Exploring metabolic effects of dipeptide feed media on CHO cell cultures by in silico model-guided flux analysis.

There is a growing interest in perfusion or continuous processes to achieve higher productivity of biopharmaceuticals in mammalian cell culture, specifically Chinese hamster ovary (CHO) cells, towards advanced biomanufacturing. These intensified bioprocesses highly require concentrated feed media in order to counteract their dilution effects. However, designing such condensed media formulation poses several challenges, particularly regarding the stability and solubility of specific amino acids. To address the difficulty and complexity in relevant media development, the biopharmaceutical industry has recently suggested forming dipeptides by combining one from problematic amino acids with selected pairs to compensate for limitations. In this study, we combined one of the lead amino acids, L-tyrosine, which is known for its poor solubility in water due to its aromatic ring and hydroxyl group, with glycine as the partner, thus forming glycyl-L-tyrosine (GY) dipeptide. Subsequently, we investigated the utilization of GY dipeptide during fed-batch cultures of IgG-producing CHO cells, by changing its concentrations (0.125 × , 0.25 × , 0.5 × , 1.0 × , and 2.0 ×). Multivariate statistical analysis of culture profiles was then conducted to identify and correlate the most significant nutrients with the production, followed by in silico model-guided analysis to systematically evaluate their effects on the culture performance, and elucidate metabolic states and cellular behaviors. As such, it allowed us to explain how the cells can more efficiently utilize GY dipeptide with respect to the balance of cofactor regeneration and energy distribution for the required biomass and protein synthesis. For example, our analysis results uncovered specific amino acids (Asn and Gln) and the 0.5 × GY dipeptide in the feed medium synergistically alleviated the metabolic bottleneck, resulting in enhanced IgG titer and productivity. In the validation experiments, we tested and observed that lower levels of Asn and Gln led to decreased secretion of toxic metabolites, enhanced longevity, and elevated specific cell growth and titer. KEY POINTS: • Explored the optimal Tyr dipeptide for the enhanced CHO cell culture performance • Systematically analyzed effects of dipeptide media by model-guided approach • Uncovered synergistic metabolic utilization of amino acids with dipeptide.

Effect of essential oils on pathogenic and biofilm-forming Vibrio parahaemolyticus strains.

In this study, the effect of three essential oils (EOs) - clove oil (CO), thyme oil (TO), and garlic oil (GO), which are generally recognized as safe - on the planktonic growth, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), motility, biofilm formation, and quorum sensing (QS) of Vibrio parahaemolyticus was investigated. All three EOs showed bacteriostatic activity, with MICs in the range 0.02%-0.09% (v/v). CO and TO completely controlled planktonic growth at 0.28% and 0.08% (v/v), which is four times their MIC (4 × MIC), after 10 min, whereas GO completely controlled growth at 0.36% (v/v) (4 × MIC) after treatment for 20 min. V. parahaemolyticus motility was significantly reduced by all three EOs at 4 × MIC (0.28% for CO, 0.08% for TO, and 0.36% for GO), whereas QS was controlled and biofilm formation reduced by all three EOs at 8 × MIC (0.56% for CO, 0.16% for TO, and 0.72% for GO) after 30 min of treatment. These results suggest that CO, TO, and GO have a significant inhibitory effect on V. parahaemolyticus cells in biofilm sand thus represent a promising strategy for improving food safety. These results provide the evidence required to encourage further research into the practical use of the proposed EOs in food preparation processes.

Surface Deformation of Biocompatible Materials: Recent Advances in Biological Applications.

The surface topography of substrates is a crucial factor that determines the interaction with biological materials in bioengineering research. Therefore, it is important to appropriately modify the surface topography according to the research purpose. Surface topography can be fabricated in various forms, such as wrinkles, creases, and ridges using surface deformation techniques, which can contribute to the performance enhancement of cell chips, organ chips, and biosensors. This review provides a comprehensive overview of the characteristics of soft, hard, and hybrid substrates used in the bioengineering field and the surface deformation techniques applied to the substrates. Furthermore, this review summarizes the cases of cell-based research and other applications, such as biosensor research, that utilize surface deformation techniques. In cell-based research, various studies have reported optimized cell behavior and differentiation through surface deformation, while, in the biosensor and biofilm fields, performance improvement cases due to surface deformation have been reported. Through these studies, we confirm the contribution of surface deformation techniques to the advancement of the bioengineering field. In the future, it is expected that the application of surface deformation techniques to the real-time interaction analysis between biological materials and dynamically deformable substrates will increase the utilization and importance of these techniques in various fields, including cell research and biosensors.

Debottlenecking and reformulating feed media for improved CHO cell growth and titer by data-driven and model-guided analyses.

Designing and selecting cell culture media along with their feeding are a key strategy to maximize culture performance in biopharmaceutical processes. However, the sensitivity of mammalian cells to their culture environment necessitates specific nutritional requirements for their growth and the production of high-quality proteins such as antibodies, depending on the cell lines and operational conditions employed. In this regard, previously we developed a data-driven and in-silico model-guided systematic framework to investigate the effect of growth media on Chinese hamster ovary (CHO) cell culture performance, allowing us to design and reformulate basal media. To expand our exploration for media development research, we evaluated two chemically defined feed media, A and B, using a monoclonal antibody-producing CHO-K1 cell line in ambr15 bioreactor runs. We observed a significant impact of the feed media on various aspects of cell culture, including growth, longevity, viability, productivity, and the production of toxic metabolites. Specifically, the concentrated feed A was inadequate in sustaining prolonged cell culture and achieving high titers when compared to feed B. Within our framework, we systematically investigated the major metabolic bottlenecks in the tricarboxylic acid cycle and relevant amino acid transferase reactions. This analysis identified target components that play a crucial role in alleviating bottlenecks and designing highly productive cell cultures, specifically the addition of glutamate to feed A and asparagine to feed B. Based on our findings, we reformulated the feeds by adjusting the amounts of the targeted amino acids and successfully validated the effectiveness of the strategy in promoting cell growth, life span, and/or titer.

