Gradient immobilization of a cell adhesion RGD peptide on thermal responsive surface for regulating cell adhesion and detachment

Using surface initiated atomic transfer radical polymerization (ATRP) and an injection method, a poly(N-isopropylacrylamide)-b-poly(acrylic acid)-g-RGD (PNIPAAm-b-PAA-g-RGD) gradient surface was prepared. First, a thermoresponsive surface with a constant thickness of PNIPAAm was fabricated, onto which the AA monomers were block copolymerized using the PNIPAAm macromolecules as initiators. During this process, a continuous injection method was employed to yield a molecular weight gradient of PAA on the underlying uniform PNIPAAm layer. RGD peptide was finally covalently immobilized onto the PAA gradient by carbodiimide chemistry. In vitro culture of HepG2 cells showed that immobilization of the RGD peptide could accelerate cell attachment, while the thermoresponsive layer beneath could effectively release the cells by simply lowering temperature. Thus, the PNIPAAm-b-PAA-g-RGD gradient surface, combining the thermal response with cell affinity properties, can well regulate the cell adhesion and detachment, which may thus be useful for investigation of cell-substrate interactions with a smaller number of samples.     

Fabrication of thermoresponsive polymer gradients for study of cell adhesion and detachment

A poly(N-isopropylacrylamide) (PNIPAAm) gradient covalently anchored on a silicon substrate with a linear variation of thickness was fabricated by continuous injection of the reaction mixture (NIPAAm, CuBr and its ligand, methanol, and water) into a glass chamber containing a silicon wafer, whose surface had been homogeneously immobilized with bromoisobutyryl bromide (BIBB). Because of the good control of the surface-initiated atom transfer radical polymerization (SI-ATRP) technique, the thickness of the PNIPAAm brushes was linearly proportional to the polymerization time. As a result, the gradient length and sharpness could be easily controlled by the experimental parameters such as the polymerization time and the injection rate. The as-prepared PNIPAAm gradients were characterized by ellipsometry, water contact angle, and atom force microscopy to detect their alteration of the thickness, surface wettability, and morphology, confirming the gradient structure. X-ray photoelectron spectroscopy confirmed the surface composition of the PNIPAAm. In vitro culture of HepG2 cells was implemented on the gradient surfaces, revealing that the cells could adhere at 37 degrees C and could be detached at 24 degrees C when the gradient thickness was in the range of 20-45 nm. The work thus develops a method to fabricate the stable gradient surface with better quality control, and clarifies in a facile manner the appropriate thickness of the PNIPAAm brushes in terms of cell adhesion and detachment.     

Thermoresponsive copolymer nanofilms for controlling cell adhesion, growth, and detachment

This study reports the development and use of a novel thermoresponsive polymeric nanofilm for controlling cell adhesion and growth at 37 °C, and then cell detachment for cell recovery by subsequent temperature drop to the ambient temperature, without enzymatic cleavage or mechanical scraping. A copolymer, poly(N-isopropylacrylamide-co-hydroxypropyl methacrylate-co-3-(trimethoxysilyl)propyl methacrylate) (abbreviated PNIPAAm copolymer), was synthesized by free radical polymerization. The thermoresponses of the copolymer in aqueous solution were demonstrated by dynamic light scattering (DLS) through detecting the sensitive changes of copolymer aggregation against temperature. The DLS measurements revealed the lower critical solution temperature (LCST) at approximately 30 °C. The PNIPAAm film stability and robustness was provided through silyl cross-linking within the film and with the hydroxyl groups on the substrate surface. Film thickness, stability, and reversibility with respect to temperature switches were examined by spectroscopic ellipsometry (SE), atomic force microscopy (AFM), and contact angle measurements. The results confirmed the high extent of thermosensitivity and structural restoration based on the alterations of film thickness and surface wettability. The effective control of adhesion, growth, and detachment of HeLa and HEK293 cells demonstrated the physical controllability and cellular compatibility of the copolymer nanofilms. These PNIPAAm copolymer nanofilms could open up a convenient interfacial mediation for cell film production and cell expansion by nonenzymatic and nonmechanical cell recovery.     

Temperature‐sensitive chitosan membranes as a substrate for cell adhesion and cell sheet detachment

A series of temperature‐sensitive poly(CSA‐co‐NIPAAm) membranes that were suitable for cell culture and confluent cell sheets detachment were prepared. The membranes with thermo‐responsive surface properties were synthesized by the copolymerization of acrylic acid‐derivatized chitosan (CSA) and N‐isopropylacrylamide (NIPAAm) in aqueous solution. Characterization of the membranes were carried out by means of the Fourier transform infrared (FTIR), X‐ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM), and water contact‐angle (WCA) measurements. The adhesion and detachment of mouse fibroblast (L929) cells on these membranes have been investigated. The study showed that poly(CSA‐co‐NIPAAm) membranes could not only enhance fibroblasts attachment but also harvest confluent cell sheets by simply lowering the temperature. Furthermore, the detached cells retained high viability and could proliferate again after transferred to a new culture surface. Copyright © 2011 John Wiley & Sons, Ltd.     

Differentiation-on-a-chip: a microfluidic platform for long-term cell culture studies

Here we demonstrate a microfluidic perfusion system suitable for a long-term (>2 week) culture of muscle cells spanning the whole process of differentiation from myoblasts to myotubes. Cell-adhesive surface microdomains alternating with a robust cell-repellent coating mimic in vivo spatial cues for muscle cell assembly and allow for confining the fusion of myoblasts into aligned, isolated multinucleated myotubes. The microfluidic system provides accurate control of the perfusion rates and biochemical composition of the environment surrounding the cells. Comparing muscle cell-specific differentiation markers and the timing of fusion, we observed no differences in differentiation between microfluidic and traditional cultures. All differentiation assays were fully microfluidic, i.e. they were performed by sequentially changing the fluids in the micro-channels. By delivering fluorescent markers using heterogeneous laminar flows, it was possible to confine a membrane receptor labeling assay to a region smaller than a myotube. Our method can serve as an improved in vitro model for studying muscle cell differentiation and for characterizing extracellular molecules and mechanisms involved in neuromuscular differentiation.     

Lanthanide-containing polymer nanoparticles for biological tagging applications: nonspecific endocytosis and cell adhesion

We describe the synthesis and characterization of element-encoded polystyrene nanoparticles with diameters on the order of 100 nm and a narrow size distribution. Individual particles contain ca. 10(3) chelated lanthanide ions, of either a single element or a mixture of elements. These particles were effectively internalized by nonspecific endocytosis into three cell lines associated with human leukemia. Using an assay based upon ICP-MS detection, we could monitor quantitatively cell adhesion induced by cell differentiation of THP-1 cells in response to phorbol ester stimulation (PMA) in single cell type or mixed cultures.     

Circular, nanostructured and biofunctionalized hydrogel microchannels for dynamic cell adhesion studies

We report on a method to fabricate biofunctionalized polyethylene glycol hydrogel microchannels with adjustable circular cross-sections. The inner channel surfaces are decorated with Au-nanoparticle arrays of tunable density. These Au-nanoparticles are functionalized with biomolecules whereas the hydrogel material provides an inert and biocompatible background. This technology provides control over flow conditions, channel curvature and biomolecule density on the channel surface. It can be applied for biophysical studies of cell-surface interactions mimicking, for example, leukocyte interactions with the endothelial lining in small vessels.     

Ultrathin poly(N-isopropylacrylamide) grafted layer on polystyrene surfaces for cell adhesion/detachment control

