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

Published: April 30, 2023

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 …

Açıklamalar

The authors have nothing to disclose.

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

Etiketler

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Bu Makaleden Alıntı Yapın
Detrusor Smooth Muscle Cell Isolation: An Enzymatic Digestion Procedure to Obtain DSM Cells from Human Urinary Bladder Specimens. J. Vis. Exp. (Pending Publication), e20558, doi: (2023).

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