Abstract
Introduction
Cell-based therapies become more and more prominent for the treatment of intervertebral disc (IVD) injuries. Different strategies are under current development and address the restoration of either annulus fibrosus (AF) or nucleus pulposus (NP). Application of such Advanced Therapy Medicinal Products (ATMPs) is strictly regulated. One requirement is to show the identity of the cells, to make sure the cells are indeed AF or NP cells and retained their IVD cell character during manufacturing process before injection to the site of injury. Therefore, we recently identified novel marker genes that discriminate AF and NP cells on tissue level. However, expression of these AF and NP tissue markers has not been investigated in cultured cells, yet. The aim of this study was to proof the tissue marker”s specificity to discriminate cultured AF and NP cells. Furthermore, we evaluated the tissue markers robustness to different cell culture conditions.
Materials & Methods
AF and NP tissue was obtained from human lumbal IVD of five donors (31–45 years) with mild to moderate degenerative changes (Pfirrmann≤3). Cells were isolated by enzymatic digestion and expanded in culture medium containing 10% human serum and 1% antibiotics. To address specificity, AF and NP cells were cultured separately. To address robustness, 1) cells were cultured up to passage P2, 2) cell culture was performed using two different cell culture media and 3) cells were cryopreserved in an optional intermediate step. Gene expression analysis was performed for 11 novel AF and NP tissue marker: LDB2, ADGRL4, EMCN, ANKRD29, OLFML2A, SPTLC3, DEFB1, DSC3, FAM132B, ARAP2, CDKN2B (patent pending).
Results & Discussion
In cell culture, AF and NP cells were indistinguishable by eye. Both AF and NP cells showed same cell morphology and cell growth through monolayer expansion. For most of the tested novel AF and NP tissue marker genes no difference was seen in cultured cells AF and NP cells on mRNA level. Overall marker expression was lower in cultured cells compared to tissue level. Hence, cultured AF and NP cells lost distinct characteristics that they showed before on tissue level. However, three tissue marker genes showed distinct expression in cultured AF and NP cells: LDB2, ARAP2 and DSC3. Furthermore, expression level was not changed by serial monolayer passaging, intermediate cryopreservation or different nutrition supplied by culture media. Hence, cell marker gene expression was robust to different cell culture conditions.
Conclusion
We defined three markers to discriminate cultured AF and NP cells. Gene expression was specific for either AF or NP cells and robust. These novel AF and NP cell markers can be used to test cell identity and to show preservation of cell character in quality control of cell-therapeutic products. Morever markers are of high value for development of new ATMPs for targeted treatment of eigher AF or NP, as well as tissue engineered discs.
