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Journal ArticleDOI

Microfabricated multilayer parylene‐C stencils for the generation of patterned dynamic co‐cultures

TL;DR: In this paper, a micropatterning technique based on microfabricated multilayer parylene-C stencils was proposed to generate dynamic co-cultures of murine embryonic stem cells.
Abstract: Co-culturing different cell types can be useful to engineer a more in vivo-like microenvironment for cells in culture. Recent approaches to generating cellular co-cultures have used microfabrication technologies to regulate the degree of cell-cell contact between different cell types. However, these approaches are often limited to the co-culture of only two cell types in static cultures. The dynamic aspect of cell-cell interaction, however, is a key regulator of many biological processes such as early development, stem cell differentiation, and tissue regeneration. In this study, we describe a micropatterning technique based on microfabricated multilayer parylene-C stencils and demonstrate the potential of parylene-C technology for co-patterning of proteins and cells with the ability to generate a series of at least five temporally controlled patterned co-cultures. We generated dynamic co-cultures of murine embryonic stem cells in culture with various secondary cell types that could be sequentially introduced and removed from the co-cultures. Our studies suggested that dynamic co-cultures generated by using parylene-C stencils may be applicable in studies investigating cellular interactions in controlled microenvironments such as studies of ES cell differentiation, wound healing and development.

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Citations
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Journal ArticleDOI
TL;DR: This review highlights the recent progress in the field of microscale tissue engineering and discusses the use of various biomaterials for generating engineered tissue structures with microscale features for regenerative medicine and biorobotics.
Abstract: Each year, millions of patients suffer from organ failure or damage. Many of these patients die while they are on organ transplantation waiting lists due to insufficient number of donors. The field of tissue engineering has emerged to address this need by creating transplantable tissues or organs in the laboratory. It is an interdisciplinary field which exploits living cells through integration of engineering, materials science, biological sciences and medicine to maintain, restore and enhance normal tissue and organ function.[1, 2] Almost two decades after Langer and Vacanti’s seminal article “Tissue Engineering” [3] in which they described the principles of tissue engineering, off-the-shelf engineered tissues are becoming more and more realistic. The market for tissue engineering and regenerative medicine in the United States has grown to $6.9 billion in 2009 (Fig. 1).[4] This market is estimated to further grow to about $32 billion in 2018. The potential market spans a wide range of tissues from degenerative or trauma caused orthopedic or nervous system therapies to cardiovascular and diabetes applications, and even to dental or ophthalmological problems. However, currently available engineered tissues are largely limited to skin epidermis, corneal epithelium and cartilage.[4] The common point of these tissues is their relatively simple architecture; in other words the simplicity in their tissue architecture and cellular organization. For example, there is less need for a vasculature in an engineered cartilage tissue or corneal epithelium compared to metabolically active and large tissues such as the heart or liver. Most tissues are composed of more than one cell type, usually with quite regular and microscale organization of these cells and extracellular matrix (ECM) components secreted by them, in order to perform a specific function. Since the functionality of a certain tissue is related with this complex architecture, tissue engineers have been trying to mimic and recapitulate this complexity in vitro. Although there are other approaches such as organizing cell sheets (which will not be covered in this review article), modulating the scaffold microarchitecture is one of the most potent ways of achieving biomimetic tissues. A three dimensional (3D) version of this biomimicry is a relatively recent phenomenon, and this research field is currently under intense exploration.[5] Another advantage of incorporating 3D microfeatures within tissue engineering scaffolds is the spatial organization of different types of cells in the same way as in the target tissue. Similarly, various ECM macromolecules or ECM-mimicking synthetic molecules can be spatially distributed in a predetermined manner to enhance biomimicry. Open in a separate window Figure 1 Worldwide tissue engineering, cell therapy and transplantation market. Size and growth by region, 2009.[4]

