Elsevier

Biomaterials

Volume 27, Issue 16, June 2006, Pages 3044-3063
Biomaterials

Review
Surface engineering approaches to micropattern surfaces for cell-based assays

https://doi.org/10.1016/j.biomaterials.2005.12.024Get rights and content

Abstract

The ability to produce patterns of single or multiple cells through precise surface engineering of cell culture substrates has promoted the development of cellular bioassays that provide entirely new insights into the factors that control cell adhesion to material surfaces, cell proliferation, differentiation and molecular signaling pathways. The ability to control shape and spreading of attached cells and cell–cell contacts through the form and dimension of the cell-adhesive patches with high precision is important. Commitment of stem cells to different specific lineages depends strongly on cell shape, implying that controlled microenvironments through engineered surfaces may not only be a valuable approach towards fundamental cell-biological studies, but also of great importance for the design of cell culture substrates for tissue engineering. Furthermore, cell patterning is an important tool for organizing cells on transducers for cell-based sensing and cell-based drug discovery concepts. From a material engineering standpoint, patterning approaches have greatly profited by combining microfabrication technologies, such as photolithography, with biochemical functionalization to present to the cells biological cues in spatially controlled regions where the background is rendered non-adhesive (“non-fouling”) by suitable chemical modification. The focus of this review is on the surface engineering aspects of biologically motivated micropatterning of two-dimensional (flat) surfaces with the aim to provide an introductory overview and critical assessment of the many techniques described in the literature. In particular, the importance of non-fouling surface chemistries, the combination of hard and soft lithography with molecular assembly techniques as well as a number of less well known, but useful patterning approaches, including direct cell writing, are discussed.

Introduction

Microfabrication techniques combined with surface chemistry and material science knowledge has provided new tools to further explore, in vitro, the interactions of anchorage-dependent cells with their environment. Cellular developments such as proliferation, differentiation, migration or apoptosis are guided by multiple surface cues that are potentially remodeled during cell culture assays. The cell responses are controlled by intra-cellular signaling pathways that are originally triggered by transmembrane proteins interacting with the engineered surface [1]. The surface chemistry characterized by the type of cell-binding ligands (peptides, proteins, etc.), their surface density [2], [3], [4] and spatial distribution [1], [5], [6] as well as their conformation [7], have been demonstrated to be important surface cues. In order to be viable, anchorage-dependent cells require an adhesive surface to exert forces and consequently spread. The ability to constrain the spreading to a specific cell-surface contact area has been shown to dramatically affect cellular development [8], [9], [10]. Mechanical compliance of cell-adhering substrates can also substantially affect the cellular response and development [11]. The ability to spatially and temporally control the chemistry, the pattern geometry and the local substrate stiffness will continue to provide new insights into the fundamental aspects of cell–surface interactions [12], [13].

Apart from its use in fundamental cell-surface investigations, arrays of living cells (individual or multiple) have found major applications in both cell-based sensors and drug discovery. Cell-based sensor devices contain living cells that monitor perturbations of the environment such as toxic or pathogenic agents [14], [15], [16]. Cell-based assays in drug discovery are considered promising screening approaches, intermediate between gene- or protein-based studies and whole animal models [15], [17]. The use of cell-based assays that mimic specific in vivo behavior is believed to decrease costs while leading to more accurate prediction in the drug discovery process [18].

The different engineering approaches aiming at a precise control of cell adhesion and spreading, through chemically and spatially designed surfaces, are the main focus of this review. It primarily addresses engineers new in the field of cell patterning/cell-based sensors as well as cell biologists interested in exploiting new tools to solve specific questions related to cell–surface interaction. Therefore, the concept, the required infrastructure, and the limitations of each technique are presented, along with selected scientific results, while highlighting the novelty or the originality of each approach.

Note that this review exclusively presents two-dimensional chemical micropatterning techniques (down to 1 μm pattern dimensions) but does not include the engineering of surface topographies. Several reviews on topographical structuring have been recently published [19], [20], [21], [22]. Chemical nanopatterns have recently gained significant attention because they provide tools to study fundamental aspects of cell-adhesion at the level of single protein/receptor molecules [23]. Nanopatterned surfaces are not included in this review due to space limitation; however, several excellent reviews on nanopatterns are available [24], [25], [26], [27], [28].

