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In vitro chondrocyte differentiation using costochondral chondrocytes as a source of primary rat chondrocyte cultures: an improved isolation and cryopreservation method
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Authors
Gartland, AlisonMechler, Joshua
Mason-Savas, April
MacKay, Carole A.
Mailhot, Genevieve
Marks, Sandy C. Jr.
Odgren, Paul R.
UMass Chan Affiliations
Department of Cell BiologyDocument Type
Journal ArticlePublication Date
2005-10-02Keywords
AnimalsAnimals, Newborn
Base Sequence
*Cell Differentiation
Cells, Cultured
Chondrocytes
Cryopreservation
DNA Primers
Gene Expression
Immunohistochemistry
Microscopy, Electron
Rats
Reverse Transcriptase Polymerase Chain Reaction
Cell Biology
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INTRODUCTION: 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.Source
Bone. 2005 Oct;37(4):530-44. Link to article on publisher's siteDOI
10.1016/j.bone.2005.04.034Permanent Link to this Item
http://hdl.handle.net/20.500.14038/42869PubMed ID
16054883Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1016/j.bone.2005.04.034