Non-adhesive Cells Research Articles

High transfection efficiency and cell viability of immune cells with nanomaterials-based transfection reagent.

Gene manipulation in non-adhesive cells, especially lymphocytes, was difficult due to their low efficiency and high toxicity. Electroporation was reported as a highly efficient method for human and mouse lymphocytes. However, this method requires expensive equipment and causes severe cell damage. Here, the authors present a simple and efficient method to deliversiRNA into lymphocytes with high efficiency and cell viability. This nanomaterials-based transfection reagent was simple and cost-effective and can perform multiple transfections, which further increase the overall efficiency. This method should be applicable for many cell lines and can be used to decipher gene functions of lymphocytes.

Integrated analysis of lncRNAs and mRNAs reveals key trans-target genes associated with ETEC-F4ac adhesion phenotype in porcine small intestine epithelial cells

BackgroundLong non-coding RNAs (lncRNAs) play crucial roles in gene regulation at the transcriptional and post-transcriptional levels. LncRNAs are belonging to a large class of transcripts with ≥200 nt in length which do not code for proteins, have been widely investigated in various physiological and pathological contexts by high-throughput sequencing techniques and bioinformatics analysis. However, little is known about the regulatory mechanisms by which lncRNAs regulate genes that are associated with Enterotoxigenic Escherichia coli F4 fimbriae (ETEC-F4ac) adhesion phenotype in small intestine epithelial cells of Large White piglets. To address this, we used RNA sequencing to profile lncRNAs and mRNAs of small intestine epithelial cells in Large White piglets differing in their ETEC-F4 adhesion phenotypes and ITGB5 genotypes. Eight male piglets were used in this study and were divided into two groups on the basis of their adhesion phenotype and ITGB5 genotypes, a candidate gene for F4ac receptor. Non-adhesive group (n = 4) with CC genotype and adhesive group (n = 4) with TT genotype.ResultsIn total, 78 differentially expressed lncRNAs (DE-lncRNA) and 223 differentially expressed mRNAs (log2 |FC| > 1, P < 0.05) were identified in the comparison of non-adhesive vs. adhesive small intestine epithelial cells. Furthermore, cis- and trans-regulatory target genes of DE-lncRNAs were identified, then interaction networks of lncRNAs and their cis- and trans-target differentially expressed genes (DEGs) were constructed separately. A total of 194 cis-targets were involved in the lncRNAs-cis genes interaction network and 61 trans-targets, were involved in lncRNA-trans gene interaction network that we constructed. We determined that cis-target genes were involved in alcoholism, systemic lupus erythematosus, viral carcinogenesis and malaria. Whereas trans-target DEGs were engaged in three important pathways related to the ETEC-F4 adhesion phenotype namely cGMP-PKG signaling pathway, focal adhesion, and adherens junction. The trans-target DEGs which directly involved in these pathways are KCNMB1 in cGMP-PKG signaling pathway, GRB2 in focal adhesion pathway and ACTN4 in focal adhesion and adherens junction pathways.ConclusionThe findings of the current study provides an insight into biological functions and epigenetic regulatory mechanism of lncRNAs on porcine small intestine epithelial cells adhesion to ETEC-F4-ac and piglets’ diarrhea susceptibility/resistance.

Transcriptomic Study of Porcine Small Intestine Epithelial Cells Reveals Important Genes and Pathways Associated With Susceptibility to Escherichia coli F4ac Diarrhea.

BackgroundDiarrhea represents one of the most frequent major problems during piglets' neonatal and post-weaning periods leading to tremendous economic losses in the swine industry. Enterotoxigenic Escherichia coli (ETEC) F4 is regarded as the most important cause of diarrhea in piglets. However, some pigs are naturally resistant to those diarrheas caused by ETEC-F4, because they have no F4 receptors (F4R) on their small intestine epithelial cells that allow F4 fimbriae attachment. Thus, our study characterized a complete transcriptome of small intestine epithelial cells of Large White piglets using RNA-Seq. The aim of the study was to identify DEGs with regard to differences in the F4R phenotypes and SNP (C/T) genotypes at ITGB5 and important pathways associated with ETEC-F4ac susceptibility in small intestine epithelial cells of Large White piglets and derive molecular markers as a result of loss of F4acR in swine.MethodsA total of eight samples of small intestine epithelial cells obtained from Large White piglets (35 days old) used in this study were selected on the basis of two criteria. One was the adhesion phenotype to ETEC-F4ac fimbriae, and the other was the comparison of ITGB5 SNP (C > T) genotype sequences across all the samples. The samples were then divided into two groups, non-adhesive with CC genotype (n = 4), and adhesive with TT genotype (n = 4).ResultsMore down-regulated DEGs (p < 0.05, |log2FC| > 2) were detected in the comparison of non-adhesive vs. adhesive small intestine epithelial cells in the present study. Six genes, of which two (CNGA4, SLC25A31) exclusively expressed and four (HCN4, MYLK, KCNMA1, and KCNMB1) DEGs with up-regulation pattern in adhesive (F4R positive) pigs were involved in two pathways associated with diarrhea. The DEGs with up-regulation pattern in non-adhesive (F4R negative) pigs were mostly engaged in multiple immune response-related pathways.ConclusionThe results provide insights on the biology of the phenotypes of F4R positive and negative pigs. One gene (MYLK) located on SSC13 locus for F4acR strongly support that it might have played a role in the adhesion phenotype which was obviously detected by adhesion assay in adhesive (F4R positive) group.

Effect of PAK Inhibition on Cell Mechanics Depends on Rac1.

Besides biochemical and molecular regulation, the migration and invasion of cells is controlled by the environmental mechanics and cellular mechanics. Hence, the mechanical phenotype of cells, such as fibroblasts, seems to be crucial for the migratory capacity in confined 3D extracellular matrices. Recently, we have shown that the migratory and invasive capacity of mouse embryonic fibroblasts depends on the expression of the Rho-GTPase Rac1, similarly it has been demonstrated that the Rho-GTPase Cdc42 affects cell motility. The p21-activated kinase (PAK) is an effector down-stream target of both Rho-GTPases Rac1 and Cdc42, and it can activate via the LIM kinase-1 its down-stream target cofilin and subsequently support the cell migration and invasion through the polymerization of actin filaments. Since Rac1 deficient cells become mechanically softer than controls, we investigated the effect of group I PAKs and PAK1 inhibition on cell mechanics in the presence and absence of Rac1. Therefore, we determined whether mouse embryonic fibroblasts, in which Rac1 was knocked-out, and control cells, displayed cell mechanical alterations after treatment with group I PAKs or PAK1 inhibitors using a magnetic tweezer (adhesive cell state) and an optical cell stretcher (non-adhesive cell state). In fact, we found that group I PAKs and Pak1 inhibition decreased the stiffness and the Young’s modulus of fibroblasts in the presence of Rac1 independent of their adhesive state. However, in the absence of Rac1 the effect was abolished in the adhesive cell state for both inhibitors and in their non-adhesive state, the effect was abolished for the FRAX597 inhibitor, but not for the IPA3 inhibitor. The migration and invasion were additionally reduced by both PAK inhibitors in the presence of Rac1. In the absence of Rac1, only FRAX597 inhibitor reduced their invasiveness, whereas IPA3 had no effect. These findings indicate that group I PAKs and PAK1 inhibition is solely possible in the presence of Rac1 highlighting Rac1/PAK I (PAK1, 2, and 3) as major players in cell mechanics.

Nanowire-array-based gene electro-transfection system driven by human-motion operated triboelectric nanogenerator

Electro-transfection serves as a promising non-viral gene delivery approach, but its performance faces several drawbacks associated with the high-voltage electrical pulses involved, which may adversely affect the cell viability. Here we developed a novel TENG (triboelectric nanogenerator)-driven nanowire electrode array (T-NEA) platform to facilitate the electroporation-based siRNA delivery into both adhesive and non-adhesive mammalian cells. Unlike conventional electroporation technology, the T-NEA system is powered by a TENG working in a vertical contact-separation mode, harvesting energy from simple human tapping motion. Since lower voltage is involved in the T-NEA system as compared to conventional electroporation method, high (>90%) cell viability was achieved for all of the six kinds of cell lines used in the experiments. Highly efficient siRNA electro-transfection was achieved at a tiny amount of energy consumption. The results exhibit that siRNA molecules could be successfully delivered into more than 95% of MiaPaCa-2 cell. Even for K562 cells, which is well-known as difficult-to-transfect cell, the delivery efficiency of siRNA reaches 84%. With the T-NEA system, the K-ras gene expression in MiaPaCa-2 cells was down-regulated by 46 ± 6.29%, and the bcr-abl gene expression was reduced by 31.6 ± 6.67% in K562 cells. When the expressions of the targeted mutation genes were significantly down-regulated, the cell proliferation and anti-apoptosis ability were significantly inhibited. Overall, the T-NEA system is demonstrated as an effective and versatile platform for high-throughput gene delivery in biological research and clinical cancer treatments due to its versatility, efficiency, and uniformity.

Clonal yeast biofilms can reap competitive advantages through cell differentiation without being obligatorily multicellular.

How differentiation between cell types evolved is a fundamental question in biology, but few studies have explored single-gene phenotypes that mediate first steps towards division of labour with selective advantage for groups of cells. Here, we show that differential expression of the FLO11 gene produces stable fractions of Flo11+ and Flo11- cells in clonal Saccharomyces cerevisiae biofilm colonies on medium with intermediate viscosity. Differentiated Flo11+/- colonies, consisting of adhesive and non-adhesive cells, obtain a fourfold growth advantage over undifferentiated colonies by overgrowing glucose resources before depleting them, rather than depleting them while they grow as undifferentiated Flo11- colonies do. Flo11+/- colonies maintain their structure and differentiated state by switching non-adhesive cells to adhesive cells with predictable probability. Mixtures of Flo11+ and Flo11- cells from mutant strains that are unable to use this epigenetic switch mechanism produced neither integrated colonies nor growth advantages, so the condition-dependent selective advantages of differentiated FLO11 expression can only be reaped by clone-mate cells. Our results show that selection for cell differentiation in clonal eukaryotes can evolve before the establishment of obligate undifferentiated multicellularity, and without necessarily leading to more advanced organizational complexity.

Array-Based Platform To Select, Release, and Capture Epstein-Barr Virus-Infected Cells Based on Intercellular Adhesion.

Microraft arrays were developed to select and separate cells based on a complex phenotype, weak intercellular adhesion, without knowledge of cell-surface markers or intracellular proteins. Since the cells were also not competent to bind to a culture surface, a method to encapsulate nonadherent cells within a gelatin plug on the concave microraft surface was developed, enabling release and collection of the cells without the need for cell attachment to the microraft surface. After microraft collection, the gelatin was liquified to release the cell(s) for culture or analysis. A semiautomated release and collection device for the microrafts demonstrated 100 ± 0% collection efficiency of the microraft while increasing throughput 5-fold relative to that of manual release and collection. Using the microraft array platform along with the gelatin encapsulation method, single cells that were not surface-attached were isolated with a 100 ± 0% efficiency and a 96 ± 4% postsort single-cell cloning efficiency. As a demonstration, Epstein-Barr virus-infected lymphoblastoid cell lines (EBV-LCL) were isolated based on their intercellular adhesive properties. The identified cell colonies were collected with a 100 ± 0% sorting efficiency and a postsort viability of 87 ± 3%. When gene expression analysis of the EBV latency-associated gene, EBNA-2, was performed, there was no difference in expression between blasting or weakly adhesive cells and nonblasting or nonadhesive cells. Microraft arrays are a versatile method enabling separation of cells based on complicated and as yet poorly understood cell phenotypes.

Hydrodynamic nonadhesive cell retention in a microfluidic circuit for stressless suspension culture

Cell collection based on deterministic lateral displacement (DLD) and cell circulation with a loop channel are two component technologies for stressless cell retention which have been developed with a view to working toward suspension culture in a microfluidic channel.

Small-molecule axon-polarization studies enabled by a shear-free microfluidic gradient generator.

A deep understanding of the mechanisms behind neurite polarization and axon path-finding is important for interpreting how the human body guides neurite growth during development and response to injury. Further, it is of great clinical importance to identify diffusible chemical cues that promote neurite regeneration for nervous tissue repair. Despite the fast development of various types of concentration gradient generators, it has been challenging to fabricate neuron-friendly (i.e. shear-free and biocompatible for neuron growth and maturation) devices to create stable gradients, particularly for fast diffusing small molecules, which typically require high flow and shear rates. Here we present a finite element analysis for a polydimethylsiloxane/polyethylene glycol diacrylate (PDMS/PEG-DA) based gradient generator, describe the microfabrication process, and validate its use for neuronal axon polarization studies. This device provides a totally shear-free, biocompatible microenvironment with a linear and stable concentration gradient of small molecules such as forskolin. The gradient profile in this device can be customized by changing the composition or width of the PEG-DA barriers during direct UV photo-patterning within a permanently bonded PDMS device. Primary rat cortical neurons (embryonic E18) exposed to soluble forskolin gradients for 72 h exhibited statistically significant polarization and guidance of their axons. This device provides a useful platform for both chemotaxis and directional guidance studies, particularly for shear sensitive and non-adhesive cell cultures, while allowing fast new device design prototyping at a low cost.

Isolation of B-1 subtype B lymphocytes from human cord blood

Introduction: B-1 lymphocytes with high levels of CD5 markers are capable of differentiation and transformation into macrophage-like cells, as B-1 cells. This study aims to isolate and purify this unique subset from umbilical cord blood cell. Materials and Methods: 14 umbilical cord samples from healthy neonates of natural or elective cesarean deliveries were used. White blood cells were separated from red blood cells using hydroxyethyl starch or gelatin 3%. Cell culture was performed to separate adhesive and non-adhesive cells. Umbilical cord lymphocytes subsets were separated from the remaining leukocyte contents by Ficoll densitygradient centrifugation. Lymphocyte rosette test was used to assess purification stage. Results: Despite lack of difference in cell count between the two blood dilution methods, cell vitality in hydroxylethyl starch method was less than 87%, while cell vital activity in the dilution method by gelatin 3% was measured between 90 and 95%. Lymphocyte rosette test results indicate B-1 cell purification rate of approximately 99% by using the above method. Conclusion: This process indicates most of the extracted B-cells from human umbilical cord blood belong to the B-1 cell subtype, and gelatin 3% procedure is the best and most appropriate method in terms of number and vitality of cells.

Characterization and Differential Gene Expression between Two Phenotypic Phase Variants in Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium strain 798 has previously been shown to undergo phenotypic phase variation. One of the phenotypes expresses virulence traits such as adhesion, while the other phenotype does not. Phenotypic phase variation appears to correlate with the ability of this strain to cause persistent, asymptomatic infections of swine. A new method to detect cells in either phenotypic phase was developed using Evans Blue-Uranine agar plates. Using this new assay, rates of phenotypic phase variation were obtained. The rate of phase variation from non-adhesive to adhesive phenotype was approximately 10−4 per cell per generation while phase variation from the adhesive to the non-adhesive phenotype was approximately 10−6 per cell per generation. Two highly virulent S. Typhimurium strains, SL1344 and ATCC 14028, were also shown to undergo phase variation. However, while the rate from adhesive to non-adhesive phenotype was approximately the same as for strain 798, the non-adhesive to adhesive phenotype shift was 37-fold higher. Differential gene expression was measured using RNA-Seq. Eighty-three genes were more highly expressed by 798 cells in the adhesive phenotype compared to the non-adhesive cells. Most of the up-regulated genes were in virulence genes and in particular all genes in the Salmonella pathogenicity island 1 were up-regulated. When compared to the virulent strain SL1344, expression of the virulence genes was approximately equal to those up-regulated in the adhesive phenotype of strain 798. A comparison of invasive ability demonstrated that strain SL1344 was the most invasive followed by the adhesive phenotype of strain 798, then the non-adhesive phenotype of strain 798. The least invasive strain was ATCC 14028. The genome of strain 798 was sequenced and compared to SL1344. Both strains had very similar genome sequences and gene deletions could not readily explain differences in the rates of phase variation from non-adhesive to the adhesive phenotype.

Conformational epitopes at cadherin calcium-binding sites and p120-catenin phosphorylation regulate cell adhesion

We investigated changes in cadherin structure at the cell surface that regulate its adhesive activity. Colo 205 cells are nonadhesive cells with a full but inactive complement of E-cadherin-catenin complexes at the cell surface, but they can be triggered to adhere and form monolayers. We were able to distinguish the inactive and active states of E-cadherin at the cell surface by using a special set of monoclonal antibodies (mAbs). Another set of mAbs binds E-cadherin and strongly activates adhesion. In other epithelial cell types these activating mAbs inhibit growth factor-induced down-regulation of adhesion and epithelial morphogenesis, indicating that these phenomena are also controlled by E-cadherin activity at the cell surface. Both types of mAbs recognize conformational epitopes at different interfaces between extracellular cadherin repeat domains (ECs), especially near calcium-binding sites. Activation also induces p120-catenin dephosphorylation, as well as changes in the cadherin cytoplasmic domain. Moreover, phospho-site mutations indicate that dephosphorylation of specific Ser/Thr residues in the N-terminal domain of p120-catenin mediate adhesion activation. Thus physiological regulation of the adhesive state of E-cadherin involves physical and/or conformational changes in the EC interface regions of the ectodomain at the cell surface that are mediated by catenin-associated changes across the membrane.

A novel composite construct increases the vascularization potential of PEG hydrogels through the incorporation of large fibrin ribbons

Developing a mechanism to vascularize tissue-engineered constructs is imperative for transplant function and integration, particularly when delivering hypoxia-sensitive tissues, such as pancreatic islets. Previous efforts have focused on bulk modifications of scaffold materials rendering the entire construct permissive to vessel penetration or the formation of a porous structure where vessels can infiltrate the empty spaces. Here, we describe a novel construct composed of large fibrin ribbons encapsulated within a poly(ethylene glycol) (PEG) hydrogel. The PEG/fibrin ribbon composite scaffold facilitates coculture of adhesive and nonadhesive cell types, thus providing closely neighboring environments with distinct material properties specific to the needs of two clinically relevant cell populations. This advantage is demonstrated here by the successful coculture of pancreatic islets in the PEG component and vessel-forming endothelial cells in entrapped fibrin ribbons. Transplanted endothelial cells can form anastomosies with host vasculature, suggesting that our cocultures may lead to more rapid scaffold vascularization. Additionally, we show that surface-seeded endothelial cells form multicellular projections that migrate into nonadhesive PEG hydrogels along permissive fibrin ribbons, further demonstrating composite construct vascularization potential. Distribution of large fibrin ribbons throughout PEG hydrogels provide a potential mechanism for vascularization of a well-established biomaterial without inherently changing its desirable properties.

Feasibility study on cellular network analysis with patterned cell culture microdevice

In order to realize cellular network analysis on a chip-based system, our group has been developing a patterned cell culture microdevice with pillars in an array for tapping cells into space surrounded by the pillars. The pillar structures has advantages to trap both adhesive and non-adhesive cells and to precisely control positions of cells and distances between cells for understanding effects of various cell patterns on functions of a cellular network such as cell proliferation, differentiation, and network formation. In this paper, HeLa cell cultivation with the patterned cell culture microdevice having a pillar array fabricated by dry film of thick negative photoresist SU-8 on a glass substrate was executed as a feasibility study on a cellular network analysis. The results revealed that the device performance was found to be enough to culture HeLa cells for more than 48 h. In addition, relative extensibility of blocks of multiple cells compared with single cells tapped on the device was observed. Thus, the patterned cell culture microdevice proposed here could be applicable to analysis of cellular functions.

Analysis of Nonadherent Apoptotic Cells by a Quantum Dots Probe in a Microfluidic Device for Drug Screening

This technical note describes a facile technique to screen some anticancer drugs and evaluate their effects on nonadhesive leukemic cells in an easily fabricated microfluidic device by utilizing the Annexin V conjugated quantum dots as apoptosis detection probes. The cell immobilizing structures and gradient-generating channels were integrated within the device which was fabricated in one-single step. The nonadhesive leukemic HL-60 cells can be felicitously immobilized and cultured on the dam structures at a proper lateral pressure. We then delivered Annexin V functionalized quantum dots which can readily bind to the outer membrane of apoptotic cells and distinguish the apoptosis from unaffected cells with single cell level resolution. The diffusion time of quantum dots reduced to 5 min before imaging. The capabilities of evaluating drug effect on HL-60 cell line have been shown in both population way and individual cell level. The technique presented herein can bridge the gap between the quantum dots based in vitro cell imaging and the analysis of individual apoptotic cell in a microfluidic system, allows an easy operating protocol to screen some clinically available anticancer drugs.

A Novel Peptide Mediates Aggregation and Migration of Hemocytes from an Insect

Insect blood cells (hemocytes) comprise an essential arm of the immune system [1-7]. Several factors mediating recognition and phagocytosis of foreign intruders by hemocytes have been identified, but the mechanisms regulating hemocyte movement remain fragmentary. Embryonic hemocytes from Drosophila migrate along stereotypical routes in response to chemotactic signals from PVF ligands, members of the platelet-derived growth factor family [8-12]. Embryonic and larval hemocytes also accumulate at external wounds [11-13], but PVFs are not required for this response, suggesting involvement by other, unknown factors. Here we report the identification of hemocyte chemotactic peptide (HCP) from the moth Pseudaletia separata and present evidence that it stimulates aggregation and directed movement of phagocytic hemocytes. Spatiotemporal studies revealed that HCP is expressed in both epidermal cells and hemocytes, whereas structure-function studies identified post-translational modifications important for activity. HCP also shares similarities with another group of cytokines from moths called ENF peptides [14-17]. Taken together, our results identify HCP as a chemotactic cytokine that enhances clotting at wound sites in larvae.

Loss of Fibulin-2 Expression Is Associated with Breast Cancer Progression

Fibulin-2, an extracellular matrix protein expressed by normal epithelia, was found to be down-regulated in several breast cancer cell lines. Fibulin-2 protein expression was also decreased in breast cancer tissue samples as evaluated by immunohistochemistry. Reintroduction of Fibulin-2 into breast cancer cell lines that do not express Fibulin-2 reduced cancer cell motility and invasion in vitro but had no effect on cell growth and adhesion properties. Together with evidence that Fibulin-2 contributes to wound healing and inhibits smooth muscle cell migration, our findings suggest that loss of Fibulin-2 expression may facilitate migration and invasion in breast cancer.

Identification of an Invasion Regulatory Domain within the Core Protein of Syndecan-1

Among the four members of the syndecan family there exists a high level of divergence in the ectodomain core protein sequence. This has led to speculation that these core proteins bear important functional domains. However, there is little information regarding these functions, and thus far, the biological activity of syndecans has been attributed largely to their heparan sulfate chains. We have previously demonstrated that cell surface syndecan-1 inhibits invasion of tumor cells into three-dimensional gels composed of type I collagen. Inhibition of invasion is dependent on the syndecan heparan sulfate chains, but a role for the syndecan-1 ectodomain core protein was also indicated. To more closely examine this possibility and to map the regions of the ectodomain essential for syndecan-1-mediated inhibition of invasion, a panel of syndecan-1 mutational constructs was generated, and each construct was transfected individually into myeloma tumor cells. The anti-invasive effect of syndecan-1 is dramatically reduced by deletion of an ectodomain region close to the plasma membrane. Further mutational analysis identified a stretch of 5 hydrophobic amino acids, AVAAV (amino acids 222-226), critical for syndecan-1-mediated inhibition of cell invasion. This invasion regulatory domain is 26 amino acids from the start of the transmembrane domain. Importantly, this domain is functionally specific because its mutation does not affect syndecan-1-mediated cell binding to collagen, syndecan-1-mediated cell spreading, or targeting of syndecan-1 to specific cell surface domains. This invasion regulatory domain may play an important role in inhibiting tumor cell invasion, thus explaining the observed loss of syndecan-1 in some highly invasive cancers.

MICROBEAM ANALYSIS AT TOHOKU UNIVERSITY FOR BIOLOGICAL STUDIES

A microbeam analysis system at Tohoku University has been improved in detection efficiency for application to single cell analysis. The system is applicable to STIM analysis and to simultaneous PIXE and RBS analysis. Sample preparation methods suitable for non-adhesive single cell analysis were developed and first results with the improved analysis system are shown.

Controlling cell attachment selectively onto biological polymer-colloid templates using polymer-on-polymer stamping.

A new patterning approach using polymer-on-polymer stamping (POPS) has been developed to fabricate polymer-colloid templates for controlling selective cell attachment. In this paper, a polyamine surface patterned onto a poly(acrylic acid)/poly(allylamine hydrochloride) (PAA/PAH) cell resistant multilayer platform serves as a template for the deposition of close- or loose-packed colloidal particles. Peptides containing the RGD adhesion sequence were used to modify the PAH/colloid surface for specific cell attachment. Cell behavior was studied by varying colloidal packing array density, pattern geometry, and surface chemistry. It was found that loose-packed RGD-modified colloidal arrays enhance cell adhesion, as observed through the development of focal adhesion contacts and orientation of actin stress fibers, but close-packed colloidal arrays induce a rounded and nonadhesive cell morphology and yield a smaller number of attached cells. On loose-packed arrays, cells adjust their shapes to the pattern geometry when the stripe width is smaller than 50 microm and increase their extent of attachment when the concentration of surface RGD peptides is increased. This new biomaterials system allows the examination of cell behavior as a function of RGD surface distribution on the molecular to micrometer scale and reveals cellular response to different surface roughnesses.