Time-related effects of enzymatic disaggregation on model human lung carcinomas.
Enzyme cocktails used to prepare tumor cell suspensions may influence yield, viability, and cytology, thus time-related cocktail effects on model human lung carcinomas were examined. A549, NCI-H125, and NCI-H460 carcinomas were completely disaggregated at 25 degrees C over 2 h with either (mg/ml) collagenase/DNAase (C/D, 1/0.1), collagenase/hyaluronidase/DNAse (C/H/D, 1/0, 1/0.1), or polymyxa protease/DNAse (PP/D, 3/0.1). Trypan blue viabilities, total yields, viable yields, and flow cytometric percent tumor cells (TC) were measured every 20-30 min (n = 4-7 per tumor type). The final percentages of TC, mononuclear cells (MN), polymorphonuclear cells (PMN), lymphocytes, and necrotic cells were determined by cytology (n = 4-5 per tumor type). The time-dependent measurements showed that 1) disaggregation was progressive and complete with all cocktails; 2) viability was stable or increasing with all cocktails; 3) percent TC was stable for all cocktails, but lower for PP/D than C/D in final suspensions; and 4) PP/D gave lower final total yields, higher final viabilities, but the same final viable yields as the C cocktails, suggesting selective elimination of dead cells by PP/D. Final cytology measurements showed that PP/D gave a lower percent MN and a higher percent PMN than C cocktails. Cocktail effects may importantly influence cell suspension properties.
Howard
R B
RB
Department of Surgery, University of Colorado Cancer Center, Denver, USA.
Shroyer
K R
KR
Marcell
T
T
Swanson
L E
LE
Pagura
M E
ME
Mulvin
D W
DW
Cowen
M E
ME
Johnston
M R
MR
D000818:Animals; D002469:Cell Separation; D002470:Cell Survival; D003584:Cytological Techniques; D005434:Flow Cytometry / Q000295:instrumentation; D006801:Humans; D006867:Hydrolases / Q000378:metabolism*; D008175:Lung Neoplasms / Q000473:pathology*; D009336:Necrosis; D051381:Rats; D013997:Time Factors; D014407:Tumor Cells, Cultured
D006867:Hydrolases