Astrocyte‐Encapsulated Hydrogel Microfibers Enhance Neuronal Circuit Generation

Abstract

Astrocytes, the most representative glial cells in the brain, play a multitude of crucial functions for proper neuronal development and synaptic‐network formation, including neuroprotection as well as physical and chemical support. However, little attention has been paid, in the neuroregenerative medicine and related fields, to the cytoprotective incorporation of astrocytes into neuron‐culture scaffolds and full‐fledged functional utilization of encapsulated astrocytes for controlled neuronal development. In this article, a 3D neurosupportive culture system for enhanced induction of neuronal circuit generation is reported, where astrocytes are confined in hydrogel microfibers and protected from the outside. The astrocyte‐encapsulated microfibers significantly accelerate the neurite outgrowth and guide its directionality, and enhance the synaptic formation, without any physical contact with the neurons. This astrocyte‐laden system provides a pivotal culture scaffold for advanced development of cell‐based therapeutics for neural injuries, such as spinal cord injury.

Generation of Functional Mouse Hippocampal Neurons

“…After the mounting medium is dry the samples can be analyzed by confocal imaging. Note: Although in this protocol we show that differentiated hippocampal neurons can be obtained by 6 days in culture and we recommend the use of Ara-C for improving neuronal culture purity ( Figure 8 and Figure 9 ), in some cases co-culturing neurons with glial cells can further support neuronal maturation and long-term survival ( Kaech and Banker, 2006 ; Kim et al, 2020 ). Functional phenotypic characterization of hippocampal neurons by calcium imaging ( Figure 10 and Video 1 ) A further test of functionally differentiated neurons includes imaging of the calcium dynamic occurring in vitro following spontaneous or glutamate-induced depolarization ( Grienberger and Konnerth, 2012 ).…”

“…After the mounting medium is dry the samples can be analyzed by confocal imaging. Note: Although in this protocol we show that differentiated hippocampal neurons can be obtained by 6 days in culture and we recommend the use of Ara-C for improving neuronal culture purity ( Figure 8 and Figure 9 ), in some cases co-culturing neurons with glial cells can further support neuronal maturation and long-term survival ( Kaech and Banker, 2006 ; Kim et al, 2020 ).…”

“…Many protocols have been developed for generating cortical and hippocampal neuron cultures by co-culturing neurons with glial feeders ( Kaech and Banker, 2006 ) describing three-dimensional neuronal culture systems with astrocytes encapsulated in hydrogel microfibers ( Kim et al , 2020 ), supplementing culture medium with growth factors for long-term neuron cultures ( Ray et al , 1993 ), or generating neurons from post-natal mice ( Beaudoin et al , 2012 ). Each of these methodologies presents advantages and disadvantages depending on the goals of the experiment.…”

Colonization versus encapsulation in cell-laden materials design: porosity and process biocompatibility determine cellularization pathways

“…electrospun methacrylated hyaluronate; MeHA fibres for meniscus [269], gelatin methacryloyl; GelMA hydrogels [270] and freeze-dried mineralized collagen scaffolds for bone [271], and bioprinted GelMA and GelMA-Hap hydrogels for osteochondral repair [272]), and encapsulation (e.g. hydrogels for intervertebral disc [273], wet-spun GelMA yarns for tendon [274] and hydrogel microfibres for spinal cord [275] repair, respectively).…”

Challenges and Opportunities of Targeting Astrocytes to Halt Neurodegenerative Disorders

“…The transplantation of astrocytes genetically modified to overexpress trophic and transcription factors along with neural progenitor cells (NPCs) was tested in a rat model of Parkinson's disease, where it ameliorated the phenotype by promoting differentiation and survival of NPCs into dopaminergic neurons [88,103]. Related to these approaches, it should be mentioned that they may profit soon from the technological advancements provided by research in biomaterials aiming at boosting the astrocytes neuroprotective properties [104] as well as shielding the implanted cells from the immune system, thereby enhancing their survival [105].…”