Date of Defense
William Jackson, Biological Sciences
John Spitsbergen, Biological Sciences
Susan Stapleton, Biological Sciences
A previous method of enzymatic arteriolar digestion was developed to isolate single vascular smooth muscle (VSM) cells that retain the contractile and electrophysiological phenotype of the original vessels (Jackson et al., 1996). Although this method has proven to be useful, high variability in the contractile response to norepinephrine (NE), reactivity to NE over a 2-hour period, and physiological status of cells was observed among cell isolates. Therefore, the purpose of the present study was to modify this method to reduce the first two sources of variability, and, therefore, increase viability of isolated vascular smooth muscle cells. Hand-dissected arterioles of hamster cremaster muscles were digested with enzyme solutions of papin PAP) and dithioerythritol (DTE) for 35 minutes and collagenase (COLL), trypsin inhibitor (TI) and elastase (ELAST) for 22 minutes. The author varied the enzyme concentrations, incubation times, and rinsing procedure and observed the cell isolates for contractile reactivity (viability). When PAP and DTE were excluded from the procedure no individual VSM cells were isolated. However, reducing the time of PAP/DTE incubation produced the highest yield of cells that also exhibited contractile response to NE. Altering the times of COLL/TI/ELAST incubation and rinsing had no significant effect on the cells. Overall, the author was unable to reduce the variability of the cell viability or eliminate the time-dependent loss of response to NE with any of the modified procedures. Further modification will be required to eliminate this variability and provide us with a more reliable single cell model for study of VSM physiology and the microcirculation.
Carr, Mianda, "Isolation of Cremasteric Arteriolar Cells by Enzymatic Digestion" (2000). Honors Theses. 166.
Honors Thesis-Campus Only