Compositions and methods for producing antibody-producing immune organoids

Abstract

The present disclosure relates to the generation of antibody-producing three-dimensional immune organoid.The present disclosure also provides the method useful for the generation of such immune organoid.In particular, provided herein is three-dimensional immune organoid, which comprises a plurality of self-assembled primary immune cells and a plurality of stem cells obtained from one or more secondary lymphoid organs, and described cell is CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+ and CD105+.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 389,787, filed July 15, 2022, the disclosure of which is incorporated herein by reference in its entirety, including any drawings.

[0002] Field The present disclosure relates to the generation of antibody-producing immune organoid.For example, the present disclosure relates to the three-dimensional immune organoid that can produce antibody.The present disclosure also provides the method that is useful for producing such immune organoid. [Background technology]

[0003] background Secondary immune organs, such as lymph nodes, tonsils, and spleens, are highly structured tissues that dynamically change their mechanical and biological functionality in response to antigens. Particularly important is the formation of germinal centers (GCs), quasi-anatomical structures that activate naive B cells in these lymphoid tissues and program their conversion into antibody-producing cells, which are crucial for the development of immunological memory. To date, live animal models have primarily been used to understand immune cell development and function and to screen immunotherapies for disease, but such approaches are costly due to their long turnaround times. To address this issue, those in the art have attempted to effectively generate ex vivo organoids derived from secondary lymphoid tissues. However, such organoids have been limited by, among other issues, their source tissue (e.g., donor age, tissue type), the need for stimulation, the need for a large number of starting cells, the limited cell types present, the limited functionality of the antibodies produced, and the low number of organoids that can be generated.

[0004] The disclosure provided herein provides 3D in vitro immune organoids that fully recapitulate the cellular complexity and critical functions of in vivo secondary lymphoid organs and are capable of generating complete and robust humoral and cellular responses to antigenic stimulation. Summary of the Invention [Means for solving the problem]

[0005] overview The present disclosure demonstrates the development of a novel method for generating immune organoids capable of producing antibodies from secondary lymphoid tissue.

[0006] Provided herein, inter alia, is 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, said cells being 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 organoids are human immune organoids.

[0009] In one embodiment, the immune organoids further comprise peripheral blood mononuclear cells.

[0010] In one embodiment, the plurality of immune cells is obtained from a living patient, a surgical resection, a fine needle aspirate, a biopsy, and a deceased patient.

[0011] In one embodiment, the plurality of immune cells include B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages, and combinations thereof. In one embodiment, the B cells include one or more naive B cells, pre-GC B cells, GC B cells, memory B cells, or combinations thereof. In one embodiment, the T cells include naive CD4 T cells, memory CD4 T cells, regulatory T cells, follicular helper T cells, naive CD8 cells, memory CD8 cells, gamma delta T cells, or combinations thereof. In one embodiment, the dendritic cells include conventional dendritic cells, plasmacytoid dendritic cells, myeloid dendritic cells, or combinations thereof.

[0012] In one embodiment, the plurality of primary immune cells further include one or more 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 includes a germinal center and / or a B / T cell area. In one embodiment, the immune organoid is CXCR4 + , CD83 + , Ki67 + and IgD + . In one embodiment, the immune organoid is CD3 + and CD20 + .

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

[0016] In one embodiment, the antibodies have full humoral functionality. In one embodiment, the antibodies bind to human and non-human targets. In one embodiment, the human targets include proteins, sugars, and nucleic acids. In one embodiment, the non-human targets include infectious disease antigens, venoms, poisons, small molecules.

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

[0018] In one embodiment, the plurality of immune cells is obtained from one or more secondary lymphoid organs, hi 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 is 2×10 6 Contains 10 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 a living patient, a surgical resection, a fine needle aspirate, a biopsy, and a deceased patient.

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

[0024] The present disclosure further provides a method for producing multiple three dimensional immune organoids, the method comprising the steps of: (a) dissociating tissue from one or more secondary lymphoid organs to produce a plurality of single primary immune cells; (b) generating 1×10 6 (c) contacting a plurality of single primary immune cells, or fewer, with a solid support; and (c) culturing the plurality of single primary immune cells for 24 hours to produce a plurality of three-dimensional immune organoids, wherein the plurality of immune organoids remain viable for at least 30 days.

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

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

[0027] In one embodiment, the immune organoids are human immune organoids.

[0028] In one embodiment, the secondary lymphoid organs are the 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, the plurality of primary immune cells comprises B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages, and combinations thereof. In one embodiment, the plurality of primary immune cells further comprises one or more of stromal cells, fibroblastic reticular cells, and stem cells.

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

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

[0032] In one embodiment, the immune organoids comprise germinal centers and / or B / T cell areas. In one embodiment, the immune organoids comprise CXCR4 + , CD83 + , Ki67 + and IgD + In one embodiment, the immune organoids are CD3 + and CD20 + is.

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

[0034] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0035] [Figure 1A] Figures 1A-1B show that stem cell populations are represented in immune organoids. Figure 1A shows representative flow cytometry staining of stem cell populations contained in representative immune organoids at day 7 of culture. The cells shown are pre-gated on viable single CD45+CD34+CD45RA- cells. Data shown spans two donors. Figure 1B shows quantification of stem cell types in representative immune organoids at day 7 of culture. Cell frequencies were determined by flow cytometry. Values plotted span two different donors. [Figure 1B] Same as above.

[0036] [Figure 2A]Figures 2A-2D show the diverse immune cell populations, including both innate and adaptive cells, contained in human immune organoids. Figure 2A shows representative flow cytometry staining of B cells and T cells contained in representative immune organoids at day 7 in culture. Cells shown are pre-gated on viable single cells. Data shown spans two donors. Figures 2B and 2C show representative flow cytometry staining of immune cell types contained in representative immune organoids at day 7, including dendritic cells, monocytes, macrophages, plasmablasts, and NK cells. Cells shown are pre-gated on viable single cells. Data shown spans two donors. Figure 2D shows quantification of immune cell subsets represented in immune organoids at day 7. Cell frequencies were determined by flow cytometry. Values plotted span two different donors. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above.

[0037] [Figure 3A] Figures 3A-3B show immune organoids undergoing B cell differentiation upon stimulation. Figure 3A shows representative images of flow cytometry staining demonstrating B cell differentiation in representative immune organoids. All cells shown were previously gated on total B cells (CD3-CD19+CD45+). Phenotypes shown are: CD38-CD27- naive B cells, CD38-CD27+ memory B cells, CD38+CD27- pre-GC B cells, and CD38+CD27+ GC B cells. Plots from one representative donor are shown. Figure 3B shows quantification of B cells present in immune organoids on day 7 after immunization with hemagglutinin protein. Cell frequencies were determined by flow cytometry. Cells were previously gated on total B cells (CD3-CD19+CD45+). [Figure 3B] Same as above.

[0038] [Figure 4A]Figures 4A-4E show immune organoids composed of T cell subtypes. Figure 4A shows representative images of flow cytometry staining demonstrating 7-day-old immune organoids consisting of T cells. All cells shown were pre-gated on total T cells (CD19-CD3+). Cell types include CD4+ T cells and CD8+ T cells. Plots from two representative donors. Figures 4B and 4C show representative images of flow cytometry staining demonstrating immune organoids composed of T cell subtypes. All cells shown were pre-gated on total T cells (CD19-CD3+), and then gated on the respective naive and memory T cell populations of CD4+ T cells and CD8+ T cells. Phenotypes shown: 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. Plots are from two representative donors. Figures 4D-4E show representative images of flow cytometry staining demonstrating immune organoids composed of additional T cell subtypes. All cells shown were pre-gated on total T cells (CD19-CD3+). Cell types shown: CD3+CD4+CD25+ Tregs, CD3+CXCR5+CD25+ follicular helper T cells, CD3+CD27+ gamma delta T cells. Plots are from two representative donors. [Figure 4B] Ibid. [Figure 4C] Ibid. [Figure 4D] Ibid. [Figure 4E] Ibid.

[0039] [Figure 5A]Figures 5A-5B show that immune organoids are composed of other immune cell types, including dendritic cells, stromal cells, and fibroblastic reticular cells. Figure 5A shows a representative image of flow cytometry staining demonstrating immune organoids composed of dendritic cells (DCs). Cell types include myeloid DCs (CD14+CD11c+), plasmacytoid DCs (CD123+), conventional DCs (CD11b+CD45+), and CD14+ DCs (CD14+CD11c+). Plots are from immune organoids at day 7 across two representative donors. Figure 5B shows a representative image of flow cytometry staining demonstrating immune organoid cellular composition at day 7. Cell types include stromal cells (CD45-) and fibroblastic reticular cells (CD31+PDPN+). Plots are from immune organoids at day 7 across two representative donors. [Figure 5B] Same as above.

