Enhanced 3D Osteogenic Differentiation of Encapsulated Human MSCs in Hyaluronic Acid Core Alginate Shell Capsules under Dynamic Culture Conditions

Abstract

The growing demand for three-dimensional (3D) in vitro models that accurately replicate human tissue to investigate disease mechanisms, enable tissue replacement, and support drug testing is driving the need for more advanced manufacturing platforms. Mesenchymal stem/stromal cells (MSCs) are promising candidates for tissue engineering and stem cell therapies due to their regenerative potential and ethical advantages. However, the reproducible and scalable production of 3D models that truly mimic the human complexity and function poses a major challenge. To address these challenges, we have developed a scalable and semi-automated 3D differentiation process for generating osteogenic microtissue derived from MSCs encapsulated in core-shell capsules (CSCs). To validate material-dependent, osteo-inductive effects of hyaluronic acid (HA), we systematically characterised our encapsulation process with regard to CSC integrity, spheroid formation and viability, as well as diffusion and shear thinning properties. Osteogenic differentiation was performed in static and dynamic culture, using either conventional 6-well plate format or a rotating wall vessel bioreactor. HA-CSCs were compared with inert carboxymethyl cellulose (CMC)-CSCs. In addition, hypoxic culture condition and the implementation of human platelet lysate (hPL) further supported physiological relevance. HA led to the formation of larger spheroids with high cell viability and increased metabolic activity compared to CMC. Calcium phosphate deposition within the extracellular matrix (ECM) and elevated ALP activity confirmed successful osteogenesis in all conditions. Furthermore, both HA and dynamic culture promote osteogenesis, and when combined, synergistically enhance the osteogenic differentiation. In conclusion, we present a semi-automated, high-throughput and xeno-free platform process for the 3D differentiation of MSCs towards osteogenic lineage. By integrating HA, hypoxia and dynamic culture conditions, this capsule-based system enhances the physiological relevance, enabling advanced in vitro disease modelling, drug testing, and scalable microtissue generation while reducing reliance on animal models.