Imaging optical sensor arrays

Imaging optical fibres have been etched to prepare microwell arrays. These microwells have been loaded with sensing materials such as bead-based sensors and living cells to create high-density sensor arrays. The extremely small sizes and volumes of the wells enable high sensitivity and high information content sensing capabilities.     

Evaluation of homo- and hetero-functionally activated glass surfaces for optimized antibody arrays

Antibody arrays hold great promise for biomedical applications, but they are typically manufactured using chemically functionalized surfaces that still require optimization. Here, we describe novel hetero-functionally activated glass surfaces favoring oriented antibody binding for improved performance in protein microarray applications. Antibody arrays manufactured in our facility using the functionalization chemistries described here proved to be reproducible and stable and also showed good signal intensities. As a proof-of-principle of the glass surface functionalization protocols described in this article, we built antibody-based arrays functionalized with different chemistries that enabled the simultaneous detection of 71 human leukocyte membrane differentiation antigens commonly found in peripheral blood mononuclear cells. Such detection is specific and semi-quantitative and can be performed in a single assay under native conditions. In summary, the protocol described here, based on the use of antibody array technology, enabled the concurrent detection of a set of membrane proteins under native conditions in a specific, selective, and semi-quantitative manner and in a single assay.     

Biochemical functionalization of polymeric cell substrata can alter mechanical compliance

Biochemical functionalization of surfaces is an increasingly utilized mechanism to promote or inhibit adhesion of cells. To promote mammalian cell adhesion, one common functionalization approach is surface conjugation of adhesion peptide sequences such as Arg-Gly-Asp (RGD), a ligand of transmembrane integrin molecules. It is generally assumed that such functionalization does not alter the local mechanical properties of the functionalized surface, as is important to interpretations of macromolecular mechanotransduction in cells. Here, we examine this assumption systematically, through nanomechanical measurement of the nominal elastic modulus of polymer multilayer films of nanoscale thickness, functionalized with RGD through different processing routes. We find that the method of biochemical functionalization can significantly alter mechanical compliance of polymeric substrata such as weak polyelectrolyte multilayers (PEMs), increasingly utilized materials for such studies. In particular, immersed adsorption of intermediate functionalization reagents significantly decreases compliance of the PEMs considered herein, whereas polymer-on-polymer stamping of these same reagents does not alter compliance of weak PEMs. This finding points to the potential unintended alteration of mechanical properties via surface functionalization and also suggests functionalization methods by which chemical and mechanical properties of cell substrata can be controlled independently.     

Novel cell culture device enabling three-dimensional cell growth and improved cell function

A better understanding of cell biology and cell-cell interactions requires three-dimensional (3-D) culture systems that more closely represent the natural structure and function of tissues in vivo. Here, we present a novel device that provides an environment for routine 3-D cell growth in vitro. We have developed a thin membrane of polystyrene scaffold with a well defined and uniform porous architecture and have adapted this material for cell culture applications. We have exemplified the application of this technology by growing HepG2 liver cells on 2- and 3-D substrates. The performance of HepG2 cells grown on scaffolds was significantly enhanced compared to functional activity of cells grown on 2-D plastic. The incorporation of thin membranes of porous polystyrene to create a novel device has been successfully demonstrated as a new 3-D cell growth technology for routine use in cell culture.     

Dimensionality Controls Cytoskeleton Assembly and Metabolism of Fibroblast Cells in Response to Rigidity and Shape

Background

Various physical parameters, including substrate rigidity, size of adhesive islands and micro-and nano-topographies, have been shown to differentially regulate cell fate in two-dimensional (2-D) cell cultures. Cells anchored in a three-dimensional (3-D) microenvironment show significantly altered phenotypes, from altered cell adhesions, to cell migration and differentiation. Yet, no systematic analysis has been performed that studied how the integrated cellular responses to the physical characteristics of the environment are regulated by dimensionality (2-D versus 3-D).

Methodology/Principal Findings

Arrays of 5 or 10 µm deep microwells were fabricated in polydimethylsiloxane (PDMS). The actin cytoskeleton was compared for single primary fibroblasts adhering either to microfabricated adhesive islands (2-D) or trapped in microwells (3-D) of controlled size, shape, and wall rigidity. On rigid substrates (Young''s Modulus = 1 MPa), cytoskeleton assembly within single fibroblast cells occurred in 3-D microwells of circular, rectangular, square, and triangular shapes with 2-D projected surface areas (microwell bottom surface area) and total surface areas of adhesion (microwell bottom plus wall surface area) that inhibited stress fiber assembly in 2-D. In contrast, cells did not assemble a detectable actin cytoskeleton in soft 3-D microwells (20 kPa), regardless of their shapes, but did so on flat, 2-D substrates. The dependency on environmental dimensionality was also reflected by cell viability and metabolism as probed by mitochondrial activities. Both were upregulated in 3-D cultured cells versus cells on 2-D patterns when surface area of adhesion and rigidity were held constant.

Conclusion/Significance

These data indicate that cell shape and rigidity are not orthogonal parameters directing cell fate. The sensory toolbox of cells integrates mechanical (rigidity) and topographical (shape and dimensionality) information differently when cell adhesions are confined to 2-D or occur in a 3-D space.     

A simple microsphere-based mold to rapidly fabricate microwell arrays for multisize 3D tumor culture

Three-dimensional (3D) tumor has been considered as the best in vitro model for cancer research. In recent years, various methods have been developed to controllable prepare multisize 3D tumors. Nonetheless, reported technologies are still problematic and difficult to produce 3D tumors with highly uniform size and cell content. Here, a novel and simple microsphere-based mold approach is proposed to rapidly fabricate spherical microwell arrays for multisize 3D tumors formation, culture, and recovery. Larger amounts of HepG2 3D tumors with excellent quality and uniformity can be efficiently generated using this method. In addition, the tumor size can also be simply controlled by adjusting the diameter of the microwell arrays. All experimental results indicated that the proposed method offers a promising platform to generate and recover highly controlled multisize 3D tumors for various cell-based biomedical research.     

Microfabrication of Bubbular Cavities in PDMS for Cell Sorting and Microcell Culture Applications

We describe a novel technique, low surface energy Gas Expansion Molding （GEM）, to fabricate microbubble arrays in polydimethylsiloxane （PDMS） which are incorporated into parallel plate flow chambers and tested in cell sorting and microcell cuTture applications. This architecture confers several operational advantages that distinguish this technology approach from currently used methods. Herein we describe the GEM process and the parameters that are used to control microbubble formation and a Vacuum-Assisted Coating （VAC） process developed to selectively and spatially alter the PDMS surface chemistry in the wells and on the microchannel surface. We describe results from microflow image visualization studies conducted to investigate fluid streams above and within microbubble wells and conclude with a discussion of cell culture studies in PDMS.     

Vinyl sulfone functionalization: a feasible approach for the study of the lectin-carbohydrate interactions

Carbohydrate-mediated molecular recognition is involved in many biological aspects such as cellular adhesion, immune response, blood coagulation, inflammation, and infection. Considering the crucial importance of such biological events in which proteins are normally involved, synthetic saccharide-based systems have emerged as powerful tools for the understanding of protein-carbohydrate interactions. As a new approach to create saccharide-based systems, a set of representative monosaccharides (D-mannose, D-glucose, N-acetyl-D-glucosamine, and L-fucose) and disaccharides (lactose, maltose, and melibiose) were derivatized at their anomeric carbon with a vinyl sulfone group spanned by an ethylthio linker. This vinyl sulfone functionalization is demonstrated to be a general strategy for the covalent linkage of a saccharide in mild conditions via Michael-type additions with the amine and thiol groups from functionalized supports and those naturally present in biomolecules. The introduction of the ethylthio linker between the biorecognizable element (i.e., saccharide) and the reactive group (i.e., vinyl sulfone) was found to preserve the functionality of the former. The capability of the vinyl sulfone saccharides for the study of lectin-carbohydrate interactions was demonstrated by (i) immobilizing them on both amine-functionalized supports (glass slides and microwell plates) and polylysine-coated glass slides to create sugar arrays that selectively bind lectins (ii) coupling to model proteins to yield neoglycoproteins that are recognized by lectins and (iii) using vinyl sulfone saccharides as tags to allow the detection of the labeled biomolecule by HRP-lectins. The above results were further put tothe test with a real case: detection of carbohydrate binding proteins present in rice ( Oryza sativa ).     

