Abstract: BACKGROUND: Currently, methods for obtaining primary intervertebral disc cells are mostly cumbersome and there is a lack of relevant reports on the simultaneous extraction of three types of cells. Therefore, it is crucial to find a method to simultaneously extract three types of cells.
OBJECTIVE: To explore a method for simultaneously extracting and culturing three types of intervertebral disc cells from rats, to identify them, and to investigate the effects of monolayer versus micromass cultures on the extracellular matrix.
METHODS: The cartilaginous endplate (CEP), nucleus pulposus (NP), and annulus fibrosus (AF) tissues were separated from 3-week-old male Sprague-Dawley rat intervertebral disc tissue. For the nucleus pulposus and annulus fibrosus tissues, 0.1% pronase E was used for 30 minutes of digestion at 37 °C followed by 0.2% collagenase type II digestion for 4 hours to release the cells; for the cartilaginous endplate tissue, direct digestion with 0.2% collagenase type II for 4 hours was performed to release the cells. The cells, after removal of the digestion enzymes, were seeded into culture dishes containing culture medium, and their morphology was observed. Real-time fluorescence quantitative PCR and western blot assay were performed to detect the expression levels of various cell markers. Rat primary annulus fibrosus cells and cartilaginous endplate cells were cultured in monolayer or micromass cultures, and Alcian blue and Safranin O staining were used to assess the extracellular matrix expression capability.
RESULTS AND CONCLUSION: After 4 days of culture, all three types of intervertebral disc cells began to adhere to the bottom of the dish and gradually showed proliferative vitality. By the 8th day of culture, significant proliferation of the three types of cells was observed, with a spindle-shaped morphology. Notably, the nucleus pulposus cells exhibited multivesicular notochord-like cells. Through real-time fluorescence quantitative PCR and/or western blot assay, it was found that primary nucleus pulposus cells highly expressed Cytokeratin 19 (K19) and Carbonic anhydrase 3 (Car3), primary annulus fibrosus cells highly expressed Secreted protein acidic and cysteine rich (Sparc) and Biglycan (Bgn), and primary cartilaginous endplate cells highly expressed Parathyroid hormone receptor 1 (Pth1r) and Leucyl-tRNA synthetase 2 (Lars2). After micromass culture of primary annulus fibrosus and cartilaginous endplate cells, Alcian blue and Safranin O staining demonstrated that this culture method could enhance the expression capability of the extracellular matrix compared with monolayer culture. These results indicate that primary intervertebral disc cells extracted and cultured by this method have a good morphology and a high level of extracellular matrix expression. This method holds promise as a research tool to aid researchers in understanding the biology of intervertebral discs.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: primary nucleus pulposus cells, primary annulus fibrosus cells, primary cartilaginous endplate cells, cell extraction, cell identification, micromass culture