Microarray with Micro‐ and Nano‐topographies Enables Identification of the Optimal Topography for Directing the Differentiation of Primary Murine Neural Progenitor Cells

Abstract

During development and tissue repair, progenitor cells are guided by both biochemical and biophysical cues of their microenvironment, including topographical signals. The topographical cues have been shown to play an important role in controlling the fate of cells. Systematic investigation of topographical structures with different geometries and sizes under the identical experimental conditions on the same chip will enhance the understanding of the role of shape and size in cell-topography interactions. A simple customizable multi-architecture chip (MARC) array is therefore developed to incorporate, on a single chip, distinct topographies of various architectural complexities, including both isotropic and anisotropic features, in nano- to micrometer dimensions, with different aspect ratios and hierarchical structures. Polydimethylsiloxane (PDMS) replicas of MARC are used to investigate the influence of different geometries and sizes in neural differentiation of primary murine neural progenitor cells (mNPCs). Anisotropic gratings (2 μm gratings, 250 nm gratings) and isotropic 1 μm pillars significantly promote differentiation of mNPCs into neurons, as indicated by expression of β-III-tubulin (59%, 58%, and 58%, respectively, compared to 30% on the control). In contrast, glial differentiation is enhanced on isotropic 2 μm holes and 1 μm pillars. These results illustrate that anisotropic topographies enhance neuronal differentiation while isotropic topographies enhance glial differentiation on the same chip under the same conditions. MARC enables simultaneous cost-effective investigation of multiple topographies, allowing efficient optimization of topographical and biochemical cues to modulate cell differentiation.

Enhanced differentiation of neural progenitor cells into neurons of the mesencephalic dopaminergic subtype on topographical patterns

“…Substrate topographies were previously demonstrated to affect neuronal specification and differentiation propensity during neural stem/progenitor cell differentiation [20,23]. Most studies however have focused on comparing single topographical patterns with the same dimension and shape.…”

“…The Multi-Architecture (MARC) chip is a versatile customizable topography microarray with a size of 2.2 cm  2.2 cm which can incorporate different topographies [20]. Each of the different topographies has a field area of approximately 4 mm 2 .…”

“…The mNPCs (passages 12e19) were isolated from the hippocampus of 5-day old mice [20] and maintained on 8 mg/ml of laminin (Life Technologies) in a neural progenitor expansion medium. This medium contained Dulbecco's modified Eagle medium (DMEM)/nutrient mixture F12 (Biological Industries, Israel) in a 1:1 ratio, 1Â N2 supplements (Life Technologies) and 1Â penicillin-streptomycin (Caisson Biotech, Texas, USA).…”

“…By taking into account biophysical influences in neuronal differentiation, other studies have shown that certain topographies can enhance differentiation of neural progenitors toward neural lineages [20,21]. Additional studies have also shown that topography can enhance neuronal differentiation of other stem cell types [21e25].…”

Topography Design Concept of a Tissue Engineering Scaffold for Controlling Cell Function and Fate Through Actin Cytoskeletal Modulation

“…The techniques for fabrication of such hierarchical scaffolds have now been developed. 4,[97][98][99][100] Their effectiveness in enhancing MSC adhesion and proliferation has been shown using a scaffold consisting of microscale strands with deposited micro/nano-sized fibers. 99 Microspheres with nanowires improve adhesion to a wide range of cell types and ensure excellent in vivo biocompatibility while retaining high loading capacity inherent in micron-sized particles.…”