Biotechnology and Bioengineering最新文献

Here, we employed human embryonic stem cell spheroid (3D hESCs)-derived exosomes to assess inflammatory responses of macrophages in liver fibrosis, and found that 3D hESC-exosomes promoted the transformation of macrophages from M1 to M2 phenotype in vitro. The transplantation of 3D hESC-exosomes exhibited a notable decrease of pro-inflammatory factors, and significant increase of anti-inflammatory factors, and led to a reduction of CD86⁺M1 macrophages and an increase of Arg-1⁺M2 macrophages in the livers of treated fibrotic mice, restricting the advancement of liver fibrosis. hsa_circ_0076798, derived from 3D hESC-exosomes, acts as a molecular sponge that sequesters miR-1184, thereby mitigating miR-1184-mediated repression of DICER1 expression within macrophages. Further investigation revealed that DICER1 ultimately mitigated macrophage inflammation by deactivating TNF/NF-κB signaling pathway. Therefore, our findings demonstrated that 3D hESC-exosomes alleviated inflammatory responses by suppressing TNF/NF-κB pathway through hsa_circ_0076798/miR-1184/DICER1 axis, and offers insights into the targeted treatment of liver fibrosis via exosome-based cell-free therapy.

Citronellal and citronellol are monoterpenoids that serve as key ingredients in the flavor and fragrance industry and hold significant potential for pharmaceutical applications. Currently, these compounds are primarily obtained through chemical synthesis or extraction from plant essential oils. However, these methods raise concerns regarding environmental sustainability, carbon emissions, and the growing attentions for natural production. A promising alternative is microbial biosynthesis, which enables the natural and carbon-neutral production of these compounds in an environmentally friendly manner. In this study, we developed bienzymatic cascades for the efficient production of citronellal and citronellol from geraniol, utilizing E. coli's endogenous glycerol dehydrogenase (GLDA), NADPH-dependent aldehyde reductase (AHR), and Catharanthus roseus iridoid synthase (CrIS). The highest in vitro production reached 92 mg/L of citronellal and 170 mg/L of citronellol in a 5-h enzymatic reaction. We also engineered these enzymatic cascades into E. coli strains for in vivo production, achieving up to 714 mg/L of citronellol. This study establishes new bienzymatic cascades for the efficient biosynthesis of citronellal and citronellol, demonstrating a possible bio-based production for natural flavor and fragrance compounds.

Peripheral nerve regeneration relies on repair Schwann cells (SCs) to support axonal regrowth and functional recovery. This study aimed to identify drugs that promote this repair phenotype, which is regulated by the expression of the transcription factor c-Jun. Purmorphamine (PUR) and Smoothened agonist (SAG) are both Sonic Hedgehog (SHH) agonists that have been implicated in promoting regeneration after neurological injury in animal models. Here, we have demonstrated that SHH agonists significantly increased c-Jun expression in rat primary SCs and promoted morphological and functional changes consistent with the repair SC phenotype, including an elongated bipolar morphology and increased secretion of neurotrophic factors. Notably, PUR consistently demonstrated a greater potency in driving these effects compared with SAG at the same concentrations. We also identified 2.5 µM PUR as an effective dosage producing these measurable effects in vitro. Coculturing dorsal root ganglion (DRG) neurons with PUR-treated SCs resulted in a marked increase in neurite elongation, suggesting that cell-based or contact-dependent features of repair SCs contribute to axon growth. These findings demonstrate that SHH agonists effectively reprogram SCs into a repair phenotype, which constitutes a potential therapeutic strategy for enhancing nerve regeneration and functional recovery in peripheral nerve injury treatment.

The future of healthcare depends on leveraging state-of-the-art advancements in biopharmaceutical manufacturing across the world. A near end-to-end distributed biomanufacturing setup will enable production to occur closer to points-of-care or points-of-need, thereby reducing lead times, improving adaptability to unseasonal demands and ensuring accessibility in rural and resource-limited settings. However, the current distributed biomanufacturing systems typically produce lower volumes compared to conventional facilities. This perspective discusses the present status of distributed biomanufacturing and envisions futuristic models on portable platforms providing high mobility and production capacity. This conceptual framework for deployable biomanufacturing could facilitate an impartial global distribution of biotherapeutics, improved pandemic preparedness, reduced production costs, provide medical tourism and even utilization in space exploration. By integrating advanced technologies such as continuous manufacturing, machine learning, sensors, and real-time process control, these platforms would enable rapid, agile, and efficient production of biologics. Furthermore, we discuss key considerations such as regulatory pathways, quality management systems and secure cloud-based data handling for one such mobile platform housed on a ship. This vision for distributed biomanufacturing aims to spark innovation and drive efforts toward the futuristic goal of providing affordable and high-quality biopharmaceuticals with equitable access around the world.

Secretome derived from mesenchymal stromal cells (MSCs) holds strong therapeutic potential for regenerative medicine. However, the large-scale production of MSC secretome still presents scientific and technical challenges. This study presents a novel approach using bead-to-bead (BTB) transfer in stirred-tank bioreactors (STR) to scale up the secretome production by turbulence stimulation. By applying three successive microcarrier additions, we could achieve 428 million MSCs in 16 days, corresponding to 5.3 cumulative population doublings (CPD). The BTB transfer significantly outperformed conventional batch culture by extending cell culture duration without aggregate formation. Indeed, a 2.4-fold increase in particle/cell yield, an 88-fold increase in total particle production, and a 113-fold enhancement in particle productivity were obtained in comparison with production in static flasks, demonstrating substantial improvements while reducing the initial number of cells required. The produced EVs display the presence of CD9, CD81 and CD63 tetraspanins and positive MSC markers, validating the identity of the produced EVs. This scalable and enzyme-free BTB transfer method thus offers an efficient and reproducible strategy for high-yield secretome production while maintaining its main characteristics.

A recurring root cause for polysorbate degradation and associated particle observations in liquid biotherapeutics is the presence of residual host cell proteins (HCPs). Some of these HCPs can hydrolyze the polysorbate surfactant to release poorly-soluble fatty acids that accumulate over time and form particles. To tackle this industry-wide issue, we disrupted the genes for a plurality of potential polysorbate-degrading HCPs in CHO cells via two CHO cell engineering approaches. First, we sequentially knocked out six hydrolase genes in a stable recombinant mAb-expressing cell line and substantially decreased polysorbate degradation (up to ~90% in purified samples) without negatively impacting cell culture performance and mAb product quality. Second, we applied a more efficient multiplex gene disruption method to knockout (KO) 3, 7, and 12 hydrolase-associated genes from a CHO blank (null) host. The resulting KO hosts were stably transfected to express six different mAbs, and the purified mAb samples exhibited decreased polysorbate degradation (up to ~60%) while maintaining consistent mAb product quality. These representative CHO cell engineering studies demonstrate the feasibility of modulating CHO cells to mitigate product quality risks in biotherapeutics without deleterious effects on the production cells, even with up to 12 hydrolase genes knocked out.