Up-scaled production and differentiation of chondrogenic aggregates for endochondral bone regeneration

Chondrogenic aggregates have emerged as promising building blocks for the development of implants intended to facilitate bone defect regeneration through the endochondral bone regeneration process. While most protocols rely on static culture systems to create these cell aggregates, scalability limitations have impeded their broader application. In this regard, dynamic culture systems offer an advantageous approach by enabling automated aggregate production, which not only ensures scalability and workload reduction but also aligns with good manufacturing practices (GMP) and facilitate clinical translation. In this study, we investigated the feasibility of collision-based self-assembly of human bone marrow-derived mesenchymal stromal cell (hBM-MSC) aggregates and their subsequent chondrogenic differentiation within the same spinner flask setup. Moreover, qualitative analyses of neural cell adhesion molecule (NCAM) protein expression were performed to study the effect of the hydrodynamic environment on cell aggregation in comparison to static culture. Given that stirring rate influences cell-cell collisions, in this study different velocities of the spinner flask were examined to determine the most favourable one for spontaneous hBM-MSC aggregate formation. The identification of appropriate stirring rate allowed dynamic spinner flask culture (1x10^6 cells/mL) of the spontaneously formed aggregates for up to 21 days using two different donors. Dynamically cultured aggregates showed significant variation in aggregates size and shape as opposed to static culture. Importantly, dynamic culture did not affect aggregate viability, although cell death was observed due to the large size of the aggregates. When assessing cell differentiation, staining of dynamically cultured aggregate sections for glycosaminoglycans (GAGs) and collagen II showed inter- and intra-aggregate heterogeneity in comparison to static culture. Moreover, NCAM protein expression was not modulated by the hy