MicroRNA profiling and analysis of primary OPLL cell derived exosomes

In order to investigate the function and mechanism of Ossification of Posterior Longitudinal Ligament (OPLL) primary ligament cell derived exosomes, we take advantages of high throughput sequencing technology to fully reveal the small RNA content of both OPLL and normal posterior longitudinal ligament (PLL) cell derived exosomes. Overall design: We collect the posterior longitudinal ligament tissues of OPLL and PLL patients during surgery, PLL samples were collected from myelopathy patients who underwent ACCF operation. The collected samples were washed with PBS and subjected to primary culture immediately. The samples were cut into small pieces and attached to culture flasks for primary culture, 10%FBS high-glucose DMEM were used as culture medium for primary ligament cell culture. When cells reach 80% confluent, the cell were lysed with trypsin for passage. After 2 passages, the primary cells were ready to perform exosome collection. For exosome collection, we use 1% exosome free FBS/DMEM for culture, the medium were collected at a 3-days interval, and the collected culture medium of both OPLL and PLL primary cell were subsequently subjected to ultracentrifugation. After the ultarcentrifugation, the pellet were washed with PBS and ultarcentrifugated again before use.