A recent study published in Stem Cell Research reports that researchers successfully created three-dimensional (3D) bioprinted human brain tissue, enabling the formation of functioning neural networks that could simulate network activity in both normal and pathological situations.
Understanding neural networks in the human brain is crucial for understanding brain health and disease. However, animal-based models can’t effectively replicate the high-level data processing of the human brain due to variations in cell composition, neural networks, and synaptic integration. 3D bioprinting offers a more accurate method for creating human brain tissue by physically repositioning hydrogels and living cells within a physiologically complex cytoarchitecture. Bioprinting soft tissues like the brain, however, raises concerns as soft biomaterials can’t maintain complex 3D architectures or rigid gels.
About the Study
In the study, researchers developed a 3D bioprinting platform to manufacture tissue with defined human brain cell types in any desired dimension. They aimed to build layered brain tissue, including neural precursor cells (NPCs) that establish connections within and between brain layers to keep the structure intact. They developed a bioink for printing and used fibrin gel to print the tissues. Bioprinting methods included extrusion-based, laser-based, and droplet-based techniques. The extrusion-based 3D bioprinting technique involved layering gel to simulate brain structures like human cortex laminations.
For each layer, the researchers chose a 50mm thickness and built multilayered tissues by placing the layers horizontally side by side. They designed 3D-printed brain tissue that is relatively thin but functional, multilayered, establishes cell compositions and desired dimensions, and is easy to maintain and test in a standard laboratory environment.
The researchers determined that 2.50 mg/ml fibrinogen and 0.50 to 1.0 U thrombin were optimal concentrations for hydrogel formation, resulting in a gelation time of 145 seconds, enabling printing on 24-well plates. The majority (85%) of cells were viable and survived for seven days. The team created gamma-aminobutyric acid (GABA)- and cortical-glutamate precursor-derived from human pluripotent stem cells (hPSCs) to investigate whether GABAergic interneurons and glutamatergic neurons form synaptic connections when introduced into printed tissue before printing. Before printing, they combined the two precursor populations in a 1:4 ratio to match the ratio of interneurons to cortical projection neurons in the cerebral cortex.
The researchers recorded electrophysiological data from tissues printed with glutamatergic cortical GFP+-precursors, unfilled GABAergic MGE precursors, and hPSC-derived astrocyte precursors incorporated into glutamate neurons and GABA interneurons. The printed tissue was immunostained with an axonal marker, SMI312. They examined Alexander’s disease (AxD), a neurodegenerative disorder caused by anomalies in the GFAP gene, to study pathological mechanisms. They used live imaging of glutamate uptake through glutamate-sensitive fluorescent reporters (iGluSnFR) to investigate neuron-astrocyte interactions and neuron-glia connections in AxD.
The printed neuronal precursors developed into neurons within weeks and formed functional neural networks within and across tissue layers. Printed astrocyte precursors matured with complex processes to function in neuron-astrocyte networks. Conventional culture techniques could preserve the 3D brain tissue, making it easier to study in physiological and pathological environments. The viability of cells decreased with increasing thrombin concentrations at a fibrinogen concentration of 2.50 mg/ml, remained unchanged at a constant concentration of 0.50 E fibrinogen, and cells aggregated at increased fibrinogen levels.
The bioprinted nerve cells matured and maintained tissue form, with GFP-expressing cells transforming into microtubule-associated protein 2 neurons (MAP2+) in a band a week after printing. The printed tissue maintained a stable configuration where neuronal precursors proliferated and built neural networks. The neuronal subtypes established functional networks within the bioprinted tissue, with hPSC-derived MGE cells expressing NK2 homeobox 1 (NKX2.1) and GABA, and cortical precursors positive for Forkhead-Box G1 (FOXG1) and Paired Box 6 (PAX6). The bioprinted neuronal tissue constructions promote the growth of cortical glutamatergic neurons and GABAergic interneurons.
The researchers used a highly concentrated potassium chloride solution to print tissue with neurons and astrocytes to demonstrate functional connections. Astrocytes expressed glutamate transporter 1 (GLT-1), indicating maturation. The printed cortical and striatal neuronal bands remained intact 15 days after printing, and GFP and mCherry neurites developed towards each other. The printed human brain tissue could replicate diseased processes, with AxD astrocytes showing intracellular GFAP aggregation. After 30 days, MAP2+ neurons and GFAP+ astrocytes exhibited complex morphology and synapsin expression.
Overall, the study’s results demonstrate the ability of 3D printing to create functioning brain tissue for simulating network activity in both normal and pathological environments. The tissues generated by bioink establish functional synaptic connections between neuronal subtypes and neuron-astrocyte networks within two to five weeks. The 3D platform provides a defined environment for studying human brain networks in healthy and pathological environments; however, there are limitations, such as the softness of the gel and the 50 mm printed tissue thickness.