Modelling tissues in 3D: the next future of pharmaco-toxicology and food research?

The development and validation of reliable in vitro methods alternative to conventional in vivo studies in experimental animals is a well-recognised priority in the fields of pharmaco-toxicology and food research. Conventional studies based on two-dimensional (2-D) cell monolayers have demonstrated their significant limitations: the chemically and spatially defined three-dimensional (3-D) network of extracellular matrix components, cell-to-cell and cell-to-matrix interactions that governs differentiation, proliferation and function of cells in vivo is, in fact, lost under the simplified 2-D condition. Being able to reproduce specific tissue-like structures and to mimic functions and responses of real tissues in a way that is more physiologically relevant than what can be achieved through traditional 2-D cell monolayers, 3-D cell culture represents a potential bridge to cover the gap between animal models and human studies. This article addresses the significance and the potential of 3-D in vitro systems to improve the predictive value of cell-based assays for safety and risk assessment studies and for new drugs development and testing. The crucial role of tissue engineering and of the new microscale technologies for improving and optimising these models, as well as the necessity of developing new protocols and analytical methods for their full exploitation, will be also discussed.     

Fiber optic array biosensors

Optical fiber arrays provide a powerful substrate for creating high-density sensing systems that can address a variety of biological problems. The fiber substrate can be used to create femtoliter wells that can be loaded with individual beads to create high-density arrays for multiplexed screening and analysis. In addition, living cells can be loaded into the arrays, and their individual responses monitored over long time periods, enabling functional screening of biologically active compounds. Adherent cells can be attached to the fiber substrate to provide a rapid method for observing cell migration and for screening anti-migratory compounds. Finally, individual enzyme molecules can be loaded into the array wells enabling single molecule detection via enzyme-catalyzed signal amplification.     

A multiwell platform for studying stiffness-dependent cell biology

Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA) hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes.     

Imaging immune surveillance of individual natural killer cells confined in microwell arrays

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.     

Micron-scale positioning of features influences the rate of polymorphonuclear leukocyte migration

Microfabrication technology was used to create regular arrays of micron-size holes (2 microm x 2 microm x 210 nm) on fused quartz and photosensitive polyimide surfaces. The patterned surfaces, which possessed a basic structural element of a three-dimensional (3-D) network (i.e., spatially separated mechanical edges), were used as a model system for studying the effect of substrate microgeometry on neutrophil migration. The edge-to-edge spacing between features was systematically varied from 6 microm to 14 microm with an increment of 2 microm. In addition, collagen was used to coat the patterned quartz surfaces in an attempt to change the adhesive properties of the surfaces. A radial flow detachment assay revealed that cell adhesion was the strongest on the quartz surface (approximately 50% cell attached), whereas it was relatively weaker on polyimide and collagen-coated quartz (approximately 25% cell attached). Cell adhesion to each substrate was not affected either by the presence of holes or by the spacing between holes. A direct visualization assay showed that neutrophil migration on each patterned surface could be characterized as a persistent random walk; the dependence of the random motility coefficient (mu) as a function of spacing was biphasic with the optimal spacing at approximately 10 microm on each substrate. The presence of evenly distributed holes at the optimal spacing of 10 microm enhanced mu by a factor of 2 on polyimide, a factor of 2.5 on collagen-coated quartz, and a factor of 10 on uncoated quartz. The biphasic dependence on the mechanical edges of neutrophil migration on 2-D patterned substrate was strikingly similar to that previously observed during neutrophil migration within 3-D networks, suggesting that microfabricated materials provide relevant models of 3-D structures with precisely defined physical characteristics. In addition, our results demonstrate that the microgeometry of a substrate, when considered separately from adhesion, can play a significant role in cell migration.     

Pristine soils mineralize 3-chlorobenzoate and 2,4-dichlorophenoxyacetate via different microbial populations.

