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Protocol no. 41
RABBIT ARTICULAR CHONDROCYTE FUNCTIONAL TOXICITY TEST

In this test rabbit articular chondrocytes are cultured in the presence of test compound, the toxicity of which is then determined by its effect on the production of proteoglycan by the cells, as detected by the dye Alcian Blue.

CONTACT

Dr Monique Adolphe and Odile Blein
Laboratoire de Pharmacologie Cellulaire Ecole Pratique des Hautes Etudes 15,
Rue de l'Ecole M‚dicine F-75006 Paris France
Telephone: France - 43 29 28 69 or 43 29 28 70

RATIONALE

Healthy, freshly isolated, articular chondrocyte cells can be successfully maintained for a short period of time but still retain certain characteristics of cells in vivo, such as their ability to secrete of the components which make up the background matrix of cartilage. In this test system deleterious effects on cell function are determined by monitoring the secretion of proteoglycans by healthy cells, which can be stained by the dye Alcian Blue.

BASIC PROCEDURE

Freshly isolated rabbit articular chondrocytes are cultured and exposed to varying concentrations of test compounds. The cultures are incubated for 72 hours after which time the cells are stained with Alcian Blue. After rinsing, the dye taken up by the proteoglycan matrix is liberated and measured spectrophotometrically. Comparison between the optical density values of cells exposed to test compounds and control cultures provide an index of toxicity.

CRITICAL ASSESSMENT

Cell culture This procedure requires the isolation of cells from rabbit joints, since: 1. At present chondrocyte cell lines are unavailable. 2. Although chondrocytes can be maintained in culture for several weeks, they rapidly lose their differentiated phenotype i.e. their ability to secrete type II collagen and proteoglycans. However, it should be noted that the isolation of such cells is a time consuming procedure, which requires fresh animal tissue and considerable practical experience. Alcian Blue staining Alcian Blue is a very specific dye, which associates with the sulphate groups found along the length of proteoglycan molecules. Absorbance obtained after Alcian Blue staining is proportional to the amount of proteoglycans secreted in chondrocyte monolayers. Thus, any chemical having an effect on proteoglycan synthesis can be detected with this method.

TEST STATUS

Undergoing in-house development.

CHEMICALS TESTED

Retinoic acid
Vitamin E

REFERENCES

  1. Hassell, J.R. & Horigan, E.A. (1982) Chondrogenesis: A model developmental system for measuring the teratogenic potential of compounds. Teratogenesis, Carcinogenesis and Mutagenesis, 2, 325-331.
  2. Paulsen, D.F. & Solrush, M. (1988) Microtiter micromass cultures on limb-bud mesenchymal cells. In vitro cellular and developmental biology, 24 no. 2, 138-147.

IP-4©August 1990