A method for isolating smooth muscle cells from pig urinary bladder with low concentrations of collagenase and papain: the relation between calcium concentration and isolated cell length.
The present study describes a method for isolating single smooth muscle cells from pig urinary bladder using a continuous resuspension device. Low concentrations of collagenase and papain were sufficient to obtain a high yield of viable smooth muscle cells, which remained viable for about 3-4 h as tested with fluorescein diacetate. Addition of fetal calf serum increased the lifespan of the isolated cells and the percentage of contractile smooth muscle cells, but caused spontaneous shortening of the cells. The length and volume of the isolated smooth muscle cells depended on the calcium concentration used in the isolation buffer solution. The isolated muscle cells were apparently relaxed if a calcium concentration less than 1.0 mmol/l was used in the isolation medium. In higher calcium concentrations the isolated cells were significantly shorter, probably as a result of a contraction caused by mechanical stimulation of the cells during the isolation procedure.
Schot
R
R
Department of Urology, Erasmus University Rotterdam, The Netherlands.
van Asselt
E
E
van Mastrigt
R
R
D000818:Animals; D002021:Buffers; D002118:Calcium; D002469:Cell Separation / Q000379:methods*; D048429:Cell Size; D017364:Collagenases; D003470:Culture Media; D005069:Evaluation Studies as Topic; D066298:In Vitro Techniques; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000166:cytology* / Q000502:physiology; D010206:Papain; D013552:Swine; D001743:Urinary Bladder / Q000166:cytology* / Q000502:physiology
D002021:Buffers; D003470:Culture Media; D010206:Papain; D017364:Collagenases; D002118:Calcium