We investigated physicochemical properties of two types of poly(N-isopropylacrylamide) (PIPAAm)-grafted tissue culture polystyrene (TCPS) surfaces, to elucidate the influential factors for thermally regulated cell adhesion and detachment to PIPAAm-grafted surfaces. The two types of PIPAAm-grafted surfaces were prepared by the electron beam polymerization method. Attenuated total reflection Fourier transform infrared spectroscopy revealed that amounts of the grafted polymers were 1.4 +/- 0.1 microg/cm2 for PIPAAm-1.4 and 2.9 +/- 0.1 microg/cm2 for PIPAAm-2.9. Both PIPAAm-grafted surfaces showed hydrophobic/hydrophilic property alterations in response to temperature. However, PIPAAm-1.4 surfaces were more hydrophobic (cos theta = 0.21 at 37 degrees C and cos theta = 0.35 at 20 degrees C) than PIPAAm-2.9 (cos theta = 0.42 at 37 degrees C and cos theta = 0.50 at 20 degrees C) both above and below the PIPAAm's transition temperature. Thicknesses of the grafted PIPAAm layers were estimated to be 15.5 +/- 7.2 nm for PIPAAm-1.4 and 29.5 +/- 8.4 nm for PIPAAm-2.9, by the use of UV excimer laser and atomic force microscope. Bovine carotid artery endothelial cells (ECs) adhere to the surfaces of PIPAAm-1.4 and proliferate to form confluent cell monolayers. The cell monolayers were harvested as single cell sheets by temperature decrease from 37 to 20 degrees C. On the contrary, ECs did not adhere to the surfaces of PIPAAm-2.9. This phenomenon was correlated with an adsorption of cell adhesion protein, fibronectin, onto surfaces ofPIPAAm-1.4 and -2.9. In the case of nano-ordered thin grafted surfaces, the surface chain mobility is strongly influenced by the thickness of PIPAAm grafted layers because dehydration of PIPAAm chains should be enhanced by the hydrophobic TCPS surfaces. PIPAAm graft amounts, that is, thickness of the PIPAAm grafted layers, play a crucial role in temperature-induced hydrophilic/hydrophobic property alterations and cell adhesion/detachment behavior.     

Pyridine-Si-xanthene: A novel near-infrared fluorescent platform for biological imaging

A novel near-infrared fluorescent platform with intrinsic lysosome-targeting was reported capable of detecting cysteine in living cells and in vivo.     

Chemoenzymatic synthesis of sialooligosaccharides on arrays for studies of cell surface adhesion

Sialooligosaccharides were generated by direct enzymatic glycosylation on arrays and the resulting surfaces were suitable for the study of carbohydrate-specific cell adhesion.     

A digital microfluidic platform for primary cell culture and analysis

Digital microfluidics (DMF) is a technology that facilitates electrostatic manipulation of discrete nano- and micro-litre droplets across an array of electrodes, which provides the advantages of single sample addressability, automation, and parallelization. There has been considerable interest in recent years in using DMF for cell culture and analysis, but previous studies have used immortalized cell lines. We report here the first digital microfluidic method for primary cell culture and analysis. A new mode of "upside-down" cell culture was implemented by patterning the top plate of a device using a fluorocarbon liftoff technique. This method was useful for culturing three different primary cell types for up to one week, as well as implementing a fixation, permeabilization, and staining procedure for F-actin and nuclei. A multistep assay for monocyte adhesion to endothelial cells (ECs) was performed to evaluate functionality in DMF-cultured primary cells and to demonstrate co-culture using a DMF platform. Monocytes were observed to adhere in significantly greater numbers to ECs exposed to tumor necrosis factor (TNF)-α than those that were not, confirming that ECs cultured in this format maintain in vivo-like properties. The ability to manipulate, maintain, and assay primary cells demonstrates a useful application for DMF in studies involving precious samples of cells from small animals or human patients.     

A robust small-molecule microarray platform for screening cell lysates

Herein we report the expanded functional group compatibility of small-molecule microarrays to include immobilization of primary alcohols, secondary alcohols, phenols, carboxylic acids, hydroxamic acids, thiols, and amines on a single slide surface. Small-molecule "diversity microarrays" containing nearly 10,000 known bioactive small molecules, natural products, and small molecules originating from several diversity-oriented syntheses were produced by using an isocyanate-mediated covalent capture strategy. Selected printed bioactive compounds were detected with antibodies against compounds of interest. The new surface of the diversity microarrays is highly compatible with approaches involving cellular lysates. This feature has enabled a robust, optimized screening methodology using cellular lysates, allowing the detection of specific interactions with a broad range of binding affinity by using epitope-tagged or chimeric fluorescent proteins without prior purification. We believe that this expanded research capability has considerable promise in biology and medicine.     

