To model complex biological tissue in vitro, a specific layout for the position and numbers of each cell type is necessary. Establishing such a layout requires manual cell placement in three dimensions (3D) with micrometric precision, which is complicated and time-consuming. Moreover, 3D printed materials used in compartmentalized microfluidic models are opaque or autofluorescent, hindering parallel optical readout and forcing serial characterization methods, such as patch-clamp probing. To address these limitations, we introduce a multi-level coculture model realized using a parallel cell seeding strategy of human neurons and astrocytes on 3D structures printed with a commercially available non-autofluorescent resin at micrometer resolution. Using a two-step strategy based on probabilistic cell seeding, we demonstrate a human neuronal monoculture that forms networks on the 3D printed structure and can establish cell-projection contacts with an astrocytic-neuronal co-culture seeded on the glass substrate. The transparent and non-autofluorescent printed platform allows fluorescencebased immunocytochemistry and calcium imaging. This approach provides facile multi-level compartmentalization of different cell types and routes for pre-designed cell projection contacts, instrumental in studying complex tissue, such as the human brain.

Probabilistic cell seeding and non-autofluorescent 3D-printed structures as scalable approach for multi-level co-culture modeling

Enrico, Alessandro
Conceptualization
;
Zeglio, Erica
Writing – Review & Editing
;
2023-01-01

Abstract

To model complex biological tissue in vitro, a specific layout for the position and numbers of each cell type is necessary. Establishing such a layout requires manual cell placement in three dimensions (3D) with micrometric precision, which is complicated and time-consuming. Moreover, 3D printed materials used in compartmentalized microfluidic models are opaque or autofluorescent, hindering parallel optical readout and forcing serial characterization methods, such as patch-clamp probing. To address these limitations, we introduce a multi-level coculture model realized using a parallel cell seeding strategy of human neurons and astrocytes on 3D structures printed with a commercially available non-autofluorescent resin at micrometer resolution. Using a two-step strategy based on probabilistic cell seeding, we demonstrate a human neuronal monoculture that forms networks on the 3D printed structure and can establish cell-projection contacts with an astrocytic-neuronal co-culture seeded on the glass substrate. The transparent and non-autofluorescent printed platform allows fluorescencebased immunocytochemistry and calcium imaging. This approach provides facile multi-level compartmentalization of different cell types and routes for pre-designed cell projection contacts, instrumental in studying complex tissue, such as the human brain.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/1489379
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