Biodegradation of two chlorinated aromatic compounds was found to be a common capability of the microorganisms found in the soils of undisturbed, pristine ecosystems. We used 2,4-dichlorophenoxyacetate (2,4-D) and 3-chlorobenzoate (3CBA) as enrichment substrates to compare populations of degrading bacteria from six different regions making up two ecosystems. We collected soil samples from four Mediterranean (California, central Chile, the Cape region of South Africa, and southwestern Australia) and two boreal (northern Saskatchewan and northwestern Russia) ecosystems that had no direct exposure to pesticides or to human disturbance. Between 96 and 120 samples from each of the six regions were incubated with 50 ppm of [U-14C]2,4-D or [U-14C]3CBA. Soils from all regions samples mineralized both 2,4-D and 3CBA, but 3CBA was mineralized without a lag period, while 2,4-D was generally not mineralized until the second week. 3CBA degradative capabilities were more evenly distributed spatially than those for 2,4-D. The degradative capabilities of the soils were readily transferred to fresh liquid medium. 3CBA degraders were easily isolated from most soils. We recovered 610 strains that could release carbon dioxide from ring-labeled 3CBA. Of these, 144 strains released chloride and degraded over 80% of 1 mM 3CBA in 3 weeks or less. In contrast, only five 2,4-D degraders could be isolated, although a variety of methods were used in an attempt to culture the degraders. The differences in the distribution and culturability of the bacteria responsible for 3CBA and 2,4-D degradation in these ecosystems suggest that the two substrates are degraded by different populations. We also describe a 14C-based microtiter plate method that allows efficient screening of a large number of samples for biodegradation activity.     

Optical-fiber bundles

Optical-fiber bundles have been employed as a versatile substrate for the fabrication of high-density microwell arrays. In this minireview, we discuss the application of optical-fiber-bundle arrays for a variety of biological problems. For genomics studies and microbial pathogen detection, individual beads have been functionalized with DNA probes and then loaded into the microwells. In addition, beads differentially responsive to vapors have been employed in an artificial olfaction system. Microwell arrays have also been loaded with living cells to monitor their individual response to biologically active compounds over long periods. Finally, the microwells have been sealed to enclose single enzyme molecules that can be used to measure individual molecule catalytic activity.     

Reactive polymer multilayers fabricated by covalent layer-by-layer assembly: 1,4-conjugate addition-based approaches to the design of functional biointerfaces

We report on conjugate addition-based approaches to the covalent layer-by-layer assembly of thin films and the post-fabrication functionalization of biointerfaces. Our approach is based on a recently reported approach to the "reactive" assembly of covalently cross-linked polymer multilayers driven by the 1,4-conjugate addition of amine functionality in poly(ethyleneimine) (PEI) to the acrylate groups in a small-molecule pentacrylate species (5-Ac). This process results in films containing degradable β-amino ester cross-links and residual acrylate and amine functionality that can be used as reactive handles for the subsequent immobilization of new functionality. Layer-by-layer growth of films fabricated on silicon substrates occurred in a supra-linear manner to yield films ≈ 750 nm thick after the deposition of 80 PEI/5-Ac layers. Characterization by atomic force microscopy (AFM) suggested a mechanism of growth that involves the reactive deposition of nanometer-scale aggregates of PEI and 5-Ac during assembly. Infrared (IR) spectroscopy studies revealed covalent assembly to occur by 1,4-conjugate addition without formation of amide functionality. Additional experiments demonstrated that acrylate-containing films could be postfunctionalized via conjugate addition reactions with small-molecule amines that influence important biointerfacial properties, including water contact angles and the ability of film-coated surfaces to prevent or promote the attachment of cells in vitro. For example, whereas conjugation of the hydrophobic molecule decylamine resulted in films that supported cell adhesion and growth, films treated with the carbohydrate-based motif D-glucamine resisted cell attachment and growth almost completely for up to 7 days in serum-containing media. We demonstrate that this conjugate addition-based approach also provides a means of immobilizing functionality through labile ester linkages that can be used to promote the long-term, surface-mediated release of conjugated species and promote gradual changes in interfacial properties upon incubation in physiological media (e.g., over a period of at least 1 month). These covalently cross-linked films are relatively stable in biological media for prolonged periods, but they begin to physically disintegrate after ≈ 30 days, suggesting opportunities to use this covalent layer-by-layer approach to design functional biointerfaces that ultimately erode or degrade to facilitate elimination.     

