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In JoVE (2)
- Chip-op basis van drie-dimensionale Cultuur van de Cel in doorbloed Micro-bioreactoren
- Microfabricage van Chip-en kleinbedrijf Stellingen voor drie-dimensionale celkweek
Other Publications (17)
- Journal of Biomaterials Science. Polymer Edition
- Journal of Neuroscience Methods
- Biomolecular Engineering
- Biomaterials
- World Journal of Stem Cells
- Langmuir : the ACS Journal of Surfaces and Colloids
- Biomedical Microdevices
- Molecular Pharmaceutics
- Biosensors & Bioelectronics
- Journal of Biomedical Optics
- European Journal of Pharmaceutical Sciences : Official Journal of the European Federation for Pharmaceutical Sciences
- Drug Delivery
- Lab on a Chip
- Biomaterials
- Biomedical Microdevices
- Biointerphases
- Angewandte Chemie (International Ed. in English)
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Articles by Alexander Welle in JoVE
JoVE General
Chip-op basis van drie-dimensionale Cultuur van de Cel in doorbloed Micro-bioreactoren
Institute for Biological Interfaces, Forschungszentrum Karlsruhe
Beschrijven we een chip-gebaseerde platform voor de drie-dimensionale kweken van cellen in micro-bioreactoren. Een chip kan maximaal huis tot 10 miljoen. cellen die kunnen worden geteeld onder nauwkeurig omschreven voorwaarden met betrekking tot stroming, zuurstofspanning enz. in een steriele, gesloten circulatie lus.
JoVE General
Microfabricage van Chip-en kleinbedrijf Stellingen voor drie-dimensionale celkweek
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
We presenteren twee processen voor de microfabricage van poreuze polymeer chips voor drie-dimensionale celkweek. De eerste is hot embossing gecombineerd met een damp van oplosmiddelen lasproces. De tweede maakt gebruik van een recent ontwikkelde microthermoforming proces in combinatie met ion-Track technologie leidt tot een aanzienlijke vereenvoudiging van de productie.
Other articles by Alexander Welle on PubMed
Competitive Plasma Protein Adsorption on Modified Polymer Surfaces Monitored by Quartz Crystal Microbalance Technique
This paper describes the effects of photochemical modifications of polymer surfaces on the competitive adsorption of serum proteins and cell adhesion (hepatoma cell line HepG2, L929 fibroblasts and others). The UV modification of polystyrene, poly(methylmethacrylate) and polycarbonate alters the physico-chemical properties of these polymers in a way that allows the formation of micrometer scaled cellular patterns in vitro by controlling the composition and properties of the protein adsorbate. Using a quartz microbalance technique, capable to extract viscoelastic data in addition to the mass load of the polymer coated sensor, we have demonstrated the importance of the thickness and the viscosity of an albumin adsorbate for the observed cell adhesion in vitro. The quantity and viscosity of surface bound albumin on polystyrene, being a cell repellent material in its native state, is lowered when the surface is exposed to UV of lambda = 185 nm in air prior to the contact with albumin solutions or cell culture media. This promotes the deposition of cell adhesion proteins and explains the observed cell patterns. Apart from this special application the described quartz microbalance with dissipation monitoring provides a useful tool for general biocompatibility studies based on surface phenomena of biomaterials.
Photo-chemically Patterned Polymer Surfaces for Controlled PC-12 Adhesion and Neurite Guidance
The in vitro assembling of cellular networks offering control over cell positions and connectivities by patterned culture substrates is a valuable tool for neuroscience research and other applications in cell biology. We developed a versatile technique based on polymer surface modification which allows the patterning of different cell lines for advanced tissue engineering, among them are Pheochromocytoma cells (PC-12). In contrast to other techniques applied for surface patterning, the presented photo patterning by deep UV irradiation is applicable to the widely used cell culture substrate material polystyrene (PS) and should be easily performed in most laboratories. Irradiation of polystyrene with UV radiation of lambda = 185 nm yields mainly carboxyl groups at the polymer surface which can be used to control the spontaneous competitive protein adsorption from serum containing culture media [Welle A, Gottwald E. UV-based patterning of polymeric substrates for cell culture applications. Biomed. Microdev. 2002;4:33-41] or to serve as defined coupling sites for controlled protein/peptide immobilization. Extending our previous studies on patterning hepatoma cells and fibroblasts via spatially defined plasma protein adsorption, we here describe an advanced application to produce patterns of cell repellent albumin domains and cell attractive laminin regions for the patterning of Pheochromocytoma cells.
