Reduction in Interferon-Stimulated Genes Contributes to High-Yield Production of Influenza Virus in Suspension MDCK Cells.

Compared with the traditional vaccine produced in embryonated chicken eggs, cell-based manufacturing represented by the Madin-Darby canine kidney (MDCK) cell line has a larger production scale and reduces the risk of egg shortage in a pandemic. Establishing a culture system that enables high production of the influenza virus is a key issue in influenza vaccine production. Here, a serum-free suspension culture of MDCK (sMDCK) cells was obtained from adherent MDCK (aMDCK) cells by direct adaptation. Viral infection experiments showed that viral yields of influenza A/B virus in sMDCK cells were higher than in aMDCK cells. Transcriptome analysis revealed that numerous interferon-stimulated genes (ISGs) exhibited reduced expression in sMDCK cells. To further clarify the mechanism of high viral production in sMDCK cells, we demonstrated the antiviral role of RIG-I and IFIT3 in MDCK cells by knockdown and overexpression experiments. Furthermore, suppression of the JAK/STAT pathway enhances the viral accumulation in aMDCK cells instead of sMDCK cells, suggesting the reduction in the JAK/STAT pathway and ISGs promotes viral replication in sMDCK cells. Taken together, we elucidate the relationship between the host innate immune response and the high viral productive property of sMDCK cells, which helps optimize cell production processes and supports the production of cell-based influenza vaccines.

High-Yield Production of Selected 2D Materials by Understanding Their Sonication-Assisted Liquid-Phase Exfoliation.

Liquid-phase exfoliation (LPE) is a widely used and promising method for the production of 2D nanomaterials because it can be scaled up relatively easily. Nevertheless, the yields achieved by this process are still low, ranging between 2% and 5%, which makes the large-scale production of these materials difficult. In this report, we investigate the cause of these low yields by examining the sonication-assisted LPE of graphene, boron nitride nanosheets (BNNSs), and molybdenum disulfide nanosheets (MoS2 NS). Our results show that the low yields are caused by an equilibrium that is formed between the exfoliated nanosheets and the flocculated ones during the sonication process. This study provides an understanding of this behaviour, which prevents further exfoliation of nanosheets. By avoiding this equilibrium, we were able to increase the total yields of graphene, BNNSs, and MoS2 NS up to 14%, 44%, and 29%, respectively. Here, we demonstrate a modified LPE process that leads to the high-yield production of 2D nanomaterials.

Achieving High-Yield Production of Functional AAV5 Gene Delivery Vectors via Fedbatch in an Insect Cell-One Baculovirus System.

Despite numerous advancements in production protocols, manufacturing AAV to meet exceptionally high demand (1016-1017 viral genomes [VGs]) in late clinical stages and for eventual systemic delivery poses significant challenges. Here, we report an efficient, simple, scalable, robust AAV5 production process utilizing the most recent modification of the OneBac platform. An increase in volumetric yield of genomic particles by ∼6-fold and functional particles by ∼20-fold was achieved by operating a high-cell-density process in shake flasks and bioreactors that involves an Sf9-based rep/cap stable cell line grown at a density of about 10 million cells/mL infected with a single baculovirus. The overall volumetric yields of genomic (VG) and bioactive particles (enhanced transducing units [ETUs]) in representative fedbatch bioreactor runs ranged from 2.5 to 3.5 × 1014 VG/L and from 1 to 2 × 1011 ETU/L. Analytical ultracentrifugation analyses of affinity-purified AAV vector samples from side-by-side batch and fedbatch production runs showed vector preparations with a full and empty particle distribution of 20%-30% genomic and 70%-80% empty particles. Moreover, the stoichiometric analysis of capsid proteins from fedbatch production in shake flask and bioreactor run samples demonstrated the incorporation of higher VP1 subunits, resulting in better functionality.

High-yield production of scutellaria radix flavonoids (baicalein, baicalin and wogonin) by liquid-culture of Scutellaria baicalensis root-derived cells

Production of baicalein, baicalin and wogonin by liquid culture of Scutellaria baicalensis cells derived from the plant root was studied. The maximum production obtained were 119 mg/L of baicalein at two week, 1372 mg/L of baicalin at eight week, and 14 mg/L of wogonin at two week. In addition, the production of baicalin was drastically increased to 1000 mg/L level at 3-week culture, and the extremely high production rate (339 mg/L•week) was obtained. In the comparison of total antioxidative activities among baicalein, baicalin and wogonin, evaluated by thiocyanate method, it was suggested that the location of hydroxyl groups both at 5- and 6-position contributed to enhancement of radical scavenging activity, and/or methoxylation at 8-position diminished the activity. The possibility of utilizing these flavonoids for natural antioxidants and medicine is also discussed.