Engineering in Life Sciences最新文献

The research field of cellular agriculture has developed rapidly in recent years. Despite many successes, there is an urgent need for innovative methods to culture adherent cells. Edible scaffolds offer a promising solution for anchorage-dependent cells from agriculturally relevant species. In this study, we present a novel approach using plant-based scaffolds for the production of cultivated fat. Our findings indicate that coating of electrospun-derived plant-based scaffolds with poly-L-lysine significantly enhances cell adhesion and proliferation, offering a more cost-effective alternative to coating with extracellular matrix (ECM) components. Furthermore, we investigated the influence of various adipogenic media formulations on the fatty acid composition of the cultivated fat. Notably, the incorporation of intralipid significantly changed the lipid profile, leading to an increased proportion of stearic acid with a simultaneous reduction in the proportions of oleic, linoleic, and alpha-linolenic acid. This modulation allows for the customization of lipid profiles to satisfy diverse user requirements. However, our analysis showed that both types of matrices and the basal media formulations exerted only moderate to negligible effects on the overall fatty acid composition of the cultivated fat.

Practical application: In this study, we evaluated the impact of cold plasma and coating treatments on plant-based scaffold materials to improve porcine mesenchymal stem cell adhesion and growth. Additionally, the influence of different basal media formulations and the addition of intralipid on the fatty acid composition of the cultivated fat accumulated in differentiated adipocytes were examined. Our results provide valuable insights into how these variables can be adjusted to influence the fatty acid profile of differentiated cells, to meet the requirements of customers with variable nutritional and functional needs. Discovered findings can be used for further development of sustainable alternatives within the food technology sector.

Glycans, a diverse group of complex oligosaccharides, are critical to human physiology and hold significant potential in medical applications and as food additives. However, their synthesis by glycosyltransferases produces intricate mixtures comprising saccharides, nucleosides, and reaction buffer components, posing substantial challenges for downstream processing and purification. This study aims to establish a continuous, single-pass nanofiltration process for purifying oligosaccharide-nucleoside mixtures using a novel dual-membrane module. We investigated the influence of a static mixer, along with varying flow rates for both the diafiltration and feed streams, on the recovery rate and purity of the final product. Measurements using ESI-MS assessed product recovery and purity, while buffer removal was monitored through online conductivity measurement. The results demonstrate that incorporating a static mixer nearly doubled the saccharide recovery rate, achieving product purities exceeding 99% and 95%, along with high product recovery rates. Additionally, the reaction buffer system was found to significantly impact the overall process performance. These findings suggest that our novel dual-membrane module can be effectively utilized for the purification of enzymatically synthesized glycan products.

Summary

The growing demand for large quantities of high-purity lentiviral vectors (LVs) has driven the development of scalable, cost-effective purification strategies. Membrane chromatography is particularly well-suited for purifying large biological entities like LVs due to its low mass transfer resistance and minimal back pressure. Among these technologies, anion exchange (AEX) chromatography serves as a key unit operation during the intermediate purification stage of industrial-scale bioprocesses. Sartobind Convec D, a weak AEX membrane with reduced ligand density, lack of hydrogel grafting, and adjusted pore size distribution, was specifically designed for LVs capture. While AEX is commonly employed immediately after clarification to capture LVs, incorporating a tangential flow filtration (TFF) step beforehand can offer several advantages, including product concentration, buffer exchange, and removal of low-molecular-weight impurities. Hydrosart High-Performance TFF membranes, composed of regenerated cellulose, are characterized by high flux rates and are marketed as well-suited for viral vector purification. This study investigated the integration of these two technologies into intermediate purification workflows for LVs. Specifically, the impact of introducing a TFF step prior to AEX chromatography (TFF-AEX workflow) was compared to a process utilizing only AEX chromatography following harvest and clarification (AEX-only workflow). In the AEX-only workflow, Sartobind Convec D membranes were used directly after clarification to capture LVs. These membrane adsorbers can accommodate high flow rates, making them suitable for early downstream processing. In the TFF-AEX workflow, clarified LVs harvests were first concentrated and diafiltrated using Hydrosart TFF membranes, allowing for product enrichment, buffer conductivity adjustment, and removal of smaller contaminants prior to AEX loading. As a result, the TFF-AEX workflow demonstrated improved dynamic binding capacity and enhanced overall impurity removal compared to the AEX-only approach.

In vitro measurement of protein diffusion within matrices that simulate the subcutaneous (SQ) environment is of interest, given that protein-based therapeutics formulated for SQ injection comprise the largest class of biologics. To mimic the in vivo transport of a biologic from the SQ injection site through the extracellular matrix (ECM), in vitro diffusion assays typically utilize hyaluronic acid (HA) matrices, as it is the principal component of ECM. However, broad utility has been hampered by inherent lot-to-lot variability in commercially sourced HA, wherein key properties that impact protein diffusion (for example, molecular weight distribution and viscosity) differ across lots, even when nominal molecular weights are identical, making it challenging to compare results across matrices prepared from different HA lots. To address this gap, we report a facile approach wherein binary HA blends generated from individual HA matrices derived from distinct HA lots are functionally equivalent with respect to protein diffusion, that is, the diffusion of a representative set of proteins matches that in a previously reported single HA lot-derived matrix that served as a representative reference. Taken altogether, our protocols enable preparing blended HA matrices with consistent diffusion properties, enabling the use of in vitro assays that leverage this capability.

