Lymphoid-lymphatics-integrated organ-on-chip device and method

Abstract

Organ-on-chip system, including a bio-engineered lung region that is functionally connected to a bio-engineered lymphatic region via a functionalized connection region. The bio-engineered lung region includes cultured or transplanted lung cells / tissue, which can mimic in vivo lung function, and the bio-engineered lymphatic region includes lymphatic cells / tissue, which can mimic in vivo lymphatic function. The connection region allows for stimuli and cell secretions to be transported between the lung region and the lymphatic region to confer immune competency to the lung region, as the lymphatic region can generate an immune response based on stimuli received from the lung region, for example, in response to infections or therapeutic agents

Description

LYMPHOID-LYMPHATICS-INTEGRATED ORGAN-ON-CHIP DEVICE ANDMETHODCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 578,339, filed August 23, 2023, which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Pulmonary infections are diseases that infect the lungs and which can range from mild to life-threatening. Infection by bacterial or viral agents can result in inflammation of lung tissue and, subsequently, physiological symptoms such as cough, fever, and shortness of breath. Much research is dedicated to studying the mechanisms, treatments, and preventions for these diseases. However, it is challenging to accurately extrapolate how the response in an in vitro model will translate to an in vivo application due to the influence of the immune system.

[0003] There is a benefit for a more immune-competent lung model.SUMMARY

[0004] Disclosed herein is an organ-on-chip system, including a bio-engineered lung region that is functionally connected to a bio-engineered lymphatic region via a functionalized connection region. The bio-engineered lung region includes cultured or transplanted lung cells / tissue, which can mimic in vivo lung function, and the bio-engineered lymphatic region includes lymphatic cells / tissue, which can mimic in vivo lymphatic function. The connection region allows for stimuli and cell secretions (e.g., cytokines, antigens, antibodies, etc.) to be transported between the lung region and the lymphatic region to confer immune competency to the lung region, as the lymphatic region can generate an immune response based on stimuli received from the lung region, for example, in response to infections or therapeutic agents. The system may be used as a screening platform for pulmonary infections and stimulants, vaccine candidates and routes of vaccination, among other applications.

[0005] In an aspect, an organ-on-chip system is disclosed comprising a chip substrate defining a first compartment, a second compartment, a third compartment formed between the first compartment and the second compartment, and at least one input for directing media through the first compartment, wherein the first compartment houses a lung region that can simulate lung function and comprising lung cells or lung tissue, wherein the secondcompartment houses a lymphoid region that can simulate lymphatic function and comprising immune response invoking cells, wherein the third compartment houses a connection region formed between the lung region and the lymphoid region, and wherein, upon exposure to a stimulus, the lymphoid region invokes an immune response.

[0006] In another aspect, an organ-on-chip system is disclosed comprising: a chip substrate (e.g., PDMS substrate) defining: a first compartment comprising (i) a first cell scaffold (e.g., hydrogel) comprising first cells (e.g., wherein the hydrogel is located at the interstitial side, e.g., as the lower region, of the first compartment, and wherein the hydrogel provides a matrix for cell culture, growth, adhesion, placement) in a first sub-compartment and (ii) second cells in a second sub-compartment formed over the first cells across a membrane; a second compartment comprising a second cell scaffold with third cells; a third compartment formed between the first compartment and the second compartment (e.g., layered with Lymphatic endothelial cells (LECs)) as a connection region; and at least one input for directing media through the first compartment, wherein the first cell scaffold with the first cells and the second cells, collectively forms a lung region and simulate lung function, wherein the second subcompartments (e.g., upper region) forms an epithelial / airway region forms an air-liquid interface, and wherein the first sub-compartment (e.g., lower region) forms a perfusable, vascularized (e.g., microvascularized) network of the first cells, wherein the second cell scaffold forms a lymphoid region and simulates immune function, the third cells of the second cell scaffold comprise immune response invoking cells, wherein the connection region defined by the third compartment is formed between the lung interstitium region and the lymphoid region; and wherein, upon exposure to a stimulus, the lymphoid region invokes an immune response.

[0007] In some embodiments, the first cell scaffold and the second cell scaffold each comprise a hydrogel.

[0008] In some embodiments, the first cell scaffold comprises a first hydrogel, and the second cell scaffold comprises a second hydrogel, wherein the first hydrogel and the second hydrogel are different.

[0009] In some embodiments, the first cell scaffold as lung compartment hydrogel comprises fibrin / collagen hydrogel (e.g., in ratio 9: 1) (e.g., homing endothelial cells, fibroblast, interstitial macrophages, DCs, or a combination thereof).

[0010] In some embodiments, the second cell scaffold as a lymphoid compartment hydrogel comprises a PEG-based hydrogel (e.g., Peg-4-Malemide functionalized withfibronectin and collagen-mimicking peptides and crosslinked with a non-degradable crosslinker (DTT) and a degradable peptide crosslinker).

[0011] In some embodiments, the first cells and / or second cells of the lung region comprise endothelial cells, fibroblasts, airway macrophages, interstitial macrophages, interstitial dendritic cells, peripheral mononuclear cells, or a combination thereof.

[0012] In some embodiments, the lung region comprises all healthy cells.

[0013] In some embodiments, the lung region comprises, at least in part, cancerous or abnormal cells.

[0014] In some embodiments, the lung region is configured to simulate lung interstitium and lung epithelium function.

