Hydrogel encapsulation and micro-aggregation of co-culture MSC/AC for tissue regeneration

Abstract

Event Abstract Back to Event Hydrogel encapsulation and micro-aggregation of co-culture MSC/AC for tissue regeneration Mylene De Ruijter1, Ferry P. Melchels1, 2, Riccardo Levato1 and Jos Malda1, 3 1 University Medical Center, Department of Orthopaedics, Netherlands 2 School of Engineering and Physical Sciences, Heriot-Watt University, Institute of Biological Chemistry, Biophysics and Bioengineering, United Kingdom 3 Faculty of Veterinary Medicine, Utrecht University, Department of Equine Sciences, Netherlands Introduction: Cell-laden hydrogels are promising and versatile biomaterial platforms to generate three-dimensional (3D) engineered cartilage tissue. 3D culture systems, beneficial for tissue regeneration, can be created using hydrogels such as gelatine-methacrylamide (gelMA), and might be improved by adding native matrix components such as hyaluronic acid[1],[2]. However, to achieve effective matrix synthesis by encapsulated cells, high cell densities are currently used. Both cell-cell contact and co-culture systems improve matrix production and aggregation of chondrocytes, ensuring cell-cell contact, can increase cartilage regeneration, using a feasible amount of cells[3],[4]. The aim of this study is to create a micro-aggregate system with co-culture of mesenchymal stromal cells (MSCs) and articular chondrocytes (ACs) that can be encapsulated in a hydrogel to enhance matrix deposition using a limited amount of cells, and with potential for biofabrication. Thus, gelMA hydrogels modified with hyaluronic acid methacrylate (HAMA) were synthesized, and tested for printability as micro-aggregate laden bioinks. Methods: First, a stamp consisting of 3924 pyramids (base area = 0.044 mm2, height = 0.31 mm) was made via stereolithography. Second, pyramid shaped micro-wells were stamped into an agarose gel (4% w/v) (Fig 1A & B). ACs and MSCs (20:80 ratio) were harvested from equine donors (n=3), and co-cultured in the micro-wells. Cell seeding densities of 25, 50, 75 and 100 cells/aggregate were used to evaluate aggregation reproducibility. After 6 days the aggregates were harvested and encapsulated in gelMA/HAMA (5%/1.5% w/v) hydrogels. The gels were UV-crosslinked, and cultured in co-culture medium[3] for 8 weeks. Viability and diameter of the aggregates was assessed via a live/dead assay. Distribution of MSC/AC was investigated using 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate membrane staining. Finally, printability of aggregates in gelMA/HAMA was assessed. Results and Discussion: Diameter of the micro-aggregates was 27.8±2.6, 34.6±6.5, 46.1±3.3, 61.9±11 µm for 25, 50, 75, 100 cells/aggregate, respectively. Furthermore, cells showed high viability after aggregation (Fig 1C). Moreover, gelMA/HAMA hydrogel supported cell biosynthetic activity for the 8 weeks culture period, and micro-aggregates were not affected by the printing process, showing a viability of 89.26% ± 1.43. The advantageous effect of cell aggregation, in combination with co-culturing and 3D encapsulation, makes the micro-aggregate system a promising tool for tissue regeneration. Furthermore, the excellent printability of the aggregates in gelMA/HAMA (Fig 1D) provides opportunities for the biofabrication field. Conclusion: Fabricating micro-aggregates of MSC/AC succeeded with a high reproducibility and cell viability. Moreover, the printability of gelMA/HAMA 5%/1.5% is good, and shows great potential as aggregate carrier for tissue engineering applications. Dutch Arthritis Foundation