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.
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved