Detrusor smooth muscle cells form the wall of the urinary bladder that ultimately facilitates the urine voiding and storage. Our protocol provides a validated and reliable method for freshly isolation of single smooth muscle cells. Freshly isolated human detrusor smooth muscle cells provides an opportunity for electrophysiological and molecular examinations at single cell level.
By studying single detrusor smooth muscle cells from control, normal, and diseased human urinary bladders, we have the opportunity to validate pharmacological targets such as ion channels. Single human detrusor smooth muscle cells are ideal for electrophysiological experiments such as patch-clamp electrophysiology. Here we will present an example of studying a single ion channel named TRPM4, transient receptor potential melastatin four channel, which is a nonselective cation channel.
Novices may struggle with dissection and enzymatic treatment steps which require efficiency and familiarity including recognizing critical important steps for obtaining fresh isolated DSM cells. It is important to be familiar with human bladder histology, protocol steps for isolating single cells remain resilient, and initially expect to get suboptimal cells using this protocol. Begin by examining the whole thickness urinary bladder specimen.
Pin the specimen, mucosa facing upwards and serosa down onto a silicone enantiomer-coated 150 mm 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 three to six DSM pieces into a tube with one to two 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 one to two milliliters of DS containing collagenase type II, or DS-C, to the tube.
And incubate it at 37 degrees Celsius with occasional shaking. Discard the DS-C and wash the enzyme-treated DSM pieces five 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. Pipette 0.25 to one milliliter of cell suspension onto a glass bottom chamber sitting on the stage of an inverted microscope and incubate it for at least 45 minutes to allow the cells to adhere.
Then, remove the DS from the bath and replace it with E solution via superfusion. Pull multiple patch electrodes, fire-polish the electrode tips, and coat the tips in dental wax if needed. Fill the tip of a patch electrode with the pipette solution without amphotericin-B by briefly dipping the electrode in the solution.
Success of patch clamp electrophysiology depends on DSM cell quality and amphotericin-B solubilization. Backfill the electrode with the same pipette solution containing amphotericin-B and mount the electrode onto a holder connected to a patch clamp amplifier head stage. Use a micromanipulator to place the electrode just below the surface of the extracellular solution so that the tip of the electrode is just submerged.
Then, determine the electrode resistance using the membrane test window function of the commercial acquisition software and advance the electrode toward the cell of interest. When touching the cell surface with the electrode, form a giga-seal by applying gentle rapid negative pressure to the electrode via tubing. This results in negative pressure at the tip of the electrode resulting in a successful giga-seal as confirmed by the membrane test.
Allow 30 to 60 minutes for the amphotericin-B to diffuse down the pipette and be inserted into the plasma membrane, forming pores primarily selective to monovalent cations. Continue monitoring the giga-seal with the membrane test function. When the patch perforation is optimal, cancel out the capacitance transients by adjusting the dials for cell capacitance and series resistance on the amplifier.
Once the stable voltage-step cation currents are observed, record currents with the routing voltage-step protocol as described in the manuscript. Apply a compound or a physiological test condition to test by superfusion and record the responses for the control and test conditions as well as the washout. This protocol can be used to isolate healthy, fresh DSM cells for functional and molecular studies.
Healthy single DSM cells are characterized by spindle shaped morphology, crisp well defined edges, a well-defined halo around the cell, and semi-contractile appearance when viewed under the microscope. Furthermore, the freshly isolated DSM cells respond to mechanical stimulation and to carbachol. Cell fragments and non-viable or over-digested cells should be avoided in subsequent patch-clamp experiments.
The isolated cells have been used in amphotericin-B perforated patch-clamp experiments where whole-cell currents were measured with either a voltage-step induced or ramp protocol in three different human DSM cells. Experiments revealed that 9-phenanthrol, a TRPM4 channel inhibitor, effectively and reversibly inhibited human DSM cation currents. The 9-phenanthrol-sensitive current component illustrates a stronger inhibition at positive voltages and outward rectification.
Condition of enzymatic treatment steps, steps 2.2 and 2.3, including temperature, duration of enzyme treatments, enzyme lot sources and quality of DSM cells, are key determinants for obtaining high quality human DSM cells. Human DSM cells are also ideally suited for evaluating intracellular second messenger levels including calcium and cyclic AMP, detecting protein expressions by immunocytochemistry, and co-expression by in situ proximal litigation assay, and mRNA expression by RT-PCR, qRT-PCR, microarrays, and next generation sequencing. Human DSM cells have advanced our understanding of the roles of BK, calcium, TRPM4 channels in the urinary bladder.
Future developments will allow to specifically link electrophysiological or pharmacological properties with the transcriptome or proteome profiles.