[0040] [Figure 6A] Figures 6A-6C show the size of immune organoids. Figure 6A shows a representative bright-field image of immune organoids on day 14. The blue line indicates the area of a representative subset of organoids, which was used to calculate the diameter of each organoid. Figure 6B shows the longitudinal diameter of immune organoids calculated from the area of organoids ranging from day 1 to day 21. Median values and error bars are shown (n=4). Figure 6C shows the viable cell count of immune organoids on day 7, ranging from 120,000 cells to 2,000,000 cells. [Figure 6BC] Same as above.

[0041] [Figure 7A]Figures 7A-7B show that the immune organoids form germinal centers that are consistent with lymph node function. Figure 7A shows a representative bright-field image of 14-day-old immune organoids stimulated with the hepatitis B vaccine. The brighter structures outlined in red within the organoids are consistent with the germinal center morphology. Figure 7B shows confocal microscopy images of germinal centers within D14 immune organoids, including B cells (CD20) and T cell (CD3) composition, plasmablasts (CD138), BCL6+ cells, and PD1+ cells. [Figure 7B] Same as above.

[0042] [Figure 8A] Figures 8A-8G show that the immune organoids have cells that are consistent with germinal center function. Figure 8A shows a representative image of flow cytometry staining demonstrating immune organoids consisting of B cell and T cell areas. The cells shown are CD19+ B cells and CD3+ T cells. The plot is from a representative 14-day-old immune organoid. Figure 8B shows a representative image of flow cytometry staining demonstrating immune organoids consisting of germinal center B cells. The cells shown are gated on CD19+, CD3-, CD45+ cells, and germinal center B cells are CD27+CD38+. The plot is from four representative 14-day-old immune organoids. Figures 8C-8F show representative images of flow cytometry staining demonstrating 14-day-old immune organoids consisting of cells found in germinal centers, including CD83+, Ki67+ B cells, Ki67+ T cells, IgD+ cells, and CXCR4+ cells. Figure 8G shows the quantification of germinal center cells within a representative 14-day-old immune organoid. Cell frequency was determined by flow cytometry. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above. [Figure 8F] Same as above. [Figure 8G] Same as above.

[0043] [Figure 9A] Figures 9A-9B show that immune organoids can be modulated in response to various stimulation conditions and are composed of both B cells and T cells. Figure 9A shows the quantification of immune organoid T cells in immune organoids on day 9 and day 16 over non-stimulated and six different stimulation conditions. N = 4 organoids per stimulation condition for a representative 1 donor. Cell frequencies were determined by flow cytometry. Cells were pre-gated on total T cells (CD3+CD19-CD45+). Figure 9B shows the quantification of immune organoid B cells in immune organoids on day 9 and day 16 over non-stimulated and six different stimulation conditions. N = 4 organoids per stimulation condition for a representative 1 donor. Cell frequencies were determined by flow cytometry. Cells were pre-gated on total B cells (CD3-CD19+CD45+). [Figure 9B] The same as above.

[0044] [Figure 10A]Figures 10A-10D show that immune organoids elicit a complete adaptive immune response. Figure 10A shows representative images of flow cytometry staining demonstrating plasmablast differentiation in immune organoids in response to vaccination. Plots are from vaccinated immune organoids at representative D0 and D14. All cells shown were pre-gated on total B cells (CD19+CD3-CD45+). Phenotypes shown: CD38-CD27- naive B cells, CD38-CD27+ memory B cells, CD38+CD27- pre-GC B cells, CD38+CD27+ GC B cells, CD38+CD27+ plasmablasts. Plots from a representative 1 donor are shown. Figure 10B shows antigen-specific IgG antibodies from immune organoids at day 4, 8, 11, 15 across 12 different stimulation conditions. Data shown from a representative 1 donor. Figure 10C shows antigen-specific IgM antibodies from immune organoids at day 4, 8, 11, 14 that were stimulated twice with 6 different stimulation conditions. Plots from a representative 1 donor are shown. Figure 10D shows antigen-specific IgG antibodies from immune organoids at day 4, 8, 11, 14 that were stimulated twice with 6 different stimulation conditions. Plots from a representative 1 donor are shown. [Figure 10B] Ibid. [Figure 10C] Ibid. [Figure 10D] Ibid.

[0045] [Figure 11A]Figures 11A-11D show that immune organoids can break tolerance and generate antibodies against human targets. Figure 11A shows influenza-specific IgM and IgG antibodies from immune organoids on day 11 from unstimulated control organoids and 12 different influenza hemagglutinin protein stimulation conditions. Plots from a representative 1 donor are shown. Figure 11B shows SARS-CoV-2-specific IgM and IgG antibodies from immune organoids on day 11 from unstimulated control organoids and 12 different SARS-CoV-2 spike protein stimulation conditions. Plots from a representative 1 donor are shown. Figure 11C shows myelin-specific IgM and IgG antibodies from immune organoids on day 11 from unstimulated control organoids and 8 different myelin protein stimulation conditions. Plots from a representative 1 donor are shown. Figure 11D shows NK cell-specific IgM and IgG antibodies from immune organoids on day 11 from unstimulated control organoids and 12 different NK cell protein stimulation conditions. Plots from a representative 1 donor are shown. [Figure 11B] Ibid. [Figure 11C] Ibid. [Figure 11D] Ibid.

[0046] [Figure 12A] Figures 12A-B show that immune organoids form within 24 hours and survive for at least 30 days. Figure 12A shows a representative brightfield image of immune organoids on day 1 demonstrating complete formation after 24 hours. Figure 12B shows longitudinal brightfield images of representative immune organoids on days 1, 3, 4, 11, 14, 17, 21, 24, 28. [Figure 12B] Ibid.

[0047] [Figure 13]Figure 13 shows that immune organoids can also be cultured at the air-liquid interface. Representative bright-field images of air-liquid interface (ALI) immune organoids on days 2 and 4 are shown, as an alternative approach to culturing immune organoids. DETAILED DESCRIPTION OF THE INVENTION

[0048] Detailed Description The present disclosure provides, among others, three-dimensional immune organoids, 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+.These immune organoids address the problems of previous attempts to form immune organoids, due to the difficulty of producing organoids containing complete immune cell repertoire, and the inability to produce a large number of organoids that can also generate immune responses to many antigens.In particular, provided herein are three-dimensional immune organoids that are produced with excellent characteristics and are suitable for producing antibodies against a wide variety of human targets.Some embodiments of the present disclosure relate to a composition comprising a plurality of primary immune cells that 100% self-assemble into three-dimensional immune organoids within 24 hours.Further provided are methods useful for producing a plurality of three-dimensional immune organoids with the above-mentioned characteristics.

[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, those features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may, for clarity, be described herein in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment. definition

[0051] The singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. 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] As used herein, the terms "comprising", "consisting of", and "consisting essentially of" with respect to the aspects and embodiments of the disclosure described herein are to be understood to include those aspects and embodiments. As used herein, "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended, and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic and novel characteristics of the claimed composition or method. In particular, any recitation herein of the term "comprising" in the recitation of components of a composition or steps of a method is to be understood to include compositions and methods consisting essentially of and consisting of the recited components or steps.

[0053] Where a range of values is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, is included within the scope of the disclosure, unless the context clearly dictates otherwise, and that any other specified or intervening value in that specified range is also included within the scope of the disclosure. Unless there are specifically excluded limits in a specified range, the upper and lower limits of these smaller ranges may independently be included in the smaller ranges, which are also included within the scope of the disclosure. When a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0054] All ranges disclosed herein include all possible subranges and combinations of those subranges. Any recited range is fully descriptive, and it is recognizable that the same range can be divided into at least 2, 3, 4, 5, 10, etc. divisions. As a non-limiting example, each range discussed herein can be easily divided into a lower third, a middle third, and an upper third, etc. Similarly, those skilled in the art will understand that all terms, such as "up to," "at least," "greater than," "less than," etc., refer to ranges that are inclusive of the recited numbers and that can be subsequently divided into the subranges discussed above. Finally, those skilled in the art will understand that a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, etc.

[0055] For clarity, it is recognized that certain features of the present disclosure described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features of the present disclosure described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of embodiments of the present disclosure are specifically encompassed by the present disclosure and are disclosed herein as if each and every combination were individually and explicitly disclosed. Further, all sub-combinations of the various embodiments and their elements are also specifically encompassed by the present disclosure and are disclosed herein as if each and every such sub-combination were individually and explicitly disclosed herein.

[0056] The features of the present disclosure may be described in the context of a single embodiment, but those features may also be provided separately or in any suitable combination. Conversely, the present disclosure may be described herein in the context of separate embodiments for clarity, but the present disclosure may also be implemented in a single embodiment. Any published patent application cited herein, as well as any other published reference, document, manuscript, and scientific literature are hereby incorporated by reference for all purposes. In case of conflict, this specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and not intended to be limiting. The compositions of the present disclosure

[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-assembling primary immune cells obtained from one or more secondary lymphoid organs, as well as a plurality of stem cells, the stem cells being CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+ and CD105+. Also provided is a composition comprising a plurality of primary immune cells that self-assemble 100% into three-dimensional immune organoids within 24 hours. Immune organoid

[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 live immune cells arranged in a three-dimensional or multi-layer configuration (as opposed to a monolayer).

