Table 1 Protocols used for establishment of urinary bladder smooth muscle cells culture

I

McCoy 2013 [9]

Tissue source

Human

Pig

Quantity of the tissue

1g

1 cm²

Digestion solution

Collagenase IV (450U/ml), Dispase II, CaCl₂ 5mM

Collagenase II (0.15%, 275U/mg), Dispase II (0.2%, 175U/mg), CaCl₂ 5mM, HBSS^a

Volume of the digestion solution

40ml

5ml

Digestion conditions

1h, 37°C

1.5h, 37°C

Seeding density

All cells/300–350cm²

20 000 cells/cm²

Growth media

DMEM High glucose^b, FBS^c (10%), Gentamycin (5μg/ml)

A: DMEM High glucose^b, FBS^c Pan-Biotech (10%), Gentamycin (100μg/ml), Amphotericin B (5μg/ml), Penicillin (100U/ml), Streptomycin (100μg/ml)

B: DMEM High glucose^b, FBS^c Sigma (10%), Gentamycin (100μg/ml), Amphotericin B (5μg/ml), Penicillin (100U/ml), Streptomycin (100μg/ml)

C: SmGM-2

II

Cheng et al. 2011 [10]

Tissue source

Pig

Pig

Quantity of the tissue

No data

1 cm²

Digestion solution

Trypsin-EDTA (0,25%)

Collagenase II (0.15%)

Trypsin (0.25%), EDTA (0.02%), Collagenase II (0.15%, 275U/mg), CaCl₂ 5mM

Volume of the digestion solution

No data

5ml

Digestion conditions

30min., 37°C

1h, 37°C

Seeding density

10⁶ cells/well (48-well plate)

20 000 cells/cm²

Growth media

RPMI 1640^d, FBS^c (10%)

A, B, C

III

Lau et al. 1996 [11]

Tissue source

Rabbit

Pig

Quantity of the tissue

Whole urinary bladder muscle layer

1 cm²

Digestion solution

Collagenase I (0.1%), SFNM^e

Collagenase II (0.1%, 275U/mg), CaCl₂ 5mM, DMEM High glucose^b

Volume of the digestion solution

20ml

5ml

Digestion conditions

~16h, 37°C

~16h, 37°C

Seeding density

10⁵ cells/35mm culture dish

20 000 cells/cm²

Growth media

SFNM^e

(M199, Sodium bicarbonate (2.2g/l), ×100 BME vitamins (10ml/l), ×50 BME amino acids (20 ml/1), Fungizone (1 ml/l),Penicilin/Streptomycin solution (100 U/ml Penicillin G and 100pg/ml Streptomycin)), FBS^c(10%)

A, B, C

IV

Ma et al. 2002 [12]

Tissue source

Rat

Pig

Quantity of the tissue

Whole urinary bladder muscle layer

1 cm²

Digestion solution

1. trypsin (0.2%)

2. Collagenase (0,1%), RPMI 1640^d

1. trypsin (0.2%)

2. Collagenase II (0.1%, 275U/mg) CaCl₂ 5mM, DMEM High glucose^b

Volume of the digestion solution

No data

5ml

Digestion conditions

1. 30min., 37°C

2. 30min., 37°C

1. 30min., 37°C

2. 1h, 37°C

Seeding density

No data

20 000 cells/cm²

Growth media

RPMI 1640^d, FCS (10%)

A, B, C

V

McCoy 2013 [9]

Tissue source

Human

Pig

Number of explants

20–25 tissue explants (1mm diameter)

explants from 1 cm² of tissue (1mm diameter)

Culture dish

100 mm tissue culture Petri dish

60 mm tissue culture Petri dish

Growth media

DMEM High glucose^b, FBS^c (10%), Gentamicin (5μg/ml)

A, B, C