解離マウス皮質ニューロンの培養の準備

Hello, my name is Hilgenberg. I work here at the university in California at Irvine in the Department of Anatomy Neurobiology. Today I'm going to talk to you about how we prepare cortical cultures from mice.

These are postnatal day mice that we prepare those neurons from. And we typically use those cultures for either electrophysiology, for protein or RNA preparations. We use the same culture preparation for calcium imaging or sodium imaging.

The nice thing about these cultures really it's, it's an adaptation of the banker protocol, but we grow these cultures in the absence of glial feeder cells, which makes it a lot more convenient and the cultures are usually more rich in neurons than glial cells. So let's get started and I'll show you how we prepare Those neurons. Alright, Now we are at the point where we can remove the brain and start doing the actual dissection.

But before it's important to mention that the cover slips that you want to plate these cells in need to be coated. And we used to do that, usually do that overnight at room temperature at a polylysine concentration of 0.1 milligram per mil. And the next day we come and remove the polylysine and wash the coverer slips three times consecutively.

Depending on the application you use big dishes or small dishes or you can use glass cover slips. Alright, so this is the typical setup for the hood and I briefly explained to you what solutions you need to have ready and also which instruments come in handy for the isolation of the, or the dissection of the brain and then the actual sectioning of the brain. What we have here is the dissection solution that's cold.

So usually it's, it's on ice. We have have a Viber slicer that we need to section the brains. I have a, a boat that contains dissect solution that the brain is being mounted on and then sliced.

And I have various tools that I need for removal of the brain. I have an auger plate. It's 4%auger that I use to hold the brain in place.

We need a syringe, a couple of sterile filters, Rotman paper dishes, Petri dishes of various sizes. And this is my mouse pipe hitting tool. I also have pulled glass pipettes in here.

Those are glass capillaries that I use as dissecting tools as well as to pick up the brain pieces and basically iterate these. So our enzyme solution contains dissection solution. There is L cysteine in there to activate the pepane as well as 0.1 normal sodium hydroxide to balance the pH.

Once you add the pepane, the solution is a bit opaque and it needs to clear before we can actually use it for enzymatically digesting the tissue. And now we're basically ready To start the procedure. The fibro Slicer has been prepared.

I've inserted a blade that sections my brain and I've also stuck the vessel underneath that contains the buffer and supports the brain. And I'm ready to get the AGA block mounted onto my support plate that will also support the brain. So this aga has cured, it's a 4%aga and I'll just go and cut right through an area like this.

And on the sides basically just get a rectangular auger cut out. Then I'm using a spatula and I'm just popping this piece out of the auger block. And now I'm going to glue this auger onto my support plate with the flat side of the auger facing forward.

So this is the support that the brain is being glued onto and then is held in place by this auger. So when we do the sectioning this way around, the brain doesn't move Backwards. Now we're Ready to remove the brain.

Usually we use a P zero or a P one mouse. Alright, so this is the decapitated mouse head and I'm just going to quickly remove the the sculp. I am first going to cut the muscle band that's right here and on the other side.

And then I'm going in with my scissors. And I'm very careful that the inner side of the scissors is not going to damage the brain. And I'm cutting around the skull on both sides.

And now we can base just pick up the cartilage and I'm going to use my small ULA and I'm removing the brain like this. So this brain then goes into my Petri dish that I filled with the section solution and it has a watman filter paper on the bottom. And what I'd like to do now is cut the base of the brain so I have a nice solid flat area to mount the brain onto my shoulder.

So I'm gonna put a, a very small drop of crazy onto my support plate. I'm going to pick up my brain now with the spatula and I'm using my, my curve forceps to help me with this. And what I want to do is basically drop off a blood of the excess liquid that I have from the dissection solution and I'm going to mount the brain against my auger support.

We're now ready to transfer the brain into the buffer bath. So I'm adding dissection solution to that little vessel and I'm going to add the support that holds the brain. It's being mounted with the brain facing the blade.

Alright, so the brain is now mounted and I'm going to use the fibro slicer and I'm trying to find the beginning of the brain, moving the brain towards the blade. And I just cut a little bit of the olfactory. Now I'm moving my, my blade about 600 micrometers down and I'm doing another cut, which should go right through the cortex.

There goes the cortex and it rides up onto the, onto the blade. And I'm moving my blade another 600 micrometers down and I'm cutting the second section. It's important that this is done continuously without interruption and nice and smooth.

And with when you withdraw the blade, you stop the fiber slicer. Now going to use a a glass pipet that I used the wrong way. I actually use the back end and I'm going to move this cortical section up in here and transferring it into a clean Petri dish.

I proceed through the brain, always 600 micrometers until I've collected about five to six slices for a P zero or P one mouse. And until I get to the very end of the cortex. And we're ready now to isolate the cortex from the rest of the Brain.

Alright, so I'm Using these glass pipettes that are pulled glass pipettes as sharp as dissecting tools. And I'm usually starting with the first section I got, which is the smallest and the olfactory we have to remove. And I'm also trying to remove the meninges from this prep.

