Vertically-Aligned Functionalized Silicon Micropillars for 3D Culture of Human Pluripotent Stem Cell-Derived Cortical Progenitors

Abstract

Silicon is a promising material for tissue engineering since it allows to produce micropatterned scaffolding structures resembling biological tissues. Using specific fabrication methods, it is possible to build aligned 3D network-like structures. In the present study, we exploited vertically-aligned silicon micropillar arrays as culture systems for human iPSC-derived cortical progenitors. In particular, our aim was to mimic the radially-oriented cortical radial glia fibres that during embryonic development play key roles in controlling the expansion, radial migration and differentiation of cortical progenitors, which are, in turn, pivotal to the establishment of the correct multilayered cerebral cortex structure. Here we show that silicon vertical micropillar arrays efficiently promote expansion and stemness preservation of human cortical progenitors when compared to standard monolayer growth conditions. Furthermore, the vertically-oriented micropillars allow the radial migration distinctive of cortical progenitors in vivo. These results indicate that vertical silicon micropillar arrays can offer an optimal system for human cortical progenitors' growth and migration. Furthermore, similar structures present an attractive platform for cortical tissue engineering.

Keywords: 3D culture; cell growth; cerebral cortex; hiPSC-derived neural progenitors; human cortical progenitors; silicon pillars.

Figure 1

Design, size and morphology of 3D silicon micropillar array slides. (A) Schematic structure of a silicon slide containing vertically-aligned micropillars (micropillars are not in scale). (B) The image shows a 3D silicon slide. Scale bar: 1 mm.

Figure 2

Silicon micropillars can be arranged with different topographies. Silicon micropillar arrays are perfectly vertically-aligned with a height that can be tuned in a 200–600 μm range. Specific spacing, density and morphology of the silicon pillars can be arranged by changing the mask of lithographic process. Low and high magnification of cross-sectional SEM images of aligned (A) and staggered (B) micropillar arrays. Scale bar: 50 µm.

Figure 3

hCPs seeded on silicon micropillar arrays maintain their viability. MTT assay performed on hCPs plated on 3D silicon slide and in standard 2D monolayer shows that hCPs efficiently maintain their viability. Different cell densities were assessed 48 h after seeding. p < 0.01 (**), not significant (ns).

Figure 4

hCPs seeded on silicon slide establish close interactions with micropillars. (A) Top-view SEM image showing hCPs seeded at standard density on silicon micropillars. (B,C) Cross-sectional SEM images showing hCPs seeded at low density on silicon structures to visualize single cells-micropillars interactions. Scale bar: 20 μm (A,B) and 10 μm (C).

Figure 5

hCPs seeded on silicon slides maintain their proliferation capability. Cell growth analysis (MTT assays) performed on hCPs seeded on oxidized silicon (OxSi) or nitride silicon (NiSi) micropillar arrays. Standard 2D monolayer cultures were used as control. Cultures were assessed at different days in vitro (DIV) after seeding. p < 0.01 (**), p < 0.0001 (****), not significant (ns).

Figure 6

hCPs cultured on silicon micropillar arrays establish layered structures. (A) Picture of eGFP^+ve hCPs cultured for 14 days on silicon slides showing the establishment of a high-density 3D culture. Inset shows the same culture stained with Hoechst. (B,C) SEM images of hCPs maintained on silicon pillars device for 14 days. Cultures exhibit the generation of multiple cell layers. Scale bar: 50 μm (A) and 20 μm (B,C).

Figure 7

hCPs seeded on silicon slide maintain their neural immature identity. Immunostaining of hCPs for NESTIN (green), SOX2 (red) and nuclear staining with Hoechst (blue) in 3D culture (A) and 2D culture (B) cultured for 14 days. (C) Quantitative RT-PCR assay showing the expression levels of Nestin and β3-Tubulin transcripts in hCPs grown in 2D or 3D cultures. Scale bar: 100 μm (A) and 50 μm (B). p < 0.01 (**), not significant (ns).

Figure 8

hCPs seeded on silicon micropillar arrays preserve their cortical regional identity and upon exposure to differentiative conditions, generate cortical glutamatergic neurons. (A) eGFP^+ve hCPs cultured for 14 days on 3D silicon devices preserve the expression of the cortical progenitor marker TBR2 (red). Nuclei are stained with Hoechst (blue). Scale bar: 50 μm. (B) hCPs cultured for 5 days on 3D silicon devices and then exposed for 35 days to differentiative conditions maturate into cortical glutamatergic neurons. Left: cultures stained for the pan-neuronal neuronal marker β3-TUBULIN (red). Nuclei are stained with Hoechst (blue). Scale bar: 10 μm. Right: cultures stained for the mature neuronal marker MAP2 (red) and for the cortical neuronal marker CUX1 (green). Nuclei are stained with Hoechst (blue). Scale bar: 10 μm.