Detrusor Smooth Muscle Cell Isolation: An Enzymatic Digestion Procedure to Obtain DSM Cells from Human Urinary Bladder Specimens

Detrusor smooth muscle, or DSM, cells present within the urinary bladder wall constitute the primary functional unit of the bladder. These cells relax to facilitate urine storage in the bladder and contract to release the urine.

To isolate DSM cells, begin by taking a harvested human urinary bladder specimen. Wash the specimen to remove any traces of blood and debris. Subsequently, secure the tissue with its mucosal surface facing up. Excise the fat, blood vessels, and mucosal layer to expose the underlying detrusor muscle layer. Then, cut the mucosa-free tissue into small fragments.

Treat the tissue fragments with a  digestion cocktail containing papain and incubate. Papain, a proteolytic enzyme, promotes the digestion of proteins in the extracellular matrix that hold the cells together and initiates the loosening of the cells in the matrix. Now, remove the papain-containing supernatant.

Add collagenase enzyme suspension to the tissue fragments and incubate at physiological temperature. Collagenase breaks down the extracellular matrix and facilitates the dissociation of DSM cells. Incubation at physiological temperature helps preserve DSM cell integrity. Discard the collagenase-containing supernatant.

Wash the tissue fragments with an ice-cold buffer to remove any residual enzymes. Then, resuspend them in buffer. Gently triturate the enzyme-treated tissue fragments to ensure complete dissociation of any unreleased cells and obtain a single-cell suspension of DSM cells. Store the DSM cells at low temperatures until further use.

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.