For decades, two-dimensional (2D) cell culture has been the standard model for biological research due to its simplicity, scalability, and ease of use. However 2D culture does not reproduce the complex three-dimensional structure found in living tissues. As a result, cellular behavior observed in vitro often differs from that observed in vivo, limiting the translational relevance of experimental findings. The emergence of spheroids and organoids has significantly improved our ability to model human tissues by recreating key aspects of tissue architecture and cell–cell interactions. However, current 3D culture methods still face important challenges, including poor reproducibility, variability in organoid size and morphology, organoid fusion, hypoxia-induced necrosis, and the need for large volumes of culture media and continuous agitation.
The work presented in this thesis aimed to address these limitations through the development of AirLiwell, a novel culture platform that combines non-adhesive microwells with an air–liquid interface. This approach allows spheroids and organoids to be cultured individually while providing more controlled oxygen access throughout the tissue. As a result, AirLiwell reduces hypoxic stress, prevents fusion between organoids, improves culture standardization, and supports long-term viability. The platform also simplifies handling, reduces media consumption, and is compatible with GMP manufacturing requirements.
The first application focused on regenerative medicine, specifically autologous fat grafting. Although widely used in reconstructive surgery, fat graft survival remains unpredictable because of partial graft resorption over time. Adipose-derived stem cell (ASC) spheroids generated with AirLiwell maintained their stem-cell characteristics and multipotent differentiation potential while exhibiting an enhanced regenerative profile. Their secretome was enriched in regenerative factors compared with ASCs cultured in monolayer. When incorporated into fat grafts in a murine transplantation model, ASC spheroids significantly improved long-term graft retention, demonstrating their potential to enhance current reconstructive procedures. The second application explored the production of dopaminergic midbrain organoids for cell replacement therapy in Parkinson’s disease. Conventional culture methods often generate organoids with heterogeneous sizes and necrotic cores. In contrast, AirLiwell produced organoids with highly consistent morphology and no detectable necrosis. Transcriptomic and immunohistochemical analyses confirmed efficient neuronal and dopaminergic differentiation. Single-cell RNA sequencing further revealed a markedly improved cellular composition, with AirLiwell organoids consisting of 99% neural cells compared with only 61% in conventional immersion cultures. In addition, electrophysiological recordings showed highly synchronized neuronal activity, whereas standard culture conditions produced more heterogeneous and poorly coordinated responses. These characteristics are particularly important for generating standardized and clinically relevant dopaminergic grafts for future cell replacement therapies.
Overall, AirLiwell provides a robust platform for spheroid and organoid culture. Beyond the applications described here, its successful use in liver, bone marrow, and cancer models highlights its versatility.
