Influence of insulin immobilization to thermoresponsive culture surfaces on cell proliferation and thermally induced cell detachment

Temperature-responsive culture dishes immobilized with insulin have been fabricated and studied to shorten cell culture periods by facilitating more rapid cell proliferation. Cells are recovered as contiguous cell sheets simply by temperature changes. Functionalized culture dishes were prepared by previously reported electron beam grafting copolymerization of N-isopropylacrylamide (IPAAm) with its carboxylate-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), having similar molecular structure to IPAAm but with carboxylate side chains to tissue culture polystyrene dishes. Insulin was then immobilized onto culture dishes through standard amide bond formation with CIPAAm carboxylate groups. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on these insulin-immobilized dishes. Insulin immobilization was shown to promote cell proliferation in serum-supplemented medium. Increasing the grafted CIPAAm content on the tissue culture surfaces reduces cell adhesion and proliferation, even though these surfaces contained increased amounts of immobilized insulin. This result implies that a discrete balance exists between the amount of CIPAAm-free carboxylate groups and immobilized insulin for optimum cell proliferative stimulation. Cells grown on the insulin-immobilized surfaces can be recovered as contiguous cell monolayers simply by lowering culture temperature, without need for exogenous enzyme or calcium chelator additions. In conclusion, insulin-modified thermoresponsive culture dishes may prove useful for advanced cell culture and tissue engineering applications since they facilitate cell proliferation, and cultured cells can be recovered as viable contiguous monolayers by merely reducing culture temperature.     

Control of cell adhesion and detachment using temperature and thermoresponsive copolymer grafted culture surfaces

The hydrophobic monomer, n-butyl methacrylate (BMA) has been incorporated into thermoresponsive poly(N-isopropylacrylamide) (PIPAAm) to lower PIPAAm phase transition temperatures necessary for systematically regulating cell adhesion on and detachment from culture dishes at controlled temperatures. Poly(IPAAm-co-BMA)-grafted dishes were prepared by electron beam irradiation methods, systematically changing BMA content in the feed. Copolymer-grafted surfaces decreased grafted polymer transition temperatures with increasing BMA content as shown by water wettabilities compared to homopolymer PIPAAm-grafted surfaces. Bovine endothelial cells readily adhered and proliferated on copolymer-grafted surfaces above collapse temperature at 37 degrees C, finally reaching confluence. Cell sheet detachment behavior from copolymer-grafted surfaces depended on the culture temperature and BMA content. In conclusion, cell attachment/detachment can be controlled to an arbitrary temperature by varying the content of hydrophobic monomer incorporated into PIPAAm grafted to culture surfaces.     

PEG-variant biomaterials as selectively adhesive protein templates: model surfaces for controlled cell adhesion and migration

Our study focused on the role of poly(ethylene glycol) (PEG) in actively regulating the biological responsiveness of protein-adsorbed biomaterials. To this end, we designed PEG-variant biomaterials from a family of tyrosine/PEG-derived polycarbonates to present surfaces ranging from low to intermediate levels of PEG concentration, below the PEG level requisite for complete abolition of protein adsorption. We analyzed the effect of PEG concentration on the amount, conformation and bioactivity of an adsorbed model protein, fibronectin, and on the attachment, adhesion strength and motility of L929 fibroblasts. Our results demonstrate that low levels of PEG can regulate not only the extent but also the conformation and specific bioactivity of adsorbed fibronectin. As the PEG concentration was increased from 0 to 6 mol%, the amount of adsorbed fibronectin decreased linearly yet the fibronectin conformation was altered such that the overall bioactivity of adsorbed fibronectin was uncompromised. We report that the degree of cell attachment varied with PEG concentration in a manner similar to the dependence of fibronectin bioactivity on PEG. In contrast, the nature of cell adhesion strength dependence on PEG paralleled the pattern observed for fibronectin surface concentration. Our studies also indicated that the rate of cell migration was inversely correlated with PEG concentration over a narrow range of PEG concentration. Overall, these results highlight the striking ability of PEG-variant biomaterials to systematically regulate the behavior of adsorbed cell adhesion proteins and, consequently, effect cell functions.     

