This lab was derived from: Animal Cell Culture - A Guide to Basic Techniques (5th Edition)

Operation method

Scheme 23.13 Isolation and culture of smooth muscle cells

Materials and Instruments

0.25% Trypsin and EDTA mixture
Smooth muscle culture solution
Petri Petri dishes Scalpels and #10 scalpel blades Dissecting scissors Tissue forceps

Move

1. Taking the thoracic aorta from a calf.

2. Immerse the thoracic aorta in Hanks' solution.

3. Place the artery on ice before isolating the cells.

4. Place the artery in a 150 mm × 25 mm Petri dish.

5. Cut the artery along the long axis with dissecting scissors.

6. The inner luminal surface of the artery is lightly scraped with a scalpel blade to remove endothelial cells.

7. Separate the middle membrane of the artery from the inner and outer membranes.

8. The intima is cut into tissue pieces approximately 1 ^cm2 in size.

9. Place the intima-media block into a 60 mm × 15 mm Petri dish with the intima-media side facing down.

10. Allow the block to adhere to the wall for approximately 10 min.

11. Inject 1 ml of SMCM (smooth muscle cell medium (SMCM): DMEM with 20 % FBS, 100 U/ml penicillin and 100 μg/ml streptomycin) directly onto the block.

12. Place the tissue blocks in a humidified incubator and incubate at 37°C and 10% _CO2.

13. Add 5 ml of SMCM on day 2, taking care not to de-attach the tissue blocks.

14. Continue to incubate the blocks for 10 d. Thereafter, the smooth muscle cells migrate out of the blocks and become monolayers.

15. When cell growth is close to forming a monolayer, passaging is performed with a mixture of 0.25 % trypsin and EDTA.

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