The Effects of Mono layer Subculture on the Phenotype and Growth of Degenerated Human Intervertebral Disc Cells

This study investigated the changes in the growth and phenotype of human degenerated intervertebral disc cells depending on the frequency of subculture in vitro monolayer culture system. The annulus fibrosus (AF) and nucleus pulposus (NP) cells were cultured in a monolayer manner. At each subculture, we performed an analysis of the morphological changes, the adhesion rate, the proliferation rate and the viability. The expressions of type I, II collagen and proteoglycan were analyzed at the mRNA gene level. All three times of subculture were performed. Both the AF and NP cells gradually showed a fibroblast-like spindle shape from their irregular, polygonal shape while undergoing the third time of subculture. The cell proliferation was the highest at the second subculture time. The viability was markedly lower before the first subculture, but there was no significant difference of viability between the three times of subculture. On RT-PCR, the type II collagen expression was gradually decreased and almost unexpressed at the third subculture, but the type I collagen expression was gradually increased in the NP cells. In the AF cells, the type I collagen expression did not show a significant difference depending on the frequency of subculture. Type II collagen was not expressed from the second time of subculture. The expression of proteoglycan was gradually decreased in both the AF and NP cells and it was almost unexpressed at the third time of subculture. In conclusions, after three times of subculture, the disc cells had completely changed their original growth and phenotypic characteristics.