3D alveolar in vitro model based on epithelialized biomimetically curved culture membranes.

There is increasing evidence that surface curvature at a near-cell-scale influences cell behaviour. Epithelial or endothelial cells lining small acinar or tubular body lumens, as those of the alveoli or blood vessels, experience such highly curved surfaces. In contrast, the most commonly used culture substrates for in vitro modelling of these human tissue barriers, ion track-etched membranes, offer only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically curved track-etched membranes, preserving the mainly spherical geometry of the cells' native microenvironment. The curved membranes were created by a combination of three-dimensional (3D) micro film (thermo)forming and ion track technology. We could successfully demonstrate the formation, the growth and a first characterization of confluent layers of lung epithelial cell lines and primary alveolar epithelial cells on membranes shaped into an array of hemispherical microwells. Besides their application in submerged culture, we could also demonstrate the compatibility of the bioinspired membranes for air-exposed culture. We observed a distinct cellular response to membrane curvature. Cells (or cell layers) on the curved membranes reveal significant differences compared to cells on flat membranes concerning membrane epithelialization, areal cell density of the formed epithelial layers, their cross-sectional morphology, and proliferation and apoptosis rates, and the same tight barrier function as on the flat membranes. The presented 3D membrane technology might pave the way for more predictive barrier in vitro models in future.

Colorectal tumor-on-a-chip system: A 3D tool for precision onco-nanomedicine.

Awareness that traditional two-dimensional (2D) in vitro and nonrepresentative animal models may not completely emulate the 3D hierarchical complexity of tissues and organs is on the rise. Therefore, posterior translation into successful clinical application is compromised. To address this dearth, on-chip biomimetic microenvironments powered by microfluidic technologies are being developed to better capture the complexity of in vivo pathophysiology. Here, we describe a "tumor-on-a-chip" model for assessment of precision nanomedicine delivery on which we validate the efficacy of drug-loaded nanoparticles in a gradient fashion. The model validation was performed by viability studies integrated with live imaging to confirm the dose-response effect of cells exposed to the CMCht/PAMAM nanoparticle gradient. This platform also enables the analysis at the gene expression level, where a down-regulation of all the studied genes (MMP-1, Caspase-3, and Ki-67) was observed. This tumor-on-chip model represents an important development in the use of precision nanomedicine toward personalized treatment.

Novel three-dimensional Boyden chamber system for studying transendothelial transport.

The rapid development in combinatorial chemistry of millions of novel potential drug candidates requires in vitro devices for reliable testing of their transendothelial transport and the uptake in specific cells. To date, this is often achieved in vitro by the use of regular planar Boyden chambers, which are not reflecting the three dimensionality of the blood vessel. This technical note describes the fabrication and biological validation of a novel three-dimensional Boyden chamber system for studying transendothelial transport. The key element of this new system is a porous thin-walled microchannel produced by a SMART (substrate modification and replication by thermoforming) process comprising a combination of microthermoforming and ion track technology. The membrane-like microstructure offers the opportunity to grow endothelial cells on the inner side of the channel resembling a more natural curved organization of vessels. After establishment of a confluent HUVECs layer in the porous microchannel this novel Boyden chamber was successfully applied to study the transendothelial transport of a polycationic cell penetrating peptoid through the 3D- or curved endothelial cell layer. Thus, this system will enable the investigation of such synthetic compounds as drug delivery systems with regard to their bioavailability and functionality under organotypic conditions.

The three-dimensional cultivation of the carcinoma cell line HepG2 in a perfused chip system leads to a more differentiated phenotype of the cells compared to monolayer culture.

We describe a polymer chip with a grid-like architecture that it is intended for the three-dimensional cultivation of cells with an active nutrient and gas supply. The chip is typically made from polymethyl methacrylate or polycarbonate but can also be manufactured from biodegradable polymers, such as poly(lactic-co-glycolic acid). Different designs of the chip can be realized. In this study, we evaluated a chip with 506 microcontainers of the size of 300 x 300 x 300 microm that are capable of housing up to 6 million cells, and its suitability as a tissue-specific culture system for the carcinoma cell line HepG2 instead of primary liver cells. Related to an earlier study, where we could show the principal suitability of the system for rat primary cells, we here investigated the system's suitability for the human carcinoma cell line HepG2. The carcinoma cells were used in two different types of chip-containing bioreactors. By confocal laser scanning microscopy, we could show that cellular integrity in the chip culture was maintained and that there were no signs of apoptosis as confirmed by the absence of K18 fragmentation. Gene expression analysis of some liver-specific genes revealed a significantly higher expression of the phase II metabolism genes uridine-diphosphate- glucosyl-transferase (UGT1A1) and glutathione-S-transferase (GSTpi1) as a marker. Therefore, we conclude that by using a three-dimensional instead of a conventional monolayer culture system, hepatocellular carcinoma cells display a phenotype that resembles more closely the tissue of origin.

Flexible fluidic microchips based on thermoformed and locally modified thin polymer films.

This paper presents a fundamentally new approach for the manufacturing and the possible applications of lab on a chip devices, mainly in the form of disposable fluidic microchips for life sciences applications. The new technology approach is based on a novel microscale thermoforming of thin polymer films as core process. The flexibility not only of the semi-finished but partly also of the finished products in the form of film chips could enable future reel to reel processes in production but also in application. The central so-called 'microthermoforming' process can be surrounded by pairs of associated pre- and postprocesses for micro- and nanopatterned surface and bulk modification or functionalisation of the formed films. This new approach of microscale thermoforming of thin polymer film substrates overlaid with a split local modification of the films is called 'SMART', which stands for 'substrate modification and replication by thermoforming'. In the process, still on the unformed, plane film, the material modifications of the preprocess define the locations where later, then on the spatially formed film, the postprocess generates the final local modifications. So, one can obtain highly resolved modification patterns also on hardly accessible side walls and even behind undercuts. As a first application of the new technology, we present a flexible chip-sized scaffold for three dimensional cell cultivation in the form of a microcontainer array. The spatially warped container walls have been provided with micropores, cell adhesion micropatterns and thin film microelectrodes.