370 citations

Journal ArticleDOI
TL;DR: The applicability of parylene C as an encapsulation material for implanted neural prostheses was characterized and optimized and an electrochemical impedance spectroscopy was used to test if a pARYlene C layer was able to protect a metallic structure against corrosion on a Si(3)N(4) substrate.
Abstract: The applicability of parylene C as an encapsulation material for implanted neural prostheses was characterized and optimized. The adhesion of parylene C was tested on different substrate materials, which were commonly used in neural prostheses and the efficiency of different adhesion promotion methods was investigated. On Si(3)N(4), platinum, and on a first film of parylene C, a satisfactory adhesion was achieved with Silane A-174, which even withstood standard steam sterilization. The adhesion to gold and polyimide could not be improved sufficiently with the tested methods. Furthermore, tensile tests and measurements of the degree of crystallinity were performed on untreated, on steam sterilized, and on annealed parylene C layers to investigate the influence of thermal treatment. This led to more brittle and stiffer films due to an increase in the crystalline portion in the parylene layers. Finally, an electrochemical impedance spectroscopy was used to test if a parylene C layer was able to protect a metallic structure against corrosion on a Si(3)N(4) substrate. The results indicated that this could be only possible by treating the substrate with Silane A-174. To receive parylene C layers with a good encapsulation performance, it is important to consider the materials, which are used in the neural prosthesis, to find the best suited process parameters.

205 citations

Journal ArticleDOI
TL;DR: Using the HSC niche as a model, microscale engineering strategies capable of systematically examining and reconstructing individual niche components are discussed, suggesting synthetic stem cell-niche engineering may form a new foundation for regenerative therapies.
Abstract: Enabling stem cell-targeted therapies requires an understanding of how to create local microenvironments (niches) that stimulate endogenous stem cells or serve as a platform to receive and guide the integration of transplanted stem cells and their derivatives. In vivo, the stem cell niche is a complex and dynamic unit. Although components of the in vivo niche continue to be described for many stem cell systems, how these components interact to modulate stem cell fate is only beginning to be understood. Using the HSC niche as a model, we discuss here microscale engineering strategies capable of systematically examining and reconstructing individual niche components. Synthetic stem cell-niche engineering may form a new foundation for regenerative therapies.

172 citations


Additional excerpts

  • ...In a proof-of-principle experiment, mouse ES cells were sequentially micropatterned with fibroblasts and hepatocytes (69)....

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Journal ArticleDOI
TL;DR: The emerging role and applications of parylene as a biomaterial for surface chemical modification and a future outlook are reviewed.
Abstract: Parylene is a family of chemically vapour deposited polymer with material properties that are attractive for biomedicine and nanobiotechnology. Chemically inert parylene “peel-off” stencils have been demonstrated for micropatterning biomolecular arrays with high uniformity, precise spatial control down to nanoscale resolution. Such micropatterned surfaces are beneficial in engineering biosensors and biological microenvironments. A variety of substituted precursors enables direct coating of functionalised parylenes onto biomedical implants and microfluidics, providing a convenient method for designing biocompatible and bioactive surfaces. This article will review the emerging role and applications of parylene as a biomaterial for surface chemical modification and provide a future outlook.

146 citations


Cites background or methods from "Microfabricated multilayer parylene..."

  • ...Multilayer parylene stencils [50,51] and ultra-thick reusable parylene stencils [52] have been also developed....

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  • ...For example, the total number of different cell types that could be sequentially patterned as co-cultures was increased from three to five using a three-layers parylene stencils compared to just a single layer stencil [51]....

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Journal ArticleDOI
TL;DR: The key applications of microwell arrays, which comprise high-density arrays of micron-sized cavities with desirable geometry, are highlighted, hopefully inspiring biologists to apply these systems for their own studies.
Abstract: Microwell arrays have emerged as robust and versatile alternatives to conventional mammalian cell culture substrates. Using standard microfabrication processes, biomaterials surfaces can be topographically patterned to comprise high-density arrays of micron-sized cavities with desirable geometry. Hundreds to thousands of individual cells or cell colonies with controlled size and shape can be trapped in these cavities by simple gravitational sedimentation. Efficient long-term cell confinement allows for parallel analyses and manipulation of cell fate during in vitro culture. These live-cell arrays have already found applications in cell biology, for example to probe the effect of cell colony size on embryonic stem cell differentiation, to dissect the heterogeneity in single cell proliferation kinetics of neural or hematopoietic stem/progenitor cell populations, or to elucidate the role of cell shape on cell function. Here, we highlight the key applications of these platforms, hopefully inspiring biologists to apply these systems for their own studies.

