Establishment of a cell micropatterning method for the quantitative assessment of the organization of the keratin filament network

1.2 Cell micropatterning: a tool to regulate cell shape
Cell micropatterning refers to a technique within cell culture methods whereby the assembly of ECM-proteins e.g. fibronectin (FN) on a specific surface is controlled in order to achieve an accurate positioning of cells and control over cell size and spatial arrangement. This requires the ability to fabricate adhesive regions which mediate cell attachment and non-adhesive regions preventing cell attachment. Adhesive regions, commonly called micropatterns, can be fabricated in different shapes and sizes and can have a resolution reaching micro-scale.
One of the techniques most used in order to fabricate micropatterns on a planar substrate is micro-contact printing (µCP), in which a polydimethylsiloxane (PDMS) stamp is fabricated based on a replica molding process by casting the liquid prepolymer of PDMS over a rigid master with patterned structures. After curing, the PDMS stamp is peeled off the mold, inked with a protein of choice and transferred onto the surface of a planar substrate by placing the microstructured, inked surface of the stamp in contact with the substrate. Upon removing the stamp, the desired pattern is left behind on the surface (Fig. 2 a). Alternatively, a stencil containing through holes can be used to fabricate micropatterns. The stencil can be fabricated either by casting a thin film of the liquid material to be used directly on the master without covering it wholly so that through holes can form or by depositing a curable material on the edges of a PDMS stamp placed on a planar substrate. Via capillary suction the gaps between the stamp and the substrate is filled. After curing and the removal of the stamp, the stencil can be peeled off and applied to the final substrate to be used. Adhesive proteins deposited on top of the stencil can come in contact with the underlying substrate only at regions left exposed by the though holes, resulting in the formation of adhesive micropatterns (Fig. 2 b).

µCP and stencil patterning

Fig. 2 Schematic procedure for two different techniques of protein patterning. a) In micro-contact printing (µCP), a PDMS stamp with a microstructured surface is used to transfer extracellular matrix protein (ECM-protein) onto a glass coverslip. b) During stencil patterning, a microstructured stencil is placed on a PDMS-coated coverslip. ECM-protein is subsequently added and incubated of sometime before the stencil is removed after aspirating the ECM solution.

The protein of choice can range from adhesive ECM proteins, e.g. fibronectin, collagen or laminin to amine-terminated self-assembled monolayers (SAMs) or Arg-Gly-Asp (RGD) peptide.
Upon seeding cells on a micropatterned substrate cells adapt a similar shape and size as the adhesive micropatterns thus enabling the manipulation of cell architecture, cell-ECM and cell-cell interaction.
In the past decades it has been revealed that cell morphology regulate different cellular processes like differentiation and cell fate, cell cycle control, proliferation, apoptosis and migration (Chen et al., 1997; de Juan-Pardo et al., 2006; Huang et al., 1998; Huang and Ingber, 2000; Ingber et al., 1995; LeDuc and Bellin, 2006, Parker et al., 2002). For example, Chen et al. (1997) demonstrated that human and bovine capillary endothelial cells were limited from spreading when micropatterns decreased in size and that cell fate shifted from growth to apoptosis, regardless of the type of adhesive protein used to promote attachment. Recent studies demonstrate how far-reaching geometric influences can be: It has been hypothesized that changes in cell architecture and associated changes in nuclear shape cause changes in chromatin structure and organization and thus alternating transcriptional activity. Stiles et al. (2013) aimed to uncover the influence of different cell shapes on global gene expression patterns of human coronary artery endothelial cells. Stiles and colleagues shown that morphological restriction in general significantly affect global gene expression patterns compared to non-restricted cells. These and many other examples demonstrate the influence of the extracellular physical environment in regard to cell response. Apart from their usefulness in exploring alternations in cellular morphology and physiology adhesive micropatterns ensure reproducibility, enable the detection of drug effects and facilitate automated image acquisition and analysis.

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