2022, Vol. 26 ›› Issue (23): 3633-3637

Isolation, culture and identification of rabbit auricular chondrocytes

Zhong Ziling1, 2, Qu Shenhong2, Han Xing2, Wu Di2   

Abstract: BACKGROUND: Auricle deformity remodeling based on three-dimensional printing scaffold materials has become a focus in recent years, and whether chondrocytes can grow normally on a scaffold is the key issue.  
OBJECTIVE: To investigate the primary culture methods of rabbit auricular chondrocytes, thereby laying a solid foundation for the application of chondrocytes combined with tissue engineering.
METHODS: Rabbit auricular chondrocytes were obtained by trypsin and type II collagenase sequential digestion method. The chondrocytes were cultured and subcultured in vitro. The chondrocytes were identified by morphological observation, growth curve drawing, safranin O-fast green staining, toluidine blue staining, Victoria blue staining, and glycosaminoglycan content determination.
RESULTS AND CONCLUSION: The normal morphology of auricular chondrocytes was fusiform and polygonal under inverted phase contrast microscope. After six generations of subculture, the cells became obviously long fusiform, and the proliferation rate slowed down or even stopped. The growth curve showed that the chondrocytes from the 2nd to 5th generations could proliferate in a straight line. Results of safranin O-fast green staining showed clear nucleus and cytoplasm structures of rabbit auricular chondrocytes, with good morphological characteristics. Results of toluidine blue staining indicated that the secretion of cartilage matrix was less at the initial stage of culture and increased gradually with the increase of cell fusion. Results of Victoria blue staining showed the formation of collagen fibers in chondrocytes, suggesting the normal function of chondrocytes. Results of glycosaminoglycans content determination showed the specific secretion of glycosaminoglycans in chondrocytes. Overall, the isolation and identification system of rabbit auricular chondrocytes in primary culture was successfully established, and the auricular chondrocytes with biological activity were obtained, which could be further used in tissue engineering research.
Key words: auricle reconstruction, chondrocyte, primary culture, cell identification, sequential digestion