title: Centrifugation assay for measuring adhesion of serially passaged bovine chondrocytes to polystyrene surfaces.
abstract: A major obstacle in chondrocyte-based therapy for cartilage repair is the limited availability of cells that maintain their original phenotype. Propagation of chondrocytes as monolayer cultures on polystyrene surfaces is used extensively for amplifying cell numbers. However, chondrocytes undergo a phenotypic shift when propagated in this manner and display characteristics of more adherent fibroblastic cells. Little information is available about the effect of this phenotypic shift on cellular adhesion properties. We evaluated changes in adhesion property as bovine chondrocytes were serially propagated up to five passages in monolayer culture using a centrifugation cell adhesion assay, which was based on counting of cells before and after being exposed to centrifugal dislodgement forces of 120 and 350 g. Chondrocytes proliferated well in a monolayer culture with doubling times of 2-3 days, but they appeared more fibroblastic and exhibited elongated cell morphology with continued passage. The centrifugation cell adhesion assay showed that chondrocytes became more adhesive with passage as the percentage of adherent cells after centrifugation increased and was not statistically different from the adhesion of the fibroblast cell line, L929, starting at passage 3. This increased adhesiveness correlated with a shift to a fibroblastic morphology and increased collagen I mRNA expression starting at passage 2. Our findings indicate that the centrifugation cell adhesion assay may serve as a reproducible tool to track alterations in chondrocyte phenotype during their extended propagation in culture.
authors.affiliation: Food and Drug Administration's Center for Devices and Radiological Health (FDA, CDRH), Silver Spring, MD 20993, USA. david.kaplan@fda.hhs.gov
authors.forename: David S
authors.initials: DS
authors.lastname: Kaplan
authors.forename: Victoria M
authors.initials: VM
authors.lastname: Hitchins
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Vegella
authors.forename: Richard A
authors.initials: RA
authors.lastname: Malinauskas
authors.forename: Kimberly M
authors.initials: KM
authors.lastname: Ferlin
authors.forename: John P
authors.initials: JP
authors.lastname: Fisher
authors.forename: Carmelita G
authors.initials: CG
authors.lastname: Frondoza
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002448:Cell Adhesion / Q000502:physiology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D002498:Centrifugation; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism*; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D007124:Immunoenzyme Techniques; D051379:Mice; D010641:Phenotype; D011137:Polystyrenes / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012692:Serial Passage
chemical_list: D024043:Collagen Type II; D011137:Polystyrenes; D012333:RNA, Messenger