Density Of Adherent Cells Research Articles

Interface of Phospholipid Polymer Grafting Layers to Analyze Functions of Immobilized Oligopeptides Involved in Cell Adhesion.

The aim of this study was to design a material surface for use in the analysis of the behavior of biomolecules at the interface of direct cell contact. A superhydrophilic surface was prepared with poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), which was grafted onto a substrate with controlled polymer chain density. An arginine-glycine-aspartic acid (RGD) peptide was immobilized at the surface of the polymer graft surface (PMPC-RGD surface). Initial adhesion of the cells to this substrate was observed. The PMPC-RGD surface could enable cell adhesion only through RGD peptide-integrin interactions. The density and movability of the RGD peptide at the terminal of the graft PMPC chain and the orientation of the RGD peptide affected the density of adherent cells. Thus, the PMPC graft surface may be a good candidate for a new platform with the ability to immobilize biomolecules to a defined position and enable accurate analysis of their effects on cells.

Enhancement of corrosion resistance and biocompatibility of Mg-Nd-Zn-Zr alloy achieved with phosphate coating for vascular stent application

We have developed a convenient chemical conversion method of preparing a Mg3(PO4)2 coating with a lamellar structure of about 1–5μm width on Mg-Nd-Zn-Zr (denoted as JDBM) alloy, aiming to improve the corrosion resistance and biological response. The protective effect of the phosphate coating on JDBM alloy leads to an increase in corrosion resistance of about 25% from 0.337±0.021 to 0.253±0.047mmy−1. A systematic study on in vitro cytocompatibility test shows that the phosphate coated JDBM has minimal negative effect on human umbilical vein endothelial cells (HUVECs) viability, growth and proliferation, and the coating also presents a favorable affinity for HUVECs by supporting higher adherent cell density and sufficient cell spreading. In addition, the investigation of in vitro hemocompatibility confirms reduced hemolysis ratio and excellent anti-platelet adhesion property of the phosphate coated JDBM. Furthermore, this study also presents our more recent efforts to develop an in-depth understanding of the mechanism which is involving anti-platelet adhesion on the surface of biodegradable Mg substrate.

Author Correction: Photoinduced self-initiated graft polymerization of methacrylate monomers on poly(ether ether ketone) substrates and surface parameters for controlling cell adhesion

One of the super engineering plastics, poly(ether ether ketone) (PEEK), was functionalized by photoinduced self-initiated graft polymerization of various methacrylate monomers. Anionic, cationic, zwitterionic, nonionic hydrophilic, and nonionic hydrophobic polymer layers were formed onto PEEK substrates. Physical and chemical surface characterizations of the resultant polymer-grafted PEEK substrates were performed through measurements of their surface free energies and ζ potentials. The values of these parameters varied in the ranges of 39–71 mJ/m2 and −69 to 46 mV, respectively. These parameters reflected the chemical structures of the grafted polymers. To understand the effects of these surface parameters on cell adhesion behavior at the substrate surface, the amount of fibronectin adsorbed on the plasma-contacting surface and the density of fibroblast cells adhered to the surface were determined. The adherent cell density showed a good linear correlation with the amount of fibronectin adsorbed on the plasma-contacting surface. The polymer surface with zero ζ potential showed a lower adsorbed fibronectin density. Both anionic and cationic polymer layers had increased cell adhesion compared with that on the original PEEK substrate, whereas the zwitterionic polymer layers significantly prevented cell adhesion. In conclusion, grafting zwitterionic polymers onto a PEEK substrate is anticipated to be useful in the development of a biomedical PEEK substrate. To obtain functionalization of super-engineering plastic, poly(ether ether ketone) (PEEK) substrate, photoinduced self-initiated graft polymerization of several kinds of functional methacrylate was carried out. Graft polymerization proceeded well, and 50–250 nm in thickness graft polymer layers were generated on the PEEK substrate. Surface characteristics such as surface free energy and surface ζ potential on the polymer-grafted PEEK substrate were corresponded to the chemical structure of the graft polymers. Cell attachment on the polymer-grafted PEEK substrate was examined. The surface ζ potentials of the polymer-grafted PEEK substrates governed the cell adhesion density.

Label-free self-referenced sensing of living cells by terahertz metamaterial-based reflection spectroscopy.