[0060] In some embodiments, immune organoid is produced ex vivo.Those skilled in the art will easily understand that the immune organoid described herein does not exist in nature.

[0061] Organoid is generally the artificial construct that is created in vitro to mimic or resemble the function and / or histological structure of organ, tissue or their part.Organoid as used herein can be the cell structure that is obtained by the expansion of immune cell and stem cell, and is composed of tissue-specific cell types that self-assemble.In the present disclosure, the term " organoid " can be used to refer to normal (for example, non-tumor) organoid.Organoid can comprise one or more (for example, one, two, three, four or more) differentiated cell types according to the specific tissue and / or organ that is model 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 origin of the primary cells that form the organoids. In some embodiments, "derived from one or more secondary lymphoid organs" may mean that the organoids are formed from the secondary lymphoid organs without any passage of primary cells. In some embodiments, "derived from one or more secondary lymphoid organs" may mean that the organoids are formed after one passage of primary cells. In some embodiments, "derived from one or more secondary lymphoid organs" may mean that the organoids are formed after more than one passage of primary cells. Secondary lymphoid organs are sites where adaptive immune responses are initiated and lymphocytes are maintained. Exemplary secondary lymphoid organs include lymph nodes (LNs), spleens, Peyer's patches (PPs), and mucosa-associated lymphoid tissue (MALT), adenoids, and tonsils. In some embodiments, the primary immune cells are obtained from the spleen, lymph nodes, Peyer's patches, and / or MALT.

[0063] Secondary lymphoid tissue can be obtained from mammal (i.e., donor or patient), for example, human, dog, cat, rabbit, monkey, chimpanzee, cow, pig or goat.Secondary lymphoid tissue can be obtained from living patient, surgical resection, needle aspirate, biopsy or directly from dead patient.In some embodiments, secondary lymphoid tissue is obtained from human, thereby resulting in human immune organoid.

[0064] In some embodiments, secondary lymphoid tissue is obtained and dissociated mechanically, enzymatically, or both. In some embodiments, secondary lymphoid tissue is dissociated using proteolytic and / or collagenolytic enzymes. 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), TrypLE Select (Thermo Fisher), or any combination thereof. In some embodiments, secondary lymphoid tissue is mechanically dissociated, for example, by trituration. In some embodiments, the resulting single cell suspension of cells is filtered to remove any undissociated cell clumps.

[0065] Primary immune cells derived from secondary lymphoid tissue can be any cells of hematopoietic origin that are functionally involved in the initiation and / or execution of innate and / or adaptive immune responses, for example, typically 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, the plurality of immune cells includes B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages, and combinations thereof.

[0066] In some embodiments, the B cells include one or more naive B cells, pre-GC B cells, GC B cells, memory B cells, or combinations thereof. In some embodiments, the B cells mainly include naive B cells. In some embodiments, the B cells undergo B cell differentiation upon stimulation to give rise to pre-GC B cells, GC B cells, memory B cells, or combinations 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 include naive CD4 T cells, memory CD4 T cells, regulatory T cells, follicular helper T cells, naive CD8 cells, memory CD8 cells, gamma delta T cells, or combinations 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+ or CD4+CD45RA-CD45RO+. In some embodiments, memory CD8 T cells are CD8+CD45RA-CD45RO+. In some embodiments, regulatory T cells are CD3+CD4+CD25+. In some embodiments, follicular helper T cells are CD3+CXCR5+CD25+. In some embodiments, gamma delta T cells are CD3+CD27+.

[0068] In some embodiments, the dendritic cells include conventional dendritic cells, plasmacytoid dendritic cells, myeloid dendritic cells, or combinations 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 plasmablasts are CD38+CD27+ plasmablasts.

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

[0073] In some embodiments, immune organoids as described herein produce and / or modify T cells.For example, new T cell phenotype changes can occur in immune organoids, for example, the increase of helper T cells.In some embodiments, new cytotoxic T cells and / or memory T cells can be produced in immune organoids as described herein.This phenotype change can be identified by using assays, including but not limited to, flow cytometry, to detect the distinguishing marker on T cell.Cytotoxic T cells can be further identified by using T cell cytotoxicity assays, for example, as described in Examples below.

[0074] In some embodiments, the amount of immune cells present in the immune organoid 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 organoid of the present disclosure comprises primary immune cells in the 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. When 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, any suitable amount of stem cells, stromal cells and fibroblastic reticular cells may make up the remaining number / percentage of cells.

[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 described above, immune organoids also contain multiple stem cells. Stem cells can be characterized by both the presence of markers associated with specific epitopes identified by antibodies and the absence of certain markers identified by the lack of binding of specific antibodies. Stem cells can also be identified by functional assays both in vitro and in vivo, particularly assays related to the ability of stem cells to give rise to multiple differentiated progeny. The stem cells of the immune organoids described herein can be identified as CD34+, CD45RA-, ITGA3+, EPCR+, CD90+, CD73+ and CD105+. Thus, stem cells can include hematopoietic stem cells. Hematopoietic stem cells relate to a subset of pluripotent stem cells that give rise to all blood cell types or immune cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), as well as lymphoid lineages (T cells, B cells, NKT cells, NK cells). "Stem cells" can refer to cells that retain the ability to regenerate themselves by mitotic cell division and can differentiate into a diverse range of specialized cell types. Stem cells can also include mesenchymal stem cells. Mesenchymal stem cells include, for example, stem cells that can be obtained from bone marrow, peripheral blood, skin, hair follicles, muscle tissue, endometrium, blood, umbilical cord blood, and primary cultures of various tissues. Mesenchymal stem cells can differentiate into all or some of osteocytes, chondrocytes and adipocytes.

[0077] As described later in the examples, stem cells can be identified using flow cytometry and immunofluorescence. These methods are known in the art and include 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 surface of cells. Other cell types

[0078] In some embodiments, the immune organoids further comprise peripheral blood mononuclear cells (PBMCs). PBMCs can be isolated from peripheral blood and can be 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 repertoire of the organoids. By way of example, in some embodiments, the PBMCs are isolated from the same donor as the secondary lymphoid tissue. In some embodiments, the PBMCs are isolated from different donors.

[0079] In other embodiments, the immune organoids further comprise one or more stromal cells and fibroblastic reticular cells. Stromal cells are a certain type of cell that make up a particular type of connective tissue in the body. Fibroblastic reticular cells are stromal cells found in secondary lymphoid organs. The presence of both types of cells in the immune organoids described herein contributes to the deposition of the extracellular matrix and the overall architecture of the three-dimensional immune organoids. By way of example, using the methods described in the examples herein, stromal cells can be identified by CD105+, CD29+, CD44+, CD90+ and CD45-, and fibroblastic reticular cells can be identified by PDPN+ and CD31+.

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

[0081] In some embodiments, the immune organoids of the present disclosure 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. In some embodiments, the organoids can contain a total of about 1,500, 2,000 or 5,000 to about 10,000, 25,000 or 50,000 cells, or a total of about 1,000, 5,000, 10,000 or 50,000 to about 75,000, 100,000, 150,000, 250,000, 500,000, 750,000, 1,000,000, 50,000,000 or 100,000,000 cells. In some embodiments, the immune organoids of the present disclosure can 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 can contain about 10 million cells per mL, or about 20 million cells per mL. In some embodiments, the organoids of the present disclosure can contain from about 5 million or 10 million cells per mL to about 15 million or 20 million cells per mL. The organoids of the present disclosure can be in any suitable three-dimensional or multi-layered shape. In some embodiments, the organoids of the present disclosure are in the form of spheroids. In some embodiments, the organoids of the present disclosure may self-assemble in suspension or in a medium.