Dual delivery of stem cells and insulin-like growth factor-1 in coacervate-embedded composite hydrogels for enhanced cartilage regeneration in osteochondral defects.

Exogenous dual delivery of progenitor cell population and therapeutic growth factors (GFs) is one of alternative tissue engineering strategies for osteochondral tissue regeneration. In the present study, an implantable dual delivery platform was developed using coacervates (Coa) (i.e., a tertiary complex of poly(ethylene argininylaspartate diglyceride) (PEAD) polycation, heparin, and cargo insulin-like growth factor-1 (IGF-1), in thiolated gelatin (gelatin-SH)/ poly(ethylene glycol) diacrylate (PEGDA) interpenetrating network (IPN) hydrogels. Since Coa is able to protect cargo GF and maintain its long-term bioactivity, it is speculated that Coa-mediated delivery of chondrogenic factor IGF-1 with the aid of adipose-derived stem cells (ADSCs) would synergistically facilitate osteochondral tissue repair during physiological regeneration process. Our results indicate that gelatin-SH/PEGDA IPN hydrogels demonstrated biocompatibility and mechanical properties for a possible long-term transplantation, and PEAD-base Coa exhibited a sustained release of bioactive IGF-1 over 3 weeks. Subsequently, released IGF-1 from Coa could effectively induce chondrogenic differentiation of embedded ADSCs in the hydrogel, by showing enhanced glycosaminoglycan deposition and expression of chondrogenesis-associated genes. More importantly, at 12 weeks post-implantation in a rabbit full thickness osteochondral defect model, the quality of regenerative tissues in both chondral and subchondral layers was significantly improved in dual delivery of ADSC and IGF-1 in Coa encapsulated in gelatin-SH/PEGDA IPN hydrogels, as compared with a single delivery of ADSC only and a dual delivery without Coa. Therefore, we conclude that our Coa-embedded composite hydrogel platform could effectively augment osteochondral tissue regeneration holds promise for a feasible osteoarthritis therapeutic application.

The effect of serum types on Chondrogenic differentiation of adipose-derived stem cells.

BACKGROUND: Fetal bovine serum (FBS) is the most essential supplement in culture media for cellular proliferation, metabolism, and differentiation. However, due to a limited supply and subsequently rising prices, a series of studies have investigated a biological feasibility of replaceable serums to substitute FBS. Along with the increasing interests to manufacture stem cell-based cellular products, optimizing the composition of culture media including serums and exogenous growth factors (GFs) is of importance. In this experiment, the effect of bovine serum (BS) and newborn calf serum (NCS) on proliferation and chondrogenic differentiation capacity of human adipose derived stem cells (ADSCs) was evaluated, especially in the chondrogenically supplemented culture condition. METHODS: ADSCs were chondrogenically cultured with FBS, BS, and NCS for 14 days. For the acceleration of in vitro chondrogenesis, exogenous insulin-like growth factor and transforming growth factor-ß3 were added. Viability and proliferation of ADSCs were evaluated using Live/Dead fluorescence staining and DNA amount, respectively. To investigate a chondrogenic differentiation, a series of assays were performed including a quantification of glycosaminoglycan deposition, alcian blue staining, and RT-PCR analysis for type II collagen, aggrecan and Sox-9 genes. RESULTS: The results demonstrated that proliferation of ADSCs was facilitated in FBS condition as compared with other serum types. For chondrogenic marker gene expression, serum substitutes enhanced Sox-9 expression level on day 14. The deposition of glycosaminoglycan was more facilitated in BS condition regardless of additional chondrogenic GFs. CONCLUSION: It could be presumably speculated that serum types and exogenous supplements of GFs could also be important parameters to optimize culture media composition, especially in order to maintain the enhanced levels of both proliferation and chondrogenic differentiation of ADSCs during expansion.

Direct Dehydrogenation of n-Butane Over Pt/Sn/Zn-K/Al2O3 Catalyst: Effect of Hydrogen in the Feed.

Al2O3 was prepared by a sol-gel method for use as a support. Pt/Sn/Zn-K/Al2O3 catalyst was then prepared by a sequential impregnation method, and it was applied to the direct dehydrogenation of n-butane to n-butenes and 1,3-butadiene. Physicochemical properties of Pt/Sn/Zn-K/Al2O3 catalyst were examined by X-ray diffraction (XRD), nitrogen adsorption-desorption isotherm, inductively coupled plasma atomic emission spectroscopy (ICP-AES), temperature-programmed reduction (TPR), CO chemisorption, and temperature-programmed oxidation (TPO) measurements. In order to improve the catalyst stability, the effect of hydrogen in the feed on the catalytic performance in the direct dehydrogenation of n-butane was studied. The catalyst stability and reusability in the direct dehydrogenation of n-butane was also investigated. Experimental results revealed that the addition of hydrogen in the feed decreased conversion of n-butane and yield for total dehydrogenation products but improved the stability of the catalyst. The catalytic activity and stability of regenerated Pt/Sn/Zn-K/Al2O3 catalyst in the presence of hydrogen slightly decreased compared to those of fresh Pt/Sn/Zn-K/Al2O3 catalyst due to the slight sintering of platinum particles.