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- ItemA hydroquinone-specific screening system for directed P450 evolution(Berlin ; Heidelberg ; New York : Springer, 2018) Weingartner, Alexandra M.; Sauer, Daniel F.; Dhoke, Gaurao V.; Davari, Mehdi D.; Ruff, Anna Joëlle; Schwaneberg, UlrichThe direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 μM) and a broad linear detection range (5 to 250 μM) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M). © 2018, The Author(s).
- ItemTowards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes(New York, NY [u.a.] : Hindawi Publ. Corp., 2017) Menzel, Sarah; Finocchiaro, Nicole; Donay, Christine; Thiebes, Anja Lena; Hesselmann, Felix; Arens, Jutta; Djeljadini, Suzana; Wessling, Matthias; Schmitz-Rode, Thomas; Jockenhoevel, Stefan; Cornelissen, Christian GabrielIn patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.
- ItemWetting-Induced Polyelectrolyte Pore Bridging(Basel : MDPI, 2021) Kalde, Anna; Kamp, Johannes; Evdochenko, Elizaveta; Linkhorst, John; Wessling, MatthiasActive layers of ion separation membranes often consist of charged layers that retain ions based on electrostatic repulsion. Conventional fabrication of these layers, such as polyelectrolyte deposition, can in some cases lead to excess coating to prevent defects in the active layer. This excess deposition increases the overall membrane transport resistance. The study at hand presents a manufacturing procedure for controlled polyelectrolyte complexation in and on porous supports by support wetting control. Pre-wetting of the microfiltration membrane support, or even supports with larger pore sizes, leads to ternary phase boundaries of the support, the coating solution, and the pre-wetting agent. At these phase boundaries, polyelectrolytes can be complexated to form partially freestanding selective structures bridging the pores. This polyelectrolyte complex formation control allows the production of membranes with evenly distributed polyelectrolyte layers, providing (1) fewer coating steps needed for defect-free active layers, (2) larger support diameters that can be bridged, and (3) a precise position control of the formed polyelectrolyte multilayers. We further analyze the formed structures regarding their position, composition, and diffusion dialysis performance.
- ItemTrypsin-Free Cultivation of 3D Mini-Tissues in an Adaptive Membrane Bioreactor(Weinheim : Wiley-VCH, 2020) Djeljadini, Suzana; Lohaus, Theresa; Gausmann, Marcel; Rauer, Sebastian; Kather, Michael; Krause, Bernd; Pich, Andrij; Möller, Martin; Wessling, MatthiasThe production of large scaffold-free tissues is a key challenge in regenerative medicine. Nowadays, temperature-responsive polymers allow intact tissue harvesting without needing proteolytic enzymes. This method is limited to tissue culture plastic with limited upscaling capacity and plain process control. Here, a thermoresponsive hollow fiber membrane bioreactor is presented to produce large scaffold-free tissues. Intact tissues, rich in cell-to-cell connections and ECM, are harvested from a poly(N-vinylcaprolactam) microgel functionalized poly(ether sulfone)/poly(vinylpyrrolidone) hollow fiber membrane by a temperature shift. The harvested 3D tissues adhere in successive cultivation and exhibit high vitality for several days. The facile adsorptive coating waives the need for extensive surface treatment. The research is anticipated to be a starting point for upscaling the production of interconnected tissues enabling new opportunities in regenerative medicine, large-scale drug screening on physiological relevant tissues, and potentially opening new chances in cell-based therapies. © 2020 The Authors. Advanced Biosystems published by Wiley-VCH GmbH
- ItemAll-Conjugated Polymer Core-Shell and Core-Shell-Shell Particles with Tunable Emission Profiles and White Light Emission(Weinheim : Wiley-VCH, 2021) Haehnle, Bastian; Schuster, Philipp A.; Chen, Lisa; Kuehne, Alexander J. C.Future applications of conjugated polymer particles (CPP) in medicine, organic photonics, and optoelectronics greatly depend on high performance and precisely adjustable optical properties of the particles. To meet these criteria, current particle systems often combine conjugated polymers with inorganic particles in core-shell geometries, extending the possible optical characteristics of CPP. However, current conjugated polymer particles are restricted to a single polymer phase composed of a distinct polymer or a polymer blend. Here, a synthetic toolbox is presented that enables the synthesis of monodisperse core-shell and core-shell-shell particles, which consist entirely of conjugated polymers but of different types in the core and the shells. Seeded and fed-batch dispersion polymerizations based on Suzuki-Miyaura-type cross-coupling are investigated. The different approaches allow accurate control over the created interface between the conjugated polymer phases and thus also over the energy transfer phenomena between them. This approach opens up completely new synthetic freedom for fine tuning of the optical properties of CPP, enabling, for example, the synthesis of individual white light-emitting particles.
