Isolation of smooth muscle cells from bovine aorta
Procedure: Adapted from E. Edelman’s laboratory, which was adapted from A. Gotlieb’s laboratory, U. of Toronto, Canada, as described by E. Koo.
Source of aortas: Slaughter house
A Arena & Sons
159 Ash Street, Hopkinton, MA 01748
Phone: 508-435-3673 Fax: 508-435-3673
Contact person = John (Research 87) 508-481-8420
Delivery charge = $ 40.00
Bovine Aorta = $ 5.00/each
Bovine Heart = $ 7.00/each
Every Wednesday. (1st time, call 1 week in advance)
Materials:
Sterile 10 in. hemostats, one per aorta
Sterile scissors, 2
Sterile forceps, 4
Sterile coverslips
one liter beakers, 2 (sterility optional)
250 mL beakers, 2 (sterility optional)
sterile 50 mL flask for collagenase
sterile 35 mm, 100 mm and large sized 150 mm petri dishes
sterile 6 well plates
Type I collagenase (Sigma C-9891)
Dental wax (NeoWax 0.05” Baseplate wax, Dentsply Intern., Inc., York PA 17405, Polysciences Inc., cat # 00403)
26G (or higher) 0.5 in. sterile syringe needles
sterile pre-packaged surgical stainless steel blades
Solutions
Sterile PBS-PS, 2-3 liters, with 2% PenStrep. Note that the conc is 2 times higher than in cell culture.
DMEM-10% CS. 1% PS should be added as usual.
Collagenase solution: Dissolve 15 mg collagenase in 20 mL PBS-PS (= 0.75 mg/mL). Made fresh before use, and filter through 0.2 um sterile filter into a Al-foil lined sterile 50 mL flask (light sensitive). 25 mL for every 6 aortas.
Procedure (asceptically in cell culture hood)
Explant method
1.Fill the two large and two small beakers half way with PBS-PS
2.Once aortas arrive, store them in a large PBS-PS filled beaker that is sitting on ice.
3.Cut off bottom 1/3 of aorta and place on an opened large petri dish.
4.Cut off all of the adventitia and remove all blood clots using sterile scissors and forceps.
5.Locate the area with the fewest branches, which is usually near the aortic arch, and cut off all other sections of the aorta, keeping the section that had the greatest length between the branches, but not including the branches.
6.Place section in large PBS-PS filled beaker, and place scissors and forceps in PBS-PS filled small beaker to soak when not using.
7.Repeat steps 3-6 for all aortas.
8.in large petri dish place at least 2 sheets of dental wax and rinse with 70% ethanol, aspirate off, and rinse with PBS-PS, aspirate off.
9.Longitudinally cut open section and lay it down onto the wax sheet with endothelial side / inside of vessel up, and pin down the four corners with syringe needles.
10.Rinse with PBS-PS (always keep aorta wet).
11.With sterile blade, scrape surface (with a light touch) to remove endothelial cell layer, rinse surface with PBS-PS.
12.With another blade score the surface about 1/2 way down into the vessel. Cut little squares in a square, checkerboard fashion, each square is about 5 x 5 mm.
13.With sterile forceps peel off the scored squares and place in a 35 mm petri dish, 3-4 squares per dish.
14.Place a sterile coverslip with sterile forceps over the squares.
15.With sterile transfer pipet drop 10% CS-DMEM next to the coverslip, so the liquid by capillary action fills the space between the cover slip and dish while surrounding the squares, incubate at 37°C in cell culture incubator. SMC will migrate out from the squares as they grow within 2-3 weeks, these are P0 cells.
