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
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).