Microstructured Scaffolds for Liver Tissue Cultures of High Cell Density: Morphological and Biochemical Characterization of Tissue Aggregates

Journal of Cellular Biochemistry. May, 2005  |  Pubmed ID: 15770659

Very high cell densities and optimal vascularization characterize among others organs and tissues in vivo. In order to study organ-specific functions in vitro or to make use of them in medical devices/treatments in the future, this natural architecture should be rebuilt. An important aspect in this context is the appropriate ratio of medium to cell volume being so far not optimally reestablished in most of the currently available in vitro systems. To improve such culture conditions, we constructed a microstructure to culture hepatocytes and (without any addition of extracellular matrix material) characterized liver tissue in the form of evenly sized aggregates. The liver-specific differentiation status of such aggregates was monitored by their ability to perform CYP450 dependent xenobiotic metabolism along with the measurement of albumin secretion. Freshly isolated adult rat hepatocytes show an initial loss of total CYP450 content and of associated activities (mixed function oxidases). However, in the aggregate system, this level did not decrease further but remained stable or even increased throughout the culture period of 10-13 days. The CYP450 dependent metabolism of the hepatocytes is able to respond to classic inducing agents. The described culture efficiently supports liver-specific functions of adult rat hepatocytes and seems to be suited not only for use in an extracorporeal liver device but also for the formation of evenly sized small aggregates to be of use in transplantation of differentiated liver tissue. Moreover, after design variations, the microstructure can be applied for functional analysis of metabolically active hepatocytes as well as for toxicological and pharmacological validation.

Expression of the Cochaperone HspBP1 is Not Coordinately Regulated with Hsp70 Expression

Cell Biology International. Jun, 2006  |  Pubmed ID: 16677834

Intracellular levels of the heat stress protein Hsp70 are elevated following exposure to elevated temperature. The cochaperone HspBP1 is an intracellular protein that is known to bind to and regulate Hsp70 activity. The purpose of this study was to determine if HspBP1 levels changed when Hsp70 levels were altered. Heat stress resulted in an increase in Hsp70 levels but no change in HspBP1 levels. Treatment of cells with the apoptosis inducing drug camptothecin lowered Hsp70 levels but again had no effect on HspBP1 levels. Cells treated with camptothecin plus heat stress did not exhibit an increase in Hsp70 levels. Over-expression in cells stably transfected with HspBP1 cDNA resulted in a 290% increase in HspBP1 levels without a similar change in Hsp70 levels. These results demonstrate that Hsp70 and HspBP1 are not coordinately regulated but provide evidence that an increase in the ratio of HspBP1 to Hsp70 correlates with apoptosis, in a similar way to reducing the amount of Hsp70.

Expression of HSP72 After ELF-EMF Exposure in Three Cell Lines

Bioelectromagnetics. Oct, 2007  |  Pubmed ID: 17508393

It has been reported that magnetic fields with flux densities ranging from microT to mT are able to induce heat shock factor, HSP72 mRNA or heat shock proteins in various cells. In this study we investigated changes in the HSP72 mRNA transcription level in three cell lines (HL-60, H9c2, and Girardi heart cells) and in the intracellular HSP72 protein content in two cell lines (HL-60 and Girardi heart cells) after treatment schemes using electromagnetic fields with a flux density of 2 microT to 4 mT, a frequency of 50 Hz and exposure times from 15 to 30 min. None of the treatments or modalities showed any significant effect on the HSP72 protein level, although HSP72 mRNA could be induced, at least to some extent, with one of the parameter combinations in all cell lines tested. Obviously, HSP72 mRNA transcription and translation are not necessarily coupled in certain cells. This leads to the conclusion that electromagnetic field effects on HSP72 mRNA levels are not indicative for downstream effects unless increased mRNA levels can be correlated with increased HSP72 protein levels as well.

A Chip-based Platform for the in Vitro Generation of Tissues in Three-dimensional Organization

Lab on a Chip. Jun, 2007  |  Pubmed ID: 17538721

We describe a multi-purpose platform for the three-dimensional cultivation of tissues. The device is composed of polymer chips featuring a microstructured area of 1-2 cm(2). The chip is constructed either as a grid of micro-containers measuring 120-300 x 300 x 300 microm (h x l x w), or as an array of round recesses (300 microm diameter, 300 microm deep). The micro-containers may be separately equipped with addressable 3D-micro-electrodes, which allow for electrical stimulation of excitable cells and on-site measurements of electrochemically accessible parameters. The system is applicable for the cultivation of high cell densities of up to 8 x 10(6) cells and, because of the rectangular grid layout, allows the automated microscopical analysis of cultivated cells. More than 1000 micro-containers enable the parallel analysis of different parameters under superfusion/perfusion conditions. Using different polymer chips in combination with various types of bioreactors we demonstrated the principal suitability of the chip-based bioreactor for tissue culture applications. Primary and established cell lines have been successfully cultivated and analysed for functional properties. When cells were cultured in non-perfused chips, over time a considerable degree of apoptosis could be observed indicating the need for an active perfusion. The system presented here has also been applied for the differentiation analysis of pluripotent embryonic stem cells and may be suitable for the analysis of the stem cell niche.

Chondrogenesis of Mesenchymal Stem Cells in Gel-like Biomaterials in Vitro and in Vivo

Frontiers in Bioscience : a Journal and Virtual Library. 2008  |  Pubmed ID: 18508526

Gel-like carrier materials were introduced into cell therapy of cartilage lesions to improve chondrocyte retention and distribution in the defect. Mesenchymal stem cells (MSC) are now discussed as an alternative cell source for repair. We here asked whether distinct gel-like carriers can support chondrogenesis of MSC in vitro and lead to stable cartilage-like transplants in vivo. Chondrogenesis of MSC embedded in collagen type I gel, fibrin glue, Matrigel and PuraMatrix peptide hydrogel was assessed and gene expression analysis, proteoglycan content, and collagen synthesis were quantified. Differentiated constructs were transplanted subcutaneously into SCID mice. All carriers supported chondrogenesis in vitro, but displayed material-dependent differences on COL2A1 gene expression, total collagen synthesis and proteoglycan deposition. The undesired calcification and microossicle formation in ectopic transplants in vivo was consistently suppressed by Matrigel. In sum, gel-like biomaterials were suitable carriers for MSC and promoted chondrogenesis. Suppression of calcification by particular gel-like materials makes their use even more attractive for MSC-based tissue engineering approaches in cartilage repair.

Tissue Reconstruction in 3D-spheroids from Rodent Retina in a Motion-free, Bioreactor-based Microstructure

Lab on a Chip. Dec, 2008  |  Pubmed ID: 19023488

While conventional rotation culture-based retinal spheroids are most useful to study basic processes of retinogenesis and tissue regeneration, they are less appropriate for an easy and inexpensive mass production of histotypic 3-dimensional tissue spheroids, which will be of utmost importance for future bioengineering, e.g. for replacement of animal experimentation. Here we compared conventionally reaggregated spheroids derived from dissociated retinal cells from neonatal gerbils (Meriones unguiculatus) with spheroids cultured on a novel microscaffold cell chip (called cf-chip) in a motion-free bioreactor. Reaggregation and developmental processes leading to tissue formation, e.g. proliferation, apoptosis and differentiation were observed during the first 10 days in vitro (div). Remarkably, in each cf-chip micro-chamber, only one spheroid developed. In both culture systems, sphere sizes and proliferation rates were almost identical. However, apoptosis was only comparably high up to 5 div, but then became negligible in the cf-chip, while it up-rose again in the conventional culture. In both systems, immunohistochemical characterisation revealed the presence of Müller glia cells, of ganglion, amacrine, bipolar and horizontal cells at a highly comparable arrangement. In both systems, photoreceptors were detected only in spheroids from P3 retinae. Benefits of the chip-based 3D cell culture were a reliable sphere production at enhanced viability, the feasibility of single sphere observation during cultivation time, a high reproducibility and easy control of culture conditions. Further development of this approach should allow high-throughput systems not only for retinal but also other types of histotypic spheroids, to become suitable for environmental monitoring and biomedical diagnostics.

The Famous Versus the Inconvenient - or the Dawn and the Rise of 3D-culture Systems

World Journal of Stem Cells. Dec, 2009  |  Pubmed ID: 21607106

One of the greatest impacts on in vitro cell biology was the introduction of three-dimensional (3D) culture systems more than six decades ago and this era may be called the dawn of 3D-tissue culture. Although the advantages were obvious, this field of research was a "sleeping beauty" until the 1970s when multicellular spheroids were discovered as ideal tumor models. With this rebirth, organotypical culture systems became valuable tools and this trend continues to increase. While in the beginning, simple approaches, such as aggregation culture techniques, were favored due to their simplicity and convenience, now more sophisticated systems are used and are still being developed. One of the boosts in the development of new culture techniques arises from elaborate manufacturing and surface modification techniques, especially micro and nano system technologies that have either improved dramatically or have evolved very recently. With the help of these tools, it will soon be possible to generate even more sophisticated and more organotypic-like culture systems. Since 3D perfused or superfused systems are much more complex to set up and maintain compared to use of petri dishes and culture flasks, the added value of 3D approaches still needs to be demonstrated.

