פורסים תרבות Organotypic של E18 מוח החולדה

My name is Laura Elias. I'm A neuroscience graduate student working in Arnold k Stein's lab. We're in the institute for re Regeneration medicine here at UCSF, and today I'm going to be showing you how to do organic typic slice culture of E 18 rat brains.

So this procedure involves dissecting the brains out, embedding them in low melt agros, then slicing them on the viome and taking your corona sections, and then placing them in the Slice culture system. So there are a few important tools in reagents That we use for this slice culture procedure. You'll need a dissecting microscope to dissect out the brains.

We also use this V like a VT 1000 S vibram, and that's a good system for cutting your slices. The glue can be important. We use this lock type for for glue that glues the brains very well because sometimes if you use the wrong type of glue, the brains will will come off when you're trying to cut them, and that can be problematic.

We also use these milli cell filters that these are the filters that we plate are our slices on while they're in culture, and this is also an important Part of the process. All right, so before you start the Slice of culture process, there are a couple of of steps that you'll wanna take care of. So first, you need to make your A CSF, and you want to, after you finish making your A CSF, you wanna get this on ice so that it chills as well as start bubbling it with carbogen.

You'll also want to make your 4%agros. So for this, you wanna use low melt agros and dissolve it in a CSF, microwave it, and then keep it at at 50 degrees until you'll embed your brains. You also want to make your slice culture media, and after you make your slice culture media, you should put it at 37 degrees so it starts warming up so that when you put your slices in it at the end, it'll be at the right temperature.

And at this point, you should be ready to collect Your tools and start the slice culture process. All right, now we're gonna make slice Cultures from the brains of embryonic day 18 rat embryos. So first I'm gonna check that the mother is fully anesthetized by toe pinch.

Here's an example where the, the rat still has a tiny reflex and is pulling it hind leg back a little bit when I squeeze on its foot. And so we'll have to wait longer or Re anesthetize a bit, and we don't see any reaction. So I'm gonna go ahead and open her up to retrieve the embryos.

And I just want to pull up the skin so that I can get a nice clean cut. Remove some of the embryos here, and I'm going to cut them out. So you can cut in between two embryos and then we'll cut along.

And now we can remove this embryo and detach it from the placenta by making an incision right adjacent to the placenta. Now we'll move the embryo to cold A CSF and remove the remainder of the, of the sack here. Yeah, that's and snip off there.

So now this embryo is Ready and we'll go make brain sections. All right, so now we have our embryo, And I'm going to remove the head here and scissors. Okay, now I'm ready to dissect out the brain.

First we have to immobilize the head, and we do that by placing pins through the eye sockets as well as in the hind brain. And this helps us immobilize the brain in the dish. And so as you see here now, we have an immobilized brain in our dish, and we'll start by making an incision in the hind brain and then going forward more roly.

So I'm gonna start by making an incision here near the cerebellum. I'm going to get my scissors under the skin and the skull, and then move my incision up to the Roswell area. Then I'm gonna make two more incisions laterally near at the very end of the cortex, near the sero meum.

I'm gonna make a incisions that laterally in that direction, and then an incision there. And then I'll use my forceps to peel off the skull as such. And now we've exposed the braid, and I'll then scoop it out from the head As such.

So there you have your fully dissected Brain. All right, now we're gonna embed these brains in Agros, and I've pre-made 4%low melt agros. And so you make this up in a CSF, microwave it, and then keep it at about 50 degrees so that it stays liquid.

And so now I'm going to start the embedding process. So I'm gonna take some of this agros, put it into these dishes, and we'll fill these up pretty much to the top, these ones. Alright, so then the first step is to take your brains and just to basically rinse them off in a dish of agro.

You don't want excess liquid on the brains because it prevents them from, from embedding well and sticking well to the agros. And so if you don't wash them well in the first step, they tend to fall out of the agros when you try and slice them on the viome. And so we just swirl them around and try and get off excess liquid.

So then we'll transfer these brains into these other dishes that are full of the 4%agros shown here. So we're now positioning the brains in the agros. As such, you don't want it too close to the bottom of it or the top of the dish so that it, that it'll be surrounded by agros on all sides.

And then we're gonna place the top over this dish, cover it up, and then we're gonna place this dish on ice so that the ris will harden and the brain will be embedded nicely in the eyebrows and the agro should Harden in about five or 10 minutes. All right, so now we have our brains Embedded in agros and where we're going to start sectioning them on the viome here to make approximately 300 micron thick sections that we, we will be using for sliced culturing. We're gonna do this as well as all of the other things we've done in this cold A CSF that is being bubbled with carbogen.

And so now we're ready to start. All right, so now we're going to begin by removing the brain from the agros. So we're gonna just be taking off the top here, and sometimes you have to help make sure that the, the block of agros comes out of the top.

