Development of Three-Dimensional Culture System for Maintenance of Bovine Nucleus Pulposus Cells
Authors: Li H. Y. , Ma J. N. , Chooi W. H. , Chan D. , Cheah K. S. E. , Chan B. Pui 
Published: May 2014

Introduction Nucleus pulposus (NP) is located at the center of the intervertebral disc (IVD) and rich in collagen type II and proteoglycans. The glycosaminoglycans (GAGs) in the proteoglycans enable the retention of a high amount of water, which is important for the mechanical function of the IVD. In the pathology of degenerative disc disease (DDD), the loss of proteoglycan and water may be related to changes in NP cells (NPCs). To enable studies in disc cell biology, developing a 3D culture system for NPCs with maintained phenotype is critical. In this study, we compared two microencapsulation systems using collagen alone and collagen-glycosaminoglycan (GAG) co-precipitate, respectively, in maintaining NPC phenotype and used a micropatterning platform to screen for the type of extracellular matrix components. Materials and Methods NP from bovine caudal spines was digested to isolate bovine NPCs (bNPCs). In the first 3D culture system, the bNPCs were encapsulated in microspheres made of collagen and then cultured up to 2 weeks before evaluation. The second 3D culture system was based on collagen-GAG co-precipitate. In brief, it was fabricated by mixing chondroitin-6-sulfate, aminated type I collagen, and bNPCs, followed by centrifugation to collect the constructs, which were then cultured up to 2 weeks followed by evaluation. Lastly, protein microstructures were fabricated using a laser-based crosslinking technique. Bovine serum albumin (BSA), together with extracellular matrix (ECM) proteins such as collagen, was crosslinked using laser and rose bengal. Cells were seeded onto these microstructures and evaluated. Cell morphology and distribution were revealed by histology and actin staining. Viability was examined by live/dead staining. Phenotype and components in the ECM were analyzed by immunohistochemistry and histology. Results Contraction of microspheres was monitored over time, especially from day 3 to day 7. Increase in cell density was observed over time, with a ring of dense cells at periphery starting to form since day 7. Some rounded cells with large vacuoles, simulating the morphology of bNPC in native NP tissue, were observed on day 7 in collagen microspheres. However, cells with such morphology were not observed in later time points. Most cells were alive throughout the culture period. Alcian blue staining became stronger over time and was particularly strong in later time points at the periphery region. IHC of collagen type II was intense in both the cells and extracellular matrix except on day 3. The local and global mechanical properties of the microspheres may affect the cell morphology. Data on collagen-GAG co-precipitate and protein micropatterns will also be presented. Conclusion The collagen microspheres supported the survival and proliferation of bNPCs and partially maintained some phenotypic characteristics of bNPCs, including the production of collagen type II throughout the microspheres. The local and global mechanical properties of the microspheres may affect the distribution of actin and morphology of the cells. Disclosure of Interest None declared References Chan BP, Ma JN, Xu JY, Li CW, Cheng JP, Cheng SH. Femto-second laser-based free writing of 3D protein microstructures and micropatterns with sub-micrometer features: a study on voxels, porosity, and cytocompatibility. Adv Funct Mater 2014;24(3):277–294 Chan BP, Hui TY, Yeung CW, Li J, Mo I, Chan GC. Self-assembled collagen-human mesenchymal stem cell microspheres for regenerative medicine. Biomaterials 2007;28(31):4652–4666 Choy AT, Leong KW, Chan BP. Chemical modification of collagen improves glycosaminoglycan retention of their co-precipitates. Acta Biomater 2013;9(1):4661–4672 Yuan M, Leong KW, Chan BP. Three-dimensional culture of rabbit nucleus pulposus cells in collagen microspheres. Spine J 2011;11(10):947–960