Competitive Protein Adsorption on Micro Patterned Polymeric Biomaterials, and Viscoelastic Properties of Tailor Made Extracellular Matrices
Cell adhesion on biomaterial surfaces and the vitality of anchorage dependent cells is affected by several parameters of an adsorbate layer which is intentionally or spontaneously formed. Surface pre-treatments and several conditioning steps prior and during to the cell/biomaterial contact affect the composition, orientation, quantity and viscoelasticity of the interfacing layer between cells and biomaterial. This work was performed to elucidate the response of cells on two modified biomaterial surfaces based on protein or carbohydrate adsorbates: (a) Masked UV irradiations opened a simple route to obtain chemically patterned substrates controlling serum protein adsorption and cell adhesion. It is possible to achieve structures of subcellular size and to produce immobilized gradients. In order to examine the protein matrix deposited on these substrates we applied a quartz microbalance technique (QCM-D) capable to extract viscoelastic data in addition to the mass uptake during plasma protein deposition. It was found that the quantity and viscosity of surface bound albumin is lowered when the surface is modified (patterned) by UV exposure. Hence, the UV modification promotes the competitive adsorption of cell adhesion proteins from the media or upon secretion by the cells and yields to the observed cell patterns. (b) Another tissue engineering technique, using immobilized, modified and/or cross linked hyaluronic acid (HA), an important extra cellular matrix component in vivo, is also examined by QCM-D. Our data demonstrate that HA can be modified by an activation with a carbodiimide, followed by the application of an alpha,omega-bisamino polyethyleneglycol. The QCM-D data can be interpreted as a stiffening of the HA layer combined with the release of hydration water. Further, the hydration state and the viscoelastic behaviour of surface bound ultrathin HA hydrogels was examined. Quantification of viscoelastic parameters of thin films of ECM by QCM-D is valuable for the interpretation of durotaxis, describing effects of mechanical substrate parameters on the adhesion and motility of cells.
Electrospun Aliphatic Polycarbonates As Tailored Tissue Scaffold Materials
Two different aliphatic polycarbonates were synthesised from CO(2) and the respective epoxides. Poly(propyl carbonate) (PPC) was prepared by heterogeneous catalysis with zinc glutarate. Poly(cyclohexyl carbonate) (PCHC) was prepared via living copolymerisation homogeneously catalysed by a 3-amino-2-cyanoimidoacrylate zinc acetate complex and subjected to electrospinning. The obtained nanofibres had a well-defined morphology free of beads along the fibres and with slightly porous structures on their surface. Subsequently, low-power deep UV irradiations, previously applied for photochemical surface modifications of two-dimensional and three-dimensional scaffolds from biostable polymers, were performed. Here, an effect on surface and bulk properties of PPC nanofibres was observed. Surface modifications of both polymers affected plasma protein adsorption. Photochemical bulk modifications observed for the first time on PPC nanofibres are indicating the possibility of spatial control of biodegradation rates, hence allow for control of the progression of host/implant interactions in vivo. In particular PPC was used for cell culture of L929 fibroblasts and primary rat hepatocytes. Even delicate primary cells showed good adhesion to the scaffolds and high viability.
The Famous Versus the Inconvenient - or the Dawn and the Rise of 3D-culture Systems
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.
Benzylguanine Thiol Self-assembled Monolayers for the Immobilization of SNAP-tag Proteins on Microcontact-printed Surface Structures
The site-selective, oriented, covalent immobilization of proteins on surfaces is an important issue in the establishment of microarrays, biosensors, biocatalysts, and cell assays. Here we describe the preparation of self-assembled monolayers consisting of benzylguanine thiols (BGT) to which SNAP-tag fusion proteins can be covalently linked. The SNAP-tag, a modified O(6)-alkylguanine-DNA alkyltransferase (AGT), reacts with the headgroup of BGT and becomes covalently bound upon the release of guanine. Bacterially produced recombinant His-tag-SNAP-tag-GFP was used to demonstrate the site-specific immobilization on BGT surface patterns created by microcontact printing (microCP). With this versatile method, any SNAP-tag protein can be coupled to a surface.
Spatially Controlled Cell Adhesion on Three-dimensional Substrates
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.
