Immunofluorescence staining of live lymph node tissue slices.

Explants of lymphoid tissue provide a rare opportunity to assess the organization of the immune system in a living, dynamic environment. Traditionally, ex vivo immunostaining is conducted in fixed tissue sections, while live tissues are analyzed using genetically engineered fluorescent reporters or adoptively transferred, pre-labelled cell populations. Here, we validated a protocol for immunostaining and imaging in live, thick slices of lymph node tissue, thus providing a spatial "map" of the lymph node while maintaining the viability and functionality of the slices. Using anti-B220/CD45R (B cell) as a prototype antibody, the procedure for immunostaining was tested for sufficient signal to noise with respect to staining time, temperature, and wash time, and the specificity was verified in comparison to isotype controls. Immunostaining signal in live tissue slices was detectable to atleast 120 µm deep for both whole antibodies and F(ab')2 fragments using the staining procedure. This procedure revealed the expected changes in B cell organization in lymph nodes from immunized mice. Cell surface staining with most antibodies did not induce cytokine secretion, and cytokine secretion in response to T cell stimulation was unaffected by immunostaining. Staining with known a mitogenic antibody (anti-CD3) simultaneously labelled the cells and activated the tissue, confirming that reagents for live immunostaining must be selected judiciously. As a proof of concept, this method was used to reveal the dynamic distribution of CD69, a T cell activation marker, in lymph node slices before and after ex vivo stimulation.

Spatially resolved microfluidic stimulation of lymphoid tissue ex vivo.

The lymph node is a structurally complex organ of the immune system, whose dynamic cellular arrangements are thought to control much of human health. Currently, no methods exist to precisely stimulate substructures within the lymph node or analyze local stimulus-response behaviors, making it difficult to rationally design therapies for inflammatory disease. Here we describe a novel integration of live lymph node slices with a microfluidic system for local stimulation. Slices maintained the cellular organization of the lymph node while making its core experimentally accessible. The 3-layer polydimethylsiloxane device consisted of a perfusion chamber stacked atop stimulation ports fed by underlying microfluidic channels. Fluorescent dextrans similar in size to common proteins, 40 and 70 kDa, were delivered to live lymph node slices with 284 ± 9 µm and 202 ± 15 µm spatial resolution, respectively, after 5 s, which is sufficient to target functional zones of the lymph node. The spread and quantity of stimulation were controlled by varying the flow rates of delivery; these were predictable using a computational model of isotropic diffusion and convection through the tissue. Delivery to two separate regions simultaneously was demonstrated, to mimic complex intercellular signaling. Delivery of a model therapeutic, glucose-conjugated albumin, to specific regions of the lymph node indicated that retention of the drug was greater in the B-cell zone than in the T-cell zone. Together, this work provides a novel platform, the lymph node slice-on-a-chip, to target and study local events in the lymph node and to inform the development of new immunotherapeutics.