[0082] In some embodiments, the immune organoid contains germinal centers and / or B / T cell zones. Germinal centers (GCs) are quasi-anatomical structures that program B cell transformation into antibody-producing cells. Within GCs, B cells undergo somatic mutations in genes encoding their B cell receptors, which, following effective selection, can lead to the emergence of B cell clones that bind to antigens with high affinity. As described in Stebegg et al., "Regulation of the Germinal Center Response," Front. Immunol. 9 (2018), GCs are divided into two distinct compartments: light zones and dark zones. In some embodiments, the immune organoids of the present disclosure contain both light zones and dark zones. The dark zone (DZ) contains a network of CXCL12-producing reticular cells and is the site of GC B cell proliferation and somatic hypermutation (SHM). Centroblasts then follow a CXCL13 gradient and invade the light zone (LZ) as central cells due to their expression of CXCR5. In the LZ, central cells capture, incorporate, and process antigens presented on follicular dendritic cells, then present them to follicular helper T cells for selection.This process is regulated by follicular regulatory T cells, which also reside in the LZ.Upon receiving survival signals from Tfh cells, central cells re-enter the DZ for further rounds of proliferation and SHM, and then exit GC as memory B cells or high-affinity antibody-secreting plasma cells.Therefore, the presence of both dark and bright areas in the immune organoids of the present disclosure can be identified using techniques including, but not limited to, immunofluorescence as described in Examples.In some embodiments, germinal centers in immune organoids are CXCR4 + , CD83 + , Ki67 + and IgD + In some embodiments, the B cell and T cell areas in the germinal centers of the immune organoids are characterized as CD3 + and CD20 + It can be characterized as: antibody production

[0083] In some embodiments, the immune organoid of the present disclosure can produce antibodies.Antibodies are proteins used by the immune system to identify and neutralize foreign substances, such as pathogenic bacteria and pathogenic viruses.Antibodies recognize the specific molecules of pathogens, called antigens.Antibodies can appear in various types known as isotypes or classes.In placental mammals, there are five antibody classes known as IgA, IgD, IgE, IgG and IgM, which are further subdivided into subclasses such as IgA1 and IgA2.Therefore, the immune organoid of the present disclosure can produce IgA, IgD, IgE, IgG, IgM, and their combinations.The prefix "Ig" represents immunoglobulin, while the suffix indicates the type of heavy chain that the antibody contains, with heavy chain types α (alpha), γ (gamma), δ (delta), ε (epsilon), and μ (mu) resulting in IgA, IgG, IgD, IgE, and IgM, respectively.

[0084] B cell antibody isotypes change during cell development and activation. Immature B cells, which have never been exposed to antigen, express only the cell surface-bound IgM isotype. This ready-to-respond form of B lymphocytes is known as "naive B lymphocytes." Naive B lymphocytes express both IgM and IgD on their surface. The coexpression of both immunoglobulin isotypes renders B cells ready to respond to antigens. Following engagement of cell-bound antibody molecules with antigen, B cell activation occurs, leading to cell division and differentiation into antibody-producing cells called plasma cells. These activated B cells begin producing secreted, rather than membrane-bound, antibodies. Some daughter cells of activated B cells undergo isotype switching, a mechanism that triggers antibody production, shifting from IgM or IgD to other antibody isotypes with defined roles in the immune system: IgE, IgA, or IgG. Thus, the immune cells of the immune organoids of the present disclosure may also include cells that have undergone or will undergo isotype switching.

[0085] Antibodies are important for the development of humoral immune responses, in which antibodies are produced by B cells and secreted into the blood and / or lymph in response to antigenic stimulation. In a properly functioning immune response, antibodies specifically bind to antigens (e.g., pathogens) on the surface of cells, marking the cells for destruction by phagocytotic cells and / or complement-mediated mechanisms. Briefly, antibodies, once bound to antibody-bound target cells, are involved in several important functions, including antibody-dependent cellular cytotoxicity (ADCC), phagocytosis (opsonization), and complement-dependent cytotoxicity (CDC).

[0086] Therefore, in some embodiments, immune organoid has complete humoral function.Specifically, in some embodiments, the antibody produced by immune organoid of the present disclosure can function in ADCC.ADCC is the in vitro or in vivo process in which antibody binds to antigen on the surface of cell, and then can be combined with immune effector cell through the sequence in the Fc domain of the antibody, which then causes the release of toxin that can kill the bound cell.ADCC activity can be measured by methods known in the art, including but not limited to, by using the in vitro method described in the examples herein.

[0087] In some embodiments, the antibody produced by immune organoid of the present disclosure can function in CDC.CDC refers to the in vitro or in vivo process that antibody binds to the antigen on the surface of eukaryotic or prokaryotic cell, and then can be combined with C1q protein through the sequence in the Fc domain of the antibody, which then leads to the initiation of the classical complement cascade, which can kill the bound cell.CDC activity can be measured by using known methods in the art, including but not limited to, by using the in vitro method described in the examples herein.

[0088] In some embodiments, the antibody produced by immune organoid of the present disclosure can function in opsonization.Opsonization is the process that antibody binds to the antigen on the surface of cell, and then can be combined with immune cell through the sequence in its Fc domain, which then leads to the phagocytosis, consumption and ultimately killing of antibody-bound cell by immune cell.Opsonization activity can be measured by methods known in the art, including but not limited to, by using the in vitro method described in the examples herein.

[0089] In some embodiments, immune organoid as described herein exhibits at least one of ADCC, CDC and opsonization activity.In some embodiments, immune organoid as described herein exhibits at least two of ADCC, CDC and opsonization activity.In some embodiments, immune organoid as described herein exhibits all three of ADCC, CDC and opsonization activity.

[0090] In some embodiments, the antibody produced by the immune organoid of the present disclosure can also undergo the process of somatic hypermutation.Somatic hypermutation refers to the process of increased gene mutation, and is thought to require activation-induced cytidine deaminase (AID) and error-prone DNA repair.SHM was first described by observing that, following antigen stimulation, the immunoglobulin gene region encoding the variable region of light and heavy chains increases in B lymphocytes.AID is discussed, for example, in Smith et al., Trends Genet. 20:224-227 (2004).The existence of somatic hypermutation can be identified by using methods including, but not limited to, measuring AID upregulation, for example, by quantitative PCR.B cell receptor sequencing can also be used to identify the accumulation of mutation.

[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 (recall response) and against targets to which the patient is naive.

[0092] In some embodiments, the immune organoids described herein produce an autoimmune response against an autoantigen.

[0093] In some embodiments, the antibody produced by immune organoid as described herein binds to human target and non-human target.Exemplary human target includes but is not limited to protein, sugar and nucleic acid.Exemplary non-human target can include but is not limited to infectious disease antigen, venom, poison, small molecule. composition

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

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

[0096] Secondary lymphoid tissue can be obtained from mammal (i.e., donor or patient), for example, human, dog, cat, rabbit, monkey, chimpanzee, cow, pig or goat.Secondary lymphoid tissue can be obtained from living patient, surgical resection, fine needle aspirate, biopsy or directly from dead patient.In some embodiments, secondary lymphoid tissue is obtained from human, thereby resulting in human immune organoid.

[0097] In one embodiment, the immune cells obtained from one or more secondary lymphoid organoids from a single donor produce about 1000, about 100,000, about 20,000, about 30,000, about 40,000, about 50,000 organoids.In some embodiments, the immune cells obtained from one or more secondary lymphoid organs from a single donor produce about 50,000 organoids.This is in contrast to the approximately 25 organoids that are produced per donor using previously known technology.

[0098] In some embodiments, the plurality of primary immune cells self - assemble 100% into three - dimensional immune organoids in 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 can be present among the plurality of immune cells are as described above. The plurality of immune cells can contain a total of about 2×10 6 cells, 1.5×10 6 cells, 1×10 6 cells, 0.5×10 6 cells, 0.4×10 6 cells, 0.3×10 6 cells, 0.2×10 6 cells, 0.1×10 6 cells or fewer.

[0100] As described above, the inclusion of additional cell types is contemplated in the compositions of the present disclosure. Additional cell types include, but are not limited to, PBMC, stromal cells, and fibroblastic reticular cells. The methods of the present disclosure

[0101] The present disclosure provides, among other things, a method for producing a plurality of immune organoids. The method includes: (a) dissociating tissue from one or more secondary lymphoid organs to produce a plurality of single primary immune cells; (b) contacting 1×10 6 or fewer single primary immune cells with a solid support; and (c) culturing the plurality of single primary immune cells for 24 hours to produce a plurality of three - dimensional immune organoids, wherein the plurality of immune organoids continue to survive for at least 30 days.

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

[0103] Tissues can be dissociated by a variety of methods including, but not limited to, both mechanical and / or enzymatic processing. For example, tissue dissociation can be mechanical (mincing or shearing), or can use single or combined proteolytic enzymes, such as matrix metalloproteases and / or neutral proteases, such as collagenase, trypsin, dispase, LIBERASE (Boehringer Mannheim), Accumax. It can be dissociated by enzymatic digestion with hyaluronidase and / or pepsin, or by a combination of mechanical and enzymatic methods. Methods of tissue dissociation are also described above.

[0104] Using the methods described herein, dissociation of secondary lymphoid tissues produces a suspension of single primary immune cells. Those primary immune cells can be any cells of hematopoietic origin that are functionally involved in the initiation and / or execution of the innate and / or adaptive immune response, such as, typically, 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 includes, prior to step (b), a step of freezing a plurality of single primary immune cells.

[0106] In some embodiments, prior to step (c), the cryopreserved immune cells are thawed and treated with a ROCK inhibitor. ROCK is a serine / threonine kinase that functions as a target protein of Rho (which has three isoforms, namely RhoA, RhoB, and RhoC). Exemplary ROCK inhibitors include, but are not limited to, antibodies against ROCK, dominant inhibitory ROCK variants, as well as siRNA and antisense nucleic acids that suppress 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, hydroxyl-HA-1077, GSK2699962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, pirinotecan, and brevystatin.

[0107] As described herein, 1×10 6A single or less single primary immune cell is contacted with the solid support. The solid support can include one reservoir and / or multiple reservoirs (for example, the wells of a well plate). The reservoir(s) can be any suitable reservoir or container that holds organoids. In some embodiments, the reservoir is a well of a well plate, for example, but not limited to, a well in a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, a 384-well plate, a transwell, a ULA plate, a flat-bottom plate, or a V-bottom plate. The solid support can also include a base (pedestal) for use in an air-liquid interface. The air-liquid interface can be the interface that primary immune cells are exposed to in the culture described herein. The primary tissue can be mixed with a gel solution, which is then poured onto the layer of gel formed in a container that includes a lower semipermeable support, for example, a membrane. The container is placed in an outer container that contains a medium, so that the gel containing the tissue is not submerged in the medium. The primary tissue is exposed to air from above and liquid medium from the bottom.

[0108] In certain embodiments, the cell culture medium that organoid is cultured in comprises serum-supplemented cell culture medium.The characteristics of the medium that cell is cultured in can also include, for example, any growth factor or differentiation inducer that can exist, and if present, supporting structure (for example, substrate on solid surface).

[0109] According to the method of the present disclosure, a plurality of single primary immune cells are cultured for 24 hours to produce a plurality of immune organoids, so that a plurality of immune organoids continue to live for at least 30 days.In some embodiments, a plurality of immune organoids continue to live for 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 days.In some embodiments, the tissue from a single donor as described herein is used to produce about 1000, about 100,000, about 20,000, about 30,000, about 40,000 or about 50,000 organoids.

[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 with respect to the composition.

[0111] In some embodiments, using the methods described herein, multiple immune organoids have diameters and / or maximum dimensions 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 comprises the germinal center and / or B / T cell area described above. In some embodiments, the plurality of immune organoids further comprises CXCR4 + , CD83 + , Ki67 + and IgD + In some embodiments, the plurality of immune organoids are CD3 + and CD20 + is.

[0113] In some embodiments, by using the method provided herein, a plurality of immune organoids produce antibody.The types and functions of various antibodies and the humoral immunity that results are described in detail above. [Example]

[0114] While certain 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. Accordingly, no limitation to the precise summary and disclosure presented herein is intended. Example 1 method

[0115] Tissue collection and freezing. For example, whole spleens and lymph nodes from deceased or living donors (of any age) were collected and thinly sliced by a certified clinician into sections, placed in hypothermosol + 2x Pen / Strep, 1x Normocin, and stored at 4°C (4°C) for shipping and further processing. Hypothermosol medium was prepared and the workspace was set up. Cryovials were labeled with the date, tissue type, batch ID number, and name [link to cryovial sticker template]. Upon arrival at the laboratory, tissues were completely immersed in fresh hypothermosol + 2x Pen / Strep, 1x Normocin, and unprocessed sections were kept on ice (4°C). Using a disposable scalpel, tissues were carefully dissected into 5mm x 5mm x 5mm pieces and placed in 24-well plates for dissection. The tissue is rinsed with 1 ml of 0.5 mM EDTA and then incubated in 1 ml of 0.5 mM EDTA at 37°C for 1 hour (modulate accordingly and record changes). 1 ml of Accumax is added to the tissue and incubated at 37°C for 1 hour, and 1 ml of complete medium is added to stop the reaction. Count the cells using an NC-200 and record the number of viable cells and viability. Centrifuge the cells at 250 x g for 10 minutes. Aspirate the cells and store in 1 ml of Cryostore at 10 x 10 cells per vial. 6 The cells are then resuspended at 1000 cells / mL. The cells are then transferred to pre-barcoded cryovials. The vials are transferred to Mr. Frosty and stored at -80°C overnight, and transferred to LN2 the next day. The barcode is recorded in the LN2 inventory (LN2 number, hotel number, box number, location in box).

[0116] Organoid formation. Once thawed, add a ROCK inhibitor (e.g., Y-27632) to the complete medium. Partially immerse the cryovial in a 37°C bath and thaw until ice chips remain. Transfer the cells from the cryovial to a 50 ml conical tube. Add 1 ml of complete medium dropwise, swirling between each drop, and add 9 ml of complete medium. 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 respin the tube (200 x g, 5 minutes) while counting. Add 100 μl (total of 1 x 10 cells) per well in the upper chamber. 6 ~2×10 6 Calculate the resuspension volume to achieve a total cell count (100 cells / well). Add 1 μg / ml BAFF to complete medium, and add 200 μl of supplemented medium per well or platform, e.g., in a transwell, ULA plate, flat-bottom plate, or V-bottom plate. Add stimuli directly to the cultures and then incubate overnight at 37°C. Replenish organoids with BAFF-supplemented complete medium every 3 days.

[0117] Addition of PBMCs. Once organoids have formed, replenish with PBMC-derived adaptive cells.

[0118] Flow cytometry. Prepare staining buffer (1x DPBS + 0.5% BSA or 1x DPBS + 1% FBS) and design flow panels using Biolegend's Spectra Analyzer tool (https: / / www.biolegend.com / en-us / spectra-analyzer). Remove organoid-containing plates from the incubator and place in a cleaned safety cabinet. Disrupt organoids to generate a single-cell suspension. Count cells and place in flow tubes. Add 2 mL of staining buffer to each tube. Centrifuge cells at 500 g for 5 minutes and resuspend 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 for 30 minutes protected from light. Wash samples twice with 2 mL of flow staining buffer, aspirate the supernatant, and resuspend cells in the remaining buffer. Add secondary antibody according to the manufacturer's instructions and incubate at 4°C for 30 minutes protected from light. 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. If necessary, fix the cells at this point using 1% paraformaldehyde. Analyze the samples with single-color compensation controls.

[0119] Immunofluorescence. Thaw slides with sections and allow to dry completely. To remove OCT, immerse slides in PBS for 15 minutes at room temperature. Place slides into Sequenza clips while still submerged in PBS. Carefully place clips with slides attached into a Sequenza rack. Pour PBS over the slides, ensuring they are properly sealed and that the PBS does not drain through too quickly. For permeabilization and blocking, add 150 μl of permeabilization buffer to each slide and incubate for 1 hour. Wash sections three times for 5 minutes with 150 μl of PBS. Add 150 μl of blocking buffer to each section and incubate for 1 hour at room temperature with the rack fully covered.

[0120] The primary antibody is prepared by diluting it to its appropriate concentration with an appropriate volume of blocking buffer. 150 μl of the antibody solution is added to each section and incubated overnight at 4°C with complete coverage.

[0121] The secondary antibody is prepared by diluting it to its appropriate concentration in blocking buffer, protected from light using aluminum foil, and kept at 4°C. DAPI is prepared by diluting it to its appropriate concentration in PBS, protected from light using aluminum foil, and kept at 4°C.

[0122] The slides are washed three times with PBS for 5 minutes each, 150 μl of the antibody solution is added to each section and incubated at room temperature for 2 hours with complete coverage. Then, the slides are washed three times with PBS for 5 minutes each, 150 μl of DAPI is added to each section and incubated at room temperature for 10 minutes with complete coverage. The slides are washed three times with PBS for 5 minutes each.

[0123] ELISA. First, prepare assay diluent (1% BSA in 1×PBS without +Mg and +Ca) and wash buffer (1×PBS + 0.05% Tween®-20). Coat the ELISA plate with coating buffer diluted from 5×ELISA coating buffer to 1× working solution in DI water. Coat wells “1” and “2” with coating buffer mixed with IgG capture Ab at a dilution of 1:300 (100 μL / well of diluted mix capture antibody). Coat the remaining wells of the plate with coating buffer mixed with influenza A recombinant protein at a concentration of 0.1 μg / well (pipette 100 μL per well). Cover the plate with film and place it in a shaker at 20 - 25 °C for 1 hour, or cover it with film and incubate overnight at 4 °C. Block the ELISA plate by washing each well with 300 μL of the prepared wash buffer. Repeat this a total of 4 times to complete. Add 200 μL of assay diluent to each well of the plate and place it on a shaker at room temperature for a total of 1 hour.

[0124] Prepare all standards while the plate is being blocked. Dilute the standards to a concentration of 1000 ng / mL using assay diluent. Add 500 μL of assay diluent to each tube. Add 500 μL of AD from the prepared first standard (200 ng / mL). This becomes tube 1 with a final concentration of 100 ng / mL. Continue with 2× serial dilutions to the final tube. One tube contains assay diluent only as a positive control.

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

[0126] For the addition of the detection antibody HRP, prepare the detection antibody diluent at a 1:100,000 dilution. Remove the plate from the shaker and wash it 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 it on the shaker for 1 hour.