3D tissue culture substrates produced by microthermoforming of pre-processed polymer films.

We describe a new technology based on thermoforming as a microfabrication process. It significantly enhances the tailoring of polymers for three dimensional tissue engineering purposes since for the first time highly resolved surface and bulk modifications prior to a microstructuring process can be realised. In contrast to typical micro moulding techniques, the melting phase is avoided and thus allows the forming of pre-processed polymer films. The polymer is formed in a thermoelastic state without loss of material coherence. Therefore, previously generated modifications can be preserved. To prove the feasibility of our newly developed technique, so called SMART = Substrate Modification And Replication by Thermoforming, polymer films treated by various polymer modification methods, like UV-based patterned films, and films modified by the bombardment with energetic heavy ions, were post-processed by microthermoforming. The preservation of locally applied specific surface and bulk features was demonstrated e.g. by the selective adhesion of cells to patterned microcavity walls.

Microthermoforming as a novel technique for manufacturing scaffolds in tissue engineering (CellChips).

The CellChip is a microstructured polymer scaffold, which favours a three-dimensional cultivation of cells within an array of cubic microcontainers. The manufacturing process used so far is microinjection moulding combined with laser-based perforation. In a first attempt to simplify the process, costly perforation was avoided by using commercially available, inexpensive microfiltration membranes for the bottom of the microcavities. Microthermoforming is a promising novel technique which allows the CellChip to be produced from thin film. Working pressures of approximately 4000 kPa were required for the adequate moulding of 50 microm thick films from three different polymers (polystyrene, polycarbonate, cyclo-olefin polymer). Integrating drafts and chamfers in micromoulds is not going to eliminate an uneven thickness profile, but reduces demoulding forces. Microthermoformed CellChips of polycarbonate were perforated by an ion track technique to guarantee a sufficient supply of medium and gases to the cells. The prestructured CellChips were irradiated with 1460 MeV xenon ions at a fluence of a few 10(6) ions/cm2. The tracks were etched in an aqueous solution of 5 N NaOH at 30 degrees C, which resulted in cylindrical pores approximately 2 microm in diameter. Microinjection-moulded, membrane-bonded and thermoformed CellChips were subjected to comparative examination for viability in a cell culture experiment with parenchymal liver cells (HepG2). The cells stayed viable over a period of more than 20 days. No significant differences in viability between injection-moulded, membrane-bonded, and thermoformed CellChips were observed.

Microthermoforming of flexible, not-buried hollow microstructures for chip-based life sciences applications.

A new method is presented for the manufacturing of flexible, not buried and thin-walled hollow microstructures from polymer films. This low-cost method seems to be especially suited for the fabrication of plastic microstructures for fluidic one-way applications in the field of life sciences. It is based on a thermoforming process adapted to microstructure technology and is called 'microthermoforming'. Inside a hot embossing press, a heated thin thermoplastic film is formed into the evacuated microcavities of a plate-shaped metal mould using a compressed gas. The film may be heat-sealed on to a thicker plastic film substrate inside the same press without demoulding the thermoformed film. To demonstrate the performance of the new manufacturing method, flexible capillary electrophoresis and cell culture chips from polystyrene, polycarbonate and a cyclo-olefin polymer with 16 and 625 parallel microstructures each, respectively, have been fabricated.

Further development of microstructured culture systems and their use in tissue engineering.

The Forschungszentrum Karlsruhe aims at improving its CellChip. Its main feature is the 1 cm2 core, subdivided into 900 cubic microcontainers (300 x 300 x 300 microns). It is manufactured by injection molding using biodegradable (polylactide) as well as non-degradable (PMMA or PC) polymers. The CellChips will be modified such that membranes will be mounted at the bottom of the CellChip, thus facilitating backend processing. Furthermore, the membranes can be adapted ideally to the assay system of interest by various surface modification techniques.

Conventional PKC-alpha, novel PKC-epsilon and PKC-theta, but not atypical PKC-lambda are MARCKS kinases in intact NIH 3T3 fibroblasts.

Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in intact cells has been employed as an indicator for activation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological conditions, however, remained to be identified. In our present study using stably transfected NIH 3T3 cell clones we demonstrate that expression of constitutively active mutants of either conventional cPKC-alpha or novel nPKC-epsilon increased phosphorylation of endogenous MARCKS in the absence of phorbol 12,13-dibutyrate in intact mouse fibroblasts, implicating that each of these PKC isoforms itself is sufficient to induce enhanced MARCKS phosphorylation. Similarly, ectopic expression of a constitutively active mutant of PKC-theta significantly increased MARCKS phosphorylation compared to vector controls, identifying PKC-theta as a MARCKS kinase. The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduced MARCKS phosphorylation in intact cells at a similar dose-response as enzymatic activity of recombinant isoenzymes cPKC-alpha, nPKC-epsilon, and nPKC-theta in vitro. Consistently, phorbol 12,13-dibutyrate-dependent MARCKS phosphorylation was significantly reduced in cell lines expressing dominant negative mutants of either PKC-alpha K368R or (dominant negative) PKC-epsilon K436R. The fact, that the constitutively active PKC-lambda A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not involved in this process. We conclude that under physiological conditions, conventional cPKC-alpha and novel nPKC-epsilon, but not atypical aPKC-lambda are responsible for MARCKS phosphorylation in intact NIH 3T3 fibroblasts.