Advancing the cell culture landscape:the instructive potential of artificial and natural geometries

This research focuses on how surface structures can influence the behaviour of cells. There is a great diversity of surface structures, which makes the identification of an optimal physical environment for a specific phenotype difficult. Therefore, platforms that allow screening of many different designs at the same time facilitate the identification of an optimal cultural environment. Using the TopoChip, which contains 2176 unique microtopographies, structures have been identified that support the tenocyte phenotype, the primary cell type of the tendon. In addition, this also applies to mesenchymal stem cells (MSCs), which experience an activation of tendon-related genes. Furthermore, the library has been creatively expanded by using natural surface topographies that cause unique cell behaviour, such as promoting osteogenesis

Accelerated cell line development and improved characterisation of lentiviral vector production through application of MALDI-ToF mass spectrometry and multivariate data analysis.

Ph. D. Thesis.Several cell and gene therapies will be commercially launched within the next few years using lentiviral vectors as the gene delivery vehicle. Oxford BioMedica’s Lentivector® platform is an advanced lentiviral-based gene delivery system designed for improved safety and efficacy. The growing interest in these vectors has created a strong demand for large scale production of lentiviral vectors as well as for development of packaging and producer cell lines. This EngD project used a combination of matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS) and multivariate data analysis (MVDA) to analyse cell and lentiviral vector samples. A comparison between mass spectra of samples produced across small and large scale in adherent and suspension culture was used to identify what aspects of the manufacturing process had the biggest impact on cell and vector variation. Principal component analysis was applied to compare different lentiviral vector production methods, assess data structure of the process parameters and examine whole cell and vector mass spectrometry data. This approach led to improved characterisation of lentiviral vectors and HEK293T cells. It demonstrated the capability to differentiate between adherent and suspension cells as well as cell lines of different levels of performance as defined by lentiviral vector infectious titre. Partial least squares discriminant analysis (PLS-DA) was used to calibrate and validate a predictive model of cell line performance based on mass spectrometry and viral vector titre data obtained from multiple HEK293T cell lines. PLS-DA model validation resulted in 87.5% accuracy in classification of cell lines as high or low producers based on a discrimination threshold determined by viral vector titre. The results of PLS-DA modelling indicated that this method can be used for accurate cell line performance prediction, accelerating cell line development by several weeks, improving cell selection and reducing campaign timelines.EPSR

Chemical Information Bulletin

Periodic supplement for "the regular journals of the American Chemical Society," containing annotated bibliographies of chemical documentation literature as well as information about meetings, conferences, awards, scholarships, and other news from the American Chemical Society (ACS) Division of Chemical Literature

IL-12 priming during initial antigen contact increases generation of effector and memory CD8 T cells by changing properties

open11sciescopu

IL-15 and its role in rheumatoid arthritis

Background: IL-15 is involved in all phases of rheumatoid arthritis. Recently we have shown that rheumatoid arthritis synovial fibroblasts (RASF) express both IL-15 and functional IL-15 receptor [1]. Objective: The aim of present study was to identify pathways that are regulated by autocrine IL-15 (IL-15R) in RASF. Methods: RASF were transfected with plasmid encoding IL-15R antagonist (CRB-15, Cardion AG) or control constructs. RNA from transient transfectants were used for Microarray analysis. The differential expression of genes obtained by microarray analysis was verified by SYBR Green real-time PCR. The expression of IL-15Rα, cell proliferation and the expression of p16 and p21 were evaluated in stably transfected cells. Results: The IL-15R antagonist produced by transfected RASF blocked the endogenous IL-15/IL-15Rα interaction, which resulted in an inhibition of cell proliferation (45 ± 10%) via an increase of the expression of p16. In addition, we found that inhibition of IL-15Rα induced the expression of mRNA for FGFR-3. Since two isoforms of FGFR-3 have been identified (FGFR-3b and FGFR-3c) [2], we tested the effect of IL-15Rα inhibition on their expression. In contrast to FGFR-3b, the level of mRNA for FGFR-3c was strongly increased in cells transfected with the IL-15R antagonist (4.71 ± 2.5 in transient transfectants and 6.1 ± 1 fold in stable transfectants). FGFR-3c isoform binds specifically FGF-9, but also FGF-2 [2]. Besides FGFR-3, FGF-2 that is abundant in RA joints binds to FGFR-1. In vitro studies revealed that FGFR-1 transmits a potent mitogenic signal, whereas FGFR-3 usually has no stimulatory effect or inhibits cell proliferation. In contrast to FGFR-3c, blocking of IL-15Rα did not change the mRNA expression for FGFR-1 in RASF. Moreover, we checked whether FGF-2 affects the expression of IL-15Rα. Indeed, FGF-2 strongly decreased the spontaneous and tumor necrosis factor alpha-triggered expression of IL-15Rα at the mRNA and protein levels. Conclusion: Our findings raise the possibility of a negative loop between FGF-2/FGFR-3c and IL-15/IL-15R signaling in RASF. Moreover, the activation of RASF by FGFs could depend on the ratio of FGFR-1/FGFR-3 expression, which is controlled by the endogenous IL-15/IL-15R system

Antigen-specific suppression of inflammatory arthritis by dendritic cells

Purpose Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. We have shown that NF-κB inactivation in dendritic cells (modified DC) converts them into cells that tolerize rather than immunize to specific antigen [1]. Antigen-exposed modified DC prevent priming of immunity, and they suppress previously primed immune responses. Regulatory CD4+ T cells, which can transfer antigen-specific tolerance in an IL-10-dependent fashion, mediate the tolerance. We hypothesized that modified DC exposed to arthritogenic antigen would suppress clinical arthritis after disease onset. Methods Antigen-induced arthritis was induced in C57/Bl6 mice by priming to methylated bovine serum albumin (mBSA) antigen followed by challenge injection of mBSA to one knee. Knee swelling was apparent within 2 days, with peak clinical signs apparent at 5 days. Mice were treated with antigen-exposed modified DC between 2 and 6 days after mBSA challenge to the knee joint. Results Clinical arthritis was suppressed in each group receiving mBSA-exposed modified DC within 4 days compared with mice that received either no DC or keyhole limpet hemocyanin-exposed modified DC. Clinical improvement was associated with mBSA-specific tolerance in mice receiving mBSA-exposed modified DC. Tolerance induction was not impaired by concomitant administration of anti-tumor necrosis factor alpha monoclonal antibody. Subsequent rechallenge with intra-articular IL-1 induced flare of arthritis in all groups, which could be effectively suppressed by a second administration of mBSA-exposed modified DC. Conclusions The data indicate that modified DC induce antigen-specific immune suppression in this model of inflammatory arthritis, even after full clinical expression of the disease. These observations have important implications for antigen-specific therapy of autoimmunity