Flow cytometric evaluation of cell dispersion from human head and neck tumors.
The preparation of single-cell suspensions from 25 human head and neck tumors is described. Dispersal was performed overnight at 4 degrees C under slight agitation of the tissue suspensions using various combinations of enzymes and additives. The cell suspensions were examined for number of cells released, viability, amount of debris, and DNA distribution by means of flow cytometry (FCM). It was shown that both trypsin/dithioerythritol (TD) and collagenase/D Nase (CDse) were of value in dispersing single cells from tumor tissue. In contrast to CDse, incubation with TD appeared to be cytolytic to normal lymphocytes. In a number of cases, DNA-FCM revealed ploidy abnormalities in a TD-suspension, which were not discernible in the concurrent CDse-suspension. Cell culture of primary cell suspensions corroborated the reliability of the DNA-FCM measurements. Pretreatment with CDse improved tumor disaggregation by TD and indicated a different dispersal capacity. Addition of Ca2+ and Mg2+ ions to the dispersal mixtures and preincubation of tumor slices in complete medium for 1 day before initiation of cell dispersion influenced favorably the quality of the cell suspension.
Bijman
J T
JT
Wagener
D J
DJ
van Rennes
H
H
Wessels
J M
JM
van den Broek
P
P
D000328:Adult; D000368:Aged; D002469:Cell Separation; D002712:Chlorides; D004273:DNA, Neoplasm / Q000032:analysis; D003850:Deoxyribonuclease I; D004226:Dithioerythritol; D005260:Female; D005434:Flow Cytometry; D006258:Head and Neck Neoplasms / Q000032:analysis / Q000473:pathology*; D006801:Humans; D006821:Hyaluronoglucosaminidase; D008094:Lithium; D018021:Lithium Chloride; D008297:Male; D003012:Microbial Collagenase; D008875:Middle Aged; D010434:Pepsin A; D012259:Ribonuclease, Pancreatic; D014357:Trypsin
D002712:Chlorides; D004273:DNA, Neoplasm; D004226:Dithioerythritol; D008094:Lithium; D003850:Deoxyribonuclease I; D012259:Ribonuclease, Pancreatic; D006821:Hyaluronoglucosaminidase; D014357:Trypsin; D010434:Pepsin A; D003012:Microbial Collagenase; D018021:Lithium Chloride