Fluid shear induced endothelial cell detachment from glass - influence of adhesion time and shear stress

In this study, human umbilical vein and human saphenous vein endothelial cells were seeded on glass and exposed to fluid shear in a parallel-plate flow chamber. Cell retention, morphology and migration were studied as a function of shear stress and of adhesion time prior to exposure to shear. Three-hour and 24-h adhesion times gave rise to comparable cell retention values after 2 h of flow for both cell types. Cell retention decreased from 85 to 20% as shear stress increased from 88 to 264 dynes cm⁻² (8.8 to 26 Pa). Mean spreading areas decreased after the onset of flow, but subsequently stabilized to plateau values, which were smaller at higher shear stresses. Shape factors increased faster to higher values as cells were exposed to higher shear stresses, without any obvious preference in orientation of the cells with respect to the direction of flow. Migration was unidirectional with flow and linear with time. Migration was faster for cells which had adhered for 24 h than for cells which had adhered for 3 h and was accompanied by the presence of fibrillar structures left behind on the surface upstream of migrating cells. It is concluded that after 3 h adhesion to glass, cells have adhered with an adhesion strength that does not substantially increase during the next 21 h. However, during this time changes in cell-substratum interactions seem to occur judging by the differences in, e.g., migration rates.     

Micropatterned surfaces for controlling cell adhesion and rolling under flow

Cell adhesion and rolling on the vascular wall is critical to both inflammation and thrombosis. In this study we demonstrate the feasibility of using microfluidic patterning for controlling cell adhesion and rolling under physiological flow conditions. By controlling the width of the lines (50–1000 μm) and the spacing between them (50–100 μm) we were able to fabricate surfaces with well-defined patterns of adhesion molecules. We demonstrate the versatility of this technique by patterning surfaces with 3 different adhesion molecules (P-selectin, E-selectin, and von Willebrand Factor) and controlling the adhesion and rolling of three different cell types (neutrophils, Chinese Hamster Ovary cells, and platelets). By varying the concentration of the incubating solution we could control the surface ligand density and hence the cell rolling velocity. Finally by patterning surfaces with both P-selectin and von Willebrand Factor we could control the rolling of both leukocytes and platelets simultaneously. The technique described in this paper provides and effective and inexpensive way to fabricate patterned surfaces for use in cell rolling assays under physiologic flow conditions.     

The use of patterned dual thermoresponsive surfaces for the collective recovery as co-cultured cell sheets

Heterotypic cell interactions are critical to achieve and maintain specific functions in many tissues and organs. We have focused on patterned structure surfaces to enable co-culture of heterotypic cells and recovery of patterned co-cultured cell sheets for applications in tissue engineering. Thermoresponsive polymers exhibiting different transition temperatures in water comprise both poly(N-isopropylacrylamide) (PIPAAm) and n-butyl methacrylate (BMA) co-grafted as side chains to PIPAAm main chains. These copolymers were surface-grafted in patterns to obtain patterned dual thermoresponsive cell culture surfaces using electron beam polymerisation method and porous metal masks. On patterned surfaces, site-selective adhesion on and growth of rat primary hepatocytes (HCs) and bovine carotid endothelial cells (ECs) allowed patterned co-culture, exploiting hydrophobic/hydrophilic surface chemistry regulated by culture temperature as the sole variable. At 27 degrees C, seeded HCs adhered exclusively onto hydrophobic, dehydrated P(IPAAm-BMA) co-grafted domains (1-mm laser dot), but not onto neighbouring hydrated PIPAAm domains. Sequentially seeded ECs then adhered exclusively to hydrophobised PIPAAm domains upon increasing culture temperature to 37 degrees C, achieving patterned co-cultures. Reducing culture temperature to 20 degrees C promoted hydration of both polymer-grafted domains, permitting release of the co-cultured, patterned cell monolayers as continuous cell sheets with heterotypic cell interactions. Recovered co-cultured cell sheets can be manipulated, moved and sandwiched with other structures, providing new useful constructs both for basic cell biology research and preparation of tissue-mimicking multi-layer materials through overlaying co-cultured cell sheets.     

Effect of cell anisotropy on differentiation of stem cells on micropatterned surfaces through the controlled single cell adhesion

Micropatterns of arginine-glycine-aspartic acid (RGD) on poly(ethylene glycol) (PEG) hydrogels were fabricated. Under an appropriate size of microislands on this strong and persistent non-fouling background, single mesenchymal stem cells (MSCs) from rats were well localized, keeping the same adhesive area but different shapes. The cell shapes influenced the differentiation of MSCs, and the osteogenic and adipogenic differentiations exhibited different trends. According to comparison between square and rectangular cells, optimal adipogenic differentiation occurred at aspect ratio (AR) 1, but the optimal osteogenic differentiation was found when AR was about 2. We further interpreted the optimal ratios as reflecting the inherent global anisotropy of free adipoblasts and osteoblasts on unpatterned culture plates. According to comparison between globally isotropic circular, square, triangular, and star cells, the optimal adipogenic and osteogenic differentiations happened in circular and star cells, respectively. In this case we found that extents of both adipogenic and osteogenic differentiations were linearly related to cell perimeter, which reflects the non-roundness or local anisotropy of cells. Hence, the present study makes semi-quantitative investigations of the effects of cell shape on differentiation of stem cells based on a material technique, and reveals that the shape anisotropy is very important in directing the lineage commitments of stem cells.     

The use of electron beam lithographic graft-polymerization on thermoresponsive polymers for regulating the directionality of cell attachment and detachment

A simple process for nano-patterned cell culture substrates by direct graft-polymerization has been developed using an electron beam (EB) lithography system requiring no photo-masks or EB-sensitive resists. The compound N-isopropylacrylamide (IPAAm) was locally polymerized and grafted directly by EB lithographic exposure onto hydrophilic polyacrylamide (PAAm)-grafted glass surfaces. The size of the surface grafted polymers was controlled by varying the area of EB dose, and a minimal stripe pattern with a 200 nm line-width could be fabricated onto the surface. On the stripe-patterned surfaces, above the lower critical solution temperature (LCST), the cells initially adhered and spread with an orientation along the pattern direction. The magnitude of the spreading angle and elongation of adhered cells depended on the pattern intervals of the grafted PIPAAm. When culture temperature was lower than the LCST, cultured cells detached from the surfaces with strong shrinkage along the pattern direction, and sometimes folded and became parallel with the stripe pattern. This patterned cell recovery technique may be useful for the construction of muscle cell sheets with efficient shrinkage/relaxation in a specific direction and spheroidal 3D cell structures, with application to tissue engineering and microfluidic cellular devices.     

Assembly and characterization of biofunctional neurotransmitter-immobilized surfaces for interaction with postsynaptic membrane receptors

Herein, we report progress toward the development of bioactive surfaces based on gamma-aminobutyric acid (GABA), a major neurotransmitter in the nervous system. Whereas immobilization techniques have focused largely on antibodies, enzymes, and receptors, to our knowledge, this is the first report of a prototype neurotransmitter-immobilized surface. Biosurfaces were assembled onto either mica or glass using passive adsorption of avidin and subsequent attachment of a derivatized form of GABA via a biotin-avidin affinity bond. Surface characterization of these prepared bimolecular surfaces was determined using atomic force microscopy in tapping mode. The data reveal that passive adsorption of avidin is uniformly dispersed and cluster densities can be controlled through the concentration of the avidin incubation solution. GABA tethered via biotin to these avidin surfaces displayed a unique surface topology; in addition, histograms of surface heights suggest two different types of molecular cluster populations. Functional assays were performed to test the biological activity of the synthesized GABA. Anti-GABA antibody directed to these bimolecular surfaces result in morphological topologies and histograms that indicate antibody-antigen binding. However, nonspecific anti-immunoglobulin G antibodies directed to these surfaces show low binding affinity. Taken together, the data support the idea that the synthesized surfaces are biofunctional.     

Biomimetic surfaces for control of cell adhesion to facilitate bone formation

Cell adhesion to extracellular matrix ligands through integrin receptors plays a central role in bone formation and maintenance by anchoring cells and triggering signals that direct osteoblast proliferation and differentiation. Moreover, osteoblast adhesion to adsorbed, synthesized, or engineered extracellular ligands on synthetic surfaces is critical to numerous biomedical and biotechnological applications. Considerable research efforts have concentrated on the development of surfaces that promote osteoblast differentiation and bone formation. Emerging surface engineering approaches have focused on creating biomimetic substrates that target integrins to activate signaling pathways directing the osteoblast differentiation program. These initiatives generally rely on controlling the adsorption of extracellular matrix ligands or engineering synthetic supports presenting bioadhesive motifs from extracellular matrix proteins. These biomolecular approaches provide promising strategies for the engineering of robust biofunctional matrices that control cell adhesion and signaling and promote osteoblast proliferation, differentiation, and matrix mineralization.     