Terahertz (THz) metamaterial-based reflection spectroscopy is proposed for label-free sensing of living cells by a self-referenced method. When sensing the living Madin-Darby canine kidney cell monolayer and phosphate buffered saline solution, self-referenced signals showed significant differences in peak intensity because of inherent discrepancy in the imaginary part of their complex refractive indices, as confirmed by 3D-FDTD simulations. The resonance peak intensity was unaffected by cell monolayer thickness variation, demonstrating feasibility for sensing various cells. Simulations and experiments showed that saponin-induced changes in cell permeability could be monitored in real-time. The self-referenced signal was linearly dependent on the adherent cell density, illustrating a label-free in situ THz metamaterial-based cell sensor.

Cell Density Detector Based on Light Beam Focusing.

Although the lab-on-a-chip system has been successfully applied in a wide variety of fields, the goal of achieving a cell counter with simple operation, low cost, and high accuracy still attracts continuous research efforts. In this paper, the authors explore a cell counter based on light beam focusing to measure the density of adherent cells. In this sensor, the light emitted from the optical fibers is collimated by the collimating lens formed in polydimethylsiloxane (PDMS). The uniformly attached adherent cells act as a convex lens, focusing the collimated light propagated through them. The intensity of the focused light indicates the density of the adherent cells. For Hela cells, a detection limit of 8.3 × 104 cells/mL with a detection range from 0.1 × 106 cells/mL to 1.0 × 106 cells/mL is achieved. This sensor is particularly useful for drug screening, cell pathology analysis, and cancer pre-diagnosis.

Novel approach for the prediction of cell densities and viability in standardized translucent cell culture biochips with near infrared spectroscopy.

Nearinfraredspectroscopy is a rapid and nondestructive method for compositional analysis of biological material. The technology is widely used within bioreactors and possesses potential as a standardized method for quality control in miniaturized microfluidic cell culture systems. Here, we established a method for quantification of cell density and viability of adherent HepaRG cells cultured in a translucent, miniaturized cell culture biochip. The newly developed statistical models for interpretation of nearinfraredspectroscopy from biochips are the basis for a novel method of fast, continuous, and contact-free analysis of cell viability and real-time monitoring of cell growth. The technique thus paves the way for a robust and reliable high-throughput analysis of biochip-embedded cell cultures.

Improvement of biofouling resistance on bacterial cellulose membranes

Bacterial cellulose possesses excellent biocompatibility and mechanical strength that show great potentials for biomaterial applications. In this study, the surface modifications of bacterial cellulose (BC) membranes were facilitated using either simple coating or chemical grafting methods. The surface coating method is to simply immobilize BC membranes with poly(ethylene glycol) (PEG) solutions of concentration from 1 to 10%, followed by post-treatment with argon (Ar) plasma. The chemical method involved grafting mPEG (monofunctional methyl ether PEG) on BCs. The outcomes of surface modifications were characterized by surface chemical compositions (electron spectroscopy for chemical analysis (ESCA), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), thermogravimetry analysis (TGA), and surface morphology (atomic force microscopy (AFM) and scanning electron microscopy (SEM)). The effects of resistance to biofouling were verified by quantifying the adsorption of proteins and mammalian cells. The results showed that the PEG coating on BCs improved the resistance to cell adhesion by more than 30%. On the other hand, the specific chemical grafting resulted in a particularly high resistance to biofouling that the density of adherent cells reduced by more than 70% when compared to that on pristine BC. We have demonstrated that the two proposed methods were effective for the preparation of bioinert BC membranes with great potentials for applications in biomaterials and tissue engineering.

