Method for generating organoids in hydrogel microparticles

Abstract

The present invention relates to the generation of organoids using granular hydrogel, which is a compacted assembly of hydrogel microparticles.

Description

[0001] METHOD FOR GENERATING ORGANOIDS IN HYDROGEL MICROPARTICLES

[0002] Field of invention

[0003] The invention relates to organoids, methods of generating them and uses thereof.

[0004] *ound of the invention

[0005] Physiologically relevant human tissue models are crucial for translational research, including discovery and validation of novel therapies.

[0006] The emergence of organoids as an alternative to human models has the potential to address this. An organoid is an in vitro, 3D organotypic culture which captures architectural, cellular and molecular features of a target organ or tissue. Organoids are also referred to in the art as microphysiological systems or an organ-on-a-chip. Organoids have proven transformative in a number of different fields, including cardiovascular, neurological and cancer research. In the field of bone and bone marrow related diseases, however, there has been limited progress in the generation of culture systems that faithfully capture the architectural and cellular complexity of the bone and bone marrow microenvironments .

[0007] Creating an organoid that captures the complexity of the bone and bone marrow in vitro remains challenging because of the range of different cell types which are carefully arranged into microenvironments. For example, the central, myelopoietic bone marrow is thought to be primarily composed of sinusoidal and arteriolar endothelial cells supporting the production of myeloid lineage cells. Conversely, the endosteal niche, invested with its own specific vascular cells (e.g. Type H capillaries) is thought to maintain the generation of lymphoid lineage progenitors and quiescent, long lived haematopoietic stem cells (HSCs). Bone marrow mesenchymal stem cell (BM MSC) derived perivascular cells, adipocytes, and osteolineage cells are also critical components of the different niches. The bone architecture is maintained through a dynamic equilibrium facilitated by the balance of osteolineage cells (osteoclasts and osteoblasts).

[0008] WO 2023 / 156774 reports a method of generating bone marrow organoids that involves embedding mesodermal aggregates in bulk hydrogel to drive vascular sprouting and the subsequent generation of 3D structures with a vascular and haematopoietic architecture. WO 2023 / 194370 reports a method of generating bone marrow organoids that involves embedding mesodermal aggregates in a polymerized 3D collagen 1 / Matrigel matrix. However, these methods are costly, labour intensive, time consuming, and are not possible to automate. Reducing the usage of Matrigel and manual steps would be hugely beneficial to improve variation within and across experiments in terms of the size and composition. The use of excessive extracellular matrix can also be deleterious in the context of the biology of cells within the organoid, despite its utility in supporting the development of elaborate vascular networks. However, despite the limitations of using bulk hydrogels, this has been the gold standard in generating self-organising vascular structures in vitro for decades.

[0009] It is therefore an object of the invention to provide further and / or improved methods of generating organoids that substantially reduces cost and improves utility and scalability.

[0010] Summary of the invention

[0011] The inventors identified a method that uses a granular hydrogel, which is a compacted assembly of hydrogel microparticles, to effectively generate organoids that capture the physiological features of native human tissues. In particular, the method involves simply generating a single preparation comprising cells and granular hydrogel. After supplementation of various factors over time, such as growth factors, cytokines and / or differentiation factors, organoids are generated. The resulting organoids are useful as models (e.g. in vitro, in vivo or ex vivo) with many uses, such as for studying tissue biology (e.g. development, homeostasis or regeneration), regenerative medicine (the production of cell therapies), and disease modelling (e.g. disease mechanism, drug screening, or personalised medicine).

[0012] Granular hydrogels have been primarily used in 3D bioprinting for their shear thinning behaviour and self-healing properties. They have not been used to generate complex self-organising vascularised organoid cultures as the current state of the art in the field applies bulk hydrogels, where cross-linked matrix components create a scaffold, and a bulk hydrogel is thought to be needed to drive angiogenic sprouting and vasculogenesis.

[0013] Unexpectedly, the inventors found that using a granular hydrogel generated organoids that capture the full physiological complexity of the bone marrow, similar to traditional ‘bulk’ hydrogel methods, and also led to several major advantages. In particular, the organoids generated using granular hydrogels comprise a lumen-forming vessel network, and contain a diverse range of cell types, e.g. haematopoietic cells and stromal cells, as well as mineral deposits (see Figures 3 to 6). The organoids also have substantially improved cell density compared to their previous method, facilitating biologically relevant cell-cell and cell-matrix interactions allowing true simulation of the cell functions and signalling pathways that occur in vivo. The inventors also showed that the bone marrow organoids are particularly useful for engrafting cells from other sources e.g. adult donors (see Figures 7 to 9), including cell types derived from patients that have proven difficult to culture ex vivo in order to model disease and evaluate therapies in the relevant human tissue setting.

[0014] In addition to increased throughput and lower costs from the granular method, the inventors also show an increase in the expression of native extracellular matrix (ECM) proteins within organoids generated by the granular hydrogel -based method compared with the bulk hydrogel-based method, while maintaining the same range of cell types (see Figures 10 and 11).

[0015] Key ECM proteins known to be essential to the native biology of the bone marrow, collagen types I and III (COL1A1, COL3A1), fibronectin and periostin are significantly upregulated in the relevant stromal cells (fibroblasts, MSCs, osteolineage cells) generated in granular hydrogel-derived organoids when compared to the bulk method (Figure 11). Hence, organoids generated by the granular hydrogel-based method better mimic the native expression of key ECM proteins (Bandyopadhyay, Shovik et al., (2024), Cell, Volume 187, Issue 12, 3120-3140. E29), facilitating the improved mineralisation observed (Figures 5 and 17).

[0016] The inventors also demonstrate the utility of the granular hydrogel-derived bone marrow organoids to model human diseases by engraftment with cells from patients e.g. those with blood malignancies such as multiple myeloma (exemplified in Figures 12 to 15), and in generating blood cells, including immune cells, for example for cell therapy (see Figure 16).

[0017] The granular hydrogel-based method for generating organoids is cost-effective, involves simple liquid handling steps, is highly reproducible, is scalable, can be automated, and results in consistent production of even, vascularized organoids. In particular, compared to the prior art method which uses bulk hydrogel, e.g. as disclosed in WO 2023 / 156774, the granular hydrogel-based method uses a significantly reduced gel volume per organoid. The granular hydrogel-based method is extremely feasible to automation as it involves simple liquid handling steps and is highly reproducible. The granular hydrogelbased method also provides significant scalability as the average volume and diameter of the organoids are reduced at no cost to complexity or cellularity, making the approach amenable to high throughput / automated pipetting technologies that have a hard size limit.

