To date, pharmaceutical progresses in central nervous system (CNS) diseases are clearly hampered by the lack of suitable disease models. Indeed, animal models do not faithfully represent human neurodegenerative processes and human in vitro 2D cell culture systems cannot recapitulate the in vivo complexity of neural systems. The search for valuable models of neurodegenerative diseases has recently been revived by the addition of 3D culture that allows to recreate the in vivo microenvironment including the interactions among different neural cell types and the surrounding extracellular matrix (ECM) components. In this review, the new challenges in the field of CNS diseases in vitro 3D modeling are discussed, focusing on the implementation of bioprinting approaches enabling positional control on the generation of the 3D microenvironments. The focus is specifically on the choice of the optimal materials to simulate the ECM brain compartment and the biofabrication technologies needed to shape the cellular components within a microenvironment that significantly represents brain biochemical and biophysical parameters.
Revealing the 3D composition of intact tissue specimens is essential for understanding cell and organ biology in health and disease. State-of-the-art 3D microscopy techniques aim to capture tissue volumes on an ever-increasing scale, while also retaining sufficient resolution for single-cell analysis. Furthermore, spatial profiling through multi-marker imaging is fast developing, providing more context and better distinction between cell types. Following these lines of technological advance, we here present a protocol based on FUnGI (fructose, urea and glycerol clearing solution for imaging) optical clearing of tissue before multispectral large-scale single-cell resolution 3D (mLSR-3D) imaging, which implements 'on-the-fly' linear unmixing of up to eight fluorophores during a single acquisition. Our protocol removes the need for repetitive illumination, thereby allowing larger volumes to be scanned with better image quality in less time, also reducing photo-bleaching and file size. To aid in the design of multiplex antibody panels, we provide a fast and manageable intensity equalization assay with automated analysis to design a combination of markers with balanced intensities suitable for mLSR-3D. We demonstrate effective mLSR-3D imaging of various tissues, including patient-derived organoids and xenografted tumors, and, furthermore, describe an optimized workflow for mLSR-3D imaging of formalin-fixed paraffin-embedded samples. Finally, we provide essential steps for 3D image data processing, including shading correction that does not require pre-acquired shading references and 3D inhomogeneity correction to correct fluorescence artefacts often afflicting 3D datasets. Together, this provides a one-week protocol for eight-fluorescent-marker 3D visualization and exploration of intact tissue of various origins at single-cell resolution.
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