Compositions and methods for producing antibody-producing immune organoids

Abstract

The present disclosure relates to the generation of three-dimensional immune organoids that produce antibodies. The present disclosure also provides methods useful for producing such immune organoids.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 389,787, filed on July 15, 2022, the disclosure of which (including any drawings) is incorporated herein by reference in its entirety. Technical Field

[0002] The present disclosure relates to the generation of immune organoids that produce antibodies. For example, the present disclosure relates to three-dimensional immune organoids that can produce antibodies. The present disclosure also provides methods for producing such immune organoids. Background Art

[0003] Secondary immune organoids (such as lymph nodes, tonsils and spleens) are highly structured tissues that dynamically change mechanisms and biological functions in response to antigens. More importantly, the activation of immature B cells in these lymphoid tissues forms subanatomical structures, germinal centers (GCs), which convert B cells into cells that produce antibodies, which is crucial for the development of immune memory. At present, in vivo animal models have been mainly used to understand the development of immune cells, function and screening of immunotherapies for diseases, but this method is costly and has a long turnaround time. In order to solve this problem, those skilled in the art have tried to successfully generate in vitro organoids from secondary lymphoid tissues. However, such organs are subject to source tissues (such as the age of the donor, the type of tissue), stimulation requirements, the need for a large number of starting cells, limited existing cell types, limited antibody function production, and the number of organoids that can be made.

[0004] The disclosure provided herein provides 3D in vitro immune organoids that fully recapitulate the cellular complexity and key functions of in vivo secondary lymphoid organs and can generate complete and robust humoral and cellular responses to antigenic stimulation. Summary of the invention

[0005] The present disclosure presents the research and development results of a new method for developing immune organoids from secondary lymphoid tissue, wherein the immune organoids have the ability to produce antibodies.

[0006] Provided herein are, inter alia, a three-dimensional immune organoid comprising a plurality of self-assembled primary immune cells and a plurality of stem cells obtained from one or more secondary lymphoid organs, wherein the cells are CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+, and CD105+.

[0007] In one embodiment, the one or more secondary lymphoid organs are from the spleen, lymph nodes, Peyer's patches, and MALT.

[0008] In one embodiment, the immune organoid is a human immune organoid.

[0009] In one embodiment, the immune organoid further comprises peripheral blood mononuclear cells.

[0010] In one embodiment, the plurality of immune cells is obtained from living patients, surgical resections, fine needle aspirates, biopsies, and deceased patients.

[0011] In one embodiment, a plurality of immune cells include B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages, or a combination thereof. In one embodiment, B cells include one or more immature B cells, pre-GC B cells, GC B cells, memory B cells, or a combination thereof. In one embodiment, T cells include immature CD4 T cells, memory CD4 T cells, T regulatory cells, T follicular helper cells, immature CD8 cells, memory CD8 cells, γδ T cells, or a combination thereof. In one embodiment, dendritic cells include conventional dendritic cells, plasmacytoid dendritic cells, myeloid dendritic cells, or a combination thereof.

[0012] In one embodiment, the plurality of primary immune cells further comprises one or more of stromal cells and fibroblastic reticular cells.

[0013] In one embodiment, the immune organoid is 8000 μm or less in diameter.

[0014] In one embodiment, the immune organoid comprises a germinal center and / or a B / T cell zone. In one embodiment, the immune organoid is a CXCR4 + 、CD83 + 、Ki67 + and IgD + In one embodiment, the immune organoid is a CD3 + and CD20 + .

[0015] In one embodiment, the immune organoid produces antibodies.

[0016] In one embodiment, the antibody has full humoral function. In one embodiment, the antibody binds to both human and non-human targets. In one embodiment, human targets comprise proteins, sugars, and nucleic acids. In one embodiment, non-human targets comprise infectious disease antigens, venoms, poisons, small molecules.

[0017] The present disclosure also provides a composition comprising a plurality of primary immune cells, 100% of which self-assemble into three-dimensional immune organoids within 24 hours.

[0018] In one embodiment, the plurality of immune cells are obtained from one or more secondary lymphoid organs. In one embodiment, the one or more secondary lymphoid organs are from the spleen, lymph nodes, Peyer's patches, and MALT.

[0019] In one embodiment, the plurality of immune cells are human immune cells.

[0020] In one embodiment, the plurality of immune cells comprises 2×10 6 or fewer cells.

[0021] In one embodiment, the composition further comprises peripheral blood mononuclear cells.

[0022] In one embodiment, the plurality of immune cells is obtained from living patients, surgical resections, fine needle aspirates, biopsies, and deceased patients.

[0023] In one embodiment, the plurality of immune cells comprises B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages or a combination thereof. In one embodiment, the plurality of primary immune cells further comprises one or more stromal cells, fibroblastic reticular cells and stem cells.

[0024] The present disclosure further provides a method for producing a plurality of three-dimensional immune organoids. The method comprises: (a) dissociating tissue from one or more secondary lymphoid organs to produce a plurality of single primary immune cells; (b) 6 (c) culturing the individual plurality of single primary immune cells for 24 hours to generate a multiplex three-dimensional immune organoid, wherein the multiplex immune organoid remains viable for at least 30 days.

[0025] In one embodiment, the method comprises freezing a plurality of single primary immune cells prior to (b).

[0026] In one embodiment, the method comprises thawing a plurality of single primary immune cells and treating them with a ROCK inhibitor after freezing.

[0027] In one embodiment, the immune organoid is a human immune organoid.

[0028] In one embodiment, the secondary lymphoid organs are spleen, lymph nodes, Peyer's patches and MALT. In one embodiment, the secondary lymphoid organs are obtained from living patients, surgical resections, fine needle aspirates, biopsies and deceased patients.

[0029] In one embodiment, a plurality of primary immune cells include B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages or a combination thereof. In one embodiment, a plurality of primary immune cells also include one or more stromal cells, fibroblasts and stem cells.

[0030] In one embodiment, the immune organoid is 8000 μm or less in diameter.

[0031] In one embodiment, the immune organoid is a three-dimensional structure.

[0032] In one embodiment, the immune organoid comprises a germinal center and / or a B / T cell zone. In one embodiment, the immune organoid is a CXCR4 + 、CD83 + 、Ki67 + and IgD + In one embodiment, the immune organoid is a CD3 + and CD20 + .

[0033] In one embodiment, the immune organoid produces antibodies. In one embodiment, the antibodies have full humoral function. In one embodiment, the antibodies bind to human and non-human targets. In one embodiment, human targets include proteins, sugars, and nucleic acids. In one embodiment, non-human targets include infectious disease antigens, venoms, poisons, small molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The features of the present disclosure are particularly set forth in the appended claims. The features and advantages of the present disclosure may be better understood by referring to the following detailed description (which sets forth an illustrative embodiment utilizing the principles of the present disclosure) and the accompanying drawings:

[0035] Figure 1A-1B Showing that stem cell populations exist in immune organoid tissues. Figure 1A Representative flow cytometry staining of stem cell populations contained in representative immune organoid tissues at day 7 culture is shown. Cells shown are pre-gated for viable, single CD45+CD34+CD45RA- cells. Data are shown for two donors. Figure 1B Quantification of stem cell population types in representative immune organoid tissues at day 7 of culture is shown. The frequency of cells was determined by flow cytometry. The values ​​plotted cover two different donors.

[0036] Figures 2A-2D Shown are the various immune cell populations contained in human immune organoid models, including innate and adaptive cells. Figure 2ARepresentative flow cytometric staining of B and T cells contained in a representative immune organoid tissue in 7-day culture is shown. Cells shown are pre-gated for live, single cells. Data are shown for two donors. Figure 2B and 2C Representative flow cytometric staining of immune cell types contained in representative immune organoid tissues at day 7 culture, including dendritic cells, monocytes, macrophages, plasmablasts, and NK cells. Cells shown are pre-gated for live, single cells. Data are shown for two donors. Figure 2D Shown is the quantification of subsets of immune cells in immune organoid tissue at day 7. The frequencies of cells were determined by flow cytometry. The values ​​plotted cover two different donors.

[0037] Figure 3A-3B Showing that immune organoids undergo B cell differentiation upon stimulation. Figure 3A Representative images of flow cytometry staining are shown, which demonstrate representative immune organoid B cell differentiation. All cells shown were previously gated for total B cells (CD3-CD19+CD45+). Phenotypes are shown as: CD38-CD27- naive B cells, CD38-CD27+ memory B cells, CD38+CD27- pre-GC B cells, CD38+CD27+ GC B cells. Drawings from a representative donor are shown. Figure 3B Quantification of B cells present in immune organoids at day 7 immunized with hemagglutinin protein is shown. The frequency of cells was determined by flow cytometry. Cells were previously gated for total B cells (CD3-CD19+CD45+).

[0038] Figures 4A-4E Shown that immune organoids are composed of T cell subtypes. Figure 4A Representative images of flow cytometry staining are shown, demonstrating that day 7 immune organoids are composed of T cells. All cells shown were previously gated for total T cells (CD19-CD3+). Cell types include CD4+T cells and CD8+T cells. Plots are from 2 representative donors. Figure 4B and 4CRepresentative images of flow cytometry staining are shown, showing that immune organoids are composed of T cell subtypes. All cells shown were previously gated for their naive and memory T cell populations for total T cells (CD19-CD3+), and then gated for CD4+T cells and CD8+T cells. Phenotypes are shown as: CD4+CCR7+CD45RA+ naive CD4 T cells, naive CD8 T cells, CD4+CCR7+CD45RA+, CD4+CD45RA-CD45RO+ memory CD4 T cells, CD8+CD45RA-CD45RO+ memory CD8 T cells. Drawings are from 2 representative donors. Figure 4D-4E Representative images of flow cytometry staining are shown, demonstrating that immune organoids are composed of additional T cell subtypes. All cells shown were previously gated for total T cells (CD19-CD3+). Cell types shown are: CD3+CD4+CD25+Tregs, CD3+CXCR5+CD25+T follicular helper cells, CD3+CD27+γδT cells. Plots are from 2 representative donors.

[0039] Figure 5A-5B Immune organoids were shown to be composed of other immune cell types, including dendritic cells, stromal cells, and fibroblastic reticular cells. Figure 5A Representative images of flow cytometry staining are shown, demonstrating that immune organoids are composed of dendritic cells (DCs). Cell types include myeloid DCs (CD14+CD11c+), plasmablastoid DCs (CD123+), conventional DCs (CD11b+CD45+), and CD14+ DCs (CD14+CD11c+). Plots are from day 7 immune organoids, involving 2 representative donors. Figure 5B Representative images of flow cytometry staining are shown, which demonstrate the intracellular composition of immune organoids at day 7. Cell types include stromal cells (CD45-) and fibroblastic reticular cells (CD31+PDPN+). Plots are from representative immune organoids involving 2 representative donors.

