MicroRNA profiling of normal and imflammation induced nuclues pulposus and annulus fibrosis primary cells

In order to investigate the changes and different function of normal and degenerated nuclues pulposus (NP) and annulus fibrosis (AF) derived exosomes, we take advantages of high through-put sequencing technology to fully reveal the small RNA content of both normal and degenerated NP and AF primary cell derived exosomes. Overall design: We collect the normal NP and AF tissues samples from spine fracture patients who underwent discectomy. The collected samples were washed with PBS and subjected to primary culture immediately. The samples were first operated under magnifier to separate NP and AF tissue, the tissues were then lysed using type II collagenase and the lysed cells were put to culture flasks for primary culture. 10%FBS high-glucose DMEM were used as culture medium for primary 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 were subsequently subjected to ultracentrifugation. After the ultarcentrifugation, the pellet were washed with PBS and ultarcentrifugated again before use.