Optimisation of isolation of richly pure and homogeneous primary human colonic smooth muscle cells.
BACKGROUND
Inherent properties of gastrointestinal smooth muscle can be assessed using isolated cell suspensions. Currently available isolation techniques, based on short 2-h enzymatic digestion, however, present the disadvantage of low cellular yield with brief viability. These features are an important limiting factor especially in studies in humans in which tissue may not be available daily and mixing of samples is not recommended.


OBJECTIVE
To optimise the isolation procedure of cells from human colon to obtain a richly pure primary smooth muscle cell preparation.


METHODS
Slices of circular muscle layer, obtained from surgical specimens of human colon, were incubated overnight in Dulbecco's modified eagle's medium supplemented with antibiotics, foetal bovine serum, an ATP-regenerating system and collagenase. On the following day, digested muscle strips were suspended in HEPES buffer, and spontaneously dissociated smooth muscle cells were harvested and used either immediately or maintained in suspension for up to 72 h. Cell yield, purity, viability, contractile responses, associated intracellular calcium signals and RNA and protein extraction were evaluated and compared to cell suspensions obtained with the current short digestion protocol.


CONCLUSIONS
The overnight isolation protocol offers the advantage of obtaining a pure, homogeneous, long-life viable cell suspension that maintains a fully differentiated smooth muscle phenotype unchanged for at least 72 h and that allows multiple functional/biochemical studies and efficient RNA extraction from a single human specimen.
Tattoli
I
I
Department of Clinical Sciences, University La Sapienza, Viale del Policlinico, 00161 Rome, Italy.
Corleto
V D
VD
Taffuri
M
M
Campanini
N
N
Rindi
G
G
Caprilli
R
R
Delle Fave
G
G
Severi
C
C
D002469:Cell Separation / Q000379:methods*; D003106:Colon / Q000166:cytology*; D006801:Humans; D009130:Muscle, Smooth / Q000166:cytology*