Ultrathin Ceramic Membranes as Scaffolds for Functional Cell Coculture Models on a Biomimetic Scale.

Epithelial tissue serves as an interface between biological compartments. Many in vitro epithelial cell models have been developed as an alternative to animal experiments to answer a range of research questions. These in vitro models are grown on permeable two-chamber systems; however, commercially available, polymer-based cell culture inserts are around 10 µm thick. Since the basement membrane found in biological systems is usually less than 1 µm thick, the 10-fold thickness of cell culture inserts is a major limitation in the establishment of realistic models. In this work, an alternative insert, accommodating an ultrathin ceramic membrane with a thickness of only 500 nm (i.e., the Silicon nitride Microporous Permeable Insert [SIMPLI]-well), was produced and used to refine an established human alveolar barrier coculture model by both replacing the conventional inserts with the SIMPLI-well and completing it with endothelial cells. The structural-functional relationship of the model was evaluated, including the translocation of gold nanoparticles across the barrier, revealing a higher translocation if compared to corresponding polyethylene terephthalate (PET) membranes. This study demonstrates the power of the SIMPLI-well system as a scaffold for epithelial tissue cell models on a truly biomimetic scale, allowing construction of more functionally accurate models of human biological barriers.

Comparison of human olfactory and skeletal MSCs using osteogenic nanotopography to demonstrate bone-specific bioactivity of the surfaces.

Recently we identified a novel population of mesenchymal stem cells (MSCs) from human olfactory mucosa (OM-MSCs), a tissue which promotes neurogenesis throughout life, and demonstrated that they promoted CNS myelination to a greater extent than bone marrow-derived (BM)-MSCs. Previous data demonstrated that nanotopographies with a degree of disorder induce BM-MSC osteogenic differentiation. Thus, using biomaterials as non-chemical tools, we investigated if MSCs from a completely different cellular niche could be induced to differentiate similarly to nanoscale cues alone. Both MSCs differentiated into bone when cultured on nanotopographically embossed polycaprolactone (PCL) with a disordered pattern and heights but not on a "smooth" non-embossed PCL control substrate, but OM-MSC changes were at lower expression levels. Both MSCs showed similar increases in differentiation markers at the protein and mRNA level when plated on the two patterned surfaces. Thus, topographical cues from substrates with disordered patterns can up-regulate several MSC resident genes in both BM-MSCs and OM-MSCs. Moreover, antibody purified BM-MSCs had similar properties to non-purified BM-MSCs. These data suggest that MSCs from a neural cellular niche express similar bone-induced cues to BM-MSCs, suggesting that MSCs that inherently support nervous tissue can differentiate along the bone lineage in a similar manner to MSCs from a skeletal environment.

Delineating fibronectin bioadhesive micropatterns by photochemical immobilization of polystyrene and poly(vinylpyrrolidone).

Bioadhesive micropatterns, capable of laterally confining cells to a 2D lattice, have proven effective in simulating the in vivo tissue environment. They reveal fundamental aspects of the role of adhesion in cell mechanics, proliferation, and differentiation. Here we present an approach based on photochemistry for the fabrication of synthetic polymer micropatterns. Perfluorophenyl azide (PFPA), upon deep-UV exposure, forms a reactive nitrene capable of covalently linking to a molecule that is in close proximity. PFPA has been grafted onto a backbone of poly(allyl amine), which readily forms a self-assembled monolayer on silicon wafers or glass. A film of polystyrene was applied by spin-coating, and by laterally confining the UV exposure through a chromium-on-quartz photomask, monolayers of polymers could be immobilized in circular microdomains. Poly(vinylpyrrolidone) (PVP) was attached to the background to form a barrier to nonspecific protein adsorption and cell adhesion. Micropatterns were characterized with high-lateral-resolution time-of-flight secondary ion mass spectrometry (TOF-SIMS), which confirmed the formation of polystyrene domains within a PVP background. Fluorescence-microscopy adsorption assays with rhodamine-labeled bovine serum albumin demonstrated the nonfouling efficiency of PVP and, combined with TOF-SIMS, allowed for a comprehensive characterization of the pattern geometry. The applicability of the micropatterned platform in single-cell assays was tested by culturing two cell types, WM 239 melanoma cells and SaOs-2 osteoblasts, on micropatterned glass, either with or without backfilling of the patterns with fibronectin. It was demonstrated that the platform was efficient in confining cells to the fibronectin-backfilled micropatterns for at least 48 h. PVP is thus proposed as a viable, highly stable alternative to poly(ethylene glycol) for nonfouling applications. Due to the versatility of the nitrene-insertion reaction, the platform could be extended to other polymer pairs or proteins and the surface chemistry adapted to specific applications.

Increased plasticity of the stiffness of melanoma cells correlates with their acquisition of metastatic properties.

