title: In vitro chondrocyte differentiation using costochondral chondrocytes as a source of primary rat chondrocyte cultures: an improved isolation and cryopreservation method.
abstract: BACKGROUND
  Isolating and culturing primary chondrocytes such that they retain their cell type and differentiate to a hypertrophic state is central to many investigations of skeletal growth and its regulation. The ability to store frozen chondrocytes has additional scientific and tissue engineering interest. Previous work has produced approaches of varying yield and complexity but does not permit frozen storage of cells for subsequent differentiation in culture. Investigations of growth plate dysplasias secondary to defective osteoclastogenesis in rodent models of osteopetrosis led us to adapt and modify a culture method and to cryopreserve neonatal rat costochondral chondrocytes.
  
  
  METHODS
  Chondrocytes were isolated from dissected ribs of 3-day-old rat pups by collagenase, hyaluronidase, and trypsin serial digestions. This was done either immediately or after the isolation was interrupted following an initial protease treatment to allow the chondrocytes, still in partially digested rib rudiments, to be frozen and later thawed for culture. Cells were plated in flat-bottom wells and allowed to adhere and grow under different conditions. Choice of media permitted cells to be maintained or induced to differentiate. Cell growth was monitored, as was expression of several relevant genes: collagen types II and X; osteocalcin, Sox9, adipocyte FABP, MyoD, aggrecan, and others. Mineralization was measured by alizarin red binding, and cultures were examined by light, fluorescence, and electron microscopy.
  
  
  RESULTS
  Cells retained their chondrocyte phenotype and ability to differentiate and mineralize the collagen-rich extracellular matrix even after freezing-thawing. RT-PCR showed retention of chondrocyte-specific gene expression, including aggrecan and collagen II. The cells had a flattened, "proliferating zone" appearance initially, and by 2 weeks post-confluence, exhibited swelling and other salient features of hypertrophic cells seen in vivo. Collagen fibrils were abundant in the extracellular matrix, along with matrix vesicles. The switch to collagen type X as marker for hypertrophy was not rigidly temporally regulated as happens in vivo, but its expression increased during hypertrophic differentiation.
  
  
  CONCLUSIONS
  This method should prove valuable as a means of studying chondrocyte regulation and has the advantages of simpler initial dissection, yields of a purer chondrocyte population, and the ability to stockpile frozen raw material for subsequent studies.
authors.affiliation: Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Avenue N., Worcester, MA 01655, USA.
authors.forename: Alison
authors.initials: A
authors.lastname: Gartland
authors.forename: Joshua
authors.initials: J
authors.lastname: Mechler
authors.forename: April
authors.initials: A
authors.lastname: Mason-Savas
authors.forename: Carole A
authors.initials: CA
authors.lastname: MacKay
authors.forename: Geneviève
authors.initials: G
authors.lastname: Mailhot
authors.forename: Sandy C
authors.initials: SC
authors.lastname: Marks
authors.forename: Paul R
authors.initials: PR
authors.lastname: Odgren
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D015925:Cryopreservation; D017931:DNA Primers; D015870:Gene Expression; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers