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.

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.

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.

Neuronal outgrowth of PC-12 cells after combined treatment with nerve growth factor and a magnetic field: influence of the induced electric field strength.

In view of possible therapeutic applications of magnetic fields, the effect of an enhancement of neuronal outgrowth at higher figures of flux density and induced field strength was investigated. On the average sinusoidal magnetic field treatment at 100 microTrms/50 Hz did not change nerve growth factor (NGF) induced neurite outgrowth to a statistically significant extent. These results suggest that further increasing the induced field strength by using either higher flux densities and/or more sophisticated wave forms might be necessary to cause the neuronal response of PC-12 cells, as seen in other experiments.

Quantification of NGF-dependent neuronal differentiation of PC-12 cells by means of neurofilament-L mRNA expression and neuronal outgrowth.

We demonstrate that the degree of neuronal development of PC-12 cell differentiation can be quantified by the expression of neurofilament-L (NF-L) mRNA, when an optimal concentration of NGF (50 ng/ml) is used. During the first 7 days of NGF treatment, the relative amount of NF-L mRNA was found to increase continuously and to correlate with the outgrowth of neurites in a statistically significant way. Thus, mRNA expression is, under these conditions, a suitable means for reliably monitoring the differentiation of PC-12 cells as early as after 3 days of NGF treatment. The results obtained with 5 ng/ml NGF differ from those with 50 ng/ml: during the first 3 days of NGF treatment, neuronal outgrowth was less than with 50 ng/ml, although the NF-L mRNA levels did not depend significantly on NGF concentration. Beyond day 3, NF-L mRNA levels did not increase further at 5 ng/ml as opposed to 50 ng/ml NGF. These differences point to different signal transduction processes involved in neuronal differentiation at high and low NGF concentration. Expression of NF-L protein in response to NGF treatment was also demonstrated. In summary, our results stress that stable and sustained differentiation of PC-12 cells can only be achieved with 50 ng/ml NGF.

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.

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.

Patterned polymer surfaces for cell culture applications.

We studied the physico/chemical effects of deep UV irradiation of polystyrene, PMMA and polycarbonate with respect to cell adhesion and protein immobilization. Photochemical modifications of the polymer surfaces yielded unstable peroxides and carboxylic acid groups. Patterned enzyme and antibody adsorbates were realized by coupling via carbodiimid activation of the COOH-moities. Hepatoma cells (HepG2) and fibroblasts (L929) adhered in the presence of serum proteins in the culture medium on the irradiated regions of the substrate without any further treatment.

A tissue-like culture system using microstructures: influence of extracellular matrix material on cell adhesion and aggregation.

Special microenvironmental conditions are required to induce and/or maintain specific qualities of differentiated cells. An important parameter is the three-dimensional tissue architecture that cannot be reproduced in conventional monolayer systems. Advanced tissue culture systems will meet many of these demands, but may reach their limits, especially when gradients of specific substances over distinct tissue layers must be established for long-term culture. These limitations may be overcome by incorporating microstructures into tissue-like culture systems. The microstructured cell support presented consists of a flat array of 625 cubic microcontainers with porous bottoms, in which cells can be supplied with specific media from both sides of the tissue layer. Permanent cell lines and primary rat hepatocytes have been used to test the culture system. In order to define reproducible conditions for tissue formation and for cell adherence to the structure, several ECM (extracellular matrix) components were tested for coating of microstructured substrata. The described tissue culture system offers great flexibility in adapting the cell support to specific needs.

Cyclic AMP response in cells exposed to electric fields of different frequencies and intensities.

The action on intracellular cyclic AMP (cAMP) of therapeutically used 4000-Hz electric fields was investigated and compared with 50-Hz data. Cultured mouse fibroblasts were exposed for 5 minutes to 4000-Hz sine wave internal electric fields between 3 mV/m and 30 V/m applied within culture medium. A statistically significant decrease in cellular cAMP concentration relative to unexposed cells was observed for fields higher than 10 mV/m. The drop in cAMP was most pronounced at lower field strengths (71% of controls at 30 mV/m) and tended to disappear at higher field strengths. An increase of cAMP content was observed with 50-Hz electric fields, as was also the case when 4000-Hz fields were modulated with certain low frequencies.

The DNA content of some mammalian cells measured by flow cytometry and its influence on radiation sensitivity.

The DNA content of nine mammalian cell lines was determined by flow cytometry. Using radiobiological data from this and other laboratories a correlation between DNA mass and 1/D0 for X-rays, alpha-particles, and heavy ions could be established when the quantities were plotted on a log-log scale. The slopes of the regression lines amounted to 0.65 (X-rays), 0.64 (alpha-particles) and 0.74 (heavy ions). A similar correlation was found between DNA content and mean inactivation dose. The rather uniform slopes close to 2/3 suggest that radiosensitivity may depend on the surface area of the sensitive target, (cell nucleus) indicating a possible non-uniform distribution of radiosensitive sites within the nucleus.

Radioresistance of rat glioma cell lines cultured as multicellular spheroids. Correlation with electrical cell-to-cell-coupling.