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

[0128] Obtain data using a plate reader. For data analysis, the absorbance values of duplicates must be within 10% of each other. Plot a standard curve for the IgG standard (known concentration) samples and plot the average absorbance value minus (-) the blank value on the vertical (Y) axis as the respective standard concentration for each standard. Plot the corresponding human IgG concentration, which correlates with the absorbance value, on the horizontal (X) axis. This is used to estimate the concentration of the unknown sample by extrapolation.

[0129] Somatic hypermutation. Cells are collected at day 0 and at the relative endpoint. Cells are washed with FACS buffer and incubated with the biotinylated recombinant protein of interest at a concentration of 4 μg / mL in the presence of Fc block. Further, to determine the B cell lineage (CD38+CD27+), cells are incubated with a fluorescent-labeled antibody panel. +Protein B cells of the GC or plasmablast phenotype are sorted into 96-well plates. From individual B cells, cDNA is harvested, tagged with unique DNA barcodes, and pooled per plate. Gene-specific PCR is used to amplify the immunoglobulin heavy chain variable region and the light chain variable region. The library is shipped for sequencing and the sequences are analyzed. Fastq files are generated by inverse multiplexing using the MiSeq reporter and quality filtered. Paired reads are stitched and separated by well ID and consensus sequence. The well ID reads are clustered into operational taxonomic units. The operational taxonomic unit sequences are analyzed by IMGT HighV-Quest. Clone families are defined by the same V and J gene usage and at least 70% amino acid identity in both the heavy chain and light chain CDR3 loci.

[0130] Exemplary primer sequences used for the detection of somatic hypermutation are shown in Table 1 below.

Table 1

[0131] Antibody function. ADCC. To prepare target cells, 2×10 cells 5Wash the cells three times with 5 ml of RPMI 1640 containing 2 μg / ml TPCK trypsin. Remove the 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 medium (RPMI 1640) containing 2 μg / ml TPCK trypsin. Add 5 ml of medium (RPMI 1640) containing 2% calf serum and 2 μg / ml TPCK trypsin to the T-25 flask. Incubate the cells at 37°C for 48 hours. After incubation, a sample of target cells is taken and the incidence of infected cells is assessed by their ability to hemagglutinate with turkey RBCs and bind polyclonal antibodies. 80-95% of the cells should be infected. 2 x 10 6 The cells are washed twice with PBS and are labeled with PKH67.

[0132] For effector cell preparation (PBMC isolation), PBMCs are thawed from the biobank. PBMCs are washed twice with PBS. Antibody-dependent cell-mediated cytotoxicity assay (ADCC) is performed using 5.0 x 10 4 Labeled target cells are used to dispense 50 μl of RPMI 1640 medium into each well in duplicate in a round-bottom 96-well plate according to the layout shown below. 50 μl of antibody is added to the wells according to the layout and incubated for 15 minutes at 37°C in a CO2 incubator. Unlabeled normal PBMC effector cells in 100 μl of RPMI 1640 / 0.5% Pen / Strep are added at 2.5 x 10 cells. 7Cells were re-incubated at 37°C for 2 hours in a CO2 incubator. After 2 hours, 1 μl of the fluorescent dead cell dye 7-amino-actinomycin-D (7-AAD) was added and incubated for 20 minutes at 4°C in the dark. Cells were analyzed using a flow cytometer to acquire a total of 5,000 target cells. Cell death percentages were determined by software analysis of four identifiable cell populations: viable effector cells (no dye), dead effector cells (7-AAD only), viable target cells (PKH-67 only), and dead target cells (PKH-67 and 7-AAD).

[0133] Antibody function. CDC. 20 μL of target cells were added to 2 x 10 cells. 5 Add 10 μg / ml of antibody to each well of a 96-well assay plate. A 10-point titration curve of the test antibody is generated using 1:2.5 serial dilutions starting at 1 μg / ml. Each antibody dilution is added to the plate to initiate 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 opsonize the cells. Dilute complement 1:18 in complete medium and add 25 μl to the appropriate wells. Place the plate on an orbital shaker for an additional 30 seconds and then 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. Incubate the plate on the benchtop at room temperature for 5 minutes. Add 125 μl of medium to each well. Centrifuge the plate at 750 RPM for 1 minute. Add 50 μl of enzyme assay diluent to the appropriate wells of the luminescence white plate. Add 50 μl of reaction supernatant to the wells containing assay diluent. Add 100 μl of 2× enzyme assay reagent to each diluted supernatant. Add 50 μl of 1× detection reagent to each diluted supernatant. Shake the plate for 30 seconds and then read the luminescence immediately on a plate reader.

[0134] Antibody function: Antibody-dependent cell-mediated phagocytosis. Target cells are labeled with PKH67 and seeded at 25,000 cells per well into 96-well plates containing 10% human AB serum. Serial dilutions of the antibody of interest are made to eight half-log concentrations and added to the target cells. Macrophages are added at a 4:1 effector:target ratio. Cells are centrifuged at 500g for 5 minutes and incubated at 37°C for 4 hours. Cells are resuspended by flicking the tube and incubated with the recommended dilutions of CD11b-APC, CD14-APC, and CD66-PE for 45 minutes, protected from light. Cells are washed twice with PBS and the supernatant is aspirated. 1% PFA is 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 resuspending in culture medium. Target cells are prepared by density gradient centrifugation to remove dead cells and resuspending at the appropriate density. Cells are resuspended in culture medium containing Cell Tracker Deep Red at 1:1000 for 30 minutes at 37°C. Cells are washed and resuspended in culture medium. Co-cultures are set up. Cells are seeded at the desired effector:target ratio. Desired co-culture time points are set for testing. To control for spontaneous target cell death, ensure that wells contain only labeled target cells. At each desired incubation time point, cells are harvested, pelleted, and resuspended in Annexin V binding buffer mixed with Annexin V PE and 7-AAD. Cells are incubated at RT for 15 minutes, and labeled cells are analyzed by flow cytometry within 1 hour of staining.

[0136] Gas-liquid interface immune organoids. To create a partitioned cell culture system such as a Transwell, a permeable membrane support is inserted into a cell culture plate. The gel matrix was prepared by mixing the collagen matrix with 10× concentrated sterile culture medium (Ham’s F12) and sterile reconstitution buffer (2.2 g of NaHCO3 in 100 ml of 0.05 N NaOH and 200 mM HEPES) at a ratio of 8:1:1 on ice. The reconstitution buffer was added and mixed to avoid foaming. 1 ml of the reconstituted collagen solution was added to the plate and allowed to solidify by placing it in an incubator at 37 °C for 30 minutes. 1×10 6 ~2×10 6 cells were seeded on top of the collagen matrix in the upper chamber of the plate. Complete medium was added to the cells such that the liquid height matched the cell height. The medium was changed when it appeared to have changed color.

[0137] Automatic image segmentation for area and diameter. The brightfield image was opened and its histogram equalized. The saturated pixel portion was set to 0%. Canny edge detection was performed using a Gaussian kernel radius pixel resolution set according to the settings. The maximum filter was performed using a radius set for the value according to the settings. This filter creates an execution window that replaces the central pixel with the maximum value of the adjacent pixels. Morphological operation closing was performed using an iteration count set to 10 and a count set to 3. This filled the remaining small holes in the image. Morphological opening was performed using an iteration count set to 10 and a count set to 3. This removed small structures such as fragments localized outside the organoids. The contour of the segmented area was overlaid with the original brightfield image to check the accuracy of the segmentation. If necessary, the parameters of the procedure were adjusted, paying particular attention to the high threshold setting of the Canny edge detection. The area was calculated using Analyze > Measure. The diameter was calculated using the formula d = 2√(A / π).

[0138] Antigen-specific IgG sandwich ELISA. On the day of transferring the samples, the plates containing the supernatant were thawed by placing them on ice for several hours. An assay diluent, which is also the blocking buffer (1% BSA in 1× PBS without +Mg and +Ca), was prepared. 4 ml of 7.5% BSA was added to 26 ml of DPBS (total = 30 ml). The assay diluent was stored at 4°C for short-term storage and at -20°C for long-term storage. A washing buffer (1× PBS + 0.05% Tween®-20) was prepared. The 10× washing buffer (10× PBS + 0.5% Tween®-20) was prepared by adding 2.5 ml of Tween®-20 to 500 ml of 10× PBS. To prepare 1 l of 1× washing buffer, 900 ml of MiliQ water was added to 100 ml of the 10× buffer. The remaining 10× washing buffer was stored at 4°C for later use.

[0139] Coat the ELISA plates. The coating solution (1 μg / ml antibody in 1× coating buffer) was prepared as follows to ensure a total volume of 100 μl / well. The 5× ELISA coating buffer was diluted to 1× working solution in DI water. The capture antibody was added to a final concentration of 1 μg / ml. 100 μl / well was added. The plates were covered with film and incubated at 20 - 25°C for 1 hour or overnight at 4°C with gentle shaking on a shaker.