Use of a special bioreactor for the cultivation of a new flexible polyurethane scaffold for aortic valve tissue engineering

BackgroundTissue engineering represents a promising new method for treating heart valve diseases. The aim of this study was evaluate the importance of conditioning procedures of tissue engineered polyurethane heart valve prostheses by the comparison of static and dynamic cultivation methods.MethodsHuman vascular endothelial cells (ECs) and fibroblasts (FBs) were obtained from saphenous vein segments. Polyurethane scaffolds (n = 10) were primarily seeded with FBs and subsequently with ECs, followed by different cultivation methods of cell layers (A: static, B: dynamic). Group A was statically cultivated for 6 days. Group B was exposed to low flow conditions (t1= 3 days at 750 ml/min, t2= 2 days at 1100 ml/min) in a newly developed conditioning bioreactor. Samples were taken after static and dynamic cultivation and were analyzed by scanning electron microscopy (SEM), immunohistochemistry (IHC), and real time polymerase chain reaction (RT-PCR).ResultsSEM results showed a high density of adherent cells on the surface valves from both groups. However, better cell distribution and cell behavior was detected in Group B. IHC staining against CD31 and TE-7 revealed a positive reaction in both groups. Higher expression of extracellular matrix (ICAM, Collagen IV) was observed in Group B. RT- PCR demonstrated a higher expression of inflammatory Cytokines in Group B.ConclusionWhile conventional cultivation method can be used for the development of tissue engineered heart valves. Better results can be obtained by performing a conditioning step that may improve the tolerance of cells to shear stress. The novel pulsatile bioreactor offers an adequate tool for in vitro improvement of mechanical properties of tissue engineered cardiovascular prostheses.

Impact of microcarrier coverage on using permittivity for on-line monitoring high adherent Vero cell densities in perfusion bioreactors

The paper presents the interest of on-line permittivity monitoring to estimate the density of Vero cells growing on microcarriers (MCs), even when high cell densities were reached in perfusion bioreactors (4.5×106cellsml−1). Cultures were performed with various MCs concentrations in a reactor equipped with a settling tube. A linear correlation between on-line permittivity and off-line volumetric cell concentration was observed provided that MCs are not fully covered by cells. High permittivities such as 250pFcm−1 could be measured without signal saturation of the Fogale Biomass system®. The correlation was no longer linear when cell density per carrier exceeded 100% cell confluency corresponding to 150cellsMC−1 (0.15×106cellscm−2). This behaviour was attributed to the decrease of cell volume when cells saturated MCs surface. It mainly happened when low MCs concentration and continuous medium renewing were used. Therefore, permittivity sensor can be considered as a reliable tool to monitor on-line adherent cell densities not exceeding total cell confluency. Moreover, it could be useful to detect when cell confluency occurs.

In VitroEndothelialization of Electrospun Terpolymer Scaffolds: Evaluation of Scaffold Type and Cell Source

A family of methacrylic terpolymer biomaterials was electrospun into three-dimensional scaffolds. The glass transition temperature of the polymer correlates with the morphology of the resulting scaffold. Glassy materials produce scaffolds with discrete fibers and large pore areas (1531±1365 μm(2)), while rubbery materials produce scaffolds with fused fibers and smaller pore areas (154±110 μm(2)). Three different endothelial-like cell populations were seeded onto these scaffolds under static conditions: human umbilical vein endothelial cells (HUVECs), adult human peripheral blood-derived outgrowth endothelial cells, and umbilical cord blood-derived human blood outgrowth endothelial cells. Cellular behavior depended on both cell type and scaffold topography. Specifically, cord blood-derived outgrowth endothelial cells showed more robust adhesion and growth on all scaffolds in comparison to other cell types as measured by the density of adherent cells, the number of proliferative cells, and the enzymatic activity of the adherent cells. Peripheral blood-derived outgrowth cells exhibited less ability to inhabit the terpolymer interfaces in comparison to their cord blood-derived counterparts. HUVECs also exhibited less of a capacity to colonize the terpolymer interfaces in comparison to the cord blood-derived cells. However, the mature endothelial cells did show scaffold-dependent behavior. Specifically, we observed an increase in their ability to populate the low-porosity scaffolds. All cells maintained an endothelial phenotype after 1 week of culture on the electrospun scaffolds.

Real-time monitoring of adherent Vero cell density and apoptosis in bioreactor processes

This study proposes an easy to use in situ device, based on multi-frequency permittivity measurements, to monitor the growth and death of attached Vero cells cultivated on microporous microcarriers, without any cell sampling. Vero cell densities were on-line quantified up to 10(6)cellmL(-1). Some parameters which could potentially impact Vero cell morphological and physiological states were assessed through different culture operating conditions, such as media formulation or medium feed-harvest during cell growth phase. A new method of in situ cell death detection with dielectric spectroscopy was also successfully implemented. Thus, through permittivity frequency scanning, major rises of the apoptotic cell population in bioreactor cultures were detected by monitoring the characteristic frequency of the cell population, f(c), which is one of the culture dielectric parameters. Both cell density quantification and cell apoptosis detection are strategic information in cell-based production processes as they are involved in major events of the process, such as scale-up or choice of the viral infection conditions. This new application of dielectric spectroscopy to adherent cell culture processes makes it a very promising tool for risk-mitigation strategy in industrial processes. Therefore, our results contribute to the development of Process Analytical Technology in cell-based industrial processes.