Nanoparticles Based on PLGA:Poloxamer Blends for the Delivery of Proangiogenic Growth Factors
New blood vessel formation is a critical requirement for treating many vascular and ischemia related diseases, as well as for many tissue engineering applications. Angiogenesis and vasculogenesis, in fact, represent crucial processes for the functional regeneration of complex tissues through tissue engineering strategies. Several growth factors (GFs) and signaling molecules involved in blood vessels formation have been identified, but their application to the clinical setting is still strongly limited by their extremely short half-life in the body. To overcome these limitations, we have developed a new injectable controlled release device based on polymeric nanoparticles for the delivery of two natural proangiogenic GFs: platelet derived growth factor (PDGF-BB) and fibroblast growth factor (FGF-2). The nanoparticle system was prepared by a modified solvent diffusion technique, encapsulating the GF both in presence and in the absence of two stabilizing agents: bovine serum albumin (BSA) and heparin sodium salt (Hp). The developed nanocarriers were characterized for morphology, size, encapsulation efficiency, release kinetics in vitro and GF activity in cell cultures. The results have indicated that the coencapsulation of stabilizing agents can preserve the GF active structure and, in addition, increase their encapsulation efficiency into nanoparticles. Through this optimization process, we were able to raise the encapsulation efficiency of FGF-2 to 63%, and that of PDGF-BB to 87%. These PLGA:poloxamer blend nanoparticles loaded with GFs were able to release PDGF-BB and FGF-2 in a sustained fashion for more than a month. This work also confirms other positive features of PLGA:poloxamer nanoparticles. Namely, they are able to maintain their stability in simulated biological medium, and they are also nontoxic to cell culture models. Incubation of nanoparticles loaded with FGF-2 or PDGF-BB with endothelial cell culture models has confirmed that GFs are released in a bioactive form. Altogether, these results underline the interest of PLGA:poloxamer nanoparticles for the controlled delivery of GFs and substantiate their potential for the treatment of ischemic diseases and for tissue engineering applications.
Biosensors Coated with Sulfated Polysaccharides for the Detection of Hepatocyte Growth Factor/scatter Factor in Cell Culture Medium
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.
Fluorescence Excitation on Monolithically Integrated All-polymer Chips
All-polymer chips with integrated optical waveguides and microfluidic channels were built as a platform for fluorescence excitation of biological samples. Their functionality has been shown for four cases: (i) fluorescence of labeled phospholipids inside a microfluidic channel, (ii) fluorescence of stained cells inside a microfluidic channel, (iii) fluorescence of labeled phospholipids on top of a polymer waveguide, and (iv) fluorescence of stained cells on top of a polymer waveguide.
PLGA:poloxamer Blend Micro- and Nanoparticles As Controlled Release Systems for Synthetic Proangiogenic Factors
Tissue engineering is one of the most promising research areas in bioregenerative medicine. However, the restoration of biological functionalities by implanting bioartificially engineered tissues is still highly limited because of their lack of vascular networks. The use of proangiogenic molecules delivered from a controlled release device is a promising strategy to induce tissue vascularization. Indeed, the controlled release system can enhance the therapeutic effect in vivo of many short half-life drugs, while circumventing the need for repeated administrations. In this work, PLGA:poloxamer blend based micro- and nanoparticles have been developed for the sustained delivery of a recently developed synthetic proangiogenic compound: SHA-2-22. Drug-loaded PLGA:poloxamer blend microparticles were prepared by an oil-in-oil solvent extraction/evaporation technique. Drug-loaded PLGA:poloxamer nanoparticles were prepared by a modified solvent diffusion technique. These drug carriers were characterized with regard to their physicochemical properties, morphology, drug encapsulation efficiency and release kinetics in vitro. The results show that by adjusting the formulation conditions, it is possible to obtain PLGA:poloxamer micro- and nanoparticles with very high drug loadings, and with the capacity to release the active compound in a controlled way for up to one month. In vitro cell assays performed in an endothelial cell model confirmed the bioactivity of SHA-22-2 encapsulated in PLGA:poloxamer microparticles.
Hyaluronic Acid/Chitosan Nanoparticles As Delivery Vehicles for VEGF and PDGF-BB
The development of a vascular network in tissue-engineered constructs is a fundamental bottleneck of bioregenerative medicine, particularly when the size of the implant exceeds a certain limit given by diffusion lengths and/or if the host tissue shows a very active metabolism. One of the approaches to achieve the vascularization of tissue constructs is generating a sustained release of proangiogenic factors from the ischemic site. This work describes the formation and characterization of hyaluronic acid-chitosan (HA/CS) nanoparticles for the delivery of two pro-angiogenic growth factors: vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF-BB). These nanoparticles were prepared by an ionic gelification technique, and different formulations were developed by encapsulating the growth factors in association with two stabilizing agents: bovine serum albumin or heparin sodium salt. These carriers were characterized with regard to their physicochemical properties, their stability in biological media, and their cytotoxicity in the C3a hepatoma cell line. The results show that nanoparticles around 200 nm can be prepared by this method. HA/CS nanoparticles were stable when incubated in EMEM cell culture medium or in water at 37°C for 24 h. Cell culture tests confirmed that HA/CS nanoparticles are not cytotoxic within the concentration range used for growth factor delivery. Moreover, HA/CS nanoparticles were able to entrap efficiently both growth factors, reaching association values of 94% and 54% for VEGF and PDGF, respectively. In vitro release studies confirm that PDGF-BB is released from HA/CS nanoparticles in a sustained manner over approximately 1 week. On the other hand, VEGF is completely released within the first 24 h.
Rapid Prototyping of Microstructures in Polydimethylsiloxane (PDMS) by Direct UV-lithography
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.
Selective Immobilization of Sonic Hedgehog on Benzylguanine Terminated Patterned Self-assembled Monolayers
Patterned two-component, self-assembled monolayers on gold were produced by UV lithography. An oligo(ethylene glycol) terminated disulfide served as inert matrix reducing unspecific protein adsorption and cell adhesion. The second component of the self-assembled monolayer (SAM) presented a benzylguanine moiety for the immobilization of Sonic hedgehog (Shh) fused to a mutant O(6)-alkylguanine-DNA alkyltransferase (SNAP-tag™). The enzymatic activity of the SNAP-tag allows selective and covalent immobilization of the linked Shh. Time-of-flight secondary ion mass spectrometry verified the correct lateral distribution of the benzylguanine head groups in the patterned SAM. The quantification of unspecific and specific protein binding to mixed SAMs showed increased adsorption of albumin with increasing benzylguanine/(ethylene glycol) ratios. However, the immobilization of SNAP-tagged Shh was not blocked by pre-adsorbed albumin. Furthermore, the obtained micro-patterned substrates permitted direct immobilization of SNAP-tagged Shh even in the presence of many competing proteins from conditioned media of transfected HEK293 cells. Therefore, the presented system is suited for the controlled immobilization of fusion proteins from complex mixtures avoiding purification steps.
Microstructuring of Multiwell Plates for Three-dimensional Cell Culture Applications by Ultrasonic Embossing
Since three-dimensional (3D) cell culture models better reflect tissues in vivo in terms of cell shape and microenvironment compared to conventional monolayer cultures, 3D tissue culture substrates gain more importance for a wide range of biological applications like drug discovery, toxicological studies, cancer and stem cell research. In this study we developed a method for the fabrication of 3D cell culture substrates in a multiwell plate format by microstructuring the bottom of 96-well cell culture plates using an ultrasonic embossing process. The resulting microstructured area consists of cubic microcavities in which adherent multicellular aggregates can be formed. We performed the biological evaluation of the system with the liver-derived human cell-line HepG2 and compared the novel substrate with a commercially available 3D culture system comprising porous alginate sponges. Metabolic activity (alamarBlue® reduction) and induction of four biotransformation enzymes (EROD, ECOD, UGT, SULT) were determined by fluorimetry or HPLC. Our results revealed that HepG2 cells in microstructured plates showed a higher mitochondrial activity, as well as enzyme activity of ECOD and UGT after treatment with an inducer when compared to cells cultured in alginate sponges at otherwise comparable conditions. Since we have modified standard cell culture plates, the obtained system is adaptable to automated screening and might be useful for all kinds of cultures including adult, progenitor and stem cells which need a 3D culture configuration to restore or maintain the differentiated status.
In Vitro Observation of Dynamic Ordering Processes in the Extracellular Matrix of Living, Adherent Cells
Collecting information at the interface between living cells and artificial substrates is exceedingly difficult. The extracellular matrix (ECM) mediates all cell-substrate interactions, and its ordered, fibrillar constituents are organized with nanometer precision. The proceedings at this interface are highly dynamic and delicate. In order to understand factors governing biocompatibility or its counterpart antifouling, it is necessary to probe this interface without disrupting labels or fixation and with sufficient temporal resolution. Here the authors combine nonlinear optical spectroscopy (sum-frequency-generation) and microscopy (second-harmonic-generation), fluorescence microscopy, and quartz crystal microgravimetry with dissipation monitoring in a strategy to elucidate molecular ordering processes in the ECM of living cells. Artificially (fibronectin and collagen I) and naturally ordered ECM fibrils (zebrafish, Danio rerio) were subjected to nonlinear optical analysis and were found to be clearly distinguishable from the background signals of diffusive proteins in the ECM. The initial steps of fibril deposition and ordering were observed in vitro as early as 1 h after cell seeding. The ability to follow the first steps of cell-substrate interactions in spite of the low amount of material present at this interface is expected to prove useful for the assessment of biomedical and environmental interfaces.
Adding Spatial Control to Click Chemistry: Phototriggered Diels-Alder Surface (Bio)functionalization at Ambient Temperature
A photoconjugation strategy based on light-triggered Diels-Alder addition of o-quinodimethanes is compatible with biomolecules and proceeds rapidly at ambient temperature without the need of a catalyst. Spatial control was confirmed by photopatterning of a small-molecule ATRP initiator, a polymer, and a peptide in a time-of-flight secondary-ion mass spectrometry investigation.