[0040] Figures 6A-6C The sizes of immune organoids are shown. Fig. 6A Shown are representative bright field images of immune organoids at day 14. Blue lines indicate the area of ​​a representative subset of organoids, from which the diameter of each organoid was calculated. Figure 6B Shown are the longitudinal diameters of immune organoids calculated from organoid area from day 1 to day 21. Medians and error bars are shown (n=4). Figure 6C Viable cell counts of immune organoids at day 7 are shown, with cell counts ranging from 120,000 cells to 2,000,000 cells.

[0041] Figure 7A-7B Immune organoids were shown to form germinal centers consistent with lymph node function. Fig. 7A Shown are representative bright field images of immune organoids stimulated with hepatitis B vaccine on day 14. The lighter structures outlined in red in the organoids are consistent with germinal center morphology. Figure 7B Confocal microscopy images of germinal centers with B cell (CD20) and T cell (CD3) tissue, plasmablasts (CD138), BCL6+ cells, and PD1+ cells in immune organoids at day 14 are shown.

[0042] Figures 8A-8G Shown that immune organoids have cells consistent with germinal center function. Fig. 8A Representative images of flow cytometry staining are shown, demonstrating that immune organoids consist of B cell and T cell regions. Cells are shown as CD19+ B cells and CD3+ T cells. Plots are from a representative day 14 immune organoid. Figure 8B Representative images of flow cytometry staining are shown, demonstrating that immune organoids are composed of germinal center B cells. Cells are shown gated for CD19+, CD3-, CD45+ cells, and germinal center B cells are CD27+CD38+. Plots are from four representative day 14 immune organoids. Figures 8C-8F Representative images of flow cytometry staining are shown, demonstrating that day 14 immune organoids are composed of cells found in germinal centers, including CD83+, Ki67+ B cells, Ki67+ T cells, IgD+ cells, and CXCR4+ cells. Figure 8G Shown is the quantification of germinal center cells in representative day 14 immune organoids. The frequency of cells was determined by flow cytometry.

[0043] Figures 9A-9B It was shown that immune organoids are composed of both B cells and T cells, which can be regulated under various stimulation conditions. Fig.9A Quantification of immune organoid T cells at day 9 and 16 in unstimulated and 6 different stimulation conditions is shown. N=4 organoids per stimulation condition for one representative donor. The frequency of cells was determined by flow cytometry. Cells were previously gated for total T cells (CD3+CD19-CD45+). Fig. 9B Quantification of immune organoid B cells at day 9 and 16 in unstimulated and 6 different stimulation conditions is shown. N=4 organoids per stimulation condition for one representative donor. The frequency of cells was determined by flow cytometry. Cells were previously gated for total B cells (CD3-CD19+CD45+).

[0044] Figures 10A-10D Demonstrated that immune organoids undergo a complete adaptive immune response. Fig. 10A Shown are representative images of flow cytometry staining, which show that immune organoids experience plasmablast differentiation after vaccination. Drawings are from representative immune organoids of the 0th day and the 14th day of vaccination. All cells shown are previously gated to total B cells (CD19+CD3-CD45+). Phenotype is shown as: CD38-CD27- immature B cells, CD38-CD27+ memory B cells, CD38+CD27- front GC B cells, CD38+CD27+ GC B cells, CD38+CD27+ plasmablasts. Shown are drawings from a representative donor. Fig. 10B Shown are antigen-specific IgG antibodies from organoids immunized under 12 different stimulation conditions at day 4, day 8, day 11, and day 15. Data are shown from one representative donor. Fig. 10C Shown are antigen-specific IgM antibodies from immune organoids stimulated twice under 6 different stimulation conditions at days 4, 8, 11, and 14. Plots from one representative donor are shown. Fig. 10D Shown are antigen-specific IgG antibodies from immune organoids stimulated twice under 6 different stimulation conditions at days 4, 8, 11, and 14. Plots from one representative donor are shown.

[0045] Figures 11A-11D We show that immune organoids can overcome tolerance to generate antibodies against human targets. Fig.11A Shown are influenza-specific IgM and IgG antibodies from day 11 immune organoids from unstimulated control organoids and 12 different influenza hemagglutinin protein stimulation conditions. A plot of one representative donor is shown. Fig. 11B Shown are SARS-CoV-2-specific IgM and IgG antibodies from day 11 immune organoids from unstimulated control organoids stimulated with 12 different SARS-CoV-2 spike protein conditions. A plot of one representative donor is shown. Fig. 11C Shown are myelin-specific IgM and IgG antibodies from day 11 immune organoids from unstimulated control organoids and 8 different myelin protein stimulation conditions. A plot of one representative donor is shown. Fig.11D Shown are NK cell-specific IgM and IgG antibodies from day 11 immune organoids from unstimulated control organoids and 12 different NK cell protein stimulation conditions. A plot of one representative donor is shown.

[0046] Fig. 12A-B shows that immune organoids were formed within 24 hours and remained active for at least 30 days. Fig. 12A Shown are representative bright field images of day 1 immune organoids demonstrating complete formation after 24 hours. Fig. 12B Longitudinal bright-field images of representative immune organoids at days 1, 3, 4, 11, 14, 17, 21, 24, and 28 are shown.

[0047] Fig.13 Shown that immune organoids can also be cultured at an air-liquid interface. Shown are representative bright field images of day 2 and day 4 air-liquid interface (ALI) immune organoids as an alternative method to culture immune organoids. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present disclosure provides, inter alia, three-dimensional immune organoids, comprising a plurality of self-assembled primary immune cells and a plurality of stem cells obtained from one or more secondary lymphoid organs, wherein the stem cells are CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+ and CD105+. These immune organoids solve the problems caused by past attempts at forming immune organoids, namely the difficulty in producing organoids containing a complete immune cell lineage and producing a large number of organoids that can also produce immune responses to multiple antigens. The three-dimensional immune organoids provided herein in particular have excellent properties and are suitable for producing antibodies against a variety of human targets. Some embodiments of the present disclosure relate to a composition comprising a plurality of primary immune cells, 100% of which self-assemble into three-dimensional immune organoids within 24 hours. A method for producing a plurality of three-dimensional immune organoids having the above-mentioned properties is also provided.

[0049] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0050] Although various features of the present disclosure may be described in the context of a single embodiment, such features may also be provided separately or in any suitable combination. Conversely, although the content of the present disclosure may be described herein in terms of different embodiments for clarity, the content of the present disclosure may also be implemented in a single embodiment. definition

[0051] Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural references. For example, the term "a cell" includes one or more cells, including mixtures thereof. "A and / or B" is used herein to include all of the following alternatives: "A", "B", "A or B", and "A and B".

[0052] It will be understood that the various aspects and embodiments of the present disclosure described herein include aspects and embodiments of "comprising," "consisting of," and "consisting essentially of." As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unmentioned elements or method steps. As used herein, "consisting of" does not include any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that have no substantial effect on the basic and novel characteristics of the claimed composition or method. Any description of the term "comprising / including" herein, particularly in the description of the composition of the composition or the description of the steps of the method, is understood to include compositions and methods consisting essentially of the described composition or steps.

[0053] When a numerical range is provided, it is understood that, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of the range, separated by one-tenth of the unit of the lower limit, and any other explicitly indicated values ​​or intervening values ​​within the stated range, are included in the disclosure. The upper and lower limits of these smaller ranges may independently be included in these smaller ranges and are also covered by the disclosure, subject to any explicitly excluded limits in the stated range. When the stated range includes one or both limits, ranges excluding either or both of the included limits are also included in the disclosure.

[0054] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of these sub-ranges. Any listed range can be considered to be able to fully describe and make the range be subdivided into at least two equal parts, three equal parts, four equal parts, five equal parts, ten equal parts, etc. As a non-limiting example, each range discussed herein can be easily subdivided into the lower third, the middle third, and the upper third, etc. As will be understood by those skilled in the art, all expressions such as "at most", "at least", "greater than", "less than" and the like include the listed numbers and refer to the ranges that can be subsequently subdivided into sub-ranges as described above. Finally, as will be understood by those skilled in the art, a range contains separate members. Therefore, for example, a group with 1-3 articles refers to a group with 1, 2 or 3 articles. Similarly, a group with 1-5 articles refers to a group with 1, 2, 3, 4 or 5 articles, and so on.

[0055] It should be understood that certain features of the present disclosure, which are described in different embodiments for the sake of clarity, may also be provided in combination in a single embodiment. Conversely, individual features of the present disclosure, which are described in the context of a single embodiment for the sake of brevity, may also be provided separately or in any suitable sub-combination. All combinations of embodiments related to the present disclosure are specifically covered by the present disclosure and are disclosed herein, just as each and every combination is individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and elements thereof are also specifically covered by the present disclosure and are disclosed herein, just as each such sub-combination is individually and explicitly disclosed herein.

[0056] Although the various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. On the contrary, although the present disclosure is described in the context of different embodiments for the sake of clarity, the present disclosure may also be implemented in a single embodiment. Any published patent application cited herein and any other published references, documents, manuscripts and scientific literature are incorporated herein by reference for any purpose. In the event of a conflict, the present specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be limiting. Summary of the invention

[0057] As described in more detail below, one aspect of the present disclosure relates to a three-dimensional immune organoid comprising a plurality of self-assembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality of stem cells, wherein the stem cells are CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+, and CD105+. A composition comprising a plurality of primary immune cells is also provided, 100% of which self-assemble into a three-dimensional immune organoid within 24 hours. Immune organoids

[0058] As described in more detail below, one aspect of the present disclosure relates to a three-dimensional immune organoid comprising a plurality of self-assembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality of stem cells, wherein the stem cells are CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+, and CD105+.

[0059] As used herein, a "three-dimensional immune organoid" can be a composition of living immune cells arranged in a three-dimensional or multilayered configuration (as opposed to a monolayer).

[0060] In some embodiments, the immune organoids are generated ex vivo.One skilled in the art will readily appreciate that the immune organoids described herein are also non-naturally occurring.

