Topographical and Physicochemical Modification of Material Surface to Enable Patterning of Living Cells

ABSTRACT:?

Precise control of the architecture of multiple cells in culture and in vivo via precise engineering of the material surface properties is described as cell patterning. Substrate patterning by control of the surface physicochemical and topographic features enables selective localization and phenotypic and genotypic control of living cells. In culture, control over spatial and temporal dynamics of cells and heterotypic interactions draws inspiration from in vivo embryogenesis and haptotaxis. Patterned arrays of single or multiple cell types in culture serve as model systems for exploration of cell-cell and cell-matrix interactions. More recently, the patterned arrays and assemblies of tissues have found practical applications in the fields of Biosensors and cell-based assays for Drug Discovery. Although the field of cell patterning has its origins early in this century, an improved understanding of cell-substrate interactions and the use of microfabrication techniques borrowed from the microelectronics industry have enabled significant recent progress. This review presents the important early discoveries and emphasizes results of recent state-of-the-art cell patterning methods. The review concludes by illustrating the growing impact of cell patterning in the areas of bioelectronic devices and cell-based assays for drug discovery.     

Microbial sensor cell arrays

Motivated by the advantages endowed by high-throughput analysis, researchers have succeeded in incorporating multiple reporter cells into a single platform; the technology now allows the simultaneous scrutiny of a large collection of sensor strains. We review current aspects in cell array technology with emphasis on microbial sensor arrays. We consider various techniques for patterning live cells on solid surfaces, describe different array-based applications and devices, and highlight recent efforts for live cell storage. We review mathematical approaches for deciphering the data emanating from bioreporter collections, and discuss the future of single cell arrays. Innovative technologies for cell patterning, preservation and interpretation are continuously being developed; when they all mature, cell arrays may become an efficient analytical tool, in a scope resembling that of DNA microarray biochips.     

Fabrication of cell-based arrays using micropatterned alkanethiol monolayers for the parallel silencing of specific genes by small interfering RNA

The emergence of small interfering RNA (siRNA) opened a new opportunity to study gene functions in a genome. However, large-scale loss-of-function analyses further require cell-based high-throughput methods that allow simultaneous silencing of the huge number of genes by siRNA. In this study, we aim at fabricating the cell-based siRNA arrays that facilitate parallel introduction of multiple siRNAs into cultured mammalian cells. The siRNA arrays were prepared using surface chemical processes including the micropatterning of a self-assembled monolayer and the layer-by-layer assembly of siRNA and cationic lipid. We examined the feasibility of the siRNA array for the sequence-specific gene silencing in an array format. Furthermore, the effects of siRNA loading and culture period after transfection were studied to optimize cell-based assays on the siRNA arrays. The results obtained in this study demonstrated that our method provides the siRNA arrays with spatial specificity in gene silencing, which will serve to obtain a quantitative data set from the cell-based screens on siRNA arrays.     

Application of magnetic force-based cell patterning for controlling cell-cell interactions in angiogenesis

To investigate the effects of cell-cell interactions on cellular function, the microenvironment surrounding cells should be precisely controlled. Here, we describe a cell patterning technique, which utilizes magnetic force and magnetite nanoparticles. This method was used to develop cell culture arrays for investigation of cell behaviors in angiogenesis. Pin holder devices that contain more than 6,000 pillars on the surface are used for fabricating the cell culture arrays by setting it on a magnet. The magnetically labeled cells were arranged by magnetic distribution. When the human umbilical vein endothelial cells are arranged at 250 microm intervals (5.9 cells/spot), the cells spread toward other cell cluster on adjacent spots in 4.5 h, and formed cord-like structures in 8.5 h. It was shown that cell-cell interactions were successfully investigated using magnetic cell arrangement.     

Reading the glyco-code: New approaches to studying protein–carbohydrate interactions

The surface of all living cells is decorated with carbohydrate molecules. Hundreds of functional proteins bind to these glycosylated ligands; such binding events subsequently modulate many aspects of protein and cell function. Identifying ligands for glycan-binding proteins (GBPs) is a defining challenge of glycoscience research. Here, we review recent advances that are allowing protein-carbohydrate interactions to be dissected with an unprecedented level of precision. We specifically highlight how cell-based glycan arrays and glyco-genomic profiling are being used to define the structural determinants of glycan-protein interactions in living cells. Going forward, these methods create exciting new opportunities for the study of glycans in physiology and disease.     

