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The characterisation of human detrusor smooth muscle cell cultures

Wood, Daniel Nigel; (2003) The characterisation of human detrusor smooth muscle cell cultures. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The aim of this project was to establish the contractile, biochemical and morphological phenotype of cultured human detrusor smooth muscle cells. Bladder biopsies were taken from human subjects following ethical approval and informed consent and comparisons were made between freshly isolated cells and cultured detrusor myocytes. Single detrusor myocytes were obtained by removal of urothelium, using sharp dissection and subsequent enzyme digestion. Cells were cultured in a D-valine substituted minimum essential medium, with 10 % foetal bovine serum. Cultures were maintained at 37 °C with 5 % CO 2, grown to confluence and passaged. Cultured cells stained for a-smooth muscle actin. They were also compared to fresh cells using 1H NMR spectroscopy. Enzyme digestion did not alter the biochemical phenotype of freshly isolated tissue. Cultured cells showed some differences in overall biochemical phenotype which may provide a means to optimize their contractile function. Epifluorescence microscopy was used to measure the Ca2+ responses of the cells to carbachol and ATP and their ability to restitute Ca 2+ responses. Cultured cells maintained their sensitivity, magnitude and restitution of Ca2+ response with carbachol, compared to freshly isolated counterparts. However, they were less sensitive to ATP than freshly isolated cells, this may represent a change to the purinergic receptor subtype. Using analogues of mediators involved in the production of cAMP from diacylglycerol the effect on intracellular Ca2+ was recorded. Analogues of this pathway raised the resting intracellular Ca2+ and modulated the transient response. Using a culture force monitor the contractile characteristics of these cells were measured. Incorporating these cells into a collagen gel and attaching this to a strain gauge a significant force, that could be manipulated by carbachol, was generated. Cultured detrusor myocytes maintained their morphology and response to physiological agonists. This would allow their development for use in tissue implants and models.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The characterisation of human detrusor smooth muscle cell cultures
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Human detrusor smooth muscle cel
URI: https://discovery.ucl.ac.uk/id/eprint/10098696
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