# Primary Culture and Characterization of a Crucian Carp (Carassius carassius) Osteoblast Cell Line (COBC) and the Effects of Hypoxia on Its Differentiation

## TL;DR

Researchers created and characterized an osteoblast cell line from crucian carp and studied how hypoxia affects its function, offering a new model for studying fish bone biology and environmental stress.

## Contribution

The first osteoblast cell line from Chongming crucian carp was established and its response to hypoxia was characterized.

## Key findings

- COBCs showed typical osteoblast properties and optimal growth in L-15 medium with 20% fetal bovine serum at 28°C.
- Under hypoxia, COBCs exhibited increased cell death, gene expression changes, and altered antioxidant enzyme activity.
- Osteogenic marker genes like runx2a and runx2b were significantly upregulated in COBCs compared to fish tissues.

## Abstract

In this study, an osteoblast cell line was established and characterized from Chongming crucian carp (Carassius carassius), an important aquaculture species from the lower Yangtze River region. The osteoblast cells, named Chongming Carassius carassius osteoblast cells (COBCs), were isolated from vertebral tissue and cultured under optimal conditions. The cells were characterized through various methods, including chromosomal analysis, enzyme assays, and gene expression studies. The COBCs showed typical osteoblastic properties and responded to environmental stress, such as hypoxia–reoxygenation, by altering gene expression and antioxidant enzyme activity. This study provides a valuable cell model for understanding fish bone biology and the effects of environmental stress.

In the present study, vertebral bone tissues derived from Chongming crucian carp (Carassius carassius), a dominant species during the summer and autumn seasons on Chongming Island in the lower Yangtze River, were used to establish and characterize a Carassius carassius osteoblast cell line (COBC). The established COBCs were assessed using chromosome 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 °C. The histochemical staining assay results were all positive, thereby confirming that the isolated cells display typical osteoblast characteristics. Quantitative PCR analysis revealed that osteogenic marker genes, including runx2a and runx2b, were expressed at significantly higher levels in COBCs than in fish tissues. Under hypoxic stress, COBCs exhibited morphological changes, an increase in cell death, significant alterations in gene expression, and variations in antioxidant enzyme activity. These responses facilitate adaptation to hypoxic stress. This study established the first osteoblast cell line of the Chongming crucian carp and characterized its biological properties and response to hypoxic stress. These findings offer a valuable in vitro cell model and technical support for research on fish bone tissue biology and the assessment of environmental stress effects.