[0061] Typically, an organoid is an artificial construct created in vitro to mimic or simulate the function and / or histological structure of an organ, tissue, or a portion thereof. As used herein, an organoid can be a cell structure obtained by amplification of immune cells and stem cells, and is composed of self-organized tissue-specific cell types. In the present disclosure, the term "organoid" can be used to refer to normal (e.g., non-tumor) organoids. An organoid may contain one or more (e.g., 1, 2, 3, 4, or more) differentiated cell types, depending on the specific tissue and / or organ being modeled or simulated. Immune cells

[0062] The immune cells comprising the three-dimensional immune organoids of the present disclosure are derived from one or more secondary lymphoid organs. As used herein, "derived from" generally refers to the source of the primary cells that form the organoids. In some embodiments, "derived from one or more secondary lymphoid organs" may refer to that the organoids are formed without any passage of primary cells from the secondary lymphoid organs. In some embodiments, "derived from one or more secondary lymphoid organs" may refer to that the organoids are formed after 1 passage of the primary cells. In some embodiments, "derived from one or more secondary lymphoid organs" may refer to that the organoids are formed after more than 1 passage of the primary cells. Secondary lymphoid organs are sites for initiating adaptive immune responses and maintaining lymphocytes. Exemplary secondary lymphoid organs include lymph nodes (LN), spleen, Peyer's patches (PP) and mucosa-associated lymphoid tissue (MALT), adenoids and tonsils. In some embodiments, primary immune cells are obtained from spleen, lymph nodes, Peyer's patches and / or MALT.

[0063] Secondary lymphoid tissue can be obtained from a mammal (i.e., a donor or patient), such as a human, dog, cat, rabbit, monkey, chimpanzee, cow, pig, or goat. Secondary lymphoid tissue can be taken directly from living patients, surgical resections, fine needle aspirates, biopsies, and deceased patients. In some embodiments, secondary lymphoid tissue is obtained from a human, and thereby results in a human immune organoid.

[0064] In some embodiments, secondary lymphoid tissue is obtained and decomposed by physics, enzymatic or both. In some embodiments, secondary lymphoid tissue is dissociated using proteolytic enzymes and / or collagenases. In some embodiments, secondary lymphoid tissue is enzymatically dissociated using Accutase (StemCell Technologies), Accumax (StemCell Technologies), trypsin, trypsin / EDTA, collagenase, dispase, TrypLE Express (Thermo Fisher), TrypLESelect (Thermo Fisher) or any combination thereof. In some embodiments, secondary lymphoid tissue is physically dissociated by trimerization, for example, using a pipette. In some embodiments, a single cell suspension of the obtained cells is filtered to remove any undissociated cell mass.

[0065] The primary immune cell derived from secondary lymphoid tissue can be any hematopoietic cell, which is functionally related to the initiation and / or execution of innate and / or adaptive immune responses, such as typical CD3 or CD4 positive cells. Exemplary types of primary immune cells include but are not limited to dendritic cells, killer dendritic cells, mast cells, NK cells, plasmablasts, macrophages, B cells or T cells. In some embodiments, a plurality of primary immune cells include B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages or combinations thereof.

[0066] In some embodiments, B cells include one or more naive B cells, pre-GC B cells, GC B cells, memory B cells, or a combination thereof. In some embodiments, B cells include primary naive B cells. In some embodiments, B cells undergo B cell differentiation after stimulation to produce pre-GC B cells, GC B cells, memory B cells, or a combination thereof. In some embodiments, naive B cells are CD38-CD27-. In some embodiments, memory B cells are CD38-CD27+. In some embodiments, pre-GC B cells are CD38+CD27-. In some embodiments, GC B cells are CD38+CD27+.

[0067] In some embodiments, the T cells comprise naive CD4 T cells, memory CD4 T cells, T regulatory cells, T follicular helper cells, naive CD8 cells, memory CD8 cells, γδ T cells, or a combination thereof. In some embodiments, naive CD4 T cells are CD4+CCR7+CD45RA+. In some embodiments, naive CD8 T cells are CD8+CD45RA-CD27+. In some embodiments, memory CD4 T cells are CD4+CCR7+CD45RA+, CD4+CD45RA-CD45RO+. In some embodiments, memory CD8 T cells are CD8+CD45RA-CD45RO+. In some embodiments, T regulatory cells are CD3+CD4+CD25+. In some embodiments, T follicular helper cells are CD3+CXCR5+CD25+. In some embodiments, γδ T cells are CD3+CD27+.

[0068] In some embodiments, the dendritic cells comprise conventional dendritic cells, plasmacytoid dendritic cells, myeloid dendritic cells, or a combination thereof. In some embodiments, the dendritic cells are CD45+CD11b+ dendritic cells.

[0069] In some embodiments, the monocytes are CD14+CD11b+ monocytes.

[0070] In some embodiments, the macrophages are CD14+ macrophages.

[0071] In some embodiments, the plasmablast is a CD38+CD27+ plasmablast.

[0072] In some embodiments, the NK cells are CD56+ NK cells.

[0073] In some embodiments, the immune organoids described herein generate and / or modify T cells. For example, new T cell phenotypic changes may occur in immune organoids, such as an increase in T helper cells. In some embodiments, new cytotoxic T cells and / or memory T cells may be generated in immune organoids described herein. Such phenotypic changes may be identified using assays including, but not limited to, flow cytometry to detect identification markers on T cells. For example, cytotoxic T cells may be further identified using the T cell cytotoxicity assay described in the Examples below.

[0074] In some embodiments, the amount of the plurality of immune cells present in the immune organoids of the present disclosure can be about 1%, 5%, 10%, 25%, 50% to about 55%, 60%, 75%, 80%, 90%, or 95% of the total number of cells present in the organoid. In some embodiments, the organoids of the present disclosure contain primary immune cells in an amount of about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the total number of cells present in the organoid. If the organoids of the present disclosure contain primary immune cells in an amount less than 100% of the total number of cells present in the organoid, stem cells, stromal cells, and fibroblastic reticular cells can make up the remaining number / percentage of cells in any suitable amount.

[0075] In some embodiments, B cells are present in the immune organoids of the present disclosure in an amount of about 1%, 5%, 10%, 15% or 20%. In some embodiments, T cells are present in the immune organoids of the present disclosure in an amount of about 1%, 5%, 10% or 15%. In some embodiments, NK cells are present in the immune organoids of the present disclosure in an amount of about 1%, 5%, 10% or 15%. In some embodiments, macrophages are present in the immune organoids of the present disclosure in an amount of about 1%, 2%, 3%, 4% or 5%. In some embodiments, monocytes are present in the immune organoids of the present disclosure in an amount of about 1%, 2%, 3%, 4% or 5%. In some embodiments, dendritic cells are present in the immune organoids of the present disclosure in an amount of about 1%, 2%, 3%, 4% or 5%. In some embodiments, plasmablasts are present in the immune organoids of the present disclosure in an amount of about 1%, 2%, 3%, 4% or 5%. Stem Cells

[0076] As mentioned above, immune organoids also include multiple stem cells. Stem cells are characterized by the presence of markers related to specific epitopes recognized by antibodies, as well as the absence of certain markers identified by the lack of binding of specific antibodies. Stem cells can also be identified by functional assays in vitro and in vivo, particularly assays related to the ability of stem cells to produce multiple differentiated offspring. The stem cells of immune organoids described herein are identified as CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+ and CD105+. Therefore, stem cells can include hematopoietic stem cells. Hematopoietic stem cells refer to a subset of pluripotent stem cells that produce all blood or immune cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineages (T cells, B cells, NK T cells, NK cells). "Stem cells" can refer to cells that maintain self-renewal ability and can be differentiated into a variety of specialized cell types by mitotic cell separation. Stem cells can also include mesenchymal stem cells. Mesenchymal stem cells include stem cells that can be obtained from, for example, bone marrow, peripheral blood, skin, hair root, muscle tissue, endometrium, blood, umbilical cord blood, and primary cultures of various tissues. Mesenchymal stem cells can differentiate into all or several of osteocytes, chondrocytes, and adipocytes.

[0077] Stem cells can be identified using flow cytometry and immunofluorescence as described in the examples below. These methods are known in the art and involve the use of antibodies to detect the presence or absence of various protein markers (e.g., CD34, CD45RA, ITGA3, EPCR, CD90, CD73, and CD105) on the cell surface. Other cell types

[0078] In some embodiments, immune organoids also include peripheral blood mononuclear cells (PBMCs). PBMCs can be isolated from peripheral blood and identified as any blood cell with a round nucleus (i.e., lymphocytes, monocytes, natural killer cells (NK cells) or dendritic cells). The addition of PBMCs provides an increase in the size of the immune system of the organoid. For example, in some embodiments, PBMCs are isolated from the same donor as the secondary lymphoid tissue. In some embodiments, PBMCs are isolated from different donors.

[0079] In other embodiments, the immune organoids also include one or more stromal cells and fibroblastic reticular cells. Stromal cells are cells that make up certain types of connective tissue in the body. Fibroblastic reticular cells are stromal cells found in secondary lymphoid organs. The presence of the two types of cells in the immune organoids described herein contributes to the deposition of the extracellular matrix and the overall structure of the three-dimensional immune organoids. For example, the methods described in the examples in the text can be used to identify stromal cells by CD105+, CD29+, CD44+, CD90+ and CD45-, and fibroblastic reticular cells by PDPN+ and CD31+.

[0080] In some embodiments, stromal cells are present in an immune organoid of the present disclosure at about 0.5%, 1%, 1.5%, or 2%. In some embodiments, fibroblastic reticular cells are present in an immune organoid of the present disclosure at about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. structure

[0081] In some embodiments, the diameter and / or maximum dimension of the immune organoids of the present disclosure is about 8000 μm, 7500 μm, 7000 μm, 7000 μm, 6500 μm, 6000 μm, 5500 μm, 5000 μm, 4500 μm, 4000 μm, 3500 μm, 3000 μm, 2500 μm, 2000 μm, 1500 μm, 1000 μm, 500 μm, 250 μm, 100 μm, 50 μm, or less. In some embodiments, the organoids may contain about 1500, 2000, or 5000 to about 10000, 25000, or 50000 cells in total, or about 1000, 5000, 10000, or 50000 to about 75000, 100000, 150000, 250000, 500000, 750000, 100000, 5000000, or 100000000 cells in total. In some embodiments, the immune organoids of the present disclosure may contain about 1 million, 2 million, or 5 million to about 10 million, 25 million, 50 million, or 100 million cells per mL. In some embodiments, the organoids of the present disclosure may contain about 10 million cells per mL or 20 million cells per mL. In some embodiments, the organoids of the present disclosure may comprise about 5 or 10 million cells per mL to about 15 or 20 million cells per mL. The organoids of the present disclosure may be any suitable three-dimensional shape or multilayer shape. In some embodiments, the organoids of the present disclosure are in the form of spheres. In some embodiments, the organoids of the present disclosure may self-organize in a suspension or culture medium.

[0082] In some embodiments, the immune organoid comprises a germinal center and / or a B / T cell zone. The germinal center (GC) is a subanatomical structure that programs B cells to transform into cells that produce antibodies. Within the GC, B cells undergo somatic mutations in the genes encoding their B cell receptors, which, after successful selection, can lead to the emergence of B cell clones that bind antigens with high affinity. As described in Stebegg et al., "Regulation of the Germinal Center Response," Front. Immunol. 9 (2018), the GC is divided into two different compartments: a light zone and a dark zone. In some embodiments, the immune organoids of the present disclosure comprise both a light zone and a dark zone. The dark zone (DZ) contains a network of reticular cells that produce CXCL12 and is a site for GC B cell proliferation and somatic hypermutation (SHM). Then, the centroblasts, through their expression of CXCR5, follow the CXCL13 gradient to enter the light zone (LZ) and become centroblasts. In the LZ, centrocytes capture antigens presented on follicular dendritic cells, internalize them, process them, and then present them to T follicular helper cells to undergo selection. This process is regulated by T follicular regulatory cells that are also present in the LZ. After receiving survival signals from Tfh cells, centrocytes re-enter the DZ for the next round of proliferation and SHM, and then they leave the GC as memory B cells or plasmablasts that secrete high-affinity antibodies. Therefore, the presence of dark and light areas in the immune organoids of the present disclosure can be determined using techniques including, but not limited to, immunofluorescence as described in the Examples. In some embodiments, the germinal centers in the immune organoids are characterized by CXCR4 + 、CD83 + 、Ki67 + and IgD + In some embodiments, the B and T cell regions in the germinal centers of the immune organoids are characterized by CD3 + and CD20 + . Antibody production

[0083] In some embodiments, the immune organoids of the present disclosure are capable of producing antibodies. Antibodies are proteins used by the immune system to identify and neutralize foreign matter (such as pathogenic bacteria and viruses). Antibodies recognize unique molecules of pathogens, called antigens. Antibodies can come in different types, called isotypes or classes. In placental mammals, there are five antibody classes, called IgA, IgD, IgE, IgG, and IgM, which are further subdivided into subclasses such as IgA1 and IgA2. Therefore, the immune organoids of the present disclosure are capable of producing IgA, IgD, IgE, IgG, IgM, and combinations thereof. The prefix "Ig" stands for immunoglobulin, and the suffix indicates the type of heavy chain contained in the antibody: heavy chain types α (alpha), γ (gamma), δ (delta), ε (epsilon), and μ (mu) produce IgA, IgG, IgD, IgE, and IgM, respectively.

[0084] The antibody isotype of B cells changes during cell development and activation. Immature B cells (which have never been exposed to antigens) only express IgM isotypes in cell surface binding form. B lymphocytes in this state of being ready to respond are called "immature B lymphocytes". Immature B lymphocytes express both surface IgM and IgD. The co-expression of these two immunoglobulin isotypes prepares B cells to respond to antigens. After the cell-bound antibody molecules engage with the antigen, B cells are activated, which causes cell division and differentiation into cells that produce antibodies, which are called plasmablasts. In this form of activation, B cells begin to produce antibodies in secretory form rather than membrane-bound form. Some daughter cells of activated B cells undergo isotype conversion, which is a mechanism that converts the production of antibodies from IgM or IgD to other antibody isotypes, i.e., IgE, IgA or IgG, which have a clear role in the immune system. Therefore, the immune cells of the immune organoids disclosed herein may also encompass those immune cells that have or will undergo the conversion process of isotypes.

[0085] Antibodies are important for the development of humoral immune responses, and are produced by B cells and secreted into the blood and / or lymph in response to antigenic stimuli. In a functional immune response, antibodies specifically bind to antigens on the surface of cells (such as pathogens), marking cells for destruction by phagocytes and / or complement-mediated mechanisms. In short, antibodies participate in several important functions after binding to target cells bound to the antibodies, including antibody-dependent cellular cytotoxicity (ADCC), phagocytosis (opsonization), and complement-dependent cytotoxicity (CDC).

[0086] Thus, in some embodiments, the immune organoids have full humoral functions. Specifically, in some embodiments, the antibodies produced by the immune organoids of the present disclosure can play a role in ADCC. ADCC is an in vitro or in vivo process in which antibodies can bind to antigens on the surface of cells and then engage with immune effector cells through sequences within the Fc domain of the antibody, thereby leading to the release of toxins that can kill the bound cells. ADCC activity can be measured using methods known in the art, including but not limited to the in vitro methods described in the Examples herein.

[0087] In some embodiments, antibodies produced by the immune organoids of the present disclosure can play a role in CDC. CDC refers to an in vitro or in vivo process in which an antibody can bind to an antigen on the surface of a eukaryotic or prokaryotic cell and then engage with the C1q protein through a sequence within the Fc domain of the antibody, thereby initiating the classical complement cascade reaction that can kill the bound cell. CDC activity can be measured using methods known in the art, including but not limited to using in vitro methods as described in the Examples herein.

[0088] In some embodiments, antibodies produced by immune organoids of the present disclosure can play a role in opsonization. Opsonization is a process in which an antibody can bind to an antigen on the surface of a cell and then engage with immune effector cells through sequences within the Fc domain of the antibody, thereby causing the immune cells to engulf, consume, and ultimately kill the cells bound to the antibody. Opsonization can be measured using methods known in the art, including but not limited to using in vitro methods as described in the Examples herein.

[0089] In some embodiments, the immune organoids described herein exhibit at least one of ADCC, CDC, and opsonization activities. In some embodiments, the immune organoids as described herein exhibit at least two of ADCC, CDC, and opsonization activities. In some embodiments, the immune organoids as described herein exhibit all three ADCC, CDC, and opsonization activities.

[0090] In some embodiments, antibodies produced by immune organoids disclosed herein can also undergo a process of somatic hypermutation. Somatic hypermutation refers to a process of enhanced gene mutation, which is believed to require activation-induced cytidine deaminase (AID) and error-prone DNA repair. SHM was originally described by observing that B lymphocytes increased mutations in immunoglobulin gene regions encoding light and heavy chain variable regions after antigen stimulation. For example, Smith et al., Trends Genet. 20: AID is discussed in 224-227 (2004). The presence of somatic hypermutation can be determined by methods including, but not limited to, measuring AID upregulation (such as quantitative PCR). B cell receptor sequencing can also be used to identify a collection of mutations.

[0091] In some embodiments, the immune organoids described herein produce plasmablasts and antigen-specific antibodies against targets to which the patient donor has been exposed (anamortic response) and to which the patient has not been exposed.

[0092] In some embodiments, the immune organoids described herein generate an autoimmune response against a self-antigen.

[0093] In some embodiments, antibodies produced by immune organoids described herein bind to human and non-human targets. Exemplary human targets include, but are not limited to, proteins, sugars, and nucleic acids. Exemplary non-human targets may include, but are not limited to, infectious disease antigens, venoms, poisons, small molecules. Composition

[0094] As described in more detail below, one aspect of the present disclosure relates to a plurality of primary immune cells, wherein 100% of the primary immune cells self-assemble into three-dimensional immune organoids within 24 hours.

[0095] As described above, a plurality of primary immune cells can be obtained from one or more secondary lymphoid organs. Secondary lymphoid organs useful for the compositions and methods of the present disclosure are also described above.

[0096] Secondary lymphoid tissue can be obtained from a mammal (i.e., a donor or patient, such as a human, dog, cat, rabbit, monkey, chimpanzee, cow, pig, or goat). Secondary lymphoid tissue can be obtained directly from living patients, surgical resections, fine needle aspirates, biopsies, and deceased patients. In some embodiments, secondary lymphoid tissue is obtained from a human and human immune organoids are generated therefrom.

[0097] In one embodiment, a plurality of immune cells obtained from one or more secondary lymphoid organoids from a single donor produces about 1,000, about 100,000, about 20,000, about 30,000, about 40,000, about 50,000 organoids. In some embodiments, a plurality of immune cells obtained from one or more secondary lymphoid organs from a single donor produces about 50,000 organoids. This is in contrast to the production of about 25 organoids per donor using previously known techniques.

[0098] In some embodiments, the plurality of primary immune cells self-assembles 100% into a three-dimensional immune organoid within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours.

[0099] The types of immune cells that may be present in the plurality of immune cells are described above. The plurality of immune cells may include approximately 2 x 10 6 , 1.5x10 6 , 1x10 6 , 0.5x10 6 , 0.4x10 6 , 0.3x10 6 , 0.2x10 6 , 0.1x10 6 or less total cells.

[0100] As described above, additional cell types may also be considered for inclusion in the compositions of the present disclosure. Additional cell types include, but are not limited to, PBMCs, stromal cells, and fibroblastic reticular cells. Methods of the present disclosure

[0101] The present disclosure provides a method for producing a plurality of immune organoids. The method comprises: (a) dissociating tissue from one or more secondary lymphoid organs to produce a plurality of single primary immune cells; (b) subjecting 1×10 6 (c) culturing the plurality of single primary immune cells for 24 hours to generate a plurality of three-dimensional immune organoids, wherein the plurality of immune organoids remain viable for at least 30 days.

[0102] Secondary lymphoid tissues useful in the methods of the present disclosure are described above with respect to compositions. In short, secondary lymphoid tissues can be obtained from mammals (i.e., donors or patients, such as humans, dogs, cats, rabbits, monkeys, chimpanzees, cattle, pigs, goats). Secondary lymphoid tissues can be taken directly from living patients, surgical resections, fine needle aspirates, biopsies, and deceased patients. In some embodiments, secondary lymphoid tissues are obtained from humans, and human immune organoids are thereby obtained.

[0103] Tissues can be dissociated by a variety of methods, including but not limited to physical and / or enzymatic treatments. For example, tissue dissociation can be physical (chopping or shearing), or using a single or combined proteolytic enzyme, such as matrix metalloproteinases and / or neutral proteases, such as collagenase, trypsin, dispase, LIBERASE (Boehringer Mannheim), Accumax. Enzymatic hydrolysis can be performed by hyaluronidase and / or pepsin, or a combination of physical and enzymatic methods to dissociate. Methods for tissue dissociation are also described above.

[0104] Using the methods described herein, the dissociation of secondary lymphoid tissue produces a suspension of single primary immune cells. These primary immune cells can be any hematopoietic derived cells that are functionally involved in the initiation and / or execution of innate and / or adaptive immune responses, such as typical CD3 or CD4 positive cells. Exemplary types of primary immune cells include, but are not limited to, dendritic cells, killer dendritic cells, mast cells, NK cells, plasmablasts, macrophages, B cells or T cells and combinations thereof.

[0105] In some embodiments, the method involves freezing a plurality of single primary immune cells prior to step (b).

[0106] In some embodiments, prior to step (c), the frozen immune cells are thawed and treated with a ROCK inhibitor. ROCK is a serine / threonine kinase that functions as a target protein of Rho (there are three isozymes--RhoA, RhoB and RhoC). Exemplary ROCK inhibitors include, but are not limited to, antibodies against ROCK, dominant negative ROCK variants, and siRNA and antisense nucleic acids that inhibit ROCK expression. Other exemplary ROCK inhibitors include, but are not limited to, thiazovivin, Y27632, Fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536, HA-1077, hydroxy-HA-1077, GSK2699962A, SB-772077-B, N-(4-pyridinyl)-N′-(2,4,6-trichlorophenyl)urea, 3-(4-pyridinyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridinyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, pyrintegrin, and blebbistatin.

[0107] As mentioned in this article, 1x10 6 Or less single primary immune cells are in contact with a solid support. The solid support may include a reservoir and / or multiple reservoirs (such as holes in a well plate). One or more reservoirs may be any suitable reservoir or container for containing organoids. In some embodiments, the reservoir is a hole in a well plate (for example, but not limited to a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, a 384-well plate, a perforated plate, a ULA plate, a flat bottom plate, or a V bottom plate). The solid support may also include a base for an air-liquid interface. The air-liquid interface may be an interface exposed to primary immune cells in the culture medium described herein. The primary tissue may be mixed with a gel solution and then poured onto a layer of gel formed in a container with a lower semipermeable support, such as a membrane. . The container is placed in an outer container containing a culture medium so that the gel containing the tissue is not immersed in the culture medium. The primary tissue is exposed to air from the top and to liquid culture medium from the bottom.

[0108] In certain embodiments, the cell culture medium for culturing organoids comprises a serum-supplemented cell culture medium. The characteristics of the culture medium for culturing cells may also include, for example, any growth factors or differentiation-inducing factors that may be present, and a support structure (such as a substrate on a solid surface), if present.

[0109] According to the methods disclosed herein, a plurality of single primary immune cells are cultured for 24 hours to produce a plurality of immune organoids, such that the plurality of immune organoids remain viable for at least 30 days. In some embodiments, the plurality of immune organoids remain viable for 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days. In some embodiments, about 1000, about 100000, about 20000, about 30000, about 40000, about 50000 organoids are produced using tissue from a single donor as described herein.

[0110] In some embodiments, the plurality of immune cells further comprises one or more of stromal cells, fibroblastic reticular cells, and stem cells as described above for the composition.

[0111] In some embodiments, using the methods described herein, the plurality of immune organoids have a diameter and / or maximum dimension of about 8000 μm, 7500 μm, 7000 μm, 7000 μm, 6500 μm, 6000 μm, 5500 μm, 5000 μm, 4500 μm, 4000 μm, 3500 μm, 3000 μm, 2500 μm, 2000 μm, 1500 μm, 1000 μm, 500 μm, 250 μm, 100 μm, 50 μm, or less.

[0112] In some embodiments, using the methods provided herein, the plurality of organoids further contain germinal centers and / or B / T cell regions as described above. In some embodiments, the plurality of immune organoids are CXCR4 + 、CD83 + 、Ki67 + and IgD + In some embodiments, the plurality of immune organoids are CD3 + and CD20 + .

[0113] In some embodiments, multiple immune organoids produce antibodies using the methods provided herein. The various antibody types and functions and the humoral immunity generated thereby are described in detail above. Example

[0114] Although particular alternatives of the present disclosure have been disclosed, it should be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims.Therefore, there is no intention to be limited to the precise abstract and disclosure presented herein. Example 1: Method

[0115] Tissue collection and freezing. For example, whole spleens and whole lymph nodes are collected from deceased donors (of all ages) or living donors (of all ages) and cut into pieces by an approved clinician, placed in cryopreservation solution + 2x Pen / Strep, 1x Normocin, and stored at 4°C for transportation until further processing. Prepare cryopreservation medium and organize the work area. Label the cryotubes with the date, tissue type, batch ID number, and name (link to cryotube label template). Upon arrival at the laboratory, completely immerse the tissue in fresh cryopreservation solution + 2x Pen / Strep, 1x Normocin, and keep unprocessed tissue pieces on ice (4°C). Using a disposable scalpel, carefully cut the tissue into 5mm x 5mm x 5mm pieces and place in a 24-well plate for dissociation. Rinse the tissue with 1mL 0.5mM EDTA and incubate in 1mL 0.5mM EDTA at 37°C for 1 hour (adjust and record changes based on results). The tissue was incubated with 1 mL Accumax at 37°C for 1 hour and the reaction was stopped with 1 mL Complete Medium. Cells were counted using NC-200 and viable cell counts and viability were recorded. Cells were centrifuged at 250 x g for 10 minutes. Cells were aspirated and plated at 10 x 10 6 Cells / mL were resuspended in 1 mL Cryostore. The cells were then transferred to pre-barcoded cryovials. The vials were transferred to Mr. Frosty and stored overnight at -80°C and transferred to LN2 the next day. The barcodes were recorded in the LN2 inventory (LN2 number, storage location number, box number, and arrangement in the box).

[0116] Organoid formation. After thawing, add a ROCK inhibitor (such as Y-27632) to the complete medium. Partially submerge the cryovial in a 37°C water bath and thaw until a small amount of ice remains. Transfer the cells from the cryovial to a 50 mL conical tube. Add 1 mL of complete medium dropwise, stirring between each drop, until 9 mL of complete medium has been added. Spin at 200 x g for 5 minutes. Discard the supernatant and resuspend the cells in 2 mL of complete medium. Count the cells using an NC-200 and spin the tube again while counting (200 x g for 5 minutes). Calculate the volume of the suspension so that each well in the upper chamber reaches 100 μl (a total of 1×10 6 Up to 2×10 6cells). Supplement complete medium with 1 μg / mL BAFF and add 200 μl of supplemented medium per well (e.g., perforated plate, ULA plate, flat-bottom plate, V-bottom plate, or pedestal). Add stimuli directly to the culture before incubation overnight at 37°C. Supplement organoids with complete medium supplemented with BAFF every 3 days.

[0117] Addition of PBMCs. Once established, organoids are supplemented with adaptive cells from PBMCs.

[0118] Flow cytometry. Prepare staining buffer (1x DPBS + 0.5% BSA or 1x DPBS + 1% FBS) and use the Biolegend Spectra Analyzer tool (https: / / www.biolegend.com / en-us / spectra- analyzer) Design the flow assay plate. Remove the plate containing the organoids from the incubator and place in a pre-cleaned biosafety cabinet. Break the organoids apart to form a single cell suspension. Count the cells and place them into the flow tubes. Add 2 mL of staining buffer to each tube. Centrifuge the cells at 500 g for 5 minutes and resuspend them in approximately 50 μL of staining buffer. Add the appropriate amount of primary antibody according to the manufacturer's instructions and incubate at 4°C in the dark for 30 minutes. Wash the samples twice with 2 mL of flow staining buffer, aspirate the supernatant and resuspend the cells in the remaining buffer. Add the secondary antibody according to the manufacturer's instructions and incubate at 4°C in the dark for 30 minutes. Wash the samples twice with 2 mL of flow staining buffer, aspirate the supernatant and resuspend the test samples in 250 μL of staining buffer and the unstained control in 350 μL. Fix the cells with 1% paraformaldehyde if necessary. Prepare single-color compensation controls and analyze the samples.

[0119] Immunofluorescence. Thaw the slides with sections and allow to dry completely. Submerge the slides in PBS for 15 minutes at room temperature to remove OCT. Place the slides in a Sequenza clip while submerged in PBS. Carefully place the clip with the attached slides into the Sequenza rack. Allow PBS to flow over the slides to ensure that the slides are well sealed and that the PBS is not flowing through too quickly. For permeabilization and blocking, add 150 μL of permeabilization buffer to each slide and incubate for 1 hour. Rinse sections 3 times with 150 μL of PBS for 5 minutes each. Add 150 μL of blocking buffer to each section and incubate for 1 hour at room temperature with the slides completely covered.

[0120] Prepare primary antibodies by diluting them to their appropriate concentrations in blocking buffer in an appropriate volume. Add 150 μL of antibody solution to each section and incubate overnight at 4 °C with the sections completely covered.

[0121] Secondary antibodies were prepared by diluting to their appropriate concentrations in blocking buffer, then wrapped in aluminum foil to protect from light and stored at 4° C. DAPI was prepared by diluting to the appropriate concentrations in PBS, then wrapped in aluminum foil to protect from light and stored at 4° C.

[0122] Wash the slides 3 times with PBS for 5 minutes, then add 150 μL of antibody solution to each section and incubate at room temperature for 2 hours with complete coverage. Then wash the slides 3 times with PBS for 5 minutes. Add 150 μL of DAPI to each section and incubate at room temperature for 10 minutes with complete coverage. Wash the slides 3 times with PBS for 5 minutes.

[0123] ELISA. First prepare the assay diluent (1xPBS w / o +Mg and +Ca in 1% BSA) and wash buffer (1XPBS + 0.05% Tween-20). The ELSA plate is coated with a coating buffer diluted from 5X ELISA coating buffer with DI water to 1x working solution. The "1" column and the "2" column are coated with a coating buffer mixed with IgG capture Ab at a dilution of 1:300 (100 μL / well dilution of mixed capture antibody). The remaining wells of the plate are coated with a coating buffer mixed with influenza A recombinant protein at a concentration of 0.1ug / well (100 μL inoculated per well). Cover the plate with a film and place it on a shaker at 20-25°C for 1 hour, or cover the plate with a film and incubate overnight at 4°C. The ELISA plate is blocked by washing with 300 μL of the prepared wash buffer in each well. Repeat until completed, for a total of 4 times. 200 μL of assay diluent was added to each well of the plate and placed on a shaker at room temperature for 1 hour.

[0124] While the plate is sealed, prepare all standards. Dilute the standards to a concentration of 1000 ng / mL with assay diluent. Add 500 μL of assay diluent to each tube. Add 500 μL of AD from the initially prepared standard (200 ng / mL). This is tube 1, and the final concentration is 100 ng / mL. Continue with 2-fold serial dilutions until the last tube. One tube contains only assay diluent.

[0125] Remove the plate from the shaker and wash a total of 4 times. Add the prepared standards to the wells in duplicate at 100 μL and determine the sample dilution. Add the assay diluent to all sample wells of the plate to a total volume of 100 μL / well based on the sample dilution. Place the plate on a shaker at room temperature for 2 hours.

[0126] For the addition of detection antibody-HRP, the detection antibody dilution was prepared at a 1:100,000 dilution. Remove the plate from the shaker and wash a total of 4 times. Add 100 μL of the prepared detection antibody solution to all wells of the plate. Cover the plate with film and place on the shaker for 1 hour.

[0127] The next step is 2TMB substrate incubation and then stop the reaction. Prepare TMB substrate and protect from light. Add 5.5mL of solution to each plate. Remove the plate from the shaker and wash each well with 300μL of prepared wash buffer for a total of 5 times. Add 100μL of prepared TMB solution to each well of the plate. Place the plate in the dark and monitor the color change. When the 4th standard is developed, add 100μL of ELISA STOP solution or (2N H2SO4) to each well.

[0128] Acquire data on a microplate reader. For data analysis, the absorbance values ​​of duplicates should be within 10% of each other. Draw a standard curve for the IgG standard (known concentration) samples, and plot the average absorbance value of each standard at each standard concentration minus the blank value on the vertical axis (Y axis). The corresponding human IgG concentration is plotted on the horizontal axis (X axis) associated with the absorbance value. This is used to infer the concentration of unknown samples.

[0129] Somatic hypermutation. Cells were harvested from day 0 and at comparative endpoints. Cells were washed with FACS buffer and incubated with biotinylated recombinant protein of interest at a concentration of 4 μg / mL in the presence of Fc blocking agent. Additionally, cells were incubated with a panel of fluorescently labeled antibodies to determine B cell lineage (CD38+CD27+). B cells carrying specific proteins with GC or plasmablast phenotype were sorted into 96-well plates. cDNA was collected from individual B cells and labeled with unique DNA barcodes and then pooled per plate. Gene-specific PCR was used to amplify immunoglobulin heavy and light variable regions. Libraries were sent for sequencing and sequences were analyzed. Fastq files were generated by demultiplexing using the MiSeq reporter and quality filtered. Paired sequencing reads were spliced ​​and separated by well ID as well as consensus sequence. Reads of well IDs were clustered into operational taxonomic units. Operational taxonomic unit sequences were analyzed using IMGT High V-QUEST. Clonal families are defined by identical V and J gene usage, and at least 70% amino acid identity in the CDR3 sites of the heavy and light chains.

[0130] Exemplary primer sequences for detecting somatic hypermutation are shown in Table 1 below. Table 1 FW, forward; RV, reverse; GSP, gene-specific primer

[0131] Antibody function. ADCC. To prepare target cells, wash 2x10^5 cells / mL with 5 mL of RPMI 1640 containing 2μg / mL TPCK-trypsin three times. Remove the culture medium and inoculate the virus or pathogen at an MOI of 2. Allow the inoculum to adsorb for 60 minutes at 37°C. Gently wash the cells with 6 mL of serum-free culture medium (RPMI1640) containing 2μg / mL TPCK trypsin. Add 5 mL of culture medium (RPMI1640) containing 2μg / mL TPCK trypsin and 2% calf serum to a T-25 flask. The cells are incubated at 37% for 48 hours. After incubation, extract the target cell sample to assess the incidence of infected cells by the ability of the target cells to produce hemagglutination with turkey RBCs and the ability to bind to polyclonal antibodies. 80-95% of the cells should be infected. Wash 2x10^6 cells twice with PBS. The cells are labeled with PKH67.

[0132] To prepare effector cells (isolation of PBMC), PBMCs were thawed from a biobank. PBMCs were washed twice with PBS. Antibody-dependent cell-mediated cytotoxicity assays (ADCC) were performed in duplicate according to the following layout in a round-bottom 96-well plate, using 5.0×104 labeled target cells contained in 50 μL RPMI 1640 medium per well. 50 μL of antibodies were added to the wells according to the layout and incubated in a 37°C CO2 incubator for 15 minutes. 100 μL of unlabeled normal PBMC effector cells in RPMI 1640 / 0.5% Pen / Strep at a concentration of 2.5x 10^7 cells / mL were added to each well according to the layout. The cells were incubated in a CO2 incubator at 37°C for 2 hours. After 2 hours, 1 μL of the fluorescent dead cell dye 7-aminoactinomycin-D (7-AAD) was added and incubated in the dark at 4°C for 20 minutes. Cells were analyzed on a flow cytometer and a total of 5000 target cells were harvested. The percentage of cell death was determined by analyzing four identifiable cell populations, namely live effector cells (no dye), dead effector cells (7-AAD only), live target cells (PKH-67 only), and dead target cells (PKH-67 and 7-AAD), using software.

[0133] Antibody Function. CDC. Add 20 μL of target cells at a cell concentration of 2x10^5 cells / mL to each well of a 96-well assay plate. Perform a 10-point titration curve of the test antibody using 1:2.5 serial dilutions starting at 1 μg / mL. Add each antibody dilution to the plate to start the reaction. Place the plate on an orbital shaker for 30 seconds and then transfer to a 37°C / 5% CO2 incubator for 15 minutes to allow the cells to hemolyze. Dilute complement 1:18 in complete media and add 25 μL to the appropriate wells. Place the plate on an orbital shaker for an additional 30 seconds and then place in a 37°C / 5% CO2 incubator for 30 minutes. Remove the plate from the incubator and allow it to cool to room temperature for 15 minutes. Add 10 μL of lysis buffer to the lysis control wells. Place the plate on the benchtop for 5 minutes at room temperature. Add 125 μL of media to each well. Centrifuge the plate at 750 RPM for 1 minute. Add 50 μl of enzyme assay diluent to the appropriate well of the white luminescent plate. Add 50 μl of reaction supernatant to the well containing assay diluent. Add 100 μl of 2x enzyme assay reagent to each diluted supernatant. Add 50 μl of 1x assay reagent to each diluted supernatant. Shake the plate for 30 seconds and immediately read the luminescence value on the microplate reader.

[0134] Antibody Function. Antibody-dependent cell-mediated phagocytosis. Target cells were labeled with PKH67 and seeded at 25,000 per well in 96-well plates containing 10% human AB serum. Antibodies of interest were serially diluted to 8 half-log concentrations and added to target cells. Macrophages were added at a 4:1 effector:target ratio. Cells were centrifuged at 500 g for 5 minutes and incubated at 37°C for 4 hours. Cells were resuspended by flicking the tube and incubated with CD11b-APC, CD14-APC, and CD66-PE at the recommended dilutions for 45 minutes in the dark. Cells were washed twice with PBS and the supernatant was aspirated. 1% PFA was added to the wells and stored at 4°C until analysis.

[0135] T cell cytotoxicity assay. Effector (CD8+) cells are prepared by density gradient centrifugation to remove dead cells and resuspended in culture medium. Target cells are prepared by density gradient centrifugation to remove dead cells and then resuspended at the correct density. Resuspend cells in culture medium containing 1:1000 Cell Tracker Deep Red for 30 min at 37°C. Wash cells and resuspend cells in culture medium. Establish co-cultures. Cells are plated at the desired effector:target ratio. Set up the desired co-culture time points for the assay. Make sure one well contains only labeled target cells as a control for spontaneous target cell death. At each desired incubation time point, collect cells, pellet them and resuspend them in annexin V binding buffer mixed with annexin V PE and 7-AAD. Cells are incubated for 15 min at RT and labeled cells are analyzed by flow cytometry within one hour of staining.

[0136] Air-liquid interface immune organoids. A permeable membrane support is inserted into a cell culture plate to construct a compartmentalized cell culture system similar to a transport plate. The gel matrix is ​​prepared by mixing the collagen matrix with 10X concentrated sterile culture medium (Ham's F12) and sterile reconstitution buffer (100mL 0.05N NaOH and 200mM HEPES with 2.2g NaHCO3) at a ratio of 8:1:1 on ice until use. Add reconstitution buffer and mix to avoid bubbles. 1mL of reconstituted collagen solution is added to the plate and placed in a 37°C incubator for 30 minutes to solidify. 1x10^6-2x10^6 cells are seeded into the culture plate chamber on top of the collagen matrix. Complete culture medium is added to the cells so that the liquid height matches the cell height. When the color of the culture medium changes, replace the culture medium.

[0137] Automatic image segmentation for area and diameter. A brightfield image was opened and histogram equalized. The fraction of saturated pixels was set to 0%. Canny edge detection was performed using a Gaussian kernel radius pixel resolution as set in the settings. A maximum filter was run with the radius set to the appropriate value as set in the settings. The filter creates a sliding window that replaces the center pixel with the maximum value of the neighboring pixels. A morphological close was performed with 10 iterations and 3 counts. This filled the remaining small holes in the image. A morphological open was run with 10 iterations and 3 counts. This eliminated small structures such as debris outside the organoid. The outline of the segmented area was superimposed on the original brightfield image and the accuracy of the segmentation was checked. The parameters of the protocol were adjusted if necessary, with a particular focus on adjusting the high threshold setting for the Canny edge detection. Area was calculated using Analyze > Measure. Diameter was calculated using the formula d = 2√(A / π).

[0138] Sandwich antigen-specific IgG ELISA. On the day of sample transfer, place the culture plate containing the supernatant on ice for a few hours to thaw. Prepare the assay diluent, which is also the blocking buffer (1% BSA in 1xPBS w / o +Mg and +Ca). Add 4mL of 7.5% BSA to 26mL of DPBS (30mL total). The assay diluent is stored at 4°C for short-term storage and at -20°C for long-term storage. Prepare the wash buffer (1XPBS+0.05% Tween-20). Prepare 10X wash buffer (10xPBS+0.5% Tween-20) by adding 2.5mL Tween-20 to 500mL 10xPBS. Add 900mL of MiliQ water to 100mL of 10X buffer to prepare 1L 1X wash buffer. Any remaining 10X wash buffer is stored at 4°C for subsequent use.

[0139] Coat ELISA plates. Coating solution (1 μg / mL antibody, 1x coating buffer) is prepared as follows, ensuring a total volume of 100 μL / well. Dilute 5x ELISA coating buffer with DI water to a 1x working solution. Add capture antibody to a final concentration of 1 μg / mL. Add 100 μL / well. Cover the plate with film and incubate on a shaker at 20-25°C with gentle shaking for 1 hour, or incubate overnight at 4°C.

[0140] Block the ELISA plate. Wash the plate by the following steps. Load the wash buffer into the plate washer. Wash the plate 4 times with 250 μL / well of wash buffer. Block the plate with 200 μL / well of assay diluent. Cover the plate with film and incubate on a shaker at room temperature for 1 hour or overnight at 4°C.

[0141] Capture antigen / antibody. Wash the plate 4 times with 250 μL / well of wash buffer. Prepare the capture antigen / antibody solution by following the steps below. Prepare enough solution to ensure a total volume of 100 μL per well. Add the capture antigen / antibody to the assay diluent to achieve a final concentration of 1 μg / mL. Coat the plate by adding 100 μL / well of the prepared antigen / antibody solution. Cover the plate and incubate on a shaker for 1 hour at room temperature.

[0142] Intracellular staining. 6mL fixation / permeabilization working solution was prepared as follows. 1.5mL Foxp3 fixation / permeabilization concentrate was added to 4.5mL Foxp3 fixation / permeabilization diluent. 40mL 1X permeabilization buffer was prepared as follows. 4mL 10X permeabilization buffer was added to 36mL MilliQ water. The dissociated organoid cells were added to two separate test tubes containing 9mL cell culture medium and centrifuged (5min, 350g). The supernatant was poured out. 2E6 cells from each donor were transferred to two 1.5mL test tubes (test tube 1: live / dead only, fixed; test tube 2: whole group, fixed) and centrifuged (5min, 350g). The supernatant was flicked to remove. The cells were resuspended in 200μL of Live-DeadAqua diluted 1:500 in PBS and incubated in the dark for 15 minutes at RT. 2 μL of each surface stain was added to 188 μL PBS + 0.5% BSA (CD20-Pacific Blue, CD3-FITC, CXR4-PerCP-Cy5.5, IgD-PE, CD83-APC, CD45-AF700) from the required 16 / 18 group. When the cells were finished incubating, the surface staining cocktail was added directly to tube 2. 200 μL PBS + 0.5% BSA was added to tube 1. Incubate the cells in the dark at RT for 15 minutes. When the cells were finished incubating, 200 μL PBS + 0.5% BSA was added and spun (5min, 350g). The supernatant was flicked to remove. 400 μL PBS + 0.5% BSA was added and spun again (5 minutes, 350g). The supernatant was flicked to remove. This step was then repeated. Then add 1mL Foxp3 fixation / permeabilization working solution to each test tube, pulse vortex, and incubate at 4°C for 30 minutes. Transfer the cells to two 15mL Falcon tubes. Add 2mL 1X permeabilization buffer to both test tubes and centrifuge (5min, 350g). Flick the supernatant and resuspend the cells in the remaining permeabilization buffer. Add 2μL Ki67-BV605 to the resuspended cells and mix with a pipette. Repeat this step. Flick the supernatant and resuspend the cells in 250μL PBS+0.5% BSA and then perform flow cytometry.

[0143] Sandwich antigen-specific IgM ELISA. ELISA. On the day of sample transfer, place the culture plate containing the supernatant on ice for a few hours to thaw. Prepare the assay diluent, which is also the blocking buffer (1% BSA in 1xPBS w / o +Mg and +Ca). Add 4mL of 7.5% BSA to 26mL of DPBS (30mL total). The assay diluent is stored at 4°C for short-term storage and at -20°C for long-term storage. Then prepare the wash buffer (1XPBS+0.05% Tween-20). Prepare 10X wash buffer (10xPBS+0.5% Tween-20) by adding 2.5mL Tween-20 to 500mL 10xPBS. Add 900mL of MiliQ water to 100mL of 10X buffer to prepare 1L of 1X wash buffer. Any remaining 10X wash buffer is stored at 4°C for subsequent use.

[0144] Coat ELISA plates. Coating solution (1 μg / mL antibody in 1x coating buffer) is prepared as follows, ensuring a total volume of 100 μL / well. Dilute 5x ELISA coating buffer with DI water to a 1x working solution. Add capture antibody to a final concentration of 1 μg / mL and add 100 μL / well. Cover the plate with film and incubate at 20-25°C with gentle shaking on a shaker for 1 hour, or overnight at 4°C.

[0145] Block the ELISA plate. Load the wash buffer in the plate washer and wash the plate 4 times with 250 μL / well of wash buffer. Block the plate with 200 μL / well of assay diluent. Cover the plate with film and incubate on a shaker at room temperature for 1 hour or overnight at 4 °C.

[0146] Then, wash the plate 4 times with 250 μL / well of wash buffer. Prepare the capture antigen / antibody solution by the following. Prepare enough solution to ensure a total volume of 100 μL per well. Add the capture antigen / antibody to the assay diluent to achieve a final concentration of 1 μg / mL. Coat the plate with 100 μL / well of the prepared antigen / antibody solution. Cover the plate and incubate on a shaker for 1 hour at room temperature. Example 2: Stem cell populations are represented in immune organoids

[0147] This example demonstrates that immune organoids contain a population of stem cells that are present only in secondary lymphoid organs and not in the blood. These stem cells proliferate and play a role in lymph node support and function as well as immune organoid support and function.

[0148] Briefly, immune organoids were generated using the methods described in Example 1. Day 7 cultures were stained for stem cell markers and subjected to flow cytometry. Figure 1A and Figure 1B As shown, on day 7, the immune organoids contained CD45+CD34+, CD45+CD34+CD45RA-, CD45+CD34+CD45RA-CD201(EPCR)+CD49c(ITGA3)+, and CD45+CD34+CD45RA-CD201(EPCR)+CD49c(ITGA3)+CD105+CD73+ stem cell populations. Example 3: Human immune organoids contain multiple immune cell populations, including innate cells and adaptive cells

[0149] This example shows that immune organoids are composed of the same diverse immune cell types found in human lymph nodes. Together, these cells recapitulate the structure and function of lymph nodes.

[0150] Briefly, immune organoids were generated using the methods described in Example 1. Day 7 cultures were stained for markers of B cells, T cells, NK cells, macrophages, monocytes, dendritic cells, and plasmablasts. Figure 2A As shown, B cells were identified as the CD19+ population, and T cells were identified as the CD3+ population. Figure 2B The presence of CD11b+CD45+ dendritic cells, CD11b+CD14+ monocytes and CD14+ macrophages is shown. Figure 2C The presence of CD38+CD27+ plasmablasts and CD56+ NK cells is shown. Figure 2D Quantification of immune cell subsets in day 7 immune organoid cultures is shown. Example 4: Immune organoids undergo B cell differentiation after stimulation

[0151] This example demonstrates that immune organoids undergo a variety of different responses upon stimulation, including B cell differentiation.

[0152] Briefly, immune organoids were generated using the methods described in Example 1. Figure 3A Shown are cells that were previously gated for total B cells (CD3-CD19+CD45+) and then stained for CD38-CD27- naive B cells, CD38-CD27+ memory B cells, CD38+CD27- pre-GC B cells, CD38+CD27+ GC B cells. Figure 3BThe results showed that stimulated immune organoids at day 0 were primary naive B cells, which then differentiated into pre-GC B cells, GC B cells, memory B cells, and plasmablasts. These B cell phenotypes are consistent with human B cell responses in lymph nodes to infection, vaccination, or other stimuli. Example 5: Immune organoids are composed of T cell subtypes

[0153] This example demonstrates that immune organoids are composed of T cells, including subtypes consistent with T cell subtypes found in human lymph nodes.

[0154] Briefly, immune organoids were generated using the methods described in Example 1. Figure 4A Shown is a day 7 culture previously gated for total T cells (CD19-CD3+). Cells were then stained for CD4+ and CD8+ T cells by flow cytometry.

[0155] Figure 4B-4C Flow cytometry staining is shown, which indicates that immune organoids are composed of T cell subtypes. All cells shown were previously gated for total T cells (CD19-CD3+), and CD4+T cells and CD8+T cells were subsequently gated to obtain respective naive and memory T cell populations. Phenotypes are shown as: CD4+CCR7+CD45RA+ naive CD4 T cells, naive CD8 T cells, CD4+CCR7+CD45RA+, CD4+CD45RA-CD45RO+ memory CD4 T cells, CD8+CD45RA-CD45RO+ memory CD8 T cells.

[0156] Figure 4D-4E Flow cytometry staining is shown, which indicates that immune organoids are composed of additional T cell subtypes. All cells shown are previously gated for total T cells (CD19-CD3+). Cell types are shown as: CD3+CD4+CD25+Treg, CD3+CXCR5+CD25+T follicular helper cells, CD3+CD27+γδT cells. Example 6: Immune organoids composed of other immune cell types

[0157] This example demonstrates that immune organoids are composed of rarer immune cell types that are critical to human lymph nodes.

[0158] Briefly, immune organoids were generated using the methods described in Example 1. Day 7 cultures were screened for myeloid DCs (CD14+CD11c+), plasmacytoid DCs (CD123+), conventional DCs (CD11b+CD45+), and CD14+ DCs (CD14+CD11c+) ( Figure 5AAs shown in Figure 5b, the day 7 cultures also included cell types such as stromal cells (CD45-) and fibroblastic reticular cells (CD31+PDPN+). Example 7: Size of immune organoids

[0159] This example demonstrates that immune organoid size depends on the number of cells used to form the organoid, as well as on the stimulation conditions. Organoids grow substantially after successful stimulation due to cell proliferation and activation.

[0160] Briefly, immune organoids were generated using the methods described in Example 1. Fig. 6A Day 14 cultures were imaged and diameters measured as shown. Figure 6B As shown, the diameter of the organoids increased from day 1 to day 21. The total number of cells in the organoids also increased ( Figure 6C ). Example 8: Immune organoids form germinal centers

[0161] This example demonstrates that germinal centers with light and dark zones are necessary to recapitulate lymph node biology and cannot be found with current in vitro techniques. The organoid platform described herein uniquely generates germinal centers, thereby recapitulating key features of human biology.

[0162] Briefly, immune organoids were generated using the methods described in Example 1. Day 14 cultures were stimulated with hepatitis B vaccine and organoids were observed under bright field microscopy. Fig. 7A As shown, the light-colored structures outlined in red in the organoids are consistent with the morphology of germinal centers.

[0163] Confocal microscopy was then performed to image germinal centers with B cell (CD20) and T cell (CD3) tissue, plasmablasts (CD138), BCL6+ cells, and PD1+ cells in immune organoids at day 14 ( Figure 7B ). Example 9: Immune tissue cells have cells with the same function as germinal centers

[0164] This example demonstrates that germinal centers need to exist and function. These data show that these germinal centers are fully functional and recapitulate all the necessary aspects of lymph node function, and it is the first organoid technology that can do this.

[0165] Fig. 8A Flow cytometry staining is shown, which shows that the immune organoids are composed of B cell regions and T cell regions. The cells are shown as CD19+ B cells and CD3+ T cells. The plot is from a representative day 14 immune organoid. Flow cytometry was then performed to identify germinal center B cells. Figure 8B As shown, day 14 cultures contained CD27+CD38+ germinal center B cells.

[0166] like Figures 8C-8F As shown, additional cell types were identified in culture at day 14. In particular, the immune organoids at day 14 were composed of cells found in germinal centers, including CD83+, Ki67+ B cells, Ki67+ T cells, IgD+ cells, and CXCR4+ cells.

[0167] like Figure 8G Cell types were quantified as indicated. Example 10: Immune organoids are composed of B cells and T cells that can be regulated under various stimulation conditions.

