Cell Adhesion Area Research Articles

Use of a gradient-generating microfluidic device to rapidly determine a suitable glucose concentration for cell viability test

We successfully determined a suitable glucose concentration for endothelial cells (ECs) using a gradient-generating microfluidic chip and a micro-stamper that were fabricated using micro-electro-mechanical systems (MEMS) technology. Our strategy was to generate a stable concentration gradient in the observation area based on a microfluidic network and micro-mixers, which produced a concentration gradient under various flow rates. The areas for cell adhesion were delineated on a glass slide with a micro-stamper using the micro-contact printing (μCP) method. We also discuss which glucose concentration gradients are suitable for cell viability test (i.e., 0–0.2%, 0.05–0.15%, and 0.06–0.17%). After examining various concentration gradients, the suitable glucose concentration for EC’s viability test was determined to range from 0.077% (4.2 mM) to 0.147% (8.16 mM). Higher or lower concentrations caused the ECs to atrophy or die. In this study, we describe a gradient-generating microfluidic chip that can be used to produce various drug concentrations for multi-concentration tests.

P09. Cell shape and F-actin regulate Hippo signaling pathway

The proliferation of cultured cells is regulated by cell density, a phenomenon known as cell contact inhibition of proliferation. Previously, we and others have shown that high cell density inactivates the transcription factor Tead, which controls cell proliferation, via the Hippo signaling pathway by suppressing nuclear localization of the coactivator protein Yap. Cell density affects both, cell–cell contacts and the cell shape, but the exact role of these latter cellular properties in regulating the Hippo pathway remains elusive. In this study, we examined the role of the cell shape on the Hippo pathway. To exclude the effects of cell–cell contacts, individual NIH3T3 cells were cultured in small cell–adhesive areas termed microdomains. The cell shape was changed according to the microdomains size. Nuclear localization of Yap was observed with large microdomains or corresponding flattened cells, but not with small microdomains resulting in the formation of round cells. Clear stress fiber formation was observed only in large microdomains concomitant with nuclear Yap. Disruption of F-actin by drug treatment reduced the nuclear localization of Yap and transcriptional activity of Tead irrespective of cell shape. Core components of the Hippo pathway (upstream to downstream) are Merlin, Mst, Lats, Yap and Tead. When activity of the Hippo pathway was blocked by the expression of a dominant negative form of Merlin, Yap stayed in the nucleus, even when F-actin was disrupted. These results suggest that F-actin regulates the Hippo pathway upstream of Merlin, and that the cell shape regulates the Hippo pathway independent of cell–cell contacts by controlling the amount of F-actin.

Embryoid body culture of mouse embryonic stem cells using microwell and micropatterned chips

The proliferation and differentiation properties of embryoid bodies (EB) from mouse embryonic stem (ES) cells were compared under two microchip conditions: microwell chip and micropatterned chip. The microwell chip contained 270 microwells (diameter, 600 μm; depth, 600 μm) on a polymethylmethacrylate plate and was surface-modified with polyethylene glycol (PEG) to render it non-adhesive. The micropatterned chip contained 270 gelatin spots (diameter, 200 μm) as the cell adhesion area on a glass plate; the region lacking these spots was PEG-modified to render it non-adhesive. The ES cells spontaneously formed the EBs from cell aggregates in each microwell in the chip. In contrast, cells inoculated onto the patterned chip formed a monolayer on the gelatin spots and gradually proliferated to form EBs. The EBs in the patterned chip maintained the high cell growth rate and the expression of endoderm (TTR and AFP) and mesoderm (Nkx2.5, αMHC, Flk1, and PDGFRβ) markers was increased, and these cell properties were similar to the previous methods (hanging drop and round-bottomed 96-well plate cultures). In contrast, the proliferation of ES cells in the microwell chip was lower than in the patterned chip and previous methods, and the EB differentiation proceeded slowly and only formed a small amount of endoderm. These results indicate that the difference of EB generating process in the microchip cultures may affect to the proliferation and differentiation of ES cells, and the existence of microwell structure in the microchip downregulates the cell proliferation and the differentiated progress of ES cells.

Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin‐containing focal adhesions

Actin-myosin contractility modulates focal adhesion assembly, stress fiber formation, and cell migration. We analyzed the contributions of contractility to fibroblast adhesion strengthening using a hydrodynamic adhesion assay and micropatterned substrates to control cell shape and adhesive area. Serum addition resulted in adhesion strengthening to levels 30-40% higher than serum-free cultures. Inhibition of myosin light chain kinase or Rho-kinase blocked phosphorylation of myosin light chain to similar extents and eliminated the serum-induced enhancements in strengthening. Blebbistatin-induced inhibition of myosin II reduced serum-induced adhesion strength to similar levels as those obtained by blocking myosin light chain phosphorylation. Reductions in adhesion strengthening by inhibitors of contractility correlated with loss of vinculin and talin from focal adhesions without changes in integrin binding. In vinculin-null cells, inhibition of contractility did not alter adhesive force, whereas controls displayed a 20% reduction in adhesion strength, indicating that the effects of contractility on adhesive force are vinculin-dependent. Furthermore, in cells expressing FAK, inhibitors of contractility reduced serum-induced adhesion strengthening as well as eliminated focal adhesion assembly. In contrast, in the absence of FAK, these inhibitors did not alter adhesion strength or focal adhesion assembly. These results indicate that contractility modulates adhesion strengthening via FAK-dependent, vinculin-containing focal adhesion assembly.

Effects of micrometric titanium particles on osteoblast attachment and cytoskeleton architecture

Titanium (Ti) and its alloys are widely used in biomedical devices as bone tissue replacements due to their advantageous bulk mechanical properties and biocompatibility. It is known that particles released from Ti-based implants impair essential functions of osteoblasts, which for survival require attachment to specific extracellular matrix proteins at the bone surface. This study investigates whether Ti particles of micrometric sizes affect the osteoblast attachment machinery. Exposure of human osteoblastic Saos-2 cells to Ti particles impaired their adhesion strength, migration and proliferation. Attenuation of these functions was associated with reduced cell spreading, cell membrane disruptions and loss of cell shape. Cell exposure to Ti particles led to changes in cytoskeletal structures, including reduced ventral stress fibers combined with a disorderly arrangement of β-tubulin and acetylated α-tubulin fibers. Cytoskeleton disassembly was associated with a reduction in overall cell adhesion area, characterized by fewer centrally localized focal adhesions and shorter focal contacts at the periphery. Paxillin adaptor protein redistributed to peripheral corner regions, colocalizing with poorly organized actin fibers at attachment sites. Total focal adhesion kinase (FAK) protein amounts, as well as its degree of phosphorylation on the active form p-FAK (Tyr-397), decreased, which was accompanied by a lesser extent of co-localization with paxillin in focal contacts. On the other hand, p-FAK (Tyr-407), an inhibitory form of FAK, accumulated in the focal contacts of Ti-treated cells. Pyk2 phosphorylated on Tyr-402 colocalized with paxillin in focal contacts of untreated cells, while it was barely detected upon exposure to particles. In summary, changes in the phosphorylation states of both FAK and Pyk2 tyrosine kinases at focal contacts underlie impaired bone-forming cell attachment after exposure to Ti particles of micrometric sizes.

Novel High-Resolution Micropatterning for Neuron Culture Using Polylysine Adsorption on a Cell Repellant, Plasma-Polymerized Background

The ability to organize individual neurons and their processes in culture provides important benefits to both basic neuroscience research applications and the development of biomedical microdevices. While numerous methods have been used to produce such micropatterning of neurons and cells in general, there has yet been no method to simultaneously provide high-resolution patterns with high compliance of cells to desired patterns and good manufacturability. To develop such a process, this work used a plasma polymerized, nonfouling poly ethylene oxide (PEO)-like film to provide a cell repellant substrate on which cell adhesive micropatterns can be selectively laid down. While the use of plasma polymerized, organic films have been used for cell micropatterning, this process exploits the often-overlooked tendency for the surface of this PEO-like material to adsorb polylysine from aqueous solution while remaining nonfouling with respect to other species, such as bovine serum albumin (BSA) and immunoglobulin G (IgG). When the adsorption of polylysine was enhanced by brief plasma oxidation, which slightly alters the surface chemistry of the material, simple photolithographic liftoff could be used to micropattern stable, cell adhesive areas on an otherwise cell repellant background. We showed that the application of photolithography itself on the PEO-like material did not alter its chemical properties, nor did it result in the erosion of the micropatterned polylysine on its surface. Hippocampal neurons from embryonic mice flourished on these micropatterned substrates and exhibited viability comparable to neurons cultured on polylysine coated glass. Furthermore, the compliance of cell bodies and outgrowing neurites to the micropatterns was nearly perfect. In addition to providing cell adhesive regions, the micropatterned polylysine coating also served as a template mediating the immobilization of other bioactive species such as IgG and laminin. Using this "piggybacking" of laminin on polylysine, we were also able to culture and micropattern retinal ganglion cells (RGC).

Geometric Control of Stem Cell Differentiation Rate on Surfaces

We develop a general methodology to create a patterned surface array that allows for the study of how the cell population, surface adhesion area, and pattern geometry combine to influence stem cell differentiation. We use a simple microfabrication technique to pattern human mesenchymal stem cells (hMSC's) on transparent surfaces and develop a new method to quantitate adipogenic differentiation. We found that the pattern geometry and therefore the cell population, rather than the cell adhesive area, influence the rate of adipogenic differentiation from hMSC's. Furthermore, the cells within the pattern behave more characteristically of a tissue than do individual cells because a certain critical threshold cell density is required to induce differentiation.

Micropatterned organoid culture of rat hepatocytes and HepG2 cells

The culture of liver cell organoids (multicellular aggregates) such as spheroids or cylindroids, which can strongly express liver functions, has been advocated as a useful technique that has advantages over monolayer culture. This paper describes a micropatterning technique for obtaining spheroids and cylindroids by using rat hepatocytes or HepG2 cells. We developed culture chips that comprised multiple, circular or rectangular microwells; the bottom surface of each microwell was modified with collagen to create a cell adhesion area, and the entire microwell, excluding the collagen-coated spots, was modified with polyethylene glycol (PEG) to create a nonadhesive area. Rat hepatocytes and HepG2 cells formed uniform spheroids and cylindroids on the circular and rectangular chips, respectively. Consequently, two-dimensional micropatterned chips containing homogeneous spheroids or cylindroids were generated. The expression of liver functions (protein secretion and ammonia removal) was greater in the spheroids and cylindroids than in the monolayer culture, and this expression was maintained for at least 2 weeks of culture. Thus, this chip technology has potential for use in various applications that involve organoid culture.

Laser- and UV-assisted modification of polystyrene surfaces for control of protein adsorption and cell adhesion

An appropriate choice of laser and process parameters enables new approaches for the fabrication of polymeric lab-on-chip devices with integrated functionalities. We will present our current research results in laser-assisted modification of polystyrene (PS) with respect to the fabrication of polymer devices for cell culture applications. For this purpose laser micro-patterning of PS and subsequent surface functionalization was investigated as function of laser and process parameters. A high power ArF-excimer laser radiation source with a pulse length of 19 ns as well as a high repetition ArF-excimer laser source with a pulse length of 5 ns were used in order to study the influence of laser pulse length on laser-induced surface oxidation. The change in surface chemistry was characterized by X-ray photoelectron spectroscopy and contact angle measurements. The difference between laser-assisted modification versus UV-lamp assisted modification was investigated. A photolytic activation of specific areas of the polymer surface and subsequent oxidization in oxygen or ambient air leads to a chemically modified polymer surface bearing carboxylic acid groups well-suited for controlled competitive protein adsorption or protein immobilization. Finally, distinct areas for cell growth and adhesion are obtained.

Influence of fetal calf serum on differentiation of mesenchymal stem cells to chondrocytes during expansion

Mesenchymal stem cells (MSCs) should be expanded in vitro while maintaining their multilineage potential before differentiation to one mesenchymal lineage for application to regeneration therapy. The effect of fetal calf serum (FCS) on undesirable differentiation during subcultivations for the expansion was investigated. The expression level of the aggrecan gene, which is a marker of chondrogenic differentiation, gradually and markedly increased during the subcultivations of MSCs with the addition of 10% FCS and without additional cytokines. The percentage of cells positive for CD90 and CD166, which are markers of MSCs, decreased, and the percentage of large polygonal cells and the average cell adhesion area increased during the expansion. There was a marked difference in the increase in the aggrecan expression level between the two expansion cultures employing different FCS lots, although their proliferation rates were almost the same. The decrease in FCS concentration resulted in a higher percentage of CD90(+)CD166(+) cells, a lower percentage of large polygonal cells, and a lower level of aggrecan expression. Consequently, FCS components could stimulate MSC differentiation to chondrocytes and a lower concentration could decrease this differentiation.

Cell adhesion and polarisation on molecularly defined spacing gradient surfaces of cyclic RGDfK peptide patches

In vivo cell migration and location are orchestrally guided by soluble and bound chemical gradients. Here, gradients of extracellular matrix molecules are formed synthetically by the combination of a surface nanopatterning technique called block copolymer nanolithography (BCN) and a biofunctionalisation technique. A modified substrate dip-coating process of BCN allows for the formation of precise molecular gradients of cyclic RGDfK peptide patches at interfaces, which are presented to cells for testing cell adhesion and polarisation. Surfaces formed by BCN consist of hexagonally ordered gold dot patterns with a gradient in particle spacing. Each dot serves as a chemical anchor for the binding of cyclic RGDfK peptides, which are specifically recognised by α vβ 3 integrins. Due to steric hindrance only up to one integrin binds to one functionalised gold dot which forms a peptide patch spacing. We demonstrate how cell morphology, adhesion area, actin and vinculin distribution as well as cell body polarisation are influenced by the peptide patch spacing gradient. As a consequence, these gradients of adhesive ligands induce cell orientation towards smaller particle spacing when the gradient strength is 15 nm/mm at least. This implicates that an adherent cell's sensitivity to differentiate between ligand patch spacing is approximately 1 nm across the cell body.

Correlation between cell morphology and aggrecan gene expression level during differentiation from mesenchymal stem cells to chondrocytes

A morphological parameter of polygonal index was defined as the ratio of cell adhesion area versus the square of the major cell axis, and cells that had an adhesion area larger than 4000 mum(2) and a polygonal index larger than 0.3 were considered large polygonal cells. Cell morphology tended to change from fibroblast-like to polygonal and the percentage of the large polygonal cells increased almost in proportion to aggrecan mRNA expression level during the differentiation culture of mesenchymal stem cells (MSCs) to chondrocytes. Approximately 80% of the large polygonal cells were negative for MSC marker (CD90, CD166) expression and the aggrecan mRNA expression level of the large polygonal cells was markedly higher than that of cells with other morphologies.

Morphological Study of Fibroblasts Treated with Cytochalasin D and Colchicine Using a Confocal Laser Scanning Microscopy

The role of actin filaments and microtubules in 3D cell morphology was investigated using confocal laser scanning microscopy and image analysis based on a region-growing method. Fibroblasts were treated with cytochalasin D or colchicine to disrupt the actin filaments or microtubules, respectively, and the structure and distribution of these cytoskeletal filaments were observed using a confocal laser scanning microscope. From the 3D reconstructed fluorescence images of the cytoskeleton, morphological parameters such as volume, adhesion area, height, and volume ratio of individual cells were determined. The volume ratio was defined as the ratio of the partial volume for every 10% of the height to the total cell volume. The cell volume decreased slightly after the disruption of actin filaments and microtubules, but the change was not significant. The cell adhesion area was significantly decreased after the disruption of actin filaments and microtubules, and was significantly smaller in actin filament-disrupted cells than in microtubule-disrupted cells. Cell height increased significantly after actin filament disruption, whereas it remained almost unchanged after microtubule disruption. Analysis of the volume ratio revealed that the cell shape changed from a cone to a hemisphere after disruption of actin filaments and slightly shifted toward a hemisphere-like shape after microtubule disruption. These results suggest that actin filaments are the major component responsible for the maintenance of global cell shape and that the contribution of microtubules to global cell morphology is much less than that of actin filaments.

