So this is the first step in the neural precursor cell isolation, dissociation, and culturing. First I'm going to coat a 24 well plate. It's not treated for tissue culture.
I'm gonna coat it with a laminin so the the cells will stick. So first I'm gonna coat it with a mixture of water and poly de lysine. Gonna add about 450 microliters per well.
So I'm just gonna, So I've coated half the plate, these two rows here with the poly D lysine solution. And I'm gonna incubate for five minutes at room temperature just in the hood here. So it's been incubating for five minutes.
I'm just gonna remove the PDL solution with a vacuum here. House vacuum action. Now I'm gonna rinse the wells with water again, about 450 microliters.
Really no incubation time for the water rinse. I'm just gonna go ahead and aspirate the, the water. So now I'm gonna add the laminate solution.
Laminate's dissolved into EM EM media at two micrograms per mil. So I'm gonna add 450 microliters of the laminate solution to each, well, right now I'm coating plastic. You could also have coated cover slips in these wells.
I am gonna incubate the cells at 37 degrees in a tissue culture incubator for at least four hours. So I have two Fine forceps that are bent at a 45 degree angle. I have some fine scissors also bent at a 45 degree angle.
Some blunted forceps just for the initial dissection of the uterus and some large scissors for cutting up in the female. All right, this Is A CD one, normal female. She's pregnant with embryonic day 12 and a half embryos.
I've already sacrificed her using CO2 asphyxiation and cervical dislocation. And now I'm going to remove the uterus and subsequently the embryos. So first I need to disinfect the area for abdomen with 70%ethanol.
My large forceps I grab in the middle of her abdomen and towards the lower half, make one cut, put my instruments down and I simply tear the skin, exposing the abdomen again, grabbing in the same place. Make another cut in the middle and cut laterally opening up the female. Now I'm exposing the embryos.
Here's her uterus, swollen with embryos. So I make the first cut just below the ovaries, trim the fat along the way and pulling upward. I lay that side down and I only grab in between the embryos.
Obviously grabbing on top of the embryos might damage the tissue. And that's obvious. They look like a string of grapes.
Grab in between the grapes, again, cutting above the ovary on the other side and trim the fat. Final cut will be made here in the middle. Again, I'm trimming the fat.
There's the uterus with Embryos. So my dissection media is PBS and 0.6%glucose and penicillin streptomycin. I'm first just gonna rinse the embryos in the dissection media just to get the blood off.
So my dissection is clean. So I'm gonna cut along the length of the uterus opposite the side of the fat to expose the embryos. Where's The fat?
It's on the other side because you can't see it. So there's an exposed embryo here. I can show you what this looks like in the scope.
So here's what it looks like under the scope. I'm gonna cut along this side of the uterus. Opposite side, the fat.
See the fat here on the, I can see fat real. So I'm gonna just cut along, top the length all the way down. And you can see that the embryos will pop out as I extend down the uterine down the uterus.
Little more north. Oh, there's one.Gorgeous. So you're cutting along off side opposite the fat embryos just popping out.
Yeah, they all are. A couple here that you have to go back sometimes and open something up if you missed it. So that's the amnion or is that the corion and the, this is the corion.
The amnion is, is inside, is closely associated to the embryo. See, you can't really see the amnion from here. Once I get the emyo embryo out, you'll, I can tell that these are smaller and not gonna use those.
But, so there's all the exposed embryos. So now I have the embryos exposed here. I'm going to peel the layers away from the embryo and then transfer the embryo to a clean dish for the fine dissection.
So there's the first layer. Once I get that off, I usually, I look for the umbilical cord. That's a pretty tough tissue.
And I can actually lift the animal with the umbilical cord. That's it right there. Can you point to it?
Yeah, right there. Just see the, yeah, the red vessel, the fanny thing.Yeah. So that's the umbilical cord and it attaches to the abdomen and I usually just reach in and Grab it.
There we go. So now I'm gonna remove the skin from The embryo's head. It's exposing the cerebral cortex, which is where I'll harvest the cells from.
So with my left hand, I'm going to just puncture through the, the back of the embryo to hold it just like that. I'm holding it against the bottom of the plate. I'm gonna go in with my right hand, with my forceps under the eye, up under the skin, piercing it and then tearing it off.