A centrifugation cell adhesion assay for high-throughput screening of biomaterial surfaces

A quantitative analysis of cell adhesion is essential in understanding physiological phenomena and designing biomaterials, implant surfaces, and tissue-engineering scaffolds. The most common cell adhesion assays used to evaluate biomaterial surfaces lack sensitivity and reproducibility and/or require specialized equipment and skill-intensive operation. We describe a modified centrifugation cell adhesion assay that uses simple and convenient techniques with standard laboratory equipment and provides reliable, quantitative measurements of cell adhesion. This centrifugation assay applies controlled and uniform detachment forces to a large population of adherent cells, providing robust statistics for quantifying cell adhesion. The applicability of this system to the design and characterization of biomaterial surfaces is shown by evaluating cell adhesion on substrates using different coating proteins, cell types, seeding times, and relative centrifugal forces (RCF). Results verify that this centrifugation cell adhesion assay represents a simple, convenient, and standard method for high-throughput characterization of a variety of biomaterial surfaces and conditions.     

Modulating malignant epithelial tumor cell adhesion,migration and mechanics with nanorod surfaces

The failure of tumor stents used for palliative therapy is due in part to the adhesion of tumor cells to the stent surface. It is therefore desirable to develop approaches to weaken the adhesion of malignant tumor cells to surfaces. We have previously developed SiO₂ coated nanorods that resist the adhesion of normal endothelial cells and fibroblasts. The adhesion mechanisms in malignant tumor cells are significantly altered from normal cells; therefore, it is unclear if nanorods can similarly resist tumor cell adhesion. In this study, we show that the morphology of tumor epithelial cells cultured on nanorods is rounded compared to flat surfaces and associated with decreased cellular stiffness and non-muscle myosin II phosphorylation. Tumor cell viability and proliferation was unchanged on nanorods. Adherent cell numbers were significantly decreased while single tumor cell motility was increased on nanorods compared to flat surfaces. Together, these results suggest that nanorods can be used to weaken malignant tumor cell adhesion, and therefore potentially improve tumor stent performance.     

Effect of surface roughness of hydroxyapatite on human bone marrow cell adhesion, proliferation, differentiation and detachment strength

Initial attachment of osteoblast cells and mineralization phenomena are generally enhanced on rough, sandblasted substrata. In the present work the effect of surface roughness of hydroxyapatite (HA) on human bone marrow cell response was investigated. Human bone marrow cells were plated onto HA disc-shaped pellets, prepared from synthetic HA powder. The pellets were sintered and polished with SiC paper 180-, 600- and 1200-grit, resulting in three surface roughness grades. Cell adhesion, proliferation and differentiation (evaluated with the expression of ALP activity) were determined following various incubation periods. Cell detachment strength was determined as the shear stress required to detach a given quantity of the adherent cells from the different substrata, using a rotating disc device that applied a linear range of shear stresses to the cells. The cells attached and grew faster on culture plastic in comparison with HA. No statistically significant differences were observed in the expression of ALP activity on all three HA surfaces and culture plastic. Cell adhesion, proliferation and detachment strength were surface roughness sensitive and increased as the roughness of HA increased. The percentage of the adherent cells decreased in a sigmoidal mode as a function of the applied shear stress. In conclusion, surface roughness of HA generally improved the short- and longer-term response of bone marrow cells in vitro. This behavior could be explained by the selective adsorption of serum proteins.     

Protein adsorption and cell adhesion on cationic,neutral, and anionic 2-methacryloyloxyethyl phosphorylcholine copolymer surfaces

Protein adsorption and cell adhesion on cationic, neutral, and anionic water-soluble 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer surfaces were compared. These model MPC copolymers coated SiO₂ surfaces exhibited comparable surface ζ-potentials of 26.1 mV, near 0 mV, and ?24.2 mV, respectively. X-ray photoelectron spectroscopy analyses indicated the similarities and the differences in the surface composition between the sample surfaces. Atomic force microscopy analyses revealed that the type of the charged moiety did not affect the surface roughness. Static contact angle measurements and dynamic contact angle analyses not only indicated that the surfaces were very hydrophilic in general, but also provided information on the surface mobility and the dominant role of MPC at the surface in aqueous conditions. Comparing with the SiO₂ substrates on which protein seriously adsorbed and cell heavily adhered, three MPC copolymers coated surfaces, despite their different charge properties, exhibited significantly low adsorbed amounts of different proteins having various electrical natures and totally no cell adhesion. This suggested that the incorporation of charged moieties in the MPC copolymers did not significantly inspire both the protein adsorption and cell adhesion. The MPC moieties were predominant at the surface when in contact with aqueous conditions and thereby dominated the bio-adsorptions, while the possible effect from electrostatic interactions would be too small and too limited to influence the overall situation. Therefore, these MPC copolymer surfaces can satisfy those biological applications requiring not only electrical but also non-biofouling properties.     

