Development of a hydrogel-based microfluidic model of the lymph node
Lymph nodes (LNs) are immunological hubs where antigen and naïve lymphocytes meet to mount antigen specific immune responses. In cancers, the tumour-draining lymph node (TDLN) is the site of initial anti-tumour immune response. The TDLN undergoes dramatic reprogramming in response to tumour drainage that is still partly unclear. In vivo models do not easily permit us to follow events longitudinally or to manipulate the environment, while currently there is a lack of in vitro platforms that recapitulate the LN complexity to study its role in response to antigens and in disease. First, I have tried to adapt an existing PDMS-based device to model the LN. Having encountered some limitations, I have then developed a novel hydrogel-based LN-on-chip device. I have optimized a fluidic system, cell culture conditions and hydrogel composition to obtain long term culture of lymphoid primary cultures from spleen with a stromal cell line of fibroblastic reticular cells (FRCs). Different hydrogel formulations were tested and characterized with multiple techniques focusing on maintaining physiological cell morphology in the new 3D environment. In addition, a LN-like architecture was achieved where B cells are segregated from T cells, into B cell follicles. After optimization of the 3D cellular system with built-in fluidics, I applied the model to different context. Initially, PMA/Ionomycin, a strong cell activator, was perfused to obtain a general activation response. Then, LPS was used to trigger a reaction to a bacteria-derived antigen. Finally, using the OT-I model, I have used the device to trigger an antigen specific immune response to an OVA peptide. In the final part of the thesis, I modelled a TDLN in vitro. The LN device was incubated with tumour conditioned media (TCM) and IL-7 downregulation was observed, similarly to in vivo murine melanoma models. Then, to increase the complexity of the source of the tumour cues, a new tumour model was developed through 3D bioprinting, which also includes a shell of cancer associated fibroblasts (CAFs) and immune cells to form a complex engineered tumour microenvironment (TME). Incubation of the LN device with conditioned medium from the engineered TME also induced downregulation of IL-7. v Unlike current in vivo models that require node dissection at each discrete time point, this system allowed the monitoring of tissue remodelling in real-time via live imaging and measure molecular changes at a surface markers level as well as gene expression. Overall, this platform can represent a valid tool to study in vitro the LN complexity in a more tractable way.
3D immunology, Microfluidics, Organ-on-chip
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