The quest to unravel the complexities of the human brain has long been at the forefront of scientific inquiry. Central to this pursuit is the understanding of the intricate networks of neurons and glial cells that constitute the brain’s architecture. Traditional animal models, while invaluable, fall short in mimicking the sophisticated information processing capabilities of the human brain.
This discrepancy stems from fundamental differences in neuronal composition, synaptic integration, and astrocytic complexity between humans and other animals. Recognizing these limitations, researchers have turned to advanced biotechnologies to create more accurate models of human neural tissue.
Recent advancements in stem cell research have ushered in a new era of possibilities. Scientists have successfully engineered human neural tissues with three-dimensional (3D) cytoarchitectures from human pluripotent stem cells (hPSCs), including both induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs).
These developments leverage various culture systems, such as hydrogel cultures, scaffold-based cultures, acoustic levitational assembly, self-organized brain spheroids/organoids, and brain-on-a-chip models. Among these, 3D bioprinting emerges as a leading technology, offering unparalleled precision in fabricating neural tissues by spatially depositing living cells within biologically complex structures.
However, the journey towards creating functional neural tissues is fraught with challenges. Traditional methods, while innovative, often involve the use of scaffolds or molds that are not biodegradable, hindering cell migration and neural network formation. Furthermore, achieving a balance between structural support and functional capability in soft tissue bioprinting has proven difficult. Many 3D printed neural tissues, although structurally layered, lack the functional connectivity between neurons and glia that is crucial for mimicking actual brain tissue.
In response to these challenges, a novel approach has been developed, focusing on assembling neuronal and glial subtypes into 3D neural tissues with functional connections. This method employs extrusion bioprinting, a technique that prints one layer alongside another horizontally, diverging from the traditional vertical stacking. This innovation not only maintains the structural integrity of the neural tissue but also facilitates live-cell imaging and electrophysiological recording, offering a groundbreaking platform for studying human neural networks under various conditions.
The success of this bioprinting approach hinges on the selection of an appropriate biomaterial, or bioink. Fibrin hydrogel, composed of fibrinogen and thrombin, has been identified as an ideal candidate due to its biocompatibility with neural cells. It supports cell survival, promotes functional maturation, and maintains structural stability, making it an optimal choice for neural tissue bioprinting.
The extrusion 3D bioprinting technique stands out for its ability to closely mimic the laminations of the human cortex, offering a promising avenue for constructing multi-layered brain tissues. Critical to this process is the control over the tissue’s thickness, ensuring adequate oxygen and nutrient diffusion essential for cell growth and function. By limiting each layer’s thickness to 50 μm and depositing layers horizontally, researchers have achieved a multi-layered, yet functional, neural tissue model.
This breakthrough in 3D bioprinting technology represents a significant leap forward in the field of neuroscience. It not only addresses the limitations of traditional animal models and earlier bioprinting methods but also opens new avenues for research into brain health and disease. As this technology continues to evolve, it promises to deepen our understanding of the human brain’s complexities, paving the way for novel therapeutic strategies and advancing our quest to decipher the most enigmatic organ in the human body.
Discussion
Harnessing 3D Bioprinting for Functional Human Neural Tissues: Insights and Innovations
The advent of 3D bioprinting technology has opened new avenues in neuroscience research, offering unprecedented opportunities to explore the complexities of human neural networks. Our recent achievements in printing functional human neural tissues underscore the potential of this technology to bridge the gap between conventional animal models and the intricate architecture of the human brain. Through meticulous design and innovation, we have developed a bioink conducive to neuronal survival, neurite outgrowth, and synapse formation, facilitating the assembly of neuronal and glial subtypes into functional 3D neural tissues.
A New Paradigm for Neural Tissue Engineering
Our approach diverges from traditional methodologies by printing tissue bands horizontally, enhancing live-cell imaging and electrophysiological recording capabilities. This orientation allows for the formation of functional synaptic connections between neuronal subtypes and neuron-astrocyte networks within a mere 2–5 weeks post-printing. The printed brain tissues offer a well-defined platform for analyzing human neural networks under both healthy and diseased states, marking a significant leap forward in the field.
Key Features and Advancements
The platform exhibits several distinguishing features that underscore its innovative impact:
- Rapid Establishment of Functional Neural Circuits: Within 2–5 weeks of printing, the tissues demonstrate functional synaptic activity, a feat not easily achieved with other bioprinting methods or organoid cultures due to their reliance on stiff bioinks which impede neurite sprouting.
- Diverse Neuronal Subtype Integration: Our method enables the incorporation of various neuronal subtypes at precise ratios, offering a level of control over tissue composition that surpasses current organoid or other bioprinting techniques.
- Neuron-Glial Network Formation: The platform facilitates quick establishment of neuron-glial networks, simplifying the analysis of these crucial interactions within a three-dimensional context.
- Assembly of Functional Layered Tissues: By mimicking the interactions between different brain regions, the platform retains intrinsic tissue properties such as axonal projection, offering insights into the functional connectivity between distinct neural populations.
- Optimized Tissue Dimensions: The design ensures adequate nutrient and oxygen diffusion, critical for the survival, growth, and functional integrity of the neural tissues.
- Predictable Functional Cell Types: The outcome of the printed tissues can be anticipated based on the composition of the progenitor cells used, allowing for a more targeted approach to studying neural network functions.
Addressing the Limitations
Despite its promising features, our 3D bioprinting platform is not without its challenges. The softness of the bioink limits the ability to print multiple layers vertically, and the maximum thickness of the printed tissues is capped at about 50 μm to ensure the formation of functional networks. Additionally, the current technology does not allow for the orientation of mature neurons, and while the printed tissues lack the intrinsic structural organization found in brain organoids, they retain essential anatomical and functional neuronal connections.
Future Directions
The limitations of the current prototype highlight areas for future development. Advances in bioprinting technology are expected to enable more sophisticated designs with precise cellular orientations and organization. The ability to print neural tissues with defined compositions of neural cell types at specific ratios opens new possibilities for assessing the biophysical properties of human neural circuits. Moreover, the platform offers a unique vantage point for exploring neural cell interactions and circuitry under pathological conditions, providing a valuable tool for drug development and therapeutic research.
In conclusion, our work represents a significant step toward the creation of functional human neural tissues through 3D bioprinting. By overcoming the limitations of existing models and leveraging the capabilities of this emerging technology, we are poised to unlock new insights into the functioning of the human brain, paving the way for innovative treatments and a deeper understanding of neurological diseases.
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