Immune system on a chip and uses thereof

Abstract

Provided herein are multi-immune organ chips, systems comprising such, and methods of use thereof for modeling an immune response.

Description

[0001] IMMUNE SYSTEM ON A CHIP AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to an immune system on a chip and uses thereof, e.g., for evaluating drug induced immune responses and / or drug safety and efficacy.

[0004] BACKGROUND TO THE INVENTION

[0005] The immune system plays a role in a wide range of diseases including cancer, neurodegenerative diseases, chronic infections, and autoimmunity. Given the substantial differences between the human immune system and animal immune systems, immune system chips that model human-specific immune responses to treatments targeting the immune system will enable assessments that are otherwise missed in animal models.

[0006] Single organ chips and multiorgan chips are known in the art. However, development of multi-immune-organ chips has been extremely challenging due to engineering obstacles such as combining different design requirements for different organs, integrating vascular connections to each organ, and avoiding introduction of air bubbles and infections.

[0007] It is therefore of interest to develop multiorgan immune system chips that overcome these challenges, thereby providing a model system for recapitulating human-specific immune responses.

[0008] SUMMARY OF THE INVENTION

[0009] The present disclosure is based, at least in part, on the concept that if different immune system organs are connected via a lymphatic endothelial space, such as a lymphatic endothelial space in one or more channels, in a microfluidic platform that provides controlled culturing of cells and delivery of media, then a multiorgan immune system on a chip that can recapitulate immune responses can be produced.

[0010] Accordingly, aspects of the present disclosure provide a microfluidic device (300) comprising a muscle chip (311) and a lymph node chip (313); and a plurality of channels (312) fluidly connecting the muscle chip (311) to the lymph node chip (313), wherein the lymph node chip (313) comprises lymphatic cells and immune cells; and the muscle chip (311) comprises muscle cells.

[0011] In some embodiments, the muscle chip (311) comprises extracellular matrix, myoblasts, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, monocytes, neutrophils, or combinations and sub-combinations thereof.

[0012] In some embodiments, the lymph node chip (313) comprises extracellular matrix, lymphatic endothelial cells, monocytes, T cells, B cells, dendritic cells, fibroblasts, or combinations and sub-combinations thereof.

[0013] In some embodiments, the ratio of cells in the lymph node chip (313) to cells in the muscle chip (311) is between 1:4 to 1: 10.

[0014] In some embodiments, the plurality of channels (312) comprises lymphatic endothelial cells. In some embodiments, the plurality of channels (312) is fluidly connected to a port (314).

[0015] Aspects of the present disclosure provide a microfluidic device (400) comprising a lung chip (411) and a lymph node chip (413); and a plurality of channels (412) fluidly connecting the lung chip (411) to the lymph node chip (413), wherein the lymph node chip (413) comprises lymphatic cells and immune cells; and the lung chip (411) comprises lung cells.

[0016] In some embodiments, the lung chip (411) comprises extracellular matrix, lung epithelial cells, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, or combinations and sub-combinations thereof.

[0017] In some embodiments, the lymph node chip (413) comprises extracellular matrix, lymphatic endothelial cells, monocytes, T cells, B cells, dendritic cells, fibroblasts, or combinations and sub-combinations thereof.

[0018] In some embodiments, the ratio of cells in the lymph node chip (413) to cells in the lung chip (411) is between 1:4 to 1: 10.

[0019] In some embodiments, the plurality of channels (412) comprises lymphatic endothelial cells. In some embodiments, the plurality of channels (412) is fluidly connected to a port (414).

[0020] Aspects of the present disclosure provide a microfluidic device (500) comprising a muscle chip (511), a lymph node chip (513), and a lung chip (515); and a first set of a plurality of channels (512a) fluidly connecting the muscle chip (511) to the lymph node chip (513) and a second set of a plurality of channels (512b) fluidly connecting the lung chip (515) to the lymph node chip (513); wherein the lymph node chip comprises lymphatic cells and immune cells; the muscle chip comprises muscle cells; and the lung chip comprises lung cells.

[0021] In some embodiments, the muscle chip (511) comprises extracellular matrix, myoblasts, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, monocytes, neutrophils, or combinations and sub-combinations thereof.

[0022] In some embodiments, the lung chip (515) comprises extracellular matrix, lung epithelial cells, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, or combinations and sub-combinations thereof.

[0023] In some embodiments, the lymph node chip (513) comprises extracellular matrix, lymphatic endothelial cells, monocytes, T cells, B cells, dendritic cells, fibroblasts, or combinations and sub-combinations thereof.

[0024] In some embodiments, the ratio of cells in the lymph node chip (513) to cells in the muscle chip (511) is between 1 :4 and 1 :10 and the ratio of cells in the lymph node chip (513) to cells in the lung chip (515) is between 1:4 and 1:10. In some embodiments, the ratio of cells in the lymph node chip (513), the muscle chip (511), and the lung chip (515) is between 1:4:4 and 1: 10: 10.

[0025] In some embodiments, the first set of the plurality of channels (512a) and the second set of the plurality of channels (512b) comprise lymphatic endothelial cells. In some embodiments, the first set of the plurality of channels (512a) is fluidly connected to a first port (514a) and a second port (514b) and the second set of the plurality of channels (512b) is fluidly connected to a third port (514c) and a fourth port (514d).

[0026] Aspects of the present disclosure provide a system (100) comprising a first organ chip (111) and a second organ chip (113); and a plurality of channels (112) fluidly connecting the first organ chip (111) to the second organ chip (113), a pump (120); a mixer (130); and a fluidic network (140) fluidly connecting the first organ chip (111), the second organ chip (113), the pump (120), and the mixer (130); wherein the fluidic network (140) comprises at least one inlet (142) and at least one outlet (144); and wherein the first organ chip (111) comprises a muscle chip or a lung chip and the second organ chip (113) comprises a lymph node chip; and wherein the lymph node chip comprises lymphatic cells and immune cells; the muscle chip comprises muscle cells; and the lung chip comprises lung cells. In some embodiments, the first organ chip (111) comprises the muscle chip, which comprises extracellular matrix, myoblasts, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, monocytes, neutrophils, or combinations and sub-combinations thereof.

[0027] In some embodiments, the first organ chip (111) comprises the lung chip, which comprises extracellular matrix, lung epithelial cells, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, or combinations and sub-combinations thereof.

[0028] In some embodiments, the second organ chip (113) comprises the lymph node chip, which comprises extracellular matrix, lymphatic endothelial cells, monocytes, T cells, B cells, dendritic cells, fibroblasts, or combinations and sub-combinations thereof.

[0029] In some embodiments, the first organ chip (111) comprises the muscle chip and the ratio of cells in the lymph node chip to cells in the muscle chip is between 1 :4 to 1 : 10 or the first organ chip (111) comprises the lung chip and the ratio of cells in the lymph node chip to cells in the lung chip is between 1 :4 and 1: 10.

[0030] In some embodiments, the plurality of channels (112) comprises lymphatic endothelial cells. In some embodiments, the plurality of channels (112) is fluidly connected to a port (114a).

[0031] In some embodiments, the pump (120) is one or more of a pneumatic pump, an electromagnetic pump, a peristaltic pump, or a pressure pump.

[0032] In some embodiments, the system further comprises one or more sensors and / or one or more cameras. In some embodiments, the one or more sensors are selected from the group consisting of a temperature sensor, an oxygen sensor, a pH sensor, a humidity sensor, a photodetector, a transducer, an analyte or metabolite sensor, a fluid level sensor, a flow sensor, a hydrometer, a viscometer, a velocity sensor, and an electrical sensor.

[0033] In some embodiments, the system further comprises a control module.

[0034] Aspects of the present disclosure provide a system (200) comprising a first organ chip (211), a second organ chip (213), and a third organ chip (215); and a first set of a plurality of channels (212a) fluidly connecting the first organ chip (211) to the second organ chip (213) and a second set of a plurality of channels (212b) fluidly connecting the third organ chip (215) to the second organ chip (213); a pump (220); a mixer (230); and a fluidic network (240) fluidly connecting the first organ chip (211), the second organ chip (213), the pump (220), and the mixer (230); wherein the fluidic network (240) comprises at least one inlet (242a, 242b) and at least one outlet (244a, 244b); wherein the first organ chip (211) comprises a muscle chip or a lung chip, the second organ chip (213) comprises a lymph node chip, and the third organ chip (215) comprises the muscle chip or the lung chip; and wherein the lymph node chip comprises lymphatic cells and immune cells; the muscle chip comprises muscle cells; and the lung chip comprises lung cells.

[0035] In some embodiments, the first organ chip (211) comprises the muscle chip, which comprises extracellular matrix, myoblasts, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, monocytes, neutrophils, or combinations and sub-combinations thereof; and wherein the third organ chip (215) comprises the lung chip, which comprises extracellular matrix, lung epithelial cells, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, or combinations and sub-combinations thereof.

[0036] In some embodiments, the first organ chip (211) comprises the lung chip, which comprises extracellular matrix, lung epithelial cells, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, or combinations and sub-combinations thereof; and wherein the third organ chip (215) comprises the muscle chip, which comprises extracellular matrix, myoblasts, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, monocytes, neutrophils, or combinations and sub-combinations thereof.

[0037] In some embodiments, the second organ chip (213) comprises the lymph node chip, which comprises extracellular matrix, lymphatic endothelial cells, monocytes, T cells, B cells, dendritic cells, fibroblasts, or combinations and sub-combinations thereof.

[0038] In some embodiments, the ratio of cells in the lymph node chip to cells in the muscle chip is between 1 :4 and 1: 10 and the ratio of cells in the lymph node chip to cells in the lung chip is between 1:4 and 1 : 10. In some embodiments, the ratio of cells in the lymph node chip, the muscle chip, and the lung chip is between 1 :4:4 and 1 : 10: 10.

[0039] In some embodiments, the first set of the plurality of channels (212a) and the second set of the plurality of channels (212b) comprise lymphatic endothelial cells. In some embodiments, the first set of the plurality of channels (212a) is fluidly connected to a first port (214a) and a second port (214b) and wherein the second set of the plurality of channels (212b) is fluidly connected to a third port (214c) and a fourth port (214d).

[0040] In some embodiments, the pump (220) is one or more of a pneumatic pump, an electromagnetic pump, a peristaltic pump, or a pressure pump. In some embodiments, the system further comprises one or more sensors and / or one or more cameras. In some embodiments, the one or more sensors are selected from the group consisting of a temperature sensor, an oxygen sensor, a pH sensor, a humidity sensor, a photodetector, a transducer, an analyte or metabolite sensor, a fluid level sensor, a flow sensor, a hydrometer, a viscometer, a velocity sensor, and an electrical sensor.

[0041] In some embodiments, the system further comprises a control module.

[0042] Aspects of the present disclosure provide a microfluidic device comprising a first organ chip and a second organ chip; and a plurality of channels fluidly connecting the first organ chip to the second organ chip, wherein the first organ chip is a lymph node chip and the second organ chip is a muscle chip or a lung chip; and wherein the lymph node chip comprises lymphatic cells and immune cells; the muscle chip comprises muscle cells; and the lung chip comprises lung cells.

[0043] In some embodiments, the muscle chip comprises extracellular matrix, myoblasts, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, monocytes, neutrophils, or combinations and sub-combinations thereof.

[0044] In some embodiments, the lung chip comprises extracellular matrix, lung epithelial cells, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, or combinations and sub-combinations thereof.

[0045] In some embodiments, the lymph node chip comprises extracellular matrix, lymphatic endothelial cells, monocytes, T cells, B cells, dendritic cells, fibroblasts, or combinations and sub-combinations thereof.

[0046] In some embodiments, the second organ chip is the muscle chip and the ratio of cells in the lymph node chip to cells in the muscle chip is between 1 :4 to 1 : 10 or the second organ chip is the lung chip and the ratio of cells in the lymph node chip to cells in the lung chip is between 1 :4 and 1: 10.

[0047] In some embodiments, the microfluidic device further comprises a third organ chip and an additional channel fluidly connecting the first organ chip to the third organ chip, wherein the third organ chip is a muscle chip or a lung chip.

[0048] In some embodiments, the second organ chip is the muscle chip and the third organ chip is the lung chip. In some embodiments, the second organ chip is the lung chip and the third organ chip is the muscle chip. In some embodiments, the ratio of cells in the lymph node chip to cells in the muscle chip and cells in the lung chip is between 1 : 4: 4 to 1 : 10: 10.

[0049] In some embodiments, the plurality of channels comprises lymphatic endothelial cells.

[0050] Aspects of the present disclosure provide a method comprising contacting an agent with any one of the systems described herein or any one of the microfluidic devices described herein; and detecting a cellular response; wherein the agent comprises a therapeutic agent, a pathogenic agent, or a combination thereof.

[0051] In some embodiments, the agent is selected from the group consisting of a cell, a protein, a peptide, an antibody or antigen binding fragment thereof, a nucleic acid, and a small molecule.

[0052] In some embodiments, the cellular response is selected from the group consisting of viability, proliferation, activation, respiration, metabolism, cell migration, cell contractility, differential expression of cell biomarkers, production and / or release of biomolecules, action potentials, and combinations thereof.

[0053] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The skilled artisan will understand that the drawings primarily are for illustrative purposes and are not intended to limit the scope of the inventive subject matter described herein. The drawings are not necessarily to scale; in some instances, various aspects of the inventive subject matter disclosed herein may be shown exaggerated or enlarged in the drawings to facilitate an understanding of different features. In the drawings, like reference characters generally refer to like features (e.g., functionally similar and / or structurally similar elements).

[0056] FIG. 1 is a flow chart illustrating biological processes involved in generation of a response to vaccine administration.

[0057] FIG. 2 illustrates one example of a lymph node chip.

[0058] FIG. 3A illustrates one example of a muscle chip formed with PDMS surrounding the anchorage nodes selectively surface functionalized using the heterobifunctional linker, sulfo- SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate) (SSH).

[0059] FIG. 3B illustrates one example of a muscle chip with a series of pillars for the tissue to wrap around and an inlet port in the central channel. FIG. 3C illustrates one example of a muscle chip with four anchorage nodes arranged to create a rectangularly shaped muscle tissue after sculpting between the capillary barriers.

[0060] FIG. 4 illustrates one example of a lung chip.

[0061] FIG. 5 illustrates one example of a muscle-lymph node chip.

[0062] FIG. 6 illustrates one example of a lymph node-lung chip.

[0063] FIG. 7 illustrates one example of a muscle-lymph node-lung chip.

[0064] FIG. 8 is a schematic depiction of a system comprising two organ chips (e.g., muscle and lymph node or lung and lymph node) in accordance with some embodiments described herein.

[0065] FIG. 9A is a schematic depiction of a system comprising three organ chips (e.g., muscle, lymph node, and lung) in accordance with some embodiments described herein.

[0066] FIG. 9B is another schematic depiction of a system comprising three organ chips (e.g., muscle, lymph node, and lung) in accordance with some embodiments described herein.

[0067] FIG. 10 is a schematic depiction of muscle and lymph node on a chip in accordance with some embodiments described herein.

[0068] FIG. 11 is a schematic depiction of lung and lymph node on a chip in accordance with some embodiments described herein.

[0069] FIG. 12 is a schematic depiction of lymph node connected to muscle and lung on a chip in accordance with some embodiments described herein.

[0070] DETAILED DESCRIPTION

[0071] As repeated waves of the COVID pandemic rage across the world and the possibility of future respiratory pandemics remains, it has become increasingly clear that tools to mount an equally rapid response are urgently needed. What is needed are physiologically relevant interconnected, complex in vitro models that can be used to simulate introduction of a vaccine or treatment (e.g., vaccine injection, antibody injection) into a muscle, a subsequent immune response in draining lymph nodes, and eventually protection against pathogen infection or treatment of disease. Further, with an appreciation for the increased speed in developing vaccines (e.g., COVID- 19 vaccines) and therapeutic solutions to other pathogens or diseases, there is a long-felt, unmet need for scientific tools that decrease development time while improving the translatability of biotherapeutic candidates into the clinic. Next generation adjuvants, messenger ribonucleic acid (mRNA)-based platforms, and pharmaceutical therapies (e.g., monoclonal antibodies, small molecules) need tools to better investigate mechanism of action (MoA), reactogenicity, efficacy, and / or translation from the bench to humans. There is an increasing demand for translationally relevant in vitro models that could reduce or potentially replace the reliance on in vivo models. See, e.g., Horejs, C. Organ chips, organoids and the animal testing conundrum. Nature Reviews Materials 2021, 6, 372-373. Most vaccine candidates are first screened in mouse models that often overestimate the adaptive immune response and underestimate the reactogenicity of the vaccine or treatment candidate, which leads to faulty selection of candidates with higher chance of failure in human clinical trials. See, e.g., Seok, J.; Warren, H. S.; Cuenca, A. G.; et al. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proceedings of the National Academy of Sciences 2013, 110, 3507-3512.

[0072] Biologies and vaccine candidates are also tested in non-human primates (NHPs) for efficacy, pharmacokinetic (PK) and / or pharmacodynamic (PD) modeling, and safety, which was manageable when the number of biologies and vaccines was small. However, with an everexpanding pipeline across the industry, the number of NHP experiments required by the Food and Drug Administration (FDA) is reaching unsustainable levels. Recent guidance in early 2022 from the FDA has requested sponsors to consider alternatives to NHPs when performing toxicology studies. See, e.g., “Nonclinical Considerations for Mitigating Nonhuman Primate Supply Constraints Arising from the COVID-19 Pandemic: Guidance for Industry,” U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologies Evaluation and Research (CBER), & Oncology Center for Excellence (OCE), February 2022, archived at The Internet Archive, web.archive.org / web / 20221220025415 / https: / / www.fda.gov / media / l 55950 / download, capture date December 20, 2022 (last accessed December 18, 2023). See also, e.g., Ingber, D.E. Human organs-on-chips for disease modelling, drug development and personalized medicine. Nat Rev Genet 23, 467-491 (2022).

[0073] Development of an immune system on a chip that is adaptable to multiple immunological tests across pharmaceutical and vaccine discovery efforts is highly desirable and would allow for rapid testing of small molecules, large molecules, and / or vaccine candidates before progressing into clinical development. The present disclosure describes a complex invention that develops, connects, and stabilizes into an interconnected immune multi-organ system in vitro muscle on a chip, lymph node on a chip, and / or lung on a chip organ systems. The interconnected immune multi-organ system of the present invention allows for understanding and prediction of MoA and efficacy of novel biotherapeutic (e.g., small molecules, large molecules, and / or vaccine) candidates against disease, including ongoing (e.g., influenza, respiratory syncytial virus (RSV), human rhinoviruses (HRV)) and future infectious disease outbreaks.

[0074] Skilled artisans are aware of single organ systems providing in vitro single organ systems including muscle, lymph node, and respiratory microphysiological systems (MPS) models. These single organ systems have demonstrated important organ specific features of differentiation and maturation of the tissue-specific cell types, but also have shown markers of disease specific biology captured in a dish. However, these single organ systems have not combined an immune system model with a vascularized muscle model, a lymph node model, and / or lung system(s) model(s) into interconnected two or three organ systems.

[0075] The present disclosure describes interconnected immune multi-organ systems including: a muscle on a chip unit, a lymph node on a chip unit, and / or a lung on a chip unit connected via systemic and lymphatic vascular circulation that mimics in vivo physiology of vaccination or treatment (e.g., injection), vaccine or treatment (e.g., injection response), site specific infection or reaction, and / or response derived from vaccination or treatment.

[0076] Following below are detailed descriptions of various concepts related to, and exemplary embodiments of, systems, methods, and components related to a multiorgan immune system on a chip (also referred to as an immune system chip). The immune system chips described herein are particularly useful because these models may replace animal models, thereby saving animal lives and accelerating development of new therapeutics against emerging infectious diseases such as coronaviruses.

[0077] DEFINITIONS

[0078] The following paragraphs include various definitions used in this document.

[0079] The term “about” as used herein means plus or minus 10%.

[0080] As used herein, the term “channel” refers to a pathway that allows for movement of a fluid (e.g., liquid, gas). As such, channels can connect one or more components. Channels can be any size and shape suitable for use in the immune system chips described herein. As used herein, the term “immune organ” refers to an organ involved in immune responses and / or in the immune system. Non-limiting examples of immune organs include lymphoid organs (e.g., spleen, tonsils, lymph nodes), muscle, and lung.

[0081] As used herein, the term “immune response” refers to any response produced by the immune system. An immune response can be produced against an antigen, an allergen, an agent, or a combination thereof. An immune response can be an immunostimulatory response or an immunosuppressive response. Non-limiting examples of immunostimulatory responses include increased antigen-specific antibody production; activation or proliferation of lymphocytes (e.g., natural killer (NK) cells, CD4+ T lymphocytes, CD8+ T lymphocytes, macrophages); and increased synthesis of immunostimulatory cytokines (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL- 10, IL- 12, IFN-a, IFN-0, IFN-y, TNF-a). Non-limiting examples of immunosuppressive responses include reduced production of antigen-specific antibodies (e.g., reduced IgE production); activation of lymphocytes or other cell populations; and increased synthesis of cytokines having immunosuppressive effects (e.g., increased synthesis of IFN-y).

[0082] As used herein, the term “organ on a chip” or “organ chip” refers to a microfluidic device that mimics a mammalian (e.g., human) organ. In some examples, the organ chip can mimic an organ with respect to its microstructures, functions, physiochemical environments, and combinations thereof.

[0083] As used herein, the term “fluidly connected” refers to connections between two or more components of an organ chip in a manner such that a fluid or a portion thereof (e.g., any material or medium including, but not limited to, cell culture media, cells, gas, suspensions, aerosols, and biological fluids) can directly or indirectly pass or flow from one component to another component (e.g., from muscle on a chip to lymph node on a chip).

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0085] FIG. 1 is a flow chart (8) illustrating biological processes involved in generation of a vaccine response. A vaccine is generally injected into muscle tissue (10). The vaccine is taken up by muscle cells and tissue resident antigen presenting cells (APCs), which causes an inflammatory response (e.g., TLR3, TLR7, and TLR8 on the endosome; cytosolic sensors such as RIG-I, MDA5, PKR, and OAS also recognize dsRNAs and ssRNAs in the cytoplasm result in production of proinflammatory cytokines, e.g., pro-inflammatory cytokines (e.g., IL-6, IL-8, TNF-a) and type 1 interferon response (e.g., IFN-0)) and antigen expression and production (12). The vaccine is also taken up in draining lymph nodes (dLNs) and by resident APCs and stromal cells (14).

[0086] Lymphatic endothelial cells under the inflammatory cytokines express CCL21 and activated DCs express CCR7.

[0087] Antigen expression and production then occurs, including APC uptake, toll-like receptor (TLR) response, inflammatory cytokines drawing in more monocytes and / or APCs, and uptake and / or maturation of DCs (e.g., CDlc+ / CD141+) (16). Lymphatic endothelial cells under the inflammatory cytokines express CCL21 and activated DCs express CCR7 (18). Laminar flow directed towards draining lymph nodes (dLNs) establishes a robust CCL21 gradient to which the antigen carrying DCs respond (conserved chemotaxis axis) (20). DCs move towards dLNs (22) and are pushed deep into the T-cell territory via the robust CCL21 gradient (24). Activated DCs cross present the antigen to naive T cells (26). Activated cDCls cross present, activate, and expand antigen specific CD8+ T cells, may cross transfer to lymph node resident cDCls to activate CD8+ T cells, and / or may be helped by CD4+ T cells (28).

[0088] Activated cDC2s cross present the antigen to antigen specific naive CD4+ T cells (30). Antigen-specific T-follicular helper cells are induced (CD14+ macrophages can do this too) (32). CD4+ TFH cells move to the B-cell zone (34). Binding with different B-cell receptors (BCRs), affinity maturation, somatic hypermutation, clonal selection, and isotype switching occurs (36). The best B-cell is selected (compete for T-cell interaction) for plasma cell, memory B cell generation, neutralizing antibody (Ab) production, and / or infection response (38). The plasma cell in the germinal center (GC) produces antibodies; a subset of memory B cells (slightly immature) enter circulation (S1PR1 driven) and the plasma cells can home to bone marrow (BM) (40).

[0089] Antigen specific CD8+ T-cells leave the lymph node (S1PR1 driven) and migrate to the intended site of action (e.g., muscle and lung) (42). Antigen expressing muscle cells (after mRNA vaccination) are eliminated under a CD8+ T-cell response (44). Upon re-infection in the secondary sites or even a different variant, the memory B-cells undergo somatic hypermutation (SHM) and AF locally in GC to produce neutralizing antibodies (46). An infection response is generated (48).

[0090] This external infection is simulated in the connected vascularized lung. These neutralizing antibodies are thereby protective to the exogenous infection challenge in the lung. For example, based on prior studies using a single organ lung on chip, it is expected that a vascularized lung on a chip compartment co-cultured with plasmacytoid dendritic cells (tissue resident APCs) under an exogenous infection challenge releases cytokines that are typical indicators of infection response including, but not limited to, IL-6, IP- 10, RANTES, IFN-P, MCP-1, IL-8. See, e.g., Si et al. A human-airway-on-a-chip for the rapid identification of candidate antiviral therapeutics and prophylactics. Nature biomedical engineering 2021, 5, 815- 829.

[0091] For example, an RSV challenge study in calves indicates that interferon signaling, granzyme B signaling, and pathogen pattern recognition receptors are key to draw in immune cells in response to infection. See, e.g., Johnston et al. Experimental challenge with bovine respiratory syncytial virus in dairy calves: bronchial lymph node transcriptome response. Scientific reports 2019, 9, 1-13. The interferon signaling importance is also seen in mouse studies, which suggest that type I IFNR provides the first direct stimulatory signal for local respiratory tract B cells during influenza virus infection and is responsible for local up-regulation of CD69 and CD86 on lymph node B cells within 48 hours of infection. See, e.g, Coro et al. Type I IFN receptor signals directly stimulate local B cells early following influenza virus infection. The Journal of Immunology 2006, 176, 4343-4351. In vivo studies suggest that lung dendritic cells (e.g., CD1 lblow / negCD103+) are specialized for the transport of influenza antigen from the lung to the lymph node, and for critical for processing and presentation of these viral antigens to CD8+ T-cells. See, e.g., Ho et al. Lung CD103+ dendritic cells efficiently transport influenza virus to the lymph node and load viral antigen onto MHC class I for presentation to CD8 T cells. The Journal of Immunology 2011, 187, 6011-6021. Furthermore, multiple DC subsets may be needed to model this crosstalk. For example, mouse studies suggest that during RSV infections, at least three DC subsets are involved during the activation of lymph node trafficking naive and memory CD4+ and CD8+ T-cells. See, e.g., Lukens et al. Respiratory syncytial virus-induced activation and migration of respiratory dendritic cells and subsequent antigen presentation in the lung-draining lymph node. Journal of Virology 2009, 83, 7235-7243. Antigen specific CD8+ T-cells can then migrate into the lung tissue and to the inflamed muscle tissue to target and eliminate antigen expressing muscle cells that were successfully transfected with mRNA containing lipid nanoparticles.

[0092] I. Systems Comprising Multi-Immune-Organ Chips

[0093] Aspects of the present disclosure provide systems comprising multi-immune-organ chips. In some embodiments, the systems described herein include two or more immune organs (e.g., muscle, lung, lymph node, or combinations thereof) on a chip, a plurality of channels connecting the two or more immune organs, a pump for circulating fluid throughout the system, and a mixer for removing fluid from the system.

[0094] In one embodiment, the multi-immune-organ chip includes a lymph node chip. The lymph node on a chip is created with the specific intention to receive antigen carrying activated dendritic cells (e.g, cDCls and cDC2s) from the vascularized muscle unit to the afferent lymphatic endothelial channels, e.g., under laminar flow. Next, the system ensures delivery of activated antigen presenting cells deep into the T-cell zone (via CCL21 gradient), followed by cross-presentation of antigens to the naive T-cells. Antigen specific CD8+ T-cells, memory B- cells, and / or neutralizing antibodies are produced by high antigen affinity plasma B-cells that can exit the lymph node chip to neutralize the infection in the connected lung on a chip and eliminate antigen expressing muscle cells. The key features of this lymph node on a chip, supported by inclusion of critical components of the lymph node (e.g., naive T-cells, naive B-cells, germinal center resident antigen presenting cells, germinal center B cells, follicular dendritic cells, follicular reticular cells, lymph node stroma cells, lymphatic endothelial cells, etc.) are to recapitulate important features of geminal center in vitro, including the production of antigen specific antibodies, antigen specific somatic hypermutation and affinity maturation, plasmablast differentiation, and class-switch recombination, plasma B-cell, memory B-cells, and CD8+ T- cells production.

[0095] In one embodiment, the lymph node on a chip also receives the eluent from the infected lung (e.g., cytokines and antigenic peptides) and mobilizes an immune response to neutralize the infection in the lung on a chip. In one embodiment, the chip is fabricated using at least one thermoplastic. In one embodiment, the chip is fabricated using polydimethylsiloxane (PDMS), polyetherimide (e.g., ULTEM), polyurethane (e.g., thermoplastic polyurethane), acrylic, polystyrene, polyethylene terephthalate (PET), poly(methyl methacrylate) (PMMA), polycarbonate, poly(ethylene glycol) diacrylate (PEGDA), and / or cyclic olefin copolymer (COP). In one embodiment, the chip is entirely fabricated using polydimethylsiloxane (PDMS). In one embodiment, PDMS base and curing agent are mixed thoroughly for device fabrication. In one embodiment, the PDMS base and curing agent are mixed thoroughly at a weight ratio between about 3: 1 and about 20: 1. In one embodiment, the PDMS base and the curing agent are mixed thoroughly at a weight ratio of 10: 1. The resulting mixture is then degassed (e.g., in a vacuum-based degasser). In one embodiment, the degassed mixture is then poured over at least one device mold containing the device chamber features to be fabricated. Molded PDMS is cured (e.g., at 60° C for 7 hours in a hot air oven).

[0096] In one embodiment, the lymph node chip features a central wider microfluidic channel flanked by two capillary barriers. In one embodiment, the central microfluidic channel contains a rectangular space that connected with the capillary barriers on each end. In one embodiment, the chip contains two openings that are directly above at the ends of the rectangular central chamber, allowing easy access to the body of the chip. In one embodiment, the openings in the top layer are created using biopsy punches. The bottom layer contained cuboidal space to hold the features described in the top layer with the total height of the chip being approximately 3-4 mm. In one embodiment, the upper and lower layers are plasma treated and adhered to each other using uncured, activated PDMS pre-polymerized mixture.

[0097] In an embodiment, the lymph node on a chip is created using donor tonsil tissue as described in the Examples below.

[0098] In one embodiment, the lymph node chip includes extracellular matrix (ECM), lymphatic endothelial cells, monocytes (e.g., CD14+ / CD16-, CD14+ / CD16+), T-cells, B-cells, fibroblastic reticular cells, follicular dendritic cells, conventional type 1 dendritic cells (cDCls), conventional type 2 dendritic cells (cDC2s), and / or plasmacytoid DCs.

[0099] FIG. 2 illustrates one example of a lymph node chip.

[0100] Vascularized, tissue resident antigen presenting cells containing muscle chip (in-vitro) is created with a specific intention to mimic spaces where vaccine candidates (e.g., mRNA-based vaccines, protein-based adjuvant vaccines) and pharmaceutical candidates (e.g., small molecules, large molecules) can be injected that enable antigen expression, production, uptake, inflammatory response generation, drawing in of more immune cells (e.g., macrophages) via connected vasculature, antigen uptake, and activation of professional antigen presenting cells.

[0101] In one embodiment, vascularized muscle on a chip with endothelial cells and lymphatic endothelial cells flanking a first side (e.g., left side) and a second side (e.g., right side) of the central muscle chip unit are prepared by adding a mixture of skeletal muscle myoblasts mixed with fibroblasts cells in extracellular matrix suspension into a central space trapped (e.g., attached to anchoring nodes) between small linear barriers.

[0102] Any method suitable for inducing endothelial cell (e.g., HUVEC, LEC) sprouting can be used to create vascularized muscle tissue. For example, endothelial cell sprouting can be induced using cell culture media comprising VEGFA and VEGFC. Additional agents (e.g., HGF, ANG-1 and SIP, PMA, FGFb, MCP-1, SDFla) can be included in the culture media to promote lymphangiogenesis, vascular angiogenesis, and sprouting.

[0103] In one embodiment, the chip is fabricated using at least one thermoplastic. In one embodiment, the chip is fabricated using polydimethylsiloxane (PDMS), polyetherimide (e.g., ULTEM), polyurethane (e.g., thermoplastic polyurethane), acrylic, polystyrene, polyethylene terephthalate (PET), poly(methyl methacrylate) (PMMA), polycarbonate, poly(ethylene glycol) diacrylate (PEGDA), and / or cyclic olefin copolymer (COP). In one embodiment, the chip is entirely fabricated using poly dimethylsiloxane (PDMS). In one embodiment, the PDMS base and curing agent are mixed thoroughly at a weight ratio between about 3: 1 and about 20: 1. In one embodiment, PDMS base and curing agent are mixed thoroughly at a weight ratio of 10: 1 for device fabrication. In one embodiment, this mixture is then degassed in a vacuum-based degasser. In one embodiment, the degassed mixture is then poured over a device mold containing the device chamber features to be fabricated. In one embodiment, molded PDMS is cured (e.g., at 60°C for 7 hours in a hot air oven).

[0104] In one embodiment, the mold is used to create a top layer geometry of the muscle chip featuring a central wider microfluidic channel flanked by two capillary barriers. In one embodiment, the central microfluidic channel also contains a circular space (e.g., for muscle tissue sculpting) that connects and merges with the two capillary barriers. In one embodiment, the circular space has at least one anchorage node (e.g., at least two anchorage nodes). In one embodiment, the circular space has at least two anchorage nodes, wherein each end has one or more of the at least two anchorage nodes. In one embodiment, the chip includes openings that are directly above each of the at least one anchorage node, allowing easy access to the chip. In one embodiment, the openings in the top layer are created using biopsy punches. In one embodiment, the bottom layer includes cuboidal space. In one embodiment, the chip has a total height of approximately 3-4 mm. In one embodiment, the upper and lower layers are plasma treated and adhered to each other using uncured, activated PDMS pre-polymerized mixture.

[0105] In one embodiment, in order to create the sculpted muscle tissue, PDMS surrounding the at least one anchorage node is selectively surface functionalized using the heterobifunctional linker, sulfo-SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate) (SSH) to create at least one adhesive anchorage node. In this embodiment, the rest of the chip remains untreated. In one embodiment, for the PDMS surface functionalization procedure, SSH at a concentration of 1 mg / mL in deionized water is selectively applied to the area surrounding the at least one anchorage node. In one embodiment, SSH droplets are retained in the at least one anchorage node by surface tension effects while leaving the untreated surfaces of the tissue chambers (e.g., circular central spaces) dry during this procedure. In one embodiment, after adding SSH solution, devices are exposed to UV light (e.g., at an optical output of 30 mW / cm2for 5 min). In one embodiment, the SSH solution is then aspirated, and the previous step repeated for another 5 min of UV exposure before washing the surface. In one embodiment, sculpting devices are then washed with phosphate-buffered saline (PBS), after which the SSH treated surfaces are considered functionalized. In one embodiment, only the anchorage node(s) of the device are surface functionalized, and the rest of the muscle chip (circular area) remains non adhesive to allow for ECM compaction and tissue contraction during muscle tissue sculpting. In another embodiment, the muscle chip contains at least one pillar (e.g., a singular central pillar or a series of pillars) for the muscle tissue to wrap around during the extracellular matrix (ECM) compaction and tissue contraction phase. In one embodiment, both above-described muscle chip embodiments have two openings to access the muscle chamber (e.g., central muscle chamber) to be filled with desired cell types mixed in with the extracellular matrix of interest.

[0106] In one embodiment, at least one access node (e.g., two access nodes) is created in the area outside of the contracted muscle tissue to refill the central muscle channel with extracellular matrix with any desired cell type (e.g., endothelial cells, fibroblasts, dendritic cells) of interest. In one embodiment, the at least one access node is used to create a fluid tight plug in the central section of the muscle chip after ECM compaction and tissue sculpting condensed the tissue from the capillary barrier boundary.

[0107] In one embodiment, at least one additional capillary barrier (e.g., two additional capillary barriers) is added on the lower side of the chip when the top capillary barriers are found to be insufficient to contain the muscle construct within the capillary barrier boundaries. In this embodiment, human umbilical vein endothelial cells (HUVECs) and lymphatic endothelial cells (LECs) addition beyond the capillary barrier and sprouting into the muscle tissue are achieved in a similar way.

[0108] In a certain embodiment, the CD34+ HSC derived cDCl, cDC2, and plasmacytoid DCs are mixed with the muscle cells (e.g., up to 10%-20%), introduced into the muscle unit via vascular channel or added at a later stage to create a co-culture assay system of immune cell containing vascularized muscle tissues. In one embodiment, the muscle chip contains an inlet to access the muscle tissue (e.g., circular central region) and an inlet-outlet to connect the vascular endothelial cell channel to the systemic flow.

[0109] In one embodiment, the muscle chip includes extracellular matrix (ECM), myotubes (e.g., three-dimensional myotubes), vascular endothelial cells, lymphatic endothelial cells, tissue resident antigen presenting cells, conventional type 1 dendritic cells (cDCls), conventional type 2 dendritic cells (cDC2s), and / or plasmacytoid DCs.

[0110] FIG. 3A illustrates one example of a muscle chip formed with PDMS surrounding the anchorage nodes selectively surface functionalized using the heterobifunctional linker, sulfo- SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate) (SSH).

[0111] FIG. 3B illustrates one example of a muscle chip with a series of pillars for the tissue to wrap around and an inlet port in the central channel.

[0112] FIG. 3C illustrates one example of a muscle chip with four anchorage nodes arranged to create a rectangularly shaped muscle tissue after sculpting between the capillary barriers.

[0113] In one embodiment, the multi-immune-organ chip includes a lung chip. A vascularized multicellular lung on a chip unit is created to mimic epithelial lung (e.g., alveolar or bronchial) infection or treatment, mounting of infection or treatment response, infiltration of tissue resident and / or vascular immune cells into the lung spaces, and / or transmission of the infection or treatment signal to the connected lymph node chip to mount an immune response. In one embodiment, the lung chip includes primary lung epithelial cells, patient- derived lung organoids, and / or lung cells derived from induced pluripotent stem cells (iPSCs). In one embodiment, the primary lung epithelial cells, patient-derived lung organoids, and / or lung cells are healthy. Alternatively, the primary lung epithelial cells, patient-derived lung organoids, and / or lung cells are diseased. In one embodiment, the primary lung epithelial cells, patient- derived lung organoids, and / or lung cells are used to form an in vitro lung epithelium.

[0114] In one embodiment, the primary lung epithelial cells, patient-derived lung organoids, and / or lung cells are bronchial, bronchiolar, and / or alveolar cells. In one embodiment, the lung chip serves as a model for a lung tissue that is distinct from the initial origin of the primary cell type. For example, and not limitation, the lung chip includes bronchiolar cells as the primary cell type, but the tissue evolves to have characteristics closer to bronchial tissue. In one embodiment, the lung chip further includes additional supporting cells including, but not limitation to, lung fibroblasts, endothelial cells (e.g., pulmonary microvascular endothelial cells, human umbilical vein endothelial cells, lymphatic endothelial cells), and / or pericytes (e.g., to induce vascular structures in or nearby the lung cells).

[0115] In one embodiment, the lung chip includes undifferentiated, partially differentiated, or fully differentiated primary immune cells, progenitors, or induced pluripotent stem cells. In one embodiment, the undifferentiated, partially differentiated, or fully differentiated primary immune cells, progenitors, or induced pluripotent stem cells serve as immune components of the lung chip. In one embodiment, the lung chip is cultured using media types and supplements suitable to a development stage (i.e., vascularization medium, differentiation medium, air-liquid interface medium, or a combination thereof). In one embodiment, the culture is submerged or at the airliquid interface, depending on the time point and model maturity. In one embodiment, additional supplements are added into the medium of the lung chips at certain time point to either maintain or influence the differentiation of the immune components in the model.

[0116] In one embodiment, the lung chip includes extracellular matrix (ECM), vascular endothelial cells, tissue resident antigen presenting cells, conventional type 1 dendritic cells (cDCls), conventional type 2 dendritic cells (cDC2s), plasmacytoid DCs, tissue resident immune cells, and / or lymphatic vasculature.

[0117] FIG. 4 illustrates one example of a lung chip. In one embodiment, the present invention comprises a muscle-lymph node chip. The muscle-lymph node chip comprises a muscle chip and a lymph node chip. The muscle-lymph node chip can comprise one or more chips. For example, in one embodiment, the muscle-lymph node chip is a single chip comprising both a muscle chip and a lymph node chip. Alternatively, the muscle-lymph node chip is formed of more than one chip. FIG. 5 illustrates one example of a muscle-lymph node chip.

[0118] The muscle chip and the lymph node chip are connected via a plurality of channels (e.g., one or more channels). In one embodiment, the plurality of channels comprises polydimethylsiloxane (PDMS), polyetherimide (e.g., ULTEM), polyurethane (e.g., thermoplastic polyurethane), acrylic, polystyrene, and / or polyethylene terephthalate (PET). In one embodiment, the plurality of channels comprises polydimethylsiloxane (PDMS). In one embodiment, the plurality of channels is populated (e.g., selectively populated) with lymphatic endothelial cells. In one embodiment, the plurality of channels is lined with at least one layer of the lymphatic endothelial cells. In one embodiment, the at least one layer is a monolayer. For example, and not limitation, in vivo, lymphatic endothelial cells line the blind ended afferent lymphatic endothelial capillaries in a monolayer and collect interstitial flow and drain it into the tissue resident lymph node. A plurality of blind ended afferent lymphatic capillaries merge into a few afferent lymphatic vessels and drain into a lymph node. These afferent lymphatic vessels in vivo also have internal channel valves that ensure that the flow is always unidirectional.

[0119] In one embodiment, the lymphatic drainage connection on the chip comprises lymphatic endothelial capillaries of the afferent lymphatics, lymphatic endothelial vessels of the afferent lymphatics, lymphatic endothelial vessels of the draining lymph node, and efferent lymphatics that leave the lymph node.

[0120] In one embodiment, the plurality of channels is formed using photolithography, three- dimensional (3D) printing, needle extrusion, and / or machine milling. In one embodiment, one or more of the plurality of channels have a dimension between about 25 pm and 100 pm. Afferent lymphatic vessels are described in Schineis, et al. "Cellular traffic through afferent lymphatic vessels." Vascular pharmacology 112 (2019): 31-41, which is incorporated herein by reference in its entirety.

[0121] Lymphatic endothelial cells in the lymphatic capillaries in vivo are typically oakleafshaped. Neighboring endothelial cells partially overlap and are connected by discontinuous cell- cell junctions that generate open flaps, which are very useful for dendritic cells to cross into the capillaries from which they then move towards the lymphatic vessels. Within the lymphatic vessels, the flow rate can be a bit higher, so the DCs move away and drain into the lymph node. The lymphatic endothelial cells in the collecting lymphatic vessels (that form by the merger of many lymphatic capillaries) are elongated shape and are connected by tight and continuous cellcell junctions. They are also surrounded by continuous basement membrane. See, e.g., Schineis, et al. "Cellular traffic through afferent lymphatic vessels." Vascular pharmacology 112 (2019): 31-41.

[0122] In the present invention, these structured cells of the collecting lymphatic vessels are shown in the plurality of the channels (FIGS. 5-6).

[0123] In one embodiment, the plurality of channels receives interstitial fluid (e.g., a stream of interstitial fluid). In one embodiment, the stream of interstitial fluid is a steady stream of interstitial fluid. In one embodiment, the interstitial fluid flows and / or drains from the muscle chip to the lymph node chip. This embodiment provides laminar flow generated via movement of the interstitial fluid over the lymphatic endothelial cells. In one embodiment, the laminar flow is between about 1 pm / sec to about 30 pm / sec. In one embodiment, the laminar flow is from the afferent to efferent side of the lymph node chip. This ensures movement of content from the muscle chip that will carry the immunogenic component of the vaccine or treatment to the lymph node chip and exit of lymph node resident cells into the systemic circulation.

[0124] In one embodiment, the lymphatic endothelial cells fill the entirety of the space between the capillary barriers. This embodiment creates a connective vascular network between the muscle chip and the lymph node chip.

[0125] In one embodiment, the lymphatic endothelial cells line into the lymph node chip (e.g., deep into the lymph node chip) to allow transport of activated dendritic cells into the T-cell zone of the lymph node chip. In one embodiment, components of the muscle-lymph node chip (e.g., muscle chip, lymph node chip, plurality of channels) are scaled to represent in vivo geometries. This scaling includes, but is not limited to, relative physical sizes of the chips and / or dimensions of the chips (e.g., the muscle chip and the lung chip), a number of cells plated per tissue, tissue composition (e.g., cellular, epithelial, stromal, vascular, immune, and / or lymphatic composition), a relative number of immune cells added, a number of afferent lymphatic vessels per lymph node (e.g., at least one afferent lymphatic vessel per lymph node (e.g., multiple afferent lymphatic vessels per lymph node)), and / or media volumes. For example, and not limitation, in one embodiment, the number of afferent lymphatic vessels per lymph node is between one and fifteen. Other numbers of afferent lymphatic vessels per lymph node are compatible with the present invention. In another example, the volume of fluid in the system needs to be maintained (e.g., to a physiologically scaled level) to prevent dilution of circulating communication factors through the microtissues on chips.

[0126] In one embodiment, the muscle-lymph node chip is bidirectional. The lymph node chip and the lung chip are connected in a continuous fluid circuit. In one embodiment, the continuous fluid circuit is driven by a pump (e.g., a peristaltic pump, pneumatic pump) and / or routed through gravity force.

[0127] In one embodiment, cellular matter in the lymph node-lung chip is human leukocyte antigen (HLA) matched.

[0128] The way the lymph node chip is connected to the muscle chip is important to ensure the correct level of immunogen can reach the lymph node to enable immune enhancement. In one embodiment, the muscle-lymph node chip includes a vascular connection that is separate from the tissue channels (muscle and lymph). This embodiment ensures that large molecules and immune cells can circulate freely without any physical barriers.

[0129] In one embodiment, the muscle chip comprises myotubes within contracted extracellular matrix space interspersed with vascularization networks. In one embodiment, the myotubes are terminally differentiated, multinucleated, and / or myosin heavy.

[0130] In one embodiment, a vascularization unit in the muscle chip comprises vascular and lymphatic networks interspersed within the extracellular matrix around the myotubes. This allows immune cell movement in and out of the vascularized and lymphatic networks.

[0131] In a particular embodiment, the vascular angiogenic and lymphangiogenic compartments are separated. In one embodiment, the vascular networks connect to the vascular endothelial channel and the lymphatic vessels connect to the lymphatic endothelium channel that drains into the lymph node on a chip. In one embodiment, the vascular endothelial channel further connects to the systemic circulation, allowing flowing in of immune cells (e.g., macrophages, monocytes, dendritic cells) into the vascular endothelium followed by their extravasation in the muscle tissue. The dendritic cells may include cDCls, cDC2s, and / or plasmacytoid DCs. In one embodiment, the dendritic cells are derived from CD34+ hematopoietic stem cells. In one embodiment, the immune cells are mixed directly in the muscle extracellular matrix space, resulting in tissue resident dendritic cells capable of mount a strong immune response (e.g., cytokine release) under a vaccine candidate or treatment administration. Systemic flow of fluid moves from the endothelial vascular section to the angiogenic network, through the muscle tissue on a chip, which drains the contents into the lymphatic endothelium and then into the connected lymph node chip.

[0132] Connection from the muscle chip to the lymph node chip is created via a draining lymph node system, whereby activated antigen presenting cells (e.g., after vaccination or treatment into the muscle chip) traverse from the muscle chip to the lymph node chip on a bed of lymphatic endothelial cells (LECs). In one embodiment, the activated antigen presenting cells extravasate into the lymphatic endothelium and then its lumen. In one embodiment, LECs are activated under the vaccine candidate or treatment’s specific inflammatory cytokines. These cytokines include, but are not limited to, TLR3, TLR7, and / or TLR8 on the endosome; cytosolic sensors (e.g., RIG-I, MDAS, PKR, OAS) that may recognize dsRNA and / or ssRNA in the cytoplasm; proinflammatory cytokines (e.g., IL-6, IL-8, TNE-a, IL-lb); and type 1 interferon response (e.g., ILN-P).

[0133] Lymphatic endothelium under pro-inflammatory and laminar flow conditions express adhesion receptors and chemokines (e.g., ICAM1, VCAM1, CCL21, CCL39, etc.). The activated antigen presenting cells (e.g., cDCls, cDC2s) express cognate chemokine receptors (e.g., CCR7 (cognate to CCL21 chemokine)) and migrate along the lymphatic endothelium into the draining lymph node. Activated lymphatic endothelial cells are lined in the chip such that they deliver the activated antigen presenting cells (e.g., cDCls, cDC2s) deep into the lymph node on a chip to bind with the naive T-cells to initiate targeted CD8+ T-cell, memory B-cell, and plasma B-cell generation.

[0134] The lymph node chip comprises components mimicking the lymphoid structures in vivo (e.g., germinal center, secondary lymphoid structure). Connected with the muscle chip via afferent lymphatic endothelium, the lymph node on a chip comprises components of the lymph node including, but not limited to, naive T-cells (e.g., helper T-cells (CD4+) and cytotoxic T- cells (CD8+)), B-cells, germinal center resident antigen presenting cells, follicular dendritic cells, follicular reticular cells, lymph node stromal cells, lymphatic endothelial cells, and the like. These cell types are arranged similar to in vivo tissue morphology to mimic important features of the germinal center in vitro including, but not limited to, production of antigen specific antibodies, antigen specific somatic hypermutation and affinity maturation, class-switch recombination, plasmablast differentiation, plasma B-cell, memory B-cells, and CD8+ T-cells production.

[0135] After arrival within the lymph node section, the antigen carrying activated antigen presenting cell cDC2s, for example, interact with the naive CD4+ T-cells to induce their differentiation into follicular helper T-cells that further interact with the B-cells expressing the strongest affinity B-cell receptors against the desired antigen (B-cell selection). The strongest B- cell bound to the follicular helper T-cells undergoes different processes of clonal expansion (centroblast formation), antigen specific somatic hypermutation and affinity maturation, plasmablast differentiation, class-switch recombination and differentiation, and antigen specific antibody producing plasma B-cell and memory B-cell production. eDCs cross present, activate, and expand antigen specific CD8+ T-cells via the strongest binding TCRs on CD8+ T-cells, leading to their expansion and maturation.

[0136] In one embodiment, the muscle chip is provided with a 3 -way fluid connection to switch between external media circulation versus systemic circulation between the lymph node chip and muscle chip. In one embodiment, after a vaccine candidate (e.g., an mRNA-based vaccine or any other type of vaccine) is introduced into a muscle chip and provides a subsequent immune response generation within the lymph node chip, activated antigen specific CD8+ T-cells are generated within the lymph node chip and migrate to the muscle chip via systemic circulation to eliminate the antigen expressing muscle cells.

[0137] In one embodiment, the endothelial tube within the muscle section receives its own fluid flow apart from systemic circulation. In one embodiment, the muscle-lymph node chip includes a fluidic connection providing at least one flow (e.g., systemic flow, exogenous flow). In one embodiment, the fluidic connection is a three-way fluidic connection that allows for switching between systemic flow and / or exogenous flow. In one embodiment, the muscle-lymph node chip allows for mixing of the systemic flow and the exogenous flow into one tube before flowing the muscle chip. In an alternative embodiment, the fluidic connection is a two-way fluidic connection that allows for switching between system flow and exogenous flow without mixing.

[0138] The muscle on a chip receives inlet flow into the vascular endothelial channels. The excess flow drains via an immediate outlet downstream. The interstitial flow generated travels from the muscle tissue through the blind ended afferent lymphatic endothelial capillaries into afferent lymphatic endothelial vessels which further empty into the draining lymph node, and into the lymph node chip. As needed, the immune cells are provided to the muscle chip via the vascular endothelial channel to extravasate into the muscle on a chip as needed. The higher- pressure inlet fluid flow drives interstitial fluid flow into the vascularized muscle tissue. Laminar flow is provided into the lymphatic drainage into the connected lymph node. The fluid circuit is completed beyond the lymph node chip, where the exiting fluid is collected and draining into the systemic flow. In one embodiment, the exiting fluid is recirculated into the muscle on a chip tissue. The circuit is provided with a pump to move fluid and a mixer to collect fluid during circulation. The cell rich fluid collected in the mixer can be analyzed (e.g., continuously or at predetermined intervals) to qualitatively estimate the health status of the muscle-lymph chip.

[0139] In one embodiment, the present invention comprises a lymph node-lung chip. The lymph node-lung chip comprises a lymph node chip and a lung chip. The lymph node-lung chip can comprise one or more chips. For example, in one embodiment, the lymph node-lung chip is a single chip comprising both a lymph node chip and a lung chip. Alternatively, the lymph nodelung chip is formed of more than one chip. FIG. 6 illustrates one example of a lymph node-lung chip.

[0140] The lymph node chip and the lung chip are connected via a plurality of channels (e.g., one or more channels). In one embodiment, the plurality of channels comprises polydimethylsiloxane (PDMS), polyetherimide (e.g., ULTEM), polyurethane (e.g., thermoplastic polyurethane), acrylic, polystyrene, polyethylene terephthalate (PET), poly(methyl methacrylate) (PMMA), polycarbonate, poly(ethylene glycol) diacrylate (PEGDA), and / or cyclic olefin copolymer (COP). In one embodiment, the plurality of channels comprises at least one thermoplastic. In one embodiment, the plurality of channels comprises polydimethylsiloxane (PDMS). In one embodiment, the plurality of channels is populated (e.g., selectively populated) with lymphatic endothelial cells. In one embodiment, the plurality of channels is lined with at least one layer of the lymphatic endothelial cells. In one embodiment, the at least one layer is a monolayer.

[0141] In one embodiment, the plurality of channels is formed using photolithography, three- dimensional (3D) printing, needle extrusion, and / or machine milling. In one embodiment, one or more of the plurality of channels have a dimension between about 25 pm and 100 pm. See, e.g., Schineis, et al. "Cellular traffic through afferent lymphatic vessels." Vascular pharmacology 112 (2019): 31-41.

[0142] In one embodiment, the plurality of channels receives interstitial fluid (e.g., a stream of interstitial fluid). In one embodiment, the stream of interstitial fluid is a steady stream of interstitial fluid. In one embodiment, the interstitial fluid flows and / or drains from the lung chip to the lymph node chip. This embodiment provides laminar flow generated via movement of the interstitial fluid over the lymphatic endothelial cells.

[0143] In one embodiment, the lymphatic endothelial cells fill the entirety of the space between the capillary barriers. This embodiment creates a connective vascular network between the lymph node chip and the lung chip.

[0144] In one embodiment, the lymphatic endothelial cells line into the lymph node chip (e.g., deep into the lymph node chip) to allow transport of activated dendritic cells into the T-cell zone of the lymph node chip. In one embodiment, components of the lymph node-lung chip (e.g., lymph node chip, lung chip, plurality of channels) are scaled to represent in vivo geometries. This scaling includes, but is not limited to, relative physical sizes of the chips and / or dimensions of the chips (e.g., the lymph node chip and the lung chip), a number of cells plated per tissue, tissue composition (e.g., cellular, epithelial, stromal, vascular, immune, and / or lymphatic composition), a relative number of immune cells added, a number of afferent lymphatic vessels per lymph node (e.g., at least one afferent lymphatic vessel per lymph node (e.g. multiple afferent lymphatic vessels per lymph node)), and / or media volumes. The scaling allows for the lymph node to create enough neutralizing antibodies to have a quantifiable effect at the infection control in the lung. If the proportion is not correct, for example, if the lymph node is too small in relation to lung tissue, even if the high affinity neutralizing antibodies could be created in the lymph node, a titer that is too low will provide little to no effect at controlling a viral infection in the lung. For example, and not limitation, in one embodiment, the number of afferent lymphatic vessels per lymph node is between one and fifteen. Other numbers of afferent lymphatic vessels per lymph node are compatible with the present invention. Additionally, for example, the volume of fluid in the system needs to be maintained (e.g., to a physiologically scaled level) to prevent dilution of circulating communication factors through the microtissues on chips.

[0145] In one embodiment, the lymph node-lung chip is bidirectional. The lymph node chip and the lung chip are connected in a continuous fluid circuit. In one embodiment, the continuous fluid circuit is driven by a pump (e.g., a peristaltic pump, pneumatic pump) and / or routed through gravity force.

[0146] In another embodiment, the lymph node-lung chip is unidirectional, such that fluid does not flow back to the lymph node chip after the fluid exits the lung chip. In one embodiment, this fluid flow pattern is drive by a pump (e.g., a peristaltic pump) and / or routed through gravity force.

[0147] In one embodiment, all cellular matter in the lymph node-lung chip is human leukocyte antigen (HLA) matched.

[0148] The way the lymph node chip is connected to the lung chip is important to ensure the immunogenic response to the vaccine or treatment that is developed in the lymph node compartment and can be transported effectively to the lung (e.g., where the infection occurs). For example, the infection in the lung is sensed in the lymph node and the response travels from the lymph node to the lung. The mass ratio of the organs (i.e., lymph node and lung) are important to reflect physiological proportions so that the lymph node chip can create enough neutralizing antibodies to have a quantifiable affect at the infection control in the lung. If the proportion is not right, for example, if the lymph node chip is too small in relation to lung tissue, even if the high affinity neutralizing antibodies could be created in the lymph node chip, a titer that is too low will provide little to no effect at controlling the viral infection in the lung chip.

[0149] In one embodiment, the connection from the lymph node chip to the lung chip is guarded or isolated through at least one barrier so that a live infection does not route to the lymph node unimpeded after it exits the lung chip. This is a precaution to prevent the infection spreading to the rest of the systems. In one embodiment, the at least one barrier is an endothelial barrier, a lymphatic barrier, and / or an immune barrier.

[0150] In one embodiment, the lung-lymph node chip includes a lung- epithelial air liquid interface culture above an extracellular matrix with defined vascular (e.g., left side of the lung chip) and lymphatic sprouts and / or networks (e.g., right side of the lung chip) within the extracellular matrix space. In one embodiment, the vascular endothelial side also connects to the systemic flow. The lymphatic sprouts and / or networks drain into the connected lymph node via a draining lymphatic arrangement, which act as a pathway for the antigen presenting cells and other immune cells to move from the lung on a chip to the connected lymph node chip. The content exiting the lymph node chip enters the systemic circulation loop and connects with the vascular endothelial side of the two-organ chip, completing the circulation loop. Lymph node resident CD8+ T-cells thus travel from the lymphatic network to the lung site via systemic circulation (e.g., systemic continuous circulation). Infection is restricted to the epithelial side of the chip to avoid sepsis and complete system breakdown. Infection in the lung chip releases cytokines that are typical indicators of infection response (e.g., IL-6, IP-10, RANTES, IFN-0, MCP-l, IL-8).

[0151] In one embodiment, the present invention comprises a muscle-lymph node-lung chip. The muscle-lymph node-lung chip comprises a muscle chip, a lymph node chip, and a lung chip. The muscle-lymph node-lung chip can comprise one or more chips. For example, in one embodiment, the muscle-lymph node-lung chip is a single chip comprising a muscle chip, a lymph node chip, and a lung chip. Alternatively, the muscle-lymph node-lung chip is formed of more than one chip. In one embodiment, the lung chip is added subsequent to priming of the muscle-lymph node chip units. FIG. 7 illustrates one example of a muscle-lymph node-lung chip.

[0152] While the two-organ muscle-lymph node interconnected model provides critical insight into the efficiency of a vaccine or treatment candidate (e.g., mRNA vaccine) in generating robust antigen production, immune activation and response in the muscle chip and production of CD8+ T-cells, antibody producing plasma cells, and memory B-cells in the connected lymph node chip, it only provides a limited understanding of the effectiveness of the immune response. Their connection to the lung on a chip to create a three-organ (muscle, lymph node, and lung) interconnected model allows a deeper understanding of the neutralization and infection fighting abilities of the generated immune response.

[0153] In order to ensure a robust three-organ muscle-lymph node-lung system capable of mounting an immune response (e.g., after vaccine or treatment candidate addition) and mounting a response to an infection at the lung site, appropriate cell types, maturation protocols, infection dosage, lymphatic and vascular systemic circulation, timelines, and analysis strategies are designed and assembled into the three-organ connected chip. For example, a vaccine candidate with a booster dose can be introduced to a muscle site sufficiently prior to administration of a pathogen to the lung site to generate a robust immune response prior to infection. In one embodiment, the lung chip is connected at a later stage after the vaccine candidate has primed the muscle-lymph two organ chip system. In one embodiment, the expanded lymph node chip is shared between both the muscle chip and the lung chip. Dual lymphatic drainage allows the draining of interstitial fluid from the muscle chip and the lung chip into the central shared lymph node, allowing exchange of contents, shared antigen presentation, immune response generation, and memory response activation in response to vaccination or treatment from the muscle side and infection introduction in the lung side. For example, the interstitial flow generated travels from the muscle tissue through the blind ended afferent lymphatic endothelial capillaries into afferent lymphatic endothelial vessels which further empty into the draining lymph node, and into the lymph node chip. The design then pools the content exiting the lymph node chip and distributes it to the lung chip and the muscle chip in a pseudo-unidirectional systemic flow. For example, the shared lymph node provides centralization of the vaccine or treatment response generation and infection response generation into a single lymph node section capable of mounting a strong immune response whenever needed.

[0154] Movement of fluid (e.g., directional flow) within the vascularized lymph node chip allows movement of cells out of the lymph node chip into the systemic circulation (e.g., via exit out of the chip) and subsequent delivery into muscle and lung chips. Mounting a large-scale immune response sufficient to neutralize antigen expressing muscle cells and a rapidly progressing infection in the lung chip requires proportionally sized lymph node chip that harbors resident antigen specific CD8+ T cells and antibody producing plasma cells, ready to expand and act on cue. Under an infection assault, inflammatory cytokine rich media and antigen carrying dendritic cells from lung chip drain into the shared lymph node chip activate the previously primed (e.g., by a vaccine candidate) CD8+ T-cells, memory B-cells, and neutralizing antibody producing plasma B-cells, which exit the lymph node chip into the systemic circulation and is circulated in the muscle and the lung chips to mount a response.

[0155] Similar to the muscle-lymph node two-organ chip, a vaccine or treatment candidate (e.g., mRNA vaccine (lipid nanoparticle) or adjuvanted vaccine) introduced into the multicellular differentiated, tissue resident antigen presenting cells (e.g., cDCls and cDC2s) containing vascularized muscle tissue (e.g., with endothelial vascular and lymphangiogenic segments) causes antigen expression-production, strong inflammatory response (e.g., pro- inflammatory cytokines such as IL-6, IL-8, TNF-a) and type 1 interferon response (e.g., IFN-0) within the muscle chip. Activated antigen presenting cells (e.g., cDCls and cDC2s) migrate from the muscle chip towards the lymph node chip via a draining lymph node configuration, traversing on a bed of lymphatic endothelial cells, activated under the inflammatory cytokines and laminar flow to express adhesion receptors (e.g., ICAM1, VCAM1, CCL21 (gradient), etc.).

[0156] Pushed into the lymph node chip to the T-cell zone (e.g., via CCL21 gradient on LECs), these activated antigen presenting cells cross-present their antigens to antigen specific naive CD4+ T cells and subsequently generate robust CD8+ T-cell and B-cell responses. Naive CD4+ T-cells mature into follicular helper T-cells, which bind to the highest affinity B-cell receptor containing B-cell to promote its affinity maturation, somatic hypermutation, clonal selection, and / or isotype switching to produce a robust humoral response. The B-cell maturation process generates plasma cells (e.g., produce a strong titer of neutralizing antibody) and memory B-cells ready (within the lymph node chip) to mature into plasma cells upon reinfection or infection with a different variant.

[0157] A connected lung on a chip, under an artificially generated infection assault, unleashes a cytokine response (e.g., IL-6, IP-10, RANIES, IFN-P, MCP-1, IL-8) along with several antigenic segments that escape to the underlying tissue or are carried to the shared lymph node chip by dendritic cells or professional antigen presenting cells via the draining lymph node configuration, driving the release and exit of CD8+ T-cells and high antibody titers from plasma cells from the lymph node chip (e.g., exit via a centralized outlet into systemic circulation, e.g., pseudo-unidirectional flow). The response generated within the shared lymph node chip neutralizes the infection and also eliminates the infected lung cells in the lung chip. Plasma cells entering circulation also move into second sites (e.g., lung) to aid the humoral response.

[0158] Without a full-scale immune system of a living organism, an infection may break through the epithelial barrier of the lung much faster than in vivo and may seep into the circulation. In one embodiment, connections to the lung are unidirectional. Additionally, or alternatively, exit from the lung is final to prolong stability of the overall system. In one embodiment, effective load of a pathogen introduced to the lung chip is modified to prevent infection spread from the epithelial side of the lung into the systemic circulation after breaking the endothelial barrier. For example, and not limitation, a lower titer of an infectious agent (e.g., virus, bacteria) can be applied to the lung chip (e.g., lower multiplicity of infection (MOI)). In another example, the infectious agent (e.g., virus, bacteria) is modified (e.g., reduced or prevented to enter) by presence of neutralizing or binding antibodies or anti-viral agents or anti-bacterial agents. In an alternate embodiment, fluid is recirculated into the muscle and lung (e.g., to study vaccine response dependent prevention).

[0159] Similar to the muscle-lymph node chip and the lymph node-lung chip, in one embodiment, the lymphatic endothelial cells line into the lymph node chip (e.g., deep into the lymph node chip) to allow transport of activated dendritic cells into the T-cell zone of the lymph node chip. In one embodiment, components of the muscle-lymph node-lung chip (e.g., muscle chip, lymph node chip, lung chip, plurality of channels such as a first set of channels and a second set of channels) are scaled to represent in vivo geometries. This scaling includes, but is not limited to, relative physical sizes of the chips and / or dimensions of the chips (e.g., the muscle, the lymph node chip, and the lung chip), a number of cells plated per tissue, tissue composition (e.g., cellular, epithelial, stromal, vascular, immune, and / or lymphatic composition), a relative number of immune cells added, a number of afferent lymphatic vessels per lymph node (e.g., at least one afferent lymphatic vessel per lymph node (e.g. multiple afferent lymphatic vessels per lymph node)), and / or media volumes. The scaling allows for the lymph node to create enough neutralizing antibodies to have a quantifiable effect at the infection control in the lung. If the proportion is not correct, for example, if the lymph node is too small in relation to lung tissue, even if the high affinity neutralizing antibodies could be created in the lymph node, a titer that is too low will provide little to no effect at controlling a viral infection in the lung. Additionally, for example, the volume of fluid in the system needs to be maintained (e.g., to a physiologically scaled level) to prevent dilution of circulating communication factors through the microtissues on chips. Further, lymphatic connections can be scaled to reduce or enhance the vaccine and infection load and response.

[0160] The muscle-lymph node-lung chip can provide dose dependent immune response generation. For example, and not limitation, sequentially higher doses of vaccine candidates applied to the muscle chip would result in relative increases in antibody generation (e.g., plasma cells (immediate response), memory B cell formation, effect T and memory T cell formation in the connected lymph node chip. In another example, application of different and sequentially higher doses of a viral agent to the lung side result in antigen delivery to the connected and shared lymph node, resulting in broad increases and reactivation in the memory B and T cells, resulting in an immune response mounted from the lymph node to the infected lung system. The muscle-lymph node-lung chip also provides reproducibility across multiple donors. For example, reproducibility in the complex in vitro models come from using standardized techniques and the same media and ECM materials across different donors. When feasible, automation is used to create the cell and ECM loaded chips to enable reproducibility. In one embodiment, the chips utilize Society for Biomolecular Sciences (SBS) format (in shape and dimension) to enable easier automation of the cell and ECM dispensing into the chips. In one embodiment, the chips have a dimension of 85.48 mm x 127.76 mm.

[0161] The three-organ chip can capture the in vivo biology of vaccination, immune activation, and infection response within an in vitro system.

[0162] FIGS. 8-9B illustrate systems for modeling immune responses provided herein.

[0163] FIG. 8 provides a high-level schematic of a system (100) comprising a first organ chip (111), a second organ chip (113), a pump (120), and a mixer (130). In one embodiment, the first organ chip (111) is a muscle chip or a lung chip. In one embodiment, the second organ chip (113) is a lymph node chip. For example, the first organ chip (111) can be a muscle chip and the second organ chip (113) can be a lymph node chip (also referred to as a muscle-lymph node chip). In another example, the first organ chip (111) can be a lung chip and the second organ chip (113) can be a lymph node chip (also referred to as a lung-lymph node chip).

[0164] The first organ chip (111) and the second organ chip (113) are fluidly connected via a plurality of channels (112) that are lined with lymphatic endothelial cells. The plurality of channels (112) can be fluidly connected to a port (114) for transferring fluid (e.g., adding and / or removing fluid, e.g., media, sample, cells, waste, or combinations thereof).

[0165] The first organ chip (111) and the second organ chip (113) are also fluidly connected via a fluidic network (140) through which media and / or cells are moved via the pump (120). The fluidic network (140) is fluidly connected to the mixer (130), which is fluidly connected to a port or reservoir (150). In one embodiment, the port or reservoir (150) is used for transferring fluid (e.g., sample, media, waste, cells, or combinations thereof). In one embodiment, the port or reservoir (150) includes a sampler. In one embodiment, the sampler obtains a sample of the fluid. In one embodiment, the sampler tests the fluid. In one embodiment, the testing of the fluid occurs at intervals (e.g., predetermined intervals (e.g., 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, etc.). The direction of fluid flow through the system (100) is depicted with arrows. The system (100) can include an inlet (142) and an outlet (144) for transferring fluid (e.g., adding and / or removing fluid, e.g, media, sample, cells, waste, or combinations thereof). For example, the system (100) includes the inlet (142) for adding media and immune cells into the system and the outlet (144) for removing excess immune cells from the system. The inlet (142) and the outlet (144) can be fluidly connected to the first organ chip (111) and the second organ chip (113) via the fluidic network (140).

[0166] FIG. 9A provides a high-level schematic of a system (200) comprising a first organ chip (211), a second organ chip (213), a third organ chip (215), a pump (220), and a mixer (230). The first organ chip (211) can be a muscle chip or a lung chip. The second organ chip (213) can be a lymph node chip. The third organ chip (215) can be a muscle chip or a lung chip. For example, the first organ chip (211) can be a muscle chip, the second organ chip (213) can be a lymph node chip, and the third organ chip (215) can be a lung chip (also referred to as a muscle-lymph nodelung chip).

[0167] The second chip (213) is centrally located such that it can be fluidly connected to the first chip (211) and the third chip (215) via two sets of a plurality of channels (212a, 212b), with one set of the plurality of channels (212a) connecting the first chip (211) and the second chip (213) and the other set of the plurality of channels (212b) connecting the second chip (213) and the third chip (215). The two sets of the plurality of channels (212a, 212b) are lined with lymphatic endothelial cells. The plurality of channels (212a, 212b) can be fluidly connected to one or more ports (214a, 214b, 214c, 214d) for transferring fluid (e.g., adding and / or removing fluid, e.g., media, sample, cells, waste, or combinations thereof). The plurality of channels (212a, 212b) can be fluidly connected to a first port (214a, 214c) for adding fluid (e.g., media, sample, cells, waste, or combinations thereof) and a second port (214b, 214d) for removing fluid (e.g., media, sample, cells, waste, or combinations thereof). In one embodiment, the second port (214b, 214d) includes a vacuum source to achieve a constant pressure drop across the organ chip to drive interstitial flow.

[0168] The first organ chip (211), the second organ chip (213), and the third organ chip (215) are also fluidly connected via a fluidic network (240) through which media and / or cells are moved via the pump (220). The fluidic network (240) is fluidly connected to the mixer (230), which is fluidly connected to a port (250) for transferring fluid (e.g., sample, media, waste, cells, or combinations thereof). The direction of fluid flow through the system (200) is depicted with arrows.

[0169] The system (200) can include inlets (242a, 242b) and outlets (244a, 244b) for transferring fluid (e.g., adding and / or removing fluid, e.g., media, sample, cells, waste, or combinations thereof). For example, the system (200) includes inlets (242a, 242b) for adding media and immune cells into the system and outlets (244a, 244b) for removing excess immune cells from the system. The inlets (242a, 242b) and the outlets (244a, 244b) can be fluidly connected to the first organ chip (211), the second organ chip (213), and the third organ chip (215) via the fluidic network (240).

[0170] FIG. 9B provides a high-level schematic of a system (200) comprising a first organ chip (211), a second organ chip (213), a third organ chip (215), a pump (220), and a port or reservoir (260). In one embodiment, the port or reservoir (260) includes a sampler. In one embodiment, the sampler obtains a sample of the fluid. In one embodiment, the sampler tests the fluid. In one embodiment, the testing of the fluid occurs at intervals (e.g., predetermined intervals (e.g, 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, etc.). Systems described herein can include one or more pumps. The pump can be a passive pump or an active pump. Non-limiting examples of a pump for use in the systems described herein include a pneumatic pump, an electromagnetic pump, a peristaltic pump, or a pressure pump. Alternatively, or in addition to, fluid can be moved in the system via gravity flow. In one embodiment, the pump includes one or more splitters. In one embodiment, the pump includes one or more one-way valves to direct fluid flow. In one embodiment, the pump includes one or more solenoid valves. In one embodiment, the pump includes at least one one-way valve, at least one two-way valve, or at least one three-way valve.

[0171] As previously described, in one embodiment, the second organ chip (213) is a lymph node chip. In one embodiment, the pump regulates flow into opposite arms of the fluidic lines, driving flow towards the first organ chip (211) and the third organ chip (215) that flank the second organ chip (213) (e.g., central shared lymph node space).

[0172] Systems described herein can include one or more additional components that can be used in conjunction with the systems described herein. For example, in some embodiments, the system can include a pump, a mixer, a sensor (e.g., a fluorescent sensor, a chemical sensor, an optical sensor, and / or an electrical sensor),, a camera or imaging system, a control module, a user interface (e.g., a graphical user interface (GUI)), a valve, a reservoir, a sterile filter, a membrane, a bubble trap, a light source (e.g., to provide an excitation beam), a fluorophore, a temperature controller (e.g., heater, incubator), or a combination thereof. Non-limiting examples of sensors for use in systems described herein include a temperature sensor, an oxygen sensor, a pH sensor, a humidity sensor, a photodetector, a transducer (e.g., a force transducer), an analyte sensor (e.g., a metabolite sensor), a fluid level sensor, a flow sensor (e.g., an air flow sensor), a hydrometer, a viscometer, a velocity sensor, an electrical sensor, and combinations thereof. In one embodiment, the sensor obtains measurements continuously or at predetermined intervals (e.g., every minute, every 5 minutes, every 10 minutes, every 30 minutes, every hour, etc.). For example, and not limitation, in one embodiment, the system includes at least one continuous metabolite sensor. In another example, in one embodiment, the system includes continuous monitoring of the circulating media across different vascular and lymphatic chambers using at least one of the above-described sensors. Non-limiting example of cameras for use in systems described herein include a single-lens reflex (SLR) camera, a large format camera, a medium format camera, a compact camera, a video camera, a digital camera, a confocal microscope, a multi-photon microscope, a label-free imaging imager (e.g., microscope), and combinations thereof.

[0173] In one embodiment, the camera or imaging system includes at least one magnification lens. In one embodiment, the camera or imaging system includes at least one array. In one embodiment, the camera or imaging system includes at least one filter (e.g., band pass filter). In one embodiment, the camera or imaging system includes a sample stage. In one embodiment, the sample stage includes a motor. Non-limiting examples of motors for use in camera or imaging systems herein include a stepper motor, a servomotor, a piezo motor, or a linear motor. In one embodiment, the motor includes a breaking mechanism.

[0174] Systems described herein can include a control module that is configured to operate one or more components of the system and / or the immune system chip. In some embodiments, the control module includes a processor and a computer-readable medium (e.g., memory media, storage media). In some embodiments, the control module can control fluid flow. In other embodiments, the control module can control temperature. In some embodiments, the control module can control and / or process data from the one or more sensors and / or the one or more cameras. IL Immune System Chips

[0175] Aspects of the present disclosure provide microfluidic devices comprising multi-immune organs on one or more chips, which are also referred to as immune system chips. The microfluidic device can comprise two or more immune organs, which are also referred to as organ chips, and one or more channels that fluidly connect the two or more immune organs. FIGS. 10-12 illustrate microfluidic devices for modeling immune responses provided herein. Muscle-Lymph Node Chips

[0176] FIG. 10 provides a high-level schematic of a microfluidic device (300) comprising a muscle chip (311) and a lymph node chip (313), which is also referred to as a muscle- lymph node chip. As shown in FIG. 10, muscle on a chip (311) and lymph node on the chip (313) are fluidly connected via a plurality of channels (312) that are lined with lymphatic endothelial cells.

[0177] Muscle on the chip (311) creates a vascular endothelial space (1) that is connected to a lymphatic endothelial space (2) created in the plurality of channels (also referred to as lymphatic endothelial channels) between the muscle on the chip (311) and the lymph node on the chip (313).

[0178] Lymph node on the chip (313) also creates a lymphatic endothelial space (2), which is connected to the lymphatic endothelial space (2) in the plurality of channels. As such, the vascular endothelial space (1) is connected to the lymphatic endothelial space (2) extending from the plurality of channels (312) into the lymph node on the chip (313).

[0179] Muscle-lymph node chips can include one or more ports for transferring fluid (e.g., adding and / or removing media and / or cells). The one or more ports can be fluidly connected to the muscle chip, the lymph node chip, the channels, or a combination thereof. For example, as shown in FIG. 10, the muscle-lymph node chip (300) includes a port (314) that is fluidly connected to the muscle chip (311) and the plurality of channels (312).

[0180] Muscle-lymph node chips can include any number of cell culture chambers (e.g., 1, 2, 3, 4, or more cell culture chambers) for culturing cells to produce muscle and lymph node on chips. For example, the muscle-lymph node chip includes a first chamber for culturing cells that form muscle and a second chamber for culturing cells that form lymph node.

[0181] Muscle-lymph node chips can include various ratios of lymph node and muscle (e.g., ratios of cells in the lymph node chip to cells in the muscle chip). In some embodiments, the ratio of cells in the lymph node chip to cells in the muscle chip is between 1 :4 to 1 :10, e.g. , 1:5, 1:6, 1:7, 1:8, or 1:9.

[0182] Viability and / or function of muscle and / or lymph node on the chip can be assessed by analysis of various qualifying metrics. Based on one or more assessments of one or more qualifying metrics, one of ordinary skill in the art can adjust the muscle-lymph node chip to modulate its functional outcome.

[0183] Non-limiting examples of qualifying metrics for evaluating muscle on the muscle-lymph node chip include evaluating organ integrity; cargo delivery; uptake, expression and / or release; and immune cell infiltration, movement, and / or interaction. Non-limiting examples of qualifying metrics for evaluating lymph node on the muscle-lymph node chip include germinal center formation; response to antigen and / or stimuli exposure; antibody production; functional neutralization of antigens; and somatic hypermutation. Non-limiting examples of qualifying metrics for evaluating the muscle-lymph node connection of the muscle-lymph node chip include immune cell and lymphatic endothelial cell activation; chemotaxis sensing and migration; extravasation through the lymphatic endothelial cell barrier; migration on the lymphatic endothelial cell surface; and follicular helper T cell differentiation and activation.

[0184] Lung-Lymph Node Chips

[0185] FIG. 11 provides a high-level schematic of a microfluidic device (400) comprising a lung chip (411) and a lymph node chip (413), which is also referred to as a lung-lymph node chip. As shown in FIG. 11, lung on the chip (411) and lymph node on the chip (413) are fluidly connected via a plurality of channels (412) that are lined with lymphatic endothelial cells.

[0186] Lung on the chip (411) creates a vascular endothelial space (1) that is connected to a lymphatic endothelial space (2) created in the plurality of channels (also referred to as lymphatic endothelial channels) between the lung on the chip (411) and the lymph node on the chip (413).

[0187] Lymph node on the chip (413) also creates a lymphatic endothelial space (2), which is connected to the lymphatic endothelial space (2) in the plurality of channels. As such, the vascular endothelial space (1) is connected to the lymphatic endothelial space (2) extending from the plurality of channels (412) into the lymph node on the chip (413).

[0188] Lung-lymph node chips can include one or more ports for transferring fluid (e.g., adding and / or removing media and / or cells). The one or more ports can be fluidly connected to the lung chip, the lymph node chip, the channels, or a combination thereof. For example, as shown in FIG. 11, the lung-lymph node chip (400) includes a port (414) that is fluidly connected to the lung chip (411) and the plurality of channels (412).

[0189] Lung-lymph node chips can include any number of cell culture chambers (e.g., 1, 2, 3, 4, or more cell culture chambers) for culturing cells to produce lung and lymph node on chips. For example, the lung-lymph node chip includes a first chamber for culturing cells that form lung and a second chamber for culturing cells that form lymph node.

[0190] Lung-lymph node chips can include various ratios of lymph node and lung (e.g., ratios of cells in the lymph node chip to cells in the lung chip). In some embodiments, the ratio of cells in the lymph node chip to cells in the lung chip is between 1:4 to 1: 10, e.g., 1:5, 1:6, 1 :7, 1:8, or 1:9.

[0191] Viability and / or function of lung-lymph node chips can be assessed by analysis of various qualifying metrics. Based on one or more assessments of one or more qualifying metrics, one of ordinary skill in the art can adjust the lung-lymph node chip to modulate its functional outcome.

[0192] Non-limiting examples of qualifying metrics for evaluating lung on the lung-lymph node chip include organ integrity; infection; infection response; and treatment and / or recovery. Nonlimiting examples of qualifying metrics for evaluating lymph node on the lung-lymph node chip include germinal center formation; response to antigen and / or stimuli exposure; antibody production; functional neutralization of antigens; and somatic hypermutation. Non-limiting examples of qualifying metrics for evaluating the lung-lymph node connection of the lung-lymph node chip include immune cell and lymphatic endothelial cell activation; chemotaxis sensing and migration; extravasation through the lymphatic endothelial cell barrier; migration on the lymphatic endothelial cell surface; and follicular helper T cell differentiation and activation. Muscle-Lymph Node-Lung Chips

[0193] FIG. 12 provides a high-level schematic of a microfluidic device (500) comprising a muscle chip (511), a lymph node chip (513), and a lung chip (515). As shown in FIG. 12, lymph node on the chip (513) is centrally located such that it can be fluidly connected to muscle on the chip (511) and lung on the chip (515) via two sets of a plurality of channels (512a, 512b), with one set of the plurality of channels (512a) extending between the lymph node chip (513) and the muscle chip (511) and the other set of channels (512b) extending between the lymph node chip (513) and the lung chip (515). Muscle on the chip (511) and lung on the chip (515) create vascular endothelial spaces (1) that are connected to lymphatic endothelial spaces (2) created in the lymph node chip (513) and in the plurality of channels (512a, 512b). As such, the lymphatic endothelial space (2) extends from the lymph node on the chip (513) through the plurality of channels (512a, 512b) and into the muscle chip (511) and the lung chip (515).

[0194] Muscle-lymph node-lung chips can include any number of cell culture chambers (e.g., 1, 2, 3, 4, or more cell culture chambers) for culturing cells to produce muscle, lymph node, and lung on chips. For example, the muscle-lymph node-lung chip includes a plurality of chambers, each of which is used to culture cells that form muscle, lymph node, and lung.

[0195] Muscle-lymph node-lung chips can include one or more ports for transferring fluid (e.g., adding and / or removing media and / or cells). The one or more ports can be fluidly connected to the muscle chip, the lymph node chip, the lung chip, the channels, or a combination thereof. For example, as shown in FIG. 12, the muscle-lymph node-lung chip (500) includes ports that is fluidly connected to the lymphatic channels (e.g., inlet and outlet ports).

[0196] Muscle-lymph node-lung chips can include various ratios of muscle, lymph node, and lung (e.g., ratios of cells in the lymph node chip to cells in the muscle chip and cells in the lung chip). In some embodiments, the ratio of cells in the lymph node chip to cells in the muscle chip and cells in the lung chip is between 1:4:4 to 1: 10: 10, e.g., 1 :4:5 to 1 :10:10, 1 :4:6 to 1 :10:10, 1:4:7 to 1:10: 10, 1:4:8 to 1 :10: 10, 1:4:9 to 1:10:10, 1 :5:4 to 1 :10:10, 1 :6:4 to 1 :10:10, 1:7:4 to 1:10: 10, 1:8:4 to 1:10: 10, or 1:9:4 to 1: 10:10. In other embodiments, the ratio of cells in the lymph node chip to cells in the muscle chip and cells in the lung chip is between 1 :1.5: 0.5 and 1:2:0.5.

[0197] Viability and / or function of muscle-lymph node-lung chips can be assessed by analysis of various qualifying metrics. Based on one or more assessments of one or more qualifying metrics, one of ordinary skill in the art can adjust the muscle-lymph node-lung chip to modulate its functional outcome.

[0198] Non-limiting examples of qualifying metrics for evaluating muscle on the muscle-lymph node chip include evaluating organ integrity; cargo delivery; uptake, expression and / or release; and immune cell infiltration, movement, and / or interaction. Non-limiting examples of qualifying metrics for evaluating lymph node on the lung-lymph node chip include germinal center formation; response to antigen and / or stimuli exposure; antibody production; functional neutralization of antigens; and somatic hypermutation. Non-limiting examples of qualifying metrics for evaluating lung on the muscle-lymph node-lung chip include organ integrity; infection; infection response; and treatment and / or recovery.

[0199] Non-limiting examples of qualifying metrics for evaluating the muscle-lymph node connection of the muscle-lymph node chip include immune cell and lymphatic endothelial cell activation; chemotaxis sensing and migration; extravasation through the lymphatic endothelial cell barrier; migration on the lymphatic endothelial cell surface; and follicular helper T cell differentiation and activation.

[0200] Non-limiting examples of qualifying metrics for evaluating the lung-lymph node connection of the lung-lymph node chip include immune cell and lymphatic endothelial cell activation; chemotaxis sensing and migration; extravasation through the lymphatic endothelial cell barrier; migration on the lymphatic endothelial cell surface; and follicular helper T cell differentiation and activation.

[0201] Chip Components and Fabrication

[0202] Immune system chips described herein (e.g., muscle- lymph node chips, lung-lymph node chips, muscle-lymph node-lung chips) can include a substrate having at least one cell culture chamber disposed therein and / or at least one channel disposed therein. The number and the dimension of cell culture chambers and / or channels in an immune system chip can vary depending on various factors such as the design and / or function of the immune system chip.

[0203] The one or more cell culture chambers of the muscle-lymph node chip can be any size or shape suitable for culturing cells. In some embodiments, a cell culture chamber can have a circular, oval, square, rectangular, or hexagonal shape.

[0204] In one embodiment, the plurality of channels is formed using photolithography, three- dimensional (3D) printing, needle extrusion, and / or machine milling. In one embodiment, one or more of the plurality of channels have a dimension between about 25 pm and 100 pm. See, e.g., Schineis, et al. "Cellular traffic through afferent lymphatic vessels." Vascular pharmacology 112 (2019): 31-41.

[0205] In one embodiment, the plurality of channels comprises polydimethylsiloxane (PDMS), polyetherimide (e.g., ULTEM), polyurethane (e.g., thermoplastic polyurethane), acrylic, polystyrene, polyethylene terephthalate (PET), poly(methyl methacrylate) (PMMA), polycarbonate, poly(ethylene glycol) diacrylate (PEGDA), and / or cyclic olefin copolymer (COP). In one embodiment, the plurality of channels comprises at least one thermoplastic. In one embodiment, the plurality of channels comprises polydimethylsiloxane (PDMS). In one embodiment, the plurality of channels is populated (e.g., selectively populated) with lymphatic endothelial cells. In one embodiment, the plurality of channels is lined with at least one layer of the lymphatic endothelial cells. In one embodiment, the at least one layer is a monolayer.

[0206] For example, and not limitation, in one embodiment, the plurality of channels has a width between about 5 pm and about 100 pm and / or a length between about 1 mm and about 10 mm (e.g., about 1 mm and about 5 mm).

[0207] The plurality of channels comprises at least one channel. The at least one channel may be a single channel or multiple channels. For example, and not limitation, the at least one channel may be between 1 and 15 channels. Other numbers of channels are compatible with the present invention.

[0208] Immune system chips described herein can include one or more membranes, e.g., 1, 2, 3, 4, 5, 6, or more membranes. The membrane can be rigid or at least partially flexible. The term “flexible” refers to a membrane that can be stretched and / or contracted by at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, or more of its original size without loss of structural integrity.

[0209] In one embodiment, the membrane is a polyester membrane or a polydimethylsiloxane (PDMS) membrane. In one embodiment, the membrane has a thickness between 0.1 pm and 50 pm. In one embodiment, the membrane has a thickness between 1 pm and 5 pm. In another embodiment, the membrane has a thickness between 5 pm and 15 pm. In yet another embodiment, the membrane has a thickness between 10 pm and 40 pm. In still another embodiment, the membrane has a thickness between 30 pm and 50 pm.

[0210] The membrane can be non-porous or at least partially porous. In some embodiments, the pore size of the membrane is greater than the size of cells, thereby allowing cells to pass through the membrane. In some embodiments, the pore size of the membrane is less than the size of cells, thereby preventing cells from crossing the membrane. In such instances, the pore size of the membrane can be greater than the size of other molecules (e.g., nutrient molecules), thereby allowing other molecules to pass through the membrane. In one embodiment, the pore size is between 0.1 pm and 5.0 pm. In one embodiment, the pore size is between 0.3 pm and 0.5 pm. In another embodiment, the pore size is between 1.0 pm and 5.0 pm. The membrane can be non-coated or at least partially coated. When the membrane is at least partially coated, the membrane can be coated with various molecules including, but not limited to, extracellular matrix (ECM) molecules such as fibronectin, collagen (e.g., type I collagen, type IV collagen), and laminin; proteins such as growth factors; and combinations thereof.

[0211] The membrane can be seeded with cells. In such instances, cells can be seeded on one or both sides of the membrane. The sides of the membrane can be seeded with the same type of cells or with different types of cells. The membrane can be seeded with one or more layers of cells. Each layer of cells can be the same type of cells or different types of cells.

[0212] Immune system chips described herein (e.g., muscle- lymph node chips, lung-lymph node chips, muscle-lymph node-lung chips) can be fabricated using any method known in the art. Non-limiting examples of fabrication methods include soft lithography methods, microassembly methods, bulk micromachining methods, surface micro-machining methods, standard lithographic methods, wet etching, reactive ion etching, plasma etching, stereolithography and laser chemical three-dimensional writing methods, solid-object printing, machining, modular assembly methods, replica molding methods, injection molding methods, hot molding methods, laser ablation methods, and combinations thereof.

[0213] Immune system chips described herein can be fabricated from any biocompatible material suitable for culturing cells. Non-limiting examples of biocompatible materials include glass, silicon, silicones, polyurethanes or derivatives thereof, rubber, molded plastic, polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™), polyvinylchloride (PVC), poly dimethylsiloxane (PDMS), and poly sulfone. Immune system chips can be disposable and / or sterilizable.

[0214] Cells

[0215] Immune system chips described herein (e.g., muscle- lymph node chips, lung-lymph node chips, muscle-lymph node-lung chips) can include various types of cells for recapitulating immune organs.

[0216] For example, in some embodiments, muscle chips can include muscle cells (e.g., myoblasts), vascular cells (e.g., vascular endothelial cells), lymphatic cells (e.g., lymphatic endothelial cells), immune cells (e.g., macrophages, dendritic cells, monocytes, neutrophils), and combinations thereof. For example, in some embodiments, lymph node chips can include lymphatic cells (e.g., lymphatic endothelial cells), immune cells (e.g., T cells, B cells, dendritic cells), fibroblasts, and combinations thereof.

[0217] For example, in some embodiments, lung chips can include lung cells (e.g., lung epithelial cells), vascular cells (e.g., vascular endothelial cells), lymphatic cells (e.g., lymphatic endothelial cells), immune cells (e.g., macrophages, dendritic cells), and combinations thereof. Lung epithelial cells can be bronchial, bronchiolar, or alveolar.

[0218] Immune system chips described herein can include various types of immune cells. Nonlimiting examples of immune cells include T cells, B cells, dendritic cells, granulocytes, innate lymphoid cells (ILCs), megakaryocytes, monocytes, macrophages, natural killer (NK) cells, platelets, red blood cells (RBCs), and thymocytes.

[0219] In some embodiments, immune system chips can further comprise additional cell types, e.g., parenchymal cells, stem cells, progenitor cells, stromal cells, adipocytes, and combinations thereof.

[0220] Cells used in immune system chips described herein can be isolated from a subject (e.g., from a tissue and / or a fluid of the subject) using any method known in the art. Alternatively, or in addition to, cells used in immune system chips described herein can be obtained from commercial sources. In one embodiment, the cells are not returned to a body of the subject after use in the immune system chip.

[0221] Cells used in immune system chips described herein can be undifferentiated or differentiated. Undifferentiated cells can be cultured inside the immune system chip to induce cell differentiation.

[0222] Cells used in immune system chips described herein can be genetically engineered for various purposes, e.g., to express a fluorescent protein and / or to modulate expression of a gene.

[0223] Cells can be healthy cells or diseased cells. Cells can be obtained from a mammal, e.g., a human, a mouse, a hamster, a rabbit, or a domesticated animal. Cells can be obtained from culturing or from a subject (e.g, a human). Cells can be derived from any tissue suitable for harvesting cells.

[0224] Various strategies may be used to prevent immune rejection of connected organ models. In one embodiment, the cells are human leukocyte antigen (HLA) matched. In one embodiment, the cells are matched across alleles HLA-A, HLA-B, and / or HLA-C. In one embodiment, the cells are matched across alleles HLA-A, HLA-B, and HLA-C. In one embodiment, gene editing is used to delete the HLA-A and / or the HLA-B alleles in cellular components of the single organ, two organ, and / or three organ chips. In one embodiment, gene editing is used to delete the HLA- A and the HLA-B alleles in cellular components of the single organ, two organ, and / or three organ chips. In one embodiment, iPSC cells are genetically edited (e.g., to delete the HLA-A and / or the HLA-B alleles) to derive muscle, lung epithelium, and endothelium as needed. In one embodiment, B2M (02 microglobulin) knockout in select cells (e.g., non-antigen presenting cells, e.g., endothelium) may induce NK cell activity (e.g., selectively deplete NK cells from the system). In one embodiment, a targeted knockout of class II major histocompatibility complex transactivator (CIITA) is used to deplete HLA class II.

[0225] Cells can remain viable for at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 9 weeks, at least 12 weeks, or more in the immune system chips described herein.

[0226] III. Methods of Use

[0227] Immune system chips and systems comprising such described herein can be used for a variety of applications including, but not limited to, evaluating drug induced immune responses, drug safety, drug efficacy, drug screening, disease modeling and disease-recovery modeling, and immunocompatibility assays.

[0228] To perform methods described herein, an agent is brought in contact with any of the immune system chips described herein under conditions sufficient for interaction of the agent with the immune system chip, if any. Interaction of the agent with the immune system chip can be detected by measuring a cellular response, which can be detected using any suitable method for detecting the cellular response.

[0229] As used herein, the term “contacts” refers to an exposure of an agent to an immune system chip for a period sufficient for detecting a cellular response, if any. The agent can be contacted with the immune system chip for any suitable period of time.

[0230] Any agent can be used in methods described herein. The agent can be a therapeutic agent or pathogenic agent. Methods provided herein encompass contacting a microfluidic device with one or more agents. When contacting the microfluidic device with more than one agent, the agents can be the same or different. Non-limiting examples of agents include cells, proteins, peptides, antigens, antibodies or portions thereof, enzymes, nucleic acids, vaccines (e.g., mRNA vaccines, multiple antigen-presenting system (MAPS) vaccines, adjuvants, protein vaccines), drugs, small molecules, or combinations thereof.

[0231] Any cellular response can be detected in methods described herein. Non-limiting examples of cellular responses include viability, proliferation, activation, respiration, metabolism, cell chemotaxis and migration, cell circulation, cell contractility, ECM remodeling, differential expression of cell biomarkers, production and / or release of biomolecules, action potentials, and combinations thereof.

[0232] For example, in some embodiments, methods described herein can be used the effect, if any, of a vaccine (e.g., mRNA vaccine) on an immune response (e.g., cytokine production) can be evaluated. In such instances, the vaccine is contacted with the immune system chip and the immune response, if any, is detected.

[0233] Methods described herein can be carried out using any of the systems and devices described herein. In some examples, methods described herein comprise (a) contacting an agent with the microfluidic device and (b) detecting a cellular response.

[0234] In one embodiment, the present invention includes methods used to perform quantitative and / or qualitative assessments in the multi-organ chips. These methods include, but are not limited to, confocal microscopy-based imaging, flow cytometry, single-cell sequencing, transepithelial / transendothelial electrical resistance (TEER) assays, non-invasive cell viability measurements, neutralizing antibody measurements, live cell tracking, gene expression studies, spatial genomics, cytokine profiling and / or monitoring, at least one barrier integrity assay, mucus presence, pseudostratified epithelium presence, and / or CD8+ T cell measurements.

[0235] In one embodiment, the confocal microscopy-based imaging includes, but is not limited to, quantitative and / or qualitative assessment of (a) three-dimensional (3D) organization of myotubes (e.g, myosin heavy chain staining, multinucleated cells, cell fusion); (b) endothelial cells and sprouts (e.g, CD31+, VEGFR2); (c) lymphatic endothelium (e.g., LYVE-1, VEGFR3, podoplanin, Prox-1, CCL19, CCL21 (e.g., activated lymphatic endothelial cells); (d) somatic hypermutation (e.g., in the lymph node chip, for example, using activation-induced cytidine deaminase (AID) staining); (e) T-cell and B-cell zones, germinal center B cells, antigen presenting cells, plasma B-cell production, and / or memory B-cell production (e.g., CD20, CXCR5, CD3, CD4, CD8, CXCR3, CXCR4, CD138, CD83, CD80, HLA-DR, CD86, BCL6); (f) antigen expression and / or release (e.g., for vaccine candidates, for example, mRNA vaccines); and / or (g) lung epithelium markers (e.g., basal cells, ciliated cells, goblet cells, club / Clara cells). In one embodiment, the quantitative and / or qualitative assessment of lung epithelium markers includes population (e.g., pop%) and / or distribution. In one embodiment, the quantitative and / or qualitative assessment of basal cells includes KRT5 and / or KRT14 expression. In one embodiment, the quantitative and / or qualitative assessment of ciliated cells includes alphatubulin expression. In one embodiment, the quantitative and / or qualitative assessment of goblet cells includes mucin-5AC (MUC-5AC) expression. In one embodiment, the quantitative and / or qualitative assessment of club / Clara cells includes CC10, CCSP, and / or SCGB1 Al expression.

[0236] In one embodiment, the flow cytometry and / or single cell sequencing are used to measure the media and cells in circulation. In one embodiment, contents of a muscle chip and / or lymph node chip are sampled (e.g., routinely sampled) to quantify a type of cells in motion and an activation status of the cells in motion. Flow markers that may be tested include, but are not limited to, PD1, CXCR3, CXCR4, CXCR5, CD3, CD4, CD8, CD16, CD19, CD20, CD21, CD27, CD38, CD45RA, CD56, CD80, CD83, CD86, CD123, CD138, HLA-DR, IgD, IgM, BCL6, and / or AID.

[0237] In one embodiment, the TEER assays are used to measure vascular integrity, lymphatic integrity, and / or lung epithelial integrity.

[0238] In one embodiment, the non-invasive cell viability measurements include, but are not limited to, adenosine triphosphate (ATP) assays, lactate dehydrogenase (LDH) assays, and / or formazan-based assays (e.g., XTT) assays.

[0239] In one embodiment, the neutralizing antibody measurements include enzyme-linked immunosorbent assay (ELISA) (e.g., sandwich digital ELISA), single molecule array (SIMOA), and / or infection challenge assay.

[0240] In one embodiment, the live cell tracking includes tracking from muscle to lymph node (e.g., antigen-presenting cells (APCs) carrying an antigen), lung to lymph node (e.g., APCs carrying an antigen), and / or lymph to lung (e.g., leukocyte diapedesis).

[0241] In one embodiment, the cytokine profiling and / or monitoring is performed using at least one multiplex technology (e.g., MesoScale Discovery (MSD), LUMINEX) to profile and / or monitor at least one cytokine. In one embodiment, the at least one cytokine including, but not limited to, CXCL9, CXCL12, CXCL13, GM-CSF, IgD, IgM, IFN-a2, IFN-0, IFN-y, IL-la, IL- 13, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12(p40), IL-18, IL-21, IL-22, IL-27, IP-10, HLA-DR, MCP-1, MIP-ip, , RANTES, and / or TNF-a.

[0242] In one embodiment, the barrier integrity assays compatible with the present invention include, but are not limited to, fluorescein isothiocyanate (FITC) and / or tetramethylrhodamine (TRITC). In one embodiment, the at least one barrier integrity assay is used to perform a dextran leakage study to monitor lung epithelial integrity.

[0243] In one embodiment, the methods also include neutralizing antibody, cytokine, CD8, and / or T cell measurement in at least one lymph node exit (e.g., central lymph node exit) that is recirculated (e.g., to the lung chip and / or the muscle chip).

[0244] The lymph node chip may be used to study, record, and probe immune responses (e.g., against primary antigen / adjuvant and booster dose of the antigen / vaccine / adjuvant). In one embodiment, the immune response is measured in at least one pre- determined interval. The at least one pre-determined interval may include, but is not limited to, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, 48 hours, 7 days, 14 days, 21 days, and / or 28 days. Other pre-determined intervals are compatible with the present invention.

[0245] In one embodiment, the lymph node chip may be used to monitor cytokines and / or chemokines including, but not limited to, G-CSF, IFN-a2, IFN-y, IL-la, IL-ip, IL-IRA, IL-6, IL-8, IL-10, IL-21, IP-10, MIP-ip, MCP-1, CXCL-10, CXCL-13, CXCL-9, and / or TNF-a. In one embodiment, the cytokines and / or the chemokines are measured in at least one predetermined interval (e.g., 4 hours, 12 hours, 24 hours, 48 hours, 7 days, 14 days, 21 days, 28 days).

[0246] In one embodiment, fluorescence-activated cell sorting (FACS) analysis may be used to monitor phenotypic markers, activation markers, and / or antibodies (e.g., to detect vaccine antigen expression). Examples of phenotypic markers include, but are not limited to, CD45, CD66, CD3, CD19, CD56, CD14, CD16, CD123, CDl lc, CDl lb, CDlc, and / or CD14. Examples of activation markers include, but are not limited to, HLA-DR, CD32, CD40, CD80, CD83, and / or CD86.

[0247] In one embodiment, B cell and / or T cell differentiation subclusters are evaluated (e.g., pre-germinal cell (GC), GC, plasmablast, memory B-cells). In one embodiment, the B cell and / or T cell differentiation subclusters are evaluated in at least one pre-determined interval (e.g., day 7, day 14, day 21, day 28).

[0248] In one embodiment, antigen-specific IgG and / or IgM is measured. In one embodiment, the antigen-specific IgG and / or IgM is measured in at least one pre-determined interval (e.g., day 7, day 14, day 21, day 28).

[0249] In one embodiment, cell viability and / or self-organization under agent / antigen exposure is measured. In one embodiment, the cell viability and / or the self-organization is measured in at least one pre- determined interval. The at least one pre-determined interval may include, but is not limited to, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, 48 hours, 7 days, 14 days, 21 days, and / or 28 days.

[0250] In one embodiment, AID expression is measured (e.g., to show evidence of somatic hypermutation, class switching, affinity maturation, and / or neutralizing antibody production). In one embodiment, the AID expression is measured in at least one pre-determined interval. The at least one pre-determined interval may include, but is not limited to, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, 48 hours, 7 days, 14 days, 21 days, and / or 28 days.

[0251] In one embodiment, memory response is measured using at least one B cell memory panel and / or at least one T cell memory panel.

[0252] The B cell memory panel may include, but is not limited to, CD 10, CD 19, CD20, CD23, CD24, CD27, CD28, CD38, CD80, CD86, CD 138, IgA, IgD, IgE, IgG, and / or IgM. For example, and not limitation, CD 19+ and CD20+ are identification markers, and CD27 is a convenient memory marker of reference.

[0253] In one embodiment, the B cell memory panel is classified into at least one B cell memory category. The at least one B cell memory category may include, but is not limited to, naive B cell, transitional, activated, memory, plasmablasts, and / or plasmacells. The naive B cell category may include, but is not limited to, CD27-, CD24-, CD38-, CD21+, and IgD+. The transitional category may include, but is not limited to, CD27-, CD24 high, CD38 high, CD 10 low, and IgD+. The activated category may include, but is not limited to, CD27+, IgD+, IgM+, CD80+, and CD86+. The memory category may include, but is not limited to, CD27+ / -, IgD+, IgM+, IgA+, IgE+, IgG+, CD38 low, and CD23 low. The plasmablasts category may include, but is not limited to, CD27 high, CD38 high, CD20-, and CD 138-. The plasmacells category may include, but is not limited to, CD27+, CD38 high, CD 19 low, CD138+, and CD28+.

[0254] The T cell memory panel may include, but is not limited to, CD3, CD4, CD8, CD27, CD28, CD45RA, CD57, CCR7, TCRa, TCRP, and / or PD-1. For example, and not limitation, CD3, TCRa, TCRP, CD4 (helper), and / or CD8 (cytotoxic) are identification markers for the T cell population.

[0255] In one embodiment, the T cell memory panel is classified into at least one T cell memory category. The at least one T cell memory category may include, but is not limited to, naive T cell, effector memory, central memory, TEMRA, and / or TEMRA senescent. The naive T cell category may include, but is not limited to, CD45RA+ and CCR7-. The effector memory category may include, but is not limited to, CD45RA- and CCR7-. The central memory category may include, but is not limited to, CD45RA- and CCR7+. The TEMRA category may include, but is not limited to, CD45RA+, CCR7+, and CD27- / +. For example, the majority of TEMRA population are usually CD27- and CD28-, and are also identified as high cytotoxic; some CD27+ seems to be intermediate between naive and effector memory T cells. The TEMRA senescent category may include, but is not limited to, CD57+, CD28-, and PD-1+.

[0256] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0257] EXAMPLES

[0258] In order that the invention described may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the methods and compositions provided herein and are not to be construed in any way as limiting their scope.

[0259] Example 1: Preparation of Human Dendritic Cells

[0260] CD34+ Hematopoietic Stem Cells (HSCs) can be differentiated into human dendritic cells (e.g., plasmacytoid DCs (pDCs), conventional DCs such as cDCls and cDC2s) according to methods described in Kirkling et al., Cell Reports 2018, 23, 3658-3672, which is incorporated herein by reference in its entirety. In brief, HLA matched CD34+ HSCs are obtained from HLA matched donors. OP9 and 0P9-DL1 cells and OP9 media are purchased from ATCC. Briefly, 5000 OP9 and OP9-DL1 cells are seeded in 200 pL medium in 96 well round bottom plates for 1 day before addition of CD34+ bone marrow cells. Bone marrow dendritic cell media is prepared by mixing 20 ng / mL recombinant GM-CSF, 20 ng / mL recombinant human SCF, and 100 ng / mL recombinant human Flt3L in OP9 media and 10% fetal bovine serum (FBS). CD34+ bone marrow cells derived from a consenting donor are added directly over the base feeder layer of OP9 and OP9-DL1 cells. After media change on day 7 and 14, the plasmacytoid DCs, cDCls, and cDC2s are collected and used as described herein.

[0261] Example 2: Muscle Chips

[0262] This example describes microfluidic devices that mimic muscle physiology and function, which are also referred to as muscle chips.

[0263] Cellular Content

[0264] Muscle chips can include precursor myoblasts, myotubes, muscle cells derived from iPSCs, extracellular matrix, vascular endothelial cells, lymphatic endothelial cells, macrophages, cDCls, cDC2s, plasmacytoid DCs, and combinations thereof. Muscle cells can be added to the muscle chip separately or in combination with other types of cells (e.g., cDCl, cDC2, and plasmacytoid DCs). Muscle cells and other types of cells can be added to the muscle chip once or multiple times.

[0265] Design and Characterization

[0266] Design and characterization of muscle chips is described herein as well as in Mondrinos et al., Science Advances 2021, 7, eabe9446; and Agrawal et al., Lab on a Chip 2017, 17, 3447- 3461, the contents of which are each incorporated herein by reference in their entireties.

[0267] The muscle chip can include vascularized muscle in a central space flanked by endothelial cells on one side and lymphatic endothelial cells on the other side. The muscle chip can be prepared by mixing skeletal muscle myoblasts with fibroblasts cells in an extracellular matrix suspension and adding the resulting mixture into the central space of the muscle chip. After gelation of the mixture, human umbilical endothelial cells (HUVECs) and / or human dermal microvascular endothelial cells (HDMECs) are seeded on a first side of the muscle chip (e.g., left side) and lymphatic endothelial cells (LECs) are seeded on a second side of the muscle chip (e.g., right side).

[0268] Any method suitable for inducing endothelial cell (e.g., HUVEC, LEC) sprouting can be used to create vascularized muscle tissue. For example, endothelial cell sprouting can be induced using cell culture media comprising VEGFA and VEGFC. Additional agents (e.g., HGF, ANG-1 and SIP, PMA, FGFb, MCP-1, SDFla) can be included in the culture media to promote lymphangiogenesis, vascular angiogenesis, and sprouting.

[0269] Endothelial cells could get pulled into the central space of the muscle chip by contracting muscle tissue, which produces a curved vasculature. This result can be minimized by various techniques including adding endothelial cells after contraction of muscle tissue, mixing endothelial cells with muscle cells, and configuring the central space of the muscle chip to minimize muscle tissue contraction.

[0270] Various biological responses and biological parameters can be assessed on the muscle chip including cell type, cell vascularization, cell differentiation, cell alignment, contraction in response to stimuli, antigen delivery, immune response (e.g., antigen expression, immune cell infiltration, immune cell activation), and reproducibility across human donors.

[0271] Example 3: Lymph Node Chips

[0272] This example describes microfluidic devices that mimic lymph node physiology and function, which are also referred to as lymph node chips.

[0273] Cellular Content

[0274] Lymph node chips described herein can include lymphatic endothelial cells (e.g., lymphatic sprouts and / or networks), monocytes, T cells, B cells, fibroblastic reticular cells, follicular dendritic cells, extracellular matrix, cDCls, cDC2s, plasmacytoid DCs, and combinations thereof.

[0275] B cells, T cells, and monocytes are isolated from HLA matched donor leukopaks via density centrifugation followed by negative selection using commercially available kits. Dendritic cells can be isolated from spleen, whole blood, or differentiated from bone marrow progenitor cells (CD34+ Hematopoietic stem cells) or donor matched iPSCs. Other supportive cells (e.g., fibroblasts, human lymphatic endothelial cells (HLEC), human umbilical endothelial cells (HUVECs), pericytes, and other endothelial cells) can be included in the lymph node chip to create vessel structures in the or nearby the lymph node.

[0276] Tonsil tissue can be used as starting material for construction of the lymph node chip. In such instances, cells can be differentiated and maintained through different media formulations. Cells can be cultured in chambers or wells, with or without extracellular matrix.

[0277] Design and Construction of Lymph Node Chips

[0278] Design and construction of lymph node chips from whole tonsil tissue is described herein as well as in Wagar et al., Nature medicine 2021, 27, 125-135, the contents of which is incorporated herein by reference in its entirety.

[0279] Briefly, whole tonsil tissue is collected, processed, and dissected into roughly 5 mm x 5 mm x 5 mm pieces, and manually disrupted into a suspension by processing through a 100-pm strainer with a syringe plunger. Tissue debris is reduced by Ficoll density gradient separation. After washing with complete medium (RPMI with GLUTAMAX supplement, 10% FBS, 1 x nonessential amino acids, 1 x sodium pyruvate, 1 x penicillin-streptomycin, 1 x Normocin (InvivoGen), and l x insulin / selenium / transferrin cocktail (Gibco)), cells are enumerated and frozen into aliquots in FBS + 10% DMSO. Frozen cells are stored at -140 °C until use.

[0280] Cells are resuspended, mixed with extracellular matrix (ECM) solution, and injected directly into a central space within a microfluidic device representing the lymph node on a chip section. After the ECM sets in the central chamber, lymphatic endothelial cells are added on both sides flanking the central channel. Gentle fluid flow is started to allow cells to self-aggregate and form end to end lymphatic networks that extend through the lymph node chip and connect with the lymphatic vasculature units outside of the gel. The tissue is maintained in complete relevant media.

[0281] Lymph node chips can also be prepared using T cells and B cells derived from donor PBMC leukopaks. T cells and B cells can be photocrosslinked into individual zones and then injected into the lymph node chip. Alternatively, T cells and B cells can be injected into the lymph node chip within a gel mixed with supporting stromal and lymphatic endothelial cells. Side channels of the lymph node chip can be injected with lymphatic endothelial cells, lymph node fibroblasts, and MSCs to create a lymphatic endothelial tube. Alternatively, or additionally, lymphatic endothelial cells, lymph node fibroblasts, and MSCs can be mixed with the pregel and photocrosslinked near the T cell and B cell region to create lymphatic vascular connection (e.g., on top of the T cell and B cell region, under the T cell and B cell region, around the T cell and B cell region, or a combination thereof).

[0282] Various biological responses and biological parameters can be assessed on the lymph node chip including germinal center formation, antigen delivery, immune response (e.g., antibody production, immune cell infiltration, immune cell activation), and reproducibility across human donors.

[0283] Example 4: Lung Chips

[0284] This example describes microfluidic devices that mimic lung physiology and function, which are also referred to as lung chips.

[0285] Cellular Content

[0286] Lung chips can include lung epithelial cells (e.g., alveolar lung epithelial cells, bronchial lung epithelial cells), extracellular matrix, vascular endothelial cells (e.g., sprouts and / or networks), pericytes, antigen presenting cells, dendritic cells (e.g., cDCls, cDC2s, plasmacytoid DCs), immune cells, lymphatic vasculature (e.g., sprouts and / or networks), and combinations thereof.

[0287] Lung chip can include primary lung epithelial cells, patient-derived lung organoids, or lung cells derived from iPSCs. Lung cells that can be used in the lung chip can be healthy or diseased. Lung cells can be from a lower respiratory tract (e.g., bronchial, bronchiolar, or alveolar in origin), or may incorporate cells from an upper respiratory tract (e.g., nasal or tracheal in origin).

[0288] Other supporting cells such as lung fibroblasts, endothelial cells (e.g., pulmonary microvascular endothelial cells, human umbilical vein endothelial cells, lymphatic endothelial cells), and pericytes can be included to form vascular structures in or nearby the lung cells.

[0289] Immune cells in the lung chip can include undifferentiated, partially differentiated, or fully differentiated primary immune cells, progenitors, or induced pluripotent stem cells. Undifferentiated or partially differentiated cells can be differentiated in the lung chip. In such instances, the lung chip can evolve to form lung tissue that is distinct from the initial origin of the primary cell type (e.g., lung cells that are bronchiolar in origin and evolve to have characteristics closer to bronchial tissue). The lung chip can be cultured using media types and supplements suitable to the stage of cellular development (e.g., vascularization medium, differentiation medium, air-liquid interface medium, or combinations thereof). Additional supplements can be added into the medium of the lung chip to maintain the differentiation state or to induce differentiation of cells in the lung chip. Cells can be cultured using submerged or air-liquid interface techniques.

[0290] General Design and Construction

[0291] Lung chips can be constructed using a combination of membranes, chambers, channels, wells, and scaffolds (e.g., electrospun, degradable, fibrous). Lung chips can be formed from a single chip or multiple chips (e.g., multiple chips on a plate, multiple chips on a wafer). One or more chip surfaces can be treated prior to use (e.g., coated, activated, and / or washed prior to use).

[0292] Prior to seeding within the lung chip, cells can be cultured using standard adherent or suspension culture protocols appropriate to the type of cell. Supporting cells can be co-cultured with or without extracellular matrix (e.g., natural or synthetic hydrogel) in a pre-patterned or self-organizing fashion.

[0293] To model resident immune populations, undifferentiated, partially differentiated, or fully differentiated primary immune cells, progenitors, or induced pluripotent stem cells can be embedded directly within the lung chip or seeded on top of the lung chip.

[0294] To model circulating immune populations, undifferentiated, partially differentiated, or fully differentiated immune cells, progenitors, or induced pluripotent stem cells can be introduced through the vascular compartment.

[0295] Cells used in the lung chip can be transfected and / or dyed in order to enable visualization and quantification. Infectious agents, compounds, or potential therapeutics can be introduced into the lung chip using a variety of strategies including liquid suspension or aerosolized delivery to the apical surface, or delivery via the vasculature to mimic systemic delivery.

[0296] Various biological responses and biological parameters can be assessed on the lung chip including barrier integrity, tissue quality, mucous production, antigen delivery, infection duration, immune response (e.g., cytokine production, immune cell infiltration, immune cell activation), and reproducibility across human donors. Design and Construction of Lung Chips

[0297] Design and construction of lungs chips is described herein as well as in Jung et al., Biofabrication 2022, 14, 025012, the contents of which is incorporated herein by reference in its entirety.

[0298] Lung chips can include three channels with two vascularized beds flanking an open top chamber on which airway cells are directly plated without a membrane separating the epithelial and endothelial compartments. Cells are cultured on coated flasks before being plated for coculture in the lung chip. Lung chips are seeded with gel prior to seeding cells. In some instances, the gel can include cells. After polymerization, vascular cells (e.g., human pulmonary microvascular endothelial cells (HPMECs), fibroblasts, pericytes) are seeded and cultured in the lung chip. Following seeding of the vascular network, small airway epithelial cells (SAECs) are seeded via submerged culture. SAECs are allowed to adhere and form a monolayer. The culture is induced into an air-liquid interface (ALI) culture through media change / removal. The middle chamber can be left devoid of any media. The ALI culture is maintained until use.

[0299] Design and Construction of Human Lung Airway Chips

[0300] Design and construction of human lung airway chips is described herein as well as in Benam et al., Nature methods 2016, 13, 151-157; Nawroth et al., American Journal of Respiratory Cell and Molecular Biology 2020, 63 , 591 -600; Si et al. , Human organs-on-chips as tools for repurposing approved drugs as potential influenza and COVID 19 therapeutics in viral pandemics, BioRxiv 2020, 54; and Si et al., Microbiology Spectrum 2021, 9, e00257-21, the contents of which are incorporated herein by reference in their entireties.

[0301] Human lung airway chips can include two parallel channels separated by a membrane. To prepare the chips for cell plating, channels are washed with 70% ethanol, filled with UV activable solution (e.g., 0.5 mg / mL ER1 in ER2 buffer (Emulate)) to prepare the channels for extracellular matrix protein coating, and placed under an ultraviolet lamp for 20 minutes to activate the channel surface for protein coating. Channels are then washed sequentially (e.g, with ER2 buffer and PBS) to remove the unbound protein. Each side of the membrane may be coated with biomaterial (e.g, extracellular matrix proteins such as collagen, fibrin, laminin, proteoglycans, growth factors, or any synthetic or natural hydrogel). Primary human lung bronchial-airway epithelial basal stem cells (Lonza) are expanded in tissue culture flasks using airway epithelial growth medium (Promocell) to 60-70% confluency. Primary human microvascular endothelial cells (Cell Biologies) are expanded using human endothelial cell growth medium (Cell Biologies) to 70-80% confluency.

[0302] Endothelial cells (2x107cells / mL) are then seeded in the bottom channel by inverting the chip for 4h in human endothelial cell growth medium, followed by inverting the chip again and seeding of the top channel with the lung bronchial-airway epithelial basal stem cells (2.5xl06cells / mL) for 4 hours in airway epithelial cell growth medium. The respective medium for each channel is refreshed and chips are incubated overnight under static conditions at 37 °C and 5% CO2. After overnight incubation, adherent cells are perfused with the respective cell culture medium using a peristaltic pump (Ismatec) or automated cell culture module (Emulate) at a volumetric flow rate of 60 pL / hr. After 5-7 days, apical medium is removed while allowing air to fill the channel to establish an air liquid interface culture. Then, airway epithelial cells are cultured for 3-4 additional weeks while being fed a constant flow of air-liquid interface (ALI) medium (StemCell) supplemented with 0.1% VEGF, 0.01% EGF, and ImM CaCh through the bottom channel. Chips are cultured in an incubator containing 5% CO2 and 16-18% O2 at 85- 95% humidity. The apical surface of the epithelium is rinsed with PBS once per week to remove cellular debris and mucus.

[0303] Design and Construction of Breathing Alveolus Lung Chips

[0304] Design and construction of breathing alveolus lung chips is described herein as well as in Stucki et al., Lab on a Chip 2015, 15, 1302-1310; Stucki et al. Scientific reports 2018, 8, 1-13; and Felder et al., Frontiers in bioengineering and biotechnology 2019, 7, 3, the contents of which are incorporated herein by reference in their entireties.

[0305] Breathing alveolus lung chips can include reversible bonded fluidic and pneumatic parts with six independent lung alveolar barriers. The fluidic part comprises two structured plates between which a membrane is sandwiched. The plates and membrane can be made of any material suitable for constructing the lung chip, e.g., PDMS. The pneumatic part comprises a structured plate on which a membrane (e.g, a 40-pm thick membrane) is attached by plasma O2. The plates of the fluidic and pneumatic parts can be produced by soft lithography. For example, PDMS is mixed (e.g, 10:1 PDMS), degassed, and casted in hard plastic molds obtained from structured aluminum molds. The PDMS is cured in an oven at 60 °C for at least 24 h. The membrane can be fabricated as described in Stucki et al., Lab on a Chip 2015, 15, 1302-1310. Bonding of the components of the breathing alveolus lung chip can be performed as described in Stucki et al. Scientific Reports 2018, 8, 1-13.

[0306] Primary alveolar epithelial cells can be added to the chip by filling the chip with cell culture media and seeding the primary alveolar epithelial cells on the apical side of the membrane in a droplet. After 4h, the cell culture well is filled with cell culture medium. When desired, cells can be seeded on the basal side of the membrane in a droplet and cultured. The fluidic part is assembled into the pneumatic part to produce a closed chip, which is then filled with medium. Then, alveolar cells are seeded, cultured in a submerged culture for a short time, and then cultured at the air-liquid interface for the duration of the differentiation process and until use of the chip.

[0307] Example 5: Muscle-Lymph Node Chips

[0308] This example describes microfluidic devices that mimic muscle and lymph node physiology and function, which are also referred to as muscle-lymph node chips. The musclelymph node chips can be used, for example, to investigate an immune response produced in the lymph node in response to an immunogenic composition (e.g., an immunogenic composition comprising a vaccine such as a mRNA vaccine) injected into the muscle.

[0309] Cellular Content

[0310] Muscle in the muscle-lymph node chip can include precursor myoblasts, myotubes, muscle cells derived from induced pluripotent stem cells (iPSCs), extracellular matrix, vascular endothelial cells, pericytes, lymphatic endothelial cells, macrophages, conventional type 1 dendritic cells (cDCl) cells, conventional type 2 dendritic cells (cDC2) cells, plasmacytoid dendritic cells (DCs), and combinations thereof.

[0311] Lymph node in the muscle-lymph node chip can include lymphatic endothelial cells (e.g., lymphatic sprouts and / or networks), monocytes, T cells, B cells, fibroblastic reticular cells, follicular dendritic cells, extracellular matrix, cDCls, cDC2s, plasmacytoid DCs, and combinations thereof. Supportive cells (e.g., fibroblasts, human lymphatic endothelial cells (HLEC), human umbilical endothelial cells (HUVECs), pericytes, and other endothelial cells) can be included in the muscle-lymph node chip to create vessel structures in or nearby the lymph node on the chip.

[0312] Design and Construction

[0313] A schematic depiction of an exemplary design for a system comprising a muscle-lymph node chip is shown in FIG. 8 and an exemplary design of a muscle-lymph node chip is shown in FIG. 10

[0314] The muscle and lymph node are connected in the muscle-lymph node chip in a continuous fluid circuit, which can be driven by a pump and / or by gravity flow. The musclelymph node chip includes one or more ports that can be used for adding or removing media or cells as well as for collecting samples. The muscle chip is provided with a 3 -way fluid connection that allows fluid input to be switched between external media circulation and systemic circulation. As such, the muscle chip can receive its own media supplied separately from systemic circulation.

[0315] After injection of an immunogenic composition into the muscle, an antigen is expressed by antigen presenting cells in the muscle. Directed fluid flow allows movement of the antigen presenting cells (including the activated antigen presenting cells) from the muscle to the lymph node on the muscle-lymph node chip via one or more channels. Directed fluid flow also allows antigen presenting cells to exit the lymph node into systemic circulation and then reenter the muscle.

[0316] The one or more channels in the muscle-lymph node chip can include a monolayer of lymphatic endothelial cells. The monolayer extends into the lymph node on the chip to facilitate transport of fluid and cells into the lymph node on the chip. The channels are designed to allow cells to move freely between the muscle and lymph node without obstruction.

[0317] The mass ratio of muscle to lymph node in the muscle-lymph node chip can be selected to allow physiologically relevant levels of antigen to travel from the muscle to the lymph node where it can promote an immune response.

[0318] Muscle in the muscle-lymph node chip includes cell types that allow mounting of an immune response after addition of an immunogenic composition into the muscle. For example, in some implementations, muscle in the muscle-lymph node chip can include terminally differentiated, multinucleated, myosin heavy chain expressing myotubes within a contracted extracellular matrix space. Cells can be added to the muscle-lymph node chip via the continuous fluid circuit. Any of the cells included in the muscle-lymph node chip can be added once or multiple times.

[0319] Muscle in the muscle-lymph node chip includes vascular and lymphatic networks interspersed within the extracellular matrix around the terminally differentiated myotubes. Immune cells can move into and out of the vascular and lymphatic networks. The vascular and lymphatic networks can be separated such that the vascular network connects to a channel that includes vascular endothelial cells and the lymphatic network connects to a channel that includes lymphatic endothelium cells.

[0320] Lymph node in the muscle-lymph node chip includes lymph node components such as naive T cells (e.g., helper T cells (CD4+) and cytotoxic T cells (CD8+)), B cells, germinal center resident antigen presenting cells, follicular dendritic cells, follicular reticular cells, lymph node stromal cells, and lymphatic endothelial cells. Cells can be arranged to recapitulate functional features such as production of antigen specific antibodies, antigen specific somatic hypermutation and affinity maturation, class-switch recombination, plasmablast differentiation, and production of plasma B cells, memory B cells, and CD8+ T cells.

[0321] Methods for preparing muscle and lymph node on a chip that are known in the art or described herein can be used to build a muscle-lymph node chip as described herein. See Examples above.

[0322] Various biological responses and biological parameters can be assessed on the musclelymph node chip including immune cell activation, lymphatic endothelial cell activation, cell differentiation, cell migration, antibody production, and extravasation through the monolayer of lymphatic endothelial cells.

[0323] For example, the inflammatory cytokine cascade in the muscle after antigen introduction and / or expression can be assessed. In the inflammatory cascade, TLR3 / 7 and 8 on the endosome, cytosolic sensors such as RIG-I, MDA5, PKR, and OAS recognize dsRNAs and ssRNAs in the cytoplasm (e.g., mRNA vaccine) resulting in production of pro inflammatory cytokines such as (IL-6, IL-8, TNF-alpha) (IL-lb driven) and type 1 interferon response (IFN-P)). Adjuvanted protein-based vaccines lead to the production of a myriad variety of cytokines specific to the adjuvant used such as AS01 (aka QS-1) induces CXCL10 (aka IP-10) and IFN-gamma, and to a lesser extent, IL-2 and IL-5, ALUM induces more TH2 cytokines: IL-4, IL-13, and IL-5, and AS03 induces IL-6 and IP-10.

[0324] In another example, the conserved chemokine axes of importance such as the CCL21- CCR7 axis can be assessed. In the CCL21-CCR7 axis, lymphatic endothelium under these pro- inflammatory and laminar flow conditions (e.g., interstitial fluid draining via the blind ended lymphatic endothelium) express adhesion receptors and chemokines such as ICAM1, VCAM1, CCL21, and CCL19. Activated antigen presenting cells such as cDCls and cDC2s express cognate chemokine receptors such as CCR7 (cognate to CCL21 chemokine) and migrate along the lymphatic endothelium into the draining lymph node. Activated lymphatic endothelial cells are lined in the chip such that they deliver the activated antigen presenting cells (such as cDCls and cDC2s) deep into the lymph node on a chip to bind with the naive T-cells to initiate targeted CD8+ T-cell, memory B-cell, and plasma B-cell generation.

[0325] Example 6: Lung-Lymph Node Chips

[0326] This example describes generation and characterization of lung and lymph node on a chip, which is also referred to as a lung-lymph node chip. The lung-lymph node chip can be used, for example, to investigate an immune response produced in the lymph node in response to an immunogenic composition (e.g., immunogenic composition comprising a pathogen such as a virus) injected into the lung.

[0327] Cellular Content

[0328] Lung in the lung-lymph node chip can include lung epithelial cells (e.g., alveolar lung epithelial cells, bronchial lung epithelial cells), extracellular matrix, vascular endothelial cells, pericytes, (e.g., towards forming sprouts and / or networks), antigen presenting cells, dendritic cells (e.g., cDCls, cDC2s, plasmacytoid DCs), immune cells, lymphatic vasculature (e.g., towards forming sprouts and / or networks), and combinations thereof. Lung in the lung-lymph node chip can include primary lung epithelial cells, patient- derived lung organoids, or lung cells derived from iPSCs. Lung cells that can be used in the lung chip can be healthy or diseased. Lung cells can be bronchial, bronchiolar, or alveolar in origin. Supporting cells such as lung fibroblasts, endothelial cells (e.g., pulmonary microvascular endothelial cells, human umbilical vein endothelial cells, lymphatic endothelial cells), and pericytes can be included to form vascular structures in or nearby the lung cells.

[0329] Immune cells in the lung-lymph node chip can include undifferentiated, partially differentiated, or fully differentiated primary immune cells, progenitors, or induced pluripotent stem cells. Undifferentiated or partially differentiated cells can be differentiated in the lunglymph node chip. In such instances, the lung-lymph node chip can evolve to form lung tissue that is distinct from the initial origin of the primary cell type (e.g., lung cells that are bronchiolar in origin and evolve to have characteristics closer to bronchial tissue).

[0330] Lymph node in the lung-lymph node chip can include lymphatic endothelial cells (e.g., lymphatic sprouts and / or networks), monocytes, T cells, B cells, fibroblastic reticular cells, follicular dendritic cells, extracellular matrix, cDCls, cDC2s, plasmacytoid DCs, and combinations thereof. Supportive cells (e.g., fibroblasts, human lymphatic endothelial cells (HLEC), human umbilical endothelial cells (HUVECs), pericytes, and other endothelial cells) can be included in the lung-lymph node chip to create vessel structures in or nearby the lymph node on the chip.

[0331] Design and Construction

[0332] A schematic depiction of an exemplary design for a system comprising a lung-lymph node chip is shown in FIG. 8 and an exemplary design of a lung-lymph node chip is shown in FIG. 11

[0333] The lung and lymph node are connected in the chip in a continuous fluid circuit, which can be driven by a pump and / or by gravity flow. The lung and lymph node are connected via one or more channels which provide a pathway for fluid and immune cells to move from the lung to the lymph node. Immune cells can exit the lymph node into systemic circulation and then reenter the lung.

[0334] The one or more channels in the lung-lymph node chip include a monolayer of lymphatic endothelial cells. The monolayer extends into the lymph node on the chip to facilitate transport of fluid and cells into the lymph node on the chip. The channels are designed to allow cells to circulate freely between the lung and lymph node without obstruction.

[0335] When a pathogen is introduced into the lung in the lung-lymph node chip, the spread of the infection can be prevented using a barrier between the lung and lymph node connection. Any barrier suitable for preventing the infection from entering the lymph node can be used. Nonlimiting examples of barriers include endothelial barriers, lymphatic barriers, and immune barriers.

[0336] The mass ratio of lung to lymph node in the lung-lymph node chip can be selected to enable the lymph node to produce an amount of neutralizing antibodies sufficient to have a quantifiable effect at the infection site in the lung. For example, if the neutralizing antibody is effective but the titer is too low, little to no effect on the infection in the lung will be observed.

[0337] Infection in the lung can release cytokines, and therefore cytokines can be used as a marker of an immune response on the lung-lymph node chip. Any cytokine indicative of an immune response can be used as a marker, e.g., IL-6, IL-8, IL- 10, and IFN-0.

[0338] Any type of immune cells can be included in the lung-lymph node chip. For example, immune cells that play a role in processing, presentation, and transport of pathogenic antigens can be included in the lung-lymph node chip. Non-limiting examples of immune cells in the lung-lymph node chip include CD4+ T cells, CD8+ T cells, macrophages, and dendritic cells.

[0339] Methods for preparing lung on a chip and lymph node on a chip that are known in the art or described herein can be used to build a lung-lymph node chip as described herein. See Examples above.

[0340] Various biological responses and biological parameters can be assessed on the lunglymph node chip including lung infection, immune responses in the lung including infiltration of immune cells into the lung, and transmission of the infection signal into the lymph node chip to mount an immune response.

[0341] Example 7: Muscle-Lymph Node-Lung Chips

[0342] This example describes generation and characterization of muscle, lung, and lymph node on a chip, which is also referred to as a muscle-lymph node-lung chip. The muscle-lymph nodelung chip can be used, for example, to investigate an immune response produced in the lymph node in response to an immunogenic composition injected into the muscle and the effect of the immune response on a pathogen in the lung.

[0343] Cellular Content Muscle in the muscle-lymph node-lung chip can include precursor myoblasts, myotubes, muscle cells derived from iPSCs, extracellular matrix, vascular endothelial cells, (e.g., towards forming sprouts and / or networks), pericytes, lymphatic endothelial cells (e.g., towards forming sprouts and / or networks), macrophages, cDCls, cDC2s, plasmacytoid DCs, and combinations thereof.

[0344] Lymph node in the muscle-lymph node-lung chip can include lymphatic endothelial cells (e.g., lymphatic sprouts and / or networks), monocytes, T cells, B cells, fibroblastic reticular cells, follicular dendritic cells, extracellular matrix, cDCls, cDC2s, plasmacytoid DCs, and combinations thereof. Supportive cells (e.g., fibroblasts, human lymphatic endothelial cells (HLEC), human umbilical endothelial cells (HUVECs), pericytes, and other endothelial cells) can be included in the muscle-lymph node-lung chip to create vessel structures in or nearby the lymph node on the chip.