Cellular sociology regulates the hierarchical spatial patterning and organization of cells in organisms

Advances in single-cell biotechnology have increasingly revealed interactions of cells with their surroundings, suggesting a cellular society at the microscale. Similarities between cells and humans across multiple hierarchical levels have quantitative inference potential for reaching insights about phenotypic interactions that lead to morphological forms across multiple scales of cellular organization, namely cells, tissues and organs. Here, the functional and structural comparisons between how cells and individuals fundamentally socialize to give rise to the spatial organization are investigated. Integrative experimental cell interaction assays and computational predictive methods shape the understanding of societal perspective in the determination of the cellular interactions that create spatially coordinated forms in biological systems. Emerging quantifiable models from a simpler biological microworld such as bacterial interactions and single-cell organisms are explored, providing a route to model spatio-temporal patterning of morphological structures in humans. This analogical reasoning framework sheds light on structural patterning principles as a result of biological interactions across the cellular scale and up.     

Light it up: highly efficient multigene delivery in mammalian cells

Multigene delivery and expression systems are emerging as key technologies for many applications in contemporary biology. We have developed new methods for multigene delivery and expression in eukaryotic hosts for a variety of applications, including production of protein complexes for structural biology and drug development, provision of multicomponent protein biologics, and cell-based assays. We implemented tandem recombineering to facilitate rapid generation of multicomponent gene expression constructs for efficient transformation of mammalian cells, resulting in homogenous cell populations. Analysis of multiple parameters in living cells may require co-expression of fluorescently tagged sensors simultaneously in a single cell, at defined and ideally controlled ratios. Our method enables such applications by overcoming currently limiting challenges. Here, we review recent multigene delivery and expression strategies and their exploitation in mammalian cells. We discuss applications in drug discovery assays, interaction studies, and biologics production, which may benefit in the future from our novel approach.     

Pluripotent stem cell models of early mammalian development

Pluripotent stem cells derived from the early mammalian embryo offer a convenient model system for studying cell fate decisions in embryogenesis. The last 10 years have seen a boom in the popularity of two-dimensional micropatterns and three-dimensional stem cell culture systems as a way to recreate the architecture and interactions of particular cell populations during development. These methods enable the controlled exploration of cellular organization and patterning during development, using cell lines instead of embryos. They have established a new class of in vitro model system for pre-implantation and peri-implantation embryogenesis, ranging from models of the blastocyst stage, through gastrulation and toward early organogenesis. This review aims to set these systems in context and to highlight the strengths and suitability of each approach in modelling early mammalian development.     

Hematopoietic stem cell expansion: challenges and opportunities

Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivo-expanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell population. Although hematopoietic stem cells (HSCs) will rapidly expand after in vivo transplantation, experience from in vitro studies indicates that control of HSPC self-renewal and differentiation in culture remains difficult. Protocols that are based on hematopoietic cytokines have failed to support reliable amplification of immature stem cells in culture, suggesting that additional factors are required. In recent years, several novel factors, including developmental factors and chemical compounds, have been reported to affect HSC self-renewal and improve ex vivo stem cell expansion protocols. Here, we highlight early expansion attempts and review recent development in the extrinsic control of HSPC fate in vitro.     

Peptide growth factors in amphibian embryogenesis: intersection of modern molecular approaches with traditional inductive interaction paradigms

Recent discoveries of the role peptide growth factors (PGFs) play in regulating embryonic patterning and differentiation have profoundly influenced research on the molecular biology of early amphibian embryogenesis. Several PGFs have been recognized to be present as endogenous components of amphibian eggs and early embryos, while other PGFs -- which are known from heterologous systems (e.g., Drosophila) -- exert remarkable effects when injected as either protein or mRNA into eggs/embryos or when added to cultured embryonic tissue. For a variety of reasons (reviewed herein) optimism abounds that an understanding in molecular terms of the classical Spemann and Nieuwkoop tissue interactions which are generally believed to drive embryonic patterning is within reach. A critical assessment of the interpretations of some of the contemporary data on PGFs (included herein) should, however, temper some of that optimism. Likely, multiple rather than single PGFs act in a combinatorial fashion to contribute to individual patterning events. As well, substantial redundancy in PGF regulatory circuits probably exists, so the heavy reliance on tissue culture assays and overexpression studies which characterize much recent research needs to be circumvented. Potential experimental approaches for "next generation" experiments are discussed.     

