Primary growth plate chondrocyte isolation, culture, and characterization from the modern broiler

Abstract

Lameness is a leading cause of animal welfare and production concerns for the poultry industry as fast-growing, high-yielding broilers seem more susceptible to bone disease and infections. A major limitation to the study of these disorders is the lack of a chicken immortalized chondrocyte cell. Primary cell isolation is a valid and complex method for establishing a relevant in vitro model for diseases. In this study, isolation and high-density culturing of primary chondrocytes form 1-d old chicks was followed by confirmation of cell type, identification of optimal phenotypic expression, and evaluation of cells functionality. mRNA expression, as well as protein production and secretion, of COLI, COLII, Sox9, ACAN, and COLXA1 on day 3 (d3), d7, d11, d14, d18, and d21 in culture showed that avian growth plate chondrocytes under these conditions exhibit optimal phenotypes from d3 to d7. This is evident by a shift from COLII dominant expression in early-culture to COLI dominant expression by late-culture in conjunction with a loss of other chondrocyte markers Sox9, ACAN, and COLXA1. Additionally, morphological changes seen through live cell imaging coincide with the shift of phenotype in mid- to late-culture periods indicating a dedifferentiated phenotype. The functionality of the cultured cells was confirmed using Brefeldin-A treatment which significantly reduced secretion of COLII by d7 chondrocytes. These results provide a foundation for future research utilizing avian primary chondrocytes with optimal phenotypes for disease modeling or passaging.