Engineering in Life Sciences最新文献

Recent interest has been focused on extracellular matrix (ECM)-based scaffolds totreat critical-sized bone injuries. In this study, urea was used to decellularize and solubilize human placenta tissue. Then, different concentrations of ECM were composited with 8% alginate (Alg) and 12% silk fibroin (SF) for printing in order to produce a natural 3D construct that resembled bone tissue. The physical and biological features of the printed structures were evaluated entirely in vitro. Finally, a rat model was employed to examine the optimal 3D printed scaffold (5% ECM) as a bone transplant for the healing of cranial bone lesions. The present investigation demonstrated that decellularizing placental tissue fragments led to efficient removal of cell debris. In addition, a remarkable improvement in the printed scaffolds' mechanical and biological properties was observed by increasing the ECM concentration. The histology studies and real-time PCR results demonstrated the acceleration of bone regeneration in the bone lesions treated with 5%ECM-SF/Alg at 4 and 8 weeks after implantation. Overall, these results proved that the placental ECM-printed scaffolds could potentially construct biomimetic grafts to reconstruct significant bone defects and now promise to proceed with clinical studies.

The demand for lentiviral vectors (LVs) as tools for ex vivo gene therapies is ever-increasing. Despite their promising applications, challenges in LV production remain largely due to the fragile envelope, which challenges the maintenance of vector stability. Thus, downstream processing optimization to enhance efficiency, yield, and product quality is necessary. This study investigated the influence of membrane types and filtration devices during ultrafiltration (UF). Nine different membrane materials consisting of polyethersulfone (PES), regenerated cellulose, or Hydrosart, with distinct molecular weight cutoffs, were evaluated in stirred cells, centrifugal ultrafilters, and crossflow cassettes. The evaluation was based on the ability to retain infectious LV particles and remove impurities. The analysis revealed that a reinforced 100 kDa PES and a 300 kDa Hydrosart membrane had the best overall ability to concentrate infectious LVs and remove DNA, especially when operated in a stirred cell. Challenges were seen in the nonoptimized crossflow cassette process, where infectious LV recovery was generally lower compared to other devices. We demonstrated that membrane material and filtration device have a direct impact on the efficiency of LV UF.

Clostridium pasteurianum is a microorganism for production of 1,3-propanediol (1,3-PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C. pasteurianum have mainly focused on single genomic modification. The aim of this work is the development and application of a method for modification of multiple gene targets in the genome of C. pasteurianum. To this end, a new approach for consecutive genome engineering is presented for the first time using a method based on endogenous CRISPR-Cas machineries. A total of three genome modifications were consecutively introduced in the same mutant and the effect of combined changes on the genome was observed by 39% decreased specific glycerol consumption rate and 29% increased 1,3-PDO yield in mixed substrate fermentations at laboratory scale in comparison to the wildtype strain. Additionally, examination of the phenotype of the generated mutant strain led to discovery of 2,3-butanediol (2,3-BDO) production of up to 0.48 g L^-1, and this metabolite was not reported to be produced by C. pasteurianum before. The developed procedure expands the genetic toolkit for C. pasteurianum and provides researchers an additional method which contributes to improved genetic accessibility of this strain.

Bioelectrochemical systems (BESs) offer a sustainable method for chemical production, including the enhanced production of succinic acid. By combining fermentation with BES, it could be possible to achieve sustainable succinic acid production and CO₂ fixation using Actinobacillus succinogenes. In literature, the potential application of BES is commonly associated with increased succinate yields, as it is expected to enhance the availability of NADH, thereby influencing the intracellular nicotinamide adenine dinucleotide (NADH/NAD⁺) balance. However, it remains unclear whether BES can improve NADH regeneration and achieve higher NADH/NAD⁺ ratios across all growth phases of A. succinogenes. This study investigates the impact of an applied electrical potential on the intracellular NADH/NAD⁺ ratio during an electrochemical-assisted fermentation process. Using an adapted high-performance liquid chromatography method with a Supelcosil LC-18-T column, it was demonstrated that NADH availability in BES, particularly during the stationary growth phase, improved by up to 1.98-fold compared to the control. This enhancement in reducing power led to a succinate yield of 0.747 ± 0.01 g g^-1, representing a 15.65% increase compared to a fermentation without electrochemical assistance. These findings support the expectation that the use of BES could enhance the competitiveness of bio-based succinate production.