- ItemSynthetic 3D PEG-Anisogel Tailored with Fibronectin Fragments Induce Aligned Nerve Extension(Columbus, Ohio : American Chemical Society, 2019) Licht, Christopher; Rose, Jonas C.; Anarkoli, Abdolrahman Omidinia; Blondel, Delphine; Roccio, Marta; Haraszti, Tamás; Gehlen, David B.; Hubbell, Jeffrey A.; Lutolf, Matthias P.; De Laporte, LauraAn enzymatically cross-linked polyethylene glycol (PEG)-based hydrogel was engineered to promote and align nerve cells in a three-dimensional manner. To render the injectable, otherwise bioinert, PEG-based material supportive for cell growth, its mechanical and biochemical properties were optimized. A recombinant fibronectin fragment (FNIII9*-10/12-14) was coupled to the PEG backbone during gelation to provide cell adhesive and growth factor binding domains in close vicinity. Compared to full-length fibronectin, FNIII9*-10/12-14 supports nerve growth at similar concentrations. In a 3D environment, only the ultrasoft 1 w/v% PEG hydrogels with a storage modulus of ∼10 Pa promoted neuronal growth. This gel was used to establish the first fully synthetic, injectable Anisogel by the addition of magnetically aligned microelements, such as rod-shaped microgels or short fibers. The Anisogel led to linear neurite extension and represents a large step in the direction of clinical translation with the opportunity to treat acute spinal cord injuries.
- ItemOptimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations(London : BioMed Central, 2020) Keil, Timm; Dittrich, Barbara; Lattermann, Clemens; Büchs, JochenBackground: Small-scale cultivation vessels, which allow fed-batch operation mode, become more and more important for fast and reliable early process development. Recently, the polymer-based feeding system was introduced to allow fed-batch conditions in microtiter plates. Maximum glucose release rates of 0.35 mg/h per well (48-well-plate) at 37 °C can be achieved with these plates, depending on the media properties. The fed-batch cultivation of fluorescent protein-expressing E. coli at oxygen transfer rate levels of 5 mmol/L/h proved to be superior compared to simple batch cultivations. However, literature suggests that higher glucose release rates than achieved with the currently available fed-batch microtiter plate are beneficial, especially for fast-growing microorganisms. During the fed-batch phase of the cultivation, a resulting oxygen transfer rate level of 28 mmol/L/h should be achieved. Results: Customization of the polymer matrix enabled a considerable increase in the glucose release rate of more than 250% to up to 0.90 mg/h per well. Therefore, the molecular weight of the prepolymer and the addition of a hydrophilic PDMS-PEG copolymer allowed for the individual adjustment of a targeted glucose release rate. The newly developed polymer matrix was additionally invariant to medium properties like the osmotic concentration or the pH-value. The glucose release rate of the optimized matrix was constant in various synthetic and complex media. Fed-batch cultivations of E. coli in microtiter plates with the optimized matrix revealed elevated oxygen transfer rates during the fed-batch phase of approximately 28 mmol/L/h. However, these increased glucose release rates resulted in a prolonged initial batch phase and oxygen limitations. The newly developed polymer-based feeding system provides options to manufacture individual feed rates in a range from 0.24-0.90 mg/h per well. Conclusions: The optimized polymer-based fed-batch microtiter plate allows higher reproducibility of fed-batch experiments since cultivation media properties have almost no influence on the release rate. The adjustment of individual feeding rates in a wide range supports the early process development for slow, average and fast-growing microorganisms in microtiter plates. The study underlines the importance of a detailed understanding of the metabolic behavior (through online monitoring techniques) to identify optimal feed rates. © 2020 The Author(s).