Biosensors Coated with Sulfated Polysaccharides for the Detection of Hepatocyte Growth Factor/scatter Factor in Cell Culture Medium

Biosensors & Bioelectronics. Dec, 2010  |  Pubmed ID: 20719493

Process control methods for cell culture bioreactors include on-line monitoring of protein concentrations. Bioreactor samples typically contain high amounts of different proteins. The direct detection of a single protein in this complex medium is a challenging task within the development of biosensors with label-free detection. We introduce the development of a mass-sensitive biosensor based on surface acoustic waves (SAW) for the detection of hepatocyte growth factor/scatter factor (HGF/SF) in the serum containing medium of a miniaturized bioreactor for culturing hepatocytes. The specificity of the biosensor was obtained following two approaches. In the first approach, antibodies against HGF (anti-HGF) were immobilized covalently via an intermediate layer of dicarboxy polyethylene glycol on the biosensor surface. In the second approach, dextran sulfate and fucoidan were used as sensor coatings exploiting the fact that HGF binds specifically to those sulfated polysaccharides. Performing HGF assays, similar results were obtained using biosensors coated with dextran sulfate and biosensors coated with anti-HGF. Even higher sensor signals were obtained using biosensors coated with fucoidan, particularly at 37°C. Therefore, biosensor coatings based on biospecific sulfated polysaccharides offer a simple and cost-saving alternative compared to the commonly used coating with antibodies.

Thermoforming of Film-based Biomedical Microdevices

Advanced Materials (Deerfield Beach, Fla.). Mar, 2011  |  Pubmed ID: 21400590

For roughly ten years now, a new class of polymer micromoulding processes comes more and more into the focus both of the microtechnology and the biomedical engineering community. These processes can be subsumed under the term "microthermoforming". In microthermoforming, thin polymer films are heated to a softened, but still solid state and formed to thin-walled microdevices by three-dimensional stretching. The high material coherence during forming is in contrast to common polymer microreplication processes where the material is processed in a liquid or flowing state. It enables the preservation of premodifications of the film material. In this progress report, we review the still young state of the art of microthermoforming technology as well as its first applications. So far, the applications are mainly in the biomedical field. They benefit from the fact that thermoformed microdevices have unique properties resulting from their special, unusual morphology. The focus of this paper is on the impact of the new class of micromoulding processes and the processed film materials on the characteristics of the moulded microdevices and on their applications.

Promotion of Osteoblast Differentiation in 3D Biomaterial Micro-chip Arrays Comprising Fibronectin-coated Poly(methyl Methacrylate) Polycarbonate

Biomaterials. Dec, 2011  |  Pubmed ID: 21868090

Due to the architecture of solid body tissues including bone, three-dimensional (3D) in vitro microenvironments appear favorable, since herein cell growth proceeds under more physiological conditions compared to conventional 2D systems. In the present study we show that a 3D microenvironment comprising a fibronectin-coated PMMA/PC-based micro-chip promotes differentiation of primary human osteoblasts as reflected by the densely-packed 3D bone cell aggregates and expression of biomarkers indicating osteoblast differentiation. Morphogenesis and fluorescence dye-based live/dead staining revealed homogenous cell coverage of the microcavities of the chip array, whereat cells showed high viability up to 14 days. Moreover, Azur II staining proved formation of uniform sized multilayered aggregates, exhibiting progressive intracellular deposition of extracellular bone matrix constituents comprising fibronectin, osteocalcin and osteonectin from day 7 on. Compared to 2D monolayers, osteoblasts grown in the 3D chip environment displayed differential mostly higher gene expression for osteocalcin, osteonectin, and alkaline phosphatase, while collagen type I remained fairly constant in both culture environments. Our results indicate that the 3D microenvironment, based on the PMMA biomaterial chip array promotes osteoblast differentiation, and hereby renders a promising tool for tissue-specific in vitro preconditioning of osteoblasts designated for clinically-oriented bone augmentation or regeneration.

Closer to Nature-bio-inspired Patterns by Transforming Latent Lithographic Images

Advanced Materials (Deerfield Beach, Fla.). Nov, 2011  |  Pubmed ID: 21935996