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E-cadherin is a cell-cell adhesion protein that plays a prominent role in cancer invasion. Inactivation of E-cadherin in breast cancer can arise from gene promoter hypermethylation or genetic mutation. Depending on their E-cadherin status, breast cancer cells adopt different morphologies with distinct invasion modes. The tumor microenvironment (TME) can also affect the cell morphology and invasion mode. In this paper, we used a previously developed microfluidic system to quantify the three-dimensional invasion of breast cancer cells with different E-cadherin status, namely MCF-7, CAMA-1 and MDA-MB-231 with wild type, mutated and promoter hypermethylated E-cadherin, respectively. The cells migrated into a stable and reproducible microfibrous polycaprolactone mesh in the chip under a programmed stable chemotactic gradient. We observed that the MDA-MB-231 cells invaded the most, as single cells. MCF-7 cells collectively invaded into the matrix more than CAMA-1 cells, maintaining their E-cadherin expression. The CAMA-1 cells exhibited multicellular multifocal infiltration into the matrix. These results are consistent with what is seen in vivo in the cancer biology literature. In addition, comparison between complete serum and serum gradient conditions showed that the MDA-MB-231 cells invaded more under the serum gradient after one day, however this behavior was inverted after 3 days. The results showcase that the microfluidic system can be used to quantitatively assess the invasion behavior of cancer cells with different E-cadherin expression, for a longer period than conventional invasion models. In the future, it can be used to quantitatively investigate effects of matrix structure and cell treatmen

In this work we present advances in three dimensional (3D) neuronal cell culture systems based on a reversible assembly of a microbioreactor with a microelectrode array (MEA) to create a MEMS-based 3D cell culture system for in vitro neuro-electrophysiological recordings. A batch of six molds were milled in poly (methyl methacrylate). The molds were used for soft lithography of polydimethylsiloxane (PDMS). In the center of the PDMS shape, a porous polyethersulfone (PES) cylindrical tube was press-fitted to form a growth barrier between the culture chamber inside the PES tube and the microfluidic channel surrounding the PES tube. A thin layer of partially cured PDMS was used to seal the bottom of the microbioreactor and provide reversible adhesion with the glass surface of a MEA. SH-SY5Y cells were successfully differentiated inside the microbioreactors in Matrigel and demonstrated extended neuronal networks over a height of at least 184 micrometers within the system. In previous microbioreactor designs visibility was limited due to the closed top with the dispensing holes. The new open top design allows for a better evaluation of the cell culture by optical detection methods during the experiment. Electrophysiological activity was recorded within the microbioreactor using human induced pluripotent stem cell-derived cortical neurons cultured in Matrigel, in 3D, up until 21 days in vitro. In summary, we present advances made in the design, the fabrication process and integration of microbioreactors with MEAs. Optical imaging capabilities improved significantly with an open top and the culture time was further extended from 7 to 21 DIV without leakage or degradation thanks to introducing PES as a barrier material and an enhanced assembly procedure. The latter facilitated a sufficient long-term culture for neurons to mature in an environment free from flow-induced stress and provided a proof of principle for the recording of electrophysiological activity of cortical neu

Recent advances in brain-on-a-chip technology have led to the development of modified microelectrode arrays. Previously, the authors have contributed to this exciting field of neuroscience by demonstrating a fabrication process for producing microsieve chips that contain three-dimensional (3D) micropores at the electrodes [termed microsieve electrode arrays (μSEAs)]. This chip allows us to trap hundreds of single neuronal cells in parallel onto the electrodes [B. Schurink and R. Luttge, J. Vac. Sci. Technol., B 31, 06F903 (2013)]. However, trapping the neurons reproducibly under gentle, biocompatible conditions remains a challenge. The current setup involves the use of a hand-operated syringe that is connected to the back of the μSEA chip with a polydimethylsiloxane (PDMS) construct. This makes the capture process rather uncontrolled, which can lead to either cell damage by shear stress or the release of trapped neurons when unplugging the syringe and PDMS constructs. Although, the authors could achieve an efficient capture rate of single neurons within the 3D micropores (80%-90% filling efficiency), cell culture performance varied significantly. In this paper, the authors introduce a passive pumping mechanism for the parallel trapping of neurons onto the μSEA chip with the goal to improve its biological performance. This method uses the capillary pumping between two droplets (a "pumping droplet" on one side of the chip and a "reservoir droplet" on the other side) to create a stable and controllable flow. Due to simplification of the handling procedure, omitting the use of a syringe and additional connections to the μSEA chip, the set-up is compatible with real time microscopy techniques. Hence, the authors could use optical particle tracking to study the trapping process and record particle velocities by video imaging. Analyzing the particle velocities in the passive pumping regime, the authors can confirm a gentle uniform particle flow through the 3D micropores

Commercial microelectrode arrays (MEAs) for in vitro neuroelectrophysiology studies rely on conventional two dimensional (2D) neuronal cultures that are seeded on the planar surface of such MEAs and thus form a random neuronal network. The cells attaching on these types of surfaces grow in 2D and lose their native morphology, which may also influence their neuroelectrical behavior. Besides, a random neuronal network formed on this planar surface in vitro also lacks comparison to the in vivo state of brain tissue. In order to improve the present MEA platform with the above mentioned concerns, in this paper, the authors introduce a three dimensional platform for neuronal cell culturing, where a linear nanoscaffold is patterned on a microsieve array by displacement Talbot lithography (DTL) and reactive ion etching. Good pattern uniformity is achieved by the DTL method on the topographically prepatterned nonflat surface of the microsieve array. Primary cortical cells cultured on the nanopatterned microsieve array show an organized network due to the contact guidance provided by the nanoscaffold, presenting 47% of the total outgrowths aligning with the nanogrooves in the observed view of field. Hence, the authors state that this nanopatterned microsieve array can be further integrated into microsieve-based microelectrode arrays to realize an advanced Brain-on-Chip model that allows us to investigate the neurophysiology of cultured neuronal networks with specifically organized architectures.

In the literature, several types of microneedles have been extensively described. However, porous microneedle arrays only received minimal attention. Hence, only little is known about drug delivery via these microneedles. However, porous microneedle arrays may have potential for future microneedle-based drug and vaccine delivery and could be a valuable addition to the other microneedle-based drug delivery approaches. To gain more insight into porous microneedle technologies, the scientific and patent literature is reviewed, and we focus on the possibilities and constraints of porous microneedle technologies for dermal drug delivery. Furthermore, we show preliminary data with commercially available porous microneedles and describe future directions in this field of research.

The process where cancer cells leave the primary tumor and invade to the blood vessel. As shown in figure 1, intravasation is highly regulated by the micro-environment of the tumor. An important component of the micro-environment is the extracellular matrix (ECM) which can be seen as the building structure of a LEGO house. A proper model for cancer intravasation requires a proper model for the micro-environment, or in other words, a right LEGO house for cancer cells to live in! To model the process, microfluidics is used because there is: •more control on the biochemical content, •less human error by automating the experiments, •more complex designs, •and less ethical issues, it is a LEGO house! The GOAL is to study how the mechanical properties of the extracellular matrix regulate the tumor intravasation by using a microfluidic chip.

Extracellular matrix (ECM), as a bio-chemical and -physical support for cells, is of great importance in cell migration studies. 3D migration studies, compared to 2D cultures, have proven to best represent the in vivo conditions[1]. Hydrogels are usually used in in vitro studies as the 3D ECM. However, the relevance of the architecture and controllability of gels are debatable[2]. Self standing fibrous scaffolds, which more closely mimic the in vivo condition, can be fabricated (by electro-spinning) with different fiber sizes and architecture and from different materials. In addition, microfluidic chips can intrinsically control the biochemical content of the cell micro-environment which is also important for the cell migration. In this project, we have developed a new micro-fabrication method to integrate fibrous scaffolds inside a microfluidic device to study cell migration on a chip.

Arrays of microneedles (MNAs) are integrated in an out-of-plane fashion with a base plate and can serve as patches for the release of drugs and vaccines. We used soft-lithography and micromolding to manufacture ceramic nanoporous (np)MNAs. Failure modes of ceramic npMNAs are as yet poorly understood and the question remained: is our npMNA platform technology ready for microneedle (MN) assembly into patches? We investigated npMNAs by microindentation, yielding average crack fracture forces above the required insertion force for a single MN to penetrate human skin. We further developed a thumb pressure-actuated applicator-assisted npMNA insertion method, which enables anchoring of MNs in the skin by an adhesive in one handling step. Using a set of simple artificial skin models, we found a puncture efficiency of this insertion method a factor three times higher than by applying thumb pressure on the npMNA base plate directly. In addition, this new method facilitated zero MN-breakage due to a well-defined force distribution exerted onto the MNs and the closely surrounding area prior to bringing the adhesive into contact with the skin. Owing to the fact that such parameter space exists, we can conclude that npMNAs by soft lithography are a platform technology for MN assembly into a patch.

Contains fulltext : 117782.pdf (Publisher’s version ) (Closed access), Designing biomaterial surfaces to control the reaction of the surrounding tissue is still considered to be a primary issue, which needs to be addressed systematically. Although numerous in vitro studies have described different nano-metrically textured substrates capable to influence bone cellular response, in vivo studies validating this phenomenon have not been reported. In this study, nano-grooved silicon stamps were produced by laser interference lithography (LIL) and reactive ion etching (RIE) and were subsequently transferred onto the surface of 5 mm diameter Titanium (Ti) discs by nanoimprint lithography (NIL). Patterns with pitches of 1000 nm (500 nm ridge and groove, 150 nm depth), 300 nm (150 nm ridge and groove, 120 nm depth; as well as a 1:3 ratio of 75 nm ridge and 225 nm groove, 120 nm depth) and 150 nm (75 nm ridge and groove, 30 nm depth) were created. These samples were implanted in a rabbit tibia cortical bone. Histological evaluation and histomorphometric measurements were performed, comparing each sample to conventional grit-blasted/acid-etched (GAE) titanium controls. Results showed a significantly higher bone-to-implant contact at 4 weeks for the 300 nm (1:3) specimens, compared to GAE (p = 0.006). At 8 weeks, there was overall more bone contact compared to 4 weeks. However, no significant differences between the nano-textured samples and the GAE occurred. Further studies will need to address biomechanical testing and the use of trabecular bone models.

The authors present a hydrogel/poly-dimethylsiloxane (PDMS) hybrid bioreactor. The bioreactor enables a low shear stress 3D culture by integrating a hydrogel as a barrier into a PDMS casing. The use of PDMS allows the reversible adhesion of the device to a commercially available microelectrode array. A two-step molding process facilitates this relatively simple, cost effective, and leakage-free add-on microculture system. Agarose (2%) is used as hydrogel barrier material and mass transport is evaluated by fluorescein isothiocyanate-albumin fluorescence under static conditions which yields a diffusion coefficient of average value of 2.2 × 10-7 cm 2 s-1 across the barrier. To validate our bioreactor for diffusion limited 3D cell culture, rat cortical cells were successfully cultured in Matrigel for 6 days. © 2013 American Vacuum Society.

Radboud Universiteit Nijmegen, 11 januari 2012, Promotores : Jansen, J.A., Gardeniers, J.G.E. Co-promotores : Walboomers, X.F., Luttge, R., Contains fulltext : 91454.pdf (publisher's version ) (Open Access)

1 april 2012, Item does not contain fulltext, The immune response to an implanted biomaterial is orchestrated by macrophages. In this study various nanogrooved patterns were created by using laser interference lithography and reactive ion etching. The created nanogrooves mimic the natural extracellular matrix environment. Macrophage cell culture demonstrated that interleukin 1beta and TNF-alpha cytokine production were upregulated on nanogrooved substrates. In vivo subcutaneous implantation in a validated mouse cage model for 14 days demonstrated that nanogrooves enhanced and guided cell adhesion, and few multinucleated cells were formed. In agreement with the in vitro results, cytokine production was found to be nanogroove dependent, as interleukin 1beta, TNF-alpha, TGF-beta and osteopontin became upregulated. The results indicate that biomaterial surface texturing, especially at the nanometric scale, can be used to control macrophage activation to induce a wound healing response, rather than a profound inflammatory response. From the Clinical Editor: The authors investigate various nano-grooved patterns that mimic the natural extracellular matrix environment and demonstrate (both in macrophage cultures and in vivo) that interleukin 1beta and TNF-alpha cytokine production is dependent upon surface texturing at the nanometric scale. They propose that modified surfaces may trigger macrophage activation to promote a wound healing response.

Item does not contain fulltext, Nanopatterns on titanium may enhance endosseous implant biofunctionality. To enable biological studies to prove this hypothesis, we developed a scalable method of fabricating nanogrooved titanium substrates. We defined nanogrooves by nanoimprint lithography (NIL) and a subsequent pattern transfer to the surface of ASTM grade 2 bulk titanium applying a soft-mask for chlorine-based reactive ion etching (RIE). With respect to direct write lithographic techniques the method introduced here is fast and capable of delivering uniformly patterned areas of at least 4 cm(2). A dedicated silicon nanostamp process has been designed to generate the required thickness of the soft-mask for the NIL-RIE pattern transfer. Stamps with pitch sizes from 1000 nm down to 300 nm were fabricated using laser interference lithography (LIL) and deep cryogenic silicon RIE. Although silicon nanomachining was proven to produce smaller pitch sizes of 200 nm and 150 nm respectively, successful pattern transfer to titanium was only possible down to a pitch of 300 nm. Hence, the smallest nanogrooves have a width of 140 nm. An x-ray photoelectron spectroscopy study showed that only very few contaminations arise from the fabrication process and a cytotoxicity assay on the nanopatterned surfaces confirmed that the obtained nanogrooved titanium specimens are suitable for in vivo studies in implantology research.

Contains fulltext : 110986.pdf (publisher's version ) (Open Access), Organised nanotopography mimicking the natural extracellular matrix can be used to control morphology, cell motility, and differentiation. However, it is still unknown how specific cell types react with specific patterns. Both initial adhesion and preferential cell migration may be important to initiate and increase cell locomotion and coverage with cells, and thus achieve an enhanced wound healing response around an implantable material. Therefore, the aim of this study was to evaluate how MC3T3-E1 osteoblast initial adhesion and directional migration are influenced by nanogrooves with pitches ranging from 150 nm up to 1000 nm. In this study, we used a multi-patterned substrate with five different groove patterns and a smooth area with either a concentric or radial orientation. Initial cell adhesion measurements after 10 s were performed using atomic force spectroscopy-assisted single-cell force spectroscopy, and demonstrated that nascent cell adhesion was highly induced by a 600 nm pitch and reduced by a 150 nm pitch. Addition of RGD peptide significantly reduced adhesion, indicating that integrins and cell adhesive proteins (e.g. fibronectin or vitronectin) are key factors in specific cell adhesion on nanogrooved substrates. Also, cell migration was highly dependent on the groove pitch; the highest directional migration parallel to the grooves was observed on a 600 nm pitch, whereas a 150 nm pitch restrained directional cell migration. From this study, we conclude that grooves with a pitch of 600 nm may be favourable to enhance fast wound closure, thereby promoting tissue regeneration.

Contains fulltext : 109176.pdf (publisher's version ) (Open Access), A process of micromolding, delivering micro- and nanopatterned ceramic surfaces for biomaterial applications is described in this work. To create the desired structures, tape casting of ceramic slurries on microfabricated silicon mold was used. Several tape casting slurry compositions were tested to evaluate the feasibility of transferring micro- and nano-features from silicon molds. Used ceramics were alumina (alpha-Al(2)O(3)) and yttria stabilized zirconia. Three types of polymeric binders for the green tape (PVB, PES, and PVP) were investigated using three different solvents (ethanol, n-methyl-pyrrolidone, water). Well-defined features in shapes of wells with diameters down to 2.4 mum and a depth of 10 mum and pillars with diameters down to 1.7 mum and a height of 3 mum were obtained. Morphology, grain size and porosity of the sintered bodies were characterized. Finally fibroblast cells were cultured on the surfaces in order to observe their morphology under influence of the microstructured surfaces.

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Contains fulltext : 87801.pdf (publisher's version ) (Open Access), The natural environment of a living cell is not only organized on a micrometer, but also on a nanometer scale. Mimicking such a nanoscale topography in implantable biomaterials is critical to guide cellular behavior. Also, a correct positioning of cells on biomaterials is supposed to be very important for promoting wound healing and tissue regeneration. The exact mechanism by which nanotextures can control cellular behavior are thus far not well understood and it is thus far unknown how cells recognize and respond to certain surface patterns, whereas a directed response appears to be absent on other pattern types. Focal adhesions (FAs) are known to be involved in the process of specific pattern recognition and subsequent response by cells. In this study, we used a high throughput screening "Biochip" containing 40 different nanopatterns to evaluate the influence of several nanotopographical cues like depth, width, (an)isotropy and spacing (ridge-groove ratio) on osteoblast behavior. Microscopical analysis and time lapse imaging revealed that an isotropic topography did not alter cell morphology, but it highly induced cell motility. Cells cultured on anisotropic topographies on the other hand, were highly elongated and aligned. Time-lapse imaging revealed that cell motility is highly dependent on the ridge-groove ratio of anisotropic patterns. The highest motility was observed on grooves with a ratio of 1:3, whereas the lowest motility was observed on ratios of 1:1 and 3:1. FA measurements demonstrated that FA-length decreased with increasing motility. From the study it can be concluded that osteoblast behavior is tightly controlled by nanometer surface features.