[0018] Furthermore, a granular hydrogel is particularly effective for generating organoids because the hydrogel microparticles can be readily dispersed amongst cells and cell aggregates, thereby providing good bioavailability. The material is more bioavailable than in a standard crosslinked bulk hydrogel since bulk hydrogels can collapse and lose their structure, whereas granular gel will maintain this as the particles are compacted. Granular gels are more tunable as their mechanical properties can be controlled and maintained by the level of fragmentation and method of compaction. For example, spinning at higher centrifuge speeds will increase stiffness whereas spinning at lower centrifuge speeds will decrease this. It is also easier to layer different gel compositions than in a standard bulk gel, and the stiffness of individual layers can be tuned.

[0019] The formulation of hydrogel microparticles can be tailored according to the organoid to be generated. For example, the hydrogel microparticles comprise extracellular matrix and factors that can promote cell growth and differentiation efficiently. Moreover, it is not necessary to extract the granular hydrogel from the resulting organoids, and thus the organoids are cultured throughout their life time in their encapsulated microenvironment, and they are able to able to incorporate materials and remodel to better mimic the physiological organ. Furthermore, a granular hydrogel is prepared as a suspension, and so it can be evenly and reproducibly distributed across culture vessels. The use of a granular hydrogel also results in a simple method because it involves generating a single preparation comprising cells, granular hydrogel and media (see Figure 1).

[0020] Compared to prior art methods which use bulk hydrogel, e.g. as disclosed in WO 2023 / 156774, the organoids generated from the granular hydrogel-based method are superior. Organoids generated from the bulk hydrogel-based method had cell-free spaces as a result of excess extracellular material. In contrast, organoids generated from the granular hydrogel method had an increase in cell density (see Figures 2 to 4). Furthermore, the granular hydrogel -based method overcomes issues of the bulk hydrogelbased method in terms of throughput and waste, while generating organoids which retain and enhance tissue modelling. For example, the bulk hydrogel-based method involves manual extraction of the sprouts from the hydrogel (see Figure 1), which is technically challenging and can affect downstream assays. This manual step is also a source of inter- and intra- experimental variation as a consequence of user variability and the technical challenges of isolating single organoids from a large, protein rich bulk hydrogel. In contrast, the granular hydrogel-based method has a simpler liquid handling step, requiring only a single preparation of cells and granular hydrogel, and manual extraction of the sprouts is not required. Hence, the granular hydrogel-based method results in consistent production of even, vascularized organoids because of the lack of an extraction step compared to the bulk hydrogel-based method. There is also significantly less lot-to-lot variation because the granular hydrogel structure is not driven by the kinetics of bulk hydrogel formation.

[0021] Accordingly, the invention provides a method of generating organoids comprising culturing cells in a suspension of hydrogel microparticles.

[0022] The invention also provides a method of generating organoids comprising culturing cells in a granular hydrogel.

[0023] The invention also provides a method of generating bone marrow organoids from pluripotent stem cells, e.g. iPSCs, and the method comprises: (i) driving the pluripotent stem cells, e.g. iPSCs, to form mesodermal aggregates; (ii) culturing the mesodermal aggregates from (i) into hematopoietic and vascular lineages in a medium comprising haematopoietic and stromal support cytokines, such as BMP4, FGF2, VEGFA, SCF and FLT3L; and (iii) adding the mesodermal aggregates from (ii) to a suspension of hydrogel microparticles; and (iv) culturing the mixture from (iii) to form bone marrow organoids.

[0024] The invention also provides organoids obtained or obtainable by a method of the invention, optionally wherein the organoids are bone marrow organoids.

[0025] The invention further provides bone marrow organoids comprising: (a) a lumen forming vasculature network, optionally wherein the vasculature network is comprised of subsets of endothelial cells specific to the bone marrow (e.g. arteriolar sinusoidal, Type H capillaries); (b) stromal cells comprising mesenchymal stem cells (MSCs) and osteo / adipo / CAR lineage cells differentiated from MSCs, fibroblast, and / or endothelial cells; (c) haematopoietic cells comprising, for example, haematopoietic stem and progenitor cells (HSPC), haematopoietic stem cells (HSCs), and cells of the myeloid and lymphoid lineages (including but not limited to T-lineage progenitors, B-lineage cells, natural killer cells, erythroid, eo / baso / mast, megakaryocytic, and myelomonocytic cells); and (d) osteolineage cells; and optionally (e) mineral deposits; optionally wherein the bone marrow organoids are obtainable or obtained by a method of the invention and / or wherein the bone marrow organoids have a mean vascularised area of 5% or more.

[0026] The invention also provides the use of bone marrow organoids described herein as a model for studies into healthy, ageing and diseased haematopoietic and stromal cell biology, for studies of immune response to infection and inflammation.

[0027] The invention also provides a method of making a bone marrow organoid model of disease, comprising seeding donor cells onto the bone marrow organoids of the invention, optionally wherein the donor is an individual with blood cancer, e.g. blood cancer. The invention also provides a disease model obtained or obtainable by said method.

[0028] The invention further provides a model for bone and / or bone marrow related condition, such as blood / bone marrow disorders including primary and secondary cancers, and bone marrow fibrosis, wherein the model comprises bone marrow organoids described herein that have been treated with an agent to induce said condition. The invention also provides a method of producing cells for cell therapy, wherein the method comprises bone marrow organoids generated by a method of the invention, and wherein the method further comprises collecting cells released from the bone marrow organoids. The invention also provides cells for cell therapy obtained or obtainable by said method.

[0029] The invention also provides the use of a model described herein to identify agents capable of preventing or treating the disease, comprising treating the model before, during or after induction of the disease. This may include the testing of disease-modifying or preventative therapies that involve targeting cells engrafted into the organoids. For example, the organoids can be used to test anti-cancer immunotherapy agents by engrafting patient-derived cancer cells together with autologous or allogeneic immune cells into the organoids, to evaluate therapies in the relevant human tissue or tumour microenvironment.

[0030] The invention also provides a method of identifying an agent for use in treating or preventing a disease, comprising: adding an agent to a disease model comprising organoids of the invention engrafted with diseased cells of a patient, wherein an improvement in the disease state of the disease model is indicative of the effectiveness of the agent in treating or preventing said disease; optionally wherein the method further comprises adding the agent to a reference model comprising organoids of the invention engrafted with healthy cells, and comparing the effects of the agent on the reference model and the disease model.

[0031] The invention also provides the use of a composition comprising a suspension of hydrogel microparticles to generate organoids, wherein the hydrogel microparticles are optionally compacted to form a granular hydrogel, and optionally wherein the organoids are bone and / or bone marrow organoids.

[0032] The invention also provides the use of a granular hydrogel to generate organoids, optionally wherein the organoids are bone marrow organoids.

[0033] The invention also provides a kit comprising a composition, a suspension of hydrogel microparticles, and / or medium as described herein. The kit may further comprise instructions for carrying out the methods described herein. Brief description of the figures

[0034] Figure 1: Schematic diagram showing a method of the invention using granular hydrogel compared to the prior art method using bulk hydrogel (e.g. as disclosed in WO 2023 / 156774) for generating bone marrow organoids.

[0035] Figure 2: A comparison of the average size of paraffin embedded and sectioned organoids generated by (a) bulk hydrogel-based method and (b) granular hydrogel-based method, (c) Quantification of individual organoid diameter over paraffin embedded organoids collected from two independent biological repeats, (d) a plot of the minimum, maximum, and range sizes of the organoids.

[0036] Figure 3: A comparison of cellularity (cell density) in embedded and sectioned organoids generated by (a) bulk hydrogel-based method and (b) granular hydrogel-based method, (c) A quantification of cell density (by nuclear counting in sections) across two biological repeats.

[0037] Figure 4. CD34 staining of cross sections of bone marrow organoids generated by (a) bulk hydrogel-based method and (b) granular hydrogel-based method, (c) Zoomed in images of organoids generated from the granular hydrogel-based method, (d) A quantification of the total CD34+ vessel area. Samples were analyzed across two independent biological repeats, with analysis performed within each individual organoid.

[0038] Figure 5: Von Kossa staining of paraffin embedded sections of bone marrow organoids generated by (a) bulk hydrogel-based method and (b) granular hydrogel-based method.

[0039] Figure 6: Characterisation of bulk hydrogel vs. granular hydrogel generated organoids. Organoids generated in parallel using either the bulk hydrogel or the granular hydrogel method and compared by flow cytometry at day 35 of culture. (A) stromal cell panel, identifying populations: Endothelial, Fibroblast, BM MSC, LEPR+ BM MSC, and Osteolineage cells). (B) lymphoid cell panel, identifying populations: Natural Killer (NK), B Lineage, and T-Cell progenitor (thymocyte populations). (C) myeloid panel identifying populations: erythroid cells, megakaryocytes, myelomonocytic cells as well as basophils and eosinophils. (D) stem cell specific panel identifying populations: haematopoeitic stem cells (HSCs), multipotent progenitors (MPPs), Common Myeloid Progenitors (CMPs), and myelo-erythroid progenitors (MEPs).

[0040] Figure 7: Image of donor cell engraftment. Fluorescent BAF3 cells migrated into the bone marrow organoid (indicated by arrows).

[0041] Figure 8: Image of bone marrow organoids engrafted with CD34+ haematopoeitic cells derived from a patient with myelofibrosis. The image shown is a Z-stack projection of a 150um region of an imaged organoid. Patient cells are labelled in white, and indicated by an arrow. Background signal is collagen I staining to delineate the volume of the organoid.

[0042] Figure 9: Image of bone marrow organoids engrafted with CD34+ haematopoeitic cells derived from a healthy donor or a myelofibrosis patient. Patient cells are labelled with CellTrace violet, and whole organoids are labelled with aSMA (a marker of fibroblast activation) and collagen type I.

[0043] Figure 10: (a) UMAP plots of cells from human bone marrow organoids generated using either the bulk hydrogel or granular hydrogel strategies. Data shown is derived from day 35 organoids in 4 independent differentiations, generated using single cell RNA sequencing (10X genomics, 3’ v3.4 chemistry). Data was analysed using Seurat (v5.1) and cell types annotated according to expression of canonical genes (e.g. CD3 for T cells), (b) Comparison of cells generated by bulk and granular hydrogel methods reveals that the granular hydrogel method achieves excellent representation of the key haematopoietic and stromal cell types present in human bone marrow, similar to bulk hydrogel-derived organoids.

[0044] Figure 11: Comparison of expression of extracellular matrix (ECM) proteins in stromal cell subtypes from organoids generated by bulk hydrogel vs. granular hydrogelbased methods. Violin plots show significantly increased expression of ECM genes in stromal cells from organoids generated using granular hydrogels. This includes: COL1A1 (Collagen Type I) in fibroblast, mesenchymal stromal cells (MSCs), and nestin (NES)+ MSC cells; COL3A1 (Collagen Type III) in fibroblast, MSC, NES+ MSC (Nestin + Mesenchymal Stem Cells), and osteolineage cells; fibronectin (FN1) in endothelial cells, MSC / fibroblasts, osteolineage cells and osteoblasts and periostin (POSTN) in fibroblasts, Figure 12: (A) Schematic showing the protocol used for modelling disease e.g. myeloma using granular hydrogel-derived organoids. Bone marrow organoids were seeded on day 21 with CD138+ plasma cells isolated from bone marrow aspirates from patients with myeloma (MM) or CD34+ blood stem / progenitor cells from peripheral blood of healthy, mobilised donors. (B) Donor cells were labelled with a fluorescent dye (CellTrace Violet) to enable tracking of engraftment and proliferation over time. Whole-organoid imaging is shown at day 3, day 7 and day 14 after engraftment (days 24, 28, and 35 of the whole protocol). Images show that donor cells successfully engraft into organoids by day 3, with expansion of fluorescently-labelled cells by day 14. (c & d) Images show (c) cytospins of cells from dissociated organoids and (d) H&E stained histological sections of whole organoids 14 days after engraftment of primary cells. Red arrows indicate morphologically-identifiable plasma cells.

[0045] Figure 13: UMAP plots of single cell RNA sequencing data (lOx Genomics) generated from granular hydrogel-derived organoids engrafted with healthy donor control cells (n=2 donors) or cells from patients with myeloma (n=3 donors) and cells from unengrafted organoids. As for Figure 12, organoids were dissociated for scRNAseq 14 days after engraftment of adult donor cells. Data analysis was performed using Seurat (v 5.1.0) and cell types annotated based on canonical gene expression. UMAP plots represent 45,706 haematopoietic cells and 14,577 stromal cells, (a) UMAP showing haematopoietic cells including myeloid (erythroid, megakaryocyte, eosinophil / basophil / mast cells, monocytes / macrophages, dendritic cells and T-lymphocyte progenitors. Plasma cells derived from organoids engrafted with myeloma samples were successfully captured (indicated by a circle on the plot), (b) Stromal cells captured included endothelial, osteolineage, adipolineage, fibroblast and mesenchymal stem cells.

[0046] Figure 14: Heatmap showing significantly enriched gene set pathways (GSEA analysis) comparing granular hydrogel-derived organoids engrafted with cells from patients with myeloma to control organoids (engrafted with cells from healthy donors). Black boxes highlight the cell populations (X axis) and gene sets (Y axis) with the most notable changes. This confirms increase in interferon response and inflammatory pathways in stromal cells including CD271+ MSC, osteolineage cells and early osteoblasts as well as in macrophages and dendritic cells, similar to observations made in previous studies analysing bone marrow from patients with myeloma (de Jong, M.M.E. et al., (2021), Nature Immunology, 22, 769-780), confirming that the organoids accurately model human disease.

[0047] Figure 15: Violin plots comparing the expression of genes associated with inflammation in myeloma (MM)-engrafted vs. control organoids, (a) Plots show significantly increased TNF, S1000A4, and S100A9 expression in macrophages, dendritic cells (cDC2) and neutrophils in myeloma-engrafted organoids, (b) Significantly higher expression of B2M in stromal cells isolated from myeloma-engrafted organoids, as previously shown in bone marrow of patients with myeloma, (c) Expression of NFIA and NFIB in osteoblasts from myeloma-engrafted organoids is significantly lower than in osteoblasts from control organoids, suggesting impaired osteoblast differentiation (Adamik J, et al. (2018). Journal of Bone Oncology Sep 15; 13:62-70; Terpos, E., et al. (2018) Blood Cancer Journal 8, 7).

[0048] Figure 16: To investigate the capacity of bone marrow organoids to generate blood cells, flow was induced by agitating bone marrow organoids in ultra-low attachment plates. (A) A schematic of the simple flow system generated using an incubator housed rocking system. (B) Brightfield images of a bone marrow organoid after 7 days of flow / agitation. The dark large 3D structure of the organoid is surrounded by a large number of cells that have been extruded from the organoid due to flow forces. (C) area of the well showing extruded cells. (D) A higher resolution brightfield image of the cells extruded from organoids using this system. (E) Ejected cells were collected, cytospin onto glass slides and stained using Giemsa. Numbers indicate morphologically-defined cell types including: macrophages: 1, Megakaryocytes: 2, Neutrophils: 3, Monocytes: 4, Myeloid progenitors: 5, Erythroid progenitors: 6, Fat cell / adipocytes: 7.

[0049] Figure 17: Assessment of mineralisation / ossification over time. The mineral stains alizarin red and Von Kossa were applied to paraffin embedded sections of granular hydrogel-derived organoids collected at day 35 and day 80. A clear increase in mineralisation is observed between day 35 and day 80, confirming the capacity of osteolineage cells to calcify the organoids over time. Detailed description of the invention

[0050] Methods of generating organoids using granular hydrogel

[0051] The invention relates to using a granular hydrogel, which is a compacted assembly of hydrogel microparticles. Typically, a granular hydrogel is made by fragmenting bulk hydrogel, which comprises a solid or semi-solid network of cross-linked polymers, into a suspension of hydrogel microparticles, followed by compaction of the hydrogel microparticles to make a granular hydrogel. Granular hydrogels are a type of microgel, and are also known as fragmented granular hydrogels, or granular microgels (e.g. see Muir et al., 2022, JoVE, e63867).

[0052] Hence, the invention provides a method of generating organoids comprising culturing cells with a composition comprising a suspension of hydrogel microparticles, e.g. granular hydrogel. The invention also provides the use of a composition comprising a suspension of hydrogel microparticles, e.g. granular hydrogel, to generate organoids.

[0053] Methods and uses of the invention may comprise generating granular hydrogel. For example, the methods and uses may comprise fragmenting bulk hydrogel to produce hydrogel microparticles, and optionally compacting the hydrogel microparticles to produce granular hydrogel. Alternatively, methods and uses of the invention may comprise using a suspension of hydrogel microparticles, such as granular hydrogel, which has been prepared as described herein.

[0054] Hydrogel microparticles may be prepared by any means that fragments bulk hydrogel, such as chemical and / or mechanical fragmentation. The methods and uses of the invention may use hydrogel microparticles, e.g. granular hydrogel, obtained or obtainable by the fragmentation and / or compaction methods described herein.

[0055] Chemical fragmentation may comprise hydrolytic degradation, co-acervation, or enzyme digestion, such as using proteases, e.g. lipase.

[0056] Mechanical fragmentation may comprise extrusion techniques, such as passing through syringes, meshes, filters or sieves, mechanical disruption such as grinding with mortar and pestle or through the use of blenders, or mechanical agitation, for example by vortexing with or without crushing beads. For example, the bulk hydrogel may be subjected to fragmentation by batch emulsion, where mixing of immiscible liquids can lead to the generation of droplets that undergo subsequent cross-linking to form hydrogel microparticles. A similar process can be performed in microfluidic devices and is known as microfluidic emulsions.

[0057] For example, the bulk hydrogel may be subjected to extrusion fragmentation, e.g. by serial extrusion through a series of smaller and smaller needle gauges, e.g. 18 G (838 pm in inner diameter) down to 30 G (159 pm in inner diameter) or higher. For example, the bulk hydrogel may be passed through a 23G needle, followed by a 25G needle, and then a 27G needle.

[0058] The bulk hydrogel may be subjected to electrohydrodynamic spraying wherein the hydrogel undergoes extrusion through a syringe which has a voltage passed across the needle tip.

[0059] The bulk hydrogel may have a compressive moduli from about 1 kPa to about 80 kPa, e.g. about 10 kPa to about 70 kPa, or about 50 kPa to about 60 kPa. A hydrogel that has a compressive moduli of more than 80 kPa may lead to increased clogging or overpressurisation of the syringes during the fragmentation step. A bulk hydrogel with compressive moduli lower than 1 kPa may deform during mechanical fragmentation steps.

[0060] The hydrogel microparticles useful with the invention may have regular or irregular shapes. They may be spherical. They may have jagged polygon shapes. They may have uniform or polydisperse diameters. They may comprise a mixture of fibrillar structures as well as a mix of regular or irregular spherical or polygon shapes.

[0061] The hydrogel microparticles may have a mean diameter of 10-500 pm, e.g. having an upper mean diameter of <450 pm, <400 pm, <350 pm, or <300 pm, and having a lower mean diameter of > 20 pm, > 40 pm, > 60 pm, > 80 pm, or > 100 pm.

[0062] The hydrogel microparticles may have a circularity ranging from 0.2 (not circular) to almost 1 (perfect circle).

[0063] The hydrogel microparticles may have a circular equivalent diameter ranging from 10 to 300 pm.

[0064] The hydrogel microparticles may have a feret diameter ranging from about 20 pm The hydrogel microparticles may have an aspect ratio ranging from 1 to 3.

