Isolation and Culturing of Cells from Mouse Embryo Dorsolateral Telencephalons

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Dorsolateral Telencephalon Microdissection

1. Prepare the dissection medium (100 mL): 98.5 mL Hank's Balanced Salt Solution (HBSS), 0.5 mL 5 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 1 mL penicillin/streptomycin (10,000 units/mL and 10,000 µg/mL).
2. Filter-sterilize the dissection medium.
3. Remove embryonic day 14 (E14) embryos from a pregnant mouse C57/Bl6 (Mus musculus) under anesthesia with isoflurane.
   NOTE: Consider E0 as the day of vaginal plug detection. Dissection should be completed no later than 1 h after removal from the pregnant mouse. This protocol also applies to embryos E11-E17.
4. Remove the embryos by hysterectomy under sterile conditions.
5. Transfer the embryos to a Petri dish with a cold dissection medium (4 °C).
6. Remove the brains from the skull by cutting along the longitudinal fissure. The number of embryos usually varies from five to ten. Each E14 brain yields approximately 2 x 10⁶ cells, including progenitors and postmitotic neurons (1:1 ratio)
   NOTE: Use the stereomicroscope for the following steps.
7. Remove the meninges using dissection forceps. Split the telencephalon in the middle to separate the hemispheres. To isolate the dorsolateral telencephalon, cut along the dorsomedial curve and pallial-subpallial boundary using dissection forceps. Transfer the dorsolateral telencephalon to a 2 mL tube with a cold dissection medium (4 °C) until all brains are dissected.

2. Cell Dissociation and Plating

1. Prepare the proliferation medium (50 mL of DMEM+10% FCS): 44 mL Dulbecco Modified Eagle's Medium (DMEM), 5mL Fetal Calf Serum (FCS), 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 µg/mL). Filter-sterilize the proliferation medium.
2. Prepare differentiation medium (50 mL of DMEM + 2% B27): 48 mL DMEM, 1 mL B27, 0.5 mL 40% glucose, 0.5 mL penicillin/streptomycin (10,000 units/mL and 10,000 µg/mL). Filter-sterilize the differentiation medium.
3. After micro-dissection of the dorsolateral telencephalon from E14 mice, centrifuge the tube with the collected tissue and cold dissection medium (for 5 min at 4 °C, 340 x g) to precipitate the tissue.
4. Remove the supernatant with a pipette and add 1 mL of pre-warmed (37 °C) Trypsin-Ethylenediaminetetraacetic acid (EDTA) (0.05%) for chemical digestion. Incubate for 15 min at 37 °C. Add 2 mL of proliferation medium to stop trypsin activity.
5. Polish the tip of a glass Pasteur pipette over a gas burner or Bunsen burner for a few seconds to slightly narrow the aperture. Wash the pipette by aspirating FCS to coat the tip. To avoid bubbles, dissociate the cells mechanically with a fire-polished and FCS-coated Pasteur pipette.
6. Centrifuge the cells (for 5 min at 4 °C, 340 x g). Remove the supernatant with a pipette. Add 1 mL of proliferation medium and resuspend the cells using a pipette. Repeat this step once.
7. Prepare a 1:1 dilution of the cell suspension using a 0.4% Trypan Blue solution. The non-viable cells will be blue. Using a Neubauer chamber, count the number of viable cells (unstained).
8. Dilute the cells in a proliferation medium to get 10⁶ cells/mL. Add 500 µL of the cell suspension to each well of a 24-well tissue culture plate (approximately 2.5 × 10⁵ cells/cm²). Incubate the cells at 37 °C and 5% CO².
   NOTE: Do not use glass coverslips, as they can move during the experiment and change the field of observation. Alternatively, use glass bottom tissue culture plates.
   NOTE: If you are not using cell-culture-treated multidishes, it is important to pre-treat your plates with Poly-D-Lysin (50 µg/ml) for 2 h at 37 °C. PDL-treated plates can be stored at 4 °C after washing with distilled water.