Let's see here. So you can just pop it down into the bottom portion. So now we have the agros, and what we're gonna do is we're gonna cut into the agros so that we basically cut out the brain.

So I'm gonna make four cuts, one coddly, and then one at the very front, and then two along the sides. And so I'm just basically cutting out the brain just as we embedded it. So now we've made incisions and cut our brain in the agros.

And so now we should be able to basically just pop our brain out of the agros here. So our brain just came right out and it's embedded in the agros. And now we're gonna take the glue and place it on this little stage here.Set.

So we're gonna just place a little bit of glue and place our brain right on top of this glue. So basically the cerebellum, the pine brain is, is facing the glue with the olfactory bowl or rostral end facing straight up. And so then we're gonna be moving this into the Vibram chamber.

So now I'm transferring the brain into the viome where we will section it. So here we go. We place it in the Vibram and tighten it down such.

Then we will add in the cold bubbling A CSF that I have on ice here. So this also hardens the glue, which is important. And so now our brain will be attached to the plate in the vibrato.

So now we're ready to start sectioning the brain on the vibrato. I'm gonna remove a little bit of this excess agro here. And so we're gonna just make an incision, remove that.

So now we have our brain here. I'm going to put the blade onto the vibrato head here. So that will just be inserted Here and Place the blade onto the vibrato.

And I'm also going to cut this little pipette here just so that it's large enough to pick up the slices once I cut them. So there we have that. And now we're ready to start cutting.

So as I said, I used 300 micron sections for this. So we're gonna wanna move our, our blade towards the brain Moving up the stage. Okay, so first we're just positioning our blade right at, at the before the tissue and the first few cuts will either just hit agro or maybe the very rash areas, but it's really just to position our blade.

We have the, the vibration at around seven, and our speed is at around five. And so we're cutting here to make cortical coronal sections. And we're starting at the very rostral end of the brain, and we're actually barely into the brain right now.

So we're just starting. So our first slice, but it almost has, it's very rostral at this point. So we'll keep my hand sections.

So as the sections come out, I remove them with my foot and place them in bubbling room temperature, A CSF. It's also optional to put ice in the chamber here to keep everything even colder with these embryonic brains. First ice culture.

They seem to survive perfectly well as long as your A CSF is ice cold. But for other experiments, or if your brains are slightly older, you may want to put ice in your chamber just to keep everything extremely cold. So now we have our slices in the bubbling A CSF.

They can stay here for about up to an hour, but we can also just go ahead and put them Into our slice culture system. Okay, so now we're ready to prepare the dishes and the Culture system so we can incubate the slices that we just made. So we're gonna just take a six well plate here.

And we're going to, in each of these wells, we're gonna put about a mill and a half of this slice culture media that we've pre-prepared and, and warmed up to 37 degrees about a mill and a half in each of these dishes. So then I'm gonna take my filters. And so we're actually gonna place the, the brain sections on these filters, but beforehand, these filters go on this media here.

So we're gonna place the filters on top of the media you want to get rid of, get the bubbles at least to the side. You don't have wanna have bubbles under the filter. So these filters allow the slices to soak up the nutrients from the media while remaining exposed to the air so that they, they don't suffocate basically.

So now we have our filters in the dish and we're ready to get our slices and transfer them onto the filters. Now we have our slices here in this dish I've removed most of the A CSF. And so I want to quickly add the slice culture media, because basically you want to transfer them out of the A CSF into the slice culture media.

So I'm just gonna add culture media here to my slices. Try and do it gently so that I don't destroy the slices too much. So now my slices are in the culture media, and I'll just be wanting to transfer them onto these filters, the three filters that I have here.

And so I'm gonna just take my pipet here and, and suck the slice up and I'll put it on the filter. You can put a couple slices on the same filter, or if you want, you can just have one slice. I'm gonna put a couple of slices here on this filter.

And then it's important to remove this excess media here that you transferred. And so here I'm gonna just basically pick up the extra media that I transferred with the slices because you don't want too much media in these dishes or else the slices will be covered with the media and they won't last as long as they tend not to get enough oxygen. And so there, I've removed the extra media and now my slices are just sitting on this dish and they're ready to go in the incubator.

All right, so now these slices are ready to go into the 37 degree incubator. They should be good for five to seven days. And we switch the media about every three days, switching half the media, replacing it with new media.

And so I'll just go ahead and put these cultures into the incubator. Alright, that's it. We're all done.

So we use this Technique in our lab to study various aspects of cortical development, including the progenitor divisions and neuronal migration. This technique can be particularly useful coupled with time-lapse microscopy so you can follow individual cells as they divide and migrate in your slice Culture.