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

[0141] Capture antigen / antibody. The plate was washed four times with 250 μl / well of wash buffer. The capture antigen / antibody solution was prepared as follows: Enough solution was prepared to ensure a total volume of 100 μl per well. The capture antigen / antibody was added in assay diluent to achieve a final concentration of 1 μg / ml. The plate was coated with 100 μl / well of the prepared antigen / antibody solution. The plate was covered and incubated on a shaker at room temperature for 1 hour.

[0142] Intracellular staining. 6 mL of fixation / permeabilization working solution was prepared as follows: 1.5 mL of Foxp3 fixation / permeabilization concentrate was added to 4.5 mL of Foxp3 fixation / permeabilization dilution. 40 mL of 1x permeabilization buffer was prepared as follows: 4 mL of 10x permeabilization buffer was added to 36 mL of MilliQ water. Disaggregated organoid cells were added to two separate tubes containing 9 mL of cell culture medium and centrifuged (5 min, 350 g). The supernatant was decanted. 2E6 cells were transferred to two 1.5 mL tubes per donor (Tube 1: Live / Dead Only, Fixed; Tube 2: Complete Panel, Fixed) and centrifuged (5 min, 350 g). The supernatant was gently flicked off and removed. Cells were resuspended in 200 μL of Live-Dead Aqua diluted 1:500 in PBS and incubated at RT for 15 min, protected from light. 2 μL of each surface staining solution from the Claim 16 / 18 panel was added to 188 μL of PBS + 0.5% BSA (CD20-Pacific Blue, CD3-FITC, CXR4-PerCP-Cy5.5, IgD-PE, CD83-APC, CD45-AF700). After cell incubation, the surface staining cocktail was added directly to tube 2. 200 μL of PBS + 0.5% BSA was added to tube 1. The cells were incubated for 15 minutes at room temperature, away from light. After cell incubation, 200 μL of PBS + 0.5% BSA was added and spun (5 minutes, 350 g). The supernatant was flicked off and removed. 400 μL of PBS + 0.5% BSA was added and spun again (5 minutes, 350 g). The supernatant was flicked off and removed. This step was then repeated. Then, 1 mL of Foxp3 fixation / permeabilization working solution was added to each tube, pulse-vortexed, and incubated at 4°C for 30 minutes. The cells were transferred to two 15 mL Falcon tubes. 2 mL of 1x permeabilization buffer was added to both tubes and centrifuged (5 minutes, 350 g). The supernatant was gently removed, and the cells were resuspended in the remaining permeabilization buffer. 2 μL of Ki67-BV605 was added to the resuspended cells and mixed by pipetting. This step was repeated.The supernatant was gently flicked off and the cells were resuspended in 250 μL of PBS+0.5% BSA, then the flow was run.

[0143] Antigen-specific IgM sandwich ELISA. ELISA. On the day of sample transfer, plates containing supernatants were thawed by placing on ice for several hours. Assay diluent, which also served as blocking buffer (1% BSA in 1x PBS without Mg and Ca), was made. 4 ml of 7.5% BSA was added to 26 ml of DPBS (total = 30 ml). Assay diluent was stored at 4°C for short-term storage and at -20°C for long-term storage. Next, wash buffer (1x PBS + 0.05% Tween®-20) was made. 10x wash buffer (10x PBS + 0.5% Tween®-20) was prepared by adding 2.5 ml of Tween®-20 to 500 ml of 10x PBS. To prepare 1 L of 1x wash buffer, 900 ml of MiliQ water was added to 100 ml of 10x buffer. The remaining 10x wash buffer was stored at 4°C for later use.

[0144] Coat the ELISA plate. The coating solution (1 μg / ml antibody in 1× coating buffer) was prepared as follows to ensure a total volume of 100 μl / well: 5× ELISA coating buffer was diluted in DI water to a 1× working solution. Capture antibody was added to a final concentration of 1 μg / ml, adding 100 μl / well. The plate was covered with film and incubated at 20-25°C with gentle shaking on a shaker for 1 hour or at 4°C overnight.

[0145] Block the ELISA plate. The plate washer was loaded with wash buffer and the plate was washed 4 times with 250 μl / well of wash buffer. The plate was blocked with 200 μl / well of assay diluent. The plate was covered with film and incubated on a shaker at room temperature for 1 hour or at 4°C overnight.

[0146] The plate was then washed four times with 250 μl / well of wash buffer. The capture antigen / antibody solution was prepared as follows: Enough solution was prepared to ensure a total volume of 100 μl per well. The capture antigen / antibody was added in assay diluent to achieve a final concentration of 1 μg / ml. The plate was coated with 100 μl / well of the prepared antigen / antibody solution. The plate was covered and incubated on a shaker at room temperature for 1 hour. Example 2 Stem cell populations are represented in immune organoids

[0147] This example demonstrates that immune organoids contain a stem cell population that is found only in secondary lymphoid organs, not in blood. These stem cells proliferate and play a role in the support and function of lymph nodes, as well as the support and function of immune organoids.

[0148] Briefly, immune organoid is produced by the method described in Example 1.The culture of day 7 is stained for stem cell marker, and then undergoes flow cytometry.As shown in Figure 1A and Figure 1B, the immune organoid of day 7 contains the population of CD45+CD34+, CD45+CD34+CD45RA-, CD45+CD34+CD45RA-CD201(EPCR)+CD49c(ITGA3)+, CD45+CD34+CD45RA-CD201(EPCR)+CD49c(ITGA3)+CD105+CD73+ stem cell. Example 3 Diverse immune cell populations, including both innate and adaptive cells, are contained in human immune organoids.

[0149] This example demonstrates that immune organoids are composed of the same diverse immune cell types found in human lymph nodes, which work together to recapitulate lymph node structure and function.

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

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

[0152] Briefly, the immune organoids were generated using the method described in Example 1. Figure 3A shows cells that were gated on pre-gated 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, and CD38+CD27+ GC B cells. Figure 3B shows that the immune organoids stimulated on day 0 are predominantly naive B cells and then differentiate into pre-GC B cells, GC B cells, memory B cells, and plasmablasts. These B cell phenotypes are consistent with the responses of human B cells to infection or vaccination, or other stimuli in lymph nodes. (Example 5) The immune organoids are composed of T cell subtypes

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

[0154] Briefly, the immune organoids were generated using the method described in Example 1. Figure 4A shows the 7-day culture pre-gated on total T cells (CD19-CD3+). The cells were then stained by flow cytometry for CD4+ and CD8+ T cells.

[0155] Figures 4B-4C show flow cytometry staining demonstrating immune organoids consisting of T cell subtypes. All cells shown were pre-gated on total T cells (CD19-CD3+) and then gated on the respective naive and memory T cell populations of CD4+ T cells and CD8+ T cells. Shown phenotypes: 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] Figures 4D-4E show flow cytometry staining demonstrating immune organoids consisting of additional T cell subtypes. All cells shown were pre-gated on total T cells (CD19-CD3+). Shown cell types: CD3+CD4+CD25+ Treg, CD3+CXCR5+CD25+ follicular helper T cells, CD3+CD27+ gamma delta T cells. (Example 6) The immune organoids are composed of other immune cell types

[0157] This example demonstrates that the immune organoids are composed of fairly rare immune cell types that are important for human lymph nodes.

[0158] Briefly, immune organoid is produced by the method described in Example 1. The culture of day 7 is stained for myeloid DC (CD14+CD11c+), plasmacytoid DC (CD123+), conventional DC (CD11b+CD45+) and CD14+DC (CD14+CD11c+) (Fig. 5A).As shown in Fig. 5B, the culture of day 7 also contains cell types such as stromal cells (CD45-) and fibroblastic reticular cells (CD31+PDPN+). Example 7 Immune organoid size

[0159] This embodiment demonstrates that the size of immune organoid depends on the number of cells used to form organoid and stimulation conditions.After successful stimulation, organoid grows considerably due to cell proliferation and activation.

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

[0161] This example demonstrates that germinal centers with bright and dark areas are required to recapitulate lymph node biology and cannot be found in current in vitro techniques.The organoid platform described herein uniquely produces germinal centers, thus reproducing a crucial feature of human biology.

[0162] Briefly, immune organoid is produced by the method described in Example 1. 14-day culture is stimulated with hepatitis B vaccine, and organoid is visualized under bright field microscopy.As shown in Figure 7A, the brighter structure outlined in red in organoid is consistent with germinal center morphology.

[0163] Confocal microscopy was then performed to image germinal centers, including B cell (CD20) and T cell (CD3) constituents, plasmablasts (CD138), BCL6+ cells, and PD1+ cells, within the day 14 immune organoids (Figure 7B). Example 9 Immune organoids contain cells consistent with germinal center function

[0164] This example demonstrates that germinal centers must be functional as well as present. These data show that they are fully functional and regenerate all essential aspects of lymph node function, representing the first organoid technology capable of doing so.

[0165] Figure 8A shows flow cytometry staining demonstrating immune organoids consisting of B cell and T cell regions. The cells shown are CD19+ B cells and CD3+ T cells. The plot is from a representative immune organoid on day 14. Flow cytometry was then performed to identify germinal center B cells. As shown in Figure 8B, the 14-day culture contains CD27+CD38+ germinal center B cells.

[0166] As shown in Figures 8C-8F, additional cell types were then identified in the day 14 cultures. Notably, day 14 immune organoids consisted of cells found in germinal centers, including CD83+, Ki67+ B cells, Ki67+ T cells, IgD+ cells, and CXCR4+ cells.

[0167] Cell types were quantified as shown in Figure 8G. Example 10 Immune organoids are composed of both B and T cells that can be modulated by various stimulatory conditions.

[0168] This example demonstrates that the immune organoids are composed of a mixture of a large number of different immune cells that can be activated and proliferated by stimulation. B cells and T cells are an example of the dramatic differences in cell abundance after stimulating the immune organoids under six different stimulation conditions. Some stimulation conditions preferentially increased the B cell population, and some increased the T cell population.

[0169] Briefly, the immune organoids were generated using the method described in Example 1. As shown in FIGS. 9A-9B, the cultures on days 9 and 16 were stimulated under different conditions. The stimulation was a protein stimulation of influenza (hemagglutinin protein in this case), with different combinations of adjuvants introduced across all six different conditions. The frequencies of T cells (FIG. 9A) and B cells (FIG. 9B) were determined by flow cytometry. (Example 11) The immune organoids elicit a complete adaptive immune response

[0170] This example demonstrates that the organoids produce plasmablasts and antigen-specific antibodies against targets to which the patient donor was exposed (recall response) and against targets to which the patient is naive. The antibodies produced by the organoids have the ability to class switch, for example, from IgM antibodies to IgG antibodies, which is consistent with an adaptive immune response.

[0171] Briefly, as shown in FIG. 10, the immune organoids elicit a complete immune response, including a complete adaptive immune response, upon stimulation. It includes complete B cell differentiation from naive B cells to mature cells such as germinal center B cells and memory B cells, and plasmablasts. When plasmablasts are induced, these cells produce antigen-specific antibodies against the targets that stimulated the patient. The immune organoids produce antibodies against targets to which the patient was previously exposed to a recall response and against targets to which the patient is completely naive to a naive response. The antibodies produced by the organoids are antigen-specific and have the ability to class switch, for example, from IgM antibodies to IgG antibodies, which is consistent with an adaptive immune response. (Example 12) Immune organoids can break tolerance and generate antibodies against human targets

[0172] This example demonstrates that, similar to the fact that immune tolerance is a major property of systemic immunity, tolerance breakdown (i.e., autoimmune and allergic diseases) can be modeled. Currently, there is no in vitro platform that can perform it in a way that mimics human patients. These data indicate that the organoids described herein can serve as a guide for generating an autoimmune response against self-antigens and modeling these diseases

[0173] Briefly, as shown in FIGS. 11A-11D, immune organoids produce both IgM and IgG antibodies against foreign antigens, examples of which include influenza and SARS-CoV-2 here. Immune tolerance is a major property of systemic immunity, and immune organoids reproduce that feature by not responding to stimuli. Another major property of systemic immunity is the ability to break tolerance (i.e., autoimmune and allergic diseases). Under certain stimulation conditions, immune organoids can model tolerance breakdown, which is a major feature of immune organoids for producing antibodies against human targets (i.e., cancer and autoimmune diseases) for therapeutic purposes. The organoids have the ability to generate an autoimmune response against self-antigen (myelin) and serve as a guide for modeling autoimmune and allergic diseases for the first time (Example 13) Immune organoids form within 24 hours and continue to survive for at least 30 days

[0174] This example demonstrates the ability of organoids to form their essential structure and begin to function after 1 day. Those organoids currently continue to survive in culture for 30 days or more

[0175] Briefly, immune organoids were generated using the method described in Example 1. Bright-field microscopy was used to visualize organoids after 24 hours (FIG. 12A) and through to day 28 (FIG. 12B). Example 14 Immune organoids can also be cultured at the air-liquid interface

[0176] This example demonstrates that there are numerous methods for organoid production, including but not limited to air-liquid interface (ALI), scaffolds, 3D printed plates, and hydrogels, which have been developed and tested to produce these organoids in suspension and at the ALI.

[0177] In brief, there are many methods for organoid production, including but not limited to air-liquid interface (ALI), scaffold, 3D printing plate, and hydrogel.The inventors have tested all of these methods using immune organoids, and as shown in Figure 13, have developed a method for producing these organoids in suspension and using the ALI method, which is a common method for organoid production.This method involves seeding organoids on a solid substrate (i.e., collagen) and culturing them on the surface of the medium, with most of the organoids remaining submerged, but the top part exposed to air.This method has several advantages, including increased oxygen permeability to organoids, and the use of liquid to modulate organoid morphology, and height for interesting functions, and improved imaging capabilities.This is another method that immune organoids can be produced.

[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 of the references cited in this specification constitutes prior art. The discussion of references states what their authors have asserted, and the applicant reserves the right to challenge the accuracy and appropriateness of the cited documents. Although a substantial number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to in this specification, it is clearly understood that such reference does not imply admission that any of those documents form part of the common general knowledge in the art.

[0180] The discussion of the general methods provided in this specification is intended for illustrative purposes only. Upon review of this disclosure, other alternative methods and means will be apparent to those skilled in the art and should be included within the spirit and scope of this application.

[0181] Throughout this specification, various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are referred to. The disclosures of all patents, patent applications, and other publications cited in this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A three-dimensional immune organoid comprising a plurality of self-assembling 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 according to claim 1, wherein the one or more secondary lymphoid organs are derived from the spleen, lymph nodes, Peyer's patches, and MALT.

3. The immune organoid according to any one of the preceding claims, which 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 according to any one 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 according to any one of the preceding claims, wherein the plurality of immune cells include B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages, and combinations thereof.

7. The immune organoid according to claim 6, wherein the B cells include one or more naive B cells, pre-GC B cells, GC B cells, memory B cells, or combinations thereof.

8. The immune organoid according to claim 6, wherein the T cells include naive CD4 T cells, memory CD4 T cells, regulatory T cells, follicular helper T cells, naive CD8 cells, memory CD8 cells, gamma delta T cells, or combinations thereof.

9. The immune organoid according to claim 6, wherein the dendritic cells include conventional dendritic cells, plasmacytoid dendritic cells, myeloid dendritic cells, or combinations thereof.

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

11. The immune organoid according to any one of the preceding claims, having a diameter of 8000 μm or less.

12. The immune organoid according to any one of the preceding claims, comprising germinal centers and / or B / T cell areas.

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

14. CD3 ＋ and CD20 ＋ The immune organoid according to claim 12, which is such.

15. The immune organoid according to any one of the preceding claims, which produces antibodies.

16. The immune organoid according to claim 15, wherein the antibody has complete humoral function.

17. The immune organoid according to claim 16, wherein the antibody binds to human targets and non-human targets.

18. The immune organoid according to claim 17, wherein the human targets include proteins, sugars and nucleic acids.

19. The immune organoid according to claim 17, wherein the non-human targets include infectious disease antigens, venoms, poisons, and small molecules.

20. A composition comprising a plurality of primary immune cells that self-assemble 100% into three-dimensional immune organoids within 24 hours and a composition.

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

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

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

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

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

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

27. The composition according to any one of claims 20 to 26, wherein the plurality of immune cells include B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages, and combinations thereof.

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

29. A method for producing a plurality of three-dimensional immune organoids, comprising: (a) dissociating tissue from one or more secondary lymphoid organs to produce a plurality of single primary immune cells; (b) contacting 1×10 ６ or fewer of said plurality of single primary immune cells with a solid support; and (c) culturing the plurality of single primary immune cells for 24 hours to produce a plurality of three-dimensional immune organoids, wherein the plurality of immune organoids continue to survive for at least 30 days and a method.

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

31. The method according to claim 30, comprising thawing the plurality of single primary immune cells after the freezing step and treating with a ROCK inhibitor.

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

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

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

35. The method according to any one of claims 29 to 34, wherein the plurality of primary immune cells include B cells, T cells, plasmablasts, NK cells, monocytes, dendritic cells, macrophages, and combinations thereof.

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

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

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

39. The method according to any one of claims 29 to 38, wherein the immune organoid includes germinal centers and / or B / T cell areas.

40. wherein the immune organoid is CXCR4 ＋ , CD83 ＋ , Ki67 ＋ and IgD ＋ The method according to claim 39, which is as described above.

41. wherein the immune organoid is CD3 ＋ and CD20 ＋ The method according to claim 39, wherein the method is as described above.

42. The method according to any one of claims 29 to 41, wherein the immune organoid produces an antibody.

43. The method according to claim 42, wherein the antibody has a complete humoral function.

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

45. The method according to claim 44, wherein the human targets include proteins, sugars, and nucleic acids.

46. The method according to claim 44, wherein the non-human targets include infectious disease antigens, venoms, poisons, and small molecules.

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