Organoids related to immunotherapy and methods of preparing and using the same

Abstract

To provide an in vitro model in which immunotherapy can be adequately tested generally or for specific patients.SOLUTION: Provided is an in vitro cellular construct useful as a tumor model, the construct comprising living tumor cells and at least one type of living immune cell.SELECTED DRAWING: Figure 13

Description

[Technical Field]

[0001] [Priority Statement] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 625,628, filed February 2, 2018, the entire contents of which are incorporated herein by reference.

[0002] [Government support] This invention was made with government support under Grant Nos. 1U54TR001362-01, 5UL1TR001420-03, and 5P30CA012197-43 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] [Field] The present invention relates generally to organoids, including tumor organoids and organoids containing immune cells, and methods for preparing and using the same. [Background technology]

[0004] Immunotherapy has emerged as an attractive anticancer treatment due to its ability to efficiently target tumors, but there are no in vitro models in which such treatments can be adequately tested generally or for specific patients.

[0005] Initially, animal models seemed attractive because they could provide insight into the complexity of in vivo tumor physiology. However, in addition to the infrastructure requirements and ethical issues associated with animal use, these models have limited predictive power in humans. Patient-derived tumor xenograft (PDX) technology has recently been introduced to predict how a patient's tumor will respond to drugs. Small fragments of the patient's tumor are implanted into immune-deficient mice. Once the tumor fragments have grown to a sufficient size, the tumor is removed, divided into several pieces, and reimplanted into new mice. The main advantages of PDX technology are: 1) the patient's cancer cells are expanded in vivo, recreating components of the tumor microenvironment that contribute significantly to cancer pathobiology, and 2) the ability to test drugs on the patient's growing tumor prior to clinical treatment. However, PDX technology cannot grow tumors smaller than a certain size, and most successful PDXs arise from highly invasive tumors, making this technology applicable to some, but not all, cancer patients. Furthermore, the tumor microenvironment (TME) for PDXs is of murine origin and therefore lacks human-specific stromal elements.

[0006] Cancer research has been limited by the inability to accurately model tumor progression and signaling mechanisms in a controlled in vitro environment. In addition to the issues with PDX models mentioned above, animal models allow for limited manipulation and testing and are not necessarily predictive of human outcomes. Traditional in vitro 2D cultures cannot recapitulate the 3D in vivo microenvironment. Pharmacokinetics vary dramatically, effective doses in 2D are often ineffective in patients, and cell-cell / cell-matrix interactions are imprecise. Tissue culture dishes differ from in vivo tumors in terms of tissue distribution, stiffness, and 2D vs. 3D structure. Furthermore, 2D cultures can exert selective pressure on cells, altering their molecular and phenotypic characteristics. Summary of the Invention [Means for solving the problem]

[0007] A first aspect of the present invention relates to in vitro cellular constructs (or "organoids") that comprise a plurality of immune cells and are useful as models of the immune system (e.g., useful for assessing immune activity and / or useful for modulating the immune system and / or useful for screening one or more cancer treatments and / or immunotherapies).

[0008] A further aspect of the present invention relates to in vitro cellular constructs (or "organoids") useful as tumor models (e.g., useful for assessing immune activity and / or useful for modulating the immune system and / or useful for screening one or more cancer treatments and / or immunotherapies) comprising live tumor cells and at least one type of immune cell.

[0009] Another aspect of the present invention relates to a method for screening a compound of interest for immune activity in vitro and / or modulating the immune system, comprising the steps of: providing at least one organoid comprising a plurality of cells (e.g., the at least one organoid may be a liver organoid, a cardiac organoid, a tumor organoid, an immune system organoid, etc.); contacting the compound of interest with the at least one organoid in vitro; and then determining the immune response of the at least one organoid in response to contacting the compound of interest with the at least one organoid in vitro (e.g., compared to the immune response / activity of the organoid before and / or in the absence of contact with the compound of interest).

[0010] A further aspect of the present invention relates to a method for screening a compound of interest for anti-tumor activity in vitro, comprising the steps of providing at least one construct comprising live tumor cells and at least one type of immune cell; contacting the compound of interest with the construct in vitro; and then, in response to contacting the compound of interest with the construct in vitro, determining the proliferation of the live tumor cells (e.g., compared to tumor cells of at least one similar construct that have not been contacted with the compound of interest, and / or compared to live benign cells within the construct or at least one similar construct), wherein a reduction in the proliferation of the live tumor cells (e.g., lack of tumor cell proliferation, death of tumor cells, reduction in invasion by tumor cells, etc.) indicates anti-tumor activity of the compound of interest.

[0011] Another aspect of the present invention relates to a method for screening compounds of interest in vitro for immune activity, modulating the immune system, anti-metastatic activity, and / or anti-tumor activity, comprising the steps of providing a device comprising immune cell organoids and live tumor cell organoids; contacting the immune cell organoids and live tumor cell organoids with a growth medium; contacting a compound of interest with the immune cell organoids and / or live tumor cell organoids (e.g., by adding the compound to the growth medium); and, in response to contacting the compound of interest with the immune cell organoids and / or live tumor cell organoids, inhibiting the proliferation of live tumor cells (e.g., by increasing the proliferation of live tumor cells contacted with the compound of interest). and / or compared to live benign cells within the organoids or at least one similar live tumor cell organoid (wherein a reduction in the proliferation of live tumor cells (e.g., lack of tumor cell proliferation, tumor cell death, reduction in invasion by tumor cells, etc.) indicates anti-tumor activity of the compound of interest), and / or determining the immune response of the immune cell organoids and / or live tumor cell organoids (e.g., compared to the immune response / activity of the immune cell organoids and / or live tumor cell organoids before and / or in the absence of contact with the compound of interest).

[0012] A further aspect of the present invention relates to a method of activating immune cells ex vivo, the method comprising the steps of contacting immune cells (e.g., lymphocytes) with live tumor cell organoids comprising a plurality of live tumor cells to provide activated immune cells (e.g., via expression of adaptive immune mechanisms); isolating the activated immune cells from the live tumor cell organoids to provide isolated activated immune cells; and expanding the isolated activated immune cells to provide a population of activated immune cells.

[0013] It should be noted that aspects of the invention described with respect to one embodiment may be incorporated into a different embodiment even if not specifically described therein. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination. Applicant reserves the right to modify any originally filed claims and / or file any new claims as appropriate, including the right to amend any originally filed claims to depend on and / or incorporate any feature of any other claim(s), even if not originally claimed in that manner. These and other objects and / or aspects of the invention are described in detail in the specification set forth below. Additional features, advantages, and details of the invention will be apparent to those skilled in the art from a reading of the following drawings and detailed description of the preferred embodiments, such description being merely illustrative of the invention. [Brief explanation of the drawings]

[0014] [Figures 1A-1C]Immunotherapy efficacy is shown for patient tumor organoids obtained from the same patient, with or without immune cells derived from matched lymph nodes. Figure 1A shows colorectal tumor mitochondrial metabolism measured in media alone (control), media + pembrolizumab, and media + nivolumab. Increased metabolism was observed only in the presence of immune cells along with the appropriate drug (in this case, nivolumab), indicating T cell activation. Figure 1B shows survival / death imaging of appendix tumor organoids with and without node cells. While minimal differences were observed without the drug, the PD-1 inhibitors nivolumab and pembrolizumab are effective only when immune cell (lymphoid) populations are incorporated. Figure 1C shows survival / death imaging of tumor organoids derived from a melanoma patient who demonstrated no response to checkpoint inhibitor immunotherapy ("non-responder"). Patient tumor organoids demonstrate recapitulation of the non-responder phenotype. Alternative treatment with dabrafenic / trametinib (right-most panel) showed an increased death staining response. [Figure 2] 1 shows an exemplary two-organoid system according to an embodiment of the present invention. [Figure 3A-3C] Schematic diagrams of a microfluidic device according to an embodiment of the present invention. Figure 3A shows a schematic of the assembly of the layers of the microfluidic device. Figure 3B illustrates the in situ organoid patterning technique: a microfluidic chamber is filled with a hydrogel mixture containing HA hydrogel, a photoinitiator, and patient-derived tumor cells, and then irradiated with UV light through a photomask. The exposed precursors are crosslinked into the hydrogel, trapping the cells within the region, and the uncrosslinked gel is washed out of the chamber with clean PBS. Finally, the PBS is replaced with DMEM for incubation. Figure 3C shows an overview of the overall measurement setup, featuring a low-volume, closed-loop fluidic circuit for each organoid, facilitated by a computer-controlled peristaltic pump. [Figures 4A-4C]1 is a schematic representation of a chamber containing tumor cells and immune cells provided in separate but adjacent zones, allowing the cells to migrate towards each other for interaction, according to an embodiment of the invention. [Figure 5] 1 is a graph showing the response of melanoma organoids and melanoma and lymph node organoids to various checkpoint inhibitors. [Figure 6] 1 shows a diagram of an exemplary process according to an embodiment of the present invention. [Figure 7] 1 illustrates an exemplary process for isolating cells from solid tissue, according to an embodiment of the present invention. [Figure 8] 1 shows an exemplary process for preparing organoids, according to an embodiment of the present invention. [Figure 9] 1 shows an exemplary process for preparing white blood cells for addition to culture media, according to an embodiment of the present invention. [Figure 10] 1 illustrates an exemplary process for drug screening and analysis, according to an embodiment of the present invention. [Figures 11A-11D]11A shows a schematic diagram of a tumor-on-a-chip (TOC) device that can be used for injecting T cells and priming them to an activated state, according to an embodiment of the present invention. Figure 11A shows that the parallel channels of an existing TOC system can be daisy-chained together to increase the number of patient tumor organoids (PTOs) per circulator path. Figure 11B shows a schematic diagram illustrating that many PTOs can be patterned within a single chamber of the device, which can dramatically increase the number of PTOs. Figure 11C shows an exemplary schematic diagram illustrating that after T cell infusion and / or recirculation, circulating T cells can be removed and transferred to a matched, unexposed PTO TOC to test, for example, activation levels and / or tumor killing, for example, to induce priming / activation in response to tumor antigen recognition. Non-limiting examples of device channel structures include, but are not limited to, daisy-chains, e.g., a single larger chamber. These primed and / or activated T cells can then be administered to a patient. Figure 1 ID shows another exemplary scheme illustrating that after T cell infusion and / or recirculation, e.g., to induce priming / activation in response to tumor antigen recognition, circulating T cells can be removed and transferred to a corresponding unexposed PTO TOC to test, e.g., activation levels and / or tumor killing. These primed and / or activated T cells can be administered to a patient. [Figure 11E] Figure 1 ID shows live / dead staining of PTO at different stages of the process shown in Figure 1 ID. Staining is shown before treatment (top image), after priming (middle image), and after transfer to previously unexposed PTO, which induces tumor cell killing (bottom image). [Figure 12] 1 is a schematic illustrating the incorporation of immune components into patient tumor organoids, according to some embodiments of the present invention. [Figure 13] 1 is an overview showing methods for incorporating immune components into patient tumor organoids and using them for personalized medicine, according to some embodiments of the present invention. [Figures 14A-14C]14A and 14B show the viability of tumor organoids (i.e., containing only tumor cells) and tumor / immune organoids (i.e., containing tumor cells and lymph node cells). Each set of organoids was prepared using cells obtained from one melanoma patient (Figures 14A, 14B, and 14C show individualized results for each of three different melanoma patients). The results show that tumor immune organoids immune-enhanced with lymph node cells were able to demonstrate tumor cell killing under immune checkpoint inhibitor treatment, while tumor organoids without immune enhancement did not respond. [Figures 15A-15B] 15A and 15B show the viability of tumor organoids (i.e., containing only tumor cells) and tumor / immune organoids (i.e., containing immune cells and tumor cells derived from the white blood cell fraction of patient blood). Each set of organoids was prepared using cells obtained from one melanoma patient (Figures 15A and 15B show individualized results for two different melanoma patients). The results show that tumor immune organoids immune-enhanced with cells derived from the white blood cell fraction of blood were able to demonstrate tumor cell killing under immune checkpoint inhibitor treatment, while tumor organoids without immune enhancement did not respond. [Figures 16A-16B] 16A and 16B are graphs showing the viability of tumor organoids (i.e., containing only tumor cells) and tumor / immune organoids (i.e., containing immune cells and tumor cells derived from the white blood cell fraction of patient blood). The organoids in Figure 16A were prepared using cells obtained from a patient with angiosarcoma. The organoids in Figure 16B were prepared using cells obtained from a patient with DFSP. The results show that tumor immune organoids immunopotentiated with cells derived from the white blood cell fraction of blood were able to demonstrate tumor cell killing under immune checkpoint inhibitor treatment, whereas tumor organoids without immunopotentiation did not respond. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0016] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0017] Unless otherwise specified, all terms (including 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. Terms, such as those defined in commonly used dictionaries, should be interpreted to have a meaning consistent with their meaning in the context of this application and related art, and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflicts in terminology, the present specification controls.

[0018] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0019] Unless the context dictates otherwise, it is expressly intended that the various features of the invention described herein may be used in any combination. Moreover, the invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. By way of example, if the specification states that a composite comprises components A, B, and C, it is expressly intended that any of A, B, or C, or any combination thereof, may be omitted and rejected.

[0020] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) is understood to encompass the recited materials or steps "and which do not materially affect the basic and novel characteristic(s)" of the claimed invention. See Inre Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976) (emphasis in original); see also MPEP §2111.03. Thus, as used herein, the term "consisting essentially of" should not be construed as equivalent to "comprising."

[0021] The term "about" as used herein when referring to a measurable value, such as an amount or concentration, means to encompass a variation of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value, as well as the particular value. For example, "about X," where X is a measurable value, means to encompass X and a variation of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. Ranges provided herein for measurable values ​​may include any other ranges and / or individual values ​​therein.

[0022] When an element is referred to as "on," "attached," "connected," "coupled with," or "in contact with" another element, it will be understood that it may be directly on, directly attached, directly connected, directly coupled, or directly in contact with the other element, or that there may be intervening elements. In contrast, when an element is referred to as, for example, "directly on," "directly attached," "directly connected," "directly coupled with," or "in direct contact with," another element, there are no intervening elements. It will also be understood by those skilled in the art that references to structures or features located "adjacent" to another feature may have portions overlapping or underlying the adjacent feature.

[0023] Spatially relative terms such as "under," "below," "lower," "over," and "upper" may be used herein for ease of description to describe the relationship of one element or feature to another element(s) or feature(s) shown in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device during use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, an element described as "under" or "beneath" another element or feature would be oriented "over" that other element or feature. Thus, the exemplary term "under" can encompass both an orientation of "over" and "under." The device can be otherwise oriented (rotated 90 degrees or at other orientations), and the spatially relative descriptors used herein will be interpreted accordingly. Similarly, unless expressly stated otherwise, the terms "upwardly," "downwardly," "vertical," "horizontal," etc. are used herein for descriptive purposes only.

[0024] While the terms "first," "second," etc. may be used herein to describe various elements, it will be understood that these elements should not be limited by these terms. These terms are used only to distinguish one element from another. Thus, a "first" element discussed below can also be referred to as a "second" element without departing from the teachings of the present invention. The order of operations (or steps) is not limited to the order shown in the claims or drawings unless expressly stated otherwise.

[0025] As used herein, the terms "increase," "increases," "increased," "increasing," and similar terms refer to an increase in a specified parameter of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more.

[0026] As used herein, the terms "reduce," "reduces," "reduced," "reduction," "inhibit," and similar terms refer to at least about a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% reduction in a specified parameter.

[0027] As used herein, "cells" and "cell" generally refer to animal cells, particularly mammalian and primate cells, examples of which include, but are not limited to, human, dog, cat, rabbit, monkey, chimpanzee, cow, pig, and goat. Cells may be differentiated, at least in part, into specific cell or tissue types, such as liver, intestine, pancreas, lymph node, smooth muscle, skeletal muscle, central nervous system, peripheral nervous system, skin, immune system, etc. Some cells may be cancer cells, as discussed further below, in which case they may express (naturally or recombinantly) a detectable compound, also discussed further below.

[0028] The terms "three-dimensional tissue construct" and "organoid" are used interchangeably herein and refer to a composition of living cells, typically in a carrier medium, arranged in a three-dimensional or multi-layer structure (as opposed to a monolayer), as used herein. Organoids are artificial three-dimensional constructs created in vitro to mimic or resemble the function and / or histological structure of organs, tissues, or portions thereof. Suitable carrier media include hydrogels, such as cross-linked hydrogels described below. Further exemplary hydrogels include, but are not limited to, those described in PCT / US2015 / 055699, PCT / US2017 / 058531, and U.S. Application No. 16 / 156,535, filed October 10, 2018, the contents of each of which are incorporated herein by reference in their entirety. Organoids may contain one or more (e.g., one, two, three, four, or more) differentiated cell types(s) depending on the particular tissue and / or organ being modeled or mimicked. Some organoids may comprise cancer cells, which will be further discussed below.When organoids comprise cancer cells, they may comprise tissue cells, and / or comprise cell-free tissue mimics, such as extracellular matrix (or the protein and / or polymer derived therefrom), hyaluronic acid, gelatin, collagen, alginic acid, etc. (including their combinations).Therefore, in some embodiments, cells are mixed with extracellular matrix or crosslinked matrix to form organoid, and in other embodiments, cell aggregates, such as spheroids and / or organoids, can be pre-formed, and then be combined with the composition of the present invention and / or extracellular matrix.