Again, removing the olfactory of this piece, peeling, peeling it off the of the meninges. Sometimes you have pieces that you can't separate as easily, so just move on and leave those behind the good pieces. I always move towards one end and I'm going to work my way through one section after the other.

It's important that you get rid of the meninges and also when you, when you look at these sections, sometimes you want to go and flip em over so you can better see where the fiber tract is that contains most of the non neuronal cells that you don't really want in this preparation. Okay, on this section you can clearly see this fiber tract underneath the cortical layer. So I stab right between where the cortical layer ends and I'm pointing out the fiber tract that has most of the neuro non neuronal cells that we're trying to avoid in this preparation.

So I'm cutting along this line, trying to leave the fiber tracts behind. This is the step where you mince the tissue into small cubes. All right, I'm, I'm sterilizing my enzyme solution now through a 0.2 micron filter attached to a a syringe.

And I add that straight onto my, my cortical pieces just like this. Make sure that there is enough liquid that surrounds all the cortical pieces and these cortical pieces then go into a 37 degree incubator for 30 minutes, which should loosen up the tissue quite Considerably. So it's been 30 minutes that these cortical pieces have been incubating in the incubator.

In the meantime, I warmed up some neuro basal media with B 27 supplement that these cells eventually end up in. I've also prepared some solutions here. First one is the dissection solution.

So it's plain dissection solution. Then they get transferred into a high trypsin inhibitor solution that contains BSA as a scavenger and a PV to block the NMDA receptor component. And then they go through three washes of low trypsin inhibitor BSA and A PV.I'm going to remove these pieces now from the propane solution and I transfer them into a conical tube containing DS dissection solution.

These clumps will just travel downwards by gravity and it might take a minute or two before they're all settle down in the conical part of the tube. And then I'll just pick em up again and I will transfer them into the solution that contains the high trypsin inhibitor. And again, we want to wait until these clumps settle down to the bottom of the tube.

There is one more wash through a high trypsin inhibitor solution and then these clumps get washed in low trypsin inhibitor. After my last wash and the low enzyme inhibitor, I'm going to transfer the cells in my prewarm neuro basal media and I'm starting to mechanically dissociate the cells by tri them up and down a glass capillary with decreasing bore size. I'm Going to T the cells now and the tissue clumps.

But before I do so, I will remove all the debris that is visible here. There is for example, protein and DNA that that leaks out of the cells that forms this cotton ball, like stuff that hangs onto the tissue clumps. You want to remove that first because it clogs your, your pipet tip and you're forcing the tissue into through two smaller a diameter.

And then I'm going to go and break some of these glass capillaries to get the right size opening to move my cells in and out of the of the opening. This is a good pipet. This one you can see that it's nice and round and doesn't have any sharp edges.

I'm going to stick this into my mouse pipe hitter. There is a 0.2 micron filter in between and I'm going to now move the tissues in and out of my glass capillary. So the first process should go fairly smoothly because the tissue clumps are not all that much bigger than the pipet opening.

All you want to do is loosening up the the tissue. And then I'm going in with a slightly smaller pipette tip. I would like to have a nice clean round pipette tip.

This looks good. And again, all the clumps get moved in and out the pipe at once ones. So the tissue now is very soft and loosened up that if you reus suspend these clumps in media, the cells will basically fall out.

I'm now going to transfer all my dissociated cells into a fresh 15 milli mil milliliter conical tube, and I refrigerate them maybe two or three more times, gently up and down to break up cells that are still in those larger clumps. And then we want to wait one or two minutes for the big pieces that are still in there to settle down. Also, you might have glass pieces from your glass capillaries that you broke off that should settle down onto the bottom.

And then we can use aliquots of this to plate out onto fully ly coated cover slips or dishes. And I'm plating out 80 microliters onto this glass cover slip that has previously been coated with polylysine and it forms a nice bubble. The cells will eventually settle down and adhere to the glass cover slip.

So after about an hour, the cells have settled and I'm going to add very gently going to add some more media to the entire dish. Can give it a gentle swirl like so. And then these cells go into the incubator at 37 degrees and tomorrow I'll feed them with conditioned media.

Alright though, these are the cortical cells. They're just a little bit less than one day old. They were plated out yesterday and you can see that there is plenty of neurons, all have lots of lung neurons, and there's also some cell debris in there.

Those are the cells that didn't make it. And these will wash out in the next couple of feedings. The dead cells are the very small round cells with the ruffled membranes.

I've Just shown you how to prepare these dissociated cortical cultures. It is important to remember that when you remove the cortical rind from the brain slices, that you separate them from the thumb sala cortical fiber pass as it contains lots of non neuronal cells. Also, when you treasure the cells do not over as that basically kills off most of the isolated cells that you've generated by now.

So go easy on the tissue slices and you'll have a beautiful culture. Good luck on your experiments.