Living in three dimensions

Research focused on deciphering the biochemical mechanisms that regulate cell proliferation and function has largely depended on the use of tissue culture methods in which cells are grown on two-dimensional (2D) plastic or glass surfaces. However, the flat surface of the tissue culture plate represents a poor topological approximation of the more complex three-dimensional (3D) architecture of the extracellular matrix (ECM) and the basement membrane (BM), a structurally compact form of the ECM. Recent work has provided strong evidence that the highly porous nanotopography that results from the 3D associations of ECM and BM nanofibrils is essential for the reproduction of physiological patterns of cell adherence, cytoskeletal organization, migration, signal transduction, morphogenesis, and differentiation in cell culture. In vitro approximations of these nanostructured surfaces are therefore desirable for more physiologically mimetic model systems to study both normal and abnormal functions of cells, tissues, and organs. In addition, the development of 3D culture environments is imperative to achieve more accurate cell-based assays of drug sensitivity, high-throughput drug discovery assays, and in vivo and ex vivo growth of tissues for applications in regenerative medicine.     

Mimicking dynamic in vivo environments with stimuli-responsive materials for cell culture

Traditional synthetic substrates and matrices for cell culture have proven to be of only limited utility in efforts to understand and control cell behavior, in large part because they fail to capture the multifarious biochemical, mechanical, geometric and dynamic characteristics of in vivo environments. However, recent advances in materials chemistry and engineering have begun to provide researchers with a toolbox to mimic the complex characteristics of natural extracellular matrices (ECMs), providing new pathways to explore cell-matrix interactions and direct cell fate under physiologically realistic conditions. In this review, we describe recent developments in stimuli-responsive materials as dynamic substrates and matrices for cell culture, and highlight their use in furthering our understanding of how cells respond to temporal variations in their environment.     

Microvalve-assisted patterning platform for measuring cellular dynamics based on 3D cell culture

A microfluidic platform to satisfy both 3D cell culture and cell-based assay is required for credible assay results and improved assay concept in drug discovery. In this article, we demonstrate a microvalve-assisted patterning (MAP) platform to provide a new method for investigating cellular dynamics by generating a linear concentration gradient of a drug as well as to realize 3D cell culture in a microenvironment. The MAP platform was fabricated by multilayer soft lithography and several microvalves made it possible to pattern a cell-matrix (scaffold) and to exchange media solutions without breaking cell-matrix structure in a microchannel. This approach provides not only exact fluids control, bubble removal, and stable solution exchange in a microchannel, but also reliable scaffold fabrication and 3D cell culture. In this study, hepatotoxicity tests with human hepatocellular liver carcinoma cells (HepG2) were also performed in real-time monitoring where cell morphologies exposed to different drug concentrations were observed at a time. Compared to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, the MAP platform could be used to reduce drug amount and assay time for cell-based assays as much as 10 and 3 times, respectively.     

Cell patterning using magnetite nanoparticles and magnetic force

Technologies for fabricating functional tissue architectures by patterning cells precisely are highly desirable for tissue engineering. Although several cell patterning methods such as microcontact printing and lithography have been developed, these methods require specialized surfaces to be used as substrates, the fabrication of which is time consuming. In the present study, we demonstrated a simple and rapid cell patterning technique, using magnetite nanoparticles and magnetic force, which enables us to allocate cells on arbitrary surfaces. Magnetite cationic liposomes (MCLs) developed in our previous study were used to magnetically label the target cells. When steel plates placed on a magnet were positioned under a cell culture surface, the magnetically labeled cells lined on the surface where the steel plate was positioned. Patterned lines of single cells were achieved by adjusting the number of cells seeded, and complex cell patterns (curved, parallel, or crossing patterns) were successfully fabricated. Since cell patterning using magnetic force may not limit the property of culture surfaces, human umbilical vein endothelial cells (HUVECs) were patterned on Matrigel, thereby forming patterned capillaries. These results suggest that the novel cell patterning methodology, which uses MCLs, is a promising approach for tissue engineering and studying cell-cell interactions in vitro.     

