title: Time-related effects of enzymatic disaggregation on model human lung carcinomas.
abstract: 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.
authors.affiliation: Department of Surgery, University of Colorado Cancer Center, Denver, USA.
authors.forename: R B
authors.initials: RB
authors.lastname: Howard
authors.forename: K R
authors.initials: KR
authors.lastname: Shroyer
authors.forename: T
authors.initials: T
authors.lastname: Marcell
authors.forename: L E
authors.initials: LE
authors.lastname: Swanson
authors.forename: M E
authors.initials: ME
authors.lastname: Pagura
authors.forename: D W
authors.initials: DW
authors.lastname: Mulvin
authors.forename: M E
authors.initials: ME
authors.lastname: Cowen
authors.forename: M R
authors.initials: MR
authors.lastname: Johnston
mesh_terms: 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
chemical_list: D006867:Hydrolases