Basal tissue factor expression in endothelial cell cultures is caused by contaminating smooth muscle cells. Reduction by using chymotrypsin instead of collagenase.
A discrepancy exists between basal tissue factor (TF) expression found in endothelial cell cultures and the failure to detect TF in unpertubated endothelial cells in vivo. We demonstrated that basal TF expression in endothelial cell cultures originated from contaminating cells. These cells were ultrastructurally and flowcytometrically identified as smooth muscle cells. The cell cultures had been obtained from collagenase-treated human umbilical cord vessels. Histologic studies revealed that after collagenase treatment the basement membrane was digested and underlying structures were disrupted at some areas of the vein. We selected chymotrypsin as an alternative for the isolation of endothelial cells. Using chymotrypsin, the endothelial lining was selectively lost leaving the basement membrane undisturbed. Furthermore, use of chymotrypsin instead of collagenase minimized the level of basal TF activity. Taken together, we demonstrated that basal TF expression in endothelial cell cultures is caused by contaminating smooth muscle cells. This contamination can strongly be reduced using chymotrypsin instead of collagenase for isolation of endothelial cells.
Mulder
A B
AB
Department of Haematology, University Hospital of Groningen, The Netherlands.
Blom
N R
NR
Smit
J W
JW
Ruiters
M H
MH
van der Meer
J
J
Halie
M R
MR
Bom
V J
VJ
D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000494:pharmacology*; D017364:Collagenases / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005434:Flow Cytometry; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D013925:Thromboplastin / Q000187:drug effects / Q000378:metabolism*; D014470:Umbilical Cord
D012333:RNA, Messenger; D013925:Thromboplastin; D002918:Chymotrypsin; D017364:Collagenases