Cell adhesion on chiral surface: The role of protein adsorption

Chirality is one of the basic, unique, and most appealing features of biological molecules; however, many intriguing chiral phenomena in biological world remains insufficiently revealed yet. In this research, we fabricated chiral surfaces by assembling natural chiral amino acids-cysteine of opposite configurations (D- and L-) onto gold surfaces, respectively, and investigated the adhesion of the L929 fibroblast on them. No significant differences were observed in the density of adherent cells under serum-free culture condition; while in serum-containing condition, significantly more cells adhered on the L-Cys assembled surfaces. This phenomenon suggested that serum protein might play an important role in mediating the selective adhesion of cells on chiral surfaces. Hence, we adopted both radiolabeling and surface plasmon resonance (SPR) techniques to monitor protein adsorption onto the above surfaces. The results evidently showed more proteins adsorbed onto surfaces assembled with L-Cys. We propose that the difference in protein adsorption on chiral surfaces as demonstrated in this paper might not only shed light on the ensuing investigation of bio-related chirality phenomena, but also provide a novel strategy for the rational design and fabrication of novel biomaterials and bio-related devices based on chiral effects.

Multi-scale engineering for neuronal cell growth and differentiation

In this paper we investigate the role of micropatterning and molecular coating for cell culture and differentiation of neuronal cells (Neuro2a cell line) on a polydimethylsiloxane substrate. We investigate arrays of micrometric grooves (line and space) capable to guide neurite along their axis. We demonstrate that pattern dimensions play a major role due to the deformation of the cell occasioned by grooves narrower than typical cell dimension. A technological compromise for optimizing cell density, differentiation rate and neurite alignment has been obtained for 20μm wide grooves which is a dimension comparable with the average cell dimension. This topographical engineered pattern combined with double-wall carbon nanotubes coating enabled us to obtain adherent cell densities in the order of 104cells/cm2 and a differentiation rate close to 100%.

The foreign body reaction in T‐cell‐deficient mice

The role(s) of T lymphocytes in the foreign body response has not been thoroughly elucidated. Lymphocytes are known to augment macrophage adhesion and fusion in vitro. Furthermore, T lymphocytes are a possible source of the cytokines, IL-4 and IL-13, which induce macrophage fusion. In this study, we used BALB/c mice and BALB/c (nu/nu) nude mice to investigate foreign body giant cell (FBGC) formation in a T-cell-deficient setting. Mice were implanted with Elasthane 80A (PEU), silicone rubber (SR), or poly(ethylene terephthalate) (PET) for 7, 14, or 21 days using the cage implant system. Exudate cells and IL-4 and IL-13 levels in exudate supernatants were analyzed by flow cytometry and a multiplex immunoassay, respectively, at Days 7, 14, and 21. Macrophage adhesion and fusion on material surfaces were analyzed using optical microscopy. T-cell-deficient mice had lower total leukocyte concentrations at the biomaterial implant site at all time points. Adherent cell density was comparable between normal and T-cell-deficient mice except in the PEU group at Day 21. However, percent fusion, average nuclei per FBGC, and FBGC morphology were comparable between normal and T-cell-deficient mice. IL-4 was not detected in any sample, but IL-13 levels were also comparable between normal and T-cell-deficient mice indicating Th2-polarized T-cells are not the sole source of this cytokine. We have shown that there are pathways that do not require thymus-matured T lymphocytes, which lead to a normal foreign body response to biomaterials in a murine model.

