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

Abstract

Source: Malysz, J. et al. Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents. J. Vis. Exp. (2020)

In this video, we demonstrate the isolation of detrusor smooth muscle cells from human urinary bladder specimens using a two-step sequential enzymatic treatment.

Protocol

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board. 1. Dissection of DSM Tissues and Preparation of Mucosa-free DSM Pieces Examine the whole thickness urinary bladder specimen that arrived in the lab from the operating room in a tightly sealed container filled with the cold dissection/digestion so…

Representative Results

Figure 1: Summary of dissection steps resulting in the preparation of detrusor smooth muscle (DSM) pieces and setup used for enzymatic dissociation. Shown are images of: (A) a whole thickness human urinary bladder specimen provided from an open bladder surgery as extraneous surgical material in ice-cold DS, (B) the same preparation after pinning …

Materials

5 ml polystyrene round-bottom tube	Falcon	352054	Tubes for DS containing enzymes used in digestion steps
Analog vortex mixer	VWR	58816-121
Bovine serum albumin	Sigma-Aldrich	A7906	DS
Calcium chloride	Sigma-Aldrich	C1016	Extracellular solution and DS
EGTA	Sigma-Aldrich	E3889	Ca2+ chelator, used in intracellular pipette solution
Glucose	Sigma	G8270
Collagenase type 2	Worthington Biochemical Corporation	LS004177	DS-C
Cesium hydroxide hydrate	Sigma-Aldrich	C8518	Intracellular pipette solution
CsCl	Sigma-Aldrich	203025	Extracellular and intracellular solutions
DL-Dithiothreitol (DDT)	Sigma-Aldrich	D9779	Reducing agents used together with Papain
NaOH	Sigma-Aldrich	S8045
Nifedipine	Sigma-Aldrich	N7634	L-type voltage-gated Ca2+ channel blocker
Floating foam tube rack/holder	VWR Scientific	82017-634	Used for holding tubes with enzymes for temperature control
Glutamic acid (Na salt)	Sigma-Aldrich	G1626	DS
Papain	Worthington Biochemical Corporation	LS003126	DS-P
MgCl2 (hexahydrate)	Sigma-Aldrich	M2670	Extracellular and intracellular solutions
KCl	Fisher Scientific	BP366-1	Extracellular solution
NaCl	Sigma-Aldrich	S7653	Extracellular and intracellular solutions
Non-metalic syringe needle, MicroFil	WPI	MF-34G-5	Filling of intracellular pipette solution
Pasteur pipette	FisherBrand	13-678-20A	Tips are broken off and firepolished and used for titration of enzymatically treated tissues to release single DSM cells from pieces
HEPES	Sigma-Aldrich	H3375	pH Buffer
Thermo Scientific Precision shaking water bath (model 2870)	Thermo Scientific	Discontinued	Water bath for temperature control of enzymatic digestion employed as an alternative to tissue chamber-circulating bath setup
Vinyl tubing	ColePalmer	06405-3	Multiple uses including for connecting tissue bath to circulating water bath
Water circulator bath, Haake D1 L	Haake	Discontinued	Connected to tissue bath
ZeissAxiovert 40C inverted microscope with 10x and 40x objectives	Carl-Zeiss	Discontinued	Part of patch-clamp rig setup