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The lymph node is a highly structured organ that mediates the body’s adaptive immune response to antigens and other foreign particles. Central to its function is the distinct spatial assortment of lymphocytes and stromal cells, as well as chemokines that drive the signaling cascades which underpin immune responses. Investigations of lymph node biology were historically explored in vivo in animal models, using technologies that were breakthroughs in their time such as immunofluorescence with monoclonal antibodies, genetic reporters, in vivo two-photon imaging, and, more recently spatial biology techniques. However, new approaches are needed to enable tests of cell behavior and spatiotemporal dynamics under well controlled experimental perturbation, particularly for human immunity. This review presents a suite of technologies, comprising in vitro, ex vivo and in silico models, developed to study the lymph node or its components. We discuss the use of these tools to model cell behaviors in increasing order of complexity, from cell motility, to cell-cell interactions, to organ-level functions such as vaccination. Next, we identify current challenges regarding cell sourcing and culture, real time measurements of lymph node behavior in vivo and tool development for analysis and control of engineered cultures. Finally, we propose new research directions and offer our perspective on the future of this rapidly growing field. We anticipate that this review will be especially beneficial to immunologists looking to expand their toolkit for probing lymph node structure and function.

Micropatterning techniques have enabled the use of 3D cell cultures to recreate tissue-level behavior such as hypoxia or signaling gradients, but their integration with microfluidics has been limited. To access complex, non-linear and concentric geometries seen in vivo and in high-throughput culture arrays, we developed an in-situ micropatterning strategy that integrated photolithography of crosslinkable, cell-laden hydrogels with a simple microfluidic housing. By shining 405 nm light through a photomask containing the desired design, we patterned 3D cultures directly in a 130-μm deep microfluidic chamber. As a model system, we used thiol-ene step growth polymerization with thiol-modified gelatin (GelSH) and PEG-norbornene linker; the technology was also applicable to other photo-crosslinkable chemistries, including gelatin methacryloyl (GelMA) and gelatin norbornene (GelNB) with PEG-thiol linker. The on-chip patterning strategy generated 3D cultures that were self-standing and that could be combined using serial photomasks. The method consistently generated features as small as 100 μm in diameter, and shared, non-linear boundaries between cultures were readily achieved. The modularity of the platform meant that designs were interchangeable in the same microfluidic housing, without requiring new master fabrication. As a proof-of-principle, a fragile cell type, primary human T cells, were patterned in varied geometries. Cells were patterned with high regional specificity and viability remained high. We expect that this technology will enable researchers to organize 3D cultures into geometries that were previously difficult to obtain, granting access to biomimetic tissue organizations and 3D-cultured microarray formats.