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Review
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Layer-By-Layer: The Case for 3D Bioprinting Neurons to Create Patient-Specific Epilepsy Models

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Review

Layer-By-Layer: The Case for 3D Bioprinting Neurons to Create Patient-Specific Epilepsy Models

Natasha Antill-O'Brien et al. Materials (Basel).

Abstract

The ability to create three-dimensional (3D) models of brain tissue from patient-derived cells, would open new possibilities in studying the neuropathology of disorders such as epilepsy and schizophrenia. While organoid culture has provided impressive examples of patient-specific models, the generation of organised 3D structures remains a challenge. 3D bioprinting is a rapidly developing technology where living cells, encapsulated in suitable bioink matrices, are printed to form 3D structures. 3D bioprinting may provide the capability to organise neuronal populations in 3D, through layer-by-layer deposition, and thereby recapitulate the complexity of neural tissue. However, printing neuron cells raises particular challenges since the biomaterial environment must be of appropriate softness to allow for the neurite extension, properties which are anathema to building self-supporting 3D structures. Here, we review the topic of 3D bioprinting of neurons, including critical discussions of hardware and bio-ink formulation requirements.

Keywords: 3D printing; 3D scaffolds; bioink; bioprinting; brain; neural network; organoids; patient specific disease modelling; three-dimensional (3D) models.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Developed from patient cells. (A) Organoids can be developed from patient induced pluripotent stem cells (iPSCs) for disease modelling and drug testing. Differentiated cells are self-organizing akin to organogenesis in vivo. [25]. (B) Cerebral organoid with heterogenous tissue regions observed via immunostaining: SOX2+ progenitor cells labelled red, TUJ1+ neurons labelled green, and nucleic acid Hoechst stained blue [27]. Images reproduced with permission from [25,27].
Figure 2
Figure 2
(A) Two-dimensional (2D) and three-dimensional (3D) present very different environments [50]. (B) Network formation in 2D and 3D. Bourke et al. (2018) cultured day 18 rat embryonic cortical in collagen type I 2D and 3D cultures. Schematic of 2D (B1) and 3D (B4) network formation, red indicates network activation. Immunofluoresence of 2D cultures (B2) and 3D cultures (B5) imaged at 35 days in culture. Micro electrode array (MEA) recordings from the neuronal networks of 2D (B3) cultures and 3D (B6) cultures after activation (indicated by arrows) of K-L-glutamic acid addition [58]. Inset, adapted from Magill et al. (“Copyright 2000 Society for Neuroscience”), of in vivo MEA recording from rat subthalamic nucleus similar to the 3D culture recordings [59]. Images reproduced with permission from [50,58,59].
Figure 3
Figure 3
Biomaterials for 3D neural culture. (AD) LIVE/DEAD staining of cortical neurons encapsulated in 0.5% w/v agarose gels over 10 days, scale bar 100 µm. (EH) LIVE/DEAD staining of cortical neurons encapsulated in 0.04% w/v collagen gels over 10 days scale bar 100 µm; compared to agarose collagen is more supportive of neuron survival and maturation [84]. (I) Neural stem cells (NSCs) derived from iPSCs encapsulated in hyaluronic acid methacryloyl (HAMA) 1% w/v exposed to crosslinking ultra-violet (UV) light for 60, 90, and 120 s, scale bar 200 µm. Increased duration of UV exposure increases the stiffness of the resulting gel, neuron differentiation was promoted in softer gels of 130 Pa [40]. (J) Confocal depth decoded image of embryonic hindbrain cells encapsulated in 3.5%/0.5% w/v gelatin methacryloyl (GelMA)/HAMA at day 15. Colour coding indicates the depth of different planes along the z axis, scale bar 50 µm [109]. (K) Dorsal root ganglion (DRG) with neurite extension in carboxymethyl chitosan (CMC), scale bar 500 µm [111]. (L) NPCs encapsulated in varying concentration of fibrin gel after 14 days. Immunostaining for neuronal processes (β-tubulin III+) shown in green, 4′,6-diamidino-2-phenylindole (DAPI) staining of nuclei in blue, scale bar 150 µm. Neural network formation was promoted in lower concentration fibrin gels [114]. Images reproduced with permission from [40,84,109,111,114].
Figure 4
Figure 4
Extrusion bioprinted neural and iPSC derived cultures: (A) Layered cortical neurons in top and bottom layer RGD-GG [186]; (B) iPSCs in Al-Ag-CMC [183]. (A,B) Colour coded z axis. (C) Al-Ag-CMC printed grid [182]. (D) Zebrafish injected with bioprinted fluorescently labelled neural progenitor cells (NPCs) in 25% polyurethane (PU), fb = forebrain, mb = midbrain, hb+hindbrain and tg = trigeminal ganglion [184]. (E) Bioprinted alternating oligodendrocyte progenitor cells (OPCs) (red) and NPCs (green) in silicon channel 24 h post print, image adapted from [185] and (F) after four days in culture. (G) 3D printed alginate structure, (H) bioprinted NPCs in channels during assembly of 3D printed alginate structure, top view [185]. Images reproduced with permission from [182,183,184,185,186], scale bars as shown.
Figure 5
Figure 5
Methods of bioprinting neurons. (A) Nozzle-based methods: Inkjet and microvalve printing deposit drops on demand and are more appropriate for soft viscous materials. Microfluidic printing also uses soft viscous materials but deposits a filament as crosslinking occurs prior to deposition. Extrusion printing deposits a continuous filament and requires higher viscosity materials, image adapted from [174,196]. (B) Nozzle free methods include bioacoustic levitational assembly and UV laser writing [193]. Images reproduced with permission from [174,193,196].
Figure 6
Figure 6
Inkjet and other bioprinting techniques: (A) Thermally inkjet printed rat hippocampal neurons onto collagen sheet [189], scale bar 500 µm. (B) A rat cortical neuron 13 days after being thermally inkjet printed [189], scale bar 30 µm. (C) Close up image with arrow showing neuron in piezoelectric induced jet [192]. (D) Printed neurons at seven days after piezoelectric printing [191], scale bar 100 µm. (E) Piezoelectric printed neurons had initially higher neurite formation compared to non-printed controls, which stabilized by day seven [191]. (F) Schematic explaining the rationale used to microvalve print hippocampal neurons and astrocytes in (G,H), scale bar in (G) 500 µm, inset 50 µm and (H) each grid is 500 µm by 500 µm [194]. Neurons migrate and differentiate toward vascular endothelial growth factor (VEGF) releasing fibrin gels marked by * in (I,J) [195], scale bar 200 µm. (K) Microfluidic printed NPCs differentiate into glial (GFAP+ green) and neural (β-Tubulin III+ red) lineages, scale bar 50 µm [196]. (L) Microfluidic bioprinted cylinder with diameter of 6693.0 µm [196]. Live (green) and dead NPCs (red) at (M) day zero and (N) seven days after bioacoustic levitation (BAL), scale bar 250 µm [193]. (O) One week after BAL NPCs differentiated into neurons in 3D layers (β-Tubulin III+ red), progenitor cells (Nestin+ green) [193]. Images reproduced with permission from [189,191,192,193,194,195,196].

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