"Cartilage microtissues are promising tissue modules for bottom up biofabrication of implants leading to bone defect regeneration. Most of the protocols for the development of these cartilaginous microtissues have been carried out in static setups, however in order to further achieve higher scales suspension process needs to be investigated. In the present study we explored the impact of dynamic culture in a novel stirred microbioreactor system.
In order to generate cartilaginous microtissues, we first allowed cells to aggregate for 3 days, forming stable microtissues, before inoculating the bioreactors. Moreover a coupled CBM-CFD model was used in order to estimate the magnitude of shear stress on the individual microtissues. Experiments with the miniBR setup illustrated that we could culture cells dynamically in to form of microaggregates for up to 14 days. Furthermore, we observed that spheroids were able to fuse into larger structures , we used two static controls; one where spheroids cultured with the same dimensions as the initial inoculation and a second control where multiple spheroids were fused in to larger sized at the same time point as we inoculated the bioreactor. We observed that viability was not affected ether by the fusion event on (day2) or size of the microtissues while the same applied for culture in the bioreactor. Additionally, we saw a distinct difference between static and dynamic condition with a much lower fraction of EDU+ cells present in the bioreactor condition. This difference could be linked to the commitment of cells towards ECM production rather than proliferation something that is known during chondrogenic differentiation. This can be further evidenced by the histologic sections and Alcian Blue staining. Gene expression values showed a dramatic upregulation of both Indian hedgehog (IHH, 30-fold) and Collagen type X (COLX, 23-fold) well know marker of chondrogenic hypertrophy, for the day7 and day 14 time points for the dynamically cultured microtissues. Moreover Chondromodulin also showed a large upregulation (16-fold) for the last time point. Similarly to the transcriptome level, we saw distinct metabolic profiles between static and dynamic conditions. More specifically, whereas both conditions were characterized by high glucose consumption and lactate production at Day 7 and Day 14, in the bioreactor condition there was significant increase in glucose consumption and lactate production for Day 14 and a similar trend as well at Day 7. Moreover, several amino acids such as proline and aspartate showed significant differences between the conditions.
In this study we explored the impact of bioreactor-cultured microtissues in suspension as compared to statically cultured ones and the influence of shear stress on the acceleration of chondrogenic differentiation towards hypertrophy.Our work provides insights on the effect of the process environment on critical cellular, molecular, and metabolic parameters, and a straightforward strategy for the scalable production of cartilage intermediate microtissues."