[0065] Methods of measuring the circular equivalent diameter (um), feret diameter (um), aspect ratio and circularity are known in the art, e.g. as disclosed in Muir et al. (JoVE, 2022, 183, e63867). For example, parameters may be measured by first creating a dilute suspension of microparticles that can be imaged on an epifluorescent or confocal microscope and then converting images to a binary image, e.g. in ImageJ, to analyse the particles. The equivalent circular diameter (pm) for each particle may be determinable from the scale in pm / pixel. The area of hydrogel microparticles can be converted from pixels2to pm2. The area in pm2can then be used to derive the equivalent circular diameter by taking a square root of the area, dividing by TI and then doubling the result. The pm / pixel scale can also be used to obtain Feret’s diameters for hydrogel microparticles in pm. The various other parameters, namely circularity, aspect ratio, roundness and solidity are directly derivable from the output.

[0066] The hydrogel microparticles may be compacted to produce granular hydrogel. Compaction techniques are well known in the art, and any compaction techniques may be used. For example, a compaction technique may be centrifugation. The suspension of hydrogel microparticles may be centrifuged at 10,000xg to 18,000xg (e.g. 10,000xg, 12,000xg, 14,000xg, 16,000xg, or 18,000xg) for up to 10 min (e.g. 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min). The suspension of hydrogel microparticles may be centrifuged at 5xg, lOxg or lOOOxg for between 30sec and 30 min for cell containing suspensions. The suspension of hydrogel microparticles may be centrifuged at 300xg for 4 min.

[0067] Another compaction technique may be filtration, e.g. vacuum-driven filtration, which comprise loading a suspension of hydrogel microparticles onto a membrane filter, and reducing the amount of suspension medium by filtration or vacuum suction.

[0068] Methods of the invention may comprise adding the cells before compacting the hydrogel microparticles.

[0069] Methods of the invention may comprise adding the cells after compacting the hydrogel microparticles.

[0070] Methods of the invention may comprise creating multiple layers of compacted mixture comprising hydrogel microparticles and cells by repeating the steps of adding cells to the suspension of hydrogel microparticles followed by compacting the mixture. For example, the multiple layers of the compacted mixture may comprise hydrogel microparticles of different formulations and / or different cell types.

[0071] Methods of the invention may comprise creating multiple layers of compacted mixture comprising hydrogel microparticles and cells on multiple surfaces, e.g. a functionalised surface (e.g. coated plate), followed by alternative material (e.g. ceramic), or in a microfluidic device.

[0072] The granular hydrogel may comprise about > 0.1% w / v hydrogel microparticles, e.g. about > 0.5%, > 1%, > 10%, > 20%, > 30%, > 40%, > 50%, or > 60% w / v hydrogel microparticles. For example, the granular hydrogel may comprise about 0.1 % to 60 % w / v, such as about 0.5 % to about 1.5 %, w / v, or 0.1 % to 2 % w / v, hydrogel microparticles. The granular hydrogel may have a density of hydrogel microparticles of about 0.5 or more, such as about 0.6, about 0.7, about 0.8 or about 0.9.

[0073] In the embodiments where the cells are added are after compaction of hydrogel microparticles, the compacted mixture may comprise about 0.1 % to 2 % w / v (e.g. about 0.5 % to about 1.5 %, w / v) hydrogel microparticles. The mixture may comprise about 0.2 %, 0.4 %, 0.6 %, 0.8 %, 1.0 %, 1.2 %, 1.4 %, 1.6 %, 1.8 %, or 2.0 % w / v hydrogel microparticles. The mixture may comprise about 1% to 20% w / v hydrogel microparticles. The mixture may comprise about 2% to 40% w / v hydrogel microparticles. The mixture may have a density of hydrogel microparticles of about 0.5 or more, such as about 0.6, about 0.7, about 0.8 or about 0.9.

[0074] Methods of the invention may comprise a step of removing the granular hydrogel. However, this step is not necessary. Indeed, it is beneficial to retain the granular hydrogel in the organoids because the organoids would then be cultured throughout their life time in their encapsulated microenvironment, and they are able to able to incorporate materials and remodel to better mimic the physiological organ. Hence, in certain embodiments of the invention, the methods and uses do not comprise removing the granular hydrogel, e.g. by extracting the organoids from the granular hydrogel.

[0075] The hydrogel microparticles may comprise a natural polymer, such as collagen or fibrin. The hydrogel may comprise a synthetic hydrogel, for example comprising or consisting of synthetic peptides, or peptide / adhesion functionalized polysaccharides. The hydrogel microparticles may comprise polypeptide chains, polysaccharide chains, and / or extracellular matrix proteins.

[0076] The extracellular matrix protein may be collagen, such as collagen I and / or collagen IV. The hydrogel microparticles may comprise collagen I and collagen IV. The ratio of collagen I to collagen IV may be 1:1 to 3:1, e.g. 1:1. It was found that a mix of collagen I and collagen IV in the hydrogel would yield a high proportion of myeloid cells and a population of mesenchymal stromal cells, which are important for remodelling the bone marrow space.

[0077] The hydrogel microparticles may comprise Matrigel. Matrigel comprises solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, e.g. lamin, collagen IV and nest protein. Matrigel may also comprise growth factors, e.g. IGF and FGF.

[0078] The hydrogel microparticles may comprise fibrin.

[0079] The hydrogel microparticles may comprise Matrigel, fibrin, collagen type I and collagen type IV.

[0080] The hydrogel microparticles may comprise 40% Matrigel and 60% collagen, optionally wherein the collagen is collagen type I and / or IV.

[0081] The collagen in the hydrogel microparticles may be present at a total concentration of from about 0.1 to about 3.5 mg / ml, or from about 1 to about 3.5 mg / ml. The collagen may be present at a total concentration of about 1 mg / ml.

[0082] Cells suitable for use in the methods and uses of the invention may be stem cells, progenitor cells, terminally differentiated cells, or cells of immortal cell lines. The stem cells may be pluripotent stem cells or adult stem cells. The pluripotent stem cells may be embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). The iPSCs may be human iPSCs (hiPSCs). The stem cells may be obtained from human cells cultured in vitro, or in vivo in chimeric animals.

[0083] The cells may be iPSCs.

[0084] The cells may be ESCs.

[0085] The cells may be CD34+ haematopoietic stem and progenitor cells. The cells may be leukemic blasts, or terminally differentiated cells such as T-cells and macrophages, or solid tumour cells to model bone marrow metastasis.

[0086] The cells may be cells of immortalised cell lines, such as leukemic lines (e.g. HEL, MOEM-13).

[0087] The cells may be cells obtained from a subject. The subject may be human. The subject may be healthy. The subject may have a disease or a condition, such as a blood or bone marrow disorder, or a bone marrow related condition as described herein. The subject may have myelofibrosis or other myeloid or lymphoid blood cancers.

