Home › Cell Biology › Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents
Cell Biology JoVE (Open Access) Citable · DOI

Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents

DOI: 10.3791/59884-v
What you'll learn
  • ✓ Isolate viable detrusor smooth muscle cells using enzymatic dissociation protocols
  • ✓ Apply amphotericin-B perforated patch-clamp electrophysiology to single DSM cells
  • ✓ Characterize ion channel properties and cation currents in freshly isolated cells
Protocol

We describe a method for preparation of the single freshly isolated detrusor smooth muscle cells from human urinary bladder specimens employing a two-step enzymatic procedure. The obtained viable DSM cells can be studied by various single cell techniques including the described amphotericin-B patch-clamp electrophysiology to reveal physiological and pharmacological properties.

Difficulty
advanced
Total time
~4–6 hours per specimen (dissection, enzymatic treatment, cell isolation, electrophysiology recordings)
Model organism
Human detrusor smooth muscle (urinary bladder tissue)
Biosafety
BSL-1

Steps

1
Introduction to detrusor smooth muscle cell preparation

Overview of enzymatic dissociation protocol and patch-clamp electrophysiology approach for studying ion channel function in freshly isolated human DSM cells.

▶ 00:04
2
Dissect and enzymatically dissociate mucosa-free DSM tissue

Perform mechanical dissection of urinary bladder specimen to isolate detrusor smooth muscle, then apply sequential enzymatic treatment to obtain single viable cells.

▶ 01:45
3
Record voltage-step cation currents using perforated patch-clamp

Establish amphotericin-B perforated whole-cell configuration and apply voltage-step protocols to measure ion channel currents in isolated DSM cells.

▶ 03:28
4
Analyze ion channel properties and drug sensitivity

Examine pharmacological and biophysical properties of cation currents, including response to 9-phenanthrol and characterization of channel kinetics.

▶ 05:52
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