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

Samenvatting

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

1. 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 solution (Figure 1 and Table 1 for composition of DS).
   NOTE: The specimen is usually kept in cold DS from a few hours to overnight prior to arrival in the laboratory. For longer storage, DS (Table 1) is supplemented with 1 mM CaCl₂.
2. Remove and rinse the human whole thickness DSM specimen (containing all layers including mucosa, DSM, and serosa) with ice-cold DS to wash out attached debris and blood.
3. Pin the urinary bladder specimen, mucosa facing upward and serosa down, onto a silicone enantiomer-coated (Table of Materials) 150 mm diameter round dish filled with ice-cold DS (Figure 1B).
4. Remove the adjacent adipose tissue, blood vessels, epithelium (urothelium) and muscularis mucosa from the specimen by sharp dissection using microscissors and forceps.
5. Cut out several mucosa-free DSM pieces (~2–3 mm long and 4–6 mm wide) (Figure 1C).

2. Enzymatic Dissociation of DSM Pieces Yielding Freshly Isolated Single DSM Cells

1. Place 3 to 6 DSM pieces into a tube containing 1 to 2 mL of pre-warmed (~37 °C) DS containing papain and dithiothreitol (DS-P, Table 1) and incubate DSM pieces in DS-P for 30–45 min at ~37 °C gently shaking the tube occasionally (once every 10–15 min).
   NOTE: To optimally control the temperature for enzymatic treatment, tubes with tissue pieces and enzyme solutions are placed in either a glass tissue chamber filled with water connected to a circulating heated water bath (Figure 1D) or a high-precision temperature-controlled shaking water bath (Figure 1E).
2. Remove DS-P from the tube, briefly wash DSM pieces with ice-cold DS, discard cold DS from the tube leaving DSM pieces sitting at the bottom of the tube.
3. Add 1 to 2 mL of DS-containing collagenase type II (DS-C, Table 1) to the tube with DSM pieces; gently mix and incubate for 25–40 min at ~37 °C gently shaking the tube occasionally (every 10–15 min).
4. Discard DS-C and wash enzyme-treated DSM pieces 5–10 times with ice-cold DS.
5. Following the last wash, leave DS solution inside the tube; gently triturate with a fire-polished Pasteur pipette several times to release single DSM cells.
6. Place a few drops of DS solution containing dispersed DSM cells onto a glass-bottom chamber or a coverslip and visually inspect for the quality under a microscope (using a 20x or 40x objective) after at least 5 min following the application to allow the cells to adhere to the bottom.
7. Immediately use freshly isolated DSM cells for electrophysiological experiments or store the cells in a tube containing DS at ~4 °C either on ice or in a refrigerator until use (typically for up to 8 h of preparation).
   NOTE: Within the same preparation the quality of cells varies from highly viable to over-digested, dead DSM cells (Figure 2). When the sequential papain-collagenase method yields a very high number of unviable cells, the preparation is discarded, and a new digestion of DSM pieces is carried out but with reduced incubation intervals. If the procedure results in too few DSM cells, then for the subsequent digestion of DSM pieces, the incubation intervals are increased. Positive immunoreactivity to α-smooth muscle actin confirms the identity of DSM cells (Figure 3).

Solution Type	Composition (in mM)
DS (Dissection/Digestion Solution)	80 Na-glutamate, 55 NaCl, 6 KCl, 10 HEPES, 2 MgCl2, and 11 glucose, pH adjusted to 7.4 (with 10 M NaOH)
DS-P (Papain-containing DS)	DS containing 1-2 mg/ml papain, 1 mg/ml dithiothreitol and 1 mg/ml bovine serum albumin
DS-C (Collagenase-containing DS)	DS solution containing 1-2 mg/ml collagenase type II, 1 mg/ml bovine serum albumin, 0 or 1 mg/ml trypsin inhibitor and 100-200 μM Ca2+
P (Pipette)	110 CsOH, 110 aspartic acid, 10 NaCl, 1 MgCl2, 10 HEPES, 0.05 EGTA, and 30 CsCl,pH adjusted to 7.2 with CsOH, and supplemented with amphotericin-B (300-500 μg/ml)
E (Extracellular)	10 tetraethylammonium chloride (TEA), 6 CsCl, 124 NaCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, pH adjusted to 7.3-7.4 with NaOH or CsOH, and 0.002-3 (2-3 mM) nifedipine

Table 1: Compositions of dissection/digestion solution (DS), and pipette and extracellular solutions used in perforated patch-clamp experiments.

Resultaten

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 pr...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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