Engineering in Life Sciences最新文献

Polydimethylsiloxane (PDMS) is extensively utilized for the recovery of bio-alcohols, but it encounters significant obstacles in volatile organic compounds (VOCs) removal, because of the narrow size for molecules diffusion. In this work, we designed a high-efficiency diffusion channel by introducing phenyl as a spacer into PDMS chains. The monomer divinylbenzene and vinyl-terminated PDMS (vinyl-PDMS) can be chemically crosslinked with thiol-grafted PDMS (thiol-PDMS) based on thiol-ene click reaction. The result shows that the free volume radius (r₃, r₄) has a significant increase after the introduction of divinylbenzene as a spacer, which is beneficial to the transport of phenol diffusion. After a series of optimizations involving the divinylbenzene content, pervaporation (PV) operating temperature, photoinitiator content, and viscosity of vinyl-PDMS, the prepared phenyl-PDMS showed an excellent PV performance for phenol recovery containing 10.9 of separation factor and 3959.66 g m⁻² h⁻¹ of flux as separating 0.1 wt% of phenol/water solution at 70°C. This separation performance is significantly higher than the unmodified PDMS membrane, that is, 2.05 times higher in separation factor and 3.54 times higher in flux. This study provides an effective structure design for the removal of aromatic compounds by enlarging diffusion channels and will make a great contribution to biological medicine and bioengineering.

Circulating tumor cells (CTCs) hold significant promise for cancer diagnosis, prognosis, and treatment monitoring. We previously developed a technique for a single-cell filtering device known as the microcavity array (MCA), specifically designed for the efficient recovery of CTCs from whole blood samples. Efficient enrichment and release of cells from the MCA remains challenging because of cell adhesion that occurs on the MCA surface during the enrichment phase. This study investigated the effects of surface modification with 2-methacryloyloxyethyl phosphorylcholine (MPC) on the recovery efficiency of cancer cell lines from MCA. Scanning electron microscope (SEM) demonstrated reduced cell-substrate interactions, leading to improved recovery efficiency. Comparative analyses showed that the MCA method provided superior recovery efficiency and reduced processing time compared to traditional methods such as density gradient centrifugation (DGC), while maintaining cell viability and proliferative capacity. CTCs were successfully detected in patients with gastric cancer, and short-term cultures were achieved even when fewer than 20 CTCs per milliliter of blood were isolated. These findings emphasize the importance of surface modification for enhancing CTC isolation and the need for optimized culture conditions. The optimized MCA method offers a promising approach for rapid CTC recovery and potential integration with automated systems.

Practical application: The Microcavity array (MCA) is a device specifically designed for efficient recovery of CTCs from whole blood. However cell adhesion on the MCA surface can limit release efficiency. This study demonstrated that surface modification with MPC signigicantly reduces cell-substrate adhesion, improving recovery efficiency while maintaining cell viability and proliferative capacity. Compared to traditional density gradient centrifugation, the MPC-modified MCA offers shorter processing time and better performance. CTCs were successfully detected in gastric cancer, and short-term cultures were achieved even when fewer than 20 CTCs per mL of blood were isolated. The method supports downstearm applications such as cancer cell characterization and treatment monitoring. With potential for integration into automated system, the optimized MCA provides a practical, scalable solution for clinical liquid biopsy and personalized oncology.

Marburg marburgvirus (MARV) is a highly virulent human pathogen with limited therapeutic options. Recombinant MARV glycoprotein (GP) produced in Drosophila Schneider 2 (S2) cells has been extensively investigated as potential vaccine antigen with promising efficacy demonstrated in nonhuman primate models. However, the existing production process for MARV-GP involving static batch cell cultures with limited scalability and process control show lower than desirable yields. Here, we assessed various process intensification strategies in single-use orbital shaken bioreactors (OSBs) or rocking bioreactors (WAVE) and report maximum viable cell concentrations (VCCs) of 31.6 × 10⁶ cells/mL in batch, 69.5 × 10⁶ cells/mL in fed-batch (FB), and up to 210.0 × 10⁶ cells/mL in perfusion mode. By changing from a glucose-only feed to a CellBoost5 feed, MARV-GP yields were increased by over two-fold. Implementation of perfusion cultures achieved a peak MARV-GP concentration of 57.4 mg/L and a 540% higher space-time yield compared to the FB process in the 50 L WAVE system. However, maximum cell-specific productivities were achieved at a VCC of 85 × 10⁶ cells/mL and decreased with increasing cell concentrations. Glycoanalysis revealed a uniform paucimannosidic N-glycan profile, predominantly α-1,6-core-fucosylated Man3F (F(6)M3) structures, across all production modes. Notably, transitioning pH control from CO₂ to phosphoric acid shifted glycan profiles toward higher mannose forms, highlighting the influence of culture conditions on glycosylation.

Sonochemistry has become increasingly important in bioengineering research, and many in vitro and in vivo bioapplications have been developed. Cytotoxicity is always a concern in its implementation. For in vivo treatments and studies, mechanical index (MI) is known to ensure biocompatibility, and even in vitro MI has been used. Because cell characteristics and acoustic phenomena differ in vitro and in vivo, we questioned using MI in vitro. The in vitro cytotoxicity of ultrasound exposure should be investigated to support the development of cutting-edge sonochemistry. In this study, a system for irradiating cultured cells with 1–2 MHz-range ultrasound was developed to demonstrate the invalidity of employing MI alone in vitro. The results showed that cell damage is defined by the MI, ultrasound frequency, and exposure time, which are new indices for quantifying cell damage. Furthermore, cavitation and acoustic streaming are shown to be the main scientific factors that injure cells.

