sustaining, we're first gonna fix the cells and dehydrate them using 100%methanol.

So we, we have a syringe, a three mil syringe preloaded with methanol. Again, we're gonna prime the syringe, make sure methanol is flowing, stop the syringe pump, insert it into the device, and press run. We're gonna fix them in this methanol solution for two to four minutes.

So after two to four minutes of fixing and methanol, we stop the pump, release the syringe with methanol, and here we're gonna load a syringe, three mil syringe containing eem sustain standard eem sustain from Sigma diluted one to five in deionized water. After we diluted, we, we loaded into the syringe and at the, at the end of the syringe we attach 0.8 micron filter to filter out any particles which will go into, which would end up clogging the device. The stain for five to 10 minutes.

And then we'll rinse with Deion i's water. So after five to 10 minutes of staining, we'll stop the syringe pump and we're gonna switch out the syringe with the staining buffer with the syringe that contains deionized water, we're basically gonna wash out until we see the, the blue color of the SAI has been washed from the device. Once the excess gim sustain has been rinsed from the device, we stop the washing solution.

And then what we're gonna do is we're gonna remove the, the water syringe from the inlet. Then we're gonna take the outlet tubing of the device. We're gonna grip that tubing at the end with our tweezers.

This is a flexible tigon tubing and we are going to insert it back into the inlet of the device. This seals the device and keeps the cells hydrated and their morphology Intact. Now my coworker, Dr.John Hong Chang, will show an alternate protocol for isolation of CD four positive lymphocytes, followed by immunofluorescent staining directly within the microfluidic device.

So my name is Shong Hong and today I'm going to demonstrate to you cell staining in microfluidic device at this stage can already showed you how to use a micro flic immuno affinity isolation device to specifically isolate blood cells from unprecedented whole blood. For his purpose, he wants to lice the cells and get protein and DNA content from those isolated cells. Well, for the purpose of my research, I'm interested in getting the cells and then obtain the cell count, for example, to get CD four cell count from whole blood and, and use that as an indicator for diagnostic purposes.

So the device using is very similar to Ken's device. It's made of outta P-D-M-S-A straight chamber. The geometry is slightly different, so the operation parameters are slightly different while in, in general the overall operation procedure is very similar.

So I'm going to skip all the cell capture and rinsing steps and jump directly into the cell staining step in cell counting procedures. So here I have a device that's already half cells captured from whole blood and the device is already rinsed with PBS. So the outbound cells already washed outta the device and I prepared ahead ahead of time a antibody mixture that's used specifically to label the cells am isolated into the device.

So the antibody concentration we used here has been optimized for, for staining the cells with very high fluorescent intensity under fluorescent microscope. So the procedure is quite straightforward. We load the syringe filled with the antibody solution on the syringe pump and then make sure that the flow rate is set to the right parameters that we need.

And then we'll start the syringe pump, watch the end of the tubing, make sure that solution comes out already so there's no more bubble in the tubing. And then we can plug the tubing into the microfluidic device that we have cells captured. So now antibody is flowing into the device to stain the cells we have isolated into the, in the device, the flow rate here, I'm using five microliter per minute, but depending on the device geometry there, there could be different flow parameters that you want to use for your purpose.

And we'll lape the antibody solution flow in for five minutes, which is enough to enough volume to replace all the solution within the micropho channel. So after antibody injection, we'll stop flow. So we, we float in antibody at five microliter per minute for five minutes to replace all the solution within the microchannel.

Afterwards we stop flow and let the device incubate and room temperature for 30 minutes. So this should be done in dark so the antibody doesn't get quenched while the cells, the fluorescent antibodies can get quenched while the cells is incubated with a staining antibody. So after staining for usually 15 minutes to 30 minutes, we will just unplug the tubing from the device and replace the antibody syringe with a buffer syringe to wash off unbound antibody from the device.

So the washing solution here contains 1%BSA in PBS solution. BSA is used to block non binding of antibody on the surface and we'll do the same thing. So first we adjust the flow rate on the syringe pump to make sure that it's the right flow rate we wanted and then we'll start the syringe pump and make sure there's actually liquid coming out of the tubing before we plug it into the device.

The purpose of doing that is there's no bubble remain like leftover in the tubing. So when we inject in, no bubble goes into our device. So we just plug in the tubing into the device and let let it flow for another five minutes to replace all the wash out, all the unbound antibody from the device.

After the washing step, we'll fix the cells with 1%PFA. The purpose of doing that is so the, the device can be stored for a few weeks up to a few weeks if the imaging can be performed right away. So the procedure is quite straightforward.

It's similar to what we have done before. We load the syringe on to the pump, adjust the flow rate to the one we wanted, and then we start the syringe pump and we plug in the tubing to our device to inject the paraform IDE solution, which is the fixative into our device to fix the cells. Again, we let it flow for about five minutes.

So PFA replaces all the buffer in the device to fix all the cells in the device. So after the PFA fixing step, we just take the PFA syringe off and now the device is ready for imaging. In some cases, people want to have a nucleus staining to show to overlay with their cell surface marker staining.

So for that purpose will do nucleus staining using DPI at the end. So we just load the DPI syringe on turn on the syringe pump and flowing DPI into our device. So now the nucleus will be stained in those cells to show the location of the cells.

And this the, the DPI staining image can later on be overlaid with the surface marker staining to show where the cells are. So at the end of the DPI staining, we need to again wash off the L band DPI P with buffer solution. So again, we are using PBS containing 1%BSA to wash off the outbound dpi.

And the operation is similar to what we have done before. We just plug in the buffer syringe into our device and we'll, we'll let buffer flow into the device to wash off unbound dap. So that's the whole procedure of staining cells in micro fluid device.

And after all the staining procedures, the devices are ready for imaging. And because we did PFA fixing, the devices can also be stored for a few weeks. If image imaging could not be performed right away.