Modeling Immunity In Vitro: Slices, Chips, and Engineered Tissues.

Modeling immunity in vitro has the potential to be a powerful tool for investigating fundamental biological questions, informing therapeutics and vaccines, and providing new insight into disease progression. There are two major elements to immunity that are necessary to model: primary immune tissues and peripheral tissues with immune components. Here, we systematically review progress made along three strategies to modeling immunity: ex vivo cultures, which preserve native tissue structure; microfluidic devices, which constitute a versatile approach to providing physiologically relevant fluid flow and environmental control; and engineered tissues, which provide precise control of the 3D microenvironment and biophysical cues. While many models focus on disease modeling, more primary immune tissue models are necessary to advance the field. Moving forward, we anticipate that the expansion of patient-specific models may inform why immunity varies from patient to patient and allow for the rapid comprehension and treatment of emerging diseases, such as coronavirus disease 2019.

Acute Lymph Node Slices Are a Functional Model System to Study Immunity Ex Vivo.

The lymph node is a highly organized and dynamic structure that is critical for facilitating the intercellular interactions that constitute adaptive immunity. Most ex vivo studies of the lymph node begin by reducing it to a cell suspension, thus losing the spatial organization, or fixing it, thus losing the ability to make repeated measurements. Live murine lymph node tissue slices offer the potential to retain spatial complexity and dynamic accessibility, but their viability, level of immune activation, and retention of antigen-specific functions have not been validated. Here we systematically characterized live murine lymph node slices as a platform to study immunity. Live lymph node slices maintained the expected spatial organization and cell populations while reflecting the 3D spatial complexity of the organ. Slices collected under optimized conditions were comparable to cell suspensions in terms of both 24-h viability and inflammation. Slices responded to T cell receptor cross-linking with increased surface marker expression and cytokine secretion, in some cases more strongly than matched lymphocyte cultures. Furthermore, slices processed protein antigens, and slices from vaccinated animals responded to ex vivo challenge with antigen-specific cytokine secretion. In summary, lymph node slices provide a versatile platform to investigate immune functions in spatially organized tissue, enabling well-defined stimulation, time-course analysis, and parallel read-outs.

Detergent wash improves vaccinated lymph node handling ex vivo.

Lymph nodes (LNs) are essential secondary immune organs where the adaptive immune response is generated against most infections and vaccines. We recently described the use of live ex vivo LN slices to study the dynamics of adaptive immunity. However, when working with reactive lymph nodes from vaccinated animals, the tissues frequently became dislodged from the supportive agarose matrix during slicing, leading to damage that prevented downstream analysis. Because reactive lymph nodes expand into the surrounding adipose tissue, we hypothesized that dislodging was a result of excess lipids on the collagen capsule of the LN, and that a brief wash with a mild detergent would improve LN interaction with the agarose without damaging tissue viability or function. Therefore, we tested the use of digitonin on improving slicing of vaccinated LNs. Prior to embedding, LNs were quickly dipped into a digitonin solution and washed in saline. Lipid droplets were visibly removed by this procedure. A digitonin wash step prior to slicing significantly reduced the loss of LN during slicing from 13 to 75% to 0-25%, without substantial impact on viability. Capture of fluorescent microparticles, uptake and processing of protein antigen, and cytokine secretion in response to a vaccine adjuvant, R848, were all unaffected by the detergent wash. This novel approach will enable ex vivo analysis of the generation of adaptive immune response in LNs in response to vaccinations and other immunotherapies.

Spatially Resolved Analytical Chemistry in Intact, Living Tissues.

Tissues are an exciting frontier for bioanalytical chemistry, one in which spatial distribution is just as important as total content. Intact tissue preserves the native cellular and molecular organization and the cell-cell contacts found in vivo. Live tissue, in particular, offers the potential to analyze dynamic events in a spatially resolved manner, leading to fundamental biological insights and translational discoveries. In this Perspective, we provide a tutorial on the four fundamental challenges for the bioanalytical chemist working in living tissue samples as well as best practices for mitigating them. The challenges include (i) the complexity of the sample matrix, which contributes myriad interfering species and causes nonspecific binding of reagents; (ii) hindered delivery and mixing; (iii) the need to maintain physiological conditions; and (iv) tissue reactivity. This framework is relevant to a variety of methods for spatially resolved chemical analysis, including optical imaging, inserted sensors and probes such as electrodes, and surface analyses such as sensing arrays. The discussion focuses primarily on ex vivo tissues, though many considerations are relevant in vivo as well. Our goal is to convey the exciting potential of analytical chemistry to contribute to understanding the functions of live, intact tissues.

Labelling primary immune cells using bright blue fluorescent nanoparticles.

Tracking cell movements is an important aspect of many biological studies. Reagents for cell tracking must not alter the biological state of the cell and must be bright enough to be visualized above background autofluorescence, a particular concern when imaging in tissue. Currently there are few reagents compatible with standard UV excitation filter sets (e.g. DAPI) that fulfill those requirements, despite the development of many dyes optimized for violet excitation (405 nm). A family of boron-based fluorescent dyes, difluoroboron ß-diketonates, has previously served as bio-imaging reagents with UV excitation, offering high quantum yields and wide excitation peaks. In this study, we investigated the use of one such dye as a potential cell tracking reagent. A library of difluoroboron dibenzoylmethane (BF2dbm) conjugates were synthesized with biocompatible polymers including: poly(l-lactic acid) (PLLA), poly(ε-caprolactone) (PCL), and block copolymers with poly(ethylene glycol) (PEG). Dye-polymer conjugates were fabricated into nanoparticles, which were stable for a week at 37 °C in water and cell culture media, but quickly aggregated in saline. Nanoparticles were used to label primary splenocytes; phagocytic cell types were more effectively labelled. Labelling with nanoparticles did not affect cellular viability, nor basic immune responses. Labelled cells were more easily distinguished when imaged on a live tissue background than those labelled with a commercially available UV-excitable cytoplasmic labelling reagent. The high efficiency in terms of both fluorescence and cellular labelling may allow these nanoparticles to act as a short-term cell labelling strategy while wide excitation peaks offer utility across imaging and analysis platforms.

Two-way communication between ex vivo tissues on a microfluidic chip: application to tumor-lymph node interaction.

Experimentally accessible tools to replicate the complex biological events of in vivo organs offer the potential to reveal mechanisms of disease and potential routes to therapy. In particular, models of inter-organ communication are emerging as the next essential step towards creating a body-on-a-chip, and may be particularly useful for poorly understood processes such as tumor immunity. In this paper, we report the first multi-compartment microfluidic chip that continuously recirculates a small volume of media through two ex vivo tissue samples to support inter-organ cross-talk via secreted factors. To test on-chip communication, protein release and capture were quantified using well-defined artificial tissue samples and model proteins. Proteins released by one sample were transferred to the downstream reservoir and detectable in the downstream sample. Next, the chip was applied to model the communication between a tumor and a lymph node, to test whether on-chip dual-organ culture could recreate key features of tumor-induced immune suppression. Slices of murine lymph node were co-cultured with tumor or healthy tissue on-chip with recirculating media, then tested for their ability to respond to T cell stimulation. Interestingly, lymph node slices co-cultured with tumor slices appeared more immunosuppressed than those co-cultured with healthy tissue, suggesting that the chip may successfully model some features of tumor-immune interaction. In conclusion, this new microfluidic system provides on-chip co-culture of pairs of tissue slices under continuous recirculating flow, and has the potential to model complex inter-organ communication ex vivo with full experimental accessibility of the tissues and their media.

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.