Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

1. Preparation of Reagents and Solutions

1. Coating of 35 mm culture dish: Collagen Type 1 solution is supplied as an aqueous solution in 20 mM acetic acid (~100 mg protein/vial). Dilute an appropriate volume of collagen solution to a working concentration of 0.01% using sterile tissue culture grade water. Coat dishes with 1 ml of collagen solution for 3-4 hr at room temperature (RT) or 37 °C, or overnight at 2-8 °C. Remove excess solution from the coated dish and allow it to dry overnight. Rinse the dish with tissue culture grade water or PBS before adding media. Coated dishes can be stored at 4 °C for one month.
2. Prepare complete Dulbecco's Modified Eagle Medium (DMEM). Supplement the DMEM (500 ml) with 10% FBS and antibiotic-antimycotic solution (1x concentration; 1 ml/100 ml). Complete DMEM can be stored at 4 °C for two months.
3. Prepare an aliquot (50 ml) of DMEM with DNase I (0.001 mg/ml), (referred to as DMEM-DNase I).
4. Prepare another aliquot (10 ml) of DMEM with collagenase and dispase (1 mg/ml) (referred to as DMEM-collagenase/dispase).
5. Prepare Endothelial Cell Culture Medium (ECCM, 500 ml), supplemented with EGF (5 μg), ECGS (100 mg) and 5 μl of antibiotic-antimycotic solution. ECCM can be stored at 4 °C for two months.
6. Prewarm all media before use.
7. Prepare Purification buffer: PBS, pH 7.2 with 0.5% BSA and 2 mM EDTA. Keep buffer cold (2-8 °C).

2. Removal of Embryos and Dissection of Telencephalon

All experiments using laboratory animals are approved by the animal care and use committees of McLean Hospital and conform to NIH guidelines for the care and use of laboratory animals.

1. Use timed pregnant CD1 mice (embryonic day 15). The day of vaginal plug discovery is considered embryonic day 0 (E0). CD1 dams generally produce large litters, so 10-12 embryos are expected from a pregnant dam.
2. For hysterotomy, anesthetize pregnant mice with an intraperitoneal injection of Ketamine (100 mg/kg)/Xylazine (5 mg/kg). For mice approximately 20-30 g, deliver 0.3 ml of a 10 ml/kg Ketamine/Xylazine anesthetic solution and ensure that pain and distress is minimal during intraperitoneal injections of the anesthetic. Use hand restraint for the injections.
3. Check deep anesthesia by toe pinch. Remove embryos from deeply anesthetized mice. Decapitate each embryo immediately upon removal from the mother. Place the embryonic heads in sterile Petri dishes in ice cold PBS.
4. Follow hysterectomy by immediate euthanasia with anesthetic overdose (130 mg/kg pentobarbital, i.p.), a method for euthanasia, which is consistent with the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition.
5. Under a stereo microscope, remove the brain from the head with fine microtip scissors and fine forceps. Note: A high quality dissection is essential to successfully remove the pial membrane from the embryonic brain. This is achieved by practice. Fine forceps and microtip scissors are also key tools for getting a good dissection.
6. Use the forceps to get a firm grip on the brain and the microtip scissors to peel away the pial membrane. Remove the mesencephalon and metencephalon and isolate the telencephalon. Transfer pial-free telencephalon from all embryos to a 35 mm culture dish with 2 ml PBS in ice.

3. Cell Isolation

1. Mince the telencephalon into 1-2 mm fragments with a scalpel blade and collect tissue in a 15 ml Falcon tube containing 2 ml of complete DMEM.
2. Dissociate tissue gently but thoroughly with a 1 ml pipette until clumps disappear and a milky suspension is achieved.
3. Centrifuge dissociated cells at 800-1,000 x g for 5 min at RT.
4. Carefully remove the supernatant and resuspend pellet in DMEM-DNase I. Gently dissociate the cells again using 1 ml pipette and centrifuge at 800-1,000 x g for 5 min at RT.
5. Resuspend the pellet in warm complete DMEM. Filter cells through a sterile 70 μm Nylon mesh. Collect filtered cells and keep in ice.
6. Collect the cell-portions retained on the mesh and digest further with DMEM-collagenase/dispase (2 ml) for 1-2 min at RT. Wash cells 3x in DMEM-DNase I through centrifugation at 800-1,000 x g for 5 min at RT. Pool these cells with the filtered cells collected in step 3.5. Wash total cells once with complete DMEM. Proceed to determine cell number and magnetic labeling steps. Note: It is important to obtain a single-cell suspension before magnetic labeling.

4. Magnetic Labeling

1. This protocol shows the magnetic labeling of 10⁷ cells. For cell numbers lower than 10⁷, use the same reagent volume and for cell numbers greater than 10⁷, scale up all reagent volumes and total volumes accordingly (e.g. for 2 x 10⁷ total cells, use twice the volume of all indicated reagents). Keep all the reagents and cells in ice.
2. Determine cell number with a hemocytometer.
3. Centrifuge cell suspension at 300 x g for 10 min at RT. Aspirate supernatant completely.
4. Add 90 μl/10⁷ cells of purification buffer to the cell pellet followed by 10 μl of CD31 MicroBeads. The Microbeads provided by manufacturer are conjugated to monoclonal anti-mouse CD31 antibody.
5. Mix well and incubate for 15 min in the refrigerator. Note: Higher temperatures and/or longer incubation time during magnetic labeling may result in nonspecific binding.
6. Wash cells by adding 1-2 ml/10⁷ cells of purification buffer and centrifuge at 300 x g for 10 min. Remove supernatant and resuspend cells in 500 μl of purification buffer. Proceed to magnetic separation or purification step.

5. Purification

1. Place MS column in the magnetic field of MACS Separator.
2. Rinse column with 500 μl of purification buffer.
3. Apply cell suspension onto the column. Load appropriate number of cells to MS column as higher cell number may clog the column. Caution: Each MS column can accommodate a maximum number of 2 x 10⁷ cells, for a higher cell number, use more columns.
4. Collect and discard flow-through containing unlabeled cells.
5. Wash column with 500 μl of purification buffer three times. During wash steps, make sure that the column reservoir is empty before adding subsequent purification buffer aliquots.
6. Remove column from the MACS separator and place it on a 15 ml Falcon tube.
7. A plunger is supplied along with the MS column. Pipette 1 ml of purification buffer onto the MS column and immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column.
8. Plate cells on a 35 mm culture dish precoated with collagen Type 1 and grow cells in ECCM in an incubator set to 37 °C with 95% O₂ and 5% CO₂.
9. Change the medium every four days. Use PVECs for experiments or subculture after 10-12 days when the dish is confluent.

6. Subculture of PV ECS

1. Remove the ECCM and rinse the monolayer of PVECS with 1-2 ml of sterile PBS.
2. Gently add 1.5 ml of 0.25% Trypsin-EDTA solution to cover the cell monolayer.
3. Return the dish to the incubator. Within 5-7 min, cells will detach from the surface of the dish.
4. After the cells have detached, immediately add an equal volume of trypsin inhibitor and triturate several times.
5. Transfer the cells to a sterile 15 ml Falcon tube. Centrifuge the tube at 500 x g for 5 min.
6. Remove the supernatant and resuspend the pellet in 1 ml of prewarmed ECCM.
7. Seed the cells for expanding the culture, imaging or other assays.