Detrusor Smooth Muscle Cell Isolation: An Enzymatic Digestion Procedure to Obtain DSM Cells from Human Urinary Bladder Specimens
Trascrizione
Begin by examining the whole thickness urinary bladder specimen. Pin the specimen, mucosa facing upwards and serosa down, onto a silicone enantiomer-coated 150-millimeter round dish filled with ice-cold DS, and carefully remove all of the mucosa by sharp dissection.
Cut out several mucosa-free detrusor smooth muscle or DSM pieces. Place 3 to 6 DSM pieces into a tube with 1 to 2 milliliters of pre-warmed DS containing papain and dithiothreitol or DS-P. Incubate them at 37 degrees Celsius for 30 to 45 minutes, making sure to shake the tube every 10 to 15 minutes.
After the incubation, remove the DS-P from the tube and briefly wash the DSM pieces with ice-cold DS. Transfer the DS with DSM pieces to a new tube and remove the DS, leaving the DSM pieces at the bottom of the tube. Add 1 to 2 milliliters of DS containing collagenase type II or DS-C to the tube and incubate at 37 degrees Celsius with occasional shaking.
Discard the DS-C and wash the enzyme-treated DSM pieces 5 to 10 times with ice-cold DS. After the last wash, leave the DS inside the tube and gently triturate the pieces with a fire-polished Pasteur pipette to release single DSM cells.
