3D Bioprinting of Neural Tissues

Abstract

The human nervous system is a remarkably complex physiological network that is inherently challenging to study because of obstacles to acquiring primary samples. Animal models offer powerful alternatives to study nervous system development, diseases, and regenerative processes, however, they are unable to address some species-specific features of the human nervous system. In vitro models of the human nervous system have expanded in prevalence and sophistication, but still require further advances to better recapitulate microenvironmental and cellular features. The field of neural tissue engineering (TE) is rapidly adopting new technologies that enable scientists to precisely control in vitro culture conditions and to better model nervous system formation, function, and repair. 3D bioprinting is one of the major TE technologies that utilizes biocompatible hydrogels to create precisely patterned scaffolds, designed to enhance cellular responses. This review focuses on the applications of 3D bioprinting in the field of neural TE. Important design parameters are considered when bioprinting neural stem cells are discussed. The emergence of various bioprinted in vitro platforms are also reviewed for developmental and disease modeling and drug screening applications within the central and peripheral nervous systems, as well as their use as implants for in vivo regenerative therapies.

Keywords: 3D printing; bioprinting; cortical organoids; hydrogel scaffolds; neural tissues; tissue engineered constructs.

Figure 1:

Overview of 3D Bioprinting for Neural Tissue Engineering. Computer aided design models use patient derived images to mimic the specific geometry of tissues of interest. The printing bioinks may contain a combination of biomaterials, bioactive molecules, or cells to create functionalized and personalized scaffolds. Scaffolds are then printed using the computer aided design and desired bioink(s). An extrusion bioprinter (BioAssemblyBot) is shown in this schematic. Applications for bioprinted scaffolds include high throughput drug screening, in vitro modeling, or in vivo repair and regeneration. Components of the figure created with BioRender.com.

Figure 2:

Bioprinting Methods and Their Advantages for Neural Tissue Modeling and Drug Screening. A: Microfluidic Extrusion Bioprinting. This method can be used to precisely print neural stem cell aggregates, which maintain high cell viability following printing and differentiation into neurons [96]. B: Hand-held Extrusion Bioprinting. This method has been applied to efforts intended to recapitulate the architecture of cortical layers. Cortical neurons are encapsulated within distinct layers and their subsequent differentiation and connectivity is quantified following in vitro growth and differentiation [62]. C:. Digital Light Processing Bioprinting. This high resolution modality has been applied to develop a tetraculture GBM model intended to investigate cellular interactions and serve as a high-throughput drug screening platform [115]. D: Multimaterial Extrusion Bioprinting. This method has been used to create sophisticated scaffolds that mimic spinal cord architecture. It is particularly advantageous for its ability to “point deposit” distinct cell types or growth factors in precise locations [131]. Components of the figure created with BioRender.com.

Figure 3:

3D Bioprinting for In Vitro Modeling and Drug Screening. A: Dopamine-based matrix for enhanced neural differentiation. (i) CAD model of desired 3D printed scaffold; (ii) Fluorescence micrograph of printed scaffold using Texas Red-X phalloidin; (iii) Light microscopy image of scaffold, scale bar = 200 μm; (iv) Surface plot of 3D-printed scaffold, scale bar = 200 μm; (v) Assessment of neural stem cell proliferation cultured on gelMA and gelMA-dopamine scaffolds. This was quantified by CCK-8 assay at day 2, 4, and 8. No significant difference is observed; (vi) Quantification of total neurite length and (vii) of the average length of the longest neurite on gelMA and gelMA-dopamine scaffolds at day 4, 8, and 12 of culture. * p < 0.05; Reprinted (adapted) with permission from Zhou et al. [86]. Copyright (2018) American Chemical Society; B: Lipid-Bilayer 3D Printing to Study Neural Development. (i) Left – bright field image of part of the printed droplet network, comprised of Matrigel and HepG2 cells. Right – Texas-red labeled lipids reveal droplet interface bilayers (DIB). Scale bar = 100 μm; (ii) Schematic of 7 x 7 x 8 droplet network; (iii) Images of HepG2 printed droplet network in oil and then transferred to culture medium; (iv) Immunostaining of printed network at day 28 and (v) day 56. Scaffolds immunostain positive for neural progenitors (SOX2+), young neurons (TUJ1+), and cortical neurons (CTIP2+). Reprinted and adapted with permission from Zhou et al. [108]. Copyright (2020) WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim; C: 3D Bioprinted Model to Study Glioblastoma Cellular Interactions. (i) Schematic of bioprinted mini brains. Structures are 4 x 6 x 5 mm scaffolds that contain macrophages throughout the construct and glioblastoma cells in an isolated tumor region; (ii) Cross-section of bioprinted mini-brain, highlighting the glioblastoma area in red. Scale bare = 5 mm; (iii) Migration assay for green CMFDA-labeled RAW264.7 (macrophages) towards empty (top), orange CMRA-labeled RAW264.7 (middle), and orange CMRA-labeled GL261(glioblastoma) (bottom) gels after 4 days of culture; (iv) Migration quantification. Migration of RAW264.7 cells towards GL261 is significantly higher than the control or towards RAW264.7 cells, illustrating the cellular interactions between macrophages and glioblastoma cells. Reprinted and adapted with permission from Heinrich et al. [113]. Copyright (2019) WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim; D: Stem-cell derived neural progenitor spinal cord scaffolds. (i) Comparison of transected rat spinal cord to the design of the bioprinted scaffold. The scaffold contains multiple, continuous channels. Scale = 1 mm; (ii) Top view of the bioprinted scaffold, with a resolution of about 150 μm. Scale = 1 mm; (iii) Immunohistochemistry highlighting the proximity of axon projections (green, βIII-tubulin) to oligodendrocyte progenitor cells (red, mCherry); Scale = 50 μm; (iv) Close up from panel (iii), highlighting the proximity of axons to oligodendrocyte progenitor cells. Scale = 50 μm: (v-vi) Time progression of spinal cord derived neural progenitor cell axon elongation from day 0 to day 3. Scale = 30 μm. Reprinted and adapted with permission from Joung et al. [131]. Copyright (2018) WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim; E: Glioblastoma-on-a-chip for patient specific responses to chemoradiotherapy. (i) Schematic of the 3D printing process to create the glioblastoma-on-a-chip model. This is achieved by using various bioinks and materials to form a compartmentalized structure. Photographs of mock glioblastoma-on-a-chip are shown. This includes a representation of the human umbilical vein endothelial cell encapsulation in magenta and the glioblastoma cell encapsulation in blue. Scale bar = 2 cm; (ii) Evaluation of hypoxia at the core, intermediate, and peripheral zones of the bioprinted model. Pimonidazole (PM) indicates hypoxic cells, Ki67 shows proliferating cells, and DAPI identifies all cell nuclei. Scale bar = 200 μm; (iii) Graphs quantifying the percentage survival of different glioblastoma cell lines, following different concurrent chemoradiation therapy combinations. The results highlight patient specific responses to the drug formulation. Reprinted and adapted with permission from Yi et al.[116]. Copyright (2019) Springer Nature.

Figure 4:

3D Bioprinting Techniques Applied to Peripheral Nerve and Spinal Cord Repair. A: Spheroids formed from stem cells and printed using a scaffold free bioprinting method. This results in fully cellularized nerve constructs that can be transplanted into a severed rodent facial nerve to enhance nerve regeneration [142]. B: Digital light processing bioprinting used to create a hollow scaffold containing embedded bioactive molecules to enhance regeneration following sciatic nerve injury [141]. C: Extrusion bioprinting used to create a biomimetic scaffold seeded with neural stem cells, then transplanted into severed a spinal cord to enhance axon tract formation [146, 148]. Components of the figure created with BioRender.com.

Figure 5:

3D Bioprinted Scaffolds for PNS and CNS Repair. A: 3D Bioprinted gelatin-sodium alginate/rat Schwann-cell scaffolds for in vitro and in vivo applications. (i) Front view of bioprinted gelatin/alginate scaffold; (ii) Implantation of the scaffold into a mouse model. (iii-iv) In vitro characterization of gelatin/alginate/Schwann-cell scaffold by staining for live cells at day 1 and day 7 of culture. Scale bar = 100 μm; (v) Neurotrophic release determined by qRT-PCR on day 4 of in vitro 2D and 3D culture. Compared to 2D culture, mRNA of the 3D bioprinted group is significantly enriched for NGF, BDNF, GDNF, and PDGF. Reprinted and adapted with permission from Wu et al. [140]. Copyright (2019) Elsevier B.V.; B: Collagen/heparin sulfate 3D bioprinted scaffolds for improved function after spinal cord injury in rats. (i) Pictures of 3D bioprinted collage/heparin sulfate scaffolds; (ii) H&E staining, 8 weeks after injury and scaffold implantation. SCI + C/H are control scaffolds formed by freeze dry technology that have been implanted into the spinal cord injury model. SCI + 3D-C are 3D bioprinted collagen scaffolds implanted into the SCI model and SCI+ 3D-C/H are collagen/heparin sulfate scaffolds implanted into the SCI model. 3D-C/H scaffolds do not exhibit a cavity and there appears to be a linear ordered structure; (iii-iv) Immunostaining and quantification of positive neurofilaments (NF) in the spinal cord sections 8 weeks post injury. The 3D-C/H had a significant increase in NF-positive staining compared to the SCI, SCI + C/H, and SCI + 3D-C groups. Reprinted and adapted with permission from Chen et al. [147]. Copyright (2017) WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim;