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In JoVE (2)
- चिप आधारित तीन आयामी perfused लघु बायोरिएक्टर में सेल संस्कृति
- चिप आकार scaffolds के तीन आयामी सेल की खेती के लिए Microfabrication
Other Publications (9)
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Articles by Stefan Giselbrecht in JoVE
चिप आधारित तीन आयामी perfused लघु बायोरिएक्टर में सेल संस्कृति
Eric Gottwald, Brigitte Lahni, David Thiele, Stefan Giselbrecht, Alexander Welle, Karl-Friedrich Weibezahn
Institute for Biological Interfaces, Forschungszentrum Karlsruhe
हम सूक्ष्म बायोरिएक्टर में तीन आयामी कोशिकाओं की खेती के लिए चिप आधारित एक मंच का वर्णन. एक चिप ऊपर 10 Mio के लिए घर कर सकते हैं. कोशिकाओं है कि एक बाँझ, बंद संचलन पाश में द्रव का प्रवाह है, ऑक्सीजन तनाव आदि के संबंध के साथ ठीक से परिभाषित शर्तों के तहत खेती की जा सकती.
चिप आकार scaffolds के तीन आयामी सेल की खेती के लिए Microfabrication
Stefan Giselbrecht1, Eric Gottwald1, Roman Truckenmueller2, Christina Trautmann3, Alexander Welle1, Andreas Guber4, Volker Saile4, Thomas Gietzelt5, Karl-Friedrich Weibezahn1
1Institute for Biological Interfaces, Karlsruhe Research Centre, 2Institute for BioMedical Technology, University of Twente, 3Department of Materials Research, Institute for Heavy Ion Research, 4Institute of Microstructure Technology, Karlsruhe Research Centre, 5Institute for Micro Process Engineering, Karlsruhe Research Centre
हम तीन आयामी सेल की खेती के लिए झरझरा बहुलक चिप्स के microfabrication के लिए दो प्रक्रियाओं को प्रस्तुत करते हैं. पहले एक गर्म embossing एक विलायक वाष्प वेल्डिंग की प्रक्रिया के साथ संयुक्त है. दूसरा एक एक हाल ही में विकसित microthermoforming प्रक्रिया आयन ट्रैक निर्माण का एक महत्वपूर्ण सरलीकरण प्रमुख प्रौद्योगिकी के साथ संयुक्त का उपयोग करता है.
Other articles by Stefan Giselbrecht on PubMed
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.
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.
Biomedical Microdevices. Oct, 2010 | Pubmed ID: 20480241
The microenvironment of cells in vivo is defined by spatiotemporal patterns of chemical and biophysical cues. Therefore, one important goal of tissue engineering is the generation of scaffolds with defined biofunctionalization in order to control processes like cell adhesion and differentiation. Mimicking extrinsic factors like integrin ligands presented by the extracellular matrix is one of the key elements to study cellular adhesion on biocompatible scaffolds. By using special thermoformable polymer films with anchored biomolecules micro structured scaffolds, e.g. curved and micro-patterned substrates, can be fabricated. Here, we present a novel strategy for the fabrication of micro-patterned scaffolds based on the "Substrate Modification and Replication by Thermoforming" (SMART) technology: The surface of a poly lactic acid membrane, having a low forming temperature of 60 degrees C and being initially very cell attractive, was coated with a photopatterned layer of poly(L-lysine) (PLL) and hyaluronic acid (VAHyal) to gain spatial control over cell adhesion. Subsequently, this modified polymer membrane was thermoformed to create an array of spherical microcavities with diameters of 300 microm for 3D cell culture. Human hepatoma cells (HepG2) and mouse fibroblasts (L929) were used to demonstrate guided cell adhesion. HepG2 cells adhered and aggregated exclusively within these cavities without attaching to the passivated surfaces between the cavities. Also L929 cells adhering very strongly on the pristine substrate polymer were effectively patterned by the cell repellent properties of the hyaluronic acid based hydrogel. This is the first time cell adhesion was controlled by patterned functionalization of a polymeric substrate with UV curable PLL-VAHyal in thermoformed 3D microstructures.
Lab on a Chip. Apr, 2011 | Pubmed ID: 21327278
Microstructuring of polydimethylsiloxane (PDMS) is a key step for many lab-on-a-chip (LOC) applications. In general, the structure is generated by casting the liquid prepolymer against a master. The production of the master in turn calls for special equipment and know how. Furthermore, a given master only allows the reproduction of the defined structure. We report on a simple, cheap and practical method to produce microstructures in already cured PDMS by direct UV-lithography followed by chemical development. Due to the available options during the lithographic process like multiple exposures, the method offers a high design flexibility granting easy access to complex and stepped structures. Furthermore, no master is needed and the use of pre-cured PDMS allows processing at ambient (light) conditions. Features down to approximately 5 µm and a depth of 10 µm can be realised. As a proof of principle, we demonstrate the feasibility of the process by applying the structures to various established soft lithography techniques.
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.
BioTechniques. May, 2011 | Pubmed ID: 21548893
The development of automated microscopy platforms has enabled large-scale observation of biological processes, thereby complementing genome scale biochemical techniques. However, commercially available systems are restricted either by fixed-field-of-views, leading to potential omission of features of interest, or by low-resolution data of whole objects lacking cellular detail. This limits the efficiency of high-content screening assays, especially when large complex objects are used as in whole-organism screening. Here we demonstrate a toolset for automated intelligent high-content screening of whole zebrafish embryos at cellular resolution on a standard wide-field screening microscope. Using custom-developed algorithms, predefined regions of interest-such as the brain-are automatically detected. The regions of interest are subsequently imaged automatically at high magnification, enabling rapid capture of cellular resolution data. We utilize this approach for acquiring 3-D datasets of embryonic brains of transgenic zebrafish. Moreover, we report the development of a mold design for accurate orientation of zebrafish embryos for dorsal imaging, thereby facilitating standardized imaging of internal organs and cellular structures. The toolset is flexible and can be readily applied for the imaging of different specimens in various 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.
Advanced Materials (Deerfield Beach, Fla.). Nov, 2011 | Pubmed ID: 21935996
Biomedical Microdevices. Nov, 2011 | Pubmed ID: 22048776
This paper presents cell culture substrates in the form of microcontainer arrays with overlaid surface topographies, and a technology for their fabrication. The new fabrication technology is based on microscale thermoforming of thin polymer films whose surfaces are topographically prepatterned on a micro- or nanoscale. For microthermoforming, we apply a new process on the basis of temporary back moulding of polymer films and use the novel concept of a perforated-sheet-like mould. Thermal micro- or nanoimprinting is applied for prepatterning. The novel cell container arrays are fabricated from polylactic acid (PLA) films. The thin-walled microcontainer structures have the shape of a spherical calotte merging into a hexagonal shape at their upper circumferential edges. In the arrays, the cell containers are arranged densely packed in honeycomb fashion. The inner surfaces of the highly curved container walls are provided with various topographical micro- and nanopatterns. For a first validation of the microcontainer arrays as in vitro cell culture substrates, C2C12 mouse premyoblasts are cultured in containers with microgrooved surfaces and shown to align along the grooves in the three-dimensional film substrates. In future stem-cell-biological and tissue engineering applications, microcontainers fabricated using the proposed technology may act as geometrically defined artificial microenvironments or niches.