BIOCOMPATIBILITY OF LARGE-AREA 2-DIMENSIONAL ELECTRONIC MATERIALS WITH NEURAL STEM CELLS

Abstract

Two-dimensional (2D) electronic materials hold immense promise for next-generation bio/neuro-electronic interfaces, but their biocompatibility has remained uncertain due to conflicting reports from studies focused on exfoliated flakes and suspensions. In this work, we present a comprehensive in vitro evaluation of electronic-grade large-area, chemical vapor deposition (CVD)-grown 2D materials - including platinum diselenide (PtSe₂), platinum ditelluride (PtTe₂), molybdenum disulfide (MoS₂), and graphene - as substrates for mouse neural stem cell culture. Across all CVD-grown materials, the stem cells exhibited outstanding viability, with no significant differences in metabolic activity or live/apoptotic cell ratios compared to laminin-coated glass controls (p > 0.05). Importantly, these large-area 2D materials robustly supported neuronal differentiation, as evidenced by widespread βIII-tubulin expression. Strikingly, we found that flaky MoS₂ promoted significantly greater neuronal maturation (>75% NeuN⁺ neurons) than any other substrate tested (25-50% NeuN⁺; p < 0.05), revealing the critical influence of material format on bioactivity. While PtSe₂ showed a tendency to promote glial lineage differentiation, our findings firmly establish large-area CVD-grown 2D materials as biocompatible, tunable platforms for neural interfacing, paving the way for their integration into advanced bio/neuro-electronic devices.

Keywords: ApoLive-Glo Assay; Cell-viability; Graphene; Immunocytochemistry (ICC); Live-Dead Imaging; MoS2; PtSe2; PtTe2.

Figure 1.

The preparation of samples and the experimental set-up. a) CVD growth procedure of PtSe₂, PtTe₂, and MoS₂ is depicted in a schematic containing the CVD chamber, the growth temperatures, the substrate materials, and the precursors, b) process of transferring CVD-grown graphene layers onto glass coverslips, c) process of spin-coating and annealing the flaky MoS₂-IPA suspension, d) flow-chart of the experimental set-up.

Figure 2.

Raman spectra for a) Graphene, b) PtSe₂, c) PtTe₂, d) MoS₂, and e) flaky MoS₂. f) The water contact angle measurements for all materials both bare/pre-culture (solid) and post-culture (striped) (n = 5, mean +/− standard deviation). A considerable increase in water-contact-angles (WCA) after post cell-culture can be observed. The black * and blue # refer to statistical significance of difference in WCA between conditions (pre- and post-culture) and between a given material’s post-culture sample and the post-culture bare-glass substrate (p<0.0001), respectively.

Figure 3.

Viability of mNSCs after 48 hours of culture in proliferation medium. a) An ApoLive-Glo assay was performed to calculate percentage of relative fluorescence (living cells) over luminescence (dying cells) for each material. These values were normalized to the glass coverslip control condition and plotted (N=4 repeats in each of 4 independent repeats). Plot shows mean +/− SEM. b) The fluorescence values were also equated to the number of living cells based on a standard curve of serial dilutions within each repeat of the ApoLive-Glo assay. These values were normalized to the glass coverslip control condition and plotted (N=4 repeats in each of 4 independent repeats). Plot shows mean +/− SEM. One-way ANOVA showed no significant (ns) differences in viability when applied to data shown in (a) or (b). c) Representative images of the Live/Dead Assay showing live (Calcein AM, green) and dead (ethidium bromide, red) cells. Scale bars = 200 μm.

Figure 4.

Differentiation of mNSCs towards neurons after 5 days in differentiation medium. βIII-tubulin (magenta) was used to identify both immature and mature neurons. Main scale bars = 50 μm and inset scale bars = 20 μm. Quantification shows βIII-tubulin+ area divided by total nuclei for each image (N=2 repeats in each of 2 independent repeats). One-way ANOVA showed no significant (ns) differences between the conditions. Plot shows mean +/− SEM.

Figure 5.

Differentiation of mNSCs into mature neurons after 5 days in differentiation medium. Nuclear expression of NeuN (green) was used to identify mature neurons. Main scale bars = 50 μm and inset scale bars = 20 μm. Quantification shows numbers of NeuN+ nuclei divided by total nuclei for each image (N=1,2 repeats in each of 2 independent repeats). One-way ANOVA showed that flaky MoS₂ had significantly more nuclear expression than PtTe₂ (**p < 0.01), Graphene (*p < 0.05), PtSe₂ (*p < 0.05) and glass (*p < 0.05). All other conditions had no significant (ns) differences. Plot shows mean +/− SEM.

Scheme 1.

Schematic illustration of the experimental set-up and the main results of the biocompatibility studies.