Posttreatment of the effluents from wastewater treatment plants is becoming increasingly important, as the conventional treatment cannot completely remove organic trace contaminants. Promising techniques like chemical oxidation methods, including ozonation, face the challenge of potentially generating more toxic transformation products than their parent substances due to incomplete oxidation. In this work, the laccase from Trametes versicolor was immobilized on a polyester textile to create a biocatalytic textile filter for the posttreatment of organic trace contaminants and their ozonation by-products. Different filter designs for reactive filtration with biocatalytic textiles were implemented on the laboratory scale and tested for their effectiveness in degrading the dye Remazol Brilliant Blue, the pharmaceutical diclofenac, and its ozonation products. The plate module, inspired by lamellar clarifiers and featuring the textile with covalently immobilized enzyme on the lamella surfaces, exhibited the best performance characteristics. Employing this module, a continuous process of diclofenac ozonation and subsequent posttreatment with the biocatalytic filter was conducted. This not only demonstrated the feasibility of continuous biocatalytic wastewater filtration but also highlighted improved degradation efficiencies of ozonation products compared to the batch process using laccase in solution.

Establishing manufacturing processes for cell culture-based pharmaceutical products involves managing multiple parameters that can affect yield and efficiency, as well as process robustness and product quality. Implementing Quality by Design (QbD) principles can support process optimization, while streamlining the chemistry, manufacturing, and control aspects for regulatory approval. In this study, we mimic a QbD approach based on an influenza A virus production process using two clonal suspension Madin-Darby canine kidney (MDCK) cell lines with distinct characteristics. We performed a quantitative risk assessment including biological and technical parameters to identify the Critical Process Parameters (CPPs). To comprehensively study the effects and interactions of four CPPs, we used an Ambr 15 scale-down system following a Design of Experiments (DoE) approach. After data analysis and modeling, we obtained design spaces characterized by high robustness with a less than 1% risk of failure and even some indications for virus titer and yield improvement, while keeping process-related impurities such as DNA and total protein concentration low. These findings were subsequently verified at a more than 100-fold higher working volume. Taken together, our approach may stimulate ideas for the implementation of streamlined process development and regulatory approval in the field of viral vaccine production.

To increase their throughput, reduce laboratory work and improve reproducibility, automation of bioprocesses is gaining in importance nowadays. This applies in particular to microbioreactors (MBRs), which can be easily integrated in highly parallelized and automated platforms and, therefore, be applied for screenings, cell-based assays, and bioprocess development. One promising pharmaceutical application for MBRs is the performance of phage sensitivity tests called phagograms in phage therapy. However, there is no automated and parallelized platform available so far that fulfills the requirements of phagograms. Therefore, a novel highly parallelizable capillary-wave microbioreactor (cwMBR) with a volume of 7 µL, which has already been successfully applied for phagograms, was extended by an in-house built platform for automated fluid addition in the single-digit nanoliter range. The cwMBR has a phage-repellent hydrophilic glass surface. Furthermore, a custom-made highly parallelizable device for biomass measurement in the lower microliter scale was developed and validated in the cwMBR. To prove the applicability of the platform for the generation of phagograms, a phagogram using Escherichia coli and automated phage addition was performed. The results indicate a clear lysis of the bacteria by the phages and thus confirm the applicability of performing automated phagograms in the highly parallelizable cwMBR platform.

Melt Electrowriting (MEW) is a powerful technique in tissue engineering, enabling the precise fabrication of scaffolds with complex geometries. One of the most important parameters of MEW is collector speed, which has been extensively studied in relation to critical translation speed. However, its influence on crystallinity was overlooked. Crystallinity is crucial for the mechanical properties and degradation behavior of the scaffolds. Therefore, in this study, we present how printing affects the crystallinity of fibers and the resulting mechanical properties of MEW scaffolds. In systematic analysis, we observed a significant reduction in scaffold crystallinity with increased speed, as evidenced by wide-angle X-ray scattering. This decrease in crystallinity was attributed to differences in cooling rates, impacting the polycaprolactone molecular orientation within the fibers. By using tensile testing, we observed the decrease in scaffold Young's modulus with increasing collector speed. Given the relation between crystallinity and mechanical properties of the material, we developed a finite element analysis model that accounts for changes in crystallinity by employing distinct bulk Young's modulus values to help characterize scaffold mechanical behavior under tensile loading. The model reveals insights into scaffold stiffness variation with different architectural designs. These insights offer valuable guidance for optimizing 3D printing to obtain scaffolds with desired mechanical properties.

Ferulic acid is a high-value chemical synthesized in plants. The ferulic acid biosynthesis is still affected by the insufficient methylation activity of caffeic acid O-methyltransferase (COMT). In this study, we engineered COMT from Arabidopsis thaliana to match caffeic acid, and the mutant COMT^{N129V-H313A-F174L} showed 4.19-fold enhanced catalytic efficiency for degrading caffeic acid. Then, we constructed the de novo synthesis pathway of ferulic acid by introducing tyrosine ammonia lyase from Flavobacterium johnsoniae (FjTAL), 4-hydroxyphenylacetate 3-hydroxylase from Escherichia coli (EcHpaBC), and mutant COMT^{N129V-H313A-F174L}, and further increased tyrosine synthesis. Furthermore, we overexpressed two copies of COMT^{N129V-H313A-F174L} and enhanced the supply of S-adenosyl-L-methionine (SAM) by expressed S-ribosylhomocysteine lyase (luxS) and 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (mtn) to increase the production of ferulic acid. Finally, the production of ferulic acid reached 1260.37 mg/L in the shake-flask fermentation and 4377 mg/L using a 50 L bioreactor by the engineered FA-11. In conclusion, COMT enzyme engineering combined with global metabolic engineering effectively improved the production of ferulic acid and successfully obtained a fairly high level of ferulic acid production.