[0345] Lung in the muscle-lymph node-lung chip can include lung epithelial cells (e.g., alveolar lung epithelial cells, bronchial lung epithelial cells), extracellular matrix, vascular endothelial cells (e.g., sprouts and / or networks), antigen presenting cells, dendritic cells (e.g., cDCls, cDC2s, plasmacytoid DCs), immune cells, lymphatic vasculature (e.g., sprouts and / or networks), and combinations thereof. Lung in the muscle-lymph node-lung chip can include primary lung epithelial cells, patient-derived lung organoids, or lung cells derived from iPSCs. Lung cells that can be used in the lung chip can be healthy or diseased. Lung cells can be bronchial, bronchiolar, or alveolar in origin. Supporting cells such as lung fibroblasts, endothelial cells (e.g., pulmonary microvascular endothelial cells, human umbilical vein endothelial cells, lymphatic endothelial cells), and pericytes can be included to form vascular structures in or nearby the lung cells.

[0346] Immune cells in the muscle-lymph node-lung chip can include undifferentiated, partially differentiated, or fully differentiated primary immune cells, progenitors, or induced pluripotent stem cells. Undifferentiated or partially differentiated cells can be differentiated in the lunglymph node chip. In such instances, the muscle-lymph node-lung chip can evolve to form lung tissue that is distinct from the initial origin of the primary cell type (e.g., lung cells that are bronchiolar in origin and evolve to have characteristics closer to bronchial tissue).

[0347] Design and Construction A schematic depiction of an exemplary design of a system comprising a muscle-lymph node-lung chip is shown in FIG. 9. A schematic depiction of an exemplary design of a musclelymph node-lung chip is shown in FIG. 12.

[0348] The muscle, lymph node, and lung are connected in the chip in a continuous fluid circuit, which can be driven by a pump and / or by gravity flow. The lymph node can be positioned in a central location on the chip that allows it to be connected to the muscle and the lung via one or more channels. As such, fluid drains from the muscle and lung into the centrally shared lymph node, thereby allowing crosstalk between muscle, lymph node, and lung on the chip. Thus, the design of the muscle-lymph node-lung chip allows crosstalk between the immune and infection responses in the muscle, lymph node and lung.

[0349] Fluid flow within the lymph node allows cells to move out of the central exit of the lymph node chip into systemic circulation and then into the muscle and lung. The size of the lymph node is sufficient to mount an immune response against both antigen expressing cells in the muscle and an infection in the lung.

[0350] After injection of an immunogenic composition, the muscle produces an inflammatory response in which activated antigen presenting cells migrate from the muscle to the lymph node via channels. The antigen presenting cells cross present their antigens to antigen specific naive CD4+ T cells and subsequently generate robust CD8+ T cell and B cell responses. Naive CD4+ T cells matured into follicular helper T cells bind to the highest affinity B cell receptor containing B cell to promote its affinity maturation, somatic hypermutation, clonal selection, and isotype switching to produce a robust humoral response. The B cell maturation process generates plasma cells (produce a strong titer of neutralizing antibody) and memory B cells ready (within the lymph node chip) to mature into plasma cells upon reinfection or infection with a different variant.

[0351] Under an infection assault, inflammatory cytokine rich media and antigen carrying dendritic cells from the lung drain into the shared lymph node to activate vaccine candidate primed cells (e.g., CD8+ T cells, memory B cells, and neutralizing antibody producing plasma B cells), which exit the lymph node into systemic circulation and then reenter the muscle and lung to mount a response. The response generated within the shared lymph node neutralizes the infection and also eliminates the infected lung cells in the lung chip. Plasma cells entering circulation also home into secondary sites such as the lung to aid the humoral response. The muscle-lymph node-lung chip is stable for at least 30 days. The muscle-lymph nodelung chip can be injected with a booster dose of vaccine (e.g., 21-28 days after injection of the primary dose) or an additional treatment into the muscle. In one embodiment, injection of the booster dose or the additional treatment can result in increased expansion of antigen specific antibody producing plasma B cells, which allows increased neutralizing antibody in circulation, and increased expansion of CD8+ T cells, which mount a response to the pathogen infection in the lung.

[0352] The infection in the lung may break through the epithelial barrier into the circulation. Therefore, the connection to the lung can be unidirectional and / or the exit from the lung can be final to prolong the stability of the system. Loading of the pathogen into the lung chip can be used to prevent the infection from spreading from the lung into the systemic circulation after breaking the endothelial barrier.

[0353] Methods for preparing muscle, lymph node, and lung on a chip that are known in the art or described herein can be used to build a muscle-lymph node-lung chip as described herein. See Examples above.

[0354] Various biological responses and biological parameters can be assessed on the musclelymph node-lung chip including cell activation, cell differentiation, cell migration and infiltration, antibody production, infection, and transmission of the infection signal into the lymph node chip to mount an immune response.

[0355] For example, the inflammatory cytokine cascade in the muscle after antigen introduction and / or expression can be assessed. In the inflammatory cascade, TLR3 / 7 and 8 on the endosome, cytosolic sensors such as RIG-I, MDA5, PKR, and OAS recognize dsRNAs and ssRNAs in the cytoplasm (mRNA vaccine) resulting in production of proinflammatory cytokines such as (IL-6, IL-8, TNF-alpha) (IL-lb driven) and type 1 interferon response (IFN-P)). Adjuvanted proteinbased vaccines lead to the production of a myriad variety of cytokines specific to the adjuvant used such as AS01 (aka QS-1) induces CXCL10 (aka IP-10) and IFN-gamma, and to a lesser extent, IL-2 and IL-5, ALUM induces more TH2 cytokines: IL-4, IL- 13, and IL-5, and AS03 induces IL-6 and IP- 10.

[0356] In another example, the conserved chemokine axes of importance such as the CCL21- CCR7 axis can be assessed. In the CCL21-CCR7 axis, lymphatic endothelium under these pro- inflammatory and laminar flow conditions (e.g., interstitial fluid draining via the blind ended lymphatic endothelium) express adhesion receptors and chemokines such as ICAM1, VCAM1, CCL21, and CCL19. Activated antigen presenting cells such as cDCls and cDC2s express cognate chemokine receptors such as CCR7 (cognate to CCL21 chemokine) and migrate along the lymphatic endothelium into the draining lymph node. Activated lymphatic endothelial cells are lined in the chip such that they deliver the activated antigen presenting cells (such as cDCls and cDC2s) deep into the lymph node on a chip to bind with the naive T-cells to initiate targeted CD8+ T-cell, memory B-cell, and plasma B-cell generation.

[0357] Immunocompatibility Assays

[0358] Immune multi-organ chips described herein can be used to determine immune activation in the absence of a vaccine candidate or infectious agent. For example, if the leukocytes and / or tissues introduced into the immune multi-organ chip are not HLA-compatible, an alloreactive T cell response may be generated.

[0359] Various testing approaches can be used for measuring T cell alloreactivity including T cell proliferation assays (e.g., fluorescent cellular dye dilution assays), intracellular ATP synthesis assays (e.g., colorimetric assays, fluorometric assays), and quantification of cytokineproducing cell assays (e.g., enzyme-linked immunospot (ELISpot) assays). Alloreactivity can also be assessed by measuring predictive markers such as cytokines (e.g., IL-2, IFN-y) and / or by measuring T cell activation markers (e.g., CD39, CD69, CD95, and CD 154).

[0360] Additionally, immune compatibility readouts including, but not limited to, in vitro T cell cytotoxicity tests and / or measurement of expression markers for immune cell activation and / or cytokines may be used without an infection. For example, and not limitation, compatibility may be studied outside the chip before putting the cells together into the chip.

[0361] OTHER EMBODIMENTS

[0362] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What Is Claimed Is:

1. A microfluidic device (300) comprising: a muscle chip (311) and a lymph node chip (313); and a plurality of channels (312) fluidly connecting the muscle chip (311) to the lymph node chip (313), wherein the lymph node chip (313) comprises lymphatic cells and immune cells; and the muscle chip (311) comprises muscle cells.

2. The device of claim 1, wherein the muscle chip (311) comprises extracellular matrix, myoblasts, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, monocytes, neutrophils, or combinations and sub-combinations thereof.

3. The device of claim 1 or claim 2, wherein the lymph node chip (313) comprises extracellular matrix, lymphatic endothelial cells, monocytes, T cells, B cells, dendritic cells, fibroblasts, or combinations and sub-combinations thereof.

4. The device of any one of claims 1-3, wherein the ratio of cells in the lymph node chip (313) to cells in the muscle chip (311) is between 1 :4 to 1: 10.

5. The device of any one of claims 1-4, wherein the plurality of channels (312) comprises lymphatic endothelial cells.

6. The device of any one of claims 1-5, wherein the plurality of channels (312) is fluidly connected to a port (314).

7. A microfluidic device (400) comprising: a lung chip (411) and a lymph node chip (413); and a plurality of channels (412) fluidly connecting the lung chip (411) to the lymph node chip (413), wherein the lymph node chip (413) comprises lymphatic cells and immune cells; and the lung chip (411) comprises lung cells.

8. The device of claim 7, wherein the lung chip (411) comprises extracellular matrix, lung epithelial cells, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, or combinations and sub-combinations thereof.

9. The device of claim 7 or claim 8, wherein the lymph node chip (413) comprises extracellular matrix, lymphatic endothelial cells, monocytes, T cells, B cells, dendritic cells, fibroblasts, or combinations and sub-combinations thereof.

10. The device of any one of claims 7-9, wherein the ratio of cells in the lymph node chip (413) to cells in the lung chip (411) is between 1 :4 to 1 :10.

11. The device of any one of claims 7-10, wherein the plurality of channels (412) comprises lymphatic endothelial cells.

12. The device of any one of claims 7-11, wherein the plurality of channels (412) is fluidly connected to a port (414).

13. A microfluidic device (500) comprising: a muscle chip (511), a lymph node chip (513), and a lung chip (515); and a first set of a plurality of channels (512a) fluidly connecting the muscle chip (511) to the lymph node chip (513) and a second set of a plurality of channels (512b) fluidly connecting the lung chip (515) to the lymph node chip (513); wherein the lymph node chip comprises lymphatic cells and immune cells; the muscle chip comprises muscle cells; and the lung chip comprises lung cells.

14. The device of claim 13, wherein the muscle chip (511) comprises extracellular matrix, myoblasts, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, monocytes, neutrophils, or combinations and sub-combinations thereof.

15. The device of claim 13 or claim 14, wherein the lung chip (515) comprises extracellular matrix, lung epithelial cells, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, or combinations and sub-combinations thereof.

16. The device of any one of claims 13-15, wherein the lymph node chip (513) comprises extracellular matrix, lymphatic endothelial cells, monocytes, T cells, B cells, dendritic cells, fibroblasts, or combinations and sub-combinations thereof.

17. The device of any one of claims 13-16, wherein the ratio of cells in the lymph node chip (513) to cells in the muscle chip (511) is between 1 :4 and 1 :10 and the ratio of cells in the lymph node chip (513) to cells in the lung chip (515) is between 1:4 and 1 :10.

18. The device of any one of claims 13-17, wherein the ratio of cells in the lymph node chip (513), the muscle chip (511), and the lung chip (515) is between 1 :4:4 and 1 : 10: 10.

19. The device of any one of claims 13-18, wherein the first set of the plurality of channels (512a) and the second set of the plurality of channels (512b) comprise lymphatic endothelial cells.

20. The device of any one of claims 13-19, wherein the first set of the plurality of channels (512a) is fluidly connected to a first port (514a) and a second port (514b) and wherein the second set of the plurality of channels (512b) is fluidly connected to a third port (514c) and a fourth port (514d).

21. A system (100) comprising: a first organ chip (111) and a second organ chip (113); a plurality of channels (112) fluidly connecting the first organ chip (111) to the second organ chip (113); a pump (120); a mixer (130); and a fluidic network (140) fluidly connecting the first organ chip (111), the second organ chip (113), the pump (120), and the mixer (130), wherein the fluidic network (140) comprises at least one inlet (142) and at least one outlet (144); and wherein the first organ chip (111) comprises a muscle chip or a lung chip and the second organ chip (113) comprises a lymph node chip; and wherein the lymph node chip comprises lymphatic cells and immune cells; the muscle chip comprises muscle cells; and the lung chip comprises lung cells.

22. The system of claim 21, wherein the first organ chip (111) comprises the muscle chip, which comprises extracellular matrix, myoblasts, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, monocytes, neutrophils, or combinations and subcombinations thereof.

23. The system of claim 21, wherein the first organ chip (111) comprises the lung chip, which comprises extracellular matrix, lung epithelial cells, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, or combinations and sub-combinations thereof.

24. The system of any one of claims 21-23, wherein the second organ chip (113) comprises the lymph node chip, which comprises extracellular matrix, lymphatic endothelial cells, monocytes, T cells, B cells, dendritic cells, fibroblasts, or combinations and sub-combinations thereof.

25. The system of any one of claims 21-24, wherein the first organ chip (111) comprises the muscle chip and the ratio of cells in the lymph node chip to cells in the muscle chip is between1 :4 to 1 : 10 or the first organ chip (111) comprises the lung chip and the ratio of cells in the lymph node chip to cells in the lung chip is between 1 :4 and 1: 10.

26. The system of any one of claims 21-25, wherein the plurality of channels (112) comprises lymphatic endothelial cells.

27. The system of any one of claims 21-26, wherein the plurality of channels (112) is fluidly connected to a port (114a).

28. The system of any one of claims 21-27, wherein the pump (120) is one or more of a pneumatic pump, an electromagnetic pump, a peristaltic pump, or a pressure pump.

29. The system of any one of claims 21-28, further comprising one or more sensors and / or one or more cameras.

30. The system of claim 29, wherein the one or more sensors are selected from the group consisting of a temperature sensor, an oxygen sensor, a pH sensor, a humidity sensor, a photodetector, a transducer, an analyte or metabolite sensor, a fluid level sensor, a flow sensor, a hydrometer, a viscometer, a velocity sensor, and an electrical sensor.

31. The system of any one of claims 21-30, further comprising a control module.

32. A system (200) comprising: a first organ chip (211), a second organ chip (213), and a third organ chip (215); a first set of a plurality of channels (212a) fluidly connecting the first organ chip (211) to the second organ chip (213) and a second set of a plurality of channels (212b) fluidly connecting the third organ chip (215) to the second organ chip (213); a pump (220); a mixer (230); anda fluidic network (240) fluidly connecting the first organ chip (211), the second organ chip (213), the pump (220), and the mixer (230), wherein the fluidic network (240) comprises at least one inlet (242a, 242b) and at least one outlet (244a, 244b); and wherein the first organ chip (211) comprises a muscle chip or a lung chip, the second organ chip (213) comprises a lymph node chip, and the third organ chip (215) comprises the muscle chip or the lung chip; and wherein the lymph node chip comprises lymphatic cells and immune cells; the muscle chip comprises muscle cells; and the lung chip comprises lung cells.

33. The system of claim 32, wherein the first organ chip (211) comprises the muscle chip, which comprises extracellular matrix, myoblasts, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, monocytes, neutrophils, or combinations and subcombinations thereof; and wherein the third organ chip (215) comprises the lung chip, which comprises extracellular matrix, lung epithelial cells, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, or combinations and sub-combinations thereof.

34. The system of claim 32, wherein the first organ chip (211) comprises the lung chip, which comprises extracellular matrix, lung epithelial cells, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, or combinations and sub-combinations thereof; and wherein the third organ chip (215) comprises the muscle chip, which comprises extracellular matrix, myoblasts, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, monocytes, neutrophils, or combinations and sub-combinations thereof.

35. The system of any one of claims 32-34, wherein the second organ chip (213) comprises the lymph node chip, which comprises extracellular matrix, lymphatic endothelial cells, monocytes, T cells, B cells, dendritic cells, fibroblasts, or combinations and sub-combinations thereof.

36. The system of any one of claims 32-35, wherein the ratio of cells in the lymph node chip to cells in the muscle chip is between 1:4 and 1:10 and the ratio of cells in the lymph node chip to cells in the lung chip is between 1 :4 and 1: 10.

37. The system of any one of claims 32-36, wherein the ratio of cells in the lymph node chip, the muscle chip, and the lung chip is between 1:4:4 and 1 :10:10.

38. The system of any one of claims 32-37, wherein the first set of the plurality of channels (212a) and the second set of the plurality of channels (212b) comprise lymphatic endothelial cells.

39. The system of any one of claims 32-38, wherein the first set of the plurality of channels (212a) is fluidly connected to a first port (214a) and a second port (214b) and wherein the second set of the plurality of channels (212b) is fluidly connected to a third port (214c) and a fourth port (214d).

40. The system of any one of claims 32-39, wherein the pump (220) is one or more of a pneumatic pump, an electromagnetic pump, a peristaltic pump, or a pressure pump.

41. The system of any one of claims 32-40, further comprising one or more sensors and / or one or more cameras.

42. The system of claim 41, wherein the one or more sensors are selected from the group consisting of a temperature sensor, an oxygen sensor, a pH sensor, a humidity sensor, a photodetector, a transducer, an analyte or metabolite sensor, a fluid level sensor, a flow sensor, a hydrometer, a viscometer, a velocity sensor, and an electrical sensor.

43. The system of any one of claims 32-42, further comprising a control module.

44. A microfluidic device comprising: a first organ chip and a second organ chip; and a plurality of channels fluidly connecting the first organ chip to the second organ chip, wherein the first organ chip is a lymph node chip and the second organ chip is a muscle chip or a lung chip; and1wherein the lymph node chip comprises lymphatic cells and immune cells; the muscle chip comprises muscle cells; and the lung chip comprises lung cells.

45. The device of claim 44, wherein the muscle chip comprises extracellular matrix, myoblasts, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, monocytes, neutrophils, or combinations and sub-combinations thereof.

46. The device of claim 44 or claim 45, wherein the lung chip comprises extracellular matrix, lung epithelial cells, vascular endothelial cells, lymphatic endothelial cells, dendritic cells, macrophages, or combinations and sub-combinations thereof.

47. The device of any one of claims 44-46, wherein the lymph node chip comprises extracellular matrix, lymphatic endothelial cells, monocytes, T cells, B cells, dendritic cells, fibroblasts, or combinations and sub-combinations thereof.

48. The device of any one of claims 44-47, wherein the second organ chip is the muscle chip and the ratio of cells in the lymph node chip to cells in the muscle chip is between 1 :4 to 1 : 10 or the second organ chip is the lung chip and the ratio of cells in the lymph node chip to cells in the lung chip is between 1 :4 and 1: 10.

49. The device of any one of claims 44-48, further comprising a third organ chip and an additional channel fluidly connecting the first organ chip to the third organ chip, wherein the third organ chip is the muscle chip or the lung chip.

50. The device of claim 49, wherein the second organ chip is the muscle chip and the third organ chip is the lung chip.

51. The device of claim 50, wherein the second organ chip is the lung chip and the third organ chip is the muscle chip.

52. The device of any one of claims 49-51, wherein the ratio of cells in the lymph node chip to cells in the muscle chip and cells in the lung chip is between 1 :4:4 to 1 :10:10.

53. The device of any one of claims 44-52, wherein the plurality of channels comprises lymphatic endothelial cells.

54. A method comprising: contacting an agent with the system of any one of claims 21-43 or the microfluidic device of any one of claims 1-20 or 44-53; and detecting a cellular response; wherein the agent comprises a therapeutic agent, a pathogenic agent, or a combination thereof.

55. The method of claim 54, wherein the agent is selected from the group consisting of a cell, a protein, a peptide, an antibody or antigen binding fragment thereof, a nucleic acid, and a small molecule.

56. The method of claim 54 or claim 55, wherein the cellular response is selected from the group consisting of viability, proliferation, activation, respiration, metabolism, cell migration, cell contractility, differential expression of cell biomarkers, production and / or release of biomolecules, action potentials, and combinations thereof.

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