143 citations

References
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Journal ArticleDOI
08 Sep 2000-Science
TL;DR: Miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins are developed to facilitate subsequent studies of protein function.
Abstract: Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules

2,940 citations

Journal ArticleDOI
TL;DR: Fully potent early passage R1 cells and the R1-S3 subclone should be very useful not only for ES cell-based genetic manipulations but also in defining optimal in vitro culture conditions for retaining the initial totipotency of ES cells.
Abstract: Several newly generated mouse embryonic stem (ES) cell lines were tested for their ability to produce completely ES cell-derived mice at early passage numbers by ES cell tetraploid embryo aggregation. One line, designated R1, produced live offspring which were completely ES cell-derived as judged by isoenzyme analysis and coat color. These cell culture-derived animals were normal, viable, and fertile. However, prolonged in vitro culture negatively affected this initial totipotency of R1, and after passage 14, ES cell-derived newborns died at birth. However, one of the five subclones (R1-S3) derived from single cells at passage 12 retained the original totipotency and gave rise to viable, completely ES cell-derived animals. The total in vitro culture time of the sublines at the time of testing was equivalent to passage 24 of the original line. Fully potent early passage R1 cells and the R1-S3 subclone should be very useful not only for ES cell-based genetic manipulations but also in defining optimal in vitro culture conditions for retaining the initial totipotency of ES cells.

2,430 citations

PatentDOI
13 May 2002-Science
TL;DR: In this paper, the authors proposed a method for using proteome chips to systematically assay all protein interactions in a species in a high-throughput manner, and also related to methods for making protein arrays by attaching double-tagged fusion proteins to a solid support.
Abstract: The present invention relates to proteome chips comprising arrays having a large proportion of all proteins expressed in a single species. The invention also relates to methods for making proteome chips. The invention also relates to methods for using proteome chips to systematically assay all protein interactions in a species in a high-throughput manner. The present invention also relates to methods for making and purifying eukaryotic proteins in a high-density array format. The invention also relates to methods for making protein arrays by attaching double-tagged fusion proteins to a solid support. The invention also relates to a method for identifying whether a signal is positive.

1,967 citations

Journal ArticleDOI
TL;DR: An overview of the use of microfluidics, surface patterning, and patterned cocultures in regulating various aspects of cellular microenvironment is discussed, as well as the application of these technologies in directing cell fate and elucidating the underlying biology.
Abstract: Microscale technologies are emerging as powerful tools for tissue engineering and biological studies. In this review, we present an overview of these technologies in various tissue engineering applications, such as for fabricating 3D microfabricated scaffolds, as templates for cell aggregate formation, or for fabricating materials in a spatially regulated manner. In addition, we give examples of the use of microscale technologies for controlling the cellular microenvironment in vitro and for performing high-throughput assays. The use of microfluidics, surface patterning, and patterned cocultures in regulating various aspects of cellular microenvironment is discussed, as well as the application of these technologies in directing cell fate and elucidating the underlying biology. Throughout this review, we will use specific examples where available and will provide trends and future directions in the field.

1,590 citations


"Microfabricated multilayer parylene..." refers background or methods in this paper

  • ...The in vitro deposition of ECM and the subsequent assembly of cells with control over cell position(14) and spatial organization(15) are of great importance in tissue engineering.(1) Previously, patterned co-cultures of two or more cell types have been generated using approaches such as photolithography, layer-by-layer deposition of cell-adhesive materials,(12,20) elastomeric membranes, andmicrofluidics....

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  • ...The signals in the cellular microenvironment comprise of physicochemical factors, mechanical signals as well as cell–cell, cell-soluble factor, and cell– extracellular matrix (ECM) interactions.(1) Specifically, direct or indirect cell–cell interactions have been implicated in regulating a variety of cellular responses....

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Journal ArticleDOI
TL;DR: In this paper, the authors derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage, which can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties.
Abstract: We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase–PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of α-cardiac myosin heavy chain, α-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (IKr) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at ≫+20 mV, and were highly sensitive to dofetilide (IC50 of 46.9 nM). Specific binding of [3H]dofetilide was saturable and fit a one-site binding isotherm with a Kd of 140 +/− 60 nM and a Bmax of 118 fmol per 105 cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.

1,452 citations