[0015] In some embodiments, the second sub-compartment of the first compartment forms an upper region comprising the lung cells or lung tissue, wherein the first sub-compartment of the first compartment forms a lower region comprising the vascularized and perfusable networks underlying the lung cells or lung tissue.

[0016] In some embodiments, the third compartment is formed between (i) the first subcompartment of the first compartment and (ii) the second compartment.

[0017] In some embodiments, the connection region is formed between (i) the vascularized and perfusable networks of the lung region of the first sub-compartment and the lymphoid region.

[0018] In some embodiments, the at least one input directs media through the first subcompartment of the first compartment.

[0019] In some embodiments, the lymphoid region comprises lymphatic endothelial cells, tonsil cells, dendritic cells, and / or stromal cells.

[0020] In some embodiments, the lymphoid region comprises all healthy cells.

[0021] In some embodiments, the lymphoid region comprises, at least in part, cancerous or abnormal cells.

[0022] In some embodiments, the connection region is functionalized.

[0023] In some embodiments, the connection region comprises lymphatic endothelial cells.

[0024] In some embodiments, the connection region comprises all healthy cells.

[0025] In some embodiments, the connection region comprises, at least in part, cancerous or abnormal cells.

[0026] In some embodiments, the connection region is at least partially coated with proteins and / or small molecules.

[0027] In some embodiments, the membrane is a permeable polyester track-etched (PETE) membrane.

[0028] In some embodiments, the chip substrate further comprises a fourth compartment housing fibroblasts.

[0029] In another aspect, a method is disclosed of assessing stimuli on lung tissue (e.g., screening of the therapeutics or stimuli), the method comprising a) providing the organ-on- chip system of any one of the above-discussed claims; b) establishing interstitial flow of media from the lung region to the lymphoid region via the connection region; c) introducing a stimulus to at least one region of the organ-on-chip system; and d) measuring response (e.g., presence or degree of cytokine) to the stimulus.

[0030] In some embodiments, fresh media is input through the at least one input in the chip substrate.

[0031] In some embodiments, the interstitial flow of media is maintained for at least a portion of the duration of step d).

[0032] In some embodiments, the interstitial flow of media is maintained for the entire duration of step d).

[0033] In some embodiments, the stimulus is an infectious agent and / or a treatment protocol.

[0034] In some embodiments, the infectious agent is a bacterium or a virus.

[0035] In some embodiments, the treatment protocol is the administration of a therapeutic agent, ablation, resection, exposure to radiation, or a vaccination.

[0036] In some embodiments, the organ-on-chip system is exposed to the treatment protocol before and / or after exposure to the infectious agent.

[0037] In some embodiments, step d) occurs over a period of time and / or in different locations within the organ-on-chip system.

[0038] Other systems, methods, features, and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF DRAWINGS

[0039] FIGURES 1A-1D depict an example organ-on-chip system in accordance with the present disclosure.

[0040] FIGURES 2A-2D depict example dimensions for the example organ-on-chip system depicted in FIGS. 1A-1D.

[0041] FIGURE 3 depicts an example method of assessing stimuli on lung tissue using any of the disclosed organ-on-chip systems in accordance with the present disclosure.

[0042] FIGURES 4A-4E depict a multi-compartment device configured for the culture of different cell types with control over the location and duration of culture.

[0043] FIGURES 5A-5C depict that vascularized and perfusable networks underlie the mature tight airway epithelium.

[0044] FIGURES 6A-6D depict example lymphatic endothelial cells that can connect lung and Lymphoid undergoing germinal center formation.

[0045] FIGURES 7A-7B depict that virus infection in the airway induces inflammatory phenotype in lung and B cell responses in lymphoids.

[0046] FIGURES 8A-8B depict the transfer of a tracer molecule from the lung to the lymphoid. The intensity of the molecule in the lymphoid component was measured.

[0047] FIGURES 9A-9B depict cytokine responses in the lung. Interstitial flow was established from the lung to the lymphoid. This result indicates that viral infection induces strong cytokine and interferon response in the lung, which is cleared by the interstitial flow and moved to the lymphoid.

[0048] FIGURES 10A-10C depict immune cell activation in the lymphoid. Interstitial flow post-viral infection of the lung induces immune cell activation in the lymphoid compartment, as shown by the increase in antibody-secreting cells in the lymphoid. These figures report fold changes as compared to uninfected devices.DETAILED DESCRIPTION

[0049] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.DEFINITIONS

[0050] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0051] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0052] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like.

[0053] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0054] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘aboutx’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0055] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0056] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0057] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount andtype of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics.

[0058] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0059] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0060] A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatmentmay be varied for example by increasing or decreasing the amount of a disclosed compound and / or pharmaceutical composition, by changing the disclosed compound and / or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0061] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition.

[0062] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

[0063] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0064] As used interchangeably herein, “subject,” “individual,” or “patient” can refer to a vertebrate organism, such as a mammal (e.g. human). "Subject" can also refer to a cell, a population of cells, a tissue, an organ, or an organism, preferably to human and constituents thereof.

[0065] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and / or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relievingthe disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0066] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and / or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration.

[0067] As used herein, “therapeutic” can refer to treating, healing, and / or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect.EXAMPLE SYSTEM

[0068] FIG. 1A depicts a cross-section of an example organ-on-chip system 100 according to the present disclosure. FIGS. 1B-1C depicts the construction of the example organ-on-chip system 100 without cells for clarity, with the upper layer features shown in lines 101a and the lower layer features shown in lines 101b. In the example shown in FIGS. 1A-1C, the organ- on-chip system 100 includes a chip substrate comprising cell scaffolds 102 (shown as 102a, 102b) and compartments for containing and supporting cell growth. In some embodiments, the chip substrate 102 is a polymer, e.g., poly dimethylsiloxane (PDMS) substrate for housing the scaffolds, cells and / or tissue. In some embodiments, the cell scaffold 102 (e.g., 102a, 102b) can include a polymer and / or hydrogel as a matrix for cell culture, cell growth, cell adhesion, etc., for example, polydimethylsiloxane, polyethylene, polyethylene glycol, polymethyl methacrylate, polyethylene terephthalate, polyester, polystyrene, polycarbonate, polytetrafluoroethylene, or other suitable polymers or hydrogels. The cell scaffolds 102 or chip substrate can include glass or quartz. In yet other embodiments, the chip substrate can include any combination of materials described herein.

[0069] In the example shown in Fig. 1 A, the chip substrate (e.g., PDMS substrate) defines (i) a first compartment comprising a first cell scaffold 102a (shown having “Vascularized interstitium” 112a), (ii) a second compartment comprising a second cell scaffold 102b, and (iii) a third compartment formed between the first compartment and the second compartment as a connection region.

[0070] A compartment refers to a region, sparce, chamber, channel for housing functionalized components of the organ-on-chip system, including cells, tissues, and / orscaffolds. Scaffolds are engineered material matrix for cell culture, cell growth, cell adhesion, cell placement.

[0071] In Fig. 1 A, the first compartment is defined by a first sub-compartments (shown as “lower layer”) having a first cell scaffold 102a and by a second sub-compartments (shown as “upper layer””) for cells to be disposed over the first cell scaffold 102 across a membrane. The first cell scaffold includes first cells, e.g., homing endothelial cells, fibroblast, interstitial macrophages, DCs, or a combination thereof)

[0072] The second cells in a second sub-compartment formed over the first cells across the membrane may include endothelial cell, e.g., small airway epithelium, alveolar epithelium, bronchial epithelium, healthy and diseased epithelial cells, or others described herein.

[0073] The second compartment includes a second cell scaffold 102b with third cells. The second cell scaffold 102b may be PEG-based hydrogel (e.g., Peg-4-Malemide functionalized with fibronectin and collagen-mimicking peptides and crosslinked with a non-degradable crosslinker (DTT) and a degradable peptide crosslinker). The third cells may include immune response-invoking cells, B cells, T cells, peripheral blood mononuclear cells (PBMCs), etc.

[0074] The third compartment formed between the first compartment and the second compartment as a connection region can include cells or tissues, e.g., Lymphatic endothelial cells.

[0075] In Fig. 1A, the cell scaffold 102a may be formed in a space defined by the chip substrate having an upper layer 103a and a lower layer 103b. In other embodiments, the chip substrate may be only one layer. In yet other embodiments, the chip substrate may include more than two layers (e.g., three layers, four layers, five layers, etc.). The cell scaffold 102a (e.g., hydrogel, among others described herein) is defined in the first compartment and may span the upper layer 103a, the lower layer 103b, or portions thereof. The second compartment 108 in the lower layer 103b formed between the first compartment and the second compartment 106, each of which are recessed into the chip substrate 102 (e.g., “carved out” of the chip substrate 102). The chip substrate also defines a fourth compartment 110 in the lower layer 103b, which is adjacent to the first compartment. In other embodiments, the chip substrate may not include a fourth compartment. Each of the first compartment, second compartment, third compartment, and optional fourth compartment may be formed by photolithography using one or more patterning masks.

[0076] In some embodiments, the first compartment houses the cell scaffold corresponding to the lung region, which can simulate lung function. The first compartment is split into an upper region 112b and a lower region 112a, which are separated by a permeable membrane114. This is shown most clearly in FIG. ID. In some embodiments, the permeable membrane can include a polyester track-etched (PETE) membrane, a polycarbonate track-etched membrane, or other suitable permeable membranes. The upper region 112b includes lung cells and / or lung tissue, and the lower region 112b includes vascularized and perfusable networks underlying and connecting the lung cells / tissue. Together, the lung cells / tissue and the vascularized and perfusable networks form the lung region 116 that can simulate lung function. In some specific embodiments, the lung cells / tissue and the vascularized and perfusable networks can define a lung interstitium (i.e., extravascular and extracellular space). It is considered that, in some embodiments, the first compartment may not include an upper region and a lower region, and the lung region may not be split into a lung cell / tissue layer and a vascular layer. In some such embodiments, the lung region may or may not be vascularized.

[0077] An air-liquid interface can be formed across at least a portion of the lung region to simulate an in vivo environment. At least a portion of liquid (i.e., media, water, etc.) can be removed from the apical side (i.e., the top side opposite the vascularized and perfusable networks) of the lung cells / tissue in the upper region 112a, thereby forming the air-liquid interface. In some embodiments, an air-liquid interface can be formed across at least about 20% (e.g., at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, about 100%) of the total area of the apical surface of the lung region. In some embodiments, an air-liquid interface can be formed across up to about 100% (e.g., up to about 95%, up to about 90%, up to about 85%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, up to about 60%, up to about 55%, up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20%) of the total area of the apical surface of the lung region.

[0078] It is considered that an air-liquid interface can be formed across any percentage of the total area of the apical surface of the lung region, ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, an air-liquid interface can be formed across from about 20% to about 100% (e.g., from about 25% to about 95%, from about 30% to about 90%, from about 35% to about 85%, from about 40% to about 80%, from about 45% to about 75%, from about 50% to about 70%, from about 55% to about 65%, from about 20% to about 60%, from about 25% to about 55%, from about 30% to about 50%, from about 35% to about 45%, from about 60% to about 100%,from about 65% to about 95%, from about 70% to about 90%, from about 75% to about 85%) of the total area of the apical surface of the lung region.

[0079] In some embodiments, the lung region can include endothelial cells, fibroblasts, airway macrophages, interstitial macrophages, interstitial dendritic cells, and / or peripheral mononuclear cells. In some embodiments, the lung region can include all healthy cells. In other embodiments, the lung region can include, at least in part, cancerous or abnormal cells. In some embodiments, the lung region can simulate lung interstitium function. For example, in some specific embodiments, the lung region can simulate in vivo communication between the lung interstitium and the lymphatic system.

[0080] To promote and support vascularization in the lower region 112a, the fourth compartment 110 houses a fibroblast region 117, including fibroblasts encapsulated in fibrinogen gel. These fibroblasts provide angiogenic factors to the vascularized and perfusable networks in the lower region 112a via media channels 118a, 118b disposed on either side of the first compartment.

[0081] The second compartment 106 houses a lymphoid region 120, which can stimulate lymphatic function. The lymphoid region 118 includes immune response invoking cells such that, when exposed to a stimulus (e.g., an infection agent and / or a treatment protocol), the lymphoid region 120 can invoke an immune response. In some embodiments, the lymphoid region can include lymphatic endothelial cells, tonsil cells, dendritic cells, and / or stromal cells. In some embodiments, the lymphoid region can include all healthy cells. In other embodiments, the lymphoid region can include, at least in part, cancerous or abnormal cells.

[0082] In order to facilitate communication (e.g., transport of cytokines, antigens, antibodies, etc.) between the lung region and the lymphoid region, the third compartment 108 houses a connection region 122 formed between the lung region 116 and the lymphoid region 120. In some embodiments, the connection region can be functionalized. For example, in some such embodiments, the connection region can include endothelial cells (e.g., lymphatic endothelial cells). In some embodiments, the connection region can include all healthy cells. In other embodiments, the connection region can include, at least in part, cancerous or abnormal cells. Additionally or alternatively, in other such embodiments, the connection region should be at least partially coated with proteins and / or small molecules, such as laminin, fibronectin, collagen, or integrins.

[0083] In some embodiments, the cells in each compartment may be seeded directly on the chip substrate. In other embodiments, the cells in any or all compartments may be seeded on a cell scaffold within the compartment. For example, in some such embodiments, the cellscaffold can include a hydrogel, for example, fibrinogen, collagen, polyethylene glycol, or other suitable hydrogels or combinations thereof.

[0084] In some embodiments, the cell scaffold (e.g., 102a) comprises a lung compartment hydrogel comprising fibrin / collagen hydrogel (e.g., in ratio 9:1) (e.g., having homing endothelial cells, fibroblast, interstitial macrophages, DCs, or a combination thereof).

[0085] In some embodiments, the cell scaffold (e.g., 102b) as a lymphoid compartment hydrogel comprises a PEG based hydrogel (e.g., Peg-4-Malemide functionalized with fibronectin and collagen-mimicking peptides and crosslinked with a non-degradable crosslinker (DTT) and a degradable peptide crosslinker).

[0086] The organ-on-chip system 100 is constructed to permit interstitial flow between the lung region 116 and the lymphoid region 120, thereby achieving at least partial immune competency. The chip substrate 102 includes a media input 124 which directs fresh media from the first compartment to the second compartment 106 via the third compartment 108. As the media flows through the system, it can pick up stimuli (e.g., cytokines) generated by the lung region 116 can be flowed to the lymphoid region 120, which can subsequently invoke an immune response (e.g., generate antibodies). In the organ-on-chip system 100, the media input 124 directs media through the lower region 112a of the first compartment. It is considered that, in other embodiments, particularly when the first compartment does not include an upper region and a lower region, the media input can direct media through any region of the first compartment. Media is pulled out through the media outlet 126 and can subsequently be used for testing or further applications.

[0087] In some embodiments, interstitial flow can be reversed to flow from the second compartment 106 to the first compartment via the third compartment to confer the immune response (e.g., antibodies) to the lung region. In some such embodiments, the direction of interstitial flow may be reversed any number of times, including periodically.

[0088] FIGS. 2A-2D depict example dimensions of each of the features of the upper layer 103a and the lower layer 103b of the organ-on-chip system 100. It is understood that these dimensions are merely examples and may be varied as necessary as can be determined by one of skill in the art.

[0089] In a specific example, the system can be configured as an integrated lung- lymphatics-lymphoid on-chip. In specific examples, the integrated system can include a vascularized lung connected with lymphoid via lymphatics on a single chip. The example system can be well-compartmentalized for the culture of multiple tissue niches and can allow for modulating the type of cells and duration of cell culture. The lung compartment can holdprimary differentiated airway epithelium at an air-liquid interface underlined by ECM-rich vascularized interstitium with tissue-resident stromal and immune cells. The lung compartment can connect to the lymphoid compartment via the primary human lymphatic endothelium-lined channel. The lymphoid compartment can contain functional, active, and viable immune cells in a synthetic hydrogel-based organoid.EXAMPLE METHODS

[0090] FIG. 3 shows an example method 300 of assessing stimuli on lung tissue using any of the disclosed organ-on-chip systems. First, an organ-on-chip system (e.g., the example organ-on-chip system 100) is provided 302. Next, interstitial media flow is established 304 from the lung region to the lymphoid region via the connection region. Next, a stimulus is introduced 306 to at least one region of the organ-on-chip system (e.g., the lung region, the lymphoid region, or the connection region). Finally, a response to the stimulus (e.g., presence or concentration of cytokines, antibodies, etc.) is measured 308.

[0091] As discussed above, the interstitial flow of media is established by feeding fresh media through the media input in the cell scaffold and / or chip substrate. In some embodiments, interstitial flow can be maintained for at least a portion of the method, particularly at least a portion of measuring the response. In other embodiments, interstitial flow can be maintained for the entire duration of the method, particularly the entire duration of measuring the response. In yet other embodiments, interstitial flow can be stopped before measuring the response. Additionally or alternatively, in some embodiments, interstitial flow can be reversed once or multiple times throughout the duration of the method as discussed above.

[0092] In some embodiments, the stimulus can be an infectious agent. For example, in some such embodiments, the infectious agent can be a virus, a bacterium, a fungus, or another infectious pathogen. In some embodiments, the virus can be an influenza virus, a coronavirus, RSV, or another virus of embodiments. In other embodiments, the stimulus can be a treatment protocol. For example, in some such embodiments, the treatment protocol can include the administration of a therapeutic agent, ablation, resection, exposure to radiation, and / or a vaccination. In some embodiments, the method can be used to screen treatment protocols.

[0093] In some embodiments, the method can include introducing multiple stimuli to the organ-on-chip system. For example, in some such embodiments, the method can include introducing a treatment protocol before and / or after introducing an infectious agent. In some specific embodiments, the method can include introducing a vaccination, subsequently introducing an infectious agent, and, optionally, subsequently introducing a treatment for theinfection agent. In some such embodiments, each stimulus can be introduced in the same or different regions of the organ-on-chip system.

[0094] In some embodiments, the response can be measured over a period of time (e.g., seconds, minutes, hours, days, weeks, months) and / or in different locations within the organ- on-chip system.EXAMPLESExample 1

[0095] The multichannel and multicompartment microfluidic device is developed to hold an organ-like cellular structure and immune-responsive invoking cells performing in vivo-like functions, respectively. An exemplary system is shown in FIGS. 4A-4E. As shown in FIGS. 4A-4B, the design consists of a 5-channel pattern connected with a circular compartment via a straight channel. Two outer channels in the five-channel compartment contain primary lung fibroblast encapsulated in fibrinogen gel, which are crucial for providing angiogenic factors to support the formation of vascular networks in the central channel, as shown in FIG. 4C. Adjoining channels hold media for supporting the growth of the vascular network and maintaining immune cells and stromal cells in the central channel. The central channel consists of the endothelial cells and stromal cells for vasculature formation and immune cells in fibrin / collagen-based hydrogel mimicking pulmonary interstitial composition. The central channel is covered with a PETE membrane and an air pocket that homes human airway epithelium and allows for maintaining air liquid interface, promoting differentiation. Five channel design, coupled with the airway compartments, makes the lung of the system, which is connected to a circular compartment that homes lymphoid organoids. Lymphoid organoid consists of tonsil-derived immune cells along with follicular dendritic cells and other stromal cells, as shown in FIG. 4D. Both compartments are connected via a lymphatic endothelium- lined channel, which mimics the lymphatic connection between lung and lymph node. To achieve immune competency and the effects of innate and adaptive immune responses, multiple immune cells are incorporated in different layers and compartments, which is summarized in FIG. 4E

[0096] This multi-compartment and multilayer design allows control over the incorporation of type of cells and modulates the duration of the culture of each compartment. This design allows the independent and dependent study of each compartment, thus is very beneficial for studies that are aimed at understating inter-tissue or inter-organ cross-talk.

[0097] Lung on-chip interstitial space has been well characterized for this system, as shown in FIG. 5A The presence of stromal normal human lung fibroblasts is seen in outer channels,confirmed by actin staining, and the presence of a vascular network in the central channel, confirmed by CD31 staining, which is an endothelial surface marker. Endothelial cells and stromal cells secrete the extracellular matrix required for the formation of perfusable networks, as confirmed by COL IV staining. These networks form mature lumens that allow for the circulation of immune cells / blood cells through these networks (FIG. 5B). Vascular networks underlie mature epithelium, which forms a tight monolayer and expresses markers of differentiation such as beta-tubulin, shown in FIG. 5C.

[0098] The vascularized lung is connected to the lymphoid organoid compartment via the lymphatic endothelial-lined channel. Primary human skin-derived lymphatic endothelial cells, CD31+Gp38+ expressing the characteristic marker Prox-1 were cultured on the fibronectin- coated channel (FIG. 6A) to mimic the lymphatic vessel to the lymph node. Functional connectivity of the lung with lymphoid was demonstrated by flowing 3kDa-Dextran tracer molecules through the lung interstitium at near interstitial velocities. The presence of a significant signal of tracer molecule was detected in the lymphoid compartment over time, confirming functional connectivity between two compartments (FIG. 6B). Tonsil-derived cells were encapsulated in PEG-4MAL based organoid and cultured in the circular lymphoid compartment. FIG. 6C shows the presence of B cells surrounded by lymphatic endothelial cells mimicking lymph node-like organization. Further flow cytometry analysis in FIG. 6D confirms the functionality of organoids by showing the presence of viable CD 19+ B cells and its subsequent subsets CD38-CD27+ memory b cells (MBC), CD38+CD27- activated b cells (Pre-GC) and CD27+CD38+ germinal center B cells (GC) confirming the ongoing germinal center reaction in lymphoid compartment. Lung and lymphoid were cultured for 8 days successfully which allowed for vascularization and differentiation of epithelium in lung and initiation of germinal center reaction in lymphoid.Example 2

[0099] As mentioned in FIG. 4E, tissue-resident mimics macrophages, and dendritic cells were encapsulated in a central channel, and circulating immune cells obtained from RBC lysed donor-matched human blood were flown through the perfusable vasculature to impart the characteristic and responses of circulating immune cells.

[0100] Further to study the functionality of the system and the effect of pulmonary stimulants on the immune responses, virus infection was induced in the system. Live HINl / influenza A / PR8 strain was used to infect the ALI-exposed airway epithelium and cell phenotype in lung and lymphoid compartments were analyzed 96 hours post-infection.

[0101] Lung interstitium shows the presence of more interstitial macrophage phenotype as evident by increased expression of HLA-DR in CD206+ population, it indicates initiation of inflammatory pathways in lung interstitium. Further analysis is being done to confirm the cascade of immune responses in the lung interstitium. The lymphoid compartment was analyzed and it shows an increase in the percentage of B cells getting activated and entering germinal center reaction in infected devices as compared to uninfected controls. Interestingly, there is a reduction in memory B cells and the emergence of plasma B cell phenotype in infected devices. Indicating that virus exposure to already generated memory cells promoted plasma cell production.

[0102] The present evaluation demonstrates the successful integration of the lunglymphoid system and shows its utility in studying pulmonary stimulants such as viral infection. The system can also be utilized to study the effect of other pulmonary stimulants like mucosal adjuvants or vaccine candidates.

[0103] The exemplary device can provide immune competency and lung interstitium connected to lymphoid organoids and connection between two systems that are lined by lymphatic cells mimicking in vivo connection.

[0104] The exemplary device can have specific lung and Lymphoid integration.

[0105] The exemplary device can have immune competency in the lung and integration with the lymphoid system, e.g., that can be used to explore effects of influenza, inflection on the lymphoid system.Example 3

[0106] To fulfill the need for an immune-competent human lung-on-chip system aimed at understanding pulmonary infections / stimulants on innate and adaptive immune responses, a study developed a complete human lymphoid and lymphatics integrated immune competent lung on-chip system. The multichannel and multicompartment microfluidic device was developed to hold human lung-like cellular structure and human lymph node-like organoids performing in vivo-like immune functions respectively. Below is the detailed methodology for the development of the system.

[0107] Fabrication of Microfluidic Device. A two-compartment, two-layer, and five- channel device was fabricated. The device was designed using AutoCAD software.

[0108] Device fabrication assembly and plasma bonding of two layers '. Negatives of the two layers were separately designed and etched using soft lithography. A negative of the microfluidic device design was etched using a maskless aligner with photo-crosslinked SU-8 2150 resin on a silicon (Si) wafer. Device features of height 150pm for layer 1 and 1000 pmfor layer 2 were allowed to be developed. Negatives of the device design were used for the fabrication of polydimethylsiloxane (PDMS) device layers. PDMS was poured over SU-8 etched Si-wafers and was allowed to cure overnight at room temperature, followed by curing at 65°C for 30 minutes. PDMS layers were cut out from the Si-wafer. Both PDMS layers were bonded together with a polyester PETE membrane of 5pm pore size between the two layers. For the preparation of the membrane, PETE membranes were plasma treated and incubated with 5% solution of (3 -Aminopropyl)tri ethoxy silane, APTES for 10 minutes at 80°C, and excess APTES was drained. For the complete assembly of the device, both the PDMS layers (Layer 1 and Layer 2) were plasma treated, and the membrane was sandwiched between layer 1 and layer 2, with features facing the membrane. The completely assembled device was incubated at 80°C overnight and stored at 65°C until used.

[0109] To allow for the culturing of cells in devices, the PETE membrane in devices were coated with O.lmg / ml dopamine hydrochloride for one hour followed by O. lmg / ml rat tail collagen I for one hour at room temperature. The coated devices were dried overnight and sterilized by UV exposure before culturing cells.

[0110] An example device without cells was used to examine interstitial flow in the device. These results are shown in FIGS. 8A-8B.

[0111] Culturing cells in Devices'.

[0112] Lung compartment'. The lung compartment includes fibroblast, endothelial, epithelial, macrophages, dendritic cells and PBMCs. On Day 0, human fibroblasts were cultured in the outer channels in 5mg / ml human fibrinogen hydrogel at density of 3 million per ml. The central channel was loaded with a cell mixture containing with 6.5 million per ml human vein endothelial cells and 0.3 million per ml human fibroblasts in a hydrogel with a composition of 0.5mg / ml collagen and 4.5mg / ml fibrinogen-based hydrogel. Human PBMC derived macrophages and dendritic cells were also included at cell density of 0.3 million per ml respectively in the central channel mix. The devices were cultured for two days with endothelial growth medium (EGM) containing 50ng / ml VEGF / A (Vascular endothelial growth factor A). On Day 2, small airway epithelial cells were cultured on top of the PETE membrane at a cell density of 2 million per ml. The devices were cultured for another two days (until Day 4) with EGM media containing 50ng / ml VEGF / A and lOOng / ml Angiotensin I (Angl). On Day 4, airlifting was performed on the small airway epithelium, and the culture media was changed to a 1 : 1 mixture of Pneumocult-ALI media and EGM containing 50ng / ml VEGF / A and lOOng / ml Angl. The lung compartment was maintained in this media composition.

[0113] When complete perfusion of the devices was achieved, PBMCs were flowed in the media channels on the devices at a concentration of 1 million cells per ml in the presence of IL2.

[0114] Lymphoid compartment. Lymphoid organoids were made in the device in a separate compartment. 4-arm maleimide functionalized Polyethylene glycol (PEG-4-MAL) was crosslinked with dithiothreitol, and a degradable peptide crosslinker (-VPM-) was used to make hydrogel -based organoids. The 7.5% PEG-4-MAL hydrogel was functionalized with two peptides containing the -RGD domain and -GFORGER domain to allow for cell growth. Human tonsil cell suspension was obtained by mechanical disruption of the tonsil, followed by ficoll separation of lymphocytes. Lymphocytes obtained from tonsils were encapsulated in lOul hydrogels with 200,000 lymphocytes and 40,000 CD40L-expressing stromal cells and follicular dendritic cells in a 1 :1 ratio per gel. Hydrogels were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium) containing 10% FBS, IX NEAA, IX Sodium Pyruvate, IX Penicillin-Streptomycin, IX 2-mercaptoethanol. Media was supplemented with lOOng / ml human BAFF, 20ng / ml human IL4 for first 2 days, and lOng / ml human IL21 for the rest of the culture.

[0115] Lymphatics compartment. Lymphatic endothelial cells were cultured in the devices in the region connecting the lymphoid organoid with the lung compartment on Day 6 or Day 10 based on the duration of the culture. The surface was coated with human plasma fibronectin at lOOug / ml for 90 minutes at 37°C. Following coating with fibronectin, the surface was gently rinsed with PBS, ensuring it did not disturb the lung or lymphoid compartment. Lymphatic endothelial cells were added at a density of 5 million cells per ml in the connecting region. Lymphatic cells were allowed to adhere to the surface for two days to allow for the complete connection of the system.

[0116] Virus Infection in Devices'. To induce virus infection in devices, PR8 H1N1 strain was used. Viral infection was induced in the airway epithelium. The virus was added to the epithelium layer at 10 MOI (Multiplicity of Infection). The virus was allowed to adhere to the epithelium for two hours, and then excess media was removed from the epithelium. Epithelium and other open ports were sealed with adhesive polyester films. Interstitial flow was introduced in the central channel of the lung directed towards the lymphoid organoid. Infected devices were cultured post infection for either 4 days or 8 days. Media was collected separately from the lung compartment and the lymphoid compartment at the endpoint to analyze cytokines and antibodies produced using ELISA-based methods. Cells were also collected from the lung and lymphoid compartments for analysis of cell activation using flow cytometry.

[0117] Priming and Infection of Devices'. This device design allows for pre-infection priming of the lymphoid to mimic vaccination in humans. To achieve priming of the lymphoid organoid, heat-killed PR8 virus, along with adjuvant resquimoid, was added to the media of the lymphoid compartment for the first two days of culture. Post-priming, the lymphoid was cultured regularly as described above. Infection was induced in the airway epithelium after 12 days of culture with a live virus of same strain (PR8) to mimic infection of the lungs in humans. Infection was induced for either 4 days or 8 days. Media and cells were collected from the devices, and analysis was performed similarly as described above. These results are shown in FIGS. 9A-9B and FIGS. 10A-10C

[0118] Although the figures show a specific order of method steps, the order of the steps may differ from what is depicted. Also, two or more steps may be performed concurrently or with partial concurrence. Such variation will depend on the software and hardware systems chosen and on the designer choice. All such variations are within the scope of the disclosure. Likewise, software implementations could be accomplished with standard programming techniques with rule-based logic and other logic to accomplish the various connection steps, processing steps, comparison steps and decision steps.

[0119] It is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular implementations only and is not intended to be limiting.

Claims

What is claimed is:

1. An organ-on-chip system comprising: a chip substrate defining: a first compartment; a second compartment; a third compartment formed between the first compartment and the second compartment; and at least one input for directing media through the first compartment; wherein the first compartment houses a lung region that can simulate lung function and comprising lung cells or lung tissue; wherein the second compartment houses a lymphoid region that can simulate lymphatic function and comprising immune response invoking cells; wherein the third compartment houses a connection region formed between the lung region and the lymphoid region; and wherein, upon exposure to a stimulus, the lymphoid region invokes an immune response.

2. An organ-on-chip system comprising: a chip substrate defining: a first compartment comprising (i) a first cell scaffold comprising first cells in a first sub-compartment and (ii) second cells in a second sub-compartment formed over the first cells across a membrane; a second compartment comprising a second cell scaffold with third cells; a third compartment formed between the first compartment and the second compartment as a connection region; and at least one input for directing media through the first compartment; wherein the first cell scaffold with the first cells and the second cells, collectively forms a lung region and simulate lung function, wherein the second sub-compartments forms an epithelial / airway region forms an air-liquid interface, and wherein the first sub-compartment forms a perfusable, vascularized network of the first cells; wherein the second cell scaffold forms a lymphoid region and simulates immune function, the third cells of the second cell scaffold comprise immune response invoking cells;wherein the connection region defined by the third compartment is formed between the lung interstitium region and the lymphoid region; and wherein, upon exposure to a stimulus, the lymphoid region invokes an immune response.

3. The organ-on-chip system of any one of claims 1-2, wherein the first cell scaffold and the second cell scaffold each comprises a hydrogel.

4. The organ-on-chip system of any one of claims 1-2, wherein the first cell scaffold comprise a first hydrogel and the second cell scaffold comprises a second hydrogel, wherein the first hydrogel and the second hydrogel are different.

5. The organ-on-chip system of claim 4, wherein the first cell scaffold as lung compartment hydrogel comprises fibrin / collagen hydrogel.

6. The organ-on-chip system of claim 4, wherein the second cell scaffold as a lymphoid compartment hydrogel comprises a PEG based hydrogel.

7. The organ-on-chip system of any one of claims 1-6, wherein the first cells and / or second cells of the lung region comprises endothelial cells, fibroblasts, airway macrophages, interstitial macrophages, interstitial dendritic cells, peripheral mononuclear cells, or a combination thereof.

8. The organ-on-chip system of any one of claims 1-7, wherein the lung region comprises all healthy cells.

9. The organ-on-chip system of any one of claims 1-7, wherein the lung region comprises, at least in part, cancerous or abnormal cells.

10. The organ-on-chip system of any one of claims 1-9, wherein the lung region is configured to simulate lung interstitium and lung epithelium function.

11. The organ-on-chip system of any one of claims 1-10, wherein the second subcompartment of the first compartment form an upper region comprising the lung cells or lung tissue; and wherein the first sub-compartment of the first compartment form a lower region comprising the vascularized and perfusable networks underlying the lung cells or lung tissue.

12. The organ-on-chip system of claim 11, wherein the third compartment is formed between (i) the first sub-compartment of the first compartment and (ii) the second compartment.

13. The organ-on-chip system of claim 12, wherein the connection region is formed between (i) the vascularized and perfusable networks of the lung region of the first subcompartment and (ii) the lymphoid region.

14. The organ-on-chip system of any one of claims 11-13, wherein the at least one input directs media through the first sub-compartment of the first compartment.

15. The organ-on-chip system of any one of claims 1-14, wherein the lymphoid region comprises lymphatic endothelial cells, tonsil cells, dendritic cells, and / or stromal cells.

16. The organ-on-chip system of any one of claims 1-15, wherein the lymphoid region comprises all healthy cells.

17. The organ-on-chip system of any one of claims 1-15, wherein the lymphoid region comprises, at least in part, cancerous or abnormal cells.

18. The organ-on-chip system of any one of claims 1-17, wherein the connection region is functionalized.

19. The organ-on-chip system of claim 18, wherein the connection region comprises lymphatic endothelial cells.

20. The organ-on-chip system of claim 19, wherein the connection region comprises all healthy cells.

21. The organ-on-chip system of claim 19, wherein the connection region comprises, at least in part, cancerous or abnormal cells.

22. The organ-on-chip system of any one of claims 18-20, wherein the connection region is at least partially coated with proteins and / or small molecules.

23. The organ-on-chip system of claim 22, wherein the membrance as a permeable polyester track-etched (PETE) membrane.

24. The organ-on-chip system of any one of claims 1-23, wherein the chip substrate further comprises a fourth compartment housing fibroblasts.

25. A method of assessing stimuli on lung tissue, the method comprising: a) providing the organ-on-chip system of any one of claims 1-22; b) establishing interstitial flow of media from the lung region to the lymphoid region via the connection region; c) introducing a stimulus to at least one region of the organ-on-chip system; and d) measuring response to the stimulus.

26. The method of claim 25, wherein fresh media is input through the at least one input in the chip substrate.

27. The method of claim 24 or 25, wherein the interstitial flow of media is maintained for at least a portion of the duration of step d).

28. The method of claim 26, wherein the interstitial flow of media is maintained for the entire duration of step d).

29. The method of any one of claims 25-28, wherein the stimulus is an infectious agent and / or a treatment protocol.

30. The method of claim 29, wherein the infectious agent is a bacterium or a virus.

31. The method of any one of claims 25-30, wherein the treatment protocol is administration of a therapeutic agent, ablation, resection, exposure to radiation, or a vaccination.

32. The method of any one of claims 28-31, wherein the organ-on-chip system is exposed to the treatment protocol before and / or after exposure to the infectious agent.

33. The method of any one of claims 25-32, wherein step d) occurs over a period of time and / or in different locations within the organ-on-chip system.

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