Diffusion of cytokines in live lymph node tissue using microfluidic integrated optical imaging

Highlights: • A novel method quantifies diffusion of bioactive analytes in live tissue. • A microfluidic platform delivered fluorescent analytes noninvasively to tissue. • Diffusion was monitored by timelapse, widefield fluorescence microscopy. • The method, Micro-IOI, was validated against theory and existing methods. • Micro-IOI quantified the effective diffusion of cytokines in live lymph node tissue. - Abstract: Communication and drug efficacy in the immune system rely heavily on diffusion of proteins such as cytokines through the tissue matrix. Available methods to analyze diffusion in tissue require microinjection or saturating the tissue in protein, which may alter local transport properties due to damage or rapid cellular responses. Here, we developed a novel, user-friendly method – Microfluidic Integrated Optical Imaging (micro-IOI) – to quantify the effective diffusion coefficient of bioactive proteins in live tissue samples ex vivo. A microfluidic platform was used to deliver picograms of fluorescently labelled cytokines to microscale regions within slices of murine lymph node, and diffusion was monitored by widefield fluorescence microscopy. Micro-IOI was validated against theory and existing methods. Free diffusion coefficients were within 8% and 24% of Stokes-Einstein predictions for dextrans and cytokines, respectively. Furthermore, diffusion coefficients for dextrans and proteins in a model matrix were within 1.5-fold of reported results from fluorescence recovery after photobleaching (FRAP). We used micro-IOI to quantify the effective diffusion of three cytokines from different structural classes and two different expression systems – tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukin-2 (IL-2), from human and mouse – through live lymph node tissue. This is the first method to directly measure cytokine transport in live tissue slices, and in the future, it should promote a deeper understanding of the dynamics of cell-cell communication and enable targeted immunotherapy design.