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 60 nl 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.

High-Throughput Toxicity and Phenotypic Screening of 3D Human Neural Progenitor Cell Cultures on a Microarray Chip Platform

“…The system presented herein is advantageous for several reasons. Miniaturization of 3D cultures, which may better emulate in vivo conditions, permits investigation under conditions where diffusional limitations are negligible (Ranga et al., 2014, Meli et al., 2014). Moreover, the modularity of a system with two complementary chips (one for cells and one for medium components) that stamp together to generate the high-throughput cell culture is potentially suitable for 3D parallel screening.…”

“…The growth rate of ReNcell VM cultured on-chip in 3D Matrigel was roughly three times slower than that reported for 2D cultures (Donato et al., 2007, Meli et al., 2014). The on-chip 3D cultures are prepared at a medium-to-cell ratio that is about half that typically used with larger-scale (i.e., flask) 2D cultures.…”

State-of-the-Art Metabolic Toxicity Screening and Pathway Evaluation

“…64 The stem cells were mixed with alginate, which was then printed onto a hydrophobic-modified glass slide, clamped in a cell and allowed to proliferate in cell expansion medium. Cytotoxicity was assayed utilizing a fluorescence live/dead cell assay.…”

“…It also showed that some compounds, such as acetaminophen, are nontoxic to neural cells at typically utilized concentrations. 64 …”

NanoEHS beyond toxicity – focusing on biocorona

“…47–50 It is also plausible that new HTS platforms for nanoEHS could be discovered based on proteomics and genomics-based approaches. 51 …”