[0088] The methods and uses of the invention may comprise culturing one or more type of cells with the suspension of hydrogel microparticles, e.g. more than one type of cells, such as 2, 3 or 4 types of cells. For example, the input cells may be iPSCs, or cells derived therefrom (e.g. mesoderm aggregates), and then further cells may be added to the culture. The further cells may be any of the cells described herein. The further cells may be added at any time point following adding of iPSCs, or cells derived therefrom (e.g. mesoderm aggregates), to hydrogel microparticles, e.g. from day 1 to day 21, such as at day 1, 7, 14 or 21. For example, the further cells may be added when the iPSCs, or cells derived therefrom (e.g. mesoderm aggregates), have been differentiated sufficiently to establish an architecture and / or some cell fate commitment. For example, the further cells, such as an immortalised MSC cell line (which secretes haematopoietic factors), may be added early on in the protocol.

[0089] Methods and uses of the invention may comprise growing the cells into aggregates and / or differentiating the cells prior to culturing them in the suspension of hydrogel microparticles to generate organoids. The aggregates may have a mean diameter of 50-700 pm.

[0090] In embodiments of the invention relating to the use of pluripotent stem cells (e.g. iPSCs) as the input cells, the cells may be stimulated into germ layer specification (e.g. endoderm, mesoderm or ectoderm) by culturing appropriate stimulation factors, and then inducing differentiation and maturation by culturing with specific growth and signalling factors in order to obtain an organised mixture of the specific cell types that form the Hence, methods and uses of the invention may comprise: (i) driving pluripotent stem cells (e.g. iPSCs) to form aggregates of germ layer specific cells (e.g. endoderm, mesoderm or ectoderm); (ii) culturing the aggregates from (i) into differentiated lineages in a medium comprising appropriate stimulation factors; (iii) adding the aggregates from (ii) to a suspension of hydrogel microparticles, according to a method as described herein; and (iv) culturing the mixture from (iii), e.g. according to a method as described herein, until organoids are produced.

[0091] Methods for driving pluripotent stem cells (e.g. iPSCs) to form aggregates of germ layer specific cells (e.g. endoderm, mesoderm or ectoderm) are well known in the art. For example, mesoderm induction from pluripotent stem cells is described herein.

[0092] In embodiments of the invention relating to the use of adult stem cells (ASCs) as the input cells, the cells may be stimulated with the appropriate growth factors to allow proliferation.

[0093] Supplement factors may be added to the media to drive differentiation and maturation. The supplement factors and culture conditions would depend on the organoids to be generated. The skilled person in the art would be able to determine appropriate supplement factors and culture conditions using common general knowledge in the art, e.g. see Khan et al., 2023 (Cancer Discovery, 13(2): 364-385), Huang et al., 2023 (Cells, 12(12): 1590) and Zhao et al., 2022 (Nature Reviews Methods Primers 2, 94). Examples of supplement factors for generating bone marrow organoids are described below.

[0094] The various differentiated cell types can be identified according to known markers and methods in the art, e.g. as demonstrated in the Examples. For example, mesodermal differentiation markers include Brachychury (canonical marker), BMP family proteins, TWIST, TWIST2 and / or HAND1. Ectodermal differentiation markers include NES and / or OTIX2. Endodermal differentiation markers include SOX 17 and / or FOXA2. Haematopoietic differentiation markers include CD34 and CD35. Vascular differentiation markers include CD31 and / or CD 144. Stromal differentiation markers include CD90, CD271 and / or LEPR.

[0095] The methods and uses of the invention may not comprise separating the cells from the hydrogel microparticles. For example, the methods and uses of the invention may not comprise extraction (e.g. manual extraction) of cells (e.g. aggregates, developing organoids or organoids) from the hydrogel microparticles. The methods and uses of the invention may comprise culturing the cells (e.g. aggregates, developing organoids or organoids) in the suspension of hydrogel microparticles in the same container (e.g. 96-well plate) throughout the entire culture period until organoids are formed.

[0096] Methods and uses of the invention may be for generating any organoids, for example, bone, bone marrow, bone and bone marrow, mammary gland, retina, kidney, stomach, intestine, liver, lung, prostate, bladder, endometrium, blood vessels or thyroid organoids.

[0097] The invention also provides organoids obtained or obtainable by the methods described herein. The organoids of the invention are in vitro or ex vivo organoids. The organoids of the invention are synthetically produced (i.e. in vitro) and does not comprise native tissue extract.

[0098] The organoids of the invention may have an average size from about 100 pm to about 5 mm, e.g. from about 500 pm to about 4 mm. The organoids of the invention may have a narrow size distribution, e.g. a standard deviation of 2 or below.

[0099] An organoid as described herein is a 3D organotypic culture having architectural, cellular and molecular features of a target organ or tissue. For the avoidance of doubt, an organoid, such as a bone marrow organoid, as described herein is distinct from a spheroid and other 3D cell aggregate which can be multi-lineage, but are not organised into structures (e.g. vessels, luminal spaces). An organoid as described herein may capture specific regions of an organ (e.g. myelopoietic bone marrow), or multiple regions (e.g. both lymphoid and myeloid bone marrow), or the entirety of the target organ.

[0100] Mesoderm induction from pluripotent stem cells

[0101] Methods to form mesodermal aggregates from pluripotent stem cells, such as iPSCs, are known in the art. For example, the mesodermal aggregates may be formed by: incubating induced pluripotent stem cells (iPSCs) to induce the formation of iPSC aggregates; and culturing the iPSC aggregates in a media for stem cell maintenance supplemented with factors, such as BMP4, FGF2, and VEGFA, to induce formation of mesodermal aggregates. The iPSCs may be cultured with BMP4. The iPSCs may be cultured in low oxygen (e.g. 1-5%) with BMP4, with or without a WNT inhibitor, such as CHIR99021. For example, the iPSCs may be cultured in 5% Oxygen with BMP4, and a Wnt activator, such as CHIR99021. The iPSCs may be cultured in 5% oxygen with BMP4, CHIR99021, VEGFA, and FGF2.

[0102] Prior to the induction of aggregates of the pluripotent stem cells, such as iPSCs, the pluripotent stem cells may be maintained and cultured according to common methods in the art. The iPSCs may be passaged one or more times, for example at about 20-90% confluence, e.g. at about 70-80% confluence. The pluripotent stem cells, e.g. iPSCs, may be detached for passaging and / or aggregate formation, for example using physical / mechanical detachment and / or non- physical / mechanical detachment, such as using EDTA or enzymatic detachment. For pluripotent stem cell, e.g. iPSC, aggregate formation, detached pluripotent stem cells, e.g. iPSCs, may be cultured in stem cell differentiation medium for a period of at least 5 hours, or at least 12 hours, e.g. for a period of about 5-24 hours. The stem cell differentiation medium may comprise basal media supplemented (e.g. StemFlex™) with a ROCK inhibitor (e.g. RevitaCell™ (Thermo), or equivalents thereof).

