title: Generation of functionally competent single bovine adrenal chromaffin cells from cell aggregates using the neutral protease dispase.
abstract: A simple and efficient procedure has been developed to enzymatically dissociate aggregates of bovine adrenal chromaffin cells in suspension culture into viable, responsive single cells. For dissociation, the neutral protease dispase is added directly to the culture medium for a minimum of 3 h, followed by incubation of the cells in Hank's calcium-magnesium-free balanced salt solution at 37 degrees C with intermittent trituration to facilitate dispersion. This procedure generates a population of phase-bright single cells that are round in morphology, take up the dye neutral red, exclude the dye trypan blue and readily attach to tissue culture dishes coated with collagen, fibronectin or polylysine, thereby permitting applications that require plated-down conditions. When transferred to culture medium, the cells begin to reaggregate. By altering the length of time the cells are incubated in culture medium prior to attachment, the degree of reaggregation can be controlled to obtain plate-down profiles that consist of both isolated cells and cells in aggregates of varying sizes. Returning dissociated cells to suspension culture results in the reformation of large cell aggregates. Several measures of chromaffin cell function were indistinguishable for dissociated cells placed either in monolayer culture or suspension culture versus non-dissociated cells, implying that the dissociation procedure does not alter cellular responses or cause cellular damage.
authors.affiliation: Department of Pharmacology, University of Nevada School of Medicine, Reno, NV 89557-0270, USA. gcraviso@med.unr.edu
authors.forename: Gale L
authors.initials: GL
authors.lastname: Craviso
mesh_terms: D000313:Adrenal Medulla / Q000166:cytology*; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D002449:Cell Aggregation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects; D019439:Chromaffin Cells / Q000166:cytology* / Q000378:metabolism; D004246:Dimethylphenylpiperazinium Iodide / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D018722:Nicotinic Agonists / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000814:Aniline Compounds; D002395:Catecholamines; C409648:Fluo 4; D018722:Nicotinic Agonists; D014966:Xanthenes; D004246:Dimethylphenylpiperazinium Iodide; D011189:Potassium Chloride; D010450:Endopeptidases; C023139:dispase; D002118:Calcium