The study of human brain physiology and diseases like Parkinson´s and Alzheimer’s requires precise modeling of complex tissue. The complexity derives from neurons and the other cell types present in the brain, particularly with astrocytes, in a three-dimensional (3D) environment. However, most of the current studies simplify the complexity of neuronal tissue by either using 2D co-culture models or by studying 3D hydrogels with randomly dispersed cells of different types. Recent 3D models obtained using two-photon polymerization allow a precise organization of the cells, but cell placement and biological readout are performed with complicated and time-consuming procedures. Moreover, photopolymerized structures are typically fluorescent, introducing a strong background noise for fluorescence-based microscopies. To address these issues, we present a scalable method that allows a co-culture model with control positioning of the cells in a non-fluorescent two-photon polymerized 3D structure on a glass substrate. A two-step seeding strategy allows the generation of an artificial network of human neurons on the 3D printed structure separated from the astrocytic-neuronal co-culture seeded on the glass substrate. The resin chosen for the study is transparent and not autofluorescent after printing, facilitating the detection and quantification of weak fluorescence signals. As a proof-of-concept, we recorded the calcium activity of several neuronal colonies by fluorescence miscopy, avoiding time-consuming patch-clamp recording. Therefore, this platform will help study the interactions between different cell types by decoupling contact-mediated and biochemical signaling.
Defined neuronal-astrocytic interactions enabled with a 3D printed platform
Alessandro EnricoConceptualization
;Erica Zeglio;
2022-01-01
Abstract
The study of human brain physiology and diseases like Parkinson´s and Alzheimer’s requires precise modeling of complex tissue. The complexity derives from neurons and the other cell types present in the brain, particularly with astrocytes, in a three-dimensional (3D) environment. However, most of the current studies simplify the complexity of neuronal tissue by either using 2D co-culture models or by studying 3D hydrogels with randomly dispersed cells of different types. Recent 3D models obtained using two-photon polymerization allow a precise organization of the cells, but cell placement and biological readout are performed with complicated and time-consuming procedures. Moreover, photopolymerized structures are typically fluorescent, introducing a strong background noise for fluorescence-based microscopies. To address these issues, we present a scalable method that allows a co-culture model with control positioning of the cells in a non-fluorescent two-photon polymerized 3D structure on a glass substrate. A two-step seeding strategy allows the generation of an artificial network of human neurons on the 3D printed structure separated from the astrocytic-neuronal co-culture seeded on the glass substrate. The resin chosen for the study is transparent and not autofluorescent after printing, facilitating the detection and quantification of weak fluorescence signals. As a proof-of-concept, we recorded the calcium activity of several neuronal colonies by fluorescence miscopy, avoiding time-consuming patch-clamp recording. Therefore, this platform will help study the interactions between different cell types by decoupling contact-mediated and biochemical signaling.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.