Control of cell adhesion and detachment on Langmuir-Schaefer surface composed of dodecyl-terminated thermo-responsive polymers

This study used Langmuir-Schaefer (LS) method to produce thermo-responsive poly(N-isopropylacrylamide) (PIPAAm) modified surface. Dodecyl terminated-PIPAAm (PIPAAm-C12) was synthesized by reversible addition-fragmentation chain transfer radical polymerization. PIPAAm-C12 was dropped on an air–water interface and formed Langmuir film by compressing. A surface pressure measurement revealed that PIPAAm-C12 was floated and Langmuir films were formed on the interface. And the Langmuir film was transferred on a hydrophobic substrate to produce PIPAAm-C12 transferred surface (PIPAAm-LS surface). In the results of atomic force microscope, attenuated total reflection Fourier transform infrared spectroscope, and X-ray photoelectron spectroscope measurement, the transference of Langmuir films was demonstrated and densities could be precisely controlled. Ellipsometric measurements of PIPAAm-LS surfaces showed that the thicknesses of the surfaces were less than 10?nm. Cell adhesion and detachment were observed on the PIPAAm-LS surfaces. The amount of adhered cells on all LS surfaces was found to be similar on the control hydrophobic substrate at 37?°C. In regard to cell detachment, adhering cells rapidly detached themselves with higher densities and shorter PIPAAm-C12 molecules. In this method, the effect of densities and molecular weights on cell adhesion and detachment were observed. Our method should be proved novel insights for investigating cell adhesion and detachment on thermo-responsive surfaces.     

The design of polymer microcarrier surfaces for enhanced cell growth

A variety of neutral and cationic polymers based on polyamino acids were prepared and investigated as microcarriers for cell attachment and growth. Among neutral polymer particles including the alkylated poly(gamma-methyl L-glutamate) (PG) particles, in which the hydrophobicity changes as a function of the length of the alkyl groups, and hydroxy terminal PG particles, the PG particle with the longest alkyl chain (PG-C12) demonstrated the highest cell attachment rate and highest rate of cell growth. Moreover, the introduction of hydroxyl groups (PG-OH) led to a deterioration of cell growth. Cell growth on cationic particles having primary amino groups was drastically dependent upon the anion exchange capacity (AEC). A higher AEC for aminated PG microcarriers inhibited cell growth. In contrast, a higher AEC for cross-linked poly( epsilon -lysine) (PL) microcarriers facilitated cell growth. Cell growth on cationic particles clearly showed a good correlation with the pK(a,app) of the microcarriers, but not with their AEC. The particles with low and high pK(a) values possessed toxically acidic and basic pH microenvironments near the surface, respectively. These microenvironments had cytotoxic effects. On the other hand, no correlation between attachment rate constants and high cell growth was observed. The aminated particles, in which pK(a) were controlled at neutral pH, and PG-C12 produced obviously higher cell growth than did a commercially available microcarrier.     

Cell adhesion peptides alter smooth muscle cell adhesion, proliferation, migration, and matrix protein synthesis on modified surfaces and in polymer scaffolds

The effects of cell adhesion peptides (RGDS, KQAGDV, VAPG) on vascular smooth muscle cells grown on modified surfaces and in tissue-engineering scaffolds were examined. Cells were more strongly adhered to surfaces modified with adhesive ligands than to control surfaces (no ligand or a nonadhesive ligand). Cell migration was higher on surfaces with 0.2 nmol/cm(2) of adhesive ligand than on control surfaces, but it was lower on surfaces with 2.0 nmol/cm(2) of adhesive ligand than it was on control surfaces. Further, cell proliferation was lower on adhesive surfaces than it was on control surfaces, and it decreased as the ligand density increased. Similarly, in the peptide-grafted hydrogel scaffolds, cell proliferation was lower in scaffolds containing the adhesive peptides than it was in control scaffolds. After 7 days of culture, more collagen per cell was produced in control scaffolds than in scaffolds containing adhesive peptides. In addition, collagen production decreased in the scaffolds as the ligand concentration increased. While modification of a surface or scaffold material with adhesive ligands initially increases cell attachment, it may be necessary to optimize cell adhesion simultaneously with proliferation, migration, and matrix production.     