[0029] In some embodiments, organoid can be present in and / or formed in the hydrogel that comprises thiolated hyaluronic acid (also referred to herein as thiol-modified hyaluronic acid), methacrylated collagen (also referred to herein as methacrylate-modified collagen) and water.One or more (for example, 1, 2, 3, 4, 5, 6, 7 or more) additional components can be present in the hydrogel. For example, in some embodiments, the organoids may be present within a hydrogel comprising methacrylated gelatin (GelMa), heparin sulfate, chondroitin sulfate, alginic acid sodium salt, unmodified gelatin, elastin, non-thiolated hyaluronic acid, non-methacrylated collagen (e.g., type I, type II, type III, and / or type IV collagen), one or more components for modifying the elastic modulus of the composition, one or more components for modifying the cell adhesion profile, one or more components for tissue-specific biochemical modifications, and / or one or more small molecules (e.g., small molecules that may have additional crosslinking capabilities and / or small molecules that can provide hydrogen bonding and / or non-covalent complexes). In some embodiments, the organoid of the present invention can be present in and / or formed in a hydrogel comprising a photoinitiator, about 1% w / v thiolated hyaluronic acid, and about 4mg / mL methacrylated collagen, wherein thiolated hyaluronic acid and methacrylated collagen can be mixed in a volume ratio of 1:3 (thiolated hyaluronic acid: methacrylated collagen), and can be crosslinked through photopolymerization (for example, using UV light).In some embodiments, thiolated hyaluronic acid and methacrylated collagen can be mixed in a volume ratio of about 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5 (thiolated hyaluronic acid: methacrylated collagen). The volume of hydrogel in which the organoids reside and / or are deposited may range from about 1, 5, 10, 15, or 20 μL to about 25, 30, 35, 40, 45, 50, 55, or 60 μL.In some embodiments, the volume of hydrogel in which the organoids reside and / or are deposited may be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 μL.

[0030] In some embodiments, the organoid and / or hydrogel of the present invention can be present in one reservoir and / or multiple reservoirs (for example, the well of a well plate).Reservoir(s) can be any suitable reservoir or container that can hold organoid and / or hydrogel.In some embodiments, reservoir is the well of a well plate, for example, but not limited to, the well in a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate.

[0031] In some embodiments, organoids can be present in a hydrogel that includes protein (for example, adhesive protein) and / or proteoglycan (can be modified protein and / or modified proteoglycan).In some embodiments, protein and / or proteoglycan can be modified by one or more functional groups, for example, by maleimide, and can be bound and / or crosslinked to thiolated hyaluronic acid, non-thiolated hyaluronic acid, methacrylated collagen, and / or non-methacrylated collagen.In some embodiments, organoids can be present in a hydrogel that includes fibronectin, heparin, and / or laminin (can be modified fibronectin, modified heparin, and / or modified laminin (for example, modified by maleimide)), or other cell adhesion protein(s) and / or cell adhesion protein peptide derivative(s).

[0032] One or more growth factors may be present in the hydrogel. In some embodiments, the hydrogel comprises one or more growth factors linked and / or bound by heparin pendant chains. The one or more growth factors may be present in the hydrogel and / or added to the hydrogel and / or may be suitable for the specific cells and / or the specific tissue substitute and / or organoid to be produced. In some embodiments, growth factors and / or other growth-promoting proteins may be provided in a decellularized extracellular matrix composition (ECM) derived from tissue corresponding to the tissue cells (e.g., if the living animal cells are liver cells, decellularized extracellular liver matrix; if the living animal cells are cardiomyocytes, decellularized extracellular cardiac muscle matrix; if the living animal cells are skeletal muscle cells, decellularized skeletal muscle matrix; etc.). Additional collagen, glycosaminoglycan, and / or elastin (e.g., may be added to supplement the extracellular matrix composition) may also be included.

[0033] In some embodiments, organoids can be present in a hydrogel that can be customized to match the biochemical profile of one or more (e.g., 1, 2, 3, 4, 5, or more) tissues (e.g., tissues found in mammalian bodies). In some embodiments, adhesive proteins, such as those found in specific tissues, can be synthetically modified to allow direct binding to components within the composition (e.g., thiolated hyaluronic acid and / or methacrylated collagen). Growth factors can be linked via heparin pendant chains. Fibronectin, laminin, and / or other adhesive proteins can be synthetically modified to have one or more chemical groups that directly crosslink to components within the composition (e.g., thiolated hyaluronic acid and / or methacrylated collagen), thereby enabling tissue-specific customization. In some embodiments, including covalently bound fibronectin in the composition can have a significant impact on maintaining the function of organoids (e.g., liver organoids) formed and / or provided within the composition.

[0034] Cells can be incorporated into composition and / or hydrogel in any suitable form, including as unencapsulated cells, or as cells that have been previously encapsulated in spheroids, or as pre-formed organoids (as described above).The animal tissue cells that are encapsulated or contained in polymer spheroids can be produced according to known techniques, or in some cases are commercially available (see, for example, Insphero AG, 3D Hepg2 Liver Microtissue Spheroids (2012); Inspherio AG, 3D InSight™ Human Liver Microtissues, (2012)).

[0035] In some embodiments, organoid of the present invention comprises cells that are human-derived cells, and in some embodiments, organoid comprises cells that are made up of human-derived cells.Organoid of the present invention can express and / or produce one or more (for example, 1, 2, 3, 4 or more) biomarkers that are the same as the biomarkers that are produced by cells in vivo.For example, liver cells produce albumin in vivo, and the organoid of the present invention that comprises liver cells can express albumin.In some embodiments, organoid can express biomarkers in the same amount as the average amount that is produced and / or expressed by corresponding cells in vivo, or in the amount that is ±20%, ±10%, or ±5%. Exemplary biomarkers include, but are not limited to, albumin, urea, glutathione S-transferase (GST) (e.g., α-GST), chemokines (e.g., IL-8, IL-1β, etc.), prostacyclin, SBlOOB, neuron-specific enolase (NSE), myelin basic protein (MBP), hormones (e.g., testosterone, estradiol, progesterone, etc.), inhibin A / B, lactate dehydrogenase (LDH), and / or tumor necrosis factor (TNF). The cells may be differentiated or undifferentiated cells, but in some embodiments are tissue cells (e.g., liver cells such as hepatocytes, pancreatic cells, cardiac myocytes, skeletal muscle cells, etc.).

[0036] The selection of cells depends on the specific organoid to be produced, and cells can be labeled with detectable compounds, for example, but not limited to, fluorescent compounds (for example, dyes, proteins, etc.).For example, for liver organoids, liver hepatocyte cells can be used.For peripheral or central nervous organoids, peripheral nerve cells, central nerve cells, glial cells, or a combination thereof can be used.For bone organoids, bone osteoblast cells, bone osteoclast cells, or a combination thereof can be used.For lung organoids, lung airway epithelial cells can be used.For lymph node organoids, follicular dendritic lymphocytes, fibroblastic reticular lymphocytes, leukocytes, B cells, T cells, any bone marrow cells (for example, any bone marrow origin (including dendritic cells and phagocytes)), any lymphoid origin cells, or a combination thereof can be used. For smooth muscle and / or skeletal muscle organoids, smooth muscle cells, skeletal muscle cells, or a combination thereof can be used.For skin organoids, skin keratinocytes, skin melanocytes, or a combination thereof can be used.Cells can be differentiated when first incorporated into the composition, or undifferentiated cells that differentiate later can be used.Additional cells can be added to any composition and / or hydrogel.In some embodiments, tumor cells and / or immune cells can be added to organoids (for example, liver organoids), or organoids can be originally composed of tumor cells with or without immune cells.In some embodiments, the organoids of the present invention comprise, consist essentially of, or consist of one or more types of immune cells, which can be selected from leukocytes, peripheral blood mononuclear cells, follicular dendritic lymphocytes, fibroblastic reticular lymphocytes, leukocytes, B cells, T cells, any bone marrow cells (for example, any myeloid origin (including dendritic cells and phagocytes)), and / or any lymphoid origin cells.In some embodiments, one or more types of immune cells may be collected and / or obtained from lymph nodes (e.g., a lymph node biological sample), from bone marrow, and / or from the subject's peripheral blood or a fraction thereof (e.g., a white blood cell fraction, e.g., a peripheral blood mononuclear cell (PBMC) fraction).

[0037] In some embodiments, the organoid of the present invention can be mixed organoid (also referred to herein as mixed tumor / immune organoid), for example, tumor cells and immune cells exist in the same organoid.In some embodiments, the organoid of the present invention can be symbiotic organoid.As used herein, " symbiotic organoid " refers to at least one organoid that tumor cells and immune cells are in contact with each other.In some embodiments, symbiotic organoid is composed of one organoid that comprises tumor cells and immune cells (for example, mixed organoid).In some embodiments, symbiotic organoid comprises at least two organoids, and the first organoid of the at least two organoids comprises tumor cells, and the second organoid of the at least two organoids comprises immune cells, and at least a portion of the first organoid and at least a portion of the second organoid are in contact with each other. " Contacting " used herein in referring to symbiotic organoid refers to that at least a part of tumor cells and at least a part of immune cells are close enough to be in physical contact, have intercellular communication, and / or be present in and / or on the same organoid.In some embodiments, two or more separate organoids can grow together to form symbiotic organoid.In some embodiments, to provide and / or enable the communication between immune organoid and distant tumor organoid, cell transfer can be required (for example, through circulation).

[0038] The patient-derived tumor organoid (PTO) of the present invention can reproduce the tumor microenvironment of a patient, for example, by incorporating tumor cells with related stroma and / or tumor-infiltrating lymphocytes (TIL) (each obtained from the patient's own tumor).Lymph nodes incorporate 80% of all individual patient's immune system expression, and through their abundant antigen-presenting cells (APC), play a central role in the development of adaptive immunity.In some embodiments, the immune tumor organoid of the present invention can comprise peripheral blood mononuclear cells, which may be the case when lymph node tissue is not available to the patient.

[0039] Cancer cells optionally used herein may be any type of cancer cell, including, but not limited to, melanoma, carcinoma, sarcoma (including, but not limited to, angiosarcoma, myxofibrosarcoma, leiomyosarcoma, and dermatofibrosarcoma protuberans), blastoma, glioma, appendix, myeloma (e.g., multiple myeloma), head and neck, breast, lung, and astrocytoma cells. In some embodiments, cancer cells used in the present invention express N-cadherin and / or exhibit epithelial-mesenchymal transition. Cancer cells may be cancer cells from any tissue origin, including, but not limited to, intestinal (small intestine, large intestine, colon, appendix), lung, breast, prostate, skin, bone, brain, liver, pancreatic, uterine, cervical, testicular, and ovarian cancer cells.

[0040] In some embodiments, cells can be obtained from subjects, such as subjects or patients undergoing cancer treatment, and / or subjects or patients with cancer, and / or subjects with compromised immune systems.In some embodiments, cells are tumor cells, such as tumor cells from patient biopsies, and organoids prepared from such cells can be used to screen potentially effective drugs and / or treatments.Any type of tumor cells can be used in the organoids, devices, and / or methods of the present invention, including but not limited to intestinal (small intestine, large intestine, colon, appendix), lung, breast, prostate, skin, bone, brain, liver, pancreas, uterus, cervix, testis, and ovarian tumor cells.Examples of tumor organoids derived from biopsies include but are not limited to mesothelioma, colorectal, appendix, lung, melanoma, and sarcoma organoids.In some embodiments, cells include benign cells (also called non-cancerous cells) obtained from tissue biopsies. Cells can at least partially differentiate into specific cell or tissue types, such as brain, liver, intestine, pancreas, lymph node, smooth muscle, skeletal muscle, central nervous system, peripheral nervous system, skin, immune system, etc. Cells from biopsy (e.g., tumor and / or benign) can be used to form and / or prepare the organoids of the present invention, and the resulting organoids can be prepared and / or used in the method and / or device of the present invention within about 1, 2, 3, 4, 5, 6, 7, or 8 days after biopsy. In some embodiments, cells can be labeled with detectable compounds, for example, but not limited to, fluorescent compounds (e.g., dyes, proteins, etc.). In some embodiments, organoids comprising tumor cells, devices comprising them, and / or methods of using them can be as described in International Application No. PCT / US2017 / 045277, the contents of which are incorporated herein by reference in their entirety.

[0041] In some embodiments, the organoid of the present invention is not prepared and / or does not comprise the cell derived from immortalized cell line.The organoid of the present invention can comprise and / or be prepared with high-functioning cells, such as but not limited to primary cell and / or stem cell, for example, induced pluripotent stem cell and / or differentiated iPS-derived cell.

[0042] In some embodiments, the organoid of the present invention comprises a core that is made up of living tumor cells; and a shell that surrounds (for example, encapsulates) the core, and the shell is made up of living immune cells and / or living benign cells (for example, tissue cells, non-cancerous cells, etc.).Benign cells can be obtained and / or obtained from a subject, for example, from the tissue in a subject, and can be obtained from the same subject as tumor cells and / or immune cells.In some embodiments, living benign cells can be obtained and / or obtained from tissue biopsy, and / or can be tissue-specific.The organoid that comprises living benign cells can be separated from the organoid that comprises living tumor cells and / or immune cells (for example, formed separately and / or present in different chambers of device).

[0043] When cells (e.g., immune cells, tumor cells, and / or benign cells) are obtained from a subject, for example, when obtained from a tissue sample and / or tumor biopsy from a subject, different cell populations can be separated to provide one or more distinct cell populations, and one or more distinct cell populations can be labeled and / or used to prepare the organoids described herein.Methods for separating different cell populations are known to those skilled in the art, and any suitable method can be used, such as, but not limited to, fluorescence-activated cell sorting (FACS).When two or more cell populations are labeled, the two or more cell populations can have different detection signals.In some embodiments, tissue samples and / or tumor biopsies can be partially or completely genetically sequenced to identify mutations, and any identified mutations can indicate and / or suggest one or more target compounds for therapeutic purposes (e.g., immune system regulating activity and / or anti-tumor activity) for the subject. The method of the present invention can comprise the step of screening one or more target compounds identified in genetic sequencing, and contacting one or more target compounds individually and / or their combination with the organoid that uses the cell from tissue sample to prepare.The organoid of the present invention can have the same or substantially the same heterogeneity with the tissue and / or tumor that is found in vivo in the subject.

[0044] One or more cell populations (each may be labeled) can be combined in any suitable manner. In some embodiments, one or more cell populations can be added to the same common medium and / or hydrogel. In some embodiments, one or more cell populations can be used to form the organoids described herein that are encapsulated by hydrogels of the present invention. One or more different cell populations in the organoids of the present invention can be present in amounts that are substantially the same (for example, within about ±20%) as the amount of cells in that group in tissue and / or tumor in vivo. In some embodiments, when cells are obtained, sorted, and / or labeled from tissue samples from subjects, different cell populations are combined in amounts that are substantially the same as the amount present in tissue samples. In some embodiments, the cells (for example, tumor cells) used for the organoids of the present invention can be unsorted, which can help provide organoids with inherent heterogeneity. For example, in some embodiments, tumor cells are not sorted before forming the tumor organoid and / or mixed organoid of the present invention, which can allow tumor organoid to mimic the native tumor heterogeneity by, for example, including any stromal cells, endothelial cells, immune cells, etc. that exist in original biological specimen and / or sample.Similarly, in some embodiments, immune cells are not sorted before forming the immune organoid and / or mixed organoid of the present invention, which can allow organoid to maintain other cells within organoid, such as B cells, dendritic cells, macrophages (which may be important for the education and / or activation of T cells that are required to kill tumor cells).Therefore, organoid of the present invention can comprise non-tumor and / or non-T cell support cells, which can help maintain tumor or immune (e.g., lymph node) microenvironment and / or help maintain viability through various signaling mechanisms and some natural ECM remodeling.In some embodiments, tumor organoid of the present invention reproduces tumor microenvironment by incorporating tumor cells with related stroma and / or tumor infiltrating leukocytes (TIL).In some embodiments, when immune cells are obtained from blood, erythrocyte lysis is carried out to remove erythrocyte.

[0045] In some embodiments, at least two (for example, 2, 3, 4, 5, 6, 7, 8 or more) different organoids are formed using cells obtained and / or obtained from a single subject (for example, using one or more biopsies from the subject), where at least one organoid comprises live tumor cells from the tumor biopsy from the subject, and at least one separate organoid comprises live immune cells and / or live benign (for example, liver) cells from the subject.In some embodiments, at least two (for example, 2, 3, 4, 5, 6, 7, 8 or more) organoids are formed using cells obtained and / or obtained from a single subject (for example, using one or more biopsies from the subject), where at least one organoid comprises live tumor cells from the tumor biopsy from the subject and may comprise immune cells from the subject, and at least one separate organoid comprises live benign cells from the subject that are the same tissue type as the live tumor cells and may comprise immune cells from the subject. In some embodiments, the organoids of the present invention can be prepared by combining cells obtained and / or obtained from a single subject using one or more biopsies from a subject (for example, a mixed tumor / lymph node symbiotic organoid can be prepared by combining cells obtained and / or obtained from a tumor biopsy from a subject and cells obtained and / or obtained from a separate lymph node biopsy from the same subject). In some embodiments, one or more organoids of the present invention can be prepared using tissue biopsied from a subject, and cells obtained from 2mm x 2mm minced tissue can be used. In some embodiments, the organoids of the present invention can be used as tumor models and / or immune system models. For example, organoids can be useful for evaluating immune activity and / or modulating the immune system, and / or for screening one or more cancer treatments and / or immunotherapies.For example, the tumor organoids of the present invention, such as mixed tumor / immune organoids (e.g., mixed tumor / lymph node symbiotic organoids), may enable screening of adaptive immune development by training a patient's peripheral blood T cells to recognize tumor antigens presented on the surface of antigen-presenting cells (APCs) incorporated into the patient's own lymph node / tumor symbiotic organoids.

[0046] In some embodiments, organoids have a diameter and / or minimum dimension of about 50 μm, 100 μm, or 200 μm to about 350 or 500 μm, for example, about 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μm. In some embodiments, organoids have a diameter and / or minimum dimension of less than about 100, 90, 80, 70, 60, or 50 μm. In some embodiments, organoids have a volume of about 1 μL to about 20 μL, for example, a volume of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μL. Organoids may comprise a total of about 1,500, 2,000, or 5,000 to about 10,000, 25,000, or 50,000 cells, or a total of about 1,000, 5,000, 10,000, or 50,000 to about 75,000, 100,000, 150,000, 250,000, 500,000, 750,000, 1,000,000, 50,000,000, or 100,000,000 cells. In some embodiments, organoids of the present invention may comprise about 1,000,000, 2,000,000, or 5,000,000 to about 10,000,000, 25,000,000, or 100,000,000 cells per mL. In some embodiments, the organoid of the present invention may contain about 10 million cells per mL or 20 million cells per mL.In some embodiments, the organoid of the present invention may contain about 5 million or 10 million cells per mL to about 15 million or 20 million cells per mL.The organoid of the present invention may be in any suitable shape, for example, any three-dimensional shape or multi-layer shape.In some embodiments, the organoid of the present invention may be in the form of a spheroid.In some embodiments, the organoid of the present invention may be self-organized in suspension or medium (for example, cross-linked hydrogel).

[0047] As used herein, a "subject" generally refers to a human subject, although aspects of the invention may be practiced with other animal subjects, particularly mammalian subjects (e.g., dogs, cats, horses, goats, sheep) for veterinary and / or research purposes. Subjects may be male or female and of any age, including infants, juveniles, adolescents, adults, and geriatrics.

[0048] "Growth medium" and "culture medium" are used interchangeably herein and may refer to any natural or artificial growth medium (typically an aqueous solution) that sustains cells used in the practice of the present invention. Examples include, but are not limited to, essential medium or minimal essential medium (MEM), or variations thereof, such as Eagle's minimum essential medium (EMEM) and Dulbecco's modified Eagle's medium (DMEM), as well as blood, blood serum, plasma, lymph, and the like, including synthetic mimics thereof. In some embodiments, the growth medium includes a pH color indicator (e.g., phenol red).

[0049] The terms "test compound," "candidate compound," and "compound of interest" are used interchangeably herein and may refer to any compound or drug whose pharmacological or physiological activity, such as its activity on a cell or tissue (e.g., cardiac tissue), is to be determined, and / or whose interaction between two test compounds / drugs is to be determined. For illustrative purposes, isoproterenol, quinidine, propranolol, and epinephrine are examples of test compounds. However, any compound / drug may be used, including organic compounds, such as, but not limited to, proteins, peptides, nucleic acids, and / or small organic compounds (aliphatic, aromatic, and mixed aliphatic / aromatic compounds). Candidate compounds may be produced by any suitable technique, including randomly produced by combinatorial techniques and / or rationally designed based on a specific target. When drug interactions are tested, two (or more) test compounds whose potential combined effects are to be determined may be administered simultaneously, and one (or both) may be a known compound. In some embodiments, two or more test compounds may be administered in a manner similar to in vivo administration to a subject (e.g., similar to staged infusion of two or more test compounds), and may be administered simultaneously or sequentially. In some embodiments, the test compound is a metal, such as, but not limited to, aluminum, lead, etc. In some embodiments, the test compound is a heavy metal, such as, but not limited to, arsenic, cadmium, chromium, lead, and / or mercury. In some embodiments, the test compound is a pesticide. In some embodiments, the test compound is a chemotherapeutic agent and / or an immunotherapeutic agent, such as an immune checkpoint inhibitor (ICI). In some embodiments, the immunotherapeutic agent modulates and / or affects one or more components and / or activities of the subject's immune system. In some embodiments, the immunotherapeutic agent may be vemurafenib, ipilimumab, nivolumab, and / or pembrolizumab. In some embodiments, the test compound is a checkpoint inhibitor (e.g., a PD-1 inhibitor, a CTLA-4 inhibitor, etc.). In some embodiments, the test compound comprises an engineered immune cell, for example, a CAR T cell.In some embodiments, the test compound is an immunostimulatory virus with attenuated immunogenicity. In some embodiments, drug screening can be performed and / or conducted in a high-throughput manner. For example, the test compound can be present in one reservoir and / or multiple reservoirs (e.g., wells of a well plate). In some embodiments, the reservoir is a well of a well plate, for example, but not limited to, a well in a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate.

[0050] As used herein, a "detectable compound" may be a fluorescent compound (e.g., a fluorescent protein (e.g., red fluorescent protein, green fluorescent protein, etc.)), an antigenic protein or peptide (to which an antibody linked to an enzyme, fluorescent, or radioactive group or other label specifically binds), or any other suitable detectable compound. The detectable compound may be one that occurs naturally in the cell (e.g., a cell marker protein that is expressed at higher levels in cancer cells, e.g., in cancer cells than in non-cancerous cells), or one that has been inserted into the cell by genetic engineering / recombinant DNA technology (i.e., heterologous). In some embodiments, the detectable compound is a quantum dot (QD), a fluorescent organic dye, and / or a fluorescent protein. In some embodiments, the cell may express (naturally or recombinantly) the detectable compound.

[0051] The detectable compound may be any suitable compound that provides and / or produces a detectable signal that allows for differentiation and / or identification of cells and / or cell populations. The detectable signal may be provided and / or produced by one or more detectable compounds associated with the cells. In some embodiments, the detectable signal is a signal (e.g., an optical and / or electrical signal) produced by one or more detectable compounds (e.g., chemicals, proteins, etc.) associated with the cells (e.g., added to the cells, attached to the cells, bound to the cells, complexed to the cells). The detectable signal may be optically and / or electronically detectable, may be visually perceived by the human eye, and / or may be electronically decoded, detected, and / or obtained using methods known to those of skill in the art. In some embodiments, the detectable signal for a cell and / or cell population may be the absence of a signal (i.e., the absence of a detectable signal, e.g., the absence of detectable fluorescence, from the cells). In some embodiments, the detectable signal for a cell and / or cell population may be a fluorescent signal.

[0052] The devices and / or systems of the present invention may include a detector (e.g., a camera) and / or an excitation source (e.g., an excitation light source). The detector may detect and / or image a detectable signal from a cell and / or cell population. The excitation source may be used to generate and / or produce a detectable signal, for example, to provide and / or produce light that may cause a detectable compound to fluoresce, thereby providing and / or producing a detectable signal.

[0053] In some embodiments, at least a portion of a device (e.g., a microfluidic device) of the invention is transparent. For example, in some embodiments, the top and / or bottom substrate of the device may be transparent and / or the hydrogel present within the device may be transparent. The device may include a detector (e.g., a camera) operably associated with the device. The detector may be operably associated with one or more of the chambers of the device. In some embodiments, the detector may be provided above and / or below the device, and an excitation source may be provided above and / or below the device. In some embodiments, the detector includes an excitation source (e.g., a light such as a camera flash and / or an LED). The detector may be configured to detect (e.g., image) cells in one or more chambers of the device. In some embodiments, the device may include a detector (e.g., an LED / CCD detector) positioned to allow images of labeled cells in contact with one or more chambers of the device to be captured and / or quantified in real time. In some embodiments, the detector may capture images (e.g., fluorescent images) at predetermined intervals and / or may capture images and / or incidences of colonization, migration, and / or proliferation of labeled cells within and / or from organoids present in one or more chambers, which may allow real-time observation and / or quantification of cells within the organoids and / or their proliferation, migration, migration, and / or the like.

[0054] According to an embodiment of the present invention, organoid (also referred to herein as immunological organoid) comprising at least one type of immune cell is provided.At least one type of immune cell can be collected and / or obtained from lymph node, bone marrow, and / or peripheral blood (for example, lymph node biological sample, bone marrow biopsy, and / or peripheral blood collection) in subject.In some embodiments, at least one type of immune cell is selected from follicular dendritic lymphocyte, fibroblastic reticular lymphocyte, white blood cell, peripheral blood mononuclear cell, B cell, T cell, any bone marrow cell (for example, any myeloid origin (including dendritic cell and phagocyte)), and / or any lymphoid origin cell.In some embodiments, the organoid comprising at least one type of immune cell comprises white blood cell.In some embodiments, at least one type of immune cell comprises detectable compound (for example, fluorescent compound).

[0055] In some embodiments, organoids comprising at least one type of immune cell can serve as an in vitro model immune system, which can be for a specific subject / patient.This can allow screening of drugs that can affect immune system and / or can modulate immune system, for example, immunotherapeutic drugs (for example, PD-1 and CTLA-4 inhibitors, IL-2, interferon), which can enable immune cells to attack tumor cells.In some embodiments, immunological organoids of the present invention can be used to track neutrophil population (may be in the context of homing to tissue inflammation), can be used to validate genetic analysis, and / or can be used to select and / or optimize treatment for subject / patient.

[0056] Organoid of the present invention can comprise lymph node-derived cells (for example, lymph node-derived cells of subject), bone marrow, and / or leukocytes, and / or their components (for example, derived from subject, and it can be the same subject as lymph node cells).In some embodiments, organoid of the present invention comprises the cells from the fragment of excised lymph node (can be removed during tumor resection), leukocytes (for example, obtained from peripheral blood), and / or cells obtained from bone marrow biopsy, and all these cells are obtained and / or obtained from the same subject.Subject can have tumor and / or cancer, and / or immunotherapy can be the treatment option for the subject. For example, as shown in FIG. 12, tumor cells can be obtained from a subject's tumor, and / or immune cells can be obtained from a subject's lymph nodes, and the tumor cells and immune cells can be combined and / or encapsulated in a hydrogel (e.g., a native ECM hydrogel containing gelatin, collagen, hyaluronic acid, adhesion proteins (e.g., fibronectin, laminin, etc.), and / or growth factors) to form immune-enhanced patient tumor organoids. Another example of the present invention is shown in FIG. 13, which illustrates that tumor cells can be obtained from a clinical tumor biopsy from a patient, and immune cells can be obtained from the patient's lymph nodes and / or white blood cell fraction (WBCF). As shown in FIG. 13, a portion of these patient-derived tumor cells can be used to form tumor organoids in the absence of immune cells from the lymph node or WBCF sample, and another portion of the patient-derived tumor cells can be combined with immune cells from the lymph node or WBCF sample to form immune-enhanced tumor organoids. According to some embodiments, the methods of the present invention include separately exposing and / or contacting such organoids with one or more test compounds (e.g., chemotherapeutic and / or immunotherapeutic agents), and determining and / or identifying suitable test compounds (e.g., drugs, such as immune checkpoint inhibitors (ICIs)) that can be used to treat the patient from whom the cells for the organoids were obtained (Figure 13).

[0057] In some embodiments, the organoid of the present invention can comprise one or more types of live immune cells that exist in organoid, and the amount of each type is approximately the same as that of the amount that exists in the lymph node, bone marrow, and / or blood of the target.For example, the amount of T cell that exists in organoid of the present invention can be approximately the same as that of the amount of T cell that exists in the lymph node of the target in vivo.In some embodiments, the live immune cells of two or more different types exist in organoid of the present invention with the ratio or amount that is similar to that of each immune cell in the lymph node, bone marrow, and / or blood of the target in vivo. In some embodiments, one or more types of live immune cells can be present in the organoid of the present invention in an amount of about 1,500, 2,000, or 5,000 to about 10,000, 25,000, or 50,000 cells, or about 1,000, 5,000, 10,000, or 50,000 to about 75,000, 100,000, 150,000, 250,000, 500,000, 750,000, 1,000,000, 50,000,000, or 100,000,000 cells.In some embodiments, the organoid of the present invention can contain one or more types of live immune cells in an amount of about 1,000,000, 2,000,000, or 5,000,000 to about 10,000,000, 50,000,000, or 100,000,000 cells per mL.

[0058] The organoids of the present invention (e.g., immunological organoids) can be used in a device (e.g., a microfluidic device) containing tumor organoids (e.g., organoids containing tumor cells, which may be derived from the same subject as lymph nodes and / or white blood cells). The device can be used to screen the effectiveness of one or more immunotherapeutic agents (e.g., PD-1 and / or CTLA-4 inhibitors) targeting tumors in a subject, and / or to enable immune cells in the device (e.g., in immunological organoids) to attack tumor cells. Immune cells and / or tumor cells can be fluorescently labeled to colocalize and / or track immune cells using T cell activation markers and / or those that indicate tumor cell death. In some embodiments, tumor cell viability versus cell death can be evaluated using the device of the present invention. In some embodiments, when a patient (e.g., a subject from whom one or more of the cells are obtained) undergoes treatment (e.g., an immunotherapeutic drug), the in vitro response using the device and / or organoid of the present invention can be compared to or be predictive of the outcome of a real patient. In some embodiments, the methods of the present invention may include correlating and / or predicting in vitro results with in vivo results.

[0059] In some embodiments, the organoids of the present invention comprise at least one type of immune cell described herein and live tumor cells. The live tumor cells can be collected and / or obtained from a tumor in a subject. In some embodiments, the at least one type of immune cell and the live tumor cells can be collected and / or obtained from the same subject. The live tumor cells and at least one type of immune cell can be present in the organoid at any suitable ratio. In some embodiments, the live tumor cells and at least one type of immune cell can be present in the organoid at a ratio of about 1:1 or 5:1 to about 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1 (tumor cells:immune cells). In some embodiments, the ratio can be about 10:1 to about 100:1 when only leukocytes and / or tumor-infiltrating lymphocytes (TILs) are present as immune cells. In some embodiments, the ratio of tumor cells to immune cells can be about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the ratio of live tumor cells and at least one type of immune cells in organoids is about 5:1, 10:1, 50:1, or 100:1. In some embodiments, by providing organoids and / or devices containing tumor cells and / or immune cells from the same subject, organoids and / or devices can be used to evaluate whether standard chemotherapeutic agents and / or immunotherapeutic agents can be more effective for the subject. Providing organoids and / or devices containing both immune cells and tumor cells from the same subject can provide an in vitro tumor model (e.g., tumor organoid) with a genetically matched immune system, which can be used to test and / or screen one or more immunotherapeutic agents and / or chemotherapeutic agents in vitro.

[0060] In some embodiments, the organoid of the present invention comprises benign cells. For example, the organoid of the present invention may comprise at least one type of immune cells and / or living tumor cells as described herein, and may further comprise benign cells (for example, vascular endothelial cells, stromal cells, etc.). In some embodiments, the organoid of the present invention comprises at least one type of immune cells and benign cells (for example, vascular endothelial cells, stromal cells, etc.) as described herein. The benign cells can be collected and / or obtained from the same subject from which the at least one type of immune cells and / or living tumor cells are obtained and / or obtained. The cells in the organoid (for example, tumor cells, immune system cells, and / or benign cells) can be arranged in the organoid in any suitable manner. In some embodiments, the cells (for example, tumor cells, immune system cells, and / or benign cells) can be combined and / or mixed together and then randomly distributed within the organoid. In some embodiments, the organoid of the present invention comprises a core of living tumor cells and / or living immune system cells, and a shell comprising at least one type of immune system cells and / or living benign cells.In some embodiments, benign cells (e.g., endothelial cells and / or stromal cells) can be present at least partially around the organoid of the present invention, and the organoid can comprise tumor and / or immune cells.In some embodiments, an endothelial layer and / or barrier can be present at least partially or completely around the organoid of the present invention.Cells that can be present on and / or around the organoid of the present invention (e.g., tumor organoid or mixed organoid) include, but are not limited to, cells that can be present around tumors in vivo.Examples of cells that can be present on and / or around the organoid of the present invention (e.g., tumor organoid or mixed organoid) include, but are not limited to, epithelial cells, stromal cells, fibroblasts, astrocytes, astrocytes, glial cells, etc. In some embodiments, organoids of the present invention comprise immune system cells in an amount of about 1%, 5%, 10%, 25%, 50% to about 55%, 60%, 75%, 80%, 90%, 95%, or 100% of the total number of cells present within the organoid.In some embodiments, the organoid of the present invention comprises the immune system cells of about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the total number of cells that exist in organoid.When the organoid of the present invention comprises the immune system cells of less than 100% of the total number of cells that exist in organoid, any suitable amount of tumor cells and / or benign cells can fill the remaining number / percentage of cells.

[0061] The organoid of the present invention can provide a patient-specific in vitro model system, which can be used to determine the treatment for patients, and this may be before the start of treatment and / or treatment.In some embodiments, target compounds can be screened for immune activity, immune system regulation, and / or anti-tumor activity, which can be added to other screening methods, such as but not limited to, genetic biomarker evaluation and / or genetic profiling.In some embodiments, the organoid, device, and / or method of the present invention can enable and / or provide the recognition of cellular biomarkers, the quantification of biomarker expression, and / or the real-time testing of immunotherapy and / or chemotherapy drug effectiveness (including, for example, testing the effectiveness of immunogenic viruses that can be used for therapeutic purposes in patients).

[0062] Organoid of the present invention can be identified and / or used to help identify tumor mutation, and / or to associate mutation with the drug available for targeting.In some embodiments, the method of the present invention uses organoid of the present invention to identify treatment by the results achieved.

[0063] In some embodiments, the device and / or method of the present invention can combine patient-derived tumor organoids with immune system-derived components (for example, cells derived from the patient's lymph node biological sample from which tumor cells are obtained).These organoids, devices, and / or methods can be used in precision medicine-driven drug testing, evaluating the drug efficacy of immunotherapeutic agents in vitro for individual patients (for example, to identify actionable mutations and / or gene profiles and subsequently customize treatment).In some embodiments, tumor organoids and / or mixed tumor / immune organoids (for example, mixed tumor / lymph node symbiotic organoids) can be prepared directly from fresh tumors, and these tumor organoids can replicate tumor microenvironment, allowing for the recognition of cellular biomarkers, the quantification of biomarker expression, and / or the real-time testing of immunotherapy and / or chemotherapy drug efficacy. The organoids of the present invention may be provided and / or contained within a microfluidic device, which may provide and / or enable parallel screening of multiple drugs and / or provide multiple tissues and / or tumors in fluid communication. In some embodiments, the organoids and / or devices of the present invention may supplement genetic screening and / or be used to predict the degree of malignancy and / or the optimal treatment for an individual patient, which may occur before administration to the patient. Drug screening can be performed using various test compounds at various concentrations, and results may be obtained in less than about one week (e.g., less than one week after obtaining patient-derived cells used in preparing organoids for drug screening). In contrast, typical genetic screening services do not produce actionable data sets for an average of three to four weeks.

[0064] The device of the present invention can comprise immunological organoid (for example, the organoid that comprises at least one type of immune cell) and separate tumor organoid.In some embodiments, the device of the present invention comprises the organoid that comprises both tumor cells and at least one type of immune cell.The organoid that comprises tumor cells in the device of the present invention can comprise tumor cells that are derived from the same subject as immune cells, and can also comprise other cell populations, such as but not limited to vascular endothelial cells and / or stromal cells.The inclusion of cell populations other than the organoid that comprises tumor cells can help preserve tumor heterogeneity and / or clonality, and can more realistically reproduce tumors.

[0065] In some embodiments, immunological organoids can be upstream of separate tumor organoids or can be fluidically connected with them in other ways, and can be used to evaluate T cell homing to tumor cells through fluid (e.g., microfluidic) circulation.Although there may be tumor-infiltrating lymphocytes (TILs) in vivo that can initiate a response under immunotherapy, they are not active in all tumors.Therefore, the primary immune response under immunotherapy often originates from lymph nodes through circulation.In some embodiments, the device of the present invention can be used to evaluate the ability of immune cells to home from upstream immunological organoids to tumor organoids in the device, and / or can be used to evaluate T cell activation and / or tumor killing.In some embodiments, leukocytes can be injected into the device, and this may be the case when the number of nodal tissue and / or lymph node cells is insufficient.

[0066] By providing a device that combines tumor organoids from patient-specific biopsy with immune cells (for example, lymph node cells) from the same patient and / or the organoids that contain such cells, for example, genetically matched human immune system can be provided to tumor organoids.This device can be used to screen immunotherapeutic agents, and can be performed on a patient-by-patient basis.In some embodiments, the device and / or method of the present invention can, for example, test a large number of samples, thereby reducing the possibility of error and / or false positive and / or false negative.

[0067] In some embodiments, the method of the present invention can comprise the step of ex vivo activation of immune cells, which comprises contacting immune cells (for example, lymphocytes) with a living tumor cell organoid comprising a plurality of living tumor cells to provide activated immune cells; separating the activated immune cells from the living tumor cell organoid to provide isolated activated immune cells; and expanding the isolated activated immune cells to provide a population of activated immune cells. Any type of immune cells, such as those described herein, can be contacted with living tumor cell organoid. In some embodiments, immune cell(s) can be non-passaged cells (for example, non-passaged cells obtained from patients), and / or the tumor cell(s) used to form living tumor cell organoid can be non-passaged cells (for example, non-passaged cells obtained from patients). In some embodiments, immune cells and / or tumor cells can be subcultured 0, 1, 2, 3, or 4 times or more. In some embodiments, immune cells comprise peripheral blood mononuclear cells. Therefore, in some embodiments, tumor organoids described herein and patient's own lymphocytes can be used to treat cancer.For example, for patients with melanoma, this method can comprise the step of contacting patient's lymphocytes with tumor organoids produced or formed by using patient's own melanoma cells or their products, and activating patient's lymphocytes ex vivo with or without drugs, collecting activated lymphocytes, expanding their population, and then administering them to patient.One or more steps of the method of activating immune cells ex vivo can be repeated one or more times (for example, as many times or as long as the cells in organoid continue to survive).In some embodiments, this method can be carried out for patients using cells from new tumor recurrence.

[0068] In some embodiments, activated immune cells can be immune cells with adaptive immunity (e.g., T cells with adaptive immunity) and / or immune cells with immune memory obtained from ex vivo exposure to the patient's own tumor antigen (e.g., T cells with immune memory). In some embodiments, primed and / or activated immune cells can be primed and / or activated responsive to drug exposure. In some embodiments, the method for ex vivo activation of immune cells can include: using a white blood cell fraction from a patient's blood collection, which includes the patient's lymphocytes (T cells), then contacting the patient's lymphocytes with the patient's own tumor cell organoids or their secretory products, activating the lymphocytes ex vivo with or without drugs, and then collecting activated lymphocytes (e.g., T cells). In some embodiments, contacting the patient's lymphocytes with the patient's own tumor cell organoids or their secretory products can be in the presence of one or more drugs, which can increase the activation potential of lymphocytes. Activated lymphocytes can be infused back into the original patient, and can be expanded in vitro before infusion.The method of activating immune cells ex vivo can provide the activation of T cell outside of patient, and / or can make patient avoid unnecessary drug toxicity.In some embodiments, the method of activating immune cells ex vivo can activate more cells than those that are localized near tumor cell and / or tumor organoid.

[0069] Referring to FIG. 11, the device of the present invention may comprise one or more chambers, which may be connected by one or more parallel channels that may be daisy-chained with one or more chambers containing organoids (e.g., tumor organoids) (FIG. 11A), thereby increasing the number of organoids (e.g., the number of patient tumor organoids (PTOs)) per circulator path. In some embodiments, two or more organoids may be present and / or patterned in the same chamber (FIG. 11B). In some embodiments, one or more T cells may be injected and / or circulated in the device and / or method of the present invention, for example, as shown in FIG. 11C, which may induce T cell priming and / or activation in response to tumor antigen recognition. The circulating T cells may be removed from the device after a certain period of time and / or transferred. In some embodiments, the removed circulating T cells may be contacted with unexposed matched patient tumor organoids (e.g., non-immune enhanced tumor organoids), for example, to test activation level and / or tumor killing. In some embodiments, primed and / or activated T cells may be administered to a subject / patient.

[0070] In some embodiments, the methods and / or devices of the present invention may provide for and / or enable the parallel screening of two or more (e.g., 2, 3, 4, 5, 6, 7, or more) compounds of interest, in some embodiments, at least one of the two or more compounds of interest is an immunotherapeutic agent.

[0071] In some embodiments, the method and / or device of the present invention can provide and / or provide results (e.g., drug screening results and / or therapeutic analysis) within about 1 or 2 weeks (e.g., within about 2, 3, 4, 5, 6, 7, 8, 9, or 10 days) after obtaining a biopsy (e.g., lymph node and / or tumor biopsy) from a subject, and the cells are used to prepare the organoids of the present invention used in the method and / or device. In some embodiments, the method of the present invention includes treating the subject (may use a compound of interest) within about 1, 2, or 3 weeks (e.g., within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) after obtaining a biopsy (e.g., lymph node and / or tumor biopsy) from a subject (optionally depending on the step of obtaining results (e.g., drug screening results and / or therapeutic analysis) using cells from biopsy).

[0072] Also described herein is a device useful for evaluating immune cells and / or tumor cells in vitro, comprising: (a) a microfluidic device having a chamber and a channel in fluid communication with the chamber; (b) at least one organoid (e.g., an organoid containing immune cells and / or tumor cells) in the chamber; (c) a growth medium in the channel and the chamber; (d) a pump operably associated with the chamber and the channel, the pump configured to circulate the medium from the chamber, through the channel, and back to the chamber; and (e) a microporous membrane (e.g., a TRANSWELL® microporous membrane) in the channel, arranged to allow the medium to flow therethrough. The device of the present invention may be maintained at a circulating and / or intermittent flow rate, which may be from about 1, 2, or 5 μL / min to about 7, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μL / min. In some embodiments, intermittent flow may be used to reduce interstitial flow within organoids, which may be the case when testing and / or determining migration of cells into and / or out of organoids. In some embodiments, intermittent flow may be used to stimulate in vivo drug administration (e.g., infusion). In some embodiments, the device is configured and / or suitable for analytical testing, such as, but not limited to, biochemical assays and direct imaging on a chip. According to some embodiments, cells (e.g., immune cells) may be injected into the device of the present invention at a flow rate of about 1, 2, or 5 μL / min to about 7, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μL / min. In some embodiments, the flow rate and / or cell infusion rate of the devices of the present invention may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μL / min.

[0073] In some embodiments, the device is useful for screening compounds of interest in vitro for immune activity and / or for activity in modulating the immune system and / or its activity. In some embodiments, the device is useful for assessing tumor cell metastasis in vitro, for assessing tumor cell migration and / or invasion in vitro, for assessing the growth of constructs containing tumor cells in vitro, and / or for assessing response to compounds of interest. In some embodiments, the device may be useful for assessing construct size in vitro, for assessing tumor cell number in vitro, and / or for assessing tumor cell death in vitro.

[0074] In some embodiments, the method of the present invention can determine at least one tumor organoid response (for example, tumor cell viability, cell number, volume, apoptosis, etc.), at least one immune cell activity, at least one marker activation or depletion (can be evaluated by immunofluorescence biomarker), and / or the presence of at least one T cell activation marker and / or T cell dysfunction marker.In some embodiments, the method of the present invention can comprise the step of determining the migration of nodal cells, which can be through live cell tracking.The effectiveness of test compound can be determined by cell viability (live / dead), the number of live cells, the number of live cells vs. dead cells, mitochondrial metabolism (MTS), LDH quantification (positive in tumor cells), and / or IHC (for example, Annexin V vs. KI67--apoptosis vs. proliferation marker).

[0075] In some embodiments, the methods of the present invention may be used to observe the tumor killing rate and / or efficacy imposed by the engineered immune cells (e.g., CAR T cells) in vitro and / or to calculate the appropriate dosage and / or infusion rate of the engineered immune cells (e.g., CAR T cells), which may be to prevent complications for the patient based on disease volume and efficacy.

[0076] Some embodiments of the present invention provide a method for screening target compounds for immune activity in vitro and / or modulating immune system, the method comprises: providing at least one organoid comprising a plurality of cells (for example, at least one organoid can be liver organoid, cardiac organoid, tumor organoid, immunological organoid, mixed organoid etc.); contacting target compounds with at least one organoid in vitro; and determining the immune response of at least one organoid in response to the step of contacting target compounds with at least one organoid in vitro (for example, comparing with the immune response / activity of organoid before contacting with target compounds and / or in the absence of contact).In some embodiments, at least one organoid is a separate organoid comprising tumor organoid and immune cells.In some embodiments, at least one organoid is the organoid comprising tumor cells and immune cells.In some embodiments, at least one organoid is a mixed organoid (for example, mixed symbiotic organoid).

[0077] In some embodiments, the method of the present invention comprises contacting at least one organoid with culture medium.In some embodiments, culture medium comprises at least one type of immune cell (can be leukocyte), and at least one type of immune cell can be from the same origin as the cell in at least one organoid.In some embodiments, culture medium comprises target compound.

[0078] The method of the present invention may comprise determining cell migration (for example, determining the migration of at least one type of immune cell), and may use live cell tracking. In some embodiments, the method of the present invention comprises determining the presence of activation and / or depletion markers on cells (for example, cells present in organoids and / or migratory cells), and may use immunofluorescence. In some embodiments, the method of the present invention comprises: in response to contacting at least one target compound with at least one organoid in vitro, the target compound activates and / or increases the immune activity of at least one type of cell (for example, immune cell). In some embodiments, the method of the present invention comprises: in response to contacting at least one target compound with at least one organoid in vitro, there is a reduction in the proliferation (for example, lack of cell proliferation, cell death, reduction in cell infiltration, etc.) of cells (for example, tumor cells) present in at least one organoid. When at least one organoid comprises tumor cells, the method of the present invention can comprise the step of determining the reduction in the proliferation of the tumor cells present in at least one organoid (for example, the absence of cell proliferation, cell death, reduction in cell invasion, etc.) according to the step of contacting at least one organoid with target compound in vitro.In some embodiments, determining the reduction in the proliferation of live tumor cells comprises determining the number of viable tumor cells present in at least one organoid, determining the number of live tumor cells present in at least one organoid, determining the volume of at least one organoid, and / or determining the number of dead tumor cells present in at least one organoid.

[0079] In some embodiments, the method of the present invention comprises calculating the dose and / or infusion rate of the target compound to administer to a subject.In some embodiments, the method of the present invention comprises administering a therapeutically effective amount of the target compound to the subject in response to the step of contacting the target compound with at least one organoid in vitro, and it can be determined that the target compound reduces the metastasis of tumor cell in vitro, reduces the size of organoid in vitro, reduces the number of tumor cell in vitro, induces the death of tumor cell in vitro, increases the activity of immune cell in vitro, and / or activates immune cell in vitro.

[0080] In some embodiments, the device useful for evaluating tumor cells and / or immune cells in vitro comprises one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) chambers, each chamber contains at least one organoid, and they are fluidly connected with each other.The device can be useful for evaluating immune activity, for regulating immune system and / or its activity, tumor cell metastasis in vitro, for evaluating tumor cell migration and / or invasion in vitro, for evaluating the growth of constructs containing tumor cells in vitro, and / or for evaluating the response to target compounds.In some embodiments, the device can be useful for evaluating the size of constructs in vitro, for evaluating the number of tumor cells in vitro, and / or for evaluating the death of tumor cells in vitro.One or more organoids present in the device can be prepared from cells obtained from a single subject. In some embodiments, the device comprises live tumor cell organoids in a first chamber and organoids comprising immune cells in a second chamber.In some embodiments, the device comprises live tumor cell organoids and organoids comprising immune cells in the same chamber (for example, they may be separated from each other, as shown in Figure 2).In some embodiments, when two or more organoids are provided in the same chamber, the cells of the organoids and / or organoids can be present in one or more (for example, 2, 3, 4, 5 or more) zones and / or areas of the chamber.For example, in some embodiments, the chamber comprises two or more zones and / or areas, and the cells of one organoid (for example, immune cell organoids) can be present in the first zone and / or area, and the cells of the second organoid (for example, tumor cell organoids) can be present in the second zone and / or area (for example, as shown in Figure 4A-4C).Having different cells and / or organoids in the same chamber but in different zones and / or areas (they can be adjacent to each other) can provide and / or force the migration of cells through organoid, but can not provide and / or force the migration of cells through circulation.Living tumor cell organoid and liver organoid can be formed from the cells obtained from the same subject, which can provide individualized analysis.

[0081] In some embodiments, the device may comprise: a primary chamber containing live tumor cell organoids; at least one secondary chamber containing different organoids; at least one primary channel connecting the primary chamber and the secondary chamber and providing fluid communication therebetween (such as the flow of growth medium); and the primary chamber, each secondary chamber, and primary channel may contain growth medium.In some embodiments, two or more (for example, 2, 3, 4, 5, 6, 7, 8 or more) secondary chambers are provided, and each secondary chamber contains organoids containing cells different from the others.In some embodiments, at least one secondary chamber contains liver organoids, and wherein the live tumor cell organoids and liver organoids can be prepared from cells obtained from the same subject.Additional exemplary devices include, but are not limited to, those described in PCT / US2016 / 054611 and PCT / US2017 / 045277, the contents of each of which are incorporated herein by reference in their entirety.

[0082] In some embodiments, at least a portion of the device (e.g., a microfluidic device) is transparent, and the device may include a detector (e.g., a camera) operably associated with the microporous membrane and configured to detect (e.g., image) cells (e.g., tumor and / or immune cells) on the microporous membrane.

[0083] The device body itself may be composed of any suitable material or combination of materials, examples of which include, but are not limited to, polydimethylsiloxane (PDMS), polystyrene, polymethylmethacrylate (PMMA), polyacrylamide, polyethylene glycol (PEG), including functionalized PEG (e.g., PEG diacrylate, PEG diacrylamide, PEG dimethacrylate, etc., or any of the aforementioned multi-armed forms of PEG), natural polymers or proteins that can be crosslinked or cured (e.g., hyaluronic acid, gelatin, chondroitin sulfate, alginate, etc., including derivatives thereof functionalized with chemical groups to support crosslinking, including any of the above-mentioned "crosslinkable prepolymers" in crosslinked form, and combinations thereof). The device body may be formed by any suitable process (including combinations thereof), including molding, casting, additive manufacturing (3D printing), lithography, etc.

[0084] If a structural support is included within the device, the structural support, like the hydrogel, may be patterned (e.g., a regular or irregular pattern, such as a regular or irregular lattice, grid, spiral, etc.).

[0085] In some embodiments, devices of the present invention that can be used in the methods of the present invention can be configured to provide physiological or supraphysiological fluid-to-tissue volume ratios. For example, one or more chambers in a device can have an average volume ranging from about 2 μL to about 10 μL. In some embodiments, one or more chambers in a device can have an average volume of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 μL. In some embodiments, devices of the present invention (e.g., devices with at least two or six chambers) can use and / or have a volume of liquid of less than about 100 μL, e.g., about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40 μL or less. As used herein, the volume of a device refers to the volume that fills the chamber(s) and channel(s) of the device. In some embodiments, devices of the invention (e.g., devices with at least two or six chambers) may use amounts of liquid less than about 50 μL and / or have a volume less than about 50 μL. In some embodiments, the volume of the device may be increased by integration with and / or use of an external fluid reservoir. An external fluid reservoir may increase the overall system volume and / or help control the volume of the device.

[0086] The system, device, and / or method of the present invention can comprise and / or provide one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8 or more) different tissues and / or organoids (each being viable for at least 1, 2, 3, 4, 5 weeks or longer).In some embodiments, the system, device, and / or method of the present invention comprises and / or provides one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8 or more) different organoids (being in fluid communication with each other and common aqueous growth medium, each being viable for at least 1, 2, 3, 4, 5 weeks or longer).Therefore, in some embodiments, 2, 3, 4, 5, 6 or more different organoids are in fluid communication with each other and common aqueous growth medium, each being viable for at least 1, 2, 3, 4, 5 weeks or longer. In some embodiments, one or more of the organoids may be viable and may contain at least about 75% or more (e.g., about 80%, 85%, 90%, 95% or more) living cells based on the average number of cells present in the construct at 1, 2, 3, 4, 5, or more weeks. Tissues and / or organoids may be produced by differentiation from a general cell sample (e.g., a sample such as a skin sample collected from a subject). One or more of the organoids may contain cells in a proportion similar to that present in the corresponding native (e.g., human) tissue. In some embodiments, at least one of the organoids contains metastatic and / or malignant cells. In some embodiments, the function and / or characteristics of the tissue and / or organoid may be determined and / or measured and compared with the function and / or characteristics of the corresponding native tissue (e.g., the characteristics of brain organoids may be measured and compared with the same characteristics of brain tissue in a subject). In some embodiments, the function and / or properties of the tissue and / or organoid may resemble the function and / or properties of the corresponding native tissue.

[0087] As described herein, cell and / or cell sample can be used in the method of the present invention to form the organoid of the present invention.The method of the present invention can provide viable organoid.In some embodiments, the method of the present invention can achieve at least 50% or higher, for example, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or higher engraftment rate.For example, a 90% engraftment rate means that 90% probability of viable organoid or a plurality of organoids (for example, a set of organoids) can be achieved and / or provided by the method of the present invention.That is, for a 90% engraftment rate, when prepared according to the method of the present invention, 9 out of 10 cell samples (for example, tumor cell samples) will produce viable organoid or a plurality of organoids.The organoid or a plurality of organoids can be used in other methods and / or diagnosis of the present invention. In some embodiments, the cells and / or cell samples may be derived from a tumor, such as, for example, a mesothelioma biological sample and / or a GI tumor biological sample, and / or may be derived from a lymph node biopsy.

[0088] The method for screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro can be carried out by: providing a device comprising at least one organoid comprising tumor cells as described herein; circulating a culture medium in the device; administering a compound of interest to the tumor cells (for example, by adding the compound to the culture medium); and quantitatively or qualitatively detecting the tumor cells captured on the microporous membrane, wherein a smaller number of captured tumor cells (for example, compared with other tumor cells under similar conditions and / or non-metastatic cells under similar conditions) indicates a better anti-metastatic and / or anti-tumor activity of the compound of interest. In some embodiments, at least one type of immune cell can be present in the device (for example, in the chamber of the device, in the culture medium, in at least one organoid comprising tumor cells, and / or in the organoid).

[0089] A method for screening a compound of interest for anti-metastatic and / or anti-tumor activity in vitro can be carried out by providing a device described herein (e.g., a device comprising a primary chamber containing live tumor cell organoids and at least one secondary chamber); circulating a medium within the device; administering a compound of interest to the tumor cells (e.g., by adding the compound to the medium); and quantitatively or qualitatively detecting tumor cells present in one or more secondary chambers (e.g., on and / or within the organoids present in the secondary chambers), wherein a smaller number of tumor cells present in one or more secondary chambers (e.g., compared to other tumor cells under similar conditions and / or non-metastatic cells under similar conditions) indicates a superior anti-metastatic and / or anti-tumor activity of the compound of interest. In some embodiments, at least one type of immune cell can be present within the device (e.g., within the chambers of the device, in the medium, within at least one organoid containing tumor cells, and / or within the organoids).

[0090] The methods of the present invention may include labeling cells (e.g., tumor cells and / or immune cells) with a detectable compound, such as, but not limited to, a fluorescent compound (e.g., a dye, a protein, etc.).

[0091] The foregoing and other aspects of the present invention are further illustrated in the examples that follow. [Example]

[0092] Example 1 - Integration of lymph node and leukocyte components within a fresh patient-derived lung TOC model for in vitro testing of immunotherapeutic (IT) drug efficacy. Biofabrication of Integrated Tumor and Lymph Node Organoids:Node cells and leukocytes are labeled with membrane-incorporating dyes (PKH26-red or PKH67-green) and integrated with tumor biospecimen cells. After biofabrication, the presence of immune cells is confirmed by staining for immune cell surface markers to confirm colocalization with the above-mentioned membrane dyes. Controls consisted of tumor organoids alone.

[0093] PD-L1 and CTLA-4 assessment: After tissue collection, immunohistochemistry (IHC) is performed on a portion of each tumor biopsy using a PD-L1 antibody or on a node biopsy using a CTLA-4 antibody. Established 5% thresholds are used to define high PD-L1 or CTLA-4 expression, which are classified as high or low expression, respectively, allowing hypotheses to be generated for each biological sample regarding IT sensitivity.

[0094] Compiled immunotherapy drug panel: Organoids can be produced in arbitrarily small quantities, creating nearly any number of organoids for drug screening (and sorting) and biomarker identification. Pembrolizumab (pembro) targets the PD-1 receptor. Nivolumab (nivo) targets the PD-1 receptor and is generally used when the cancer does not have a BRAF mutation. Ipilimumab (ipi) activates the immune system by targeting CTLA-4.

[0095] Drug screening output metrics:Drugs are administered for 48 hours, after which efficacy is assessed as follows: cell viability is assessed by (live / dead), viable cell count, viable / dead cell count, mitochondrial metabolism (MTS), LDH quantification (positive in tumor cells), and IHC (colocalization of immune cells immunolabeled with apoptotic, proliferative, and tumor cell markers). Figure 1A shows integrated data from colorectal tumor-node organoids, demonstrating an increase in mitochondrial metabolism (presumably through T cell activation) upon incubation with nivolumab. Similarly, live / dead staining in low-grade appendiceal tumor-node organoids (Figure 1B) demonstrates increased cell death under pembro and nivo treatment. These results are corroborated using biomarker analysis. Furthermore, Figure 1C shows data from a PTO from a previously treated melanoma patient who failed to respond to pembrolizumab and ipilimumab ("non-responder"). Live / dead staining demonstrated a recapitulation of the non-responsive phenotype, where organoids derived from these tumors also failed to respond to these immune checkpoint inhibitors but responded to dabrafenib and trametinib (far right panel). This was unexpected given the patient's wild-type BRAF status. Retrospectively, the patient was found to have a downstream MEK mutation bypassing the BRAF status. An alternative therapy was identified by PTO screening more than four weeks prior to genomic sequencing, which confirmed the MEK mutation, and thus qualified the patient for trametinib. The patient began treatment with trametinib (a MEK inhibitor) and began responding successfully almost immediately.

[0096] Example 2 - Implementation of lymph node organoids upstream from a tumor-on-a-chip (TOC) to assess T cell homing to tumor cells through microfluidic circulation. 2- Biofabrication of organoids:Tumor organoids are produced, and separate nodal organoids are produced (in a manner similar to Example 1, but without tumor cells) and placed in each chamber of the microfluidic device (Figure 2). Alternatively, if nodal organoids are included, leukocytes are injected through the circulation. The control consists of tumor only.

[0097] Drug screening output metrics: The drug conditions and output are the same as those described in Example 1. Furthermore, the location of immune cell numbers is verified. Fluorescent tags allow tracking and confirmation if immune cells can migrate downstream to tumors in the presence of treatment and expose tumor-associated antigens ( FIG. 2 ). Without wishing to be bound by any particular theory, it is believed that conditions such as PD-1 blockade can enable immune cells such as T cells to recognize tumor cells, migrate to tumors, and induce tumor cell death.

[0098] A microfluidic device architecture with biofabricated PTOs within each individual organoid channel / chamber is used, as shown in Figure 2. Furthermore, the previously parallel PTO channels are daisy-chained together so that all PTOs populate a single pathway through which T cells are perfused (Figure 11A), thus maximizing exposure to tumor antigens by circulating T cells. The number of PTOs per TOC can be significantly increased by photopatterning a larger number of PTOs within a single larger chamber (Figure 11B) or within multiple parallel channels, reducing the system fluid volume.

[0099] Example 3 - Introduction of lymph node immune system components to test the efficacy of immunotherapeutic drugs within a fresh patient-derived tumor-on-a-chip model. Cells derived from lymph node fragments removed during tumor resection were incorporated into patient-specific tumor-on-a-chip organoids, thereby facilitating screening of tumor-targeting immunotherapeutic agents. This approach recapitulates inherent heterogeneity without the need to characterize specific cell populations. By using a complete mixture of cells (e.g., tumor cells, stromal cells, endothelial cells, etc.) derived from a tissue sample, the system is essentially representative of tumor heterogeneity in vivo. Responses are compared to patient outcomes.

[0100] Biological specimen acquisition and processing: Patients are primarily consented and enrolled through surgical practices for the resection of major abdominal visceral cancers or undergoing palliative procedures to remove metastatic disease and lymph nodes for the purpose of local control of residual disease refractory to ongoing immunotherapy. Not all patients require sampling of both components (tumor and lymph nodes). Sampling is only a small part of the procedure as clinically indicated. In other words, if the indicated surgical procedure does not provide access to both components (tumor and lymph nodes), sampling is performed only from those that are accessible and present within the surgical field. Fresh tumor biopsies are minced, washed, digested, and filtered to obtain a cell suspension. All specimens are coded to prevent access to patient identity. Genetic profile data are provided anonymously with the samples.

[0101] Microfluidic Device Fabrication:Traditional use of lithographically defined polydimethylsiloxane elastomers to prototype microfluidic devices can take days due to the reliance on expensive transparency masks, photolithography for device definition, continuous casting, and precise layer placement. This presents challenges for scale-up and clinical translation. However, this approach is only necessary for extraordinary resolution (approximately 1 μm). Our 3D systems are assembled using simple thin, patterned adhesive films that can self-align and layer via folding to form microfluidic structures (Figure 3A). Chamber and channel definition is achieved through the use of a computer-controlled laser cutter. Using this platform, we have produced multiple complex devices, including valves, mixers, and other functional microfluidic systems.

[0102] Organoid-device integration:To address the challenges of integrating 3D organoids into microfluidic systems, we developed an in situ biofabrication methodology utilizing hyaluronic acid (HA) and gelatin-based hydrogels (HyStem), which are widely used in tissue engineering and various biofabrication techniques. In the general method for assembling tissue constructs, HA and gelatin / collagen components are mixed with tissue organoids and a crosslinker and photoinitiator to support thiol-acrylate / methacrylate photopolymerization. Each cell suspension is added to the gel precursor at a density such that the organoid volume ratio in the final integrated construct is comparable to the volume ratio of the organ in the human body (e.g., the mass of liver organoids is five times that of cardiac organoids). Each cell-laden substrate is sequentially introduced into the microfluidic chamber, and patterning is achieved using a positive photomask to define the shape and position of the construct (Figure 3B). The crosslinked hydrogel adheres to the top and bottom of the chamber, allowing it to be maintained under fluid flow conditions powered by a microperistaltic pump that supports multiple circuits in parallel (Figure 3C). This patterning can be performed within any number of independent microfluidic chambers. The resulting 3D constructs can then be maintained for long-term viability under circulatory flow, making the entire system suitable for analytical testing, including both biochemical assays and direct on-chip imaging. This patterning can be performed within any number of independent microfluidic chambers. The resulting 3D constructs can then be maintained for long-term viability under circulatory flow, making the entire system suitable for analytical testing, including both biochemical assays and direct on-chip imaging. Further patterning (e.g., with additional cell or organoid types) can also be used to produce multi-component structures, allowing for significant system complexity. Notably, the hydrogel itself also supports the incorporation of solubilized extracellular matrix to provide additional biomolecular factors specific to each tissue organoid.ECM profiles vary between tissue types, and the inclusion of ECM components improves cell viability and function. ECM components are easily linked within the hydrogel via covalent or heparin bonds. Because the presence of specific matrix proteins (e.g., laminin, collagen I, collagen IV) and cytokines can influence tumor growth and migration (and therefore drug efficacy), reproducing these profiles in the TOC device ensures accurate reproduction of in vivo systems. Overall, this assembly method is rapid, inexpensive, and modular, with the straightforward potential to be mass-produced for numerous parallel experiments.

[0103] Analysis Plan: Sample size and power: The primary endpoint is the feasibility of developing tumor organoids and tumor / lymph node organoids. This study will enroll 20 patients and define feasibility as the success rate of organoid development. This rate is expressed as a percentage of (successful organoid development / 20). This rate is reported with its corresponding 95% confidence interval. With a planned sample size of 20, if the observed success rate is 50%, the 95% confidence interval will be approximately + / - 22.2%.

[0104] Primary outcome analysis: The primary outcome is the rate of organoid development; this rate represents the success of organoid development.

[0105] Secondary outcome analysis: The secondary outcome is the correlation of organoid chemosensitivity with individual response to chemotherapy (classified into one of three groups: stable disease, partial response, or no response). Given the small sample sizes within these three groups, the Kruskal-Wallis test will be used to assess differences in chemosensitivity observed among the three response groups.

[0106] Biofabrication of Tumor-Lymph Node Organoids:The biofabrication method described above is used to create tumor-on-a-chip models specific to patients whose tissue has been excised. In addition to cells derived from tumor biopsies, cells isolated from the patient's lymph nodes are combined with cells derived from the tumor biospecimen during the process of biofabrication of hydrogel organoids. Lymph node cells are fluorescently labeled with membrane-incorporating dyes (PKH26-red or PKH67-green) for subsequent processing. To ensure close proximity of immune cells to tumor cells, we prefer to combine tumor cells and lymph node cells at a 5:1 ratio (cell count), although this ratio can be adjusted. After organoid biofabrication, the presence of immune cells is confirmed by staining for immune cell surface markers and verifying colocalization with the membrane dyes described above.

[0107] Compiled immunotherapy drug panel: Organoids can be produced in arbitrarily small quantities, creating nearly any number of organoids for drug screening and biomarker identification. Interferon helps existing immune cells fight cancer. Interleukin-2 helps expand the number of immune cells primed to fight cancer. Pembrolizumab targets the PD-1 receptor. Nivolumab also targets the PD-1 receptor but is generally used when the cancer does not have a BRAF mutation. Ipilimumab activates the immune system by targeting CTLA-4. Nivolumab / ipilimumab, a combination therapy. Vemurafenib / cobimetinib targets tumors with BRAF mutations.

[0108] Drug screening output metrics: The efficacy of drug treatment is assessed as follows: cell viability is assessed by (live / dead), viable cell count, viable vs. dead cell count, mitochondrial metabolism (MTS), LDH quantification (positive in tumor cells), and IHC (apoptotic vs. proliferative markers). Additionally, changes in immune cell number and status are examined. Colocalization of tumor cells with labeled immune cells is assessed by imaging.

[0109] Correlating drug efficacy to patient response: Following drug screening and a determination of which drugs are most effective for a given set of biological sample organoids, these data are correlated with patient data using only de-identified data as described above.

[0110] Statistical analysis: Experiments were performed in quadruplicate or more; data are presented as the mean ± standard error of the mean. Student's t-test was used for comparison of two groups with α = 0.05. One-way analysis of variance was used for multiple comparisons, with a 95% confidence limit considered significant.

[0111] Example 4 - Introduction of patient-matched lymph node cell populations into patient tumor biological samples ex vivo tumor-on-chip organoids. Biological specimen acquisition and processing:Patients are primarily consented and enrolled through our CRS / HIPEC surgical practice for large abdominal visceral cancer resections or extremity tumor resections, as well as stage IV melanoma patients undergoing palliative procedures to remove metastatic disease and lymph nodes for the purpose of local control of remaining disease refractory to ongoing immunotherapy. Sampling is only a small part of the clinically indicated procedure. In other words, if the surgical procedure does not provide access to both components (tumor and lymph nodes), patients will not be consented for these studies. Tissues and data from the Advanced Tumor / Tissue Bank Project (BG04-104), IPHC Research Database (BG01-372), and precision medicine reports (gene mutation analysis) will also be used in this study when available. In general, with our preliminary data, we expect to obtain colorectal, appendiceal, peritoneal mesothelioma, and melanoma tissues. All specimens will be anonymous. Fresh biopsies are minced, washed, digested, and filtered to obtain a cell suspension. Some of the nodule cells will be fluorescently labeled for tracking (QTracker probes). Our goal is to generate a set of organoids from 12 separate specimens.

[0112] Assembly of the microfluidic device: The 3D system is assembled using simple thin, patterned adhesive films that can self-align and layer via folding to form microfluidic structures (Figure 3A). Feature definition is achieved by a computer-controlled laser cutter.

[0113] Organoid-device integration:The HA and gelatin / collagen components are mixed in situ with cells, as well as a crosslinker and photoinitiator for photopolymerization. Cells are added to the gel precursor, preferably at a density of 20 million cells / mL or higher. The cell-gel mixture is then sequentially introduced into microfluidic chambers, and patterning is achieved using a positive photomask to define the shape and location of the construct (Figure 3B). For the proposed study, each device contains a series of two-chamber circuits, with tumor organoids formed in one chamber and lymph node organoids formed in the other. Alternatively, both organoids can reside separately within the same chamber (Figure 2). In another form factor, tumor cells and immune cells can be photopatterned into the device as adjacent regions, allowing them to recognize tumor cell antigens, activate, and migrate to the tumor area in order for the immune cells to successfully target the tumor cells (Figures 4A-4C). The resulting 3D constructs are typically maintained with long-term viability under a circulatory flow of 10 μL / min.

[0114] Organoid characterization and baseline cell migration studies:After initiation of the platform, the TOC device is operated under flow for 1, 7, and 14 days. At these time points, organoids are fixed for IHC analysis. Tumor organoids are subjected to a panel of markers depending on the tumor type (we have characterized organoids from each of the types indicated in preliminary data). Nodal organoids (and potentially lymphoid cells that may have migrated into tumor organoids) are evaluated using antibodies for CD8, CD4, CD45, CD25, FOXP3, l-selectin, CD44, CTLA4, and PD1 to identify lymphocytes / leukocytes, Tregs, effector types, differentiation, and negative feedback. Although FACS can also be used, IHC provides temporal and spatial information within the 3D organoids. Data are initially evaluated in organoid tissue sections for initial screening, but are subsequently evaluated in 3D by whole-mount macro-confocal microscopy (Leica LCS TSI). Cell migration is assessed in a more nuanced manner using live fluorescence and tracking of the migration of labeled nodal cells.

[0115] [Evaluation of patient-specific tumor organoid responses to immunotherapy agents, with and without nodal cell populations.] Edit the Drug Panel: Drug screening is performed and drug concentrations are based on clinical plasma levels. A tumor only TOC is used as a control alongside a tumor + node TOC. Drugs screened can be: i) Pembrolizumab, which targets the PD-1 receptor. ii) Nivolumab, which targets the PD-1 receptor but is generally used when the cancer does not have a BRAF mutation. iii) Ipilimumab (Ipi), which activates the immune system by targeting CTLA-4. iv) nivolumab / ipilimumab, combination therapy. v) Vemurafenib / cobimetinib, targeting tumors with BRAF mutations. vi) Imatinib, an antibody for tumors with activating mutations of C-KIT (a stem cell marker). vii) Interferon and interleukin-2, aiding in immune activation.

[0116] Lymph node cell activation and tracking: Activation of immune cell populations is assessed using a subset of the IHC framework described above. Tracking of migrating fluorescent cells is performed as described above. Quantitative changes in migration kinetics are determined between experiments for each set of patient TOCs.

[0117] Drug screening output metrics: Drug efficacy is determined by cell viability (live / dead), number of live cells only, number of live vs dead cells, mitochondrial metabolism (MTS), LDH quantification (positive in tumor cells), and IHC (Annexin V vs KI67 - apoptotic vs proliferative markers).

[0118] Figure 5 shows cell viability data for drug screening using melanoma tumor organoids and melanoma tumor organoids containing lymph node cells. For drug screening using the checkpoint inhibitors nivolumab (nivo) or pembrolizumab (pembro), a significant reduction in organoid viability, as measured by overall metabolic activity, was observed only when the organoids contained both lymph node-derived cells and tumor cells.

[0119] Example 5 - Production of tumor and associated tissue organoids for drug screening and disease progression analysis. Biofabricated organoids made from patient tumors and related tissues serve as in vitro mimics of the in vivo microenvironment and immune system. Such mimics can be used for drug screening, including standard chemotherapy and immunotherapy, by integrating the immune system and appropriate cells into the culture. Disease progression can be further studied using the constructs, providing greater insight into patient-specific tumor behavior for improved treatment.

[0120] The process used in practicing this exemplary method is shown in Figures 4-10. All operations involving open containers must be performed within a Biosafety Cabinet (BSC). Items placed into the BSC must first be cleaned with 70% EtOH via light spray. Any excess EtOH in the BSC should be wiped away before performing the procedure.

[0121] Cell culture vessels should be wiped with 70% EtOH before being placed in the incubator or on the microscope. Excess EtOH should be wiped off before being placed in the incubator. Conical tubes containing cells are also wiped or sprayed with 70% EtOH before being placed in the centrifuge.

[0122] [Reagent preparation] DMEM-10: Add the appropriate volume of fetal bovine serum (FBS) to Dulbecco's Modified Eagle Medium (DMEM) to make a 10% solution. Additionally, add the appropriate volumes of penicillin / streptomycin and L-glutamine to make a 1% solution. Sterile filter the combined medium through an appropriate size medium filter (500 mL, 1000 mL).

[0123] Digestion solution: Add the calculated volume of reconstituted collagenase HA and BP protease to low glucose DMEM (VitaCyte). The digestion solution should be used within 2 hours of reconstitution of the enzymes.

[0124] [Digestion medium volume:] XXmg× 20mL digestion medium 200mg of tissue

[0125] [Addition to the medium:] 6.6 μl / mL collagenase HA x volume of medium prepared = volume of collagenase HA Collagenase HA should be reconstituted in 20 mL of cold diH2O in a sterile vial and may be stored for 1 year. 6.4 μl / mL BP protease x volume of medium prepared = volume of BP protease · BP Protease should be reconstituted in 2 mL of cold diH2O in a sterile vial and may be stored for 2 years.

[0126] DPBS washing solution: Add the appropriate volume of 10 mg / mL gentamicin to DPBS in an appropriately labeled sterile container to obtain a final concentration of 5 μg / mL gentamicin. Swirl the solution to ensure adequate mixing. The DPBS wash solution may be used up to 17 days after preparation.

[0127] Antibiotic wash solution: Add the appropriate volume of amikacin (20 mg / mL final concentration), amphotericin B (1 μg / mL final concentration), gentamicin (5 μg / mL final concentration), and vancomycin (5 mg / mL final concentration) to DPBS in an appropriately labeled sterile container. Swirl the solution to ensure mixing. The antibiotic wash solution should be used the same day it is prepared.

[0128] Digestion neutralization solution: Combine high glucose DMEM and fetal bovine serum (FBS) to achieve a final concentration of 10% FBS. Sterile filter and label appropriately. Vortex the solution to ensure mixing.

[0129] Digestion neutralization capacity: The volume of digestion neutralization medium used should be the same volume as the volume of digestion solution used to digest the tissue biological sample.

[0130] Heprasil: Make a stock solution of UV photoinitiator by combining DiH2O with 0.1% w / v Irgacure. Cover the conical with tin foil and place on a shaker plate at 200 rpm in a 37°C room for 30 minutes. Remove the foil cap cover from the Heprasil vial and add 1 mL of stock UV photoinitiator. Cover the vial and place in a 37°C incubator for 45 minutes.

[0131] Methacrylated Collagen-I: Remove the foil cap from the methacrylated collagen-I bottle. Add 16.8 mL of the manufacturer's acetic acid to the bottle to obtain a concentration of 6 mg / mL. Store the bottle in a 4 °C refrigerator and remove the appropriate volume for each experiment.

[0132] [Tools and Supply Preparation] PDMS coated plate: Combine the elastomer curing agent with the elastomer base in a 1:10 ratio by weight. Mix well using a stirrer for 3-5 minutes. Using a stirrer, drizzle a thin layer of PDMS into each well of a 48- or 24-well plate. Gently rock the plate to completely cover the bottom of each well. Place the plate in a vacuum for 20 minutes. Remove from vacuum and place in 80°C for a minimum of 2 hours. Once cured, place the plate under UV light in a BSC for a minimum of 20 minutes before use with cultures.

[0133] tweezers: Forceps for use with tissue in the conical are sterilized in 70% EtOH for a minimum of 30 minutes or by autoclaving.

[0134] 1.5mL Eppendorf tube: Place the tube in an autoclave safe bag, seal, and place autoclave tape over the bag. Autoclave and use exclusively for tissue processing.

[0135] [Cell treatment] Cell isolation:The biopsy tissue is transferred to a BSC and washed three times for 5 minutes in the prepared antibiotic wash solution (followed by three washes of 5 minutes each in the prepared disinfectant solution). While the tissue is being washed, the digestion enzymes for the tissue digestion solution are reconstituted. After washing, the tissue is transferred to an appropriately sized tissue culture dish and minced into small pieces using a scalpel. Once minced, the tissue is transferred to the prepared digestion solution and placed on a shaker table set at 200 rpm in a warm room for 15 minutes. After digestion, an appropriate volume of neutralizing solution is added to the digested tissue and gently pipetted to mix. For lymph nodes, cells are manually separated using a 100 μm centrifuge insert. The cell suspension is transferred to a 100 μm SteriFlip filtration unit and vacuum filtered. The flow-through is centrifuged at 300 x g for 5 minutes. The supernatant is removed from the conical, and the cells are resuspended in 1 mL of DMEM-10 and counted.

[0136] Preparation of organoids:Reconstitute Heprasil and store in a 37°C incubator until combined with the collagen. Place methacrylated collagen-I in a conical container and neutralize with the manufacturer's neutralizing solution at 85 μL per 1 mL of methacrylated collagen-I, keeping the container on ice. Combine Heprasil with methacrylated collagen in a 1:3 ratio to produce the calculated volume required for the number of organoids needed. Mix the mixture, avoiding bubbles, and leave on ice for 45 minutes. While waiting for 45 minutes, count cells from each tissue type (tumor, lymph node, liver) and aliquot them into 1.5 mL Eppendorf tubes based on the calculations. Tumor-only, tumor and lymph node, and liver-only organoids should be generated. Tumor and liver-only organoids should contain 10 million cells per mL, with each organoid being 10 μL. Tumors containing lymph node cells should contain 10 million tumor cells per mL and 5 million lymph node cells per mL. The Eppendorf tubes containing the aliquots should be centrifuged at 500 x g for 3 minutes. The supernatant should be removed from the tubes, and an appropriate volume of Heprasil / methacrylated collagen-I should be added to each. The resuspended cells are then pipetted into each PDMS-coated well of a well plate. Each organoid is crosslinked for 3 seconds using UV light. Add 0.5 mL of DMEM-10 medium to each 48-well plate or 1 mL to each 24-well plate and incubate.

[0137] Preparation of leukocytes for addition:The blood vial should be kept at room temperature until ready to be processed. Before preparation, gently invert the vial, if already done, to mix the blood so it no longer separates. Using a 15 mL conical, add 3.5 mL of Ficoll-Paque PLUS and then carefully layer the blood on top of the Ficoll layer, ensuring the Ficoll and blood do not mix. Place the 15 mL conical in a large centrifuge that allows for individualized settings. The settings should be as follows: Start Speed ​​- 9, Break Speed ​​- 1, Temperature - 20°C, Time - 40 minutes, Speed ​​- 400 x g. When centrifugation is complete, remove the conical, avoiding mixing or shaking the separated layers. Using a pipette, remove the complete second separated layer and place it in a new 15 mL conical. Add 3 mL of DMEM-10 to the conical and centrifuge at 400 x g for 5 minutes. Remove the supernatant, resuspend the cells in 1 mL of DMEM-10, and count the cells. Calculate how many wells can accommodate 25,000 cells / well.

[0138] Drug screening and analysis Experimental plan & determination of wells containing white blood cells (WBC): Determine how many organoids are generated, including tumor and lymph node cells, and tumor-only cells. For each of these organoid types, divide by 5 to determine how many drug conditions can be performed.

[0139] Choice of immunotherapy agent: If lymph nodes or WBCs are present in the culture, immunotherapy should be administered to n=5 wells / condition. Dose- and time-dependence should be considered in favor of dose-dependence. If a previous treatment before immunotherapy was administered, that drug treatment and at least one other immunotherapy should be used. Administration should be performed with and without lymph nodes and with and without WBCs. Use n=3 wells for MTS assay and n=2 wells for histology. If time-dependence is not being tested, run for 4 days without changing the medium.

[0140] Standard chemotherapy drug choices: For tumor-only organoids, select standard chemotherapy treatment based on the cancer's origin and previous patient treatment. Dose-dependence and time-dependence should be considered with priority given to dose-dependence. If prior chemotherapy treatment was administered, use that drug treatment and at least one other chemotherapy. Use n=3 wells for MTS assay and n=2 wells for histology. If time-dependence is not being tested, do not change the medium for 4 days.

[0141] MTS assay: Follow the MTS assay instructions for solution preparation as provided by the manufacturer, using high-glucose DMEM without FBS for dilution. Remove the medium from each well and replace it with 200 μL of MTS assay dilution solution. Do this for three empty, cell-free wells, which also serve as controls. Place the plate back in the incubator for 2 hours. Remove 100 μL of solution from each well and place it into individual wells of a clean 96-well flat-bottom plate. Read the absorbance of the wells at 490 nm using a plate reader.

[0142] Paraffin processing and sectioning: Organoids should be paraffinized and sectioned into 5 μm thick layers with 4 sections per slide.

[0143] H&E and IHC section staining: H&E should be performed on one slide from each condition to ensure cellularity of the organoids. IHC should be performed if cellularity is evident. The choice of IHC should be determined based on the cancer cell type and if lymph node or blood samples are used in the culture.

[0144] Example 6 - Use of PTO for analysis of immune cell activation and tumor cell killing. Tumor biological specimen processing.Melanoma biospecimens were obtained under an IRB protocol, of which both investigators on this project are members, under which the data described in this proposal were generated. Each fresh biospecimen was isolated in the clinic / operating room, placed in RPMI medium in a sterile conical container, and immediately transported to the laboratory for processing. The biospecimens were minced, washed, and incubated with collagenase / hyaluronidase to digest the major structural components of the ECM. A portion was preserved for histology. Organoids were formed in a hydrogel containing hyaluronic acid, gelatin, and a PEG crosslinker.