The stiffness of tumor cells varies during cancer progression. In particular, metastatic carcinoma cells analyzed by Atomic Force Microscopy (AFM) appear softer than non-invasive and normal cells. Here we examined by AFM how the stiffness of melanoma cells varies during progression from non-invasive Radial Growth Phase (RGP) to invasive Vertical Growth Phase (VGP) and to metastatic tumors. We show that transformation of melanocytes to RGP and to VGP cells is characterized by decreased cell stiffness. However, further progression to metastatic melanoma is accompanied by increased cell stiffness and the acquisition of higher plasticity by tumor cells, which is manifested by their ability to greatly augment or reduce their stiffness in response to diverse adhesion conditions. We conclude that increased plasticity, rather than decreased stiffness as suggested for other tumor types, is a marker of melanoma malignancy. These findings advise caution about the potential use of AFM for melanoma diagnosis. FROM THE CLINICAL EDITOR: This study investigates the changes to cellular stiffness in metastatic melanoma cells examined via atomic force microscopy. The results demonstrate that increased plasticity is a marker of melanoma malignancy, as opposed to decreased stiffness.

Transfer of ultrasmall iron oxide nanoparticles from human brain-derived endothelial cells to human glioblastoma cells.

Nanoparticles (NPs) are being used or explored for the development of biomedical applications in diagnosis and therapy, including imaging and drug delivery. Therefore, reliable tools are needed to study the behavior of NPs in biological environment, in particular the transport of NPs across biological barriers, including the blood-brain tumor barrier (BBTB), a challenging question. Previous studies have addressed the translocation of NPs of various compositions across cell layers, mostly using only one type of cells. Using a coculture model of the human BBTB, consisting in human cerebral endothelial cells preloaded with ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) and unloaded human glioblastoma cells grown on each side of newly developed ultrathin permeable silicon nitride supports as a model of the human BBTB, we demonstrate for the first time the transfer of USPIO NPs from human brain-derived endothelial cells to glioblastoma cells. The reduced thickness of the permeable mechanical support compares better than commercially available polymeric supports to the thickness of the basement membrane of the cerebral vascular system. These results are the first report supporting the possibility that USPIO NPs could be directly transferred from endothelial cells to glioblastoma cells across a BBTB. Thus, the use of such ultrathin porous supports provides a new in vitro approach to study the delivery of nanotherapeutics to brain cancers. Our results also suggest a novel possibility for nanoparticles to deliver therapeutics to the brain using endothelial to neural cells transfer.

Optimizing the osteogenicity of nanotopography using block co-polymer phase separation fabrication techniques.

Both temporary and permanent orthopedic implants have, by default or design, surface chemistry, and topography. There is increasing evidence that controlling nanodisorder can result in increased osteogenesis. Block co-polymer phase separation can be used to fabricate a nanotopography exhibiting a controlled level of disorder, both reproducibly and cost-effectively. Two different topographies, produced through the use of block co-polymer phase separation, were embossed onto the biodegradable thermoplastic, polycaprolactone (PCL). Analysis of the topography itself was undertaken with atomic force microscopy, and the topography's effect on human osteoblasts studied through the use of immunocytochemistry and fluorescence microscopy. Planar controls had a surface roughness 0.93 nm, and the substrates a high fidelity transfer of a disordered pattern of 14 and 18 nm. Cytoskeletal organization and adhesion, and increased expression of Runx2 were significantly greater on the smallest nanotopography. Expression of osteopontin and osteocalcin protein, and alizarin red staining of bone nodules were greatest on the smallest feature nanopatterns. Highly osteogenic, disordered nanotopographies can be manufactured into thermoplastics in a rapid and cost-effective way through the use of block co-polymer phase separation. Osteogenic topographies reproducibly and cost-effectively produced have a potentially useful application to the fields of implant technology and regenerative orthopedics.

Parallel AFM imaging and force spectroscopy using two-dimensional probe arrays for applications in cell biology.

Atomic force microscopy (AFM) investigations of living cells provide new information in both biology and medicine. However, slow cell dynamics and the need for statistically significant sample sizes mean that data collection can be an extremely lengthy process. We address this problem by parallelizing AFM experiments using a two-dimensional cantilever array, instead of a single cantilever. We have developed an instrument able to operate a two-dimensional cantilever array, to perform topographical and mechanical investigations in both air and liquid. Deflection readout for all cantilevers of the probe array is performed in parallel and online by interferometry. Probe arrays were microfabricated in silicon nitride. Proof-of-concept has been demonstrated by analyzing the topography of hard surfaces and fixed cells in parallel, and by performing parallel force spectroscopy on living cells. These results open new research opportunities in cell biology by measuring the adhesion and elastic properties of a large number of cells. Both properties are essential parameters for research in metastatic cancer development.

Use of force spectroscopy to investigate the adhesion of living adherent cells.

The use of force spectroscopy to study the adhesion of living fibroblasts to their culture substrate was investigated. Both primary fibroblasts (PEMF) and a continuous cell line (3T3) were studied on quartz surfaces. Using a fibronectin-coated AFM cantilever, it was possible to detach a large proportion of the 3T3 cells from the quartz surfaces. Their adhesion to the quartz surface and the effects of topography on this adhesion could be quantified. Three parameters characteristic of the adhesion were measured: the maximum force of detachment, the work of adhesion, and the distance of detachment. Few PEMF cells were detached under the same experimental conditions. The potential and limitations of this method in measuring cell/surface interactions for adherent cells are discussed.

