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The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.
Cite this Article
Ribeiro, M. P., Relvas, R., Chiquita, S., Correia, I. J. Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC). J. Vis. Exp. (41), e1940, doi:10.3791/1940 (2010).
- Arteries are obtained from UC pieces of 3-7 cm.
- Wharton's jelly that surrounds the arteries was carefully removed by cutting it with scissors.
- A blunt-end needle of an outer diameter of 0.6-1mm was inserted about 7mm from one end of the dissected arteries.
- To remove the blood remaining inside the vessels, the arteries are vertically held and perfused with, approximately 20 to 40 mL, physiological saline solution (PSS), using a 20-mL syringe connected to a needle. If necessary, this washing step was repeated for several times.
- The same procedure was repeated using RPMI 1640 supplemented with 10% of fetal bovine serum (FBS).
- One extremity of blood vessel was closed. A 10-mL syringe was connected to the needle and perfused with 3 mL of collagenase type I (800 unit/mL in Hank's Buffered Salt Solution (HBSS)). Then the other extremityof the vessel was also closed.
- The vessel was incubated with phosphate buffered saline solution (PBS), for 15 min at 37°C with agitation.
- After the ends of the vessel were cut to collect the free SMCs. The arteries were perfuse with approximately 20 to 40 mL of DMEM-F12 supplemented with 5% of FBS and antibiotic to a 50-mL tube.
- The tube was centrifuged at room temperature (250 g, 8 min), and cell pellet was resuspended in 5 mL of DMEM-F12 supplemented with 5% of FBS.
- The cell suspension was transferred to a collagen-coated T-flask and incubated at 37°C, under a 5% CO2 humidified atmosphere.
- After 5 16 h, the non-adherent cells and cellular drebis were removed, 5 mL of culture medium was added and the cells were incubated at 37°C, under a 5% CO2 humidified atmosphere.
- The cell culture media was changed every 2 3 days, until SMCs reached confluence.
- After cells attained confluence they were seeded into two or three 25 cm2 T- flasks.
- Dispose of needles in sharps container.
- Dispose of syringes in big biohazard container.
- Put all tissue into a small biohazard bag. Close the bag and freeze at -20° Celsius until incineration.
- Soak all instruments in virucide for at least 10 min.
- Return unused media to fridge.
- Dispose of gloves in biohazard waste.
The SMC can be used as models for future assays with constrictor drugs, to isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function and to use them in tissue engineering. SMCs and ECs are fundamental for the development of new biomaterials that can be used in the production of semi synthetic blood vessels to be used in humans.
Hospital Amato Lusitano, Castelo Branco Portugal.
Hospital Sousa Martins, Guarda Portugal.
|Fetal bovine serum (FBS)||Biochrom AG||0117T|
|RPMI 1640||GIBCO, by Life Technologies||3103806|
|Antibiotic||10 μL/ 1 mL of medium|
|Needle||Diameter of 0.6-1mm|
|Syringe||10 - 20 mL|
|Physiological Saline Solution (PSS)||20-40 mL per artery|
|Collagenase type I||Sigma-Aldrich||047K1052||3 mL per artery|
|Hank’s Buffered Salt Solution(HBSS)||Diluted collagenase|
|Phosphate buffered saline solution (PBS)||Warmed to 37°C|
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- Jaffe, E. A., Nachman, R. L., Becker, C. G., Minick, C. R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 52, (11), 2745-2756 (1973).
- Cairrão, E., Santos-Silva, A., Alvarez, E., Correia, I., Verde, I. Isolation and culture of human umbilical artery smooth muscle cells expressing functional calcium channels. In Vitro Cellular & Developmental Biology-Animal. 45, (3), 175-184 (2009).
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