Isolation and culture of human umbilical artery smooth muscle cells expressing functional calcium channels.
The human umbilical cord is a biological sample that can be easily obtained just after birth. A methodology was developed to perform cultures of human umbilical artery smooth muscle cells (HUASMC) expressing contractile proteins and functional ionic channels. To avoid fibroblast and endothelial cell contamination, we mechanically separated the tunica media, which only contains HUASMC and matrix proteins. To isolate the cells, collagenase V and elastase were used as hydrolyzing enzymes. The isolated cells were plated in collagen-coated dishes to obtain cultures of HUASMC. The cells obtained after different passages (1 to 6) exhibit the characteristic vascular smooth cell morphology and express smooth muscle alpha-2 actin, myosin heavy chain SM1, and alpha subunits of L- and T-type calcium channels (Cav 1.2, Cav 1.2, and Cav 3.2). Electrophysiology recordings for L- and T-type calcium channels were made, indicating that these channels are functional in the cultured cells. In conclusion, the procedure developed allows obtaining cultures of HUASMC expressing contractile proteins and also functional ionic channels. These cells could be used to study cellular and molecular aspects about the regulation of the vascular function.
Cairrão
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CICS - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506, Covilhã, Portugal.
Santos-Silva
A J
AJ
Alvarez
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E
Correia
I
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Verde
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I
D000199:Actins / Q000235:genetics / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D055724:Electrophysiological Phenomena; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D006801:Humans; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000378:metabolism*; D009218:Myosins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014469:Umbilical Arteries / Q000166:cytology*
D000199:Actins; D015220:Calcium Channels; D009218:Myosins