1 april 2010, Contains fulltext : 87800.pdf (publisher's version ) (Closed access), To fight bone diseases characterized by poor bone quality like osteoporosis and osteoarthritis, as well as in reconstructive surgery, there is a need for a new generation of implantable biomaterials. It is envisioned that implant surfaces can be improved by mimicking the natural extracellular matrix of bone tissue, which is highly a organized nano-composite. In this study we aimed to get a better understanding of osteoblast response to nanometric grooved substrates varying in height, width and spacing. A throughput screening biochip was created using electron beam lithography. Subsequently, uniform large-scale nanogrooved substrates were created using laser interference lithography and reactive ion etching. Results showed that osteoblasts were responsive to nanopatterns down to 75 nm in width and 33nm in depth. SEM and TEM studies showed that an osteoblast-driven calcium phosphate (CaP) mineralization was observed to follow the surface pattern dimensions. Strikingly, aligned mineralization was found on even smaller nanopatterns of 50 nm in width and 17 nm in depth. A single cell based approach for real time PCR demonstrated that osteoblast-specific gene expression was increased on nanopatterns relative to a smooth control. The results indicate that nanogrooves can be a very promising tool to direct the bone response at the interface between an implant and the bone tissue.

Contains fulltext : 83872.pdf (publisher's version ) (Closed access)

The authors report on the progress of laser interference lithography at 266 nm laser wavelength with a chemical amplified resist containing a polyvinyl derivate dissolved in propylene glycol monoethyl ether ester. A continuous-wave deep-UV source combined with a Lloyd mirror is a simple and useful tool for the fabrication of nanoscale periodic structures generally called nanoarrays. Aiming for a robust pattern transfer technique to fabricate nanoarrays into magnetic materials, the authors investigated the utility of a chemical amplification positive tone resist, despite the relatively high theoretical resolution limit of 133 nm (¿2) pattern period for the laser source used. Taking advantage of this new type of resist, the authors demonstrated for the first time the fabrication of an 18 Gbit in.2 dot pattern on a platinum thin film. © 2007 American Vacuum Society.

An integrated method using field-emission to control the tip-sample distance for non-contact magnetic probe recording is presented, adopting the exponential relation between current and electric field as feedback. I/V characteristics that correspond well to field emission theory are measured using a probe coated with a 100 nm conductive diamond layer. By using feedback to control the tip-sample distance at constant current, the distance was increased by 2.8 nm per volt applied bias. The method was tested by scanning a probe coated with 20 nm chromium over a conducting nanopatterned sample, at bias voltages of 0.5V, 5.0V and 50.0V. The measurements confirm that field emission can be applied to control the tip-sample distance, with sufficient resolution and current stability for magnetic probe recording. © 2007 IOP Publishing Ltd.

This paper presents the design, fabrication and first results of a microfluidic cell trap device for analysis of apoptosis. The microfluidic silicon-glass chip enables the immobilization of cells and real-time monitoring of the apoptotic process. Induction of apoptosis, either electric field mediated or chemically induced with tumour necrosis factor (TNF-alpha), in combination with cycloheximide (CHX), was addressed. Exposure of cells to the appropriate fluorescent dyes, FLICA and PI, allows one to discriminate between viable, apoptotic and necrotic cells. The results showed that the onset of apoptosis and the transitions during the course of the cell death cascade were followed in chemically induced apoptotic HL60 cells. For the case of electric field mediated cell death, the distinction between apoptotic and necrotic stage was not clear. This paper presents the first results to analyse programmed cell death dynamics using this apoptosis chip and a first step towards an integrated apoptosis chip for high-throughput drug screening on a single cellular level., QC 20100525