Influence of cell adhesion and spreading on impedance characteristics of cell-based sensors

Impedance measurements of cell-based sensors are a primary characterization route for detection and analysis of cellular responses to chemical and biological agents in real time. The detection sensitivity and limitation depend on sensor impedance characteristics and thus on cell patterning techniques. This study introduces a cell patterning approach to bind cells on microarrays of gold electrodes and demonstrates that single-cell patterning can substantially improve impedance characteristics of cell-based sensors. Mouse fibroblast cells (NIH3T3) are immobilized on electrodes through a lysine-arginine-glycine-aspartic acid (KRGD) peptide-mediated natural cell adhesion process. Electrodes are made of three sizes and immobilized with either covalently bound or physically adsorbed KRGD (c-electrodes or p-electrodes). Cells attached to c-electrodes increase the measurable electrical signal strength by 48.4%, 24.2%, and 19.0% for three electrode sizes, respectively, as compared to cells attached to p-electrodes, demonstrating that both the electrode size and surface chemistry play a key role in cell adhesion and spreading and thus the impedance characteristics of cell-based sensors. Single cells patterned on c-electrodes with dimensions comparable to cell size exhibit well-spread cell morphology and substantially outperform cells patterned on electrodes of other configurations.     

Analytical studies of silica biomineralization: towards an understanding of silica processing by diatoms

Diatoms have continued to attract research interest over a long time. One important reason for this research interest is the amazingly beautiful microstructured and nanostructured patterning of the silica-based diatom cell walls. These materials become increasingly important from the materials science point of view. However, many aspects of diatom cell wall formation and patterning are still not fully understood. The present minireview article summarizes our recent knowledge especially with respect to two major topics related to diatom cell wall formation and patterning: (1) uptake and metabolism of silicon by living diatom cells and (2) understanding of the genetic control of cell wall formation. Analytical techniques as well as recent results concerning these two topics are highlighted in this review.     

Microchip-based high-throughput screening analysis of combinatorial libraries

In recent years, there have been significant advances in biochemical assay miniturization and integration of microchip-based technologies with combinatorial library screening for high-throughput and large-scale applications. Small-molecule microarrays, protein arrays and cell-based arrays and conventional DNA arrays as well as microfluidic approaches in HTS are discussed in this review.     

A germ cell origin of embryonic stem cells?

Because embryonic stem (ES) cells are generally derived by the culture of inner cell mass (ICM) cells, they are often assumed to be the equivalent of ICM cells. However, various evidence indicates that ICM cells transition to a different cell type during ES-cell derivation. Historically, ES cells have been believed to most closely resemble pluripotent primitive ectoderm cells derived directly from the ICM. However, differences between ES cells and primitive ectoderm cells have caused developmental biologists to question whether ES cells really have an in vivo equivalent, or whether their properties merely reflect their tissue culture environment. Here, we review recent evidence that the closest in vivo equivalent of an ES cell is an early germ cell.     

Stem cell-based composite tissue constructs for regenerative medicine

A major task of contemporary medicine and dentistry is restoration of human tissues and organs lost to diseases and trauma. A decade-long intense effort in tissue engineering has provided the proof of concept for cell-based replacement of a number of individual tissues such as the skin, cartilage, and bone. Recent work in stem cell-based in vivo restoration of multiple tissue phenotypes by composite tissue constructs such as osteochondral and fibro-osseous grafts has demonstrated probable clues for bioengineered replacement of complex anatomical structures consisting of multiple cell lineages such as the synovial joint condyle, tendon-bone complex, bone-ligament junction, and the periodontium. Of greater significance is a tangible contribution by current attempts to restore the structure and function of multitissue structures using cell-based composite tissue constructs to the understanding of ultimate biological restoration of complex organs such as the kidney or liver. The present review focuses on recent advances in stem cell-based composite tissue constructs and attempts to outline challenges for the manipulation of stem cells in tailored biomaterials in alignment with approaches potentially utilizable in regenerative medicine of human tissues and organs.