A new platform for NCN dimethylamino pincer complexes: Synthesis and structural studies

The reaction of 2,6-(2-{Me₂NCH₂}C₆H₄)₂C₆H₃I (2) with Pd₂(dba)₃ produced the NCN diamine pincer complex [2,6-(2-Me₂{NCH₂}C₆H₄)₂C₆H₃PdI] (3) by an oxidative addition route. The structural analysis of ligand precursor 2 revealed a syn-conformation in the solid state. Single crystal X-ray analysis of complex 3 revealed a conventional square planar geometry about the palladium center and a global C₂ symmetric structure. Variable temperature and concentration NMR spectroscopic studies of complex 3 suggest an equilibrium between 3 and the dinuclear species [{2,6-(2-{Me₂NCH₂}C₆H₄)₂C₆H₃Pd}₂μ²-I]I in CDCl₃ solution. An unusual carbonate complex [{2,6-(2-{Me₂NCH₂}C₆H₄)₂C₆H₃Pd}₃μ³-CO₃]I₃ (4) was also structurally characterized as a minor product during synthesis of 3.     

Adhesion and detachment of biological cellsin vitro

Adhesion between biological cells and various surfaces is explained in terms of various models, including coagulation at primary or secondary minima of free energy, macromolecular bridges or matrices, and specialized structures at the surfaces of some cells. These models are used to predict the magnitudes of force necessary to detach a cell in the limiting cases of peeling and simultaneous separation over finite areas of contact. Diverse experimental assays of cellular adhesiveness are reviewed and the forces applied to individual cells are estimated. A very wide range of the forces applied to cells in different assays suggests that different mechanisms of bonding are dominant for different types of cells and surfaces under various conditions of growth and chemical environment. The peeling mode of separation is most consistent with the magnitudes of applied force used experimentally in the detachment of cells.     

Molecularly smooth cellulose surfaces for adhesion studies

Cellulose is deposited on silicon wafer surfaces via spin coating from a solution of cellulose in dimethylacetamide (+7% lithium chloride). The resulting cellulose layers were analyzed by ellipsometry, AFM, FTIR, ICP-MS, X-ray reflectivity, and contact angle measurements. For cellulose concentrations below 0.07 wt% the wafer surfaces are covered with a network of cellulose fibrils. For concentrations between 0.07 and 0.5 wt%, closed films with thicknesses between 1.5 and approximately 10 nm are obtained. These films are molecularly smooth (rms roughness<2 nm). Higher concentrations result in thicker films with significantly rougher surfaces (rms roughness>2 nm). The cellulose layers were used to investigate cellulose/cellulose adhesion and their modification by polyelectrolytes. To this end the sticking behavior of cellulose beads was analyzed. It is demonstrated that the sticking of the beads depends on the type, amount, and adsorption symmetry of adsorbed polyelectrolyte. Low, incomplete polyelectrolyte coverage always enhances sticking, whereas for high coverage the symmetry of the polyelectrolyte coating is very important. In this case, sticking (adhesion) is enhanced if only one surface is covered with polyelectrolyte prior to contact. If both surfaces were fully covered with polyelectrolytes before contact, sticking (adhesion) is decreased.     

A novel fluorescent silica tracer for biological silicification studies.

BACKGROUND: Biological silica production has drawn intense attention and several molecules involved in biosilicification have been identified. Cellular mechanisms, however, remain unknown mainly due to the lack of probes required for obtaining information on live specimens. RESULTS: The fluorescence spectra of the compound 2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole (PDMPO) are affected by the presence of >3.2 mM silicic acid. Increase in intensity and shift in the fluorescence coincide with the polymerization of Si. The unique PDMPO-silica fluorescence is explored here to visualize Si deposition in living diatoms. The fluorophore is selectively incorporated and co-deposited with Si into the newly synthesized frustules (the outer silica shells) showing an intense green fluorescence. CONCLUSIONS: We suggest that a fluorescence shift is due to an interaction between PDMPO and polymeric silicic acid. PDMPO is an excellent probe for imaging newly deposited silica in living cells and has also a potential for a wide range of applications in various Si-related disciplines, including biology of living organisms as diatoms, sponges, and higher plants, clinical research (e.g. lung fibrosis and cancer, bone development, artificial bone implantation), and chemistry and physics of materials research.     