Using continuous porous silicon gradients to study the influence of surface topography on the behaviour of neuroblastoma cells

The effects of surface topography on cell behaviour are the subject of intense research in cell biology. These effects have so far only been studied using substrate surfaces of discretely different topography. In this paper, we present a new approach to characterise cell growth on porous silicon gradients displaying pore sizes from several thousands to a few nanometers. This widely applicable format has the potential to significantly reduce sample numbers and hence analysis time and cost. Our gradient format was applied here to the culture of neuroblastoma cells in order to determine the effects of topography on cell growth parameters. Cell viability, morphology, length and area were characterised by fluorescence and scanning electron microscopy. We observed a dramatic influence of changes in surface topography on the density and morphology of adherent neuroblastoma cells. For example, pore size regimes where cell attachment is strongly discouraged were identified providing cues for the design of low-fouling surfaces. On pore size regimes more conducive to cell attachment, lateral cell–cell interactions crosslinked the cell layer to the substratum surface, while direct substrate–cell interactions were scarce. Finally, our study revealed that cells were sensitive to nanoscale surface topography with feature sizes of < 20 nm.

Phenotypic dichotomies in the foreign body reaction

To better understand the relationship between macrophage/foreign body giant cell adhesion and activation on surface-modified biomaterials, quantitative assessment of adherent cell density (cells per mm 2) and cytokine production (pgs per mL) were determined by ELISA. Further analysis to identify cellular activation was carried out by normalizing the cytokine concentration data to provide a measure of cellular activation. This method of analysis demonstrated that hydrophobic surfaces provided statistically significantly greater adherent cell densities than hydrophilic/neutral surfaces. However, when cell activation parameters were determined by normalization to the adherent cell density, the hydrophilic/neutral surfaces demonstrated statistically significantly greater levels of activation and production of IL-10, IL-1 β, IL-6, IL-8, and MIP-1 β. With increasing time, production of the anti-inflammatory cytokine IL-10 increased, whereas IL-1 β, IL-6, and IL-8 decreased and MIP-1 β was relatively constant over the culture time period. This observed dichotomy or disparity between adhesion and activation may be related to surface-induced adherent cell apoptosis. Further evaluation of macrophage activation on biomaterial surfaces indicated that an apparent phenotypic switch in macrophage phenotype occurred over the course of the in vitro culture. Analysis of cytokine/chemokine profiles with surface-modified biomaterials revealed similarities between the classically activated macrophages and the biomaterial-adherent macrophages early (day 3) in culture, while at later timepoints the biomaterial-adherent macrophages produced profiles similar to alternatively activated macrophages. Classically activated macrophages are those commonly activated by lipopolysaccharide (LPS) or interferon- γ (IFN- γ) and alternatively activated macrophages are those activated by IL-4/IL-13 or IL-10. Surface modification of biomaterials offer an opportunity to control cellular activation and cytokine profiles in the phenotypic switch, and may provide a means by which macrophages can be induced to regulate particular secretory proteins that direct inflammation, the foreign body reaction, wound healing, and ultimately biocompatibility.

Mechanics of Cellular Adhesion to Artificial Artery Templates

We are using polymer templates to grow artificial artery grafts in vivo for the replacement of diseased blood vessels. We have previously shown that adhesion of macrophages to the template starts the graft formation. We present a study of the mechanics of macrophage adhesion to these templates on a single cell and single bond level with optical tweezers. For whole cells, in vitro cell adhesion densities decreased significantly from polymer templates polyethylene to silicone to Tygon (167, 135, and 65cells/mm2). These cell densities were correlated with the graft formation success rate (50%, 25%, and 0%). Single-bond rupture forces at a loading rate of 450 pN/s were quantified by adhesion of trapped 2-μm spheres to macrophages. Rupture force distributions were dominated by nonspecific adhesion (forces <40 pN). On polystyrene, preadsorption of fibronectin or presence of serum proteins in the cell medium significantly enhanced adhesion strength from a mean rupture force of 20 pN to 28 pN or 33 pN, respectively. The enhancement of adhesion by fibronectin and serum is additive (mean rupture force of 43 pN). The fraction of specific binding forces in the presence of serum was similar for polystyrene and polymethyl-methacrylate, but specific binding forces were not observed for silica. Again, we found correlation to in vivo experiments, where the density of adherent cells is higher on polystyrene than on silica templates, and can be further enhanced by fibronectin adsorption. These findings show that in vitro adhesion testing can be used for template optimization and to substitute for in-vivo experiments.

