Multi-compartment organoid systems and uses thereof

Abstract

Provided herein are a multi-compartment organoids and methods of generating multi-compartment organoids. The multi-compartment organoids include a first ECM compartment including a first cell type and a first extracellular matrix (ECM) composition embedded into a second ECM compartment including a second ECM composition and are an in vitro 3-dimension (3D) physiologically accurate model of a tissue or organ. Specifically provided here are multi-compartment fallopian tube organoids and methods of generating multi-compartment fallopian tube organoids. Also provided here are kits including components and instructions to generate an in vitro 3D physiologically accurate model multi-compartment organoid of a tissue or organ.

Description

MULTI-COMPARTMENT ORGANOID SYSTEMS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 459,562, filed April 14, 2023. The disclosure of the prior application is considered part of and is herein incorporated by reference in the disclosure of this application in its entirety.STATEMENT REGARDING GOVERNMENT FUNDING

[0002] This invention was made with government support under Grant Nos AG060903, CA268083 and CAI 43868 awarded by the National Institutes of Health. The government has certain right in the invention.BACKGROUND OF THE INVENTIONFIELD OF THE INVENTION

[0003] The present invention relates generally to multi-compartment organoid, and more specifically to in vitro 3-dimension (3D) physiologically accurate model of tissue or organ.BACKGROUND INFORMATION

[0004] A variety of gynecologic diseases occur in the fallopian tube. High grade serous carcinoma (HGSC) is the dominant contributor to ovarian cancer mortality and has etiology traced to early precursors in the fallopian tube epithelium that eventually shed to the ovary. Recent studies have led to the STIC hypothesis, which identifies precursor lesions in the fallopian tube as the location of origin for many, if not all, HGSCs. Cell shedding, where these precursor cells are extruded from the fallopian tube, is a commonly proposed mechanism for early HGSC metastasis; however, the molecular mechanisms contributing to cell shedding and latent precursor lesion activation have not been identified. Endometriosis, which affects 10- 15% of reproductive age women and can cause infertility, also can present as lesions on the fallopian tube and is affected by cell co-culture conditions. Thus, standard of care treatment does not reflect this evolved understanding of HGSC etiology. The standard of care treatment for these diseases, however, has not adapted to the current understandings of disease etiology and remains largely ineffective, in part due to the lack of in vitro models that are physiologically and architecturally accurate.

[0005] Previous studies have demonstrated the importance of 3D architecture on cell migration, cell polarity, and disease development. Standard organoid models of the fallopian tube, however, are architecturally simplified compared to the intricate epithelial-lumen interface in scale and structure. The standard fallopian tube organoid model cultures single-cell suspensions of fallopian tube epithelial cells in basement membrane matrix until these cells self-assemble into hollow spheres. Compared to the organ, these organoids are structurally simplified and lack the ability to co-culture with stromal or diseased cell types.

[0006] There remains a need in the art for multi -compartment fallopian tube organoid that architecturally simulates the intricate architecture of the epithelial-lumen interface in structural complexity, scale, and composition, as well as the tissue’s molecular expression patterns and function. Appropriate organoids are cell- and extracellular matrix (ECM)-tunable and can be used in studies of HGSC precursor lesions and endometriosis in 3D co-culture as a function of cell and ECM composition, and physiological architecture.SUMMARY OF THE INVENTION

[0007] The present invention is based on the seminal discovery of methods of developing 3D physiologically accurate multi-compartment organoid modelling tissues and organs.

[0008] In one embodiment, the present invention provides a method of generating a multicompartment organoid including a) contacting a first extracellular matrix (ECM) composition with a first cell type to generate a first ECM compartment, and b) contacting the first ECM compartment of a) with a second ECM composition to generate a second ECM compartment or a larger ECM compartment including the first ECM compartment embedded into the second ECM composition, wherein the multi-compartment organoid is an in vitro 3-dimension (3D) physiologically accurate model of a tissue or organ, thereby generating a multi-compartment organoid.

[0009] In one aspect, contacting the first ECM with the first cell type includes generating a droplet including the first cell type embedded in the first ECM composition in a mineral oil. In another aspect, contacting the first ECM compartment with the second ECM composition includes resuspending the droplets including the first ECM and the first cell type in the second ECM composition. In one aspect, the first ECM composition and the second ECM composition are different ECM compositions. In another aspect, the first ECM composition and the second ECM composition are a same ECM composition. In one aspect, a combination of the first and second ECM compositions mimics a tissue or organ physiological microenvironment. Inanother aspect, the second ECM composition includes a second cell type. In some aspects, the first and the second cell type are a same cell type or a different cell type. In one aspect, the tissue or organ is selected from the group consisting of a fallopian tube, an endometrium, a uterus, a mammary gland, a normal breast tissue, a cervix, and an ovary. In one aspect, the first ECM composition includes a basement membrane ECM composition. In some aspects, the basement membrane ECM composition includes collagen IV and laminin. In another aspect, the second ECM composition includes a stromal ECM composition. In some aspects, the stromal ECM composition includes collagen I. In one aspect, the multi-compartment organoid is maintained in a 3D suspension culture. In another aspect, the 3D physiologically accurate model of a tissue or organ includes a multi-compartment organoid having a physiological architecture of the tissue or organ.

[0010] In another embodiment, the invention provides a multi -compartment organoid obtained by any one of the methods described herein.

[0011] In an additional embodiment, the invention provides a method of generating a multicompartment fallopian tube organoid including a) contacting a basement membrane ECM composition with a fallopian tube epithelial cells to generate a first ECM compartment, and b) contacting the first ECM compartment of a) with a stromal ECM composition to generate a second ECM compartment including the first ECM compartment embedded into the stromal ECM composition, wherein the multi-compartment fallopian tube organoid is an in vitro 3D physiologically accurate model of a fallopian tube, thereby generating a multi-compartment fallopian tube organoid.

[0012] In one aspect, the stromal ECM composition further includes fallopian tube stromal cells. In another aspect, the multi-compartment fallopian tube organoid has a lumen-like architecture. In one aspect, the multi-compartment fallopian tube organoid includes ciliated and secretory cells in a lumen compartment and tight junctions at a basement membrane ECM- stromal ECM interface. In some aspects, ciliated cells express elevated levels of FOXJ1 and / or OLFM4, as compared to non-ciliated epithelial cells. In other aspects, secretory cells are PAX8 positive. In another aspect, the multi-compartment fallopian tube organoid is sensitive to |3- estradiol and / or to progesterone. In some aspects, in response to -estradiol, epithelial cells in the multi-compartment fallopian tube organoid become ciliated. In various aspects, ciliated cells displace objects in a multi -compartment fallopian tube organoid lumen. In one aspect, the 3D physiologically accurate model of a fallopian tube includes a multi-compartment fallopian tube organoid having a physiological architecture of a fallopian tube.

[0013] In another embodiment, the invention provides a multi-compartment fallopian tube organoid obtained by any one of the methods described herein.

[0014] In one embodiment, the invention provides a multi-compartment organoid including: a) a first ECM compartment including a first cell type and an ECM composition, and b) a second ECM compartment including a second ECM composition, wherein the first ECM compartment is embedded into the second ECM compartment, wherein the multi-compartment organoid is an in vitro 3D physiologically accurate model of a tissue or organ.

[0015] In another embodiment, the invention provides a multi-compartment fallopian tube organoid including a) a first ECM compartment including a fallopian tube epithelial cell and a basement membrane ECM composition, and b) a second ECM compartment including a stromal ECM composition, wherein the first ECM compartment is embedded into the second ECM compartment, wherein the multi -compartment fallopian tube organoid is an in vitro 3D physiologically accurate model of a fallopian tube.

[0016] In a further embodiment, the invention provides a kit including a) a first cell type, b) a first ECM composition, c) a second ECM composition, and d) instructions to generate an in vitro 3D physiologically accurate model multi-compartment organoid of a tissue or organ.

[0017] In one aspect, the kit further includes a second cell type. In another aspect, the tissue or organ is selected from the group consisting of a fallopian tube, an endometrium, a uterus, a mammary gland, a normal breast tissue, a cervix, and an ovary.

[0018] In an additional embodiment, the invention provides a kit including a) a fallopian tube epithelial cell, b) a basement membrane ECM composition, c) a stromal ECM composition, and d) instructions to generate an in vitro 3D physiologically accurate model of a multi-compartment fallopian tube organoid.

[0019] In one aspect, the basement membrane ECM composition includes collagen IV and laminin. In another aspect, the stromal ECM composition includes collagen I. In one aspect, the stromal ECM composition further includes fallopian tube stromal cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 is a schematic of organoid generation, including the protocol to generate assembloids where stromal cells can be incorporated into the corona, and the protocol to generate standard organoids.

[0021] FIGS. 2A-2B show organoid architectural assembly using the multi-compartment protocol described herein, and as compared to the standard protocol. FIG. 2A shows photographs illustrating EGFP-tagged fallopian tube epithelial cells grown in the multicompartment organoid, control (standard technique with cell culture medium), or standard (standard technique with spheroid formation medium) conditions. FIG. 2B is a graph illustrating the results of a PrestoBlue cell viability assay: net proliferation in multicompartment assembloids with timepoint images of EGFP-tagged multi-compartment assembloids. Scale bar, 250 pm. N = 3, n = 4+. Statistical test: one-way ANOVA, ns P > 0.05. Data are mean ± SEM.

[0022] FIGS. 3A-3B show organoid structural assessment. FIG. 3A illustrates the effect of the ECM composition of the organoid structure. FIG. 3B shows photographs of electron microscopy showing ciliated and secretory cells as well as tight junctions. Scale bar, 100 pm. Inset scale bar for the left image is 500 nm, and for the right image is 300 nm. All organoid / assembloid images are maximum intensity projections of stacks of confocal microscopy images. Asterisk = junction complex, filled arrow = cilia / microvilli.

[0023] FIGS. 4A-4D show organoid molecular assessment. FIG. 4A shows immunohistochemistry stained tissue sections of the fallopian tube and fallopian tube multicompartment organoid immunofluorescence illustrating localized E-cadherin, Ki67 and PAX8 protein expression. Scale bar = 75 pm. FIG. 4B shows immunofluorescence photographs illustrating localized acetylated tubulin protein expression. FIG. 4C shows immunofluorescence photographs illustrating localized E-cadherin (left), KI67 (middle), PAX8 (right). Scale bar = 20 pm. FIG. 4D shows a photograph of an immunoblot illustrating occludin expression.

[0024] FIGS 5A-5D show organoid functional assessment. FIG. 5A illustrates hormone treated RT-qPCR results, in multi-compartment organoids treated with P-estradiol (“B”) and progesterone (“P”) normalized to control (“C”). FIG. 5B is a schematic diagram representing microbead trajectories from assembloid cores with microbeads only (noise, black), microbeads with FTECs (gray), and microbeads with FTECs treated with P-estradiol (medium). Scale bar, 10 pm. FIG. 5C is a graph illustrating cells ability displace microbeads simulating the fallopian tube’s main function. FIG. 5D is a graph illustrating microbead velocity.

[0025] FIGS. 6A-6C show quantitative comparison to the organ using CODA. FIG. 6A is a schematic of CODA methods, a 3D tissue visualization technique using Al that quantifies tissue parameters at the single cell level. FIG. 6B illustrates tissue hematoxylin & eosin (H&E)staining and matching classification of tissue components. FIG. 6C is a photograph illustrating CODA Z-proj ection of a whole healthy fallopian tube.

[0026] FIGS. 7A-7C illustrate organoid iterations. FIG. 7A shows photographs of the organoid when a second fallopian tube epithelial cell type is added to the cellular compartment. FIG. 7B shows photographs of the organoid when fallopian tube stromal cells are added to the collagen compartment. FIG. 7C shows photographs of the organoid when the collagen density in the corona is increased to better match the collagen density in the tissue stroma.

[0027] FIGS. 8A-8D. FIG. 8A is an image showing a healthy human fallopian tube tissue section, including hematoxylin and eosin (H&E), nuclei (dark grey), ECM and cytoplasm (light grey). Scale bar, 5mm. Inset, 75pm. FIG. 8B is an image showing a multi-compartment fallopian tube assembloid, Two types of FTECs are mixed in the multi-compartment assembloid core. FTEC cell lines are EGFP (elongated) and mCherry (dark grey) tagged. Images are maximum intensity projections. Scale bar, 250 pm. FIG. 8C is an image showing a standard fallopian tube organoid, and FIG. 8D is a set of representative images illustrating multi-compartment assembloids with all permutations to the assembloid ECM, including MATRIGEL™:MATRIGEL™, collagen I MATRIGEL™. and collagen I: collagen I.

[0028] FIGS. 9A-9F. FIG. 9A is an H&E image showing a fallopian tube tissue section. Nuclei (dark grey), and ECM and cytoplasm (light grey). Scale bar, 5 mm. Top inset is the fallopian tube fimbriae and bottom inset is the fallopian tube ampulla. Inset scale bars, 100 pm. FIG. 9B is an H&E image of a fallopian tube assembloid (multi-compartment). Scale bar, 300 pm. Inset is the epithelial region of the assembloid. Inset scale bar, 100 pm. FIG. 9C is a graph illustrating subcellular localization of primary FTEC multi-compartment assembloid proteins viaLOPIT map. FIG. 9D is an immunofluorescence staining image illustrating Ki-67 in multicompartment fallopian tube assembloids, and FIG. 9E is an immunofluorescence staining image illustrating MUC16 in multi -compartment fallopian tube assembloids. FIG. 9F is a set of graphs illustrating the finding that some proteins identified in the proteomes are categorized in the human matrisome for all tissues (left) and fallopian tube specific (right).

[0029] FIGS. 10A-10E. FIG. 10A is a graph illustrating relative expression of PAX8, FIG. 10B is a graph illustrating relative expression of FOXJ1, and FIG. 10C is a graph illustrating relative expression of OLFM4, which are genes related to ciliated differentiation. FIG. 10D is a graph illustrating relative expression of MKI67, a gene related to proliferation. FIG. 10E is a set of graphs illustrating Gene Ontology biological processes that were upregulated in - estradiol treated FTEC cell line (left) and primary FTEC (right) multi-compartmentassembloids. Relative protein quantification assessed by DIA-MS and subsequent enrichment analysis performed using ConsensusPathDB. N = 4.

[0030] FIGS. 11A-11F. FIG. 11A is an image of a multi-compartment assembloid used in the bead displacement assay with microbeads at the assembloid’ s epithelial -lumen-like interface. EGFP-tagged FTECs (light grey) and microbeads (medium grey). Scale bar, 100 pm. FIG. 11B is a graph illustrating relative frequency of microbead velocities. FIG. 11C is a cartoon depicting the bead displacement assay protocol in assembloids. Dotted arrows indicate microbead movement. FIG. 11D is a cartoon depicting cilia motility assay with assembloid digestion. Arrows indicate cell movement in DPBS. FIG. HE is a set of phase-contrast images from the cilia-driven motility assay. Cell trajectory (line at white arrow). Scale bar, 50 pm. FIG. HF is a graph illustrating an unpaired t-test, ****P < 0.0001, **P < 0.01.

[0031] FIGS. 12A-12D. FIG. 12A is a diagram of an iterative platform compared a tissue reference map (top left) and assembloid maps (top right). (Bottom left) Architectural parameters were quantified and compared from these maps. (Bottom right) Assembloid parameters were adjusted and CODA imaging repeated until an assembloid generation closely matched the tissue architecture. FIG. 12B is a diagram of the CODA workflow, including (i) A whole fallopian tube was serial sectioned. A subset of the tissue sections was H&E stained, (ii) Sequential tissue sections were aligned to alleviate tissue processing artifacts. Individual cells were detected by their nuclei, and tissue components were annotated in a subset of images. CODA then computationally reconstructs the tissue in 3D. FIG. 12C is a Z-proj ection of fallopian tube epithelium (light grey) and vasculature (dark grey) from a reference map. Scale bar, 5 mm. A 3D reconstruction of a cross section of the fallopian tube is shown (right). Scale bar, 0.5 mm. FIG. 12D is aZ-projection of the assembloid epithelium (purple) and stroma-like region (pink) from an assembloid map. Multi-compartment assembloid parameters: 1 x 104FTECs in a growth factor reduced MATRIGEL™ core, 2 mg / mL collagen I corona. Scale bar, 0.5 mm. A 3D reconstruction of the assembloid epithelium is shown (right). Scale bar, 0.25 mm.

[0032] FIG. 13A-13K. FIG. 13A is a set of images illustrating cell seeding numbers (10k = 1 x 104, 5k = 5 x 103), cell types (E = FTECs, S = stromal cells), MATRIGEL™ type (GFR = growth factor reduced, +GF = formulation with growth factors), and collagen density (2, 4, or 6 mg / mL) that were adjusted in the multi-compartment model. Lines indicate a new parameter modification. Images are maximum intensity projections of stacks of fluorescence confocal microscopy images. EGFP-tagged FTECs and mCherry -tagged stromal cells (mediumgrey). Scale bar, 300 gm. FIG. 13B is a set of representative cartoons of the architectural parameters quantified from CODA maps. The percent deviation of each assembloid generation from the fallopian tube reference values is: FIG. 13C is a graph illustrating epithelial packing density within the epithelium wall, FIG. 13D is a graph illustrating epithelial bulk cell density across the whole tissue / assembloid, FIG. 13E is a graph illustrating stromal cell packing density within the stroma (cells / mm3), FIG. 13F is a graph illustrating volume ratio of epithelium wall to lumen, FIG. 13G is a graph illustrating epithelium wall thickness (pm), and FIG. 13H is a graph illustrating ratio of epithelium nucleus to cytoplasm. Lines and numbering coordinate with parameter modifications. Dotted line indicates maximum target deviation (25%). n = 3. Data are mean ± SD. FIG. 131 is a graph illustrating quantitative comparison of assembloids to the healthy fallopian tube via PCA. Principal component % proportion of variance listed, n = 3. FIG. 13J is a set of images illustrating maximum intensity projections of stacks of confocal fluorescence microscopy images of the 10k 2 mg / mL + S assembloid generation. FTEC (light grey), stromal cells (dark grey), nuclear DNA (medium grey), F-actin (dark grey). Scale bar, 200 pm. FIG. 13K is a set of confocal fluorescence microscopy images of the 10k 2 mg / mL + S assembloid generation at varying heights throughout the assembloid, including FTEC (light grey), stromal cells (dark grey), nuclear DNA (medium grey), F-actin (dark grey). Scale bar, 200 pm.

[0033] FIGS. 14A-14G. FIG. 14A is a set of images illustrating of multi-compartment assembloid and standard organoid molecular expression patterns from FFPE tissue sections IHC stained for E-cadherin, including whole, healthy transplantation-quality tissue sections of the fallopian tube (left) and the multi-compartment assembloid (right). Scale bars, 4 mm (left) and 300 pm (right). FIG. 14B is a set of images illustrating isolated protein of interest channel for immunofluorescence images staining for FTEC1 (light grey) and FTEC2 (dark grey), Ki- 67, and FIG. 14C is an image of MUC16 in multi-compartment fallopian tube assembloids. Images are maximum intensity projections. Scale bars, 20 pm. FIG. 14D is an image of primary FTECs grown in the standard organoid model. Image is phase-contrast microscopy. Scale bar, 250 pm. FIG. 14E is a set of images of EGFP-tagged cells (light grey) are grown at the multicompartment cell density (1 xlO4cells / pL) and normal cell culture medium but plated according to the standard organoid technique. Images are maximum intensity projections of fluorescence confocal microscopy stacks (left) and DIC (right). Scale bars, 250 pm. FIG. 14F is a set of images of EGFP-tagged (light grey) standard organoid time series. Images are maximum intensity projections of fluorescence confocal microscopy stacks (left) and DIC(right). Scale bars, 250 gm. FIG. 14G is a set of images illustrating Assembloids with MATRIGEL™ or collagen I in the core and all combinations of corona ECM. Phase-contrast images. Scale bar, 500 gm.

[0034] FIGS. 15A-15G. 15A is an image of Ki-67, and FIG. 15B is an image of MUC16 in multi-compartment fallopian tube assembloids. Images are maximum intensity projections. Scale bars, 20 pm. FIG. 15C is a western blot of global protein expression in 2D and 3D (S = standard, MC = multi-compartment) culture. GAPDH is included as a loading control. FIG. 15D is a graph illustrating subcellular localization of FTEC cell line multi-compartment assembloid and FIG. 15E is a graph that illustrates primary FTEC standard organoid proteins via LOPIT map. FIG. 15F is a schematic diagram of total protein groups with at least two distinct peptides in FTEC cell line multi-compartment assembloids (left), primary FTEC multicompartment assembloids (middle) and primary FTEC standard organoids (right), and the protein groups with secretory (medium grey), cilia (dark grey) and extracellular (light grey) Gene Ontology cellular components. FIG. 15G is a graph illustrating the number of proteins identified in the ECM control proteome that are categorized in the human matrisome for all tissues. For FIGS. 15E-15G, N = 4.

[0035] FIGS. 16A-16F. FIG. 16A is a set of images showing FFPE tissue sections IHC stained for PAX8. Whole, healthy transplantation-quality tissue sections of the fallopian tube (left) and the multi-compartment assembloid (right). Scale bars, 4 mm (left) and 300 pm (right). FIG. 16B is an image of transmission electron microscopy of a standard fallopian tube organoid. Scale bar, 2 pm (left) and 200 nm (right). Asterisk = junction complex, filled arrow = cilia / microvilli, open arrow = secretion. FIG. 16C is a graph of RT-qPCR results of P- estradiol (P) or progesterone (P) treated standard organoids. Data are log 10 (relative expression: untreated control). FIG. 16D is a set of graphs illustrating relative expression of genes related to cell differentiation into ciliated epithelial cells and proliferation in standard organoids. Data are mean ± SD. Statistical test: one-way ANOVA with multiple comparisons, **P < 0.01, *P < 0.05. For FIGs 16C-16E, N = 1, n = 3. FIG. 16E is a volcano plot of upregulated (red)Zdownregulated (blue) protein groups when primary FTEC multicompartment assembloids were treated with p-estradiol (statistical significance: q-value < 0.05 and absolute log2(fold-change) > 0.2). FIG. 16F is a graph of Gene Ontology biological processes that were downregulated in P-estradiol treated primary FTEC multi-compartment assembloids. Relative protein quantification assessed by DIA-MS and subsequent enrichment analysis performed using ConsensusPathDB. FIG. 16E-FIG. 16F: N = 4.

[0036] FIGS. 17A-17G. FIG. 17A is a set of graphs illustrating the mechanical function of ciliated epithelial cells, including persistence time and total diffusivity of microbeads in the multi-compartment bead displacement assay. N = 3, n = 4-46. FIG. 17B is a schematic diagram depicting the bead displacement assay protocol in standard organoids. FIG. 17C is an image illustrating a standard organoid used in the bead displacement assay with a microbead at the organoid’s epithelial interface. EGFP-tagged FTECs (light grey) and microbeads (arrows). Scale bar, 100 pm. FIG. 17D is a schematic diagram illustrating representative microbead trajectories from standard organoids with microbeads only (noise, top), microbeads with FTECs (middle), and microbeads with FTECs treated with |3-estradiol (bottom). Scale bar, 7.5 pm. FIG. 17E is a graph illustrating relative frequency of microbead velocities in standard organoids. FIG. 17F is a graph of microbead MSD over 8 h. Center line, median; box limits, upper and lower quartiles; whiskers, min to max. FIG. 17G is a set of graphs illustrating persistence time and total diffusivity of microbeads in the standard organoid bead displacement assay, d-g, N = 2, n = 18-45. a, and f-g, data are mean ± SEM. Statistical test: a, and e-g, oneway ANOVA with multiple comparisons, ****P < 0.0001, **P < 0.01, *P < 0.05.

[0037] FIGS. 18A-18F. FIG. 18A is a schematic diagram of CODA workflow, illustrating fallopian tube tissue and assembloids which were formalin fixed and paraffin embedded (FFPE) and the entire tissue was serial sectioned. One in every two tissue sections were stained with hematoxylin and eosin (H&E) and the H&E tissue sections were scanned at 20X magnification. FIG. 18B is a set of images showing that all scanned tissue sections were aligned by an elastic registration process in sequential order (top left). Tissue components such as the epithelium, stroma, vasculature and nerves were manually annotated in a fraction of the images to train the algorithm to segment all tissue sections (top right). Individual cell nuclei were detected from the H&E images (bottom). Noise from image scans was adjusted in the cell count. FIG. 18C is a set of images and a table illustrating that an array of tissue parameters was measured from the processed tissue sections, and fallopian tube tissue and assembloids were visualized in 3D. Shown in the middle of FIG. 18C is an epithelium (light grey) and vasculature (dark grey) map of the fallopian tube. The bottom section of FIG. 18C illustrates the architectural parameters quantified from each assembloid generation were compared to the tissue. FIGs 18D-18F is a set of images and a table showing that independent testing of the CODA computational methods validated the performance of the reference maps. FIG. 18E is a table comparing ground truth to predicted structure, and FIG. 18F is a set of images comparing manual cell count to H&E count.

[0038] FIGS. 19A-19F. FIG. 19A is a set of images showing assembloid iterations assessed by CODA, including H&E stained FFPE tissue sections of all assembloid iterations. Cell seeding numbers (10k = 1 x 104, 5k = 5 x 103), cell types (E = FTECs, S = stromal cells), MATRIGEL™ type (GFR = growth factor reduced, +GF = formulation with growth factors), and collagen density (2, 4, or 6 mg / mL) were adjusted in the multi -compartment model. Lines indicate a new parameter modification. H&E: nuclei (dark grey), and ECM and cytoplasm (light grey). Scale bar, 500 pm. Inset scale bar, 100 pm. FIG. 19B Stromal cells regulate epithelial cell proliferation in the multi-compartment assembloid. FTECs were luciferase- tagged in the assembloid core with stromal cells in the corona. Luminescence measured on day 10. N = 3, n = 3-6. Statistical test: unpaired t-test, *P < 0.05. Data are mean ± SEM. FIG. 19C is a graph of a quantitative comparison of assembloids to the healthy fallopian tube via Euclidean distance, n = 3. FIG. 19D is a set of images illustrating maximum intensity projections of stacks of confocal fluorescence microscopy images of the 10k 2 mg / mL + S assembloid generation. Two representative assembloids shown. FTEC (light grey), stromal cells (dark grey), nuclear DNA (medium grey), F-actin (medium grey). Scale bar, 200 pm. FIG. 19E and FIG 19F are confocal fluorescence microscopy images of the 10k 2 mg / mL + S assembloid generation at varying heights throughout the assembloids. Two representative assembloids shown. FTEC (light grey), stromal cells (dark grey), nuclear DNA (medium grey), F-actin (medium grey). Scale bar, 200 pm.

[0039] FIG. 20 is a schematic diagram illustrating a protocol for generating multicompartment assembloids using the oil-in-water droplet microtechnology.

[0040] FIGS. 21A-21C. FIG. 21A is a schematic diagram illustrating the standard organoid suspends epithelial cells in MATRIGEL™ grown for 6 or more days. FIG. 21B is a set of images of endometrial epithelial cells illustrating cell proliferation during epithelium formation in multi-compartment assembloids more closely matched physiological expectations than standard organoids, while maintaining a lumenized epithelium structure. FIG. 21C is a graph illustrating epithelial proliferation over 7 days.

[0041] FIGS. 22A-22D. FIG. 22A is a schematic diagram of a typical multi-compartment assembloid that has epithelial cells in a MATRIGEL™ core and a collagen corona. FIG. 22B is a set of images illustrating assembloid models of many different healthy human tissues that have been successfully generated. FIG. 22C is a schematic diagram and image of mammary epithelial cells grown in coculture with myoepithelial cells that results in distinct structural arrangements mimicking the tissue. FIG. 22D is a schematic diagram and image of JARgestational trophoblastic neoplasia cells grown in the assembloid core that form a tumor-like structure.

[0042] FIGS. 23A-23E illustrate example modifications to a multi-compartment assembloid model. FIG. 23A is a schematic diagram and set of images illustrating that increasing the collagen concentration maintains the corona integrity when an endometrial assembloid contains stromal cells in the corona. FIG. 23B is a schematic diagram and set of images illustrating that stromal cells in the collagen layer can be separated from epithelial cells in the MATRIGEL™ core by an intermediate collagen layer in this tri-compartment system. FIG. 23C is a set of images illustrating that the size of the assembloid core reduces when endometrial epithelial cells are cocultured with stromal cells with an increasing density. FIG. 23D is a set of schematic diagrams illustrating that inversing the ECM composition of the core and corona leads to less organized mammary epithelial structure. FIG. 23E is a set of images illustrating ECM composition of the core and corona.

[0043] FIGS. 24A-24D illustrate assembloid processing techniques. FIG. 24A illustrates that assembloids are compatible with common downstream applications. FIG. 24B is a schematic diagram and set of images illustrating that fluorescently -tagged assembloids can be time-lapse imaged when assembloid cores are embedded in a larger volume (100 pL) of ECM. FIG. 24C is a schematic diagram of assembloids collected and digested for downstream protein-based assays. FIG. 24D is a schematic diagram illustrating a PrestoBlue cell viability assay that can measure the relative proliferation of the same assembloid over multiple days.

[0044] FIGS. 25A-25B. FIG.25A is a set of graphs illustrating that assembloid cell seeding and size are consistent within the same batch. FIG. 25B is a set of graphs illustrating that assembloid size and viability are homogeneous when comparing assembloid batches between trainees.

[0045] FIGS. 26A-26F illustrate multi-compartment assembloids of the human endometrial tissue. FIG. 26A is an image illustrating a reference H&E-stained tissue section of healthy endometrium. FIG. 26B is an image of a CODA reconstructed healthy section of the endometrium that indicates a dense packing of stromal cells surrounding a sophisticated network of interlinked glandular epithelium. FIG. 26C is an image illustrating that the endometrial assembloid structure matures by day 14. FIG. 26D is a set of images illustrating that stromal cells are introduced into the adjacent collagen corona to mimic the tissue architecture and cell-cell interactions. FIG. 26E is a set of images illustrating that theendometrial assembloid structure is tracked over 9 days. FIG. 26F is a set of images illustrating that stromal cells regulate epithelial proliferation.DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention is based on the seminal discovery of methods of developing 3D physiologically accurate multi-compartment organoid modelling tissues and organs.

[0047] The fallopian tubes are involved in physiological and pathological processes from pregnancy to ovarian cancer. However, current in vitro models studying their pathophysiology are limited to cultures of epithelial cells that are often compared to two-dimensional (2D) tissue sections and molecularly monitored under female reproductive hormone stimulation. These organoid cultures and their validation assays often oversimplify the heterogeneity of the 3D tissue and the physiological function of the fallopian tube, e.g., the active transport of oocytes. The present disclosure describes a multi -compartment assembloid, or organoid, of the human fallopian tube that is meticulously customized to reflect the cellular and extracellular compartmentalization of the tissue. In the present disclosure, the terms “assembloid” and “organoid” are used interchangeably. The present studies validate the proteome, cilia-driven transport function, and architectural accuracy of this assembloid through a novel platform that quantitatively compares 3D assembloids to a 3D single-cell resolution reference map of a healthy, transplantation-quality human fallopian tube using CODA. This assembloid model is systematically iterated to progressively resemble the 3D microanatomy of the human fallopian tube meanwhile recreating the molecular expression patterns and physiological function of the organ.

[0048] Cell-cell interactions and cell-extracellular matrix (ECM) interactions regulate homeostasis in tissue and organ development, and dysregulation can result in disease. A variety of cell and tissue culture systems are commonly used to study these physiological and pathological processes; however, two-dimensional (2D) culture systems enable partial capture of cell-cell interactions. The cellular heterogenicity, in situ architecture, and mechanical functions of the tissue microenvironment are accurately recapitulated in vitro using 3D systems, such as organoids, where cell interactions in three dimensions are preserved. However, many organoid models are also limited to one cell type in one ECM or artificial cocultures of multiple cell types in the same ECM, which do not match the tissue anatomy. Many organoid models also experience significant size variation between organoids and introduce non-physiological crosstalk between organoids that form within the same well. Thepresent disclosure describes a protocol for generating multi-compartment assembloids, which are organoid models that incorporate multiple cell types and ECMs to match the tissue structure. The resulting assembloids are size-consistent, spatially controlled by the compartmentalized seeding of cells and ECM, and individually isolated to avoid organoidorganoid crosstalk. Realistic substrate stiffness, cell-ECM adhesion, and 3D cell interactions allow this assembloid model to accurately capture the architectural, molecular, and functional expression patterns of healthy tissues. The present protocol offers a robust alternative for modeling healthy and diseased tissues as a bridge between 2D cell culture and animal models.

[0049] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0050] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and / or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0051] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0052] As used herein, the term “about” in association with a numerical value is meant to include any additional numerical value reasonably close to the numerical value indicated. For example, and based on the context, the value can vary up or down by 5-10%. For example, for a value of about 100, means 90 to 110 (or any value between 90 and 110).

[0053] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood thatmodifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0055] The fallopian tubes are a pair of muscular tube-like organs in the female reproductive system. Their primary functions are to catch ovulating oocytes from the adjacent ovaries, provide an environment that supports the survival of sperm and eggs for fertilization, and transport a developing embryo from an ovary to the uterus for implantation. This process is achieved through peristalsis driven by the fallopian tube smooth muscle and motile cilia along the fallopian tube epithelium. Structural or functional abnormalities are responsible for tubal (ectopic) pregnancy, a life-threatening condition if left untreated. The fallopian tubes also connect the uterine and peritoneal cavities, and serve as the anatomic structure for retrograde menstruation, a leading theory of endometriosis etiology which affects 10% of reproductive age women. Recently, the fallopian tubes have received significant research attention because of a new paradigm for the onset of high-grade serous ovarian carcinoma (HGSC), a common subtype of ovarian cancer. This paradigm posits that HGSCs arise from precursor lesions in the fallopian tube epithelium, called serous tubal intraepithelial carcinomas (STICs). The exact physical and molecular mechanisms that govern these fallopian tube conditions remain largely unknown, in part due to the lack of appropriate preclinical models. HGSC and endometriosis do not spontaneously occur in mice as they do in humans, so researchers have turned to engineered mouse models and in vitro organoid models to study these human diseases in an environment with high physiological similarity.

[0056] Histologically, the fallopian tube mucosa, muscularis, and serosa regions are composed of different combinations of epithelial, stromal, and / or smooth muscle cells, the extracellular matrix (ECM) of these regions varies by collagen density and the presence of basement membranes, laminin, and other ECM proteins. Beyond this compositional complexity, the organ undergoes several dynamic changes due to peritoneal fluid, and the hormones and follicular fluid that are released at various stages of the reproductive cycle. These features and interactions are characteristic of a healthy fallopian tube.

[0057] The present disclosure describes a novel multi-compartment assembloid of the human fallopian tube. An assembloid is a type of organoid that cultures multiple cell types in spatially distinct regions, thus improving the model’s compositional accuracy compared to the in situ tissue. The ECM and cellular composition of each compartment in the model can be adjusted to closely match the tissue composition. The multi-compartment assembloid preserved the molecular expression patterns observed in histological fallopian tube tissuesections and produced a physiological response to menstrual cycle hormone stimulation according to previously published validation parameters for the state-of-the-art (standard) fallopian tube epithelial organoid. Multi-compartment assembloids and standard organoids were compared to the human fallopian tube proteome via global label-free proteomics. A novel functional assay evaluated the multi-compartment assembloid’s functional capacity for cilia- driven transport of oocyte-mimicking microbeads along the epithelium facing the assembloid’s lumen-like region. Fallopian tube assembloids were rigorously compared to a whole, transplantation-quality human fallopian tube using CODA, a novel three-dimensional (3D) pathology approach that quantitatively maps the microanatomy of organs at cellular resolution and labels epithelial cells and stroma within the fallopian tube. Biomimetic parameters were identified that could be measured from a defined volume near the fimbriated end of the fallopian tube, a common location for HGSC precursor lesions, and compared assembloid and tissue values. These architectural parameters were carefully adjusted through multicompartment assembloid modifications to iteratively converge the architecture of the assembloids toward a reference map of a healthy human fallopian tube.

[0058] Direct 3D and quantitative comparison of in vitro cell culture models to organ microarchitecture and cellular composition has not been performed for fallopian tube or other organoid / assembloid models, although the field of in vitro modeling has recognized the importance of validation through other histopathology and tissue-omics-based approaches. The traditional comparison of organoid architecture and molecular expression patterns to conventional histological sections of reference human samples provides a general 2D comparison, but neglects the complex heterogeneity of tissues that are revealed in 3D. Through the iterative process, a quantitatively optimized and functional assembloid model was produced of a human fallopian tube. This assembloid can be leveraged in future studies investigating disease states of the fallopian tubes, and the assembloid validation platform can be applied to any tissue-organoid or tissue-assembloid pair to determine the biological accuracy of in vitro models.

[0059] The present disclosure describes a tissue-informed assembloid engineering platform for the development and validation of in vitro models with quantitative comparisons between the assembloid and the organ. This multifaceted protocol can be applied to organoid / assembloid models of any tissue / organ and adapted to match the architectural, molecular, and functional parameters of the organ of interest. CODA-based analysis with multi-compartment assembloid modeling pair to form a robust platform to develop customphysiologically and anatomically accurate in vitro models for study of the intricacies of many diseases and conditions. Current morphometric-based CODA can be similarly combined with molecular features derived from single-cell and spatial transcriptomics to provide improved diagnostics or assessments of disease models.

[0060] This platform is presented in the context of a healthy human fallopian tube as a basis for future studies of gynecologic diseases, such as HGSC. The present multi-compartment assembloid is similar to the mucosal folding and ECM composition of the human fallopian tube following protocols that were previously used to validate the current state-of-the-art fallopian tube organoid model. Second, the molecular similarity of the assembloid model to the fallopian tube was evaluated via comprehensive DIA-MS proteomics. The response of fallopian tube assembloids to female reproductive hormone stimulation was demonstrated via traditional functional assays and developed a novel assay that measured the ability of the assembloid epithelium to actively transport an oocyte. Through these two functional assays, the multicompartment assembloid mimicked the molecular and mechanical functions of the fallopian tube. Finally, the multi-compartment assembloid architecture was directly compared to a reference fallopian tube using quantitative metrics via CODA. The CODA-based analysis focused on the ampulla region near the fimbriated end of the reference fallopian tube where architectural measurements could be determined from a defined volume and HGSC precursor lesions are often found. Design parameters of the multi-compartment assembloids were customized to match the post-menopausal conditions of this reference organ through iterative adjustments to assembloid design parameters and sequential CODA mapping.

[0061] The multi-compartment assembloid demonstrated improved physiological similarity to the fallopian tube compared to the current state-of-the-art organoid model via molecular, functional, and architectural assessments. Tissue molecular expression patterns were preserved, more fallopian tube-specific matrisome proteins were expressed, and both secretory and ciliated epithelial cells were polarized to face the lumen-like region in the multicompartment assembloid. Both the molecular function (hormone response) and mechanical function (transport of an oocyte) of the healthy human fallopian tube were recreated in the multi-compartment model. Architecturally, a final generation of the multi-compartment assembloid closely matched the reference fallopian tube architecture.

[0062] Customizing an assembloid model with reference to the architecture of a whole, transplantation-quality human organ provides an additional quantitative validation of the assembloid model. As parameters of the multi-compartment fallopian tube assembloid weremodulated, CODA imaging and architectural quantification identified an optimized combination of assembloid parameters. While the standard model performed similarly to some multi-compartment configurations, the multi-compartment assembloid combination of 6 x 103stromal cells in a 2 mg / mL collagen I corona with 1 x 104epithelial cells in growth factor reduced MATRIGEL™ (10k 2 mg / mL + S) closely mimicked the fallopian tube cellular and extracellular architecture (< 25% deviation for many architectural measurements). The addition of stromal cells in the assembloid corona caused a significant increase in architectural similarity of the assembloid to fallopian tube tissue. Stromal cells are physiologically added to such assembloid models when isolated from epithelial cells in a collagen-rich ECM, which uses a multi-compartment model.

[0063] The use of customizable multi-compartment assembloids allows us to decipher some of the compositional features that support the maturation, controlled growth, and maintenance of fallopian tubes. This work establishes the importance of two physiological extracellular matrices (i.e., a basement membrane and surrounding collagen compartment) for FTECs to organize into mucosal folds and stimulate the production of additional ECM proteins that match the ECM composition of the human fallopian tube. Such ECM interactions are used to polarize FTECs such that both ciliated and secretory cells face the lumen-like region where ciliated cells could reproduce the organ’s mechanical function and transport an oocyte. The importance of stromal cells was shown in a secondary compartment, as observed in fallopian tube tissue, to limit epithelial growth, so that the mucosal folding architecture could be maintained over long time scales. The multi-compartment assembloid model of the human fallopian tube is a tissue- validated, healthy model. In future work, the cell-cell and cell-ECM interactions in the assembloid model can also be customized to reflect diseased tissues (e.g., precursor lesions to HGSC) and referenced back to corresponding diseased tissue CODA maps via the iterative platform was demonstrated here. The versatility of this approach provides the opportunity to include additional cell mechanical interactions or introduce disease-related ECM changes in downstream experimental applications, or to apply similar 3D culture, proteomics-based, and CODA mapping techniques to evaluate the physiological accuracy of in vitro models of other human tissues.

[0064] The oil-in-water droplet microtechnology permits a highly controlled and precise method for consistent assembloid generation. This protocol offers an informed modeling approach for a wide range of human tissues and facilitates the exploration of disease mechanisms at the ECM, cellular and molecular levels. Preliminary investigation ofendometrial assembloids reveals the advantage of adding a physiologically separate stromal compartment, suggesting a stromal regulation in epithelial development.

[0065] In one embodiment, the present invention provides a method of generating a multicompartment organoid including a) contacting a first ECM composition with a first cell type to generate a first ECM compartment, and b) contacting the first ECM compartment of a) with a second ECM composition to generate a second ECM compartment or a larger ECM compartment including the first ECM compartment embedded into the second ECM composition, wherein the multi-compartment organoid is an in vitro 3D physiologically accurate model of a tissue or organ, thereby generating a multi-compartment organoid.

[0066] As used herein, the term “organoid” refers to a miniaturized and simplified version of an organ produced in vitro in three dimensions that mimics the functional, structural, and biological complexity of that organ. The methods described herein yield “multi-compartment organoids”, which include at least two compartments. For example, the organoids include 2, 3, 4,5 or more compartments.

[0067] The methods described herein yield multi-compartment organoids that are a physiologically accurate model of a tissue or organ. By “physiologically accurate”, it is meant that the multi-compartment organoid constitutes a model of a tissue or organ having or recapitulating the physiological architecture of the tissue or organ. The physiological architecture of an organ or tissue includes for example cell-cell interactions, as well as cell / extracellular matrix interactions. Notably, the multi-compartment organoid culture features a microenvironmental architecture that mimics both the basement membrane and stromal barriers of a tissue or organ.

[0068] The methods described herein rely on the generation of a first ECM compartment, and on the subsequent generation of a second ECM compartment encompassing the first ECM compartment.

[0069] As used herein, the terms “extracellular matrix composition” and “ECM composition” can be used interchangeably and refer to protein composition used to provide cells in culture with a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins, and hydroxyapatite that provides structural and biochemical support to the cells. Because multicellularity evolved independently in different multicellular lineages, the composition of ECM varies between multicellular structures; however, cell adhesion, cell-to-cell communication and differentiation are common functions of the ECM. The animal extracellular matrix includes the interstitial matrix (orstromal matrix) and the basement membrane. Interstitial matrix is present between various animal cells (i.e. , in the intercellular spaces). Gels of polysaccharides and fibrous proteins fill the interstitial space and act as a compression buffer against the stress placed on the ECM. Basement membranes are sheet-like depositions of ECM on which various epithelial cells rest. Each type of connective tissue in animals has a type of ECM: collagen fibers and bone mineral comprise the ECM of bone tissue; reticular fibers and ground substance comprise the ECM of loose connective tissue; and blood plasma is the ECM of blood.

[0070] “ECM compartment” is meant to refer to each compartment of the multicompartment organoid, which as described herein includes an ECM composition and optionally a cell type.

[0071] In one aspect, the generation of a first ECM compartment relies on contacting a first ECM composition with a first cell type to generate a droplet. The droplet includes the first cell type embedded in the first ECM composition in a mineral oil.

[0072] The methods described herein rely on a high-throughput oil-in-water droplet microtechnology to generate highly uniform, small-volume, multi-compartment organoids. By “contacting the first ECM composition with the first cell type”, it is meant that a preset number of cells (i.e., cells from the tissue or organ corresponding to the multi-compartment organoid to be generated) is mixed, resuspended, homogenized in an ECM composition, and that the resulting mixture is then dropped into a mineral oil and incubated at 37°C for a period of time sufficient for the ECM composition to for a gel. For example, the incubation can last 10, 15, 20, 25, 30, 35, 40, 45 or more minutes.

[0073] The sphere obtained thereby, corresponding to the first compartment of the multicompartment organoid contains the cells, and can be harvested from the mineral oil.

[0074] As used herein, the term “mineral oil” refers to any colorless, odorless, light mixture of higher alkanes obtained from a mineral source for molecular biology applications, particularly a distillate of petroleum, as distinct from usually edible vegetable oils. The name “mineral oil” may also be referred to as “white oil”, “paraffin oil”, “liquid paraffin” (a highly refined medical grade), or “liquid petroleum”. Often, mineral oil is a liquid obtained from refining crude oil to make gasoline and other petroleum products. More generally, mineral oil is a transparent, colorless oil, composed mainly of alkanes and cycloalkanes, related to petroleum jelly. It has a density of around 0.8-0.87 g / cm3 (0.029-0.031 Ib / cu in).

[0075] In another aspect, the generation of a second ECM compartment relies on contacting the first ECM compartment with a second ECM composition to generate the multi-compartment organoid. The droplets (i. e. , the first compartments or first spheres) including the first cell type embedded in the first ECM composition are resuspended in a second ECM composition. By “contacting the first ECM compartment with the second ECM composition”, it is meant that the droplets (i. e. , the first compartments or first spheres) are resuspended in the second ECM composition, and that the resulting mixture is dropped into a mineral oil 37 °C for a period of time sufficient for the second ECM composition to for a gel. For example, the incubation can last 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more minutes.

[0076] The sphere obtained thereby (i.e. , second sphere) corresponding to the second compartment of the multi-compartment organoid contains the first sphere (including the cells) embedded in a second ECM compartment or a larger ECM compartment and can be harvested from the mineral oil.

[0077] In one aspect, the first ECM composition and the second ECM composition are different ECM compositions. In such case, the first ECM compartment and the second ECM compartments have a structurally different ECM composition.

[0078] In another aspect, the first ECM composition and the second ECM composition are a same ECM composition. In such case, the first ECM compartment and the second ECM compartments have a structurally identical ECM composition, with the addition of the cells amounting to the difference in the two compartments. In such case, the second compartment can alternatively be referred to as a larger ECM compartment rather than a second ECM compartment.

[0079] In the methods described herein, the first ECM composition and the second ECM compositions can be the same or a different ECM composition. The ECM compositions combination is meant to reproduce the physiological microenvironment of the tissue or organ from which a multi-compartment organoid is being generated.

[0080] In one aspect, a combination of the first and second ECM compositions mimics a tissue or organ physiological microenvironment. By “mimicking a tissue or organ physiological microenvironment”, it is meant that the combination of the ECM compositions creates a two-compartment architecture. For example, the two-compartment architecture includes a basement membrane ECM composition juxtaposed with a stromal ECM composition.

[0081] In one aspect, the first ECM composition includes a basement membrane ECM composition.

[0082] ‘ ‘Basement membrane ECM”, “basement membrane” or “BMs” are highly specialized extracellular matrices (sECMs) with distinct components that support functions including differentiation, proliferation, migration, and chemotaxis of cells during development. The composition of these sECM is as different as the tissues to which they are localized, opening the possibility that such matrices can fulfill distinct functions. Changes in BM composition play significant roles in facilitating the development of various diseases.

[0083] Basement membranes (BMs) are specialized extracellular matrices (sECMs) BMs form thin acellular layers that underlie cells and separate the cells from and connect them to their interstitial matrix. The formation of BMs is a prerequisite for normal tissue development and function. In addition, individual components of BMs regulate different biological activities, such as the development, proliferation, differentiation, growth, and migration of cells, via cell surface receptors (e.g., integrins) and non-integrin-receptors. Furthermore, BMs control cellular functions by binding and modulating the local concentrations of growth factors and cytokines. BMs are able to regulate cell polarity, cell adhesion, spreading, and migration via their effects on the cytoskeleton. Non-limiting examples of proteins that are component of BMs include: laminins, perlecan, collagen IV, agrin, and nidogens.

[0084] In some aspects, the basement membrane ECM composition includes collagen IV and laminin.

[0085] In another aspect, the second ECM composition includes a stromal ECM composition.

[0086] “Stromal ECM” or “interstitial ECM” is generally composed of component produced intracellularly by resident cells and secreted into the interstitial space via exocytosis. Once secreted, the component aggregates into an interlocking mesh of fibrous proteins and glycosaminoglycans. Non-limiting examples of components of the interstitial ECM include proteoglycans, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, collagens, elastin, fibronectin, and laminins.

[0087] In some aspects, the stromal ECM composition includes collagen I.

[0088] The methods described herein rely on the generation of a first ECM compartment comprising a first cell type, and on the subsequent generation of a second ECM compartment encompassing the first ECM compartment.

[0089] In some aspect, the second ECM compartment includes the second ECM composition and a second cell type. In some aspects, the first and the second cell type are a same cell type or a different cell type. That is, to generate a 3D physiologically accurate modelof a tissue or organ, the methods described herein rely on the combination of two or more cell types from a tissue or organ, such that the various cell types can interact with one another in the 3D multi-compartment organoid and reproduce as accurately as possible the cell-cell interaction in the actual tissue or organ.

[0090] In one aspect, the tissue or organ is selected from the group consisting of a fallopian tube, an endometrium, a uterus, a mammary gland, a normal breast tissue, a cervix, and an ovary.

[0091] In one aspect, the multi-compartment organoid is maintained in a 3D suspension culture.

[0092] The methods described herein present the advantage of allowing the multicompartment organoid to be maintained in suspension throughout their culture. That is, the organoids are not in contact with glass, plastic or any other material that can be used for organoid culture. Rather, when the double-layered spheres (i.e., the multi-compartment organoids) are collected, they are cultured and maintained in suspension.

[0093] In another embodiment, the invention provides a multi -compartment organoid obtained by any one of the methods described herein.

[0094] In an additional embodiment, the invention provides a method of generating a multicompartment fallopian tube organoid including a) contacting a basement membrane ECM composition with a fallopian tube epithelial cells to generate a first ECM compartment, and b) contacting the first ECM compartment of a) with a stromal ECM composition to generate a second ECM compartment including the first sphere embedded into the stromal ECM composition, wherein the multi-compartment fallopian tube organoid is an in vitro 3D physiologically accurate model of a fallopian tube, thereby generating a multi-compartment fallopian tube organoid.

[0095] The fallopian tubes, also known as uterine tubes, oviducts, or salpinges, are paired tubes in the human female that connect the uterus to the ovaries. The fallopian tubes are part of the female reproductive system. Each tube is a muscular hollow organ that is on average between 10 and 14 cm (3.9 and 5.5 in) in length, with an external diameter of 1 cm (0.39 in). It has four described parts: the intramural part, isthmus, ampulla, and infundibulum with associated fimbriae. The fallopian tubes are lined with simple columnar epithelium with hairlike extensions called cilia which together with peristaltic contractions from the muscular layer, move fertilized egg (zygote) along the tube.

[0096] In one aspect, the stromal ECM composition further includes fallopian tube stromal cells. In another aspect, the multi-compartment fallopian tube organoid has a lumen-like architecture. In one aspect, the multi-compartment fallopian tube organoid includes ciliated and secretory cells in a lumen compartment and tight junctions at a basement membrane ECM- stromal ECM interface.

[0097] In some aspects, ciliated cells express elevated levels of FOXJ1 and / or OLFM4, as compared to non-ciliated epithelial cells.

[0098] In other aspects, secretory cells are PAX8 positive.

[0099] In another aspect, the multi-compartment fallopian tube organoid is sensitive to |3- estradiol and / or to progesterone. In some aspects, in response to -estradiol epithelial cells in the multi-compartment fallopian tube organoid become ciliated. In various aspects, ciliated cells displace objects in a multi-compartment fallopian tube organoid lumen.

[0100] In one aspect, the 3D physiologically accurate model of a fallopian tube includes a multi-compartment fallopian tube organoid having a physiological architecture of a fallopian tube.

[0101] In another embodiment, the invention provides a multi-compartment fallopian tube organoid obtained by any one of the methods described herein.

[0102] In one embodiment, the invention provides a multi-compartment organoid including: a) a first ECM compartment including a first cell type and a first ECM composition, and b) a second ECM compartment including a second ECM composition, wherein the first ECM compartment is embedded into the second ECM compartment, wherein the multi-compartment organoid is an in vitro 3D physiologically accurate model of a tissue or organ.

[0103] In one aspect, the first ECM composition and the second ECM composition are different ECM compositions. In another aspect, the second ECM composition includes a second cell type. In one aspect, the first and the second cell type are a same cell type or a different cell type. In various aspects, the tissue or organ is selected from the group consisting of a fallopian tube, an endometrium, a uterus, a mammary gland, a normal breast tissue, a cervix, and an ovary.

[0104] In another embodiment, the invention provides a multi-compartment fallopian tube organoid including a) a first ECM compartment including a fallopian tube epithelial cell and a basement membrane ECM composition, and b) a second ECM compartment including a stromal ECM composition, wherein the first ECM compartment is embedded into the secondECM compartment, wherein the multi -compartment fallopian tube organoid is an in vitro 3D physiologically accurate model of a fallopian tube.

[0105] In one aspect, the basement membrane ECM composition includes collagen IV and laminin. In another aspect, the stromal ECM composition includes collagen I. In one aspect, the stromal ECM composition further includes fallopian tube stromal cells. In another aspect the multi-compartment fallopian tube organoid has a lumen-like architecture. In some aspects, the multi-compartment fallopian tube organoid includes ciliated and secretory cells in a lumen compartment and tight junctions at a basement membrane ECM-stromal ECM interface. In various aspects, ciliated cells express elevated levels FOXJ1 and / or OLFM4, as compared to non-ciliated epithelial cells. In other aspects, secretory cells are PAX8 positive. In one aspect, the multi-compartment fallopian tube organoid is sensitive to |3-estradiol and / or to progesterone. In some aspects, in response to P-estradiol epithelial cells in the multicompartment fallopian tube organoid become ciliated. In various aspects, ciliated cells displace objects in a multi-compartment fallopian tube organoid lumen.

[0106] In a further embodiment, the invention provides a kit including a) a first cell type, b) a first ECM composition, c) a second ECM composition, and d) instructions to generate an in vitro 3D physiologically accurate model multi-compartment organoid of a tissue or organ.

[0107] In one aspect, the kit further includes a second cell type. In another aspect, the tissue or organ is selected from the group consisting of a fallopian tube, an endometrium, a uterus, a mammary gland, a normal breast tissue, a cervix, and an ovary.

[0108] In an additional embodiment, the invention provides a kit including a) a fallopian tube epithelial cell, b) a basement membrane ECM composition, c) a stromal ECM composition, and d) instructions to generate an in vitro 3D physiologically accurate model of a multi-compartment fallopian tube organoid.

[0109] In one aspect, the basement membrane ECM composition includes collagen IV and laminin. In another aspect, the stromal ECM composition includes collagen I. In one aspect, the stromal ECM composition further includes fallopian tube stromal cells.

[0110] Without wishing to be bound to any theory, it is believed that the methods described herein can be used for the generation of multi-compartment organoid that physiologically and accurately model several heathy tissue and organ including a fallopian tube, an endometrium, a uterus, a mammary gland, a normal breast tissue, a cervix, and an ovary. The multicompartment organoid has multiple uses ranging from the study of the natural development of cell-cell interaction during the formation of the organoid, to the study of the transformation ofthe healthy organs into diseased organ or tissue. The organoid can also be used to predict the effect of drugs or drug candidates on the normal development, on the induction of a disease, or on the responsiveness of a diseased organ. The organoid can also be used for in vitro assays examining these described applications or implanted in animal models of xenograft implantation.

[0111] Presented below are examples discussing the generation of multicomponent organoids contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.EXAMPLES EXAMPLE 1 METHODS

[0112] Organoids were generated using an oil-in-water technique (as illustrated in FIG. 1). Briefly, fallopian tube epithelial cells were suspended in growth factor reduced MATRIGEL ™ (a basement membrane ECM composition) and 1 pL droplets of fallopian tube epithelial cells in an ECM were allowed to gel in mineral oil at 37°C. These droplets were then harvested and resuspended in 10 pL droplets of another ECM (2mg / ml collagen I) and cultured under standard cell culture conditions.

[0113] These organoids were structurally, functionally, and molecularly compared to the tissue of origin by light and electron microscopy, hormone responses by RT-qPCR, and protein-level assessment. Organoids were also cultured under standard cell culture conditions and stained for tissue specific markers by immunofluorescence. Further quantitative structural analysis will include CODA, comparison of organoids and whole healthy fallopian tubes.EXAMPLE 2RESULTS

[0114] Multi-compartment organoids were generated by suspending cells in a 1 pL drop of MATRIGEL™ and embedding the MATRIGEL™ core in collagen I, as illustrated in FIG. 1 in the multi -compartment upper section. The standard protocol in which cells are in a 50 uL drop of Matrigel and then cultured on a 2D plate or dish is illustrated in the lower part of FIG.1

[0115] As illustrated in FIG. 2A fallopian tube epithelial cells grown in the multicompartment organoid conditions assembled into a complex, tissue lumen-like architecture. These cells grew in unorganized clusters when grown in a drop of MATRIGEL™ on a plate (control) or in small spherical structures when specialized spheroid formation medium is added. A network of epithelial lined mucosa protruded into the fallopian tube’s lumen and contains a mixture of secretory and ciliated epithelial cells. As illustrated in FIG. 2A, the organoid model simulated this structure at a physiological scale when the ECM composition of each compartment matched the tissue architecture (basement membrane matrix and epithelial cells contained in an inner compartment with a collagen I secondary compartment). As illustrated in FIG. 2B, the organoid architecture assembly was not driven by cell proliferation.

[0116] As illustrated in FIG. 3A, the multi-compartment organoid model is ECM tunable by compartment. A MATRIGEL™ core with a collagen corona was found physiologically accurate and supported an intricate lumen-like architecture. As shown in FIG. 3B, ciliated and secretory cells were observed in the MATRIGEL™ (lumen) compartment and tight junctions at the MATRIGEL™ and collagen (stroma) were observed at interface of the multicompartment organoid.

[0117] As illustrated in FIG. 4A, protein expression as assessed by immunohistochemistry matched protein expression in the healthy tissue. Lateral E-cadherin accumulation, PAX8+ secretory cells and scattered KI67 positive cells were observed. The requirement for ECM composition to match the tissue architecture underlines the importance of biological accuracy in organoid modeling. The molecular assessment revealed that localized protein expression matches the patterns observed in tissue sections where E-cadherin is localized at adherent junctions, dispersed cells are positive for KI67, many cells are PAX8 positive / secretory, and cells organize into columnar epithelium visualized by immunofluorescence and immunohistochemistry (FIGs 4A, 4B and 4C). There was low acetylated tubulin (cilia) expression in the organoid, although some scattered cells were ciliated (see FIG. 4B). As further illustrated in FIG. 4D, global protein assessment via western blot showed E-cadherin adherent junctions formed in multi-compartment organoids, while low occludin expression was observed in all systems. This organoid model reproduced the fallopian tube’s characteristic lumen with protruding epithelial lined mucosa and reflected the organ’s organization in scale and structure (FIG. 4B). This architecture formed when each organoid compartment contained ECM materials that were physiologically accurate: epithelial cells were suspended in a low growth factor basement membrane surrounded by a collagen rich compartment. AlternativeECM compositions in adjacent compartments fail to promote this architectural assembly, underlining the importance of biological accuracy in organoid modeling. These organoids also retained the localized expression of common fallopian tube markers as observed in tissue sections. This included columnar epithelial organization, E-cadherin localization at adherens junctions between cells, dispersed cells that are KI67 positive and PAX8 positive cells.

[0118] Functionally, the multi-compartment organoids responded to |3-estradiol treatment by differentiating some cell populations into ciliated cells (decrease PAX8, increase FOXJ1 and ESRI) and progesterone by a decrease in MKI67, which agrees with physiological expectations (see FIGs 5A-5D). Notably, as shown in FIG. 5A, the organoid response to treatment as analyzed by RT-qPCR showed that cells become ciliated in response to -estradiol (increase FOXJ1, ESRI, OLFM4; decrease PAX8, acetylated tubulin). Cells were found capable of transporting beads in the lumen when ciliated (FIG. 5C), with bead trajectories and mean squared displacements over 8 hours as well as bead velocity increasing when cells are ciliated (FIG. 5B and FIG. 5D).

[0119] These assessments demonstrated that the engineered human fallopian tube organoid model simulated in vitro the organ’s structure, molecular expression patterns and function, and can be used in studies of diseases of the fallopian tube. Biological architecture is important in organoid modeling because the tissue geometry can influence the cell-cell and cell-ECM interactions that contribute to disease development. This is especially true for studies of early HGSC development in the fallopian tube where an intricate architecture is likely to contribute to cell shedding and metastasis at a secondary site across the fallopian tube lumen or the peritoneal cavity. This study presented an engineered fallopian tube organoid model with improved structural accuracy. Precursor lesion cell shedding and reintegration across the lumen has been observed in this model, and future studies will investigate the molecular mechanisms regulating this early metastatic process.

[0120] This study presents an engineered fallopian tube organoid model with improved physiological accuracy. It was found that tissue geometry can influence the cell-cell and cell- ECM interactions that contribute to disease development. In the early stages of HGSC development, the architecture of the fallopian tube epithelium likely contributes to cell shedding and eventual metastasis to a secondary site (which has been observed in this model). Multi-compartment assembloids self-organize to mimic the mucosal folds of the human fallopian tube

[0121] The described multi-compartment fallopian tube assembloid was designed to mimic the in situ microenvironment where epithelial cells lining the fallopian tube lumen are supported by a thin basement membrane and surrounded by a collagen-rich stromal matrix (FIG. 8A). This multi-ECM microenvironment was captured in vitro by first suspending fallopian tube epithelial cells (FTECs, 1 x 104cells / pL) in a small (1 pL) droplet of MATRIGEL™, a solubilized basement membrane (FIG. 8B). This first compartment, (the assembloid core) was embedded in a larger (10 pL total assembloid volume) second compartment, (assembloid corona), composed of collagen I where stromal cells can be incorporated (FIG. 1). This protocol was based on an oil-in-water droplet microtechnology recently developed to generate multi-compartment tumor organoids. Here, the versatility of this approach was leveraged by adjusting the concentrations of cells and ECM in the core and corona compartments to fit the fallopian tube architecture. FTECs were cultured in a growth factor reduced MATRIGEL™ core surrounded by a 2-6 mg / mL collagen I corona. Compared to the standard fallopian tube organoid model (FIG. 8C), where FTECs (-400 cells / pL) were suspended in 50 pL drops of MATRIGEL™ and supplemented with inhibitors / growth factors to encourage 3D growth (FIG. 1), the multi-compartment model improved the complexity of the extracellular environment and produced an epithelial architecture that more accurately mimics the fallopian tube’s mucosal folding (FIG. 4A). This new approach reduced the ECM reagents used to establish an organoid culture from 50 pL (standard) to 10 pL (multicompartment); however, the cell-free regions in multi-compartment assembloids more closely resembled the size and shape of the fallopian tube lumen. The epithelial structures in multicompartment assembloids were also much larger than the epithelial structures produced by standard protocols or a technical control where standard organoids were grown in the same unmodified culture medium used for multi-compartment assembloids (FIG. 2A).

[0122] Spontaneous epithelial organization that mimicked the tortuosity of the human fallopian tube’s mucosal folds was supported in multi -compartment assembloid cultures of many different FTEC sources, including immortalized FTECs (FIG. 14A), a mixture of FTEC cell lines (FIG. 14B), and primary FTECs (FIG. 14C). This architecture did not form when the same cells (1 x 104cells / pL) were plated according to the standard organoid technique but grown in the same unmodified cell culture medium used in multi-compartment cultures (labeled technical control, FIG. 14E). In the standard model, organoids grow from single cells, although not all FTECs developed into standard organoids (FIG. 14D). Multiple standard organoids often stacked on top of or next to each other, causing non-physiological long-rangeinteractions between separate organoids within the same primary or immortalized FTEC culture (FIG. 14F). These standard organoids assembled into small spherical clusters of cells with a hollow center that resembled a cell-free lumen-like region, but were architecturally simplified compared to the complexity of the fallopian tube’s mucosal folds.

[0123] The spontaneous assembly of FTECs into the mucosal architecture was not primarily driven by cell proliferation. The architecture did not begin to assemble until after day 3, but the change in proliferation from day 3 to day 7 was not statistically significant (FIG. 2B). The formation of these folds therefore depended on cell re-organization and interactions with the immediate and surrounding ECM (FIG. 8D, FIG. 14G). The intricate architecture resembling the fallopian tube’s lumen formed when the matrix components were organized to resemble the fallopian tube anatomy, i.e., epithelial cells were embedded in a basement membrane (MATRIGEL™) and surrounded by a collagen I stroma-like environment. The MATRIGEL™:MATRIGEL™ combination did not allow sufficient cell growth for the assembloid architecture to mature. In the collagen I: MATRIGEL™ assembloid, cell growth was contained within the core but disorganized, and in the collagen I: collagen I assembloid, epithelial cells invaded the stroma-like region. The MATRIGEL™: collagen I core: corona combination confined FTECs to a central mucosal region and provided the extracellular structure for organized folds to form.

[0124] These multi-compartment assembloids can be further customized to match the in situ microenvironment, such as by adding a third compartment containing smooth muscle cells. Here and as further demonstrated below, the dual-compartment model was sufficient for the spontaneous organization of an assembloid architecture resembling the fallopian tube’s mucosal folds.Multi-compartment assembloids maintain in situ protein expression

[0125] In addition to the similarity between the fallopian tube’s lumen and the multicompartment assembloid’s organization, the molecular expression patterns observed in these assembloids also matched the expression patterns observed in a healthy fallopian tube. By direct comparison via immunohistochemistry (IHC) of FFPE tissue sections from a transplantation-quality human fallopian tube (FIG. 9A) and the assembloid (FIG. 9B), similar patterns of E-cadherin localization were seen to the lateral cell membranes between epithelial cells (FIG. 9C, FIG. 16A) Ki-67-positive cells were scattered along the assembloid epithelium via immunofluorescence (FIG. 9D, FIG. 15A), which matched previously reported patterns in fallopian tube tissues. In this healthy fallopian tube assembloid model, MUC16staining was faint in some clustered regions (FIG. 9E, FIG. 15B). An increase in MUC16 expression could be expected in future studies using this model to investigate disease states of the fallopian tube, for example, but limited MUC16 staining in this healthy baseline model aligns with physiological expectations.

[0126] The multi-compartment assembloid model formed adherens junctions (E-cadherin) and tight junctions (occludin), via global protein expression (FIG. 15C). Similar global protein expression was observed in the standard organoid model. Compared to FTECs grown in standard 2D culture, E-cadherin expression was significantly increased in both 3D models.

[0127] Beyond these select fallopian tube epithelial markers, label-free data-independent acquisition-mass spectrometry-based proteomics (DIA-MS) was performed for a more comprehensive evaluation of the multi -compartment assembloid and standard fallopian tube organoid models. DIA-MS provided reproducible and accurate quantification of the subcellular proteomes (Supplementary Data 1-7) and the matrisomes, which were further compared to the fallopian tube matrisome previously reported in the Matrisome database. This analysis was performed on primary FTEC (FIG. 9C) and immortalized FTEC (FIG. 15D) assembloids to analyze the behavior of FTECs from two different sources in the multi-compartment model, and primary FTEC multi-compartment assembloids were also compared to primary FTEC organoids (FIG. 15E) grown in the standard model. When primary FTECs were cultured in the multi-compartment assembloid, the total number of quantifiable protein groups with at least two distinct peptides was 2,255, which was more than three times the total number of protein groups quantified when the same primary FTECs were cultured in standard organoids with 674 proteins identified (FIG. 15F). One distinction between the multi-compartment assembloid and standard organoid proteomes is the percentage of protein groups that match with extracellular components: 43% for FTEC cell line multi-compartment assembloids, 54% for primary FTEC multi-compartment assembloids, and 77% for standard organoids. However, the total number of matrisome proteins identified in multi -compartment assembloids was more than double the number of matrisome proteins identified in standard organoids (FIG. 9F). Multi-compartment assembloids also expressed larger numbers of fallopian tube specific matrisome proteins than standard organoids. One advantage of the multi-compartment assembloid model is the ability to include multiple types of ECM compared to the single ECM present in standard organoids. The multi-compartment assembloids in this study contain MATRIGEL™ (GFR) and collagen (2 - 6 mg / mL), while standard organoids contain MATRIGEL™ (GFR) without collagen. The identified matrisome proteins for all models werein excess of ECM controls (FIG. 15G), thus these additional matrisome proteins were produced by cells as the cultures matured, and the increase in matrisome proteins expressed in multicompartment assembloids was not an artifact of the additional ECM introduced in the model design.

[0128] This comprehensive and quantitative DIA-MS approach on the timsTOF HT mass spectrometer (Bruker) referred to as diaPASEF (parallel accumulation-serial fragmentation combined with data-independent acquisition) also revealed protein groups in all models (FTEC cell line multi-compartment, primary FTEC multi-compartment, and primary FTEC standard) that are associated with ciliated and secretory Gene Ontology cellular components (FIG. 15F). These proteins are used for the system’s functional accuracy which are described in greater detail in the next sections.Epithelial differentiation can be stimulated in multi-compartment assembloids

[0129] The fallopian tube epithelium is composed of a mixture of ciliated and secretory epithelial cells, which have roles in oocyte transport and survival, respectively. IHC analysis of healthy fallopian tube tissue revealed a mixture of PAX8-positive (secretory) and PAX8- negative (ciliated) epithelial cells (FIG. 10A, FIG. 16A). However, FTECs in the assembloid were PAX8-positive. By transmission electron microscopy (TEM), secretory epithelial cells and cells with cilia or microvilli facing the MATRIGEL™ region of the assembloid (FIG. 3B) were observed, which resembles the lumen of the fallopian tube. Tight junctions formed between epithelial cells at the assembloid-MATRIGEL™ (lumen-like) and assembloid- collagen I (stroma-like) interfaces, but epithelial secretions and cilia or microvilli were found at the MATRIGEL™ interface (FIG. 3B). By contrast, standard organoids also formed tight junctions and showed both cilia and secretions (FIG. 16B); however, in standard organoids, cilia or microvilli formed on both the organoid interior (lumen-like) and exterior (stroma-like) interfaces. In multi-compartment assembloids, the cells adopted a more organized structural pattern based on the cell-lumen and cell-stroma simulated interfaces, and cells were polarized such that cilia or microvilli faced the lumen-like region of the assembloid.

[0130] While IHC demonstrated that FTECs in the assembloid were secretory, TEM revealed that some assembloid epithelial cells could become ciliated. Secretory FTECs can differentiate into ciliated FTECs upon hormone stimulation, so the assembloids are treated with P-estradiol (E2) and progesterone (P4), two steroid hormones of the menstrual cycle that elicit morphological and functional responses from the fallopian tube epithelium. The resulting molecular response was measured via RT-qPCR (FIG. 5A). Overall increases were observedin estrogen receptor 1 (ESRI) expression with p-estradiol treatment and a slight increase with progesterone treatment, matching previously reported responses in fallopian tube tissue and mouse FTEC standard organoids. In the follicular phase when estrogen is high and progesterone is low, progesterone receptor levels increase in the fallopian tube epithelium and, accordingly, progesterone receptor (PGR) expression increased when the assembloids were treated with -estradiol. P-estradiol treatment also caused a decrease in the relative expression of secretory cell marker PAX8, which is evidence of ciliated FTEC differentiation (FIG. 10A- 10C). The relative expression of markers related to cilia motility / regulatory transcription factor (FOXJ1) and stem cell differentiation into ciliated cells (0LFM4) also increased with P- estradiol stimulation. MKI67 relative expression was not significantly changed from the untreated control (FIG. 10D), which aligns with previous reports of null or limited proliferation of oviductal epithelium when stimulated with estradiol. These results demonstrated a physiological response to steroid hormone stimulation of the healthy fallopian tube assembloid.

[0131] Standard organoids did not always produce these expected responses to hormone treatment (FIG. 16C). MKI67 relative expression was significantly reduced when treated with progesterone and markers of morphological cell type (PAX8, FOXJ1) were unchanged from controls with P-estradiol stimulation (FIG. 16D). Instead, there was an unexpected shift toward ciliated cells with progesterone treatment. Progesterone also prompted an unexpected increase in TNF expression. Together, these results demonstrate that the response of multi-compartment assembloids to hormone stimulation is more physiologically consistent with fallopian tube function than was observed in the standard organoid model, and the ratio of ciliated: secretory epithelial cells in multi-compartment assembloids is tuned to better match the composition of the tissue epithelium.

[0132] While cilia and smooth muscle are both involved in the transport of an oocyte through the fallopian tube, motile cilia are useful when capturing and initiating oocyte transport in the infundibulum region near the ovary. Cilia are used for a functional fallopian tube, and thus, cilia must be present in an assembloid aiming to capture the physiological fallopian tube. As an additional confirmation, Gene Ontology biological processes were identified that were upregulated / downregulated when multi-compartment assembloids were treated with |3- estradiol via diaPASEF-MS-based proteomics and ConsensusPathDB (FIG. 10E, FIG. 16E, FIG. 16F). This analysis was performed on primary and cell line multi-compartment assembloids to demonstrate the functional capacity of the assembloid model with cells from multiple sources. |3-estradiol stimulation resulted in upregulation of cytoskeleton-relatedbiological processes including cytoskeleton organization and epithelial cell differentiation (cell line), and plasma membrane bounded cell projection organization and cell motility / migration (primary) (FIG. 10E). Among other processes regarding epithelial cell junction and ECM / basement membrane assembly, these upregulated biological processes demonstrate assembloid epithelial maturation upon P-estradiol stimulation, including the differentiation of secretory cells into ciliated cells and cilia motility.Activated multi-compartment assembloids reproduce the function of the organ

[0133] From a functional perspective, the differentiation of FTECs into ciliated epithelial cells in response to -estradiol stimulation is useful. Not only do cilia exist in the epithelium, but these cilia are also motile and able to transport an object (oocyte) along the epithelium. To test the function of cilia induced by p-estradiol treatment and demonstrate the multicompartment fallopian tube assembloid’ s mechanical function, a bead displacement assay was developed where oocyte-mimicking fluorescent microbeads were mixed in the lumen-like region of assembloids (FIG. 11A). Some of these assembloids were stimulated with P-estradiol to induce ciliated differentiation of their epithelial cells. After 7 days of growth, when the assembloids had matured, microbeads were tracked using time-lapse confocal microscopy (FIG. 11B, Supplementary Video 1). The trajectories of beads were more irregular and covered larger distances when assembloids were treated with P-estradiol than in untreated controls (FIG. 5B). The stimulated trajectories were much larger than cell movement within the assembloids or the system noise without any cells, and followed the curvature of the assembloid epithelium. This was quantitatively verified by calculating the mean squared displacement (MSD) of the microbeads over 8 hours (FIG. 5C) and the microbead velocity (FIG. 11B). While there was no significant difference in the persistence of microbead movement from the untreated control (persistence time), the total microbead diffusivity was increased in the P- estradiol treated condition (FIG. 17A).

[0134] The RT-qPCR and DIA-MS proteomics results presented earlier (FIG. 5A, FIG. 10A-FIG. 10D) indicated an increase in ciliated cells when these assembloids were treated with P-estradiol. As an additional confirmation that the increase in microbead movement could be due to motile cilia, p-estradiol treated and untreated assembloids were digested, cells were isolated from assembloid debris, and cell motility in DPBS was tracked (FIG. 1). If cells had motile cilia, the cilia acted as a motor propelling suspended cells through DPBS (FIG. HE). Cells isolated from P-estradiol treated assembloids had higher MSDs than cells isolated from the untreated assembloids (FIG. HF). Together, the data collected from P-estradiol stimulatedassembloids via RT-qPCR, DIA-MS proteomics, and the cilia-driven motility assay concluded that the increased microbead movement observed in P-estradiol stimulated assembloids was the result of functional cilia along the assembloid epithelium-lumen interface. Thus, the multicompartment assembloids capture the organ’s mechanical function.

[0135] The same bead displacement assay was performed in standard organoids (FIG. 17B). Individual standard organoids grow from a single parent cell where a lumen forms in the center of an individual organoid. Due to the nature of organoid maturation in the standard model, many standard organoids did not encase microbeads in the organoid lumen-like region. Unlike the multi -compartment model where microbeads were dispersed along the epithelial-lumen interface, many beads were not accessible by lumen-facing epithelial cells in standard organoids (FIG. 17C). However, the electron microscopy results revealed that standard organoids form cilia or microvilli on the lumen-like and external epithelial surfaces, so microbeads in contact with any surface of the organoid could still be moved by cilia if the cilia were motile. The qualitative trajectories of beads tracked in the standard organoid bead displacement assay revealed larger trajectories in the untreated organoids than in those treated with P-estradiol (FIG. 17D). The quantification of these trajectories also showed little difference in the microbead velocities of any experimental group (FIG. 17E); although, microbead MSDs in untreated organoids were increased compared to the noise of the system (FIG. 17F). There was no statistically significant difference in the persistence time or total diffusivity for the microbeads tracked in these experimental groups (FIG. 17G). Together, these results suggest that bead movement in standard organoids was more likely due to the movement of cells than motile cilia induced by hormone stimulation.

[0136] These functional assessments demonstrate that the multi-compartment assembloids resemble the fallopian tube organ in both hormone response and transport ability, thus displaying functional similarity to the organ.CODA provides architectural feedback for future assembloid iterations

[0137] To validate the 3D architecture of the multi-compartment assembloid model, a platform was developed that rigorously compared the multi-compartment assembloids to a whole healthy fallopian tube using CODA (FIG. 12A). CODA is a deep learning-based pipeline that 3D reconstructs bulk tissues at cellular resolution from serial histological sections. Here, CODA generated a 3D reference map of a whole fallopian tube received from a transplantation center from an organ donor who had no history of reproductive system disease or other disorders that would influence cellular architecture or function. Using this referencemap, architectural parameters including the lumen volume and cell concentrations were quantified and then used to meticulously improve the assembloid model to mimic the tissue architecture. Assembloid parameters were adjusted iteratively until this quantitative biomimetic comparison revealed marked improvement in structural biomimetic properties of the assembloid model (i.e. , many quantified parameters were within 25% deviation from the tissue).

[0138] The CODA workflow was first applied to reconstruct the fallopian tube of a postmenopausal (72-year-old) female donor (FIG. 12B). Briefly, the FFPE tissue sample was serially sectioned (4 pm thickness) and one in every two sections was stained with hematoxylin and eosin (H&E) and digitized for an axial resolution of 8 micron (FIG. 18A). Each H&E image was registered using nonlinear color image registration (FIG. 18B). Nuclear coordinates were generated through color deconvolution and detection of hematoxylin maxima, and validated through comparison with manually generated coordinates. Fallopian tube epithelium, stroma, vasculature, nerves, and additional structures were labeled at a resolution of 1 micron using a semantic segmentation algorithm. This fallopian tube was assessed by a trained pathologist to ensure there were no pathological abnormalities and tissue labeling was accurate. Combination of registration, cell detection and tissue segmentation allowed for the production of 3D single-cell resolution maps (FIG. 18C). Image alignment, tissue segmentation, and cell detection were all validated by eye and a manually annotated ground truth (FIG. 18D). A reference map of a whole healthy human fallopian tube was produced that allowed us to visualize the intricate folding architecture of the epithelium-lumen interface in three dimensions (FIG. 12C).

[0139] The same CODA method was also applied to the assembloids to 3D reconstruct and label the assembloid epithelium and stroma (FIG. 12C) for a rigorous side-by-side comparison. Cellular and extracellular architectural parameters were computed from tissue and assembloid maps to comprehensively compare their structures. The level of similarity was analyzed between organ and assembloids, adjusted assembloid parameters (ECM composition, cell seeding densities, addition of stromal cells), and repeated this process to produce a second generation (FIG. 12A). Additional assembloid parameters were adjusted to design subsequent assembloid generation. This iterative process concluded when the majority (four of six) of the architectural measurements were within 25% deviation from the fallopian tube organ.

[0140] This hybrid CODA-assembloid platform demonstrates a novel validation process that combines advanced computational 3D mapping methods and advanced organoid modelingto produce assembloids that are micro-anatomically correct. It takes advantage of the versatility of the multi -compartment assembloid model. The result is an iteratively customized assembloid that closely resembles the reference fallopian tube.Iterative changes to assembloid parameters converge at a next-generation assembloid that mimics the tissue architecture

[0141] CODA is a high resolution 3D mapping technique, used here to improve the assembloid model; however, CODA is expensive, time-intensive, and uses specialized computational tools, making the technique medium-throughput. To leverage the insights gained from CODA maps, seven different combinations of assembloid epithelial cell seeding densities were chosen, core MATRIGEL™ compositions, and corona collagen densities in addition to the standard organoid model (FIG. 13A, FIG. 19A).

[0142] Table 1 lists CODA quantified parameters. Values are the averages of 3 assembloids per iteration. From left to right: epithelial packing density within the epithelium wall, epithelial bulk cell density across the whole tissue / assembloid, and stromal cell packing density within the stroma (cells / mm3).

[0143] Table 2 lists CODA quantified parameters. Values are the averages of 3 assembloids per iteration. From left to right: volume ratio of epithelium wall to lumen; epithelium wall thickness (pm); ratio of epithelium nucleus to cytoplasm.

[0144] These assembloids were not stimulated with |3-estradiol to reflect the postmenopausal conditions of the reference fallopian tube donor, where estrogen levels are low and fewer epithelial cells are ciliated. Beginning with (1) the original assembloid parameters (1 x 104epithelial cells seeded in a growth factor reduced MATRIGEL™ core and embedded in a 2 mg / mL collagen I corona), the collagen density was increased in the assembloid corona (2) to 4 mg / mL and 6 mg / mL. Increasing the collagen density reduced the effect of epithelial cell contractility on shrinking the total size of the assembloid core, but did not significantly change the epithelial structure within the assembloid core (Table 1 and Table 2). The composition of the assembloid core ECM was changed by seeding epithelial cells in a MATRIGEL™ matrix containing growth factors (3). This adjustment led to a change in the epithelial architecture where epithelial structures remained unconnected within the assembloid core, as visualized by confocal microscopy. A similar difference in assembloid architecture was also observed when (4) decreasing the epithelial seeding in the assembloid core (5 x 103cells seeded in a growth factor reduced MATRIGEL™ core and 2 mg / mL collagen corona).

[0145] An advantage of the multi -compartment assembloid is that stromal cells can be added into the collagen-dense stroma-simulating corona (Table 1 and Table 2). With this modification, significant ECM remodeling was observed when stromal cells were seeded in a 2 mg / mL collagen I corona, so the collagen concentration was increased to 6 mg / mL (FIG. 13A, parameter modification 5). The standard organoid technique (6) was also imaged using CODA for a total of eight models that were analyzed using the new architectural validation platform. Computational analysis was blinded to these experimental conditions.

[0146] An array of structural parameters was quantified from each assembloid map and compared to the CODA quantifications of the whole fallopian tube (FIG. 13B, Table 1 and Table 2) The measurements from this fallopian tube reference map focused on the ampulla region near the fimbriated end where a defined volume could be measured. This platform can be adjusted, however, to focus on different tissue regions and different arrays of architectural parameters specific to the study. The epithelial and stromal cell seeding densities are two parameters of the assembloids that are easily adjusted, but have different impacts on theassembloid architecture. Cell density was calculated as a bulk parameter (epithelial cell count normalized by total assembloid volume) and as a packing density (epithelial cell count normalized by epithelial volume). While the epithelium cell density differed significantly from the reference tissue for all assembloid models when calculated as a packing density within the organ / assembloid epithelium (FIG. 13C), the epithelial bulk cell density calculated across the whole assembloid more closely matched the tissue epithelial bulk density (FIG. 13D).

[0147] The inclusion of stromal cells had a significant impact on assembloid similarity to the tissue (FIG. 13E), which highlights the use of compartmentalization in an assembloid. Stromal cells can be accurately incorporated into a fallopian tube assembloid model when they are adjacent to the epithelium and in direct contact with a collagen I matrix different from the epithelial ECM. Stromal cells were seeded at a ratio of 6 stromal cells to every 10 epithelial cells (6 x 103stromal cells in the corona and 1 x 104epithelial cells in the core). This seeding ratio accounted for the different proliferation rates of the cell types so that the final ratio of stromal cells to epithelial cells in mature assembloids closely matched the ratio counted in the tissue reference map (Tables 1 and 2). The final number of stromal cells in mature assembloids was further modulated by collagen density in the corona. By increasing the collagen density from 2 mg / mL to 6 mg / mL, the final stromal cell count was decreased, which decreased the similarity of the stromal cell density per mm3total volume of the assembloid to the stromal cell density of the tissue within a 1 mm3region surrounding the fallopian tube lumen (FIG. 13E). Incorporating stromal cells into the assembloid corona also limited the proliferation of the assembloid epithelium (FIG. 19B).

[0148] Additional quantified parameters included the epithelial wall volume and lumen volume, which was reported as a ratio to demonstrate the similarity of the relative scale of the assembloid to the relative scale of the reference fallopian tube (FIG. 13F). The thickness was measured of the epithelium wall, which is an indicator of cellular organization (FIG. 13G). As shown in FIG. 8A, the fallopian tube epithelium can be a mixture of monolayered regions and regions where the epithelium becomes multi-layered. The average epithelium thickness was determined across the whole organ epithelium and confirmed that the assembloid epithelium organized similarly. Finally, the nucleus to cytoplasm ratio of epithelial cells was to compare the intracellular dimensions of assembloid epithelial cells to in situ fallopian tube epithelial cells to ensure epithelial cells were not overpacking within the assembloid (FIG. 13H).

[0149] These measured parameters for each assembloid iteration were compared to the values for the reference fallopian tube by principal component analysis (PCA) (FIG. 131). PCIand PC2 explained 34.75% and 30.06% of the total variance, respectively (PC3: 16.45%, PC4: 12.72%, PC5: 4.74%, PC6: 1.28%). This analysis revealed that the combination of seeding 1 x 104FTECs in a growth factor reduced MATRIGEL™ core and embedding that core in a 2 mg / mL collagen I corona with 6 x 103stromal cells (10k 2 mg / mL + S) mimicked the reference fallopian tube tissue. The 10k 2 mg / mL + S condition stood apart from all other generations of the multi-compartment and standard organoid as a more accurate approximation of the fallopian tube architecture. The deviation between this assembloid and the reference fallopian tube was < 25% for four of six quantified architectural parameters (FIG. 13C-FIG. 13F), thus this generation of multi-compartment assembloid achieved the target similarity to the reference fallopian tube. Finally, Euclidean distance analysis (FIG. 19C), which gives equal weight to all metrics, also agreed that the similarity of the 10k 2 mg / mL + S condition to the reference fallopian tube was significantly improved compared to all other tested assembloid models. Via fluorescence confocal microscopy, the architecture of these assembloids was visualized (FIG. 13K, FIG. 19D). Intricate epithelial folding mimicking the tortuous architecture of the fallopian tube mucosa was observed throughout the assembloid with a surrounding but distinct stroma-like region (FIG. 13K, FIGs 19E-19F).EXAMPLE 3 METHODSFallopian tube tissue

[0150] Whole healthy fallopian tube tissues were received from organ procurement organizations from donors with no history of disease. All procedures were approved by the Johns Hopkins Internal Review Board (IRB00324065). The tissues were transported in saline on wet ice, formalin fixed (10%, VWR 16004-121) and paraffin embedded. The tissue was serial sectioned at 4 pm thickness at Johns Hopkins Oncology Tissue Services (SKCC). Tissue sections were stained with hematoxylin and eosin, or immunohistochemistry (IHC) stained. Antibodies used in IHC staining are listed in Supplementary Table 1. Tissue sections were scanned at 20X or 40X magnification (Hamamatsu NanoZoomer S210).Cell culture

[0151] Fallopian tube immortalized cells were the FT2821J (epithelial), FT194 (epithelial, CVCL_A4AW) and hSl (fibroblast-like stromal) cell lines. Cells were cultured in RPMI 1640 (Gibco 11875-093) with 10% FBS (Coming 35-010-CV) and 1% penicillin-streptomycin (Sigma P0781). Fallopian tube primary epithelial cells (LifeLine Cell Technology FC-0081)were validated and quality tested at LifeLine Cell Technology and cultured in complete ReproLife™ Reproductive Medium (LifeLine Cell Technology LL-0068) for up to 3 passages. All cells were cultured at 37°C and 5% CO2 in a humidified incubator and did not surpass 20 cell passages. All experiments were conducted using these base culture mediums for immortalized or primary cells.