Osteoblast culture on bioerodible polymers: studies of initial cell adhesion and spread

The development of systems for the growth of osteoblasts on bioerodible polymeric matrices was explored. Three classes of bioerodible polymers were studied as possible matrix supports for osteoblast growth: the poly(anhydrides), poly(phosphazenes) and poly(lactic acid/glycolic acid) copolymers. Neonatal calvarial cells from Sprague–Dawley rats were seeded onto polymer disks at a density of 1 × 10⁴ cells/cm². Initial attachment and spreading, rate of growth and morphology were determined, and retention of osteoblast-like phenotype was assessed through measurements of alkaline phosphatase activity in the presence and absence of 1,25(OH)₂ vitamin D3. All results were considered relative to tissue culture polystyrene. Cells were found to attach to all polymers at 8 hr post-seeding. By 24 hr, cell numbers on all polymers were found to be decreased, except for poly(lactic acid/glycolic acid). Rat calvarial osteoblasts seeded on poly-(lactic acid/glycolic acid) reached confluency and retained their phenotype. Successful construction of viable osteoblast–bioerodible polymer composite materials, as presented in our study, may find their usefulness as grafts for atrophic non-unions of bone, for healing craniofacial and other defects and for use as prosthetic implants or coatings. Composite systems of osteoblast cultures may also find their usefulness in furthering our understanding of bone differentiation, maturation and metabolism in a matrix environment.     

Inflammatory mimetic microfluidic chip by immobilization of cell adhesion molecules for T cell adhesion

Leukocyte adhesion to adhesion molecules on endothelial cells is important in immune function, cancer metastasis and inflammation. This cell-cell binding is mediated via cell adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) found on endothelial cells. Because these adhesion molecules on endothelial cells vary significantly across several disease conditions such as autoimmune diseases, inflammation or cancer metastasis, investigations of therapeutic agents that down-regulate leukocyte-endothelial interactions have been based on in vitro models using endothelial cell lines. Here we report a new model, an inflammatory mimetic microfluidic chip, which emulates leukocyte binding to cell adhesion molecules (CAM) by controlling the types and ratio of adhesion molecules. In our model, E-selectin was essential for the synergic binding of Jurkat T cells. Immunosuppressive drugs, such as tacrolimus (FK506) and cyclosporine A (CsA), were used to inhibit T cell interactions under the physiologic model of T cell migration at a ratio of 5?:?4.3?:?3.9 (E-selectin?:?ICAM-1?:?VCAM-1). Our results support the potential usefulness of the inflammatory mimetic microfluidic chip as a T cell adhesion assay tool with modified adhesion molecules for applications such as immunosuppressive drug screening. The inflammatory mimetic microfluidic chip can also be used as a biosensor in clinical diagnostics, drug efficacy tests and high throughput drug screening due to the dynamic monitoring capability of the microfluidic chip.     

Photocontrol of cell adhesion processes: model studies with cyclic azobenzene-RGD peptides

A photoresponsive integrin ligand was synthesized by backbone-cyclization of a heptapeptide containing the integrin binding motif Arg-Gly-Asp (RGD) with 4-(aminomethyl)phenylazobenzoic acid (AMPB). Surface plasmon enhanced fluorescence spectroscopy showed that binding of the azobenzene peptide to alpha(v)beta(3) integrin depends on the photoisomeric state of the peptide chromophore. The higher affinity of the trans isomer could be rationalized by comparing the NMR conformations of the cis and trans isomers with the recently solved X-ray structure of a cyclic RGD-pentapeptide bound to integrin.     

New spin labels and probes for biological studies

Conclusions A series of new and highly stable iminoxyl mono- and biradicals was obtained.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 6, pp. 1327–1329, June, 1973.