Time-dependent effects of pre-aging polymer films in cell culture medium on cell adhesion and spreading

We have tested the hypothesis that cell adhesion and spreading on polymer films are influenced by the amount of time that the polymer films are pre-aged in cell culture medium. Cell adhesion and spreading were assessed after a 6-h culture on poly(D,L-lactic acid) (PDLLA) films that had been pre-aged in cell culture medium for 30 min, 1, 3 or 7 d. Cell adhesion and spread area were enhanced as the duration of pre-aging PDLLA films in cell culture medium was increased. Materials characterization showed that the hydrophobicity and surface morphology of the PDLLA films changed with increasing length of pre-aging time. These results suggest that cell adhesion and spreading are sensitive to the time-dependent changes in PDLLA hydrophobicity and surface morphology that occur during exposure of the polymer to cell medium for different lengths of time. These results demonstrate that cell response to a degradable, biomedical polymer can change as a function of the amount of time that the polymer is exposed to physiological medium.

Two-dimensional microarray of HepG2 spheroids using collagen/polyethylene glycol micropatterned chip

A new cell chip technology in the form of a two-dimensional microarray of HepG2 spheroids was developed by using the microcontact printing technique. The chip consisted of several collagen spots in a triangular arrangement within a 100-mm2 area at the center of a glass plate (24 x 24 mm), which served as the cell adhesion area; the region excluding the collagen spots that was modified with polyethylene glycol (PEG) served as the non-adhesion area. HepG2 cells inoculated onto the chip gradually formed spheroids with smooth surfaces and high circularity on each collagen spot due to cell proliferation; the spheroid diameters remained constant after 10 days of culture. Such a two-dimensional microarray configuration of HepG2 spheroids could be maintained for at least 2 weeks. The spheroid diameter was directly proportional to the pitch between the collagen spots on the chip. This indicates that we can factitiously control the spheroid diameter. In addition, albumin secretion activity of HepG2 spheroids increased with the increase of spheroid diameter. This chip technology may be applicable as a cellular platform for developing two-dimensional spheroid microarrays.

On-Demand Control of Living Cell Adhesion by Local Light Irradiation

In order to meet the diversifying demand for the cell manipulation in the rapid progress of cell engineering, we developed a novel technique to control living adherent cells on a culture substrate by irradiating light onto multiple points simultaneously. In clear contrast to the conventional cell patterning using the previously patterned substrate, the cell-retaining area can be defined even after cell seeding, and the captured cells can continue to grow freely beyond the defined area afterwards. Also the patterned co-culture of living cell was demonstrated by introducing living cells sequentially onto patterned cell adhesion areas assigned and created in each step on a newly developed photo-responsive culture substrate.

Patterned cell adhesion by self-assembled structures for use with a CMOS cell-based biosensor

A strategy for patterned cell adhesion based on chemical surface modification is presented. To confine cell adhesion to specific locations, an engineered surface for high-contrast protein adsorption and, hence, cell attachment has been developed. Surface functionalization is based on selective molecular-assembly patterning (SMAP). An amine-terminated self-assembled monolayer is used to define areas of cell adhesion. A protein-repellent grafted copolymer, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG), is used to render the surrounding silicon dioxide resistant to protein adsorption. X-ray photoelectron spectroscopy, scanning ellipsometry and fluorescence microscopy techniques were used to monitor the individual steps of the patterning process. Successful guided growth using these layers is demonstrated with primary neonatal rat cardiomyocytes, up to 4 days in vitro, and with the HL-1 cardiomyocyte cell line, up to 7 days in vitro. The advantage of the presented method is that high-resolution engineered surfaces can be realized using a simple, cost-effective, dip-and-rinse process. The technique has been developed for application on a CMOS cell-based biosensor, which comprises an array of microelectrodes to extracellularly record electrical activity from cardiomyocytes.

