A three-dimensional approach to in vitro culture of immune-related cells

Sofie Bruun Hartmann

    Research output: Book/ReportPh.D. thesisResearch

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    Abstract

    T lymphocytes are key players during the initiation of an adaptive immune response. The activation of these cells in vivo requires migration within the lymph nodes until they encounter antigen presenting cells (APCs) that can activate them to secrete IFN-γ which mediates downstream effector functions. The in vitro reactivation of antigen-experienced T lymphocytes and detection of IFN-γ from cell cultures can be used in a diagnostic assay to test for disease or vaccine efficacy. Practical procedures of the IFN-γ release assay (IGRA) was investigated using bovine cells and whole blood cultures was found preferable compared to PBMC cultures, partly due to the risk of losing cell subsets after purification of PBMCs.
    The development of in vitro culture systems for more than 50 years ago revolutionized the biomedical world. It became possible to study cell behavior using cell lines or primary cells in culture and to measure cell activity such as IGRA, as described above. The traditional way of culturing cells are done using polystyrene (PS) plastic ware in flask-, Petri dish- or micro titer plate format. However, these artificial two dimensional (2D) surfaces on which the cells grow, has shown to interfere with cell morphology, gene expression and overall behavior and as such gives a poor reflection of in vivo cell behavior. Therefore, it is believed that by mimicking the in vivo conditions within the cultures, this would generate “closer-to-in vivo” results. For this purpose three dimensional (3D) culture setups have been developed including artificial scaffolds and extracellular matrix gels.
    Optimization of IGRA was attempted using solid 3D scaffolds in various formats. The purpose of the 3D scaffolds was to facilitate T lymphocyte migration and subsequently activation due to increased chances of T lymphocyte/APC encounter. However, it turned out that the addition of this extra dimension to the cultures did not translate into increased de novo secretion of IFN-γ in these cultures. Furthermore, we often observed a non-specific effect on the level of IFN-γ when cells were cultures in 3D. This suggested that cells were sensitive to the geometry surrounding them and that this was independent on antigen stimulation.
    Based on these findings and a previous discovery that the polymer PDMS, gave rise to increased differentiation of a nerve cell line in vitro, we tested the effect of PDMS on the differentiation of porcine monocytes. Monocytes are immune cells of high plasticity, and thus we speculated that they might be sensitive to culture conditions. Indeed, monocytes differentiated into monocyte-derived DC (moDCs) when cultured conventionally (2D PS) in the presence of GM-CSF and IL-4, but adopted a macrophage-like gene expression profile when cultured on PDMS. Further it was found that 3D culturing resulted in increased activation of the monocyte-derived cells.
    The work in this thesis covers several aspects within primary cell culture, but central to the work is the investigation of 3D cell culture setups for improved activation/differentiation of immune cells. Conclusively, this work highlights the importance of acknowledging the effect from external factors when analyzing data generated from in vitro cultures. This being even more important when working with immune cells since these cells adopt traits and functions simply based on the nature of the culture system.
    Original languageEnglish
    Place of PublicationFrederiksberg C
    Publisher National Veterinary Institute, Technical University of Denmark
    Number of pages155
    Publication statusPublished - 2016

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