3D bioprinted human neuron‐like spheroids as an in vitro model for neurodegenerative diseases

Abstract

AbstractBackgroundThe limited number of reproducible in vitro models for neurobiology purposes consists of a difficult challenge, primarily because naturally, in the organism, cells are organized three‐dimensionally. The concept of developing three‐dimensional (3D) models to further elucidate brain organization, mechanisms, and pathways has the importance of reaching characteristic features of native tissue that facilitate the comprehension of complex neurodegenerative diseases, such as Alzheimer’s disease (AD). Bioprinting has revolutionized the concept of three‐dimensionality, producing complex structures and sculptural constructs. This study aims to demonstrate the first steps of a novel 3D bioprinted construct, illustrating that neuron‐like cells organize themselves as spheroids when cultured in a 3D environment.MethodFirst, the bioink was prepared by adding human neuroblastoma cells (SH‐SY5Y), differentiated into a more mature neuron‐like phenotype by the addition of 2% retinoic acid, for seven days into a hydrogel consisting of an Alginate/Gelatin solution. As control cells were cultured in the absence of retinoic acid. The constructs were bioprinted and crosslinked by the addition of 2% CaCl2. The swelling degree of printed hydrogel and morphological cytoskeletal organization of the neuron‐like spheroids were evaluated.ResultThe developed hydrogel has a high swellability, increasing water retention upon long‐term immersion (Figure 1A). Swellability is an important feature, indicating the hydrophilicity of the construct, which facilitates cell‐cell interactions and culture medium diffusion into the construct core. Importantly, when cellular organization within the construct was evaluated, we observed that the SH‐SY5Y cells arranged themselves as spheroids (Figure 1B), a valuable tool for investigating the 3D cellular organization. This feature reproduces important natural tissue characteristics, such as producing extracellular matrix components and forming a network of cell‐cell interaction, which cannot be reproduced in 2D structures. Moreover, neurite outgrowth is observed inside the spheroidal construct with differentiated cells (Figure 1B).ConclusionThe 3D bioprinted construct is highly suitable and advantageous as a neurobiological 3D model. Based on this construct, further studies are being developed to better simulate AD in vitro, building an interactive disease model. Acknowledgments: This study was supported by São Paulo Research Foundation (FAPESP, grants 2022/08664‐4 and 2018/45605‐8), and Brazilian National Council for Scientific and Technological Development (CNPq, grant 406258/2022‐8).