Macrophage matrix metalloproteinase-2/-9 gene and protein expression following adhesion to ECM-derived multifunctional matrices via integrin complexation

Macrophages are commonly observed at the biomaterial-tissue interface and interact with the extracellular matrix (ECM) mainly by integrin receptors to play a critical role in ECM turnover by secreting matrix metalloproteinases (MMPs). To investigate beta1 and beta3 containing integrin-mediated adhesion and subsequent MMP-2/-9 protein and gene expression in human blood-derived monocytes, biofunctional peptides immobilized onto flexible polyethylene glycol (PEG) arms were grafted onto a gelatin-based interpenetrating network (IPN). Adherent monocyte density was dramatically greater in the presence of RGD immobilized onto flexible PEG arms of the gelatin-based IPN. Pretreatment of monocytes with either anti-integrin beta1 or beta3 led to a significant decrease in adherent cell density on RGD-PEG-grafted IPNs. MMP-2 and MMP-9 protein and MMP-9 mRNA expression increased in the presence of IPNs initially, independent of ligand identity. Anti-integrin beta1 or beta3 antibody pretreatment of monocytes led to a general decrease in MMP-2/-9 protein expression. These results demonstrate the importance of beta1 and beta3 containing integrins in mediating monocyte adhesion onto RGD immobilized onto flexible PEG arms of the IPN. The results also reveal that MMP-2/-9 protein and gene expression is influenced by the presence of gelatin and not the ligands immobilized on the PEG arms of the IPN.

Antioxidant inhibition of poly(carbonate urethane) in vivo biodegradation

This study compared the effect of an antioxidant on the in vivo biodegradation of a poly(carbonate urethane) (PCU) and a poly(ether urethane) (PEU). Unstrained PEU and PCU films with and without Santowhite were implanted subcutaneously into 3-month-old Sprague-Dawley rats for 3, 6, and 12 months. Characterization of unstabilized PEU and PCU with ATR-FTIR and SEM showed soft-segment and hard-segment degradation consistent with previous studies. In particular, evidence of chain scission and crosslinking of the surface was present in the ATR-FTIR spectra of explanted specimens. Addition of 2.2 wt % antioxidant inhibited the in vivo degradation of both PCU and PEU. Although the antioxidant probably improved polyurethane biostability by decreasing the susceptibility of the polymer to degradation, modulation of the cellular response to prevent the release of degradative agents was also possible. To differentiate the effects, the foreign-body response was investigated with the use of a standard cage implant protocol. Polyurethane films were implanted in wire mesh cages subcutaneously in rats for 4, 7, and 21 days. There were no statistical differences among materials in the inflammatory exudate cell counts, adherent cell densities, or percent fusion of macrophages into foreign-body giant cells (FBGCs). Therefore, it was concluded that the antioxidant inhibited degradation by capturing oxygen radicals that would otherwise cause polyurethane chain scission and crosslinking.

Inflammatory cell recruitment and adhesion to methyl‐terminated self‐assembled monolayers: Effect of implantation time

The contribution of methyl groups in implant-triggered inflammation was investigated in vivo using self-assembled monolayers (SAMs) of alkanethiols on gold. The CH(3)-coated implants were inserted in an air-pouch cavity induced in BALB/c mice. The in situ inflammatory response was monitored 24, 48, and 72 hours later. Inflammatory cells recovered from the air pouches were counted and observed by light microscopy. The cellularity of the implant surfaces was defined by scanning electron microscopy (SEM). In comparison with gold implants, the CH(3)-coated SAMs recruited a significantly higher number of inflammatory cells. Polymorphonuclear leukocytes (PMN) were more numerous than mononuclear cells (Mo) in the exudates recovered from the air pouches with CH(3)-coated SAMs. The opposite PMN/Mo proportion was observed in air pouches of the two control groups (mice receiving gold implants or sham-operated animals). A low density of adherent cells was seen on CH(3)-coated implants, with no significant quantitative differences during the time course of the study. In contrast, the gold-coated surfaces were covered with numerous cells during all of the 3 days of the inflammation. In conclusion, implants with CH(3) surfaces are likely to induce PMN-dominated local acute inflammation but these surfaces are not associated with a significant adherence of leukocytes to the implant.