Two chemically induced rat glioblastomas, RG2 and F98, were cultured as monolayers and as multicellular spheroids and subjected to Co-gamma-irradiation. In parallel, intercellular communication between cells was determined as electrical coupling between neighbouring cells using micro-electrode techniques. A third glioblastoma with known radiobiological response (9L) was assayed with respect to intercellular communication and included into this analysis. Electrical coupling was low for RG2, intermediate for F98, and high for 9L. Radioresistance of spheroids, as expressed in terms of the mean inactivation dose computed from the survival curves increased in the same direction (RG2: 2.4 Gy; F98: 5.1 Gy; 9L: 6.5 Gy). A comparison of these parameters demonstrates a correlation between solid tumor radioresistance and gap-junctional cell-to-cell communication, at least for the class of glioblastomas analysed in this study.

Rejoining of double strand breaks in normal human and ataxia-telangiectasia fibroblasts after exposure to 60Co gamma-rays, 241Am alpha-particles or bleomycin.

The rejoining of DNA double strand breaks (dsb) induced by 60Co gamma-rays, 241Am alpha-particles or bleomycin was measured by neutral filter elution. In agreement with their colony-forming ability, ataxia-telangiectasia cells (AT2BE) and normal fibroblasts exhibited similar dsb rejoining capacity following alpha-irradiation, but showed marked differences in the rejoining kinetics of dsb induced by gamma-rays or bleomycin.

Relationship between double strand break rejoining and G2 block formation in V79 cells.

Caffeine and hypertonicity affect the survival of gamma-irradiated Chinese Hamster V79 cells in different ways: while caffeine reduces the shoulder of the dose effect curve, hypertonic treatment mainly affects its final slope suggesting that different types of damage and (or) repair mechanisms are involved. Rejoining of DNA double strand breaks (dsb), as measured by neutral filter elution technique, exhibits a fast and a slow component, indicating that dsb rejoining consists of two different processes. Hypertonicity causes a temporary inhibition of the fast rejoining step but has no effect on the overall rejoining efficiency. Thus, it appears that its sensitizing effect on survival is not correlated with impaired dsb rejoining. Caffeine was found to inhibit the rejoining of dsb even after 6 h but the length of G2 phase was normal. By contrast, hypertonically treated cells are blocked in G2 but rejoining of dsb was normal. From these results we conclude that successful rejoining of part of the dsb involves arresting the cells reversibly in G2.

Double-strand break repair and G2 block in Chinese hamster ovary cells and their radiosensitive mutants.

Two X-ray-sensitive mutants of the CHO K1 cell line were examined for their cell-cycle progression after irradiation with gamma-rays, and for their ability to rejoin double-strand breaks (DSBs) as detected by neutral filter elution. Both mutants were impaired in DSB rejoining and both were irreversibly blocked in the G2 phase of the cell cycle as determined by cytofluorometry. From one mutant we have isolated several revertants. The revertants stem from genomic DNA transfection experiments and may have been caused by gene uptake. All revertants survived gamma-irradiation as did the wild-type CHO line. One of them has been examined for its ability to rejoin DSBs and was found to be similar to the wild type.

Radiation induced DNA double strand breaks are rejoined by ligation and recombination processes.

Using the method of filter elution of double stranded DNA under neutral conditions we have shown that most of gamma-ray induced double strand breaks (DSB) are rejoined in both mammalian and bacterial cells. Rejoining also occurs in the G1 phase in V79 Chinese hamster cells and under different growth conditions. Within 8 minutes at 37 C, half the breaks are rejoined. The rejoining in E. coli is equally fast and depends on the presence of DNA ligase. Some of the breaks in E. coli rejoin slowly, and these require rec+. The non-rejoined DSB are distributed over the DNA without any preference for the nucleosomal or the linker structure in the chromosome. Two kinds of DSB rejoining are discriminated, a fast process of DNA ligation and a slower process involving rec functions.

Rejoining of DNA double-strand breaks in human fibroblasts and its impairment in one ataxia telangiectasia and two Fanconi strains.

Using the technique of neutral elution through polycarbonate filters as a measure of DNA length, and hence of the number of double-strand breaks incurred as a result of radiation damage, we found that normal human fibroblasts rejoin 50% of all breaks within only 3 min (37 degrees C). This fast rejoining was impaired in fibroblasts from one patient with Ataxia telangiectasia and in fibroblasts from two patients with Fanconi's anemia. Also the number of residual breaks after several hours of repair was higher than in control cells. Other cases with the same diseases were normal in their rejoining of double-strand breaks.

Negative pion irradiation of mammalian cells. III. A comparative analysis of DNA strand breakage, repair and cell survival after exposure to pi-mesons and X-rays.

The yields of immediate and residual breaks in DNA of X- and peak pion irradiated Chinese hamster cells were measured by hydroxylapatite chromatography in order to investigate the r.b.e. of immediate break production and the relation between residual breaks and survival in the same low dose range of up to 10 Gy. A linear dose response for immediate break induction was observed with an r.b.e. of 0.37 for peak pions, whereas the formation of residual breaks showed a linear-quadratic dose dependence for both types of radiation. The dose-effect curves of residual break can directly be correlated with the corresponding survival curves for both radiation types used indicating that residual breaks are lethal events (most probably unrepaired double strand breaks) formed in this low dose range by single- and multi-hit interactions. Despite their lower efficiency in formation of immediate breaks peak pions produce more residual breaks per dose than X-rays.

Biological effects of negative pions in monolayers and spheroids of Chinese hamster cells.

The therapeutic value of pion irradiation was critically challenged by examining, in a cell culture system, various parameters of biological significance such as relative biological efficiency (RBE), oxygen enhancement ratio (OER), DNA-break formation, changes in cell cycle kinetics and regrowth. In all parameters, pions were superior to conventional low-LET radiation.