Microwell Cluster for Processing Electron Microscope Sections for Immunocytochemistry

A microwell cluster was manufactured with silicone rubber for incubating thin sections for postembedding electron microscopic immunocytochemistry. The small size of the wells requires only minute amounts (as little as 5 μl) of antisera and other valuable immunoreagents. The capillary action of the wells holds the incubation media and grids in place, even if the cluster is turned upside down, thus facilitating safe transport and storage of the sections to be stained. This silicone rubber microwell cluster can also be used as a die to imprint wells in Parafilm sheets to be used for the same purpose.     

A defined glycosaminoglycan-binding substratum for human pluripotent stem cells

To exploit the full potential of human pluripotent stem cells for regenerative medicine, developmental biology and drug discovery, defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed, but finding chemically defined, robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides, which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells.     

Plant lectins detect age and region specific differences in cell surface carbohydrates and cell reassociation behavior of embryonic mouse cerebellar cells

When plated at high cell density in a microwell culture system, freshly dissociated embryonic mouse cerebellar cells assemble into reproducible, 3-dimensional patterns. The addition of the dimeric lectin Succinyl Concanavalin A blocks reversibly the formation of the microwell pattern, suggesting that cell surface carbohydrates affect the reassociation behavior of embryonic mouse cerebellar cells. Agglutination studes of dissociated cell populations harvested from different regions of the embryonic brain reveal that different lectins agglutinate cell populations from different embryonic brain regions. Cells from E13 cerebellum are agglutinated with Concanavalin A, wheat germ agglutinin, Ricinus communis agglutinin, mol wt 60,000, Ricinus communis agglutinin, mol wt 120,000, and Lens culinaris, but not by soybean agglutinin or a fucose-binding protein. Cells from the midbrain are agglutinated only with Concanavalin A, Ricinus communis agglutinin, mol wt 60,000 and Ricinus communis agglutinin, mol wt 120,000; those from the cerebral cortex are agglutinated only with Lens culinaris; and those from the medulla are agglutinated only with Ricinus communis agglutinin, mol wt 60,000, and Ricinus communis agglutinin, mol wt 120,000. In addition, agglutination of cerebellar cells with Concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinin is diminished over the course of development from embryonic day 13 to postnatal day 7. These studies suggest regional differences in the cell surfaces of the developling brain that are further modulated during the differentiation of the tissues. On a poly(D-lysine) treated substrate in microwell cultures, cell migration is unique to the cerebellum of the 4 brain regions studied. Surfaces treated with carbohydrate-derivatized poly(D-lysine) are currently being tested for their efficacy as substrates for differential cell migration.     

In vitro biocompatibility of schwann cells on surfaces of biocompatible polymeric electrospun fibrous and solution-cast film scaffolds

The in vitro responses of Schwann cells (RT4-D6P2T, a schwannoma cell line derived from a chemically induced rat peripheral neurotumor) on various types of electrospun fibrous scaffolds of some commercially available biocompatible and biodegradable polymers, i.e., poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polycaprolactone (PCL), poly(l-lactic acid) (PLLA), and chitosan (CS), were reported in comparison with those of the cells on corresponding solution-cast film scaffolds as well as on a tissue-culture polystyrene plate (TCPS), used as the positive control. At 24 h after cell seeding, the viability of the attached cells on the various substrates could be ranked as follows: PCL film > TCPS > PCL fibrous > PLLA fibrous > PHBV film > CS fibrous approximately CS film approximately PLLA film > PHB film > PHBV fibrous > PHB fibrous. At day 3 of cell culture, the viability of the proliferated cells on the various substrates could be ranked as follows: TCPS > PHBV film > PLLA film > PCL film > PLLA fibrous > PHB film approximately PCL fibrous > CS fibrous > CS film > PHB fibrous > PHBV fibrous. At approximately 8 h after cell seeding, the cells on the flat surfaces of all of the film scaffolds and that of the PCL nanofibrous scaffold appeared in their characteristic spindle shape, while those on the surfaces of the PHB, PHBV, and PLLA macrofibrous scaffolds also appeared in their characteristic spindle shape, but with the cells being able to penetrate to the inner side of the scaffolds.