[0152] Fluorescently tagged cells were transduced via lentivirus delivery of the fluorescent tag. Luciferase expressing cells were transduced via lentivirus delivery of a luciferase-mCherry tag (pSLCAR-CD19-BBz [Addgene 135992] with luciferase [Addgene 46793] and mCherry [Addgene 84020] sequences inserted). Transduced populations were expanded and sorted by fluorescence-activated cell sorting (SONY SH800).Multi-compartment assembloid

[0153] The protocol for generating multi-compartment assembloids is described in greater detail by Lee, et al., where the protocol was first established as a tumor organoid model. In short, a single-cell suspension of 1 x 104or 5 x 103fallopian tube epithelial cells per pL MATRIGEL™ (standard formulation 354234 or growth factor reduced 354230, Coming) were pipetted in 1 pL volumes in columns (USA Scientific 1111-3700) containing mineral oil (Sigma 69794) and allowed to gel at 37°C. After 1 h, these cores were collected in cell culture medium (RPMI 1640, Gibco 11875-093) to wash off the mineral oil and embedded in collagen I (high concentration rat tail type I, Coming 354249). A final concentration of 2, 4 or 6 mg / mL collagen I was used according to a previously described protocol. Adjustments to the collagen I concentration in the assembloid corona were made to assess the effects of corona composition on assembloid architectural similarity to fallopian tube tissue, or to support longer-term cultures of fallopian tube epithelial cells. Drops containing one assembloid core and 10 pL of collagen I were pipetted into fresh mineral oil columns and again allowed to gel at 37°C. After 1.5 h, these assembloids were collected in cell culture medium to wash off any remaining mineral oil and plated in a 96-well round bottom plate (Genesee Scientific 25-224) with normal cell culture medium as described above. All assembloids were cultured at 37°C and 5% CO2 in a humidified incubator. All assembloids were grown for at least 6 days (immortalized FTECs) or 9 days (primary FTECs) to allow time for the epithelial architectures to mature. These multi-compartment assembloids are medium throughput, generating up to 96 assembloids per batch.Standard organoid

[0154] The protocol for generating standard organoid is described in greater detail by Kessler, et al. Briefly, fallopian tube epithelial cells were suspended in growth factor reduced MATRIGEL™ (Coming 354230) at 500 cells / pL. 50pL drops of the mixture were plated on a glass bottom plate and allowed to gel. Organoids were grown in organoid formation medium containing Advanced DMEM / F-12 (Gibco 12634010), L-Glutamine (1%, Gibco 25030081), B-27 (2%, Gibco 17504044), EGF (0.1 pg / mL, ThermoFisher PHG0311), Y-27632 ROCK Inhibitor (10 pM, Cayman Chemical 10005583), TGF- RI Kinase Inhibitor VI SB431542 (0.5 pM, Sigma 616464) and HEPES (1%, Gibco 15630080). All organoids were cultured at 37°C and 5% CO2 in a humidified incubator. Timelines used for standard organoids matched the culture timelines of multi-compartment assembloids.PrestoBlue proliferation assay

[0155] Assembloid total proliferation was assessed via the PrestoBlue (ThermoFisher A13262) proliferation assay. Assembloids were incubated with PrestoBlue (10X) diluted to IX in culture medium for 3 hours at 37°C and 5% CO2 in a humidified incubator. Red fluorescence (RFU) was read on a SpectraMax plate reader (Molecular Devices, excitation 540, emission 600, cutoff 590). The average of at least 3 background wells of IX PrestoBlue diluted in culture medium without assembloids was subtracted from all RFU values. PrestoBlue solution was washed from assembloids by replenishing culture medium twice with DPBS (Coming 21-031- CV) and twice with culture medium, and cultured until the next timepoint at 37°C and 5% CO2 in a humidified incubator.Workflow for generating multi-compartment assembloids

[0156] FIG. 20 describes a workflow for generating multi-compartment assembloids. Oil column preparation includes a 10 pL pipette tip with no filter, and a “plug” is created at the bottom of oil columns with cell culture medium. Oil columns are filled with mineral oil. For cell preparation, mammalian cell culture is spun and the cell aliquot is resuspended in ECM to form a Cell-ECM solution. To form an organoid core, 1 pL of cores are dropped into each oil column to form an oil column plus a core, which is incubated for 1 hour at 37°C. For core harvest and washing, all cores are collected from oil columns into culture medium and mineral oil is aspirated. For corona preparation, organoid cores are transferred into new ECM solution to form Core-ECM solution. For organoid corona formation, 10 pL of organoids are dropped into each oil column to form an oil column plus organoid, which is incubated for 1 hour at37°C. For organoid harvest and washing, all organoids are collected from oil columns into culture medium and mineral oil is aspirated. To plate the organoid, each organoid is plated in a well of a plate with 200 pL culture medium.Imaging

[0157] Assembloids and organoids were imaged on a Nikon Ti2 microscope (phasecontrast) and a Nikon AIR confocal / Nikon Eclipse Ti inverted microscope (DIC, fluorescence and immunofluorescence confocal). The Nikon Ti2 microscope was equipped with a DS-Qi2 camera and a 10X / 0.30 Plan Fluor Phi DL objective (Nikon). Temperature and CO2 were controlled with a Tokai Hit stage top incubator. The Nikon AIR confocal / Nikon Eclipse Ti inverted microscope was equipped with a 10X / 0.30 DIC L / Nl and 20X / 10.75 DIC M / N2 Plan Fluor objective (Nikon), a 10X and 20X DIC slider (Nikon), and aNl and N2 DIC condenser (Nikon). Temperature and CO2 were controlled with an OKO Labs stage top incubator.

[0158] Fluorescence and immunofluorescence confocal images are maximum intensity projections of z-stacks taken over 200-500 pm height of the assembloid with a 10 pm interval. Assembloids were fixed overnight in 4% PFA or grown in a glass bottom plate for imaging (EGFP, mCherry). Nuclei were stained with Hoechst 33342 (ThermoFisher H3570) and F-actin was stained with Phalloidin 488 (Invitrogen A12379) or Phalloidin 647 (Invitrogen A22287) for 1 hour. The primary and secondary antibodies used in immunofluorescence (IF) imaging are outlined in Supplementary Table 1. For IF studies, assembloids were permeabilized in 0.1 % Triton X-100 (Sigma T9284) in DPBS (Coming 21-031-CV) for 1 hour, blocked with 5% normal goat serum (NGS, Cell Signaling Technology) in DPBS for 3 hours, and primary stained overnight in 1% NGS in DPBS. Assembloids were secondary stained for 3 hours in 1% NGS in DPBS. Assembloids were cleared for 20 minutes in a fructose-glycerol clearing solution (60% glycerol [JT Baker 2136-03], 2.5M D-fructose [Sigma F0127] in DI water) and imaged in DPBS or clearing solution.Transmission electron microscopy

[0159] Assembloids were fixed (2.5% glutaraldehyde, 0.1M sodium cacolydate buffer, 3 mM magnesium chloride in DI water), and imaged via transmission electron microscopy. Images were captured and analyzed with the help of a transmission electron microscopy expert at the Johns Hopkins School of Medicine Microscope Facility.Western blot

[0160] Protein was extracted from 3D cultures and 2D cells with 2X clear sample buffer (120 mM Tris pH 6.8, 4% SDS, 20% glycerol) and IX protease / phosphatase inhibitor (CellSignaling Technology 5872). 2D cells were scraped from a 10 cm dish after coating the plate with 2X clear sample buffer. All protein samples were sonicated with a needle probe sonicator (10-15 pulses, VWR Scientific) and denatured at 100°C for 5 minutes. Samples were stored at -80°C. Protein concentration was measured by microBCA (ThermoFisher, 23235) and absorbance measured on a SpectraMax plate reader (Molecular Devices, 562nm). Samples were diluted to 1 pg / pL in 2X Lamelli (BioRad 1610737) and heated for 5 minutes at 100°C. Samples were loaded in a 4-15% mini-protean gel (BioRad 4561086) for gel electrophoresis (BioRad PowerPac). Protein bands were transferred to a PVDF membrane using a Transblot Turbo pack (BioRad 1704156). The blot was blocked in blotting-grade blocker (5%, 1706404) in TBST (IX Tris Buffered Saline [BioRad 1706435], 0.1% Tween 20 [Sigma P1379], in DI water) for 1 hour and primary stained overnight at 4°C. HRP -conjugated secondary antibodies were incubated at room temperature for 1 hour. Western blots were imaged on a BioRad ChemiDoc XRS+ imager with Prometheus™ ProSignal™ HRP substrate (Genesee Scientific 20-302). Antibodies are listed in Supplementary Table 1.Hormone stimulation and RT-qPCR

[0161] After 7 days of culture, assembloid architecture had matured, and assembloids / organoids were treated with -estradiol (E2, 500 pmol / L, Sigma E2758), progesterone (P4, 50 ng / mL, Fisher Scientific AC225650250), or an untreated control. Hormones were diluted in ethanol prior to dilution in culture medium, so this dilution was equivalently reflected in the untreated control. Assembloids and organoids were grown for another 7 days with hormone stimulation before mRNA harvested (Zymo Research R2052 Direct-zol RNA MiniPrep 11-331) and cDNA synthesized using the iScript™ cDNA Synthesis Kit (BioRad 1708891) and a Blue-Ray Biotech thermocycler. Expression of select genes (primers from Integrated DNA Technologies listed in Supplementary Table 2) was quantified by iTaq-SYBR Green (BioRad) RT-qPCR on a BioRad CFX384 Touch Real-Time PCR detection system. Relative expression was calculated according to the equations described by Pfaffl.Bead displacement assay

[0162] In the bead displacement assay, red fluorescent microbeads (Cospheric FMR-1.3 1- 5um) were suspended in MATRIGEL™ with FTECs. Both standard organoids and multicompartment assembloids were generated that contained microbeads and EGFP-tagged cells with microbeads in the MATRIGEL™. The previously outlined protocols were followed; however, the multi-compartment assembloids were plated in 100 pL collagen I in a flat glassbottom plate so that the same location on an assembloid could be imaged via time-lapse microscopy. Assembloids were treated with P-estradiol (10 nM, Sigma E2758) or control medium after 24 hours. After 7 days of culture, assembloid architecture had matured, and assembloids were imaged via confocal fluorescence microscopy (10X magnification). Timelapse images were collected every 10 minutes for 12 hours. Stage drift was removed from timelapse images and microbeads tracked across all time points. Microbead trajectories, mean squared displacements and motility information were calculated according to previously published protocols.Cilia-driven motility assay

[0163] Multi-compartment assembloids with 10 pL coronas were cultured according to the same specifications of the bead displacement assay (P-estradiol [10 nM, Sigma E2758] treated after 24 hours, grown for 7 days to allow assembloid architecture to mature) and digested with 2 mg / mL collagenase I (Gibco 17018029) for 1 hour. After digestion, cells were resuspended in DPBS (Coming 21-031-CV) and time-lapse imaged for 1 hour (phase-contrast, 10X magnification). After an initial 30 minute incubation to give cells time to settle to the bottom of the plate, cells were imaged for 30 minutes at 1 minute intervals. Individual cells were tracked and their motility parameters calculated according to previously published cell tracking protocols.Proteomics

[0164] Multi-compartment assembloids and standard organoids were grown for 18 days, washed IX with DPBS and stored in cell culture medium for proteomics. The extended 18 day timeline was selected to allow time for the architectures of primary FTEC cultures to mature in the 3D models. The FT2821J FTEC cell line was grown in multi-compartment assembloids with a GFR MATRIGEL™ assembloid core and a 2 mg / mL collagen I corona. Primary FTECs were grown in multi-compartment assembloids with a GFR MATRIGEL™ assembloid core and a 6 mg / mL collagen I corona, and standard organoids in GFR MATRIGEL™. GFR MATRIGEL™, 2 and 6 mg / mL collagen I controls without cells were also incubated for 18 days with each biological replicate. Multi-compartment assembloids were treated with P- estradiol (10 nM, Sigma E2758) or control medium from day 9 to day 18, with medium refreshed every 3 days.