Section snippets

The basis of cellular patterning—non-fouling surface chemistries

In any surface-coupled cellular patterning, the ability to suppress non-specific interactions between the surface and the protein-containing media is crucial in order to generate unbiased experimental outcomes. Advances in surface chemistry have made possible the synthesis of so-called non-fouling surfaces that significantly reduce or eliminate the non-specific adsorption of proteins and other biomolecules from biological fluids such as cell culture media. Several types of native molecules have

Soft lithography

“Soft lithography” is commonly used to create chemical structures on surfaces for controlling cell–substrate interactions [69], [70]. The name “soft lithography” does not cover one specific method but rather a group of techniques with the common feature that at some stage of the process an elastomeric (“soft”) material is used to create the chemical structures. In this review we concentrate on two related techniques of this family: microcontact printing (μCP) and microfluidic patterning (μFLP).

Patterning with photolithography

In the photolithography process, geometric features drawn on a mask are transferred via UV illumination onto a substrate. A mask is generally made of a quartz (glass) plate coated with a thin layer of non-transparent chromium, and presents the desired geometric features. The design of the mask can be created with any computer-aided design (CAD) software and can be sent to a mask manufacturing company. Such quartz/chromium masks routinely allow feature resolution down to 1–2 μm. When poorer

Plasma polymerization combined with photolithography or laser ablation

Photolithography combined with plasma thin-film polymerization has been exploited over the past years for the patterning of biomaterials dedicated to two-dimensional in vitro cell cultures [147], [148]. Plasma is an ionized gas where some or all the electrons of the outer atomic orbitals have become separated from the atoms or the molecules. Artificial plasmas are often generated by radio-frequency (MHz) glow discharge setups. Provided that plasma parameters are adequate they allow deposition

Photoimmobilization and photochemically generated patterns

Surface-immobilized photoreactive molecules have been used for generating chemical micropatterns for cell attachment. Hu and coworkers immobilized oligopeptides containing the Arg–Gly–Asp (RGD) sequence on top of ethylene glycol modified-alkanethiols via UV or laser activation of benzophenone groups [150]. They showed that a linear correlation exists between the exposure time and the amount of immobilized oligopeptide (in the range of 0–5 pmol/cm2) [151]. By scanning the laser at different

Stencil-assisted patterning

A stencil is a membrane (stiff or flexible) that is structured with through-holes of the desired size and geometry. When the stencil is brought in close contact with the substrate it can be used as a template to locally modify the surface while the areas outside the holes remain protected by the stencil (Fig. 5). Processes such as deposition or ablation of molecules for chemical patterning as well as cell seeding (without any chemical surface treatment) will therefore only occur on those

Jet patterning (ink-jet technology)

Ink-jet printing is another example where tissue-engineers have adapted existing technologies for biological research and applications. For example, it is possible by minor modification of a commercial printer to create chemical patterns by directly jetting alkanethiols or proteins onto surfaces [159], [160], [161], [162]. Wilson et al. reported direct printing of cells onto gel membranes with a reported dead-cell percentage of 25% after 72 h. Cell death was primarily attributed to dehydration

Laser-guided writing with cells

Optical force is another way to direct living cells to specific locations on surfaces. Objects such as cells or particles, with a higher refractive index than the surrounding environment, and will be confined into a so-called optical trap when submitted to focused laser light. Such “lock-ins” can be punctual and thus allow the displacement of one cell at a time (laser tweezers) [164] or they can be linear. A linear “optical trap” is defined by the laser beam and the low numerical aperture lens.

General conclusions

A number of techniques utilized for cell patterning have been reviewed; some are still at their early stage of development (e.g., laser-guided cell deposition), while others benefit from several decades of dedicated development and application (e.g., photolithography). The reviewed techniques can be separated in two categories; those where the cells are passively patterned by random seeding on surfaces modified with cytophilic and cytophobic regions, and those where the cells are actively

Acknowledgements

This work was supported by the EC Sixth Framework Programme (Project “NANOCUES”), and as part of the European Science Foundation EUROCORES Programme ‘Self-Organized NanoStructures (SONS)”, by funds from the Swiss National Science Foundation and the EC Sixth Framework Programme. Financial support by ETH Zurich and EPF Lausanne is also acknowledged.

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