Name: Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC) (Video) | JoVE Journal
Uploaded: 2010-07-03T17:00:00.000+00:00
Duration: 8 min 6 s
Description: Learn to isolate human umbilical arterial smooth muscle cells using enzymatic collagenase treatment. This method maintains high cell viability for vascular biology and tissue engineering research applications.

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5 minutes

Hospital Amato Lusitano, Castelo Branco Portugal. Hospital Sousa Martins, Guarda Portugal.

<ol>
	<li>Arteries are obtained from UC pieces of 3-7 cm.</li>
	<li>Wharton's jelly that surrounds the arteries was carefully removed by cutting it with scissors.</li>
	<li>A blunt-end needle of an outer diameter of 0.6-1mm was inserted about 7mm from one end of the dissected arteries.</li>
	<li>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.</li>
	<li>The same procedure was repeated using RPMI 1640 supplemented with 10% of fetal bovine serum (FBS).</li>
	<li>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.</li>
	<li>The vessel was incubated with phosphate buffered saline solution (PBS), for 15 min at 37&deg;C with agitation.</li>
	<li>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.</li>
	<li>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.</li>
	<li>The cell suspension was transferred to a collagen-coated T-flask and incubated at 37&deg;C, under a 5% CO2 humidified atmosphere.</li>
	<li>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&deg;C, under a 5% CO2 humidified atmosphere.</li>
	<li>The cell culture media was changed every 2 3 days, until SMCs reached confluence.</li>
	<li>After cells attained confluence they were seeded into two or three 25 cm2 T- flasks.</li>
</ol>
Cleanup
<ol>
	<li>Dispose of needles in sharps container.</li>
	<li>Dispose of syringes in big biohazard container.</li>
	<li>Put all tissue into a small biohazard bag. Close the bag and freeze at -20&deg; Celsius until incineration.</li>
	<li>Soak all instruments in virucide for at least 10 min.</li>
	<li>Return unused media to fridge.</li>
	<li>Dispose of gloves in biohazard waste.</li>
</ol>

<ol><li>Van Rijen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gap+junctions+in+human+umbilical+cord+endothelial+cells+contain+multiple+connexins%5BTitle%5D)+AND+%22The+American+journal+of+physiology%22%5BJournal%5D)">Gap junctions in human umbilical cord endothelial cells contain multiple connexins.</a> The American journal of physiology. 272 (1 PT 1), C117-C117 (1997).</li>
<li>Martín de Llano, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Procedure+to+consistently+obtain+endothelial+and+smooth+muscle+cell+cultures+from+umbilical+cord+vessels%5BTitle%5D)+AND+%22Translational+Research%22%5BJournal%5D)">Procedure to consistently obtain endothelial and smooth muscle cell cultures from umbilical cord vessels.</a> Translational Research. 149 (1), 1-9 (2007).</li>
<li>Jaffe, E. A., Nachman, R. L., Becker, C. G., Minick, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culture+of+human+endothelial+cells+derived+from+umbilical+veins.+Identification+by+morphologic+and+immunologic+criteria%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.</a> J Clin Invest. 52 (11), 2745-2756 (1973).</li>
<li>Cairrão, E., Santos-Silva, A., Alvarez, E., Correia, I., Verde, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+human+umbilical+artery+smooth+muscle+cells+expressing+functional+calcium+channels%5BTitle%5D)+AND+%22In+Vitro+Cellular+%26+Developmental+Biology-Animal%22%5BJournal%5D)">Isolation and culture of human umbilical artery smooth muscle cells expressing functional calcium channels.</a> In Vitro Cellular & Developmental Biology-Animal. 45 (3), 175-184 (2009).</li>
</ol>

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.