Cytoscription: Computer controlled micropositioning of cell adhesion proteins and cells

Methods in Cell Science - Methods are described for the computer controlled micropositioning of both mammalian cells and cell adhesion proteins. Cell adhesion proteins can be micropositioned by...     

Plasma treatment of polyurethane coating for improving endothelial cell growth and adhesion

The advantage of helium plasma treatment in enhancing endothelial cell growth and adhesion on polyurethane film coated on glass substrate is demonstrated with experimental data. Human coronary artery endothelial cell (HCAE) growth and attachment was studied on (1) bare glass substrate, used as control, (2) coated glass, with and without helium plasma treatment and (3) collagen-treated polyurethane-coated glass substrates. The untreated polyurethane film surface was rough (RMS = 690 nm) and highly hydrophobic (contact angle = 90°). Cell growth on the untreated polyurethane surface was poor (cell concentration ≈ 3750/cm²) compared to glass surface (cell concentration ≈ 17 665/cm²). The atmospheric helium plasma treatment of the polyurethane film resulted in oxidation of the surface, a slight increase in roughness (RMS = 735 nm) and a significant drop in hydrophobicity (contact angle = 79°). The critical surface tension (γ _C) of polyurethane film was also increased by 2 dynes/cm due to helium plasma treatment. These changes resulted in enhanced HCAE cell growth in polyurethane film (cell concentration ≈ 16 230/cm²) compared to the untreated polyurethane film. The cell growth was also comparable to cell growth on a glass surface (17 665/cm²) and the collagen-treated polyurethane film surfaces (cell concentration ≈ 21 645/cm²), respectively. Moreover, the strength of cell attachment on a plasma-treated surface (cell retention R = 89%) under laminar flow was significantly higher than that on a glass surface (R = 71%). While the collagen-treated polyurethane surface had the highest number of HCAE cells, the cell adhesion was found to be poor (R = 42%) compared to that of a plasma-treated surface. Thus, the overall performance of the plasma-treated polyurethane film surface on endothelial cell growth was better than other substrates studied here.     

Plasma treatment of polyurethane coating for improving endothelial cell growth and adhesion

The advantage of helium plasma treatment in enhancing endothelial cell growth and adhesion on polyurethane film coated on glass substrate is demonstrated with experimental data. Human coronary artery endothelial cell (HCAE) growth and attachment was studied on (1) bare glass substrate, used as control, (2) coated glass, with and without helium plasma treatment and (3) collagen-treated polyurethane-coated glass substrates. The untreated polyurethane film surface was rough (RMS = 690 nm) and highly hydrophobic (contact angle theta = 90 degrees). Cell growth on the untreated polyurethane surface was poor (cell concentration approximately 3750/cm2) compared to glass surface (cell concentration approximately 17 665/cm2). The atmospheric helium plasma treatment of the polyurethane film resulted in oxidation of the surface, a slight increase in roughness (RMS = 735 nm) and a significant drop in hydrophobicity (contact angle theta = 79 degrees). The critical surface tension (gamma c) of polyurethane film was also increased by 2 dynes/cm due to helium plasma treatment. These changes resulted in enhanced HCAE cell growth in polyurethane film (cell concentration approximately 16 230/cm2) compared to the untreated polyurethane film. The cell growth was also comparable to cell growth on a glass surface (17 665/cm2) and the collagen-treated polyurethane film surfaces (cell concentration approximately 21 645/cm2), respectively. Moreover, the strength of cell attachment on a plasma-treated surface (cell retention R = 89%) under laminar flow was significantly higher than that on a glass surface (R = 71%). While the collagen-treated polyurethane surface had the highest number of HCAE cells, the cell adhesion was found to be poor (R = 42%) compared to that of a plasma-treated surface. Thus, the overall performance of the plasma-treated polyurethane film surface on endothelial cell growth was better than other substrates studied here.