Raman spectroscopy, a robust and non-invasive analytical method, has demonstrated significant potential for monitoring biopharmaceutical production processes. Its ability to provide detailed information about molecular vibrations makes it ideal for the detection and quantification of therapeutic proteins and critical control parameters in complex biopharmaceutical mixtures. However, its application in Saccharomyces cerevisiae fermentations has been hindered by the inherent strong fluorescence background from the cells. This fluorescence interferes with Raman signals, compromising spectral data accuracy. In this study, we present an approach that mitigates this issue by deploying Raman spectroscopy on cell-free media samples, combined with advanced chemometric modeling. This method enables accurate prediction of protein concentration and key process parameters, fundamental for the control and optimization of biopharmaceutical fermentation processes. Utilizing variable importance in projection (VIP) further enhances model robustness, leading to lower relative root mean squared error of prediction (RMSEP) values across the six targets studied. Our findings highlight the potential of Raman spectroscopy for real-time, on-line monitoring and control of complex microbial fermentations, thereby significantly enhancing the efficiency and quality of S. cerevisiae-based biopharmaceutical production.

Immunohistochemistry (IHC) is a widely used technique in diagnostic pathology and biomedical research, but there is still a need to shorten the operation process and reduce the cost of antibodies. This study aims to assess a novel IHC technique that incorporates mechanical microvibration (MMV) to expedite the process, reduce antibody consumption, and enhance staining quality. MMV was generated using coin vibration motors attached to glass slides mounted with consecutive tissue sections. Multiple antibodies targeting various antigens were used to stain cancerous and normal tissues, with and without microvibration. Various parameters were tested, including incubation durations, temperatures, and antibody dilutions. The novel method showed the potential to achieve comparable or superior outcomes in significantly less time, utilizing over 10 times less antibody than controls. MMV improved specific staining quality, yielding stronger, and better-defined positive reactions. This was validated through a multicenter double-blind assessment and quantitative image analysis. The possible mechanisms were also investigated. MMV shortens immunohistochemical staining duration, reduces antibody usage, and enhances staining specificity, likely by accelerating antibody movement and diffusion. These improvements translate to time and cost savings, offering clinical and financial value for diagnostic pathology and biomedical research.

Ethyl acetate is at present exclusively produced from fossil resources. Microbial synthesis of this ester from sugar-rich waste as an alternative is an aerobic process. Ethyl acetate is highly volatile and therefore stripped with the exhaust gas from the bioreactor which enables in situ product recovery. Previous research on microbial formation of ethyl acetate has focused on the kinetics of ester synthesis and in part on the ester stripping, while the separation of the ester from the exhaust gas has hardly been investigated. A mixed matrix membrane was developed consisting of Silikalite-1 embedded in polydimethylsiloxane which was installed in a radial–symmetrical membrane module. Evaluation of the separation of ethyl acetate was based on the analysis of the composition of the feed and retentate gas by mass spectrometry. The separation efficiency of the membrane was first tested with varied flows of artificial exhaust gas, containing defined amounts of ethyl acetate. A model for describing the separation process was parametrized by the measured data and used to design a real separation experiment. Ethyl acetate produced from delactosed whey permeate by Kluyveromyces marxianus DSM 5422 in a stirred bioreactor gassed with 0.5 vvm air was successfully separated from the exhaust gas by membranes; 93.6% of the stripped ester was separated. Liquid ethyl acetate was recovered by cooling the permeate gas to ‒78°C, whereby 99.75% of the condensed organic compounds were ethyl acetate. This study demonstrates for the first time that microbially produced and stripped ethyl acetate can be effectively separated from the exhaust gas of bioreactors by membrane technology to obtain the ester in high yield and purity.