TopoWellPlate:a well-plate-based screening platform to study cell-surface topography interactions

The field of biomaterial engineering is increasingly using high‐throughput approaches to investigate cell–material interactions. Because most material libraries are prepared as chips, immunofluorescence‐based read‐outs are used to uniquely image individual materials. This paper proposes to produce libraries of materials using a well‐based strategy in which each material is physically separated, and thus compatible with standard biochemical assays. In this work, the TopoWellPlate, a novel system to study cell–surface topography interaction in high‐throughput is presented. From a larger library of topographies, 87 uniquely defined bioactive surface topographies are identified, which induce a wide variety of cellular morphologies. Topographically enhanced polystyrene films are fabricated in a multistep cleanroom process and served as base for the TopoWellPlate. Thermal bonding of the films to bottomless 96‐well plates results in a cell culture ready, topographically enhanced, 96‐well plate. The overall metabolic activity of bone marrow‐derived human mesenchymal stem cells is measured to show the functionality of the TopoWellPlate as a screening tool, which showed a 2.5‐fold difference range in metabolic activity per cell. TopoWellPlates of this and other topographical designs can be used to analyze cells using the wealth of standardized molecular assays available and thus disclose the mechanisms of biomaterials‐induced mechanotransduction

Micro-scaled topographies direct differentiation of human epidermal stem cells

\u3cp\u3eHuman epidermal stem cells initiate terminal differentiation when spreading is restricted on ECM-coated micropatterned islands, soft hydrogels or hydrogel-nanoparticle composites with high nanoparticle spacing. The effect of substrate topography, however, is incompletely understood. To explore this, primary human keratinocytes enriched for stem cells were seeded on a topographical library with over 2000 different topographies in the micrometre range. Twenty-four hours later the proportion of cells expressing the differentiation marker transglutaminase-1 was determined by high content imaging. As predicted, topographies that prevented spreading promoted differentiation. However, we also identified topographies that supported differentiation of highly spread cells. Topographies supporting differentiation of spread cells were more irregular than those supporting differentiation of round cells. Low topography coverage promoted differentiation of spread cells, whereas high coverage promoted differentiation of round cells. Based on these observations we fabricated a topography in 6-well plate format that supported differentiation of spread cells, enabling us to examine cell responses at higher resolution. We found that differentiated spread cells did not assemble significant numbers of hemidesmosomes, focal adhesions, adherens junctions, desmosomes or tight junctions. They did, however, organise the actin cytoskeleton in response to the topographies. Rho kinase inhibition and blebbistatin treatment blocked the differentiation of spread cells, whereas SRF inhibition did not. These observations suggest a potential role for actin polymerization and actomyosin contraction in the topography-induced differentiation of spread cells. Statement of Significance: The epidermis is the outer covering of the skin. It is formed by layers of cells called keratinocytes. The basal cell layer contains stem cells, which divide to replace cells in the outermost layers that are lost through a process known as differentiation. In this manuscript we have developed surfaces that promote the differentiation of epidermal stem cells in order to understand the signals that control differentiation. The experimental tools we have developed have the potential to help us to devise new treatments that control diseases such as psoriasis and eczema in which epidermal stem cell proliferation and differentiation are disturbed.\u3c/p\u3

NanoTopoChip:high-throughput nanotopographical cell instruction

\u3cp\u3eSurface topography is able to influence cell phenotype in numerous ways and offers opportunities to manipulate cells and tissues. In this work, we develop the Nano-TopoChip and study the cell instructive effects of nanoscale topographies. A combination of deep UV projection lithography and conventional lithography was used to fabricate a library of more than 1200 different defined nanotopographies. To illustrate the cell instructive effects of nanotopography, actin-RFP labeled U2OS osteosarcoma cells were cultured and imaged on the Nano-TopoChip. Automated image analysis shows that of many cell morphological parameters, cell spreading, cell orientation and actin morphology are mostly affected by the nanotopographies. Additionally, by using modeling, the changes of cell morphological parameters could by predicted by several feature shape parameters such as lateral size and spacing. This work overcomes the technological challenges of fabricating high quality defined nanoscale features on unprecedented large surface areas of a material relevant for tissue culture such as PS and the screening system is able to infer nanotopography – cell morphological parameter relationships. Our screening platform provides opportunities to identify and study the effect of nanotopography with beneficial properties for the culture of various cell types. Statement of Significance The nanotopography of biomaterial surfaces can be modified to influence adhering cells with the aim to improve the performance of medical implants and tissue culture substrates. However, the necessary knowledge of the underlying mechanisms remains incomplete. One reason for this is the limited availability of high-resolution nanotopographies on relevant biomaterials, suitable to conduct systematic biological studies. The present study shows the fabrication of a library of nano-sized surface topographies with high fidelity. The potential of this library, called the ‘NanoTopoChip’ is shown in a proof of principle HTS study which demonstrates how cells are affected by nanotopographies. The large dataset, acquired by quantitative high-content imaging, allowed us to use predictive modeling to describe how feature dimensions affect cell morphology.\u3c/p\u3

Immune Modulation by Design: Using Topography to Control Human Monocyte Attachment and Macrophage Differentiation

Macrophages play a central role in orchestrating immune responses to foreign materials, which are often responsible for the failure of implanted medical devices. Material topography is known to influence macrophage attachment and phenotype, providing opportunities for the rational design of "immune-instructive" topographies to modulate macrophage function and thus foreign body responses to biomaterials. However, no generalizable understanding of the inter-relationship between topography and cell response exists. A high throughput screening approach is therefore utilized to investigate the relationship between topography and human monocyte-derived macrophage attachment and phenotype, using a diverse library of 2176 micropatterns generated by an algorithm. This reveals that micropillars 5-10 µm in diameter play a dominant role in driving macrophage attachment compared to the many other topographies screened, an observation that aligns with studies of the interaction of macrophages with particles. Combining the pillar size with the micropillar density is found to be key in modulation of cell phenotype from pro to anti-inflammatory states. Machine learning is used to successfully build a model that correlates cell attachment and phenotype with a selection of descriptors, illustrating that materials can potentially be designed to modulate inflammatory responses for future applications in the fight against foreign body rejection of medical devices.status: publishe

Mining for osteogenic surface topographies:in silico design to in vivo osseo-integration

\u3cp\u3eStem cells respond to the physicochemical parameters of the substrate on which they grow. Quantitative material activity relationships – the relationships between substrate parameters and the phenotypes they induce – have so far poorly predicted the success of bioactive implant surfaces. In this report, we screened a library of randomly selected designed surface topographies for those inducing osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Cell shape features, surface design parameters, and osteogenic marker expression were strongly correlated in vitro. Furthermore, the surfaces with the highest osteogenic potential in vitro also demonstrated their osteogenic effect in vivo: these indeed strongly enhanced bone bonding in a rabbit femur model. Our work shows that by giving stem cells specific physicochemical parameters through designed surface topographies, differentiation of these cells can be dictated.\u3c/p\u3