A new enzymic method for the isolation and culture of human bladder body smooth muscle cells

Abstract

Cultured cells of the human urinary bladder smooth muscle are useful for investigating bladder function, but methods for culturing them are not well developed. We have now established a novel enzymic technique. The smooth muscle layer was separated out and incubated with 0.2% trypsin for 30 min at 37 degrees C. The samples were then minced and incubated with 0.1% collagenase for 30 min and centrifuged at 900 g. The pellets were resuspended in RPMI-1640 medium containing 10% fetal calf serum (FCS) and centrifuged at 250 g. The smooth muscle cells from the supernatant were cultured in RPMI-1640 containing 10% FCS. The cells grew to confluence after 7-10 days, forming the "hills and valleys" growth pattern characteristic of smooth muscle cells. Immunostaining with anti-alpha-actin, anti-myosin, and anti-caldesmon antibodies demonstrated that 99% of the cells were smooth muscle cells. To investigate the pharmacological properties of the cultured cells, we determined the inhibitory effect of muscarinic receptor antagonists on the binding of [3H]N-methylscopolamine to membranes from cultured cells. The pKi values obtained for six antagonists agreed with the corresponding values for transfected cells expressing the human muscarinic M2 subtype. Furthermore, carbachol produced an increase in the concentration of cytoplasmic free Ca2+ an action that was blocked by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 selective antagonist. This result suggests that these cells express functional M3 muscarinic receptors, in addition to M2 receptors. The subcultured cells therefore appear to be unaffected by our new isolation method.

Isolation and culture of smooth muscle cells from human acute type A aortic dissection

“…In explant culture, several weeks are needed to establish primary cultures and subculturing is required to acquire a large number of cells [2,4]. Additionally, isolation and culture of SMCs from acute TAAD tissues are very difficult for several reasons: firstly, acute TAAD patients are usually in acute inflammatory state according to our previous clinical observation and systematic inflammatory factors are adverse for the growth of isolated SMCs; secondly, tunica media (elastic fibers and collagen fibers) of human aorta are very compactible, which is hard for SMCs migrating from the explants; thirdly, the age of donor patients may be older [3], and the SMCs are in quiescence for a long time.…”

Overexpression of progesterone receptor B increases sensitivity of human colon muscle cells to progesterone

“…Smooth muscle cells were cultured according to a previously described method (23,24,30,31). Immediately upon resection muscle tissues were transported to the motility research laboratory in ice-cold RPMI 1640 medium and incubated with 95% ethanol for 3 min.…”

A simple, rapid, and efficient method for isolating detrusor for the culture of bladder smooth muscle cells

“…For example, Ma et al [8] used 0.2 % trypsin incubation to loosen the tight junction between serosa, mucosa, and detrusor layer, so that the mucosa and serosa were removed completely ex vivo. In other study, homemade fixing plate was used for fixing the full thickness bladder sample, making the removal of mucosa and serosa more convenient [9].…”