So now I have an edge here that I can grab.Okay. And then with my other four steps, grab the opposing edge and I'll peel off the skin and usually the underlying mesenchyme comes with it. So now you can see the right hemisphere exposed one North.
I'll flip the embryo and try to find another edge to grab. So there's another edge of skin. Flip it over, continue to peel.
There we go. Exposing the cerebral cortex. So now I'm going to simply pinch the head off, which will allow me to get into a better position for removing pieces of the cerebral cortex.
So I just pinch with my forceps, rub with my other forceps underneath, cutting it right off. And there's a dorsal view. So here you can see a dorsal view of the forebrain, the two hemispheres of the cerebral cortex, the dye cephalon, and then around here the midbrain.
Here's what be taking the pieces for the neural stem cell association. So now I'm going to grab the embryo with my left forceps to hold it down like this. Gimme a good position and angle to cut out pieces of the cortex.
So now with my fine scissors, go in and I just snip the anterior cerebral cortex like that. Gimme a hole so I can make my lateral cut and I make a cut close to the dorsal midline, but I avoid midline cells trying to isolate the neuro precursors or neuro precursors that are out in the cortex. So this is a, a piece from the cerebral cortex.
And as you can see, there is a thickening here on the right side. Now that is actually the beginning of the lateral GIC eminence, which in our prep we do not want, since we are interested in the precursors from the dorsal cortex. So I'm just going to go ahead and cut that off.
I'm leaving you with just cells from the dorsal cortex. So now I'm gonna move on to the other side. Same method.
It's a little bit more awkward because I'm right-handed, but again, make the anterior cut, put your scissors to the hole. Cut along the dorsal side, lateral side. This time I'll try to avoid the gang manic evidence.
So here are the two pieces from the hemispheres of the dorsal cortex. So now I'm gonna use a cut P 1000 pipette tip to transfer the cortices to a 15 mil conical tube that contains dissecting media where they'll sit as I dissect the rest of the litter. So I'll just suck one piece up now.
So now I'm transferring the cortices from the dish into the 15 mil conical that contains dissecting media where they will sit As I dissect the rest of the litter. So I've dissected the entire litter And now I'm going to aspirate off the excess dissecting media, leaving a small amount of liquid and the courtes. So now I'm gonna add about 500 microliters of Trix and EDTA placing the tube into a 37 degree water bath for 20 minutes.
So after the 20 minute incubation, I add 500 microliters of soybean trypan inhibitor. Now I am going to mechanically iterate or dissociate the cells using a flame polished pasture pipette with a very small bore. I don't want to introduce very many air bubbles, but if you do, that's all right too.
You go up and down about five to 10 times and the cells should disperse into a cloud. Now I will spin the cells at 1000 RPMs, 25 degrees for one minute. After spinning the cells, I will remove the excess liquid by aspiration, leaving a a small pellet, which is barely visible.
It looks like a small SA smear at the bottom of this conical tube. I believe just a small amount of liquid, being very careful not to aspirate the pellet. Obviously now I'm adding wash solution to the pellet.
Okay, another spin at a thousand RPMs, 25 degrees for one minute. Again, I'm going to aspirate off the excess liquid leaving just the cell pellet and a very small amount of liquid after aspiration. I'm going to add my desired growth media with growth factors.
Obviously specific to the culture experiment, the volume I'm going to add depends on how many embryos I dissected and at what age. So typically for an entire litter of E 12 and a half embryos, you can add up to about 700 microliters. For this, I'm going to add about 300 microliters to concentrate my cells a little bit more To taken my laminate coated Plate out of the incubator.
It's been at least four hours. And now I'm going to aspirate off the laminate media. And now I'm going to do a rinse with my growth media.
I'm rinsing with about 300 microliters and I will aspirate and add 400 microliters of the growth media. So I've counted my cells using a Hema cytometer and I'm going to plate about 100, 000 cells per well. In this 24 well plate.
Each experiment requires a different number of cells per well. For my particular assay, 100, 000 has been working fine. I'll put the cells in the tissue culture incubator, 37 degrees 5%CO2, and I let them culture for about a day.
They'll adhere to the laminate and spread out after 24 hours. I will add my desire to growth factors For my particular assay.