- ItemProspects and challenges of translational corneal bioprinting(Basel : MDPI AG, 2020) Fuest, Matthias; Yam, Gary Hin-Fai; Mehta, Jodhbir S.; Campos, Daniela F.DuarteCorneal transplantation remains the ultimate treatment option for advanced stromal and endothelial disorders. Corneal tissue engineering has gained increasing interest in recent years, as it can bypass many complications of conventional corneal transplantation. The human cornea is an ideal organ for tissue engineering, as it is avascular and immune-privileged. Mimicking the complex mechanical properties, the surface curvature, and stromal cytoarchitecure of the in vivo corneal tissue remains a great challenge for tissue engineering approaches. For this reason, automated biofabrication strategies, such as bioprinting, may offer additional spatial control during the manufacturing process to generate full-thickness cell-laden 3D corneal constructs. In this review, we discuss recent advances in bioprinting and biomaterials used for in vitro and ex vivo corneal tissue engineering, corneal cell-biomaterial interactions after bioprinting, and future directions of corneal bioprinting aiming at engineering a full-thickness human cornea in the lab. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemQuantifying ligand-cell interactions and determination of the surface concentrations of ligands on hydrogel films: The measurement challenge(Melville, NY : AIP Publishing, 2015) Beer, Meike V.; Hahn, Kathrin; Diederichs, Sylvia; Fabry, Marlies; Singh, Smriti; Spencer, Steve J.; Salber, Jochen; Möller, Martin; Shard, Alexander G.; Groll, JürgenHydrogels are extensively studied for biomaterials application as they provide water swollen noninteracting matrices in which specific binding motifs and enzyme-sensitive degradation sites can be incorporated to tailor cell adhesion, proliferation, and migration. Hydrogels also serve as excellent basis for surface modification of biomaterials where interfacial characteristics are decisive for implant success or failure. However, the three-dimensional nature of hydrogels makes it hard to distinguish between the bioactive ligand density at the hydrogel-cell interface that is able to interact with cells and the ligands that are immobilized inside the hydrogel and not accessible for cells. Here, the authors compare x-ray photoelectron spectrometry (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), enzyme linked immunosorbent assay (ELISA), and the correlation with quantitative cell adhesion using primary human dermal fibroblasts (HDF) to gain insight into ligand distribution. The authors show that although XPS provides the most useful quantitative analysis, it lacks the sensitivity to measure biologically meaningful concentrations of ligands. However, ToF-SIMS is able to access this range provided that there are clearly distinguishable secondary ions and a calibration method is found. Detection by ELISA appears to be sensitive to the ligand density on the surface that is necessary to mediate cell adhesion, but the upper limit of detection coincides closely with the minimal ligand spacing required to support cell proliferation. Radioactive measurements and ELISAs were performed on amine reactive well plates as true 2D surfaces to estimate the ligand density necessary to allow cell adhesion onto hydrogel films. Optimal ligand spacing for HDF adhesion and proliferation on ultrathin hydrogel films was determined as 6.5 ± 1.5 nm.
- ItemMulti-scale processes of beech wood disintegration and pretreatment with 1-ethyl-3-methylimidazolium acetate/water mixtures(London : BioMed Central, 2016) Viell, Jörn; Inouye, Hideyo; Szekely, Noemi K.; Frielinghaus, Henrich; Marks, Caroline; Wang, Yumei; Anders, Nico; Spiess, Antje C.; Makowski, LeeBackground: The valorization of biomass for chemicals and fuels requires efficient pretreatment. One effective strategy involves the pretreatment with ionic liquids which enables enzymatic saccharification of wood within a few hours under mild conditions. This pretreatment strategy is, however, limited by water and the ionic liquids are rather expensive. The scarce understanding of the involved effects, however, challenges the design of alternative pretreatment concepts. This work investigates the multi length-scale effects of pretreatment of wood in 1-ethyl-3-methylimidazolium acetate (EMIMAc) in mixtures with water using spectroscopy, X-ray and neutron scattering. Results: The structure of beech wood is disintegrated in EMIMAc/water mixtures with a water content up to 8.6 wt%. Above 10.7 wt%, the pretreated wood is not disintegrated, but still much better digested enzymatically compared to native wood. In both regimes, component analysis of the solid after pretreatment shows an extraction of few percent of lignin and hemicellulose. In concentrated EMIMAc, xylan is extracted more efficiently and lignin is defunctionalized. Corresponding to the disintegration at macroscopic scale, SANS and XRD show isotropy and a loss of crystallinity in the pretreated wood, but without distinct reflections of type II cellulose. Hence, the microfibril assembly is decrystallized into rather amorphous cellulose within the cell wall. Conclusions: The molecular and structural changes elucidate the processes of wood pretreatment in EMIMAc/water mixtures. In the aqueous regime with >10.7 wt% water in EMIMAc, xyloglucan and lignin moieties are extracted, which leads to coalescence of fibrillary cellulose structures. Dilute EMIMAc/water mixtures thus resemble established aqueous pretreatment concepts. In concentrated EMIMAc, the swelling due to decrystallinization of cellulose, dissolution of cross-linking xylan, and defunctionalization of lignin releases the mechanical stress to result in macroscopic disintegration of cells. The remaining cell wall constituents of lignin and hemicellulose, however, limit a recrystallization of the solvated cellulose. These pretreatment mechanisms are beyond common pretreatment concepts and pave the way for a formulation of mechanistic requirements of pretreatment with simpler pretreatment liquors. © 2016 Viell et al.