Adapting tissue-engineered in vitro CNS models for high-throughput study of neurodegeneration

Abstract

Neurodegenerative conditions remain difficult to treat, with the continuing failure to see therapeutic research successfully advance to clinical trials. One of the obstacles that must be overcome is to develop enhanced models of disease. Tissue engineering techniques enable us to create organised artificial central nervous system tissue that has the potential to improve the drug development process. This study presents a replicable model of neurodegenerative pathology through the use of engineered neural tissue co-cultures that can incorporate cells from various sources and allow degeneration and protection of neurons to be observed easily and measured, following exposure to neurotoxic compounds - okadaic acid and 1-methyl-4-phenylpyridinium. Furthermore, the technology has been miniaturised through development of a mould with 6 mm length that recreates the advantageous features of engineered neural tissue co-cultures at a scale suitable for commercial research and development. Integration of human-derived induced pluripotent stem cells aids more accurate modelling of human diseases, creating new possibilities for engineered neural tissue co-cultures and their use in drug screening.

Keywords: Neurodegeneration; drug screening; induced pluripotent stem cells; neurons; three-dimensional models.

Figure 1.

Okadaic acid induces neurite degeneration in PC12 and DRG neurons. (a) Confocal micrographs of 1 mL EngNT cultures show progressive degeneration of PC12 and DRG neurites stained for β-III tubulin (green) after exposure to okadaic acid for 24 h. Scale bar = 100 µm. Dose-dependent neurite retraction was observed in both (b) PC12 and (c) DRG neurons after 24 h of exposure to okadaic acid. Significant differences in neurite length were seen in the presence of 25 and 50 nM okadaic acid when compared to controls. N = 5 gels (PC12), N = 4 gels (DRG), mean ± SEM for each condition. One-way ANOVA with Dunnett’s post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

MPP+ induces neurite degeneration in PC12 and DRG neurons. (a) Confocal micrographs of PC12 and DRG neurite degeneration after exposure to MPP⁺ at various concentrations for 24 h within 1 mL EngNT cultures stained to detect β-III tubulin immunoreactivity (green). Scale bar = 100 µm. 24 h of exposure to MPP⁺-induced dose-dependent neurite retraction of aligned (b) PC12 and (c) primary DRG neurons. Gels treated with neurotoxins were compared to untreated control gels. N = 5 gels (PC12), N = 4 gels (DRG), mean ± SEM for each condition. One-way ANOVA with Dunnett’s post hoc test, *p < 0.05, **p < 0.01 ***p < 0.001.

Figure 3.

Neurotoxins have a minimal toxic effect on C6 glia. (a) Incubation of gels containing C6 cells with 25 and 50 nM okadaic acid resulted in a significant increase in cell death when compared to the control. However, in all other conditions, the level of cell death did not differ significantly from that in control cultures. Mean ± SEM, n = 5 gels per concentration. One-way ANOVA with Dunnett’s post hoc test, *p < 0.05, **p < 0.01. (b) Fluorescent micrograph show nuclei staining of C6 cells (blue) and PI staining (red) of dead cells in the presence of 25 nM OKA. Scale bar = 100 µm.

Figure 4.

Salvianolic acid B (SAB) prevents MPP⁺-induced neurite degeneration in PC12 cells. Following 30 min pre-treatment of 1 mL EngNT co-cultures and presence of SAB for 24 h, mean neurite length of gels treated with SAB was not significantly different from the control, while MPP⁺ exposure with no SAB was significantly different from the control. Mean ± SEM, n = 5 gels per concentration. One-way ANOVA with Dunnett’s post hoc test, **p < 0.01.

Figure 5.

Development of a 50-µL gel for generation of EngNT co-cultures. (a) An example of a tile scan of an entire gel from a 50-µL test rig with seeding density of 3 × 10⁶ C6 cells/mL using confocal microscopy. The C6 cells within the gel were stained for GFAP immunoreactivity. (b) Frequency plot of the 50-µL gel and EngNT in which the angle of deviation was measured to assess alignment, approximately 500 angles per gel were measured. The frequency plots show the % frequency of each angle of deviation in tiles from the mid and side regions of the gel. (c) Confocal projections show the alignment of PC12 cells stained for β-III tubulin immunoreactivity (green) on the surface of scaled down EngNT co-cultures. Scale bar = 100 µm.

Figure 6.

MPP+ induces neurite degeneration of iPSC-derived neurons. Neurite degeneration of aligned iPSC-derived neurons in EngNT co-cultures created using 50-µL gel was observed after 24 h of exposure to 30 µM MPP⁺. Scale bar = 100 µm. Mean ± SEM. Unpaired t-test, **p < 0.01.