Neural stem cells (NSCs) are sensitive to microenvironmental cues, including cell-cell contact, cell-extracellular matrix interaction, nutrient and waste transport, and environmental oxygen composition. How these parameters affect the stem cells’ morphology, proliferation, and differentiation remains an open area for research. In this study, we demonstrated how a new platform with microfluidic cell culture devices is capable of multi-parametric microenvironment control for NSC studies.

NSCs are self-renewable and multipotent cells capable of generating various nervous system phenotypes.¹ First described in the adult mouse brain,² NSCs have received attention due to their therapeutic potential. Research efforts generally divide into three areas: expansion, differentiation and cell-cell interactions. However, building a physiologically relevant in vitro culture model remains challenging. Researchers have attempted two-dimensional culture flasks, three-dimensional neurospheres,³ scaffolds,⁴ and microfluidics .⁵  While each has demonstrated NSCs’ sensitivity to microenvironmental cues, no unified platform has enabled researchers to systematically control the microenvironment.

EMD Millipore’s CellASIC ONIX Microfluidic Platform offers comprehensive, multi-parametric microenvironmental control for cell culture studies. The platform includes a microfluidic system, microincubator controller, microincubator manifold, and a plate with four independent units and eight wells per unit. To modulate the degree of cell-cell contact, the system can seed cells at varying density, and protocols have been developed to enable changes to cell-ECM interaction. For nutrient and waste transport, passive gravity-driven and pneumatically-driven control can provide low-shear, steady-state perfusion of various media or solution exchanges. Since the cell culture chamber plate is made of gas-permeable materials, it can be inoculated with different gas mixtures. In this study, we show the oxygen microenvironment can be tuned from severe anoxia to hyperoxia.

To characterize NSCs grown in the microfluidic-controlled microenvironment, an automated immunostaining protocol was developed to visualize nestin and Sox2. The speed at which gas conditions could be changed was also evaluated to assess the system’s utility for studying cell responses to hypoxia.

The successful combination of environment control with perfusion culture in a microfluidic platform promises to further close the gap between in vitro experiments and in vivo relevance.

Materials and Methods

Rat hippocampus neural stem cells, antibodies recognizing Nestin and Sox2, the CellASIC ONIX Microfluidic System, Microincubator Controller, Tri-Gas Mixer, Microfluidic Plates and reagents were from EMD Millipore. Poly-L-ornithine and laminin were acquired from Sigma. An Olympus IX-71 inverted fluorescence microscope and an oxygen sensor from Presense were also used.

The cell culture chamber was coated with extracellular matrices, and the cell suspension was seeded at both high and low densities. The culture medium was then added to well 1 to allow gravity-driven perfusion, and the plate was placed inside an incubator.

PBS was added to well 1 as the wash solution, 4% paraformaldehyde was added to well 2 as a fixing agent, 0.2% BSA and 0.1% TritonX-100 in 1x PBS was added to well 3 for permeation and blocking, primary antibody solution with 1% BSA was added to well 4, and secondary antibody was added to well 5. The pressure applied to each valve was programmed to conduct automated immunostaining.

To investigate the impact of oxygen microenvironment, we seeded the cells under the normoxia condition and let them stabilize. We then transferred the NSCs onto microincubators and tri-gas mixers tuned to three gas compositions: severe hypoxia (~0.1% O₂), mild hypoxia (~3% O₂) and normoxia (~20% O₂). We cultured the rat NSCs through pneumatic pumping of medium across the chamber, and bright field images were acquired with a subset of cells immunostained for nestin and Sox2.NSC behavior was evaluated with respect to gaseous microenvironment.  First, the gaseous microenvironment exchange rate was determined. A pre-calibrated 5% CO₂ gas was introduced through the microincubator and the gas exchanged thoroughly in about 15 minutes (Figure 1).

Results

Key features of NSCs include self renewal and lack of differentiation, which were examined using automated immunostaining to detect the NSC markers nestin and Sox2 (Figure 2). The cells in the chamber were stained, showing nestin in the cytosol and Sox2 in the nucleus, which confirmed the rat NSCs maintained an undifferentiated phenotype.

Cells were then seeded at high density under mildly hypoxic conditions (3% O₂). After four days, the cells formed neurospheres. Staining revealed the core contained only bright spots of Sox2 while the outer ring exhibited both nestin and Sox2 expression (Figure 3). Cells detached, presumably due to cell death, 24 hours after exposure to severe hypoxia (0.1% O₂). At low cell density, cells disaggregated under mildly hypoxic conditions, which promoted single-layer cellular growth. In contrast, under normoxic conditions, the cells aggregated into multilayer cellular masses (Figure 4).

Discussion

The oxygen microenvironment regulates NSC metabolism, proliferation, surivival and fate.⁷ Given that neural stem and progenitor cells have shown increased proliferation and self-renewal under mild hypoxic conditions,^8-10 studying the effect of gaseous microenvironment has the potential to advance research on NSC-based neurodegeneration therapies.

Protocols to perform live cell imaging of rat NSCs have been developed, and by varying the microenvironment parameters, it was determined that the rat NSCs exhibited different morphologies and proliferated best under physiologic, mildly hypoxic conditions, as reported in the literature.^8-10 Through the combination of high seeding density, polyornithine/laminin coating, continuous perfusion, and mild hypoxia gas microenvironment, the rat NSCs formed a neurosphere in 96 hours.

The effect of varying cell density was investigated. The importance of cell density has been recently established by studies showing that cell-cell signaling can regulate NSC differentiation. ^{11, 12} Through live cell imaging, we discovered that, while peripheral cells around the neurosphere were successfully immunostained for two pluripotency markers, nestin and Sox2, the image of the neurosphere itself showed condensed bright spots of Sox2 but no nestin, suggesting that increased cell-cell contacts within the neurosphere may affect NSC differentiation.

In summary, we demonstrated the combinatorial effect of microenvironment parameters on rat NSCs and the capability of imaging them with fluorescent microscopy. The platform promises to facilitate assay development for NSCs and provides a better-controlled in vitro model system for neural development research.

References

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