A robust experimental model of a lymph node (LN) is imperative for the better understanding of mechanistic processes underlying immunity in health and disease. A 3D cell culture on a microfluidic chip is an ideal middle-ground to mimic in vivo responses while providing the advantages of an in vitro cell culture. Such a model must include the necessary cell-cell and cell-matrix interactions required to sufficiently generate immune responses, and should also integrate both supportive biomaterials and fluidic control.
The LN is comprised of a compartmentalized architecture; in a simplified view, it has three main regions: the sub-capsular sinus, the B cell follicles, and the deep para-cortex (both T and Dendritic cells zone). To replicate this structure, we generated a micro-patterned 3D cell culture of primary murine splenocytes inside of a microfluidic housing, creating the first 3D patterned LN-on-chip. The platform was designed to be customizable, allowing control over cellular distribution, matrix composition in different regions, and allows both incoming and interstitial fluid flow. The LN-on-chip was composed of two microfluidics “afferent lymphatic” channels, a central culture chamber, and one microfluidic “efferent lymphatic” channel. Inside the culture chamber, the hydrogel was excluded from the peripheral area through the use of micro-fabricated posts, creating a LN “sinus.” We used UV-patterning through a photomask to pattern a human-sized LN (10 mm diameter, 100 μm thick), composed of two to six 400-µm diameter “B cell follicles,” surrounded by a “para-cortex” region containing a distinct population of cells. A hydrogel matrix, 8% gelatin methacrylate cross-linked with 0.1% and lithium phenyl-2,4,6-trimethylbenzoylphosphinate, was selected to match the mechanical properties of healthy lymph nodes. We found that the patterning process retained high cell viability and basic cellular function such as cell migration. We also achieved the same fluid flow velocities as observed in vivo for incoming (5 - 6 μm/s) and interstitial (0.10 - 1 μm/s) fluid flows.
In summary, we describe a novel LN-on-chip that contains the cell types, spatial patterning, and fluid flow rates required to reproduce simple inflammatory and adaptive immune responses. In the future, this modular platform will be used to directly test hypotheses of how spatial structure and fluid flow patterns affect efficacy of an immune response.