Search Results
Wetting-Induced Polyelectrolyte Pore Bridging
Active 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.
Optimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations
Background: 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).
Can constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo-β-glucanase
Cellulases are industrially important enzymes, e.g., in the production of bioethanol, in pulp and paper industry, feedstock, and textile. Thermostability is often a prerequisite for high process stability and improving thermostability without affecting specific activities at lower temperatures is challenging and often time-consuming. Protein engineering strategies that combine experimental and computational are emerging in order to reduce experimental screening efforts and speed up enzyme engineering campaigns. Constraint Network Analysis (CNA) is a promising computational method that identifies beneficial positions in enzymes to improve thermostability. In this study, we compare CNA and directed evolution in the identification of beneficial positions in order to evaluate the potential of CNA in protein engineering campaigns (e.g., in the identification phase of KnowVolution). We engineered the industrially relevant endoglucanase EGLII from Penicillium verruculosum towards increased thermostability. From the CNA approach, six variants were obtained with an up to 2-fold improvement in thermostability. The overall experimental burden was reduced to 40% utilizing the CNA method in comparison to directed evolution. On a variant level, the success rate was similar for both strategies, with 0.27% and 0.18% improved variants in the epPCR and CNA-guided library, respectively. In essence, CNA is an effective method for identification of positions that improve thermostability.
Engineering robust cellulases for tailored lignocellulosic degradation cocktails
Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
Trypsin-Free Cultivation of 3D Mini-Tissues in an Adaptive Membrane Bioreactor
The 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
Prospects and challenges of translational corneal bioprinting
Corneal 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.
From In Vitro to Perioperative Vascular Tissue Engineering: Shortening Production Time by Traceable Textile-Reinforcement
Background: The production of tissue-engineered vascular graft (TEVG) usually involves a prolonged bioreactor cultivation period of up to several weeks to achieve maturation of extracellular matrix and sufficient mechanical strength. Therefore, we aimed to substantially shorten this conditioning time by combining a TEVG textile scaffold with a recently developed copolymer reinforced fibrin gel as a cell carrier. We further implemented our grafts with magnetic resonance imaging (MRI) contrast agents to allow the in-vitro monitoring of the TEVG’s remodeling process. Methods: Biodegradable polylactic-co-glycolic acid (PLGA) was electrospun onto a non-degradable polyvinylidene fluoride scaffold and molded along with copolymer-reinforced fibrin hydrogel and human arterial cells. Mechanical tests on the TEVGs were performed both instantly after molding and 4 days of bioreactor conditioning. The non-invasive in vitro monitoring of the PLGA degradation and the novel imaging of fluorinated thermoplastic polyurethane (19F-TPU) were performed using 7T MRI. Results: After 4 days of close loop bioreactor conditioning, 617 ± 85 mmHg of burst pressure was achieved, and advanced maturation of extracellular matrix (ECM) was observed by immunohistology, especially in regards to collagen and smooth muscle actin. The suture retention strength (2.24 ± 0.3 N) and axial tensile strength (2.45 ± 0.58 MPa) of the TEVGs achieved higher values than the native arteries used as control. The contrast agents labeling of the TEVGs allowed the monitorability of the PLGA degradation and enabled the visibility of the non-degradable textile component. Conclusion: Here, we present a concept for a novel textile-reinforced TEVG, which is successfully produced in 4 days of bioreactor conditioning, characterized by increased ECM maturation and sufficient mechanical strength. Additionally, the combination of our approach with non-invasive imaging provides further insights into TEVG’s clinical application.
All-Conjugated Polymer Core-Shell and Core-Shell-Shell Particles with Tunable Emission Profiles and White Light Emission
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.
Recycling and Separation of Homogeneous Catalyst from Aqueous Multicomponent Mixture by Organic Solvent Nanofiltration
Organic solvent nanofiltration (OSN) has evolved to an established recycling method for homogeneous catalysts. However, commercial availability has not circumvented the need for classification and the scoping of possible applications for specific solvent mixtures. Therefore, Evonik’s DuraMem® 300 was assessed for the recycling of magnesium triflate at two transmembrane pressures from a mixture of ethanol, ethyl acetate and water. Catalyst retention up to 98% and permeability of up to 4.44·10−1∙L∙bar−1∙m−2∙h−1 were possible when less than 25% ethyl acetate was in the mixture. The retention of some of the components in the ternary mixture was observed while others were enriched, making the membrane also suitable for fractioning thereof.
3D-Printing of Structure-Controlled Antigen Nanoparticles for Vaccine Delivery
Targeted delivery of antigens to immune cells using micro/nanocarriers may serve as a therapeutic application for vaccination. However, synthetic carriers have potential drawbacks including cytotoxicity, low encapsulation efficiency of antigen, and lack of a morphological design, which limit the translation of the delivery system to clinical use. Here, we report a carrier-free and three-dimensional (3D)-shape-designed antigen nanoparticle by multiphoton lithography-based 3D-printing. This simple, versatile 3D-printing approach provides freedom for the precise design of particle shapes with a nanoscale resolution. Importantly, shape-designed antigen nanoparticles with distinct aspect ratios show shape-dependent immune responses. The 3D-printing approach for the rational design of nanomaterials with increasing safety, complexity, and efficacy offers an emerging platform to develop vaccine delivery systems and mechanistic understanding.