Culturing neuron cells on electrode with self-assembly monolayer

The success of neuronal implantable microsystems relies on the quality of the interface with neuronal cells. Depending on the application, specifically engineered surfaces may either prevent or enhance cell/tissue growth with an appropriate host response. The surface chemistry and topography have major effects on the cell adherence and the interaction between the tissue and devices. We report on a simple technique to precisely explant cortical neurons in a serum-free medium on 2D electrode arrays and investigated the pad size effect on neuron cell culture and immobilization. We produced gold patterns on glass substrates using microfabrication processes. 11-Amino-1-undecanethiol self-assembled monolayer was coated only on the gold surface. Cortical neurons were cultured on the arrays to examine the dependence of neuron growth and cells distribution on pad size. We found that the terminal functional groups of the highly oriented 11-amino-1-undecanethiol thin film are essential for generating cell-adhesive areas for the rat cortical neurons. A 50 μm × 50 μm SAM pad size was found to be suitable for single cortical neuron immobilization, while the larger pads provide excellent neuron coverage. This technology may enable precise and localized neuron stimulation and surveillance for both biological research and medical applications.

The SPR signal in living cells reflects changes other than the area of adhesion and the formation of cell constructions

Surface plasmon resonance (SPR) sensors detected large angle of resonance (AR) changes, when RBL-2H3 rat mast cells were cultured and activated on a sensor chip. Here, we demonstrated that PAM212 mouse keratinocytes also showed a large change in AR, when EGF-stimulated. We explored these changes due to intracellular reactions, through the relationship between the AR and the area of cell adhesion, using confocal microscopy for RBL-2H3 cells and PAM212 cells. The effect of Mycalolide B and Toxin B, inhibitors for cell motility, on AR was observed using RBL-2H3 cells. Measuring AR in the presence of various numbers of non-stimulated cells demonstrated that AR and cell density were proportional. However, the AR increase in response to antigen was 35% higher than that expected by solely an increase of the cell adhesion area. Moreover, the AR with PAM212 cells decreased following a transient increase in response to EGF, whilst the area of cell adhesion remained at an increased level. Furthermore, the treatment of RBL-2H3 cells with either Mycalolide B or Toxin B slightly inhibited, but never abolished the AR increase induced by antigen. These treatments abolished all morphological changes, including ruffling and the increase of cell adhesion area observed by light microscopy. These results suggest that AR changes reflect intracellular events rather than changes in the size of the area to which cells adhere.

Oktoberfest for adhesion structures

The Adhesion Meeting 2005 was organized by S. Linder, R. Fassler, E. Genot and P. Jurdic, and took place in Munich between 28 and 30 April 2005. The booklet of abstracts and a picture gallery of the meeting can be accessed at: www.adhesion‐meeting.com ![][1] Cell motility and tissue invasion depend on the interaction of the cell with its surrounding environment, such as with neighbouring cells, the extracellular matrix (ECM) and even inorganic bone components (for a review, see Geiger et al , 2001). Focal adhesions have long served as a model system to study cell–matrix interactions, but other adhesion structures such as podosomes and invadopodia are made by different cell types and are heterogeneous in their molecular composition, structure and function. This apparent complexity of adhesive structures sparked the first Adhesion Meeting earlier this year. Almost 20 years after the term ‘podosome’ was coined (reviewed in Linder & Aepfelbacher, 2003; Buccione et al , 2004), a lively crowd of international researchers from the different areas of cell adhesion convened in Munich to discuss the similarities and differences of podosomes, invadopodia and focal adhesions (Fig 1). Figure 1. Different types of cell–extracellular matrix contact structures. ( A ) Elongated focal adhesions (FAs) in a stationary adhesive cell. ( B ) Peripheral focal complexes (FXs) and anchoring FAs in a protruding cell. ( C ) Podosomes (yellow) in smooth muscle cells. ( D ) Podosome rosettes (yellow) in epithelial cells. ( E ) Podosomes can aggregate to rings. ( F ) Aggregates of podosome rings can form belt‐like structures. ( G ) Invadopodia (red; arrowheads) in a cell grown on a fluorescent extracellular matrix substrate (green) form preferentially in the centre of the cell. Images were provided by M. Gimona ( A , B , C , E , F ), S. Linder ( D ), and R. Buccione and I. A. Grande ( G ). Focal adhesions mediate the stable attachment of cells … [1]: /embed/graphic-1.gif