Colony formation and matrix production by human anulus cells - Modulation in three-dimensional culture

Study Design. Human intervertebral disc cells from the anulus were tested in a study of colony formation and extracellular matrix (ECM) production during long-term three-dimensional culture with exposure to selected cytokines. Experimental studies were approved by the authors' Human Subjects Institutional Review Board. Objectives. To quantitatively evaluate colony formation and qualitatively assess ECM production ( using immunohistochemistry and in situ hybridization) in cells derived from Thompson Grades I to V discs and tested in culture with cytokines and nutrient supplementation. Summary of the Background Data. Human intervertebral disc cells offer special in vitro challenges because of the slow-growing nature of these cells and their need for specialized three-dimensional in vitro conditions, which permit the expression and production of proteoglycans and Type II collagen, two ECM products that are important for disc cell biology. Methods. Discs from 9 human subjects ( 2 control donors and 7 surgical patients, Thompson Grades I - V), mean age 35.8 years, were used to obtain anulus cells to be tested in three-dimensional agarose culture. Tests of specialized growth conditions included treatment with ITS ( insulin-transferrin-sodium selenite supplement), insulin-like growth factor I (IGF-I), and transforming growth factor-beta1 (TGF-beta1). Cultures were evaluated after 14 to 36 days of culture for % colony formation and cell numbers/ colony; immunocytochemistry, in situ hybridization, and quantitative histology were used to evaluate colony formation and ECM production. Results. Data showed that compared with the average 17.5% colony formation observed in controls, ITS, TGF-beta1 and ITS with IGF-I significantly increased colony formation (28.4%, 30.4%, and 30.4%, respectively, P less than or equal to 0.04). Even cells derived from Thompson Grade V disc showed responsiveness to cytokines and improved production of ECM in vitro. Conclusions. Findings indicated that cells derived from discs with advanced degeneration were still responsive to cytokines and could be modulated to produce Type II collagen and proteoglycans in three-dimensional culture by the addition of enriched media and selected cytokines. Such findings are important since they advance our understanding of how to modulate disc cell behavior in vitro, and may have application to potential future biologic therapies for disc degeneration.