Practical application: The measurement of in vitro diffusion of IgG-type proteins enables calculation of diffusion coefficients that could help to guide the formulation of protein-based therapeutics, administered by subcutaneous (SQ) injection, and used for treating a range of diseases, including cancer. The side-by-side comparison of these proteins over a period of time provides confirmation of consistency of properties when in vitro hyaluronic acid matrices, within which injected protein diffusion is measured, are also consistent. However, their broad utility has been hindered by the inherent variability of commercial sources of HA used to make-up matrices that simulate the SQ environment in a predictable manner. Our research addresses this gap by defining an approach (validated with rheological and diffusion measurements) that facilitates the preparation of blended matrices from different lots of HA. The resulting matrix properties enable reliable measurement of protein diffusion from one lot to the next.

The clinical translation of CRISPR genome-editing therapies is often hindered by inefficient delivery of the CRISPR-Cas RNA-protein complex into target cells. The most widely used CRISPR-Cas9 system poses a significant challenge for efficient delivery into cells due to its large size (∼1.4 kDa). Recently reported compact Cas proteins, such as Cas12f (552 Da), Cas12k (639 Da), and Cas12m (596 Da) represent attractive alternatives as cargoes for delivery. In this brief research report, we employ efficient delivery vectors to evaluate the efficiency of cellular uptake of a compact Cas protein (Cas12f) compared to the widely used larger Cas9 in human cells. Our findings demonstrate that compact Cas proteins may facilitate more efficient cellular penetration and delivery, making them a promising alternative for the development of CRISPR-based therapies.

Practical Application:

Our study demonstrates that compact Cas proteins significantly enhance cellular uptake compared to larger Cas proteins. This improved uptake efficiency suggests that compact Cas proteins could be more effective for clinical application, where size constraints and delivery efficiency are critical challenges. Combined with the optimization and refinement of the editing efficiencies of compact Cas systems, our study provokes further exploration of compact Cas proteins in various therapeutic contexts to advance the development of more efficient CRISPR-based therapies.

Microbial bioprocessing is a key technology for the production of a wide range of biomolecules, including proteins, enzymes, antibiotics, and other bioactive compounds. In recent years, there has been an increasing interest in using microfluidic platforms for bioprocessing, due to the ability to precisely control and manipulate fluids at the microscale. Microfluidics offers a transformative platform for the manufacturing of biomolecules intended for clinical applications by addressing key technical challenges in scalability, precision, reproducibility, and the ability to study complex biological systems. In this review, various methods used to fabricate microfluidic platforms and the current state-of-the-art in the synthesis/production of biopharmaceuticals, polymers, bioactive compounds, and real-time monitoring in microscale bioprocesses are discussed. Additionally, the future trends and directions are highlighted. Overall, we envisage the utilization of microfluidic platforms to advance the field of microbial bioprocessing and applications in the biomedical field.

Menthol, a natural organic compound and the primary component of mint, exhibits diverse biological activities, including analgesic, anti-inflammatory, antibacterial, neuroprotective, and anticancer effects. The chemical modification of menthol, through processes such as esterification and amination, further enhances these activities, expanding its potential applications in drug development, agriculture, and food preservation. This review explores the structure-activity relationships (SAR) of menthol and its derivatives, emphasizing the significance of molecular modifications in enhancing their pharmacological effects. Research indicates that menthol and its derivatives can improve drug permeation, reduce inflammation, enhance memory, and even target cancer cells through various mechanisms. In addition, we examine the safety and pharmacokinetics of menthol and its derivatives to better understand their clinical potential. Although significant progress has been made in preclinical models, further research is necessary to fully elucidate their mechanisms of action and optimize their therapeutic efficacy in clinical settings. Continued innovation in drug delivery technologies and the development of novel menthol derivatives present promising prospects for future therapeutic applications.

Optical spectroscopic techniques have been successfully employed in bioprocessing as process analytical technology for real-time process monitoring in numerous applications. The implementation of spectroscopy-based PAT techniques commonly necessitates the generation of representative process data used for calibration and validation of multivariate statistical models for analyzing the sample composition in real-time. To automate the generation of such data, we present a novel assembly of a commercially available chromatography system in combination with a Raman spectrometer for fast and accurate acquisition of Raman spectra. Using the ultra-/diafiltration (UF/DF) process as a case study, our methodology involved the preparation of representative calibration and validation mixtures of phosphate and citrate buffer and lysozyme as a model protein. Chemometric PLS models were calibrated and validated using these datasets, and applied to in-line recorded Raman spectra during a UF/DF experiment. The primary results demonstrated that the novel assembly provides robust and precise offline measurement of Raman spectra, which directly compare with in-line record data. The chemometric PLS models showed good alignment in calibration and validation datasets (R2 and Q2), and could be used to simultaneously monitor the buffer and protein concentrations in real-time during UF/DF. This study provides a simple, commercially available setup for automated acquisition of Raman spectra and demonstrates its straightforward application to bioprocess monitoring.