[0103] The pluripotent stem cell, e.g. iPSC, aggregates may be incubated for a period, e.g. 5-24 hours under standard cell maintenance conditions, such as 5% CO2 at 37 °C, before adding them to mesoderm induction medium. The resulting pluripotent stem cell, e.g. iPSC, aggregates may be collected by gravitation or centrifugation and resuspended in the mesoderm-inducing medium. In one embodiment, the day pluripotent stem cell, e.g. iPSC, aggregates are transferred for culture mesoderm-inducing medium is day 0. Culturing the pluripotent stem cell, e.g. iPSC, aggregates in the mesoderm-inducing medium may be under suitable cell growth conditions, for example at 5% 02, 5% CO2, and 37 °C.

[0104] The early mesoderm induction step may comprise incubation of the pluripotent stem cell, e.g. iPSC, aggregates over a period of time sufficient to form mesodermal cells in >60% (e.g. >70%, >80% >90%) of the cell population. Culturing the pluripotent stem cell, e.g. iPSC, aggregates in the mesoderm-inducing medium may be for a period of about 2-7 days, e.g. about 3-7 days or about 3-5 days. Culturing the pluripotent stem cell, e.g. iPSC, aggregates in the mesoderm-inducing medium may be for a period until aggregates reached an average size of about 200-250 pm. The size of the aggregate is understood to be the average of the largest diameter of the pluripotent stem cell, e.g. iPSC, aggregates.

[0105] The skilled person will understand that the formation of mesoderm cells may be provided as much as necessary, with the understanding that the number or percentage of mesoderm cells formed at this stage will have an impact on the end yield of the resulting organoids. For example, the higher number of aggregates will give a higher number of bone marrow organoids, but a higher number of mesodermal cells will give a more efficient differentiation into bone marrow lineages.

[0106] The production of mesodermal tissue from the pluripotent stem cell, e.g. iPSC, aggregates may be identified by detecting the presence of mesodermal markers using any method known in the art. The mesodermal markers may comprise or consist of one or more, or all, of Brachyury, Snail, TBX6 and N-cadherin. The cell marker may be detected by protein expression, for example using immunofluorescence or mRNA expression (e.g. using qRT-PCR).

[0107] The mesoderm-inducing medium may be a chemically defined medium (CDM). The mesoderm-inducing medium comprises a basal medium, such as a stem cell differentiation culture media. The mesoderm-inducing medium may comprise a stem cell differentiation media, such as APEL2 or StemPro, or equivalents thereof. The mesoderminducing medium may comprise a ROCK inhibitor, such as Y-27632.

[0108] The mesoderm-inducing medium may comprise supplement factors in an amount suitable to promote mesoderm commitment and prime early endothelial / haematopoeitic bipotent progenitors. The supplement factors may be BMP4, FGF2, and VEGFA. BMP4 may be provided in the mesoderm- inducing medium at a concentration from about 10 to about 100 ng / ml, e.g. about 50 ng / ml. FGF2 may be provided in the mesoderm-inducing medium at a concentration from about 10 to about 100 ng / ml, e.g. about 50 ng / ml. VEGFA may be provided in the mesoderm -inducing medium at a concentration from about 10 to about 100 ng / ml, e.g. about 50 ng / ml. The supplement factors comprise Wnt activator on (SB). Bone marrow organoids

[0109] The invention relates to generating bone marrow organoids that resemble native human bone marrow. A bone marrow organoid as described herein is a 3D organotypic or organoid culture comprising of stromal cells and haematopoietic cells. The bone marrow organoid may comprise multiple lineages of haematopoietic cells (including myeloid and lymphoid cells), subsets of stromal cells (including for example mesenchymal stem cells, CAR-cells, osteolineage and / or adipocytes), and a vasculature.

[0110] The methods and uses of the invention may comprise generating bone marrow organoids from pluripotent stem cells, e.g. iPSCs, such as hiPSCs.

[0111] The methods and uses of the invention may comprise: (i) driving iPSCs to form mesodermal aggregates; (ii) culturing the mesodermal aggregates from (i) into hematopoietic and vascular lineages in a medium comprising BMP4, FGF2, VEGFA, SCF and FLT3L; (iii) adding the mesodermal aggregates from (ii) to a suspension of hydrogel microparticles (e.g. according to a method as described herein); and (iv) culturing the mixture from (iii), e.g. according to a method as described herein, until bone marrow organoids are produced.

[0112] Conditions suitable for mesoderm induction in step (i) are described above.

[0113] Step (ii) may comprise culturing the mesodermal aggregates in a mesoderminducing medium for a period until the mesodermal aggregates have an average size of 100-500 pm and / or the presence of markers of early endothelial and haematopoietic differentiation. Such markers may include one or more, or all of CD144, CD31, RUNX1, and GATA1. Markers may be detected by any suitable means, such as by qRT-PCR.

[0114] An appropriate mesoderm-inducing medium is described herein. The supplement factors BMP4, FGF2, VEGFA, SCF and FLT3L in the mesoderm-inducing medium of step (ii) may be provided in the medium at a concentration from about 10 to about 100 ng / ml, e.g. about 50 ng / ml. The medium may be further supplemented with one or more additional cytokines, such as IL7 or other interleukins to induce lymphopoiesis. The additional cytokines may comprise one or more of IL11, FLT3L, GM-CSF, M-CSF, G- CSF, EPO, IL1, IL12, IL13, IL33, TPO, IL3, IL6, IL2, IL10 and IL8. The skilled person would be able to determine an appropriate amount of any of the supplement factors according to common general knowledge in the art. For example, a further supplement factor may be provided in the medium at step (ii) at a concentration from about 10 to about 100 ng / ml, e.g. about 50 ng / ml.

[0115] The culture conditions of step (ii) may be standard cell culture conditions, such as 5% O2, 5% CO2, and 37°C. The culture conditions of step b may be normoxic conditions (e.g. about 20-21% O2). The culture condition may be at low oxygen level, e.g. 3-10%, such as 5% O2. A culture condition that resembles physiologically relevant O2 tension (such as low oxygen level, e.g. 3-10%, such as 5% O2) encourages bone marrow specification and lymphoid cell generation.

[0116] Step (iii) may comprise culturing the mesodermal aggregates in a suspension of hydrogel microparticles according to the methods described herein.

[0117] Step (iv) may comprise culturing the mixture in a medium comprising supplement factors, such as cytokine and / or growth factor cocktail for generating the desired mix of haematopoietic cell lineages. The supplement factors useful for step (iv) may comprise one or more early haematopoietic factors (pan-haematopoietic factors), such as SCF, FLT3L, IL3, and / or IL6. The supplement factors useful for step (iv) may comprise one or more factors which drive lymphoid lineage development, such as IL7. The supplement factors useful for step (iv) may comprise one or more factors which drives lineage specific development and diversify the haematopoietic arms, such as EPO, TPO, and / or CSF, and interleukins. For example, the supplement factors useful for step (iv) may comprise small molecule enhancers of hematopoietic or vascular differentiation, e.g. Forskolin, UM171, PI3K inhibitors, TGFb inhibitors and / or SRI. The supplement factors useful for step (iv) may comprise one or more factors which drives development of sinusoid specific endothelial vasculature, such as VEGFC. VEGFC may be added at a later period, for example from d3 (from the overall differentiation timeline).

[0118] The supplement factors useful for step (iv) may comprise VEGFA, VEGFC, FGF2, hSCF, FLT3L, IL3, IL6, IL7, TPO, EPO and BMP4.

[0119] The supplement factors useful for step (iv) may comprise VEGFA, FGF2, BMP4, SCF, FLT3L, IL3, IL6, cGSF, EPO and / or TPO. The supplement factors useful for step (iv) may comprise VEGFA, FGF2, BMP4, SCF, FLT3L, IL3, IL6, cGSF, EPO, TPO, IL7 and / or VEGFC.

[0120] The skilled person would be able to determine an appropriate amount of any of the supplement factors according to common general knowledge in the art. For example, a supplement factor may be provided in the medium for step (iv) at a concentration from about 10 to about 100 ng / ml, e.g. about 50 ng / ml.

[0121] The mesoderm aggregates in step (iv) may be cultured until an average size from about 100 pm to about 5 mm, e.g. from about 500 pm to about 4 mm.

[0122] The mesoderm aggregates may be cultured until a vascular network is formed, and optionally for as long as a vascular network is maintained.

[0123] The mesoderm aggregates may be cultured until haematopoietic cells are formed, e.g. CD45+ cells are identified. The culture may be maintained in basal media, such as APEL2, StemPro-34, or alternative appropriate cell culture medium.

[0124] The skilled person may adjust the concentration and / or composition of cytokines in order to alter the composition of the resulting bone marrow organoid. For example, part way (e.g. about half way) through the sprouting phase the concentration of EPO may be increased to produce more erythrocytes, or EPO may be reduced along with an increase in the SCF / FLT3L content to produce more HSPCs. The increase or decrease may be sufficient to allow the production of a desired cell composition in the bone marrow organoids. The decrease of a given cytokine may be about a 10%, 30%, 50%, 80%, or 90% decrease. The increase of a given cytokine may be about a 10%, 30%, 50%, 80%, 100%, 150% or 200% increase.

[0125] The invention also provides bone marrow organoids. The bone marrow organoids may be obtainable or obtained by the methods described herein.

[0126] The composition of the bone marrow organoids of the invention typically resembles that of the native tissue, although the composition may be engineered to include other cell types, e.g. for studying specific biological questions.

[0127] The bone marrow organoids of the invention are in vitro or ex vivo organoids. The bone marrow organoids of the invention are synthetically produced (i.e. in vitro) and does not comprise native bone and / or bone marrow tissue extract. The bone marrow organoids of the invention are synthetically produced (i.e. in vitro) and does not comprise bone marrow tissue extract.

[0128] The bone marrow organoids of the invention comprise cells from multiple lineages. The bone marrow organoids of the invention may comprise stromal cells and / or haematopoietic cells, preferably both stromal and haematopoietic cells. The stromal cells may comprise vascular cells, mesenchymal stem cells, adipocytes, and peri-vascular stroma. The haematopoietic cells may comprise myeloid and lymphoid cells and their stem cell progenitors. The bone marrow organoids of the invention may comprise osteolineage cells. The bone marrow organoids of the invention may comprise mineral deposits.

[0129] For example, the bone marrow organoids of the invention may comprise: (a) a lumen forming vasculature network and / or sinusoids, (b) stromal cells comprising mesenchymal stem cells (MSCs), fibroblast, and / or endothelial cells; (c) haematopoietic cells comprising haematopoietic stem and progenitor cells (HSPC), erythroid cells, myelomonocytic cells, and / or megakaryocytic cells; and (d) osteolineage cells. The bone marrow organoids may further comprise mineral deposits.

[0130] The bone marrow organoids of the invention are typically 3D cultures which are smaller than native human tissue. The size can be adjusted based on input cell number. For example, the bone marrow organoids of the invention may have an average size from about 100 pm to about 5 mm, e.g. from about 500 pm to about 4 mm. The bone marrow organoids of the invention may have a narrow size distribution, e.g. a standard deviation of 2 or below. The bone marrow organoids of the invention may have a mean vascularised area of >5%, e.g. >10%, >20%, >40%, or >60%.

[0131] The bone marrow organoids of the invention typically have longevity. For example, they can be maintained in culture for 30 days or more, e.g. 50 days or more.

[0132] The bone marrow organoids of the invention may have similar function as the native tissues. For example, they are capable of producing red blood cells, platelets, myelomonocytic and lymphoid progenitors. Applications / uses

[0133] The invention relates to providing organoids that are useful as in vitro, ex vivo or in vivo models for uses, such as studying tissue biology, regenerative medicine, or disease modelling. Hence, the invention also provides methods and uses of the organoids for such uses.

[0134] The organoids of the invention may be useful for studying tissue biology, such as development, homeostasis or regeneration. The organoids of the invention may be useful for modelling the bone and bone marrow environment for studies into healthy, ageing and diseased haematopoietic and stromal cell biology. The organoids of the invention may be useful in modelling immune response to infection and inflammation.

[0135] The organoids of the invention may be useful for disease modelling, e.g. disease mechanism, drug screening, or personalised medicine. Hence, the invention also provides a disease model comprising the organoids of the invention. Examples of personalised medicine includes assessing individualised therapy responses, predicting treatment resistance e.g. to immunotherapies / cellular therapies, outcome from stem cell transplantation and pharmacogenomic testing, and high throughput screening. The disease may be a bone and / or bone marrow related condition, such as myelofibrosis, bone marrow fibrosis, or blood cancers, e.g. multiple myeloma, myeloma, solid tumour metastases, leukaemia, (e.g. acute or chronic lymphoblastic leukaemias, or acute or chronic myeloid leukaemias), myelodysplastic syndrome, myeloproliferative neoplasms, lymphomas, or mast cell neoplasms.

[0136] For example, the organoids of the invention may be useful for modelling bone marrow fibrosis. Extracellular matrix (ECM) proteins are known to be linked to bone marrow fibrosis, and the organoids of the invention are effective in expressing extracellular matrix (ECM) proteins (e.g. see Figure 11). Hence, the invention also provides a model of bone marrow fibrosis comprising the organoids of the invention.

[0137] The organoids of the invention may be useful for modelling multiple myeloma. The organoids of the invention may express inflammatