Submitted:
29 December 2025
Posted:
30 December 2025
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Abstract
A reliable and reproducible method for the isolation, culture, and identification of an osteoblast cell line from crucian carp (Carassius auratus) was established in this study using vertebral bone tissue from Chongming crucian carp, a locally important aquaculture strain from the lower Yangtze River region. Osteoblast cells were isolated using a tissue explant culture method, and optimal in vitro culture conditions were systematically evaluated. The established osteoblast cell line, designated Chongming Carassius auratus osteoblast cells (COBC), was characterized through chromosomal karyotype analysis, osteocalcin enzyme-linked immunosorbent assay (ELISA), and osteogenesis-related gene expression analysis. Additionally, cellular responses to environmental stress were assessed. The results showed that COBC exhibited optimal proliferation in L-15 medium supplemented with 20% fetal bovine serum at 28 ℃ under 5% CO2. Alkaline phosphatase staining, Alizarin Red staining, and von Kossa staining all yielded positive results, thereby confirming that the isolated cells possessed typical and stable osteoblastic properties, with the osteocalcin content of 36,884 ng/L. Quantitative PCR analysis revealed that osteogenic marker genes, including runx2a and runx2b, were expressed at significantly higher levels in COBCs than in muscle tissue. Under hypoxia-reoxygenation stress, COBC exhibited enhanced apoptotic responses, marked alterations in related gene expression, and modulation of antioxidant enzyme activities, suggesting a certain degree of adaptive capacity to oxygen fluctuations. This study provides the first systematic description of the establishment and biological characterization of COBC, as well as its responses to hypoxic stress. These findings offer a valuable in vitro cell model and technical support for studies on fish bone tissue biology and the assessment of environmental stress effects.