<table><tbody><tr><td>DMEM-F12</td><td>Sigma-Aldrich</td><td>095K8311</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Biochrom AG</td><td>0117T</td><td></td></tr><tr><td>RPMI 1640</td><td>GIBCO, by Life Technologies</td><td>3103806</td><td></td></tr><tr><td>Collagen</td><td>Sigma-Aldrich</td><td>067K2302</td><td></td></tr><tr><td>Antibiotic</td><td></td><td></td><td>10 &amp;#x03BC;L/ 1 mL of medium</td></tr><tr><td>Scissors</td><td></td><td></td><td>Sterilized</td></tr><tr><td>Needle</td><td></td><td></td><td>Diameter of 0.6-1mm</td></tr><tr><td>Syringe</td><td></td><td></td><td>10 - 20 mL</td></tr><tr><td>Physiological Saline Solution (PSS)</td><td></td><td></td><td>20-40 mL per artery</td></tr><tr><td>Collagenase type I</td><td>Sigma-Aldrich</td><td>047K1052</td><td>3 mL per artery</td></tr><tr><td>Hank&amp;rsquo;s Buffered Salt Solution(HBSS)</td><td></td><td></td><td>Diluted collagenase </td></tr><tr><td>Phosphate buffered saline solution (PBS)</td><td></td><td></td><td>Warmed to 37&amp;#x00B0;C</td></tr></tbody></table>

isolation of human umbilical arterial smooth muscle cells (huasmc)

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

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.

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

To Isolate human umbilical arterial smooth MUS cells. I used the following solutions. PSS, physiologic SELINE solution FBS fatal B, serum inactivate A B, antibiotic and anti mycotic rrp, M-I-P-B-S phosphate buffer selling solution, DM A F 12 collagenase and HBSS ANGs balanced selling solution to dis solve the collagenase Type one.In terms of manipulation of the samples, all process is done in laminar airflow cabin. The amical cart is cutting into small pieces between three and seven centimeters. Next, the pieces are put into a bigger container with PSS antibiotic, anti mycotic, and anti proteases to avoid deterioration or a loss of cell viability.The isolation is done at the temperature of four cells degrees. The researcher is now indicating the vein and arteries that are present in the umbilical cord. Waterton jelly that surrounds the arteries is carefully removed by cutting.We scissors to Remove double of the remaining inside the vessels. The arteries are vertically held and profused with PSS using a serene connected to a TEFL needle if necessary, this washing step can be repeated for several times. The same processor is repeated using RPMI supplemented with 10%of FPS.Then the end of arteries are closed. Next, the arteries are profused with collagenase type one. Then the upper, upper standard arteries are also closed.The arteries Are incubated with PBS for 15 minutes at the temperature of 37 CEL degrees with some vegetation After incubation, the ends are cut to collect the free smooth muscle cells. The arteries Are profused with demand F 12 supplemented with 5%of FPS antibiotic and anti Mycotic. The medium used to peruse the arteries is Collect to a tube.Tube is centrifuged at the room temperature along eight minutes at to hundred 50 gs. The centrifugation can be done in different rotations depending on the type of cells. If the speed is excessive cell damage can occur.The S natant is aspirated with the half of a past PPE connected to a vacuum pump. The pallet is reus suspended in five milliliters of D DM F 12, supplemented with 5%of FPS so that the cells can be placed on a T flask where they will grow. Collagen Was added to the bottom of the T flask in order to allow cell growth.Collagen is used to cover the bottom of T flask because one of the great difficulties we have in this method is to be able that cells adhere so it is coated with extracellular matrix proteins. Cells Are observed with the help of the inverter, optical microscope to see before incubation. The cells are incubated at the temperature of 37 cells degrees under a OMI five atmosphere with 5%of carbon dioxide After five to 16 hours.The non-ad cells and cellular diaries are removed with either of a pasture P PET connected to a vacuum Pump. Fresh culture medium is added to the T flask. The Cells Are incubated at a temperature of 37 CEL degrees and modified atmosphere with 5%of carbon dioxide.Again, the cell culture medium is changed every two to three days until smooth muscle cells reach confluence. After cells detain confluence, they are Seeded into two or three cell culture flask.

isolation-of-human-umbilical-arterial-smooth-muscle-cells-huasmc

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Learn to isolate human umbilical arterial smooth muscle cells using enzymatic collagenase treatment. This method maintains high cell viability for vascular biology and tissue engineering research applications.

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

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