[0145] TOC PTO array design. To begin, we use the same microfluidic device architecture, containing biofabricated PTOs within each individual organoid channel / chamber, as shown in Figure 3. The PTO channels are daisy-chained together so that all PTOs are present in a single pathway through which T cells are perfused (Figure 11A), thus maximizing exposure to tumor antigens by circulating T cells. However, if minimal activation or cell killing is observed, we significantly increase the number of PTOs per TOC by photopatterning a larger number of PTOs within a single, larger chamber (Figure 11B) or within multiple parallel channels, thereby reducing the system fluid volume.

[0146] Histological analysis after culture.On day 7, each construct is stained for live / dead and examined by confocal microscopy and unbiased segmentation to determine the viability of all cells. Immediately following this, samples are photobleached to remove live / dead fluorescence and then sequentially labeled and imaged with a series of fluorescent antibodies (PNL2, HMB45, melan-A, CD4+, CD8+, PDL1, PD1, CD80 / CD86, CTLA-4) to independently determine the location of melanoma cells and immune cell subtypes. To limit the number of sequential labeling / imaging cycles, we group several spectrally resolved tags together for multispectral imaging.

[0147] Immune cell perfusion / activation.We tested unsorted immune cells from lymph node biopsies, which saves time and costs compared to FACS sorting for T lymphocytes. Furthermore, this preserves the heterogeneity of node-derived cells, allowing several populations to play a role. The first set of biopsies will be used to determine how many PTOs are required to induce T cell activation, how long immune cells must circulate within these TOCs to achieve sufficient activation, and whether immune checkpoint inhibitors are required. These factors will be assessed using the following methods and metrics for T cell status: To assess functional onboard TOCs, in vitro IFN-γ production will be assessed in response to anti-CD3+ anti-CD28 antibodies (BD) and to commercially available (Proimmune Inc) peptide pools corresponding to melanoma antigens, gp100, MART-1, and tyrosinase. Conditions will be maintained for 96 hours. IFN-γ production will be assessed by ELISA (R&D Systems). T cell proliferation is measured by CFSE flow assay, measuring the cytokines IFN-γ, IL-2, and TNFα. Organoid cell suspensions recovered from TOCs by non-enzymatic ECM lysis or alternatively collagenase / hyaluronidase are analyzed by flow cytometry. Cells are gated for CD3+, CD8+, and CD4+ to determine surface marker expression of CTLA-4, TIM-3, LAG3, PD-1, and CD160, which are associated with a T cell "exhausted-like" phenotype. In another set of experiments, organoids are fixed and stained with anti-PDL-1 along with fluorescent staining of CD3+, CD4+, and CD8+ T cells to assess T cell interaction and localization relative to tumor cells, as described above. Sections are evaluated using multispectral imaging with quantitative pathology using inForm analysis software.

[0148] Potential immune checkpoint inhibitor therapy.If necessary, we expose TOCs to an intermittent flow of one of four conditions: clean medium or medium containing clinically relevant concentrations of pembrolizumab, nivolumab, or ipilimumab. The concentrations of these two widely used PD-1 inhibitors and one widely used CTLA-4 inhibitor are derived from clinical treatment guidelines and adjusted to the dose of PTO. Currently, concentrations of approximately 100 nM are used and are not expected to negatively affect PTOs unless they activate immune cells and cause the user's intended tumor cell killing.

[0149] We establish a PTO inside a microfluidic device. In construct formation, our goal is to use unpassaged patient cells to avoid genetic mutations from the in vivo tumor state. However, without wishing to be bound by any particular theory, this may inherently limit the number of cells available for incorporation. While the majority of samples we processed in preliminary studies provided more than enough cells for the proposed PTO, we believe it is possible to achieve a target cell density of 20 million cells / mL for the same biopsy, which would be challenging. If so, we will explore approaches to further reduce the dimensions of the cell culture construct while increasing the overall PTO surface area by first reducing the overall dimensions of the microfluidic chamber to minimize the volume of cell-containing precursors that must be initially introduced. Specifically, we can use a combination of precision photomasks (chrome on quartz) and thinner glass slides to reduce optical effects that limit resolution. With these methods, we expect to obtain constructs less than 100 μm in size. We can also expand to low passage numbers, thereby increasing cell availability while hoping to minimize genetic drift.

[0150] Demonstration of the utility of educated and activated T cells used as cellular therapy for PTO.Node-derived cells are transferred from the "activation" microfluidic device to a TOC device not used in the activation experiment to test cell therapy-mediated cell killing and adaptive immunity. It is believed that PTOs not previously exposed to immune cell perfusion can be successfully treated with immune cell populations activated by perfusion through a matched PTO TOC array.

[0151] Immune cell migration. If T cell activation is confirmed, the circulating immune cell suspension for priming is pumped into a clean fluid reservoir and transferred to a fresh PTO TOC from the same patient biospecimen administered without drug or immune cell exposure (Figure 11C). These immune cell populations are infused through the TOC at 10 μL / min to allow engraftment upon recognition of the PTO.

[0152] Homing evaluation. Prior to injection into the TOC, immune cell populations are fluorescently labeled with a membrane dye (DiI / DiO, ThermoFisher). Routine fluorescence imaging tracks the motility and relative position of DiI / DiO-tagged cells to the TOC, as well as their engraftment and invasion into the TOC. We previously demonstrated the effectiveness of such tracking methods in the context of organoids in our on-chip metastasis platform and used a similar method to quantify metastasis of a metastatic colorectal cancer cell line from the gastrointestinal tract to downstream liver organoids through a microfluidic platform.

[0153] Assessment of therapeutic effectiveness.Tumor cell death and nearest neighbor quantification are performed. Each organoid is supported for 3, 7, or 10 days using constant or intermittent flow, with aliquots of media collected daily for ELISA analysis of perforin and granzyme B (secreted markers indicative of T cell activation), IL-2 (T cell growth and differentiation factor), and ATP activity. On days 3, 7, and 10, constructs are stained for live / dead and examined by confocal microscopy and image segmentation to determine the viability of all cells. Shortly after this, samples are photobleached to remove live / dead fluorescence, and then sequentially labeled and imaged with fluorescent antibodies using multispectral imaging to determine the relative location of melanoma cells and distinct immune cell subtypes. We can explore the additional use of checkpoint inhibitors to further prime PTO for cell therapy, and preliminary data indicate this is feasible.

[0154] We compare the efficacy of immune checkpoint inhibitors between cohorts of patients with low and high PD-L1 and CD80 / 86 expression in melanoma. Literature values ​​for high PD-L1 and CD80 / 86 expression in melanoma vary but typically suggest a prevalence of approximately 30-60% depending on the melanoma subtype. Experiments were performed in triplicate or more; data are expressed as mean ± standard error of the mean. Student's t-test was used to compare two groups with α = 0.05. One-way analysis of variance was used for multiple comparisons, with a 95% confidence limit considered significant.

[0155] Example 7 - Design of combined lymph node / tumor organoids from the same patient for personalized immunotherapy screening. Combined lymph node / melanoma organoids were prepared using cells from the same patient, thereby generating mixed tumor / node organoids. Experiments were conducted to evaluate whether the patient's tumor, stroma, and immune system maintained viability for personalized immunotherapy screening. The mixed organoids allowed the tumor, stroma, and immune system of each individual patient to maintain viability for personalized immunotherapy screening, and enabled the generation of adaptive immunity through training of the patient's peripheral blood T cells to recognize tumor antigens presented on the surface of APCs incorporated into the patient's own lymph node / tumor co-cultured organoids.

[0156] method: Matched melanoma and lymph node biospecimens surgically obtained from the same patient were transported to the laboratory and washed with saline, antibiotics, and red blood cell lysis buffer. The biospecimens were dissociated, incorporated into an ECM-based hydrogel system, and biofabricated into 3D patient-specific mixed melanoma / node organoids. Cells were not sorted for tumor to preserve tumor heterogeneity, including stromal and immune cell components. Sets of organoids were screened in parallel for 72 hours with nivolumab, pembrolizumab, ipilimumab, and dabrafenib / trametinib. Metabolic assays and quantification of live / dead staining documented the relative drug efficacy in killing melanoma cells for a specific patient. Light microscopy, IHC, and next-generation sequencing (NGS) were used to compare organoid melanoma cells with tumor melanoma cells.

[0157] result:Biospecimens from five patients with stage III and stage IV melanoma were used to develop mixed organoids. The rate of successful establishment of viable organoid sets (i.e., engraftment rate) was 80% (4 / 5). The median time from organoid development to the start of immunotherapy testing was 7 days. Organoid responses to immunotherapy resembled patient clinical responses in 3 / 4 patients. Organoids from a fourth patient demonstrated 50% melanoma eradication with nivolumab, but the patient clinically progressed on the drug. A response to trametinib in a patient with a BRAF-wild MEK pathway-containing melanoma was suggested by organoid testing prior to NGS and confirmed by clinical response upon treatment.

[0158] Conclusion: The development of 3D mixed immune-enhanced tumor / node organoids is a viable platform, allowing an individual patient's immune system and tumor cells to maintain viability for the study of personalized immunotherapy responses and the creation of adaptive immunity.

[0159] We prepared melanoma patient tumor organoids immunopotentiated with lymph node cells and then tested them with test compounds, such as immune checkpoint inhibitors. Organoids without immunopotentiation (tumor-only organoids) did not respond, whereas tumor-immune organoids did (Figures 14A-14C). Furthermore, we prepared melanoma patient tumor organoids immunopotentiated with immune cells derived from the white blood cell fraction of each patient's blood sample and then tested them with test compounds, such as immune checkpoint inhibitors. Organoids without immunopotentiation (tumor-only organoids) did not respond, whereas tumor-immune organoids did respond (Figures 15A-15B). Organoids were prepared as described in the previous examples using hydrogels containing thiolated HA, thiolated gelatin, and a PEGDA crosslinker, or thiolated HA and methacrylated collagen.

[0160] [Example 8] Tumor organoids of sarcoma patients were prepared from patients with angiosarcoma (Fig. 16A) and dermatofibrosarcoma protuberans (DFSP) (Fig. 16B) using immune cells derived from the white blood cell fraction of blood collected from each patient, and then tested with test compounds such as immune checkpoint inhibitors. Organoids without immune enhancement (tumor-only organoids) did not respond, but tumor-immune organoids did respond. Organoids were prepared as described in the above examples using hydrogels containing thiolated HA, thiolated gelatin, and PEGDA crosslinker, or thiolated HA and methacrylated collagen.

[0161] [Example 9] Patients' own peripheral T cells were provided into the circulation along with culture medium and exposed to tumor immune organoids for 7 days (Fig. 11D). These exposed T cells were then harvested and transferred to tumor organoids derived from the same patient's tumor biological sample (not formed with patient-derived immune cells or exposed to T cells), and T cell-mediated tumor killing was observed (as indicated by visual staining of viable cells versus dead cells) (Fig. 11E). Organoids were prepared as described in the previous examples using hydrogels containing thiolated HA, thiolated gelatin, and PEGDA crosslinker, or thiolated HA and methacrylated collagen.

[0162] The foregoing is illustrative of the invention and is not to be construed as limiting thereof. The invention is defined by the following claims, including equivalents of the claims. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is set forth.

Claims

1. 1. An in vitro cellular construct (e.g., an "organoid") useful as a tumor model, comprising: comprising live tumor cells and at least one type of live immune cell, Construction.

2. 10. The construct of claim 1, The viable tumor cells are collected and / or obtained from a tumor within the subject. Construction.

3. 3. A construct according to claim 1 or 2, The at least one type of live immune cell is collected and / or obtained from lymph nodes (e.g., lymph node biopsy), bone marrow, and / or peripheral blood within the subject. Construction.

4. 4. The construct of any one of claims 1 to 3, The live tumor cells and the at least one type of live immune cells are collected and / or obtained from the same subject. Construction.

5. 5. The construct of any one of claims 1 to 4, the at least one type of live immune cell is a follicular dendritic lymphocyte, a fibroblastic reticular lymphocyte, a leukocyte, a B cell, a T cell, any myeloid cell (e.g., any cell of myeloid origin, including dendritic cells and phagocytes), and / or any cell of lymphoid origin; Construction.

6. 6. The construct of any one of claims 1 to 5, further comprising white blood cells, Construction.

7. 7. The construct of any one of claims 1 to 6, the live tumor cells and the at least one type of live immune cells are present in the construct at a ratio of about 1:1 to about 10:1, 50:1, or 100:1 (tumor cells:immune cells), and may be present at a ratio of about 5:1 or about 10:1; Construction.

8. 8. The construct of any one of claims 1 to 7, further comprising benign cells (e.g., vascular endothelial cells, stromal cells, etc.); wherein said benign cells may be collected and / or obtained from the same subject from which said at least one type of live immune cells and / or live tumor cells are obtained and / or derived, Construction.

9. 9. The construct of any one of claims 1 to 8, a shell surrounding (e.g., encapsulating) said live tumor cell core, said shell comprising said at least one type of immune cell and / or live benign cell (e.g., tissue cell, non-cancerous cell, etc.); wherein said viable benign cells may be collected and / or obtained from the same subject from which said at least one type of immune cell and / or viable tumor cell is obtained and / or derived, Construction.

10. 10. The construct of any one of claims 1 to 9, The viable tumor cells include malignant cells. Construction.

11. 11. The construct of any one of claims 1 to 10, the tumor cells, the at least one type of live immune cells, and / or the benign cells, if present, comprise mammalian cells (e.g., human, mouse, rat, monkey, etc.); Construction.

12. 12. The construct of any one of claims 1 to 11, the tumor cells and / or the at least one type of live immune cells comprise a detectable compound (e.g., a fluorescent compound); Construction.

13. 13. The construct of any one of claims 1 to 12, The construct is cultured in culture medium for 1, 2, 3, 4, 5, 6, 7 days, or 1 to 2 or 3 weeks, or for a longer period of time; Construction.

14. 14. The construct of any one of claims 1 to 13, the tumor cells, the at least one type of live immune cells, and / or the benign cells are prepared using a method that has an engraftment rate of at least 50%, 60%, 70%, 80%, or 90%; Construction.

15. 15. The construct of any one of claims 1 to 14, the construct comprises at least 75%, 80%, 85%, or 90% viable cells (e.g., viable tumor, immune, and / or benign cells) based on the average number of cells within the construct at 1, 2, 3, or 4 weeks of culture; Construction.

16. 16. The construct of any one of claims 1 to 15, further comprising a hydrogel, Construction.

17. 17. The construct of any one of claims 1 to 16, the construct has a total cell number in the range of about 1 million to about 5 million, 10 million, 25 million, 50 million, 70 million, or 100 million cells; Construction.

18. 1. An in vitro cellular construct (e.g., an "organoid") useful as an immune system model, comprising: Contains multiple live immune cells Construction.

19. 19. The construct of claim 18, The plurality of live immune cells is collected and / or obtained from the subject's lymph nodes (e.g., lymph node biological sample), peripheral blood, and / or bone marrow. Construction.

20. 20. The construct of claim 18 or 19, the plurality of live immune cells comprises follicular dendritic lymphocytes, fibroblastic reticular lymphocytes, leukocytes, B cells, T cells, any myeloid cells (e.g., any of myeloid origin, including dendritic cells and phagocytes), and / or any cells of lymphoid origin; Construction.

21. 21. A construct according to claims 18 to 20, the plurality of immune cells comprises leukocytes; Construction.

22. 22. The construct of claims 18 to 21, further comprising benign cells (e.g., vascular endothelial cells, stromal cells, etc.); wherein the benign cells may be collected and / or obtained from the same subject from which the plurality of immune cells are obtained and / or derived; Construction.

23. 23. The construct of claims 18 to 22, the plurality of live immune cells and / or the benign cells, if present, comprise mammalian cells (e.g., human, mouse, rat, monkey, etc.); Construction.

24. 24. The construct of claims 18 to 23, the plurality of live immune cells comprises a detectable compound (e.g., a fluorescent compound); Construction.

25. 25. The construct of claims 18 to 24, The construct is cultured in culture medium for 1, 2, 3, 4, 5, 6, 7 days, or 1 to 2 or 3 weeks, or for a longer period of time; Construction.

26. 26. A construct according to claims 18 to 25, The plurality of live immune cells and / or the benign cells are prepared using a method having an engraftment rate of at least 50%, 60%, 70%, 80%, or 90%. Construction.

27. 27. The construct of claims 18 to 26, the construct comprises at least 75%, 80%, 85%, or 90% viable cells (e.g., viable immune cells and / or benign cells) based on the average number of cells in the construct at 1, 2, 3, or 4 weeks of culture; Construction.

28. 28. The construct of claims 18 to 27, further comprising a hydrogel, Construction.

