小鼠海马神经元培养

Hippocampal Neuron Cultures (and mixed cortical cultures)
Submitted by: Jean Siao, Ph.D.

Begin by timing the pregnant mouse at E17-E19 days of gestation. Have ready the following:
1.  Cold Hank’s balanced salt solution
2.  1x trypsin (0.25% in HBSS)
3.  1x soybean trypsin inhibitor (1 mg/ml)--- SBTI
4.  Neurobasal medium with:
a.  B27 supplement (2 ml per 100 ml medium)
b.  L-glutamine (0.5 mM)
c.  L-glutamate (25 M)
d.  Gentamicin (10 g/L)
This is called “G3” medium.
5.  Poly-L-lysine-coated dishes, flasks, coverslips, etc., that you want to use.
6.  For mixed cortical cultures:   DMEM + 10% FBS + 40 ug/ml gentamicin

On the day of dissection and plating, place the trypsin, SBTI, and G3 medium in the 37oC incubator to equilibrate (temperature and CO2). Uncover the tissue culture plates you want under UV light to disinfect and dry.

1.  At, place the instruments in ethanol （EtOH） and some HBSS in two petri dishes on ice. Try to bury them without contaminating with ice. Put a 15 ml tube on ice too. Ethanol down the microscope.
2.  Sacrifice the pregnant mouse by cervical dislocation or another acceptable method. Open the abdomen and remove the two horns of the uterus with ethanol-sterilised scissors into a dish of HBSS. Take out the pups which are still in their placental sacs, and open the sacs without hurting the pups’ heads, which are very soft. Remove the umbilical cord. With both fetuses and pups, use sterilised scissors to cut off their heads.
3.  Place the heads on the cover of the HBSS dish and ethanol them well.
4.  Use the blunt forceps to immobilize the heads to the dish through the eyes. (Don’t squeeze or push the brain!!) Use finer forceps to peel away the scalp and then the skull. Be careful with the fetuses because their tissues are very very soft. Make sure to get rid of all of the soft skull, else you’ll destroy the brain when you try to remove it.
5.  Using the pointed end of the spatula, separate the brain stem from the spine and place the brain into the other dish of cold HBSS. Make sure to keep that dish on ice. Continue with the rest of the heads.
6.  Put the brains on the stage of the microscope and adjust light or focus so you’re confortable with the dissection. Using the curved forceps (to immobilise the cortex on the dish) and the dissecting scalpel, take off both cortices following the circle of Willis (red vessels). Try to use a cutting motion and go directly up and down the circle. Put the rest of the brain away for disposal. Continue with the rest of the brains.
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7.  Remove the meninges from the cortices with fine forceps. Use one pair to stabilise the cortex, and the other to gently lift the meninges. If you’re lucky it’ll come away in a sheet starting at the olfactory bulb:

8.  Turn the cortex around and finish removing the meninges. The hippocampus is connected at the convex side to the cortex, and at the concave side to more meninges. Get rid of all of the meninges using fine forceps.
9.  Use fine forceps to “cut” the hippocampus away from the cortex at the convex side. In the end you will have a half-circle ring.
10.  Put the hippocampi away to one side, making sure the meninges don’t contaminate them. You might prefer to have a dish of clean HBSS on ice just for this.
11.  If you want to take the cortices for mixed cortical cultures, save the remainder of the cortex and put into another 15 ml tube that has ice-cold HBSS on ice. See below (step 19) for mixed cortical cultures.
12.  At the tissue culture hood, make sure that the hippocampi have settled to the bottom of the tube and then aspirate off the HBSS. Add 1ml of warmed trypsin per 3 brains (6 hippocampi) and vortex the tube. Put into the incubator for 10-15 minutes.
13.  Aspirate off the trypsin and add same volume of warmed SBTI to hippocampi. Vortex well and leave at room temperature（RT）for a couple of minutes.
14.  Aspirate off the SBTI and add ~2 ml of warmed G3 medium to hippocampi.
15.  Use a smoothly-fire-polished pipette to triturate. When you see no chunks of tissue (after about 15 strokes you should see very few; you can do maybe another 5 strokes but more than that you’ll need to strain through a strainer―use the 70um ones behind you), stop and add enough medium to the tube to plate. This means that for the first couple of times you’ll need to count cells before plating, to get in your head the approximate number of cells you can dissect. I find it helpful to dilute the cells to the desired concentration and then put 100ul per 12mm coverslip―it saves on medium and the cells are congregated on the coverslip.
16.  Leave the plate in the incubator. After 3-5 hours of incubation, you have to change the medium. Tilt the dish so that the old medium runs off to the side, and aspirate that off. Careful not to disturb the settling cells. Add the full amount of medium per well and return to incubator.
17.  After 3-5 days in culture the medium must be half-changed to G2 medium. This is the same as G3, minus the glutamate. To half-change, just aspirate off carefully about half of the old medium and then replace that volume with G2.
18.  Half-change the medium every 3-5 days after that with G2 until you’re ready to use them.

19.  Aspirate the HBSS out of the tube containing the cortices. Add 1ml per 3-4 brains of warm trypsin and vortex. Incubate at 37oC for 10 min.
20.  Stop the trypsinization by aspirating off the trypsin and adding equal amount of warm medium (DMEM+FBS). Vortex. Incubate at RT for a couple of minutes.
21.  Aspirate off the medium and add 2-3 ml warm medium to the cortices. Using first a plain pasteur pipette, and then a fire-polished pipette, triturate until all large chunks of tissue is broken up. This should take about 20-35 strokes.
22.  Filter through a 70um mesh. Wash the tube that you triturated in with 1 ml medium and add that to the mesh.
23.  If you want, count cells. I usually plate about 4 brains per T-125 flask that was precoated with PLL.
24.  Change the medium in a couple of days to fresh DMEM+FBS+gentamicin. After that change medium about every 5-7 days. The culture should be ready in about 10 days.