Three dimensional cellular microarray platform for human neural stem cell differentiation and toxicology

Abstract

We developed a three-dimensional (3D) cellular microarray platform for the high-throughput (HT) analysis of human neural stem cell (hNSC) growth and differentiation. The growth of an immortalized hNSC line, ReNcell VM, was evaluated on a miniaturized cell culture chip consisting of 60nl spots of cells encapsulated in alginate, and compared to standard 2D well plate culture conditions. Using a live/dead cell viability assay, we demonstrated that the hNSCs are able to expand on-chip, albeit with lower proliferation rates and viabilities than in conventional 2D culture platforms. Using an in-cell, on-chip immunofluorescence assay, which provides quantitative information on cellular levels of proteins involved in neural fate, we demonstrated that ReNcell VM can preserve its multipotent state during on-chip expansion. Moreover, differentiation of the hNSCs into glial progeny was achieved both off- and on-chip six days after growth factor removal, accompanied by a decrease in the neural progenitor markers. The versatility of the platform was further demonstrated by complementing the cell culture chip with a chamber system that allowed us to screen for differential toxicity of small molecules to hNSCs. Using this approach, we showed differential toxicity when evaluating three neurotoxic compounds and one antiproliferative compound, and the null effect of a non-toxic compound at relevant concentrations. Thus, our 3D high-throughput microarray platform may help predict, in vitro, which compounds pose an increased threat to neural development and should therefore be prioritized for further screening and evaluation.

Figure 1

Comparison of ReNcell VM cell expansion in the 3D cellular microarray platform with expansion in conventional 2D culture platforms. (A) Cells seeded at 160 cells/mm² in a 96-well microtiter plate (5000 cells/well; 2.5 × 10⁴ cells/ml medium) grow exponentially with doubling times of 22 h, and no lag phase observed (open circles, right axis). Cells seeded in microcultures in alginate spots at 5000 cells/mm³ (5.8 × 10⁴ cells/ml medium) demonstrate a 4-day lag, followed by a growth phase with slower proliferation (t_D = 84 h) (open squares, left axis). The inset shows the changes in ReNcell VM viability during 3D culture, measured indirectly by following the ratio of average red-to-green fluorescence intensity with time, normalized to the first measurement taken after printing (typically 4 h after cell detachment). The data indicate a decrease in viability during the lag phase. Data represent the mean ± SEM. (B) Bright field images of the alginate microcultures (300 cells/spot; 5 × 10⁶ cells/ml) after cell expansion on-chip using expansion medium left panel), and cells similarly expanded and subsequently differentiated for 6 days in differentiation medium (right panel). ND refers to non-differentiated cells, DIFF refers to differentiated cells.

Figure 2

Cell-based microarray immunofluorescence assay for quantification of marker proteins in ReNcell VM cells cultured on 2D platforms. (A) Bright field microscopy images of ReNcell VM cells expanded in monolayer culture for 3 days (upper panel) using expansion medium, and cells similarly expanded and subsequently differentiated for 6 days in differentiation medium (lower panel). ND indicates non-differentiated cells; DIFF indicates differentiated cells. (B) Western blot analysis showing astrocytic lineage commitment after 6 days of differentiation, as evidenced by the decrease in Nestin expression, and concomitant increase in GFAP signal (γ-Tubulin and GAPDH were used as loading control). (C) On-chip immunofluorescence assay of marker proteins in ReNcell VM cells expanded or differentiated in 2D culture platforms (off-chip) and subsequently printed for analysis with 300 cells/spot (5 × 10⁶ cells/ml). Each spot has a diameter of 600 μm, as indicated. (D) Quantification of the normalized fluorescence intensity signal obtained from microarrays such as those illustrated in (C), using GAPDH as internal control (similar results obtained with γ-Tubulin normalization). Statistically significant differences between the normalized intensity of differentiated and non-differentiated cells are represented with (*) for p < 0.05. Data represent the mean ± SEM, with n ≥ 3 experiments, each with 48 replicate microcultures. (E) Expression of F-actin (green) by ReNcell VM cells expanded in monolayer culture for 3 days (upper panel) using expansion medium, and cells similarly expanded and subsequently differentiated for 6 days in differentiation medium (lower panel). Cell nuclei were counterstained with Hoechst 33342 (blue). ND indicates non-differentiated cells; DIFF indicates differentiated cells. Scale bars: 20 μm.

Figure 3

On-chip, in-cell immunofluorescence assay for quantification of marker proteins in ReNcell VM cells cultured in the 3D microarray platform. (A) Scanning images of portions of the microcultures containing ReNcell VM cells (seeded at 300 cells/spot) after expansion or differentiation on-chip, followed by immunoassay of marker proteins. (B) Quantification of marker protein expression in differentiated and non-differentiated cells through analysis of the normalized fluorescence intensity signal obtained from the scanning microarray images using GAPDH as the internal control. Statistically significant differences between the normalized intensity of differentiated and non-differentiated cells is represented with (*) for p < 0.05. Data represent the mean ± SEM, with n ≥ 3 experiments, each with 48 replicate microcultures.

Figure 4

Comparison of the dose response of ReNcell VM cells and their differentiated progeny (following 6 days of growth factor removal) to 3 developmental neurotoxicants (retinoic acid-(A), cadmium chloride-(B) and dexamethasone-(C)), and a non-neurotoxicant (acetaminophen-(E)). Cytotoxicty was determined with a live/dead cell viability assay and the data were fitted to a sigmoidal dose response to obtain the IC₅₀ values. The figures show representative curves where each data point is the mean of 48 replicates ± SEM.