[0168] This example shows that immune organoids are composed of a large mixture of different immune cells that are activated and can proliferate after stimulation. B cells and T cells are an example of the huge difference in cell abundance after stimulating immune organoids with six different stimulation conditions, some of which preferentially expand the B cell population and some of which preferentially expand the T cell population.

[0169] Briefly, immune organoids were generated using the methods described in Example 1. Figures 9A-9B As shown, cultures on day 9 and day 16 were stimulated under different conditions. The stimulus was a protein stimulus of influenza (hemagglutinin protein in this case), and different adjuvant combinations were input in all 6 different conditions. T cells ( Fig.9A ) and B cells ( Fig. 9B ) frequencies were determined by flow cytometry. Example 11: Immune organoids undergo a complete adaptive immune response

[0170] This example shows that organoids produce plasmablasts and antigen-specific antibodies against targets that have not been exposed to patient donors (anamortic responses) and are new to patients (naive). Antibodies produced by organoids have the ability to class switch, such as from IgM to IgG antibodies, which is consistent with an adaptive immune response.

[0171] In short, as shown in Figure 10, after stimulation, immune organoids undergo a complete immune response, including a complete adaptive immune response. This includes the complete differentiation of B cells from immature B cells into germinal center B cells and mature cells such as memory B cells and plasmablasts. After the induction of plasmablasts, these cells produce antigen-specific antibodies for the targets stimulated by the patient. Immune organoids will produce antibodies for targets previously exposed to the patient in the recall response, and produce antibodies for targets completely new to the patient in the initial response. The antibodies produced by the organoids have antigen specificity and have the ability of class switching, such as from IgM to IgG antibodies, which is consistent with the adaptive immune response. Example 12: Immune organoids can break tolerance to generate antibodies against human targets

[0172] This example demonstrates that immune tolerance is a key attribute of systemic immunity, and being able to simulate tolerance breaking (i.e., autoimmune and allergic diseases) is also a key attribute. There is currently no in vitro platform that can do this in a way that mimics human patients. These data suggest that the organoids described herein generate autoimmune responses against self-antigens, which is key to being able to model these diseases.

[0173] In short, Figures 11A-11D As shown, immune organoids produce IgM and IgG antibodies against foreign antigens, examples of which include influenza and SARS-CoV-2. Immune tolerance is a key attribute of systemic immunity, and immune organoids replicate this feature by not responding to stimulation. Another key attribute of systemic immunity is the ability to break tolerance (i.e., autoimmune and allergic diseases). Under certain stimulation conditions, immune organoids are able to simulate breaking tolerance (a key feature of immune organoids to produce antibodies against human targets for therapeutic purposes (i.e., cancer and autoimmune diseases)), and the organoids have the ability to produce autoimmune responses against self-antigens (myelin), and are the key to being able to simulate autoimmune and allergic diseases for the first time. Example 13: Immune organoids formed within 24 hours and remained viable for at least 30 days

[0174] This example shows that organoids can form their necessary structures and begin to function after one day. Currently, these organs remain viable in culture for 30 days or more.

[0175] Briefly, immune organoids were generated using the methods described in Example 1. After 24 hours ( Fig. 12A ) and until the 28th day ( Fig. 12B ) and observed the organoids using bright-field microscopy. Example 14: Immune organoids can also be cultured at the air-liquid interface

[0176] This example shows that there are many ways to make organoids, including but not limited to air-liquid interface (ALI), scaffolds, 3D printed plates, and hydrogels. These methods have been tested and developed to produce these organoids in suspension as well as in ALI.

[0177] In short, there are many methods for organoid production, including but not limited to air-liquid interface (ALI), scaffolds, 3D printed plates, and hydrogels. We have tested all of these methods on immune organoids and have found that Fig.13 As shown, methods have been developed to generate these organoids in suspension as well as in ALI, a common organoid generation method. This is a method in which the organoids are plated on a solid base (such as collagen) to feed the organoids so that they are cultured at the surface of the culture medium, with the majority of the organoid submerged in the culture medium but with the top exposed to air. This method has several benefits, including increased oxygen permeation into the organoid and the use of liquid to manipulate the morphology and height of the organoid to enable some interesting functions and improved imaging capabilities. This is an additional method by which immune organoids can be made.

[0178] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0179] No admission is made that any reference cited herein constitutes prior art. The discussion of these references states what their authors assert, and the applicant reserves the right to challenge the accuracy and relevance of the cited documents. It should be expressly understood that although a number of sources of information are cited herein, including scientific journal articles, patent documents and textbooks, such citation does not constitute an admission that any of these documents constitutes part of the common general knowledge in the field.

[0180] The discussion of the general method herein is for illustrative purposes only. After reading the present disclosure, those skilled in the art will clearly understand other alternative methods and options, and these should be included in the spirit and scope of the present application.

[0181] Various patents, patent applications, and other types of publications (such as journal articles, electronic database entries, etc.) are cited throughout this specification. The disclosures of all patents, patent applications, and other publications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. A three-dimensional immune organoid, comprising a plurality of self-assembled primary immune cells obtained from one or more secondary lymphoid organs and a plurality of stem cells, wherein the stem cells are CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+ and CD105+.

2. The immune organoid of claim 1, wherein the one or more secondary lymphoid organs are from spleen, lymph nodes, Peyer's patches and MALT.

3. The immune organoid according to any one of the preceding claims, wherein the immune organoid is a human immune organoid.

4. The immune organoid according to any one of the preceding claims, further comprising peripheral blood mononuclear cells.

5. The immune organoid of any of the preceding claims, wherein the plurality of immune cells are obtained from living patients, surgical resections, fine needle aspirates, biopsies, and deceased patients.

6. The immune organoid of any of the preceding claims, wherein the plurality of immune cells comprises B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages, and combinations thereof.

7. The immune organoid of claim 6, wherein the B cells comprise one or more naive B cells, pre-GC B cells, GC B cells, memory B cells, or a combination thereof.

8. The immune organoid of claim 6, wherein the T cells comprise naive CD4 T cells, memory CD4 T cells, T regulatory cells, T follicular helper cells, naive CD8 cells, memory CD8 cells, γδ T cells, or a combination thereof.

9. The immune organoid of claim 6, wherein the dendritic cells comprise conventional dendritic cells, plasmacytoid dendritic cells, myeloid dendritic cells, or a combination thereof.

10. The immune organoid of any of the preceding claims, wherein the plurality of primary immune cells further comprises one or more stromal cells and fibroblastic reticular cells.

11. The immune organoid of any preceding claim, wherein the immune organoid has a diameter of 8000 μm or less.

12. The immune organoid of any one of the preceding claims, wherein the immune organoid comprises germinal centers and / or B / T cell zones.

13. The immune organoid of claim 12, wherein the immune organoid is a CXCR4 + 、CD83 + 、Ki67 + and IgD + .

14. The immune organoid according to claim 12, wherein the immune organoid is a CD3 + and CD20 + .

15. The immune organoid of any preceding claim, wherein the immune organoid produces antibodies.

16. The immune organoid of claim 15, wherein the antibody has full humoral function.

17. The immune organoid of claim 16, wherein the antibodies bind to both human and non-human targets.

18. The immune organoid of claim 17, wherein the human targets comprise proteins, sugars, and nucleic acids.

19. The immune organoid of claim 17, wherein the non-human target comprises an infectious disease antigen, a venom, a poison, a small molecule.

20. A composition comprising: A plurality of primary immune cells, 100% of which self-assemble into three-dimensional immune organoids within 24 hours.

21. The composition of claim 20, wherein the plurality of immune cells are obtained from one or more secondary lymphoid organs.

22. The composition of claim 21, wherein the one or more secondary lymphoid organs are from spleen, lymph nodes, Peyer's patches and MALT.

23. The composition of any one of claims 20-22, wherein the plurality of immune cells are human immune cells.

24. The composition according to any one of claims 20-23, wherein the plurality of immune cells comprises 2×10 6 or fewer cells.

25. The composition of any one of claims 20-24, further comprising peripheral blood mononuclear cells.

26. The composition of any one of claims 20-25, wherein the plurality of immune cells are obtained from living patients, surgical resections, fine needle aspirates, biopsies, and deceased patients.

27. The composition of any one of claims 20-26, wherein the plurality of immune cells comprises B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages, or a combination thereof.

28. The composition of claim 27, wherein the plurality of primary immune cells further comprises one or more stromal cells, fibroblastic reticular cells, and stem cells.

29. A method for generating a plurality of three-dimensional immune organoids, the method comprising: (a) dissociating tissue from one or more secondary lymphoid organs to produce a plurality of single primary immune cells; (b) Make 1X10 6 or less of the plurality of single primary immune cells are contacted with a solid support; and (c) culturing the plurality of single primary immune cells for 24 hours to generate a plurality of three-dimensional immune organoids, wherein the plurality of immune organoids remain viable for at least 30 days.

30. The method of claim 29, wherein the method comprises freezing the plurality of single primary immune cells prior to (b).

31. The method of claim 30, wherein the method comprises thawing the plurality of single primary immune cells and treating them with a ROCK inhibitor after freezing.

32. The method of any one of claims 29-31, wherein the immune organoid is a human immune organoid.

33. The method according to any one of claims 29-32, wherein the secondary lymphoid organs are spleen, lymph nodes, Peyer's patches and MALT.

34. The method of any one of claims 29-33, wherein the secondary lymphoid organs are obtained from living patients, surgical resections, fine needle aspirates, biopsies, and deceased patients.

35. The method of any one of claims 29-34, wherein the plurality of primary immune cells comprises B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages, or a combination thereof.

36. The method of any one of claims 29-35, wherein the plurality of primary immune cells comprises one or more stromal cells, fibroblastic reticular cells, and stem cells.

37. The method of any one of claims 29-36, wherein the immune organoid has a diameter of 8000 μm or less.

38. The method of any one of claims 29-37, wherein the immune organoid is a three-dimensional structure.

39. The method of any one of claims 29-38, wherein the immune organoid comprises a germinal center and / or a B / T cell zone.

40. The method of claim 39, wherein the immune organoid is a CXCR4 + 、CD83 + 、Ki67 + and IgD + .

41. The method of claim 39, wherein the immune organoid is a CD3 + and CD20 + .

42. The method of any one of claims 29-41, wherein the immune organoid produces antibodies.

43. The method of claim 42, wherein the antibody has full humoral function.

44. The method of claim 42, wherein the antibody binds both human and non-human targets.

45. The method of claim 44, wherein the human target comprises proteins, sugars, and nucleic acids.

46. ​​The method of claim 44, wherein the non-human target comprises an infectious disease antigen, a venom, a poison, a small molecule.

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