FluidFM: combining atomic force microscopy and nanofluidics in a universal liquid delivery system for single cell applications and beyond.

We describe the fluidFM, an atomic force microscope (AFM) based on hollow cantilevers for local liquid dispensing and stimulation of single living cells under physiological conditions. A nanofluidic channel in the cantilever allows soluble molecules to be dispensed through a submicrometer aperture in the AFM tip. The sensitive AFM force feedback allows controlled approach of the tip to a sample for extremely local modification of surfaces in liquid environments. It also allows reliable discrimination between gentle contact with a cell membrane or its perforation. Using these two procedures, dyes have been introduced into individual living cells and even selected subcellular structures of these cells. The universality and versatility of the fluidFM will stimulate original experiments at the submicrometer scale not only in biology but also in physics, chemistry, and material science.

Measuring cell adhesion forces during the cell cycle by force spectroscopy.

Force spectroscopy has been used to measure the adhesion of Saos-2 cells to a glass surface at different phases of the cell cycle. The cells were synchronized in three phases of the cell cycle: G(1), S, and G(2)M. Cells in these phases were compared with unsynchronized and native mitotic cells. Individual cells were attached to an atomic force microscope cantilever, brought into brief contact with the glass surface, and then pulled off again. The force-distance curves obtained allowed the work and maximum force of detachment as well as the number, amplitude, and position of discrete unbinding steps to be determined. A statistical analysis of the data showed that the number of binding proteins or protein complexes present at the cell surface and their binding properties remain similar throughout the cell cycle. This, despite the huge changes in cell morphology and adhesion that occur as the cells enter mitosis. These changes are rather associated with the changes in cytoskeletal organization, which can be quantified by force spectroscopy as changes in cell stiffness.

Scattering of light by a single layer of randomly packed dielectric microspheres giving color effects in transmission.

Strong scattering properties are obtained for a monolayer of randomly packed polystyrene microspheres. This gives rise to structural colors in transmission. For a sphere diameter between 0.5 and 1 micron, light is mainly scattered in the forward direction. Consequently, in-plane multiple scattering can be neglected when spheres are not too close to each others. This allows one to use a single scattering approximation to reproduce transmission spectra of the system. The film color is dependent on the sphere size, but also on the observation angle. This angular dependant color is reproduced taking into account multiple scattering between spheres. These films can be useful when low reflection is needed.

Fabrication of multiscale surface-chemical gradients by means of photocatalytic lithography.

We describe a new method for the fabrication of surface-chemical gradients. A film of titanium dioxide is brought into close proximity to a uniformly monolayer-covered surface and exposed to UV light to produce oxygen radicals. The use of a gradated grayscale mask between the UV source and the TiO2 allows the production of surface-chemical gradients via oxidation of the monolayer. The technique is demonstrated on gold surfaces bearing alkanethiol SAMs. Oxidation and subsequent replacement of the oxidized thiols has been used to produce surface-chemical gradients with lengths on the submillimeter to centimeter scales. The oxidation, removal, and replacement of the thiols during the process have been demonstrated by means of XPS. This oxidative process may be applied to other surface chemistries. Similarly, other shapes and slopes of gradients may be produced, depending on the photomask employed.

Organization of nanoparticles on hard substrates using block copolymer films as templates.

We present a technique for the organization of pre-synthesized nanoparticles on hard substrates, using block copolymer films as sacrificial templates. A thin block copolymer film is dip-coated on the substrate of interest and the sample is exposed to a solution containing nanoparticles. Spontaneous preferential adsorption of the nanoparticles on one phase of the block copolymer film results in their lateral organization. An oxygen plasma etch is used to remove the polymer film; the nanoparticles end up organized on the substrate. We demonstrate that this is a general approach for the patterning of inorganic nanoparticles on hard substrates, showing the organization of metal and semiconductor nanoparticles having different chemistries at the particle/solvent and solvent/polymer interfaces. The nanoparticle patterns that we present have typical periodicities in the nanometer scale. In some cases, microcontact printing is used to create a double length scale of organization, on the micrometer and on the nanometer level. The characteristic periodicity of the template is studied with respect to the nanoparticle size in order to optimize the organization. Finally, we describe how to extend this technique for the production of continuous gold nanowires on hard substrates. We expect that the flexibility of this approach and the degree of control that can be obtained over nanoparticle organization should make it a powerful tool for nanoscale fabrication.

Micrometer-long gold nanowires fabricated using block copolymer templates.

Micrometer-long gold nanowires were fabricated via self-assembly. Diblock copolymer films served as templates for the selective adsorption of 10 nm gold nanoparticles from solution to form well-defined nanostructures. An oxygen plasma treatment induced aggregation of the nanoparticles and the formation of continuous gold nanostructures. The electrical continuity of the nanostructures was observed using scanning electron microscopy.