Abstract Methods for studying brain function and disease heavily rely on in vivo animal models, ex-vivo tissue slices, and 2D cell culture platforms. These methods all have limitations that significantly impact the clinical translatability of results. Consequently, models able to better recapitulate some aspects of in vivo human brain are needed as additional preclinical tools. In this context, 3D hydrogel-based in vitro models of the brain are considered promising tools. To create a 3D brain-on-a-chip model, a hydrogel capable of sustaining neuronal maturation over extended culture periods is required. Among biopolymeric hydrogels, chitosan-β-glycerophosphate thermogels have demonstrated their versatility and applicability in the biomedical field over the years. In this study, we investigated the ability of this thermogel to encapsulate neuronal cells and support the functional maturation of a 3D neuronal network in long-term cultures. To the best of our knowledge, we demonstrated for the first time that chitosan-β-glycerophosphate thermogel possesses optimal characteristics for promoting neuronal growth and the development of an electrophysiologically functional neuronal network derived from both primary rat neurons and neurons differentiated from human induced pluripotent stem cells co-cultured with astrocytes. Specifically, two different formulations were firstly characterized by rheological, mechanical and injectability tests. Primary nervous cells and neurons differentiated from human induced pluripotent stem cells were embedded into the two thermogel formulations. The 3D cultures were then deeply characterized by immunocytochemistry, confocal microscopy, and electrophysiological recordings, employing both 2D and 3D micro-electrode arrays. The thermogels supported the long-term culture of neuronal networks for up to 100 days. In conclusion, chitosan-β-glycerophosphate thermogels exhibit excellent mechanical properties, stability over time under culture conditions, and bioactivity towards nervous cells. Therefore, they are excellent candidates as artificial extracellular matrices in brain-on-a-chip models, with applications in neurodegenerative disease modeling, drug screening, and neurotoxicity evaluation.
Methods for studying brain function and disease heavily rely on in vivo animal models, ex-vivo tissue slices, and 2D cell culture platforms. These methods all have limitations that significantly impact the clinical translatability of results. Consequently, models able to better recapitulate some aspects of in vivo human brain are needed as additional preclinical tools. In this context, 3D hydrogel-based in vitro models of the brain are considered promising tools. To create a 3D brain-on-a-chip model, a hydrogel capable of sustaining neuronal maturation over extended culture periods is required. Among biopolymeric hydrogels, chitosan-beta-glycerophosphate (CHITO-beta-GP) thermogels have demonstrated their versatility and applicability in the biomedical field over the years. In this study, we investigated the ability of this thermogel to encapsulate neuronal cells and support the functional maturation of a 3D neuronal network in long-term cultures. To the best of our knowledge, we demonstrated for the first time that CHITO-beta-GP thermogel possesses optimal characteristics for promoting neuronal growth and the development of an electrophysiologically functional neuronal network derived from both primary rat neurons and neurons differentiated from human induced pluripotent stem cells (h-iPSCs) co-cultured with astrocytes. Specifically, two different formulations were firstly characterized by rheological, mechanical and injectability tests. Primary nervous cells and neurons differentiated from h-iPSCs were embedded into the two thermogel formulations. The 3D cultures were then deeply characterized by immunocytochemistry, confocal microscopy, and electrophysiological recordings, employing both 2D and 3D micro-electrode arrays. The thermogels supported the long-term culture of neuronal networks for up to 100 d. In conclusion, CHITO-beta-GP thermogels exhibit excellent mechanical properties, stability over time under culture conditions, and bioactivity toward nervous cells. Therefore, they are excellent candidates as artificial extracellular matrices in brain-on-a-chip models, with applications in neurodegenerative disease modeling, drug screening, and neurotoxicity evaluation.
Long-term in vitro culture of 3D brain tissue model based on chitosan thermogel
Muzzi L.;Lagazzo A.;Andolfi A.;Martinoia S.;Pastorino L.
2024-01-01
Abstract
Methods for studying brain function and disease heavily rely on in vivo animal models, ex-vivo tissue slices, and 2D cell culture platforms. These methods all have limitations that significantly impact the clinical translatability of results. Consequently, models able to better recapitulate some aspects of in vivo human brain are needed as additional preclinical tools. In this context, 3D hydrogel-based in vitro models of the brain are considered promising tools. To create a 3D brain-on-a-chip model, a hydrogel capable of sustaining neuronal maturation over extended culture periods is required. Among biopolymeric hydrogels, chitosan-beta-glycerophosphate (CHITO-beta-GP) thermogels have demonstrated their versatility and applicability in the biomedical field over the years. In this study, we investigated the ability of this thermogel to encapsulate neuronal cells and support the functional maturation of a 3D neuronal network in long-term cultures. To the best of our knowledge, we demonstrated for the first time that CHITO-beta-GP thermogel possesses optimal characteristics for promoting neuronal growth and the development of an electrophysiologically functional neuronal network derived from both primary rat neurons and neurons differentiated from human induced pluripotent stem cells (h-iPSCs) co-cultured with astrocytes. Specifically, two different formulations were firstly characterized by rheological, mechanical and injectability tests. Primary nervous cells and neurons differentiated from h-iPSCs were embedded into the two thermogel formulations. The 3D cultures were then deeply characterized by immunocytochemistry, confocal microscopy, and electrophysiological recordings, employing both 2D and 3D micro-electrode arrays. The thermogels supported the long-term culture of neuronal networks for up to 100 d. In conclusion, CHITO-beta-GP thermogels exhibit excellent mechanical properties, stability over time under culture conditions, and bioactivity toward nervous cells. Therefore, they are excellent candidates as artificial extracellular matrices in brain-on-a-chip models, with applications in neurodegenerative disease modeling, drug screening, and neurotoxicity evaluation.File | Dimensione | Formato | |
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