29. 29. The construct of claims 18 to 28, the construct has a total cell number in the range of about 1 million to about 5 million, 10 million, 25 million, 50 million, 70 million, or 100 million cells; Construction.

30. 1. A method for screening compounds of interest in vitro for immune activity and / or modulating the immune system, comprising: providing at least one organoid comprising a plurality of cells (e.g., the at least one organoid may be a liver organoid, a cardiac organoid, a tumor organoid, an immune system organoid, etc.); contacting said compound of interest with said at least one organoid in vitro; and In response to contacting the at least one organoid in vitro with the compound of interest, determining the immune response of the at least one organoid (e.g., compared to the immune response / activity of the organoid before and / or in the absence of contact with the compound of interest); Including, method.

31. 31. The method of claim 30, The at least one organoid is a construct according to any one of claims 1 to 29. method.

32. 31. The method of claim 30, The at least one organoid comprises a plurality of benign cells (e.g., liver cells, heart cells, brain cells, etc.); method.

33. 33. The method of any one of claims 30 to 32, The at least one organoid comprises a plurality of viable tumor cells. method.

34. 34. The method of any one of claims 30 to 33, The at least one organoid comprises a plurality of at least one type of immune cell. method.

35. 35. The method of any one of claims 30 to 34, contacting the at least one organoid with a culture medium; wherein the culture medium may comprise at least one type of immune cell, method.

36. 36. The method of any one of claims 30 to 35, contacting the at least one organoid with a culture medium containing leukocytes; wherein the leukocytes may be of the same origin as the plurality of cells. method.

37. 37. The method of any one of claims 30 to 36, the target compound comprises a chemotherapeutic agent and / or an immunotherapeutic agent; wherein the target compound may be an immunotherapeutic agent (e.g., vemurafenib, ipilimumab, nivolumab, and / or pembrolizumab); method.

38. 38. The method of any one of claims 30 to 37, The target compound is a checkpoint inhibitor (e.g., a PD-1 inhibitor, a CTLA-4 inhibitor, etc.), method.

39. 39. The method of any one of claims 30 to 38, The target compound includes an engineered immune cell (e.g., a CAR T cell). method.

40. 40. The method of claim 39, the at least one organoid comprises living tumor cells, and determining a reduction in proliferation of tumor cells present within the at least one organoid (e.g., lack of cell proliferation, cell death, reduction in cell invasion, etc.) in response to contacting the at least one organoid in vitro with the compound of interest; method.

41. 41. The method of any one of claims 30 to 40, determining the immune response of the at least one organoid includes determining cell migration (e.g., determining the migration of at least one type of immune cell), which may use live cell tracking; method.

42. 41. The method of any one of claims 30 to 40, determining the immune response of the at least one organoid includes determining the presence of activated and / or depleted and / or T cell dysfunction markers on cells (e.g., cells present within the organoid and / or migratory cells), which may be performed using immunofluorescence; method.

43. 43. The method of any one of claims 30 to 42, In response to contacting the compound of interest with the at least one organoid in vitro, the compound of interest activates and / or increases the immune activity of at least one type of cell (e.g., immune cell); method.

44. 44. The method of any one of claims 30 to 43, In response to contacting the compound of interest with the at least one organoid in vitro, there is a reduction in proliferation (e.g., lack of cell proliferation, cell death, reduction in cell infiltration, etc.) of cells (e.g., tumor cells) present within the at least one organoid. method.

45. 45. The method of any one of claims 30 to 44, the at least one organoid comprises viable tumor cells; The method further includes determining a reduction in proliferation of the viable tumor cells (e.g., lack of tumor cell proliferation, tumor cell death, reduction in invasion by tumor cells, etc.), which indicates anti-tumor activity of the compound of interest. method.

46. 46. ​​The method of claim 45, determining a reduction in viable tumor cell proliferation; determining the number of viable tumor cells within said at least one organoid; determining the number of viable tumor cells present within said at least one organoid; Determining the volume of said at least one organoid; and / or Determining the number of dead tumor cells present in said at least one organoid. Including, method.

47. 47. The method of any one of claims 30 to 46, The at least one organoid comprises at least one type of immune cell, and / or the at least one organoid has been contacted with at least one type of immune cell, wherein determining the immune response of the at least one organoid comprises determining the activity of the at least one type of immune cell; method.

48. 48. The method of any one of claims 30 to 47, Determining the immune response of the at least one organoid includes determining the presence of an immunological marker (e.g., determining the presence of a T cell activation marker); method.

49. 49. The method of any one of claims 30 to 48, The method further comprises calculating the dose and / or infusion rate of the compound of interest to administer to a subject in response to determining the immune response of the at least one organoid; method.

50. 50. The method of any one of claims 30 to 49, and further comprising administering to the subject a therapeutically effective amount of said compound of interest, wherein said compound of interest is determined to reduce metastasis of said tumor cells in vitro, reduce organoid size in vitro, reduce tumor cell number in vitro, induce tumor cell death in vitro, increase immune cell activity in vitro, and / or activate immune cells in vitro. method.

51. 1. A method for screening compounds of interest for antitumor activity in vitro, comprising: Providing at least one construct according to any one of claims 1 to 17; contacting said compound of interest with said construct in vitro; and determining the proliferation of viable tumor cells in response to contacting the compound of interest with the construct in vitro (e.g., compared to tumor cells of at least one similar construct that have not been contacted with the compound of interest and / or compared to viable benign cells within the construct or at least one similar construct); Including, A reduction in the proliferation of viable tumor cells (e.g., lack of tumor cell proliferation, tumor cell death, reduction in tumor cell invasion, etc.) indicates anti-tumor activity of the compound of interest. method.

52. 52. The method of claim 51, contacting said at least one construct with a culture medium; wherein the culture medium may comprise at least one type of immune cell, method.

53. 53. The method of any one of claims 51 to 52, contacting said at least one construct with a culture medium comprising leukocytes; wherein said leukocytes may be of the same origin as said live tumor cells and / or at least one type of immune cell; method.

54. 54. The method of any one of claims 51 to 53, the target compound comprises a chemotherapeutic agent and / or an immunotherapeutic agent; wherein the target compound may be an immunotherapeutic agent (e.g., vemurafenib, ipilimumab, nivolumab, and / or pembrolizumab); method.

55. 55. The method of any one of claims 51 to 54, The target compound is a checkpoint inhibitor (e.g., a PD-1 inhibitor, a CTLA-4 inhibitor, etc.), method.

56. 56. The method of any one of claims 51 to 55, The target compound includes an engineered immune cell (e.g., a CAR T cell). method.

57. 57. The method of any one of claims 51 to 56, further comprising determining the immune response of said at least one construct; wherein determining the immune response of the at least one construct may include determining cell migration (e.g., determining the migration of at least one type of immune cell), and may use live cell tracking; method.

58. 58. The method of claim 57, determining the immune response of the at least one construct includes determining the presence of activated and / or exhausted T cell dysfunction markers on cells (e.g., cells present within the organoid and / or migratory cells), which may be performed using immunofluorescence; method.

59. 59. The method of any one of claims 51 to 58, In response to contacting the compound of interest with the at least one construct in vitro, the compound of interest activates and / or increases the immune activity of the least one type of immune cell. method.

60. 60. The method of any one of claims 51 to 59, determining a reduction in viable tumor cell proliferation; determining the number of viable tumor cells within said at least one construct; determining the number of viable tumor cells present within said at least one construct; determining the volume of said at least one construct; and / or determining the number of killed tumor cells present within said at least one construct. Including, method.

61. 61. The method of any one of claims 51 to 60, The at least one organoid comprises at least one type of immune cell, and / or the at least one organoid has been contacted with at least one type of immune cell, wherein determining the immune response of the at least one organoid comprises determining the activity of the at least one type of immune cell; method.

62. 62. The method of any one of claims 51 to 61, calculating a dose and / or infusion rate of said compound of interest for administering said subject in response to determining said viable tumor cell proliferation; method.

63. 63. The method of any one of claims 51 to 62, and further comprising the step of administering to a subject a therapeutically effective amount of said compound of interest, which may be determined to reduce tumor cell metastasis in vitro, reduce construct size in vitro, reduce tumor cell number in vitro, induce tumor cell death in vitro, increase immune cell activity in vitro, and / or activate immune cells in vitro. method.

64. 1. A method for screening compounds of interest in vitro for immune activity, modulating the immune system, anti-metastatic activity, and / or anti-tumor activity, comprising: providing a device containing immune cell organoids and live tumor cell organoids; contacting the immune cell organoids and the live tumor cell organoids with a growth medium; contacting the immune cell organoids and / or the living tumor cell organoids with a compound of interest (e.g., by adding the compound to a growth medium); and determining proliferation of the live tumor cells in response to contacting the immune cell organoids and / or the live tumor cell organoids with the compound of interest (e.g., compared to tumor cells in at least one similar live tumor cell organoid that has not been contacted with the compound of interest and / or compared to live benign cells within the organoids or at least one similar live tumor cell organoid); A reduction in the proliferation of viable tumor cells (e.g., lack of tumor cell proliferation, tumor cell death, reduction in tumor cell invasion, etc.) indicates anti-tumor activity of the compound of interest. and / or Determining the immune response of the immune cell organoids and / or the live tumor cell organoids (e.g., compared to the immune response / activity of the immune cell organoids and / or the live tumor cell organoids before and / or in the absence of contact with the compound of interest). Including, method.

65. 65. The method of claim 64, The device comprises one or more chambers, and the immune cell organoids and the live tumor cell organoids are present in the same chamber; wherein the chamber (i.e., the chamber containing the immune cell organoids and the liver tumor cell organoids) may comprise two or more zones, and at least a portion of the live tumor cells of the tumor cell organoids may be present in a first zone and at least a portion of the immune cells of the immune cell organoids may be present in a second zone; method.

66. 65. The method of claim 64, The device comprises one or more chambers, and the immune cell organoids and the live tumor cell organoids are in separate chambers that are in fluid communication. method.

67. 67. The method of any one of claims 64 to 66, The step of contacting the immune cell organoids and the live tumor cell organoids with the growth medium may comprise circulating the growth medium, and the growth medium may be circulated from a first chamber containing the immune cell organoids to a second chamber containing the live tumor cell organoids. method.

68. 68. The method of any one of claims 64 to 67, the growth medium comprises at least one type of immune cell (e.g., leukocyte); wherein said at least one type of immune cells may be collected and / or obtained from the same subject as the immune cells and / or tumor cells in said immune cell organoids and said live tumor cell organoids, respectively; method.

69. 69. The method of any one of claims 64 to 68, The device may further include a third organoid (e.g., a liver organoid) comprising cells different from the live tumor cell organoid and immune cell organoid, which may be present in a separate chamber of the device; method.

70. 70. The method of any one of claims 64 to 69, Further comprising determining a reduction in the presence (e.g., a change in number or density) of tumor cells within said live tumor cell organoids compared to the number of tumor cells present within said live tumor cell organoids when the test compound is not administered; method.

71. 71. The method of any one of claims 64 to 70, The living tumor cell organoid is a construct according to any one of claims 1 to 17; method.

72. 72. The method of any one of claims 64 to 71, The immune cell organoid is a construct according to any one of claims 18 to 29; method.

73. 73. The method of any one of claims 64 to 72, the target compound comprises a chemotherapeutic agent and / or an immunotherapeutic agent; wherein the target compound may be an immunotherapeutic agent (e.g., vemurafenib, ipilimumab, nivolumab, and / or pembrolizumab); method.

74. 74. The method of any one of claims 64 to 73, The target compound is a checkpoint inhibitor (e.g., a PD-1 inhibitor, a CTLA-4 inhibitor, etc.), method.

75. 75. The method of any one of claims 64 to 74, The target compound includes an engineered immune cell (e.g., a CAR T cell). method.

76. 76. The method of any one of claims 64 to 75, determining the immune response includes determining cell migration (e.g., determining the migration of at least one type of immune cell), which may use live cell tracking; method.

77. 77. The method of any one of claims 64 to 76, determining the immune response includes determining the presence of markers of activation and / or immune system dysfunction on cells (e.g., cells present within the organoid and / or migratory cells), and may use immunofluorescence; method.

78. 78. The method of any one of claims 64 to 77, wherein, in response to the step of contacting the target compound with the immune cell organoid and / or the living tumor cell organoid, the target compound activates and / or increases the immune activity of at least one type of immune cell present in and / or around the immune cell organoid and / or the living tumor cell organoid; method.

79. 79. The method of any one of claims 64 to 78, In response to contacting the target compound with the immune cell organoids and / or the live tumor cell organoids, there is a reduction in the proliferation of tumor cells present in the live tumor cell organoids (e.g., lack of tumor cell proliferation, tumor cell death, reduced tumor cell invasion, etc.), which indicates the anti-tumor activity of the target compound. method.

80. 80. The method of claim 79, determining a reduction in viable tumor cell proliferation; Determining the number of viable tumor cells within said live tumor cell organoids; determining the number of viable tumor cells present within said viable tumor cell organoids; Determining the volume of the live tumor cell organoids; and / or Determining the number of dead tumor cells present within said live tumor cell organoids. Including, method.

81. 81. The method of any one of claims 64 to 80, The method for determining the immune response comprises determining the activity of at least one type of immune cell present in and / or around the immune cell organoid and / or the living tumor cell organoid; method.

82. 82. The method of any one of claims 64 to 81, determining the immune response includes determining the presence of an immunological marker (e.g., determining the presence of a T cell activation marker); method.

83. 83. The method of any one of claims 64 to 82, comprising: calculating a dose and / or infusion rate of said compound of interest for administering said subject in response to determining said viable tumor cell proliferation; method.

84. 84. The method of any one of claims 64 to 83, and optionally, administering a therapeutically effective amount of said compound of interest to a subject, wherein said compound of interest is determined to reduce tumor cell metastasis in vitro, reduce organoid size in vitro, reduce tumor cell number in vitro, induce tumor cell death in vitro, increase immune cell activity in vitro, and / or activate immune cells in vitro. method.

85. 1. A method for activating immune cells ex vivo, comprising: The method comprises: contacting immune cells (e.g., lymphocytes) with live tumor cell organoids comprising a plurality of live tumor cells to provide activated immune cells (e.g., via adaptive immune mechanisms); Isolating the activated immune cells from the live tumor cell organoids to provide isolated activated immune cells; and Expanding the isolated activated immune cells to provide a population of activated immune cells. Including, method.

86. 86. The method of claim 85, The viable tumor cells are collected and / or obtained from a tumor within the subject. method.

87. 87. The method of claim 85 or 86, The immune cells are collected and / or obtained from lymph nodes (e.g., lymph node biological samples) within the subject. method.

88. 88. The method of any one of claims 85 to 87, The live tumor cells and the immune cells are collected and / or obtained from the same subject. method.

89. 89. The method of any one of claims 85 to 88, The method further comprises contacting leukocytes with the live tumor cell organoids; wherein the leukocytes and the immune cells may be mixed. method.

90. 90. The method of any one of claims 85 to 89, The living tumor cell organoid further comprises benign cells (e.g., vascular endothelial cells, stromal cells, etc.); wherein said benign cells may be collected and / or obtained from the same subject from which at least one type of immune cell and / or live tumor cells are obtained and / or derived, method.

91. 91. The method of any one of claims 85 to 90, further comprising a shell surrounding (e.g., encapsulating) said core of live tumor cells; the shell comprises living benign cells (e.g., tissue cells, non-cancerous cells, etc.); wherein said viable benign cells may be collected and / or obtained from the same subject from which at least one type of immune cell and / or viable tumor cells are obtained and / or derived; method.

92. 92. The method of any one of claims 85 to 91, The viable tumor cells include malignant cells. method.

93. 93. The method of any one of claims 85 to 92, the tumor cells, the immune cells, and / or the benign cells, if present, comprise mammalian cells (e.g., human, mouse, rat, monkey, etc.); method.

94. 94. The method of any one of claims 85 to 93, comprising: the tumor cells and / or the immune cells comprise a detectable compound (e.g., a fluorescent compound); method.

95. 95. The method of any one of claims 85 to 94, Prior to the collecting step, the live tumor cell organoids and the immune cells are cultured in culture medium for 1, 2, 3, 4, 5, 6, 7 days, or 1 to 2 or 3 weeks, or longer; method.

96. 96. The method of any one of claims 85 to 95, The living tumor cell organoid is a construct according to any one of claims 1 to 17; method.

97. 97. The method of any one of claims 85 to 96, comprising: The step of contacting the immune cells with the live tumor cell organoids is in the absence of an agent that activates and / or supports the activation of the immune cells. method.

98. 97. The method of any one of claims 85 to 96, comprising: The step of contacting the immune cells with the live tumor cell organoids is in the presence of an agent that activates and / or supports the activation of the immune cells. method.

99. 99. The method of any one of claims 85 to 98, further comprising administering the population of activated immune cells to the patient; wherein the patient may be the same subject from whom one or more cells are obtained and / or derived. method.

Images