[0165] LC-MS-grade acetonitrile (ACN), methanol and water were obtained from Burdick & Jackson. Reagents for protein chemistry, including sodium dodecyl sulfate (SDS), tri ethyl ammonium bicarbonate (TEAB), trichostatin A, nicotinamide, iodoacetamide (IAA),dithiothreitol (DTT), phosphoric acid, and formic acid (FA), were purchased from Sigma- Aldrich. Urea and phosphate buffered saline (PBS) were purchased from Thermo Fisher Scientific. Sodium chloride was purchased from VWR. Sequencing-grade trypsin was purchased from Promega.Protein digestion and desalting

[0166] Multi-compartment assembloids and standard organoids and ECM controls were washed twice with cold PBS lx and then subjected to a lysis buffer containing 8 M urea, 2% SDS, 200 mM TEAB, pH 8.5, 75 mM NaCl, 1 pM trichostatin A, 3 mM nicotinamide, and lx protease / phosphatase inhibitor cocktail (Thermo Fisher Scientific, 78442), and incubated overnight at 4°C on a rotator. Lysates were clarified by spinning at 15,700 x g for 15 min at 4°C, and the supernatant containing the soluble proteins was collected. Protein concentrations were determined using a Bicinchoninic Acid Protein (BCA) Assay (Thermo Fisher Scientific, 23225), and subsequently 100 pg of protein from each sample were aliquoted and samples were brought to an equal volume using water. Samples were solubilized with 4% SDS and 50 mM TEAB at pH 8. Proteins were reduced with 20 mM DTT (10 min at 50°C followed by 10 min at room temperature) and then alkylated with 40 mM IAA (30 min at room temperature in the dark). Samples were acidified with a final concentration of 1.2% phosphoric acid, and diluted with seven volumes S-trap buffer (90% methanol in 100 mM TEAB, pH 8). Samples were then loaded onto the S-trap mini spin columns (Protifi) and spun at 4,000 x g for 10 seconds. The S-Trap columns were washed with S-Trap buffer twice at 4,000 x g for 10 seconds each. A solution of sequencing grade trypsin in 50 mM TEAB at a 1:25 (w / w) enzyme:protein ratio was then added, and after a 1-hour incubation at 47°C, trypsin solution was added again at the same ratio and proteins were digested overnight at 37°C. Peptides were sequentially eluted with 50 mM TEAB (spinning at 1,000 x g for 1 min), 0.5% FA in water (spinning at 1,000 x g for 1 min), and 50% ACN in 0.5% FA (spinning at 4,000 x g for 1 min). After vacuum drying, samples were resuspended in 0.2% FA in water, desalted with Oasis 10-mg Sorbent Cartridges (Waters). All samples were vacuum dried and resuspended in 0.2% FA in water at a final concentration of 1 p / gL. Finally, indexed retention time standard peptides (iRT; Biognosys) were spiked in the samples according to manufacturer’s instructions.Mass spectrometric analysis

[0167] Samples (200 ng) were loaded onto EvoTips Pure (Evosep) following manufacturer’s protocol. LC-MS / MS analyses were performed on an Evosep One liquidchromatography system coupled to a timsTOF HT mass spectrometer (Bruker). The solvent system consisted of 0.1% FA in water (solvent A) and 0.1% FA in ACN (solvent B). Peptides were eluted on aPepSep C18 analytical column (150 pmx 15 cm, 1.5 pm particle size; Bruker) using the 30 SPD method (44-minute gradient length, 500-nl / min flow rate). A zero-dead volume emitter was installed in the nano-electrospray source (CaptiveSpray source, Bruker Daltonics) and the source parameters were set as follows: capillary voltage 1600 V, dry gas 3 L / min, and dry temperature 180°C. Each sample was acquired in data-independent acquisition (DIA) mode coupled to parallel accumulation serial fragmentation (PASEF) or diaPASEF mode with 1 survey TIMS MS scan and 24 diaPASEF MS / MS scans per 2.03-second cycle. The dual TIMS analyzer was operated in a 100% duty cycle with equal accumulation time and ramp time of 75 ms each. For each TIMS MS scan, the ion mobility ranged from 1 / Ko = 0.85- 1.30 Vs / cm2and the mass range covered m / z 100-1,700. The diaPASEF scheme was defined as 63 x 15 Th isolation windows from m / z 305.9 to 1,250.9 and covering the ion mobility range 1 / Ko = 0.74-1.30 Vs / cm2(Supplementary Data 1). The collision energy was defined as a linear function of mobility starting from 20 eV at 1 / Ko = 0.6 Vs / cm2to 59 eV at 1 / Ko = 1.6 Vs / cm2. For calibration of ion mobility dimension, three ions of Agilent ESI-Low Tuning Mix ions were selected (m / z [Th], 1 / K0 [Th]: 622.0289, 0.9915; 922.0097, 1.1986; 1221.9906, 1.13934).DIA-MS data processing with Spectronaut vl7

[0168] All DIA data was processed in Spectronaut version 17.6.230428.55965 (Biognosys) using directDIA. Data was searched against the Homo sapiens proteome with 20,423 protein entries (UniProtKB-SwissProt), accessed on 06 / 30 / 2023. Trypsin / P was set as digestion enzyme and two missed cleavages were allowed. Cysteine carbamidomethylation was set as fixed modification, and methionine oxidation and protein N-terminus acetylation as variable modifications. Data extraction parameters were selected as dynamic, and non-linear iRT calibration with precision iRT was selected. Identification was performed using a 1% precursor and protein q-value, and iRT profiling was selected. Quantification was based on the MS / MS peak area of the 3-6 best fragment ions per precursor ion, peptide abundances were obtained by summing precursor abundances and protein abundances by summing peptide abundances. Interference correction was selected and local normalization was applied. Finally, protein groups were used with at least two different peptides (Supplementary Data 2-7). Differential protein abundance analysis was performed using unpaired t-test, and p-values were corrected for multiple testing, specifically applying group-wise testing corrections using the Storeymethod. For differential analysis, protein groups with q-value < 0.05 and absolute log2 (foldchange) > 0.2 were considered to be significantly altered (Supplementary Data 3-4).LOPIT organellar protein distribution

[0169] The hyperLOPIT2015 u2os dataset was downloaded in R (version 4.0.2; RStudio, version 2022.07.0+548) using the pRolocData R package (Bioconductor) and plotted with the pRoloc R package. To create the LOPIT organellar protein distribution dataset, a crude membrane preparation from a U2OS cell lysate was fractionated by density gradient ultracentrifugation to separate and enrich organelles based on density. Proteins were identified via quantitative multiplexed mass spectrometry and assigned to organelles based on similarities in distribution in the density gradient to well-annotated organelle protein markers. The t-SNE machine learning algorithm was used to reduce the number of dimensions in the LOPIT proteomic data to a 2D map where proteins cluster by similarity from multiple experimental factors. All LOPIT protein assignments were used as originally determined and without additional refinement.Over-representation analysis

[0170] Gene Ontology biological processes that were upregulated and downregulated following P-estradiol stimulation were identified by ConsensusPathDB over-representation analysis with -value < 0.01 and Gene Ontology biological process level > 4. The plotted protein ratio is the corrected ratio of the count of proteins mapped to each term divided by the effective size of the dataset.CODA

[0171] The CODA protocol is described in greater detail by Ki emen, et al., where the protocol was first established. Assembloids that were grown for 10 days so that the architecture could mature and fallopian tube tissues within 12 hours of collection were formalin fixed (10%, VWR 16004-121) and paraffin embedded, then serial sectioned across the whole tissue at 4 pm section thickness. One in every two tissue slides was designated for H&E staining, while the remaining unstained slides were stored for potential application of multiplexing techniques, such as immunofluorescence, immunohistochemical, and tissue-omics. All subsequent steps were performed by a trainee who was unfamiliar with the experimental design, thus blinding the computational analysis to experimental conditions. Stained slides were scanned at 20X magnification offering a 0.5 micron resolution on the x and y dimensions. To align the H&E images, a combination of global and nonlinear techniques was implemented in all images in a sample. To statistically measure the similarity between two serially aligned H&E images,cross-correlation was used. In order to identify individual cells on the H&E-stained samples, a color deconvolution step was added which extracted the hematoxylin channel of each image, and specific sized and spatially distanced two-dimensional minima were marked as nuclei. A deep learning model was trained on an image subset using manually annotated tissue cases. To enhance the performance of the convolutional neural network on unseen data, data augmentation techniques were integrated, allowing for hue, rotation, and scale changes to the training dataset. The validation of the model was ensured using independent testing images, not used for training, with the mean testing accuracies surpassing ninety percent for true positive segmentation values. Combination of the alignment, semantic segmentation and cell detection computational methods resulted in three dimensional maps of each individual tissue label at single cell resolution. Three-dimensional renderings were performed at 4 pm and 1 pm in x and y dimensions for the whole human fallopian tube and each individual assembloid, respectively. Lastly, three-dimensional analysis of the human fallopian tube and assembloids were performed in isotropic dimensions 4x4x4 pm3.Statistical analysis

[0172] Statistical analyses are described in the figure legends with p-values. N = biological replicates, n = technical replicates.Data and materials availability

[0173] All data are available in the main or supplementary materials. All raw data, cell lines, and MATLAB codes are available upon request (wirtz@ihu.edu). Raw data and complete MS data sets have been uploaded to the MassIVE repository of the Center for Computational Mass Spectrometry at UCSD and can be downloaded using the following link: https: / / massive.ucsd. edu / ProteoSAFe / dataset.isp?task=e6f60aaad6bl41e0a081c3e4236de4ba (MassIVE ID number: MSV000092646; ProteomeXchange ID: PXD044527).

[0174] [Note: To access the data repository MassIVE (UCSD) for MS data, please use: Username: MSV000092646_reviewer; Password: winter].References

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[0284] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A method of generating a multi-compartment organoid comprising: a) contacting a first extracellular matrix (ECM) composition with a first cell type to generate a first ECM compartment, and b) contacting the first compartment of a) with a second ECM composition to generate a second ECM compartment or a larger ECM compartment comprising the first ECM compartment embedded into the second ECM composition, wherein the multi-compartment organoid is an in vitro 3-dimension (3D) physiologically accurate model of a tissue or organ, thereby generating a multi-compartment organoid.

2. The method of claim 1, wherein contacting the first ECM with the first cell type comprises generating a droplet comprising the first cell type embedded in the first ECM composition in a mineral oil.

3. The method of claim 2, wherein contacting the first ECM compartment with the second ECM composition comprises resuspending the droplets comprising the first ECM and the first cell type in the second ECM composition.

4. The method of claim 1, wherein the first ECM composition and the second ECM composition are different ECM compositions.

5. The method of claim 1, wherein the first ECM composition and the second ECM composition are a same ECM composition.

6. The method of claim 4 or 5, wherein a combination of the first and second ECM compositions mimics a tissue or organ physiological microenvironment.

7. The method of claim 1, wherein the second ECM composition comprises a second cell type.

8. The method of claim 7, wherein the first and the second cell type are a same cell type or a different cell type.

9. The method of claim 1 , wherein the tissue or organ is selected from the group consisting of a fallopian tube, an endometrium, a uterus, a mammary gland, a normal breast tissue, a cervix, and an ovary.

10. The method of claim 1, wherein the first ECM composition comprises a basement membrane ECM composition.

11. The method of claim 10, wherein the basement membrane ECM composition comprises collagen IV and laminin.

12. The method of claim 1, wherein the second ECM composition comprises a stromal ECM composition.

13. The method of claim 12, wherein the stromal ECM composition comprises collagen I.

14. The method of claim 1 , wherein the multi-compartment organoid is maintained in a 3D suspension culture.

15. The method of claim 1, wherein the 3D physiologically accurate model of a tissue or organ comprises a multi-compartment organoid having a physiological architecture of the tissue or organ.

16. A multi-compartment organoid obtained by the method of any one of claim 1-15.

17. A method of generating a multi-compartment fallopian tube organoid comprising: a) contacting a basement membrane extracellular matrix (ECM) composition with a fallopian tube epithelial cells to generate a first ECM compartment, and b) contacting the first ECM compartment of a) with a stromal ECM composition to generate a second ECM compartment comprising the first ECM compartment embedded into the stromal ECM composition, wherein the multi-compartment fallopian tube organoid is an in vitro 3-dimension (3D) physiologically accurate model of a fallopian tube, thereby generating a multi-compartment fallopian tube organoid.

18. The method of claim 17, wherein the stromal ECM composition further comprises fallopian tube stromal cells.

19. The method of claim 17, wherein the multi-compartment fallopian tube organoid has a lumen-like architecture.

20. The method of claim 17, wherein the multi-compartment fallopian tube organoid comprises ciliated and secretory cells in a lumen compartment and tight junctions at a basement membrane ECM-stromal ECM interface.

21. The method of claim 20, wherein ciliated cells express elevated levels of FOXJ1, and / or OLFM4, as compared to non-ciliated epithelial cells.

22. The method of claim 20, wherein secretory cells are PAX8 positive.

23. The method of claim 17, wherein the multi-compartment fallopian tube organoid is sensitive to P-estradiol and / or to progesterone.

24. The method of claim 23, wherein in response to P-estradiol epithelial cells in the multicompartment fallopian tube organoid become ciliated.

25. The method of claim 24, wherein ciliated cells displace objects in a multi-compartment fallopian tube organoid lumen.

26. The method of claim 17, wherein the 3D physiologically accurate model of a fallopian tube comprises a multi-compartment fallopian tube organoid having a physiological architecture of a fallopian tube.

27. A multi-compartment fallopian tube organoid obtained by the method of any one of claims 16-26.

28. A multi-compartment organoid comprising: a) a first extracellular matrix (ECM) compartment comprising a first cell type and a first ECM composition, and b) a second ECM compartment comprising a second ECM composition, wherein the first ECM compartment is embedded into the second ECM compartment, wherein the multi-compartment organoid is an in vitro 3-dimension (3D) physiologically accurate model of a tissue or organ.

29. The organoid of claim 28, wherein the first ECM composition and the second ECM composition are different ECM compositions.

30. The organoid of claim 28, wherein the second ECM composition comprises a second cell type.

31. The organoid of claim 30, wherein the first and the second cell type are a same cell type or a different cell type.

32. The organoid of claim 28, wherein the tissue or organ is selected from the group consisting of a fallopian tube, an endometrium, a uterus, a mammary gland, a normal breast tissue, a cervix, and an ovary.

33. A multi-compartment fallopian tube organoid comprising: a) a first extracellular matrix (ECM) compartment comprising a fallopian tube epithelial cell and a basement membrane ECM composition, and b) a second ECM compartment comprising a stromal ECM composition, wherein the first ECM compartment is embedded into the second ECM compartment, wherein the multi-compartment fallopian tube organoid is an in vitro 3-dimension (3D) physiologically accurate model of a fallopian tube.

34. The organoid of claim 33, wherein the basement membrane ECM composition comprises collagen IV and laminin.

35. The organoid of claim 33, wherein the stromal ECM composition comprises collagen I.

36. The organoid of claim 33, wherein the stromal ECM composition further comprises fallopian tube stromal cells.

37. The organoid of claim 33, wherein the multi-compartment fallopian tube organoid has a lumen-like architecture.

38. The organoid of claim 33, wherein the multi-compartment fallopian tube organoid comprises ciliated and secretory cells in a lumen compartment and tight junctions at a basement membrane ECM-stromal ECM interface.

39. The organoid of claim 38, wherein ciliated cells are expressing elevated levels of FOXJ1, and / or 0LFM4.

40. The organoid of claim 38, wherein secretory cells are PAX8 positive.

41. The organoid of claim 33, wherein the multi-compartment fallopian tube organoid is sensitive to p-estradiol and / or to progesterone.

42. The organoid of claim 41, wherein in response to P-estradiol cells in the multicompartment fallopian tube organoid become ciliated.

43. The organoid of claim 42, wherein ciliated cells displace objects in a multicompartment fallopian tube organoid lumen.

44. A kit comprising: a) a first cell type, b) a first extracellular matrix (ECM) composition, c) a second ECM composition, and d) instructions to generate an in vitro 3-dimension (3D) physiologically accurate model multi-compartment organoid of a tissue or organ.

45. The kit of claim 44, further comprising a second cell type.

46. The kit of claim 44, wherein the tissue or organ is selected from the group consisting of a fallopian tube, an endometrium, a uterus, a mammary gland, a normal breast tissue, a cervix, and an ovary.

47. A kit comprising: a) a fallopian tube epithelial cell, b) a basement membrane extracellular matrix (ECM) composition, c) a stromal ECM composition, and d) instructions to generate an in vitro 3-dimension (3D) physiologically accurate model of a multi -compartment fallopian tube organoid.

48. The kit of claim 47, wherein the basement membrane ECM composition comprises collagen IV and laminin.

49. The kit of claim 47, wherein the stromal ECM composition comprises collagen I.

50. The kit of claim 47, wherein the stromal ECM composition further comprises fallopian tube stromal cells.

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