From the Cover: Bioengineered functional brain-like cortical tissue

The brain remains one of the most important but least understood tissues in our body, in part because of its complexity as well as the limitations associated with in vivo studies. Although simpler tissues have yielded to the emerging tools for in vitro 3D tissue cultures, functional brain-like tissues have not. We report the construction of complex functional 3D brain-like cortical tissue, maintained for months in vitro, formed from primary cortical neurons in modular 3D compartmentalized architectures with electrophysiological function. We show that, on injury, this brain-like tissue responds in vitro with biochemical and electrophysiological outcomes that mimic observations in vivo. This modular 3D brain-like tissue is capable of real-time nondestructive assessments, offering previously unidentified directions for studies of brain homeostasis and injury.The brain possesses extraordinary connectivity of neural networks. This complexity is evident at multiple levels of structural and functional hierarchy, including microcircuits dominated by neuronal clusters and larger distinctive regions of grey matter interconnected by white matter axon tracts. These features are highlighted in the Blue Brain project (1) and the Human Connectom Project (2) that aim to compile detailed information about connectivity at various levels and ultimately, reconstruct the human brain as a large-scale network. However, at the tissue level, the complex interconnectivity is masked by the distribution of neurons, such as in the stratified laminar layers of the neocortex. Although functionally related neurons generally group together (3, 4), boundaries of functional units cannot be readily revealed with phenotype markers, necessitating electrophysiological studies and correlative functional outcomes. It is, therefore, necessary to differentiate physical and functional associations of neuronal populations to unravel complex networks.Three-dimensional tissue engineering could provide compartmentalized cultures of discrete and identifiable structures to emulate native tissues and thereby, provide insight into the complexities. By recreating cell–cell and cell–ECM interactions, 3D structures enable the formation of tissue-mimetic architectures and promote more realistic physiological responses than conventional 2D cultures (5). Toward this goal, multilayer lithography (6), 3D patterning of bulk structures (7), and 3D tissue printing (8) are used. These rationally designed structures have been generated for tissue engineering of the lung, liver, and kidney, for which the structure–function relationships are modular-based and well-defined. Recent advances in stem and progenitor cell technology have induced cells to differentiate into and produce tissue-appropriate cell compositions and ECM components and form biomimetic tissues with nascent functions, including the cerebral organoids (9). These technologies show self-organization capability of cells in tissue-mimetic environments, such as native tissue-derived decellularized scaffolds (10–13). However, densely packed brain tissue with an architecture defined by neuronal connectivity (14, 15) presents a unique challenge to define modular structures with specific functions. Rather than reconstructing a whole-brain network, we aimed at reducing the structural complexity to fundamental features that are relevant to tissue-level physiological functions.Neural connectivity at the basic level, which includes segregated neuronal and axonal compartments, is particularly relevant for brain disorders, such as diffuse axonal injury in brain trauma (16, 17). However, ECM gel-based in vitro 3D systems have not yielded tissue-level functional assessments, possibly because of their inadequate mechanical properties and fast degradation compared with brain tissue. Here, we developed 3D compartmentalized neuronal cultures with silk fibroin-based biomaterials offering tunable mechanical properties, versatile structural forms, and brain and neural culture compatibility (18–21). This brain-like tissue provides rudimentary but relevant features of brain neural networks. The physiologically relevant and responsive 3D brain-like tissue also shows capability for the assessment of brain disorders, such as traumatic brain injury (TBI).