A Dual Gradient Assay for the Parametric Analysis of Cell–Surface Interactions

Abstract

Cellular response to microgrooves is addressed using a new assay format, comprising orthogonal gradients of continuously varied groove pitch and depth. Dual layer etch masks are created using a combination of micropatterning and plasma polymer deposition. A silicon substrate with a constant groove width of 8 μm and with ridge width increasing from 8 μm in 0.5 μm steps across 10 mm is fabricated by photolithography. A plasma-polymerized hexane film which is 120 nm thick at one end of these grooves, and 10 nm at the other, is deposited under a diffusion mask. Reactive etching of the patterned sample transfers a gradient of groove pitch and groove depth into the silicon substrate. A silicon master with a gradient of groove depth spanning more than two orders of magnitude (less than 10 nm to over 1000 nm) is used to create an injection molding inlay for mass replication of the screening topography. Polycarbonate replicas are molded for use in cell culture studies, and the functionality of the topography as a high-throughput screening platform is investigated. The response of MDCK, h-TERT fibroblasts, and LE2 endothelial cells is examined, in terms of attachment and morphological response to the variation in topographical cues, with the aim of pinpointing the optimal combination of groove pitch and depth to elicit a tailored response from each cell type. When the range of topographical features screened on a single substrate is considered, this new assay represents a significant step forward in the parametric design and analysis of topographical cues at the biomaterial interface.

Enhanced Differentiation of Human Embryonic Stem Cells Toward Definitive Endoderm on Ultrahigh Aspect Ratio Nanopillars

“…DE cells may have a slightly higher affi nity to the pillar substrates compared to hES cells ( Figure 5 ). Selective cell attachment using nanotopography has previous been reported between different cell types, [ 35,36 ] but to our knowledge it has not been observed between hES cells and hES cell derived DE cells. The selective affi nity alone however cannot explain the high fraction of Sox17 on the soft nanopillars (Figure 3 ) because the ratio of hES to DE cells is ≈1:20 after the differentiation on soft pillars and only 1:1.29 using predifferentiated cells ( Figure 5 ).…”

Characterization of Pore Structure in Biologically Functional Poly(2-Hydroxyethyl Methacrylate) - Poly(Ethylene Glycol) Diacrylate (PHEMA-PEGDA)

“…al. demonstrated that cell alignment along the axis of micro- and nano-features is cell type dependent [4]. Multiple cell types displayed alignment in response to linear surface features of different widths and depths.…”

“…While the cell's response to topography has been extensively studied [1], [2], [3], [4], [5], the mechanisms that regulate cell interaction with topography remain to be clearly understood [6]. Manipulation of the cell-topography interaction can potentially be exploited for tissue engineering, including cell alignment and tissue development.…”

“…Multiple cell types displayed alignment in response to linear surface features of different widths and depths. For example, human fibroblasts displayed high levels of cell alignment at a wider range of feature dimensions [4]. More importantly, topographies can alter biological functionality.…”

“…Surface roughness also appears to regulate phenotype-specific protein production in MC3T3 osteoblasts [5]. While previous studies of the cell-topography interaction further advanced our understanding, the substrate preparation was largely labor intensive and generally required specialized equipment for casting or lithography [1], [2], [3], [4], [7]. To increase the efficiency of substrate production, an alternative fabrication process may be warranted.…”