Tech platform creates 3D neural tissues in which neurons and glia connect

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In a recently published study, Dr Cells are stem cellsResearchers have created three-dimensional (3D) bioprinted human brain tissue, which allows the creation of functional neural networks that can mimic network activity under normal and pathological conditions.

Study: 3D bioprinting of human neural tissues with functional connectivity.  Image credit: whitehoune/Shutterstock.com
Study: 3D bioprinting of human neural tissues with functional connectivity. Image credit: whitehoune/Shutterstock.com

Background

Understanding the neural networks of the human brain is critical to understanding brain health and disease. However, animal-based models cannot effectively reproduce the higher-order data processing of the human brain due to variations in cell structure, neural networks, and synaptic integration. 3D bioprinting provides a more accurate method for creating human brain tissue by physically repositioning hydrogels and living cells inside an anatomically complex cytoarchitecture. However, bioprinting soft tissues, such as the brain, is of concern because soft biomaterials cannot sustain complex 3D architectures or rigid gels.

About the study

In the current study, the researchers developed a 3D bioprinting platform to create tissue with defined human brain cell types at any desired scale.

The team’s goal was to create layered neural tissue, including neural progenitor cells (NPCs) that form connections within and between layers of the brain, keeping the structure intact. They developed a bioink for printing. They use fibrin gel to print tissue. Methods of bioprinting include extrusion-based, laser-based, and droplet-based techniques. The extrusion three-dimensional bioprinting technique deposits gel in layers to mimic brain structures similar to human cortex laminations.

The researchers selected a thickness of 50 mm for each layer and created a multi-layered tissue by placing the layers adjacent to each other in a horizontal arrangement. They designed 3D-printed brain tissues that are relatively thin but functional and multi-layered, with established cell composition and desired dimensions, and easily maintained and tested in a standard laboratory setting.

The researchers determined that 2.50 mg per mL of fibrinogen and 0.50 to 1.0 U of thrombin were the optimal concentrations for hydrogel formation, resulting in a gelation time of 145 seconds, allowing printing of 24-well plates. After six hours, most (85%) cells were viable and survived for seven days. The team generated medial ganglionic eminence (MGE)-derived gamma-aminobutyric acid (GABA) and cortical (glutamate) progenitors from green fluorescent protein-expressing (GFP).+) and GFP To investigate whether human pluripotent stem cells (hPSCs) form synaptic connections when inserted into GABAergic interneurons and glutamatergic neurons printed tissue. Before printing, they combined the two progenitor populations in a ratio of 1:4 to match the ratio of cortical projection neurons to interneurons in the cerebral cortex.

The researchers recorded electrophysiological data from tissues imprinted with GFP+ glutamatergic cortical progenitors, non-dyed MGE GABAergic progenitors, and hPSC-derived astrocyte progenitors included in glutamate neurons and GABA interneurons. The printed tissue was immunostained with an axonal marker, SMI312. They studied Alexander disease (AxD), a neurodegenerative disease caused by abnormalities in the GFAP gene, to investigate pathogenic mechanisms. They used live imaging of glutamate uptake by glutamate-sensitive fluorescent probes (iGluSnFR) to investigate neuron-astrocyte interactions and neuron-glial connections in AxD.

result

Imprinted neuronal progenitors develop into neurons within weeks, forming functional neural networks within and across tissue layers. Imprinted astrocyte progenitors develop into astrocytes with complex mechanisms to function in the neuron-astrocyte network. Conventional culture techniques can capture 3D brain tissues, making them easier to investigate in physiological and pathological settings. Cell viability decreased with increasing concentrations of thrombin at a fibrinogen concentration of 2.50 mg/mL but remained unchanged at a constant concentration of 0.50 U fibrinogen, and cells aggregated at increased fibrinogen levels.

Bioprinted neural cells mature and retain tissue form, with GFP-expressing cells in a band transforming into microtubule-associated protein 2 (MAP2+) neurons one week after printing. The imprinted tissue maintained a stable configuration where neural progenitors proliferated and formed neural networks. Neuronal subtypes establish functional networks within bioprinted tissues, hPSC-derived MGE cells express NK2 homeobox 1 (NKX2.1) and GABA and cortical progenitors positive for forkhead-box G1 (FOXG1) and paired box 6 (PAX6). Bioprinted neural tissue constructs promote growth of cortical glutamatergic neurons and GABAergic interneurons.

The researchers used a high-concentration potassium chloride solution to print tissue, neurons and astrocytes, demonstrating functional connectivity. Astrocytes express glutamate transporter 1 (GLT-1), which indicates maturation. Printed cortical and striatal neuronal bands were intact 15 days after printing, and GFP and mCherry neurites developed towards each other. As imprinted human brain tissues can replicate diseased processes, AxD astrocytes display intracellular GFAP aggregates. By 30 days, MAP2+ neurons and GFAP+ astrocytes display complex morphology and synapsin expression.

Conclusion

Overall, the study results demonstrated the ability of 3D printing to create functional brain tissue to mimic network activity in normal and pathological settings. Bioink-created tissues 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 settings; However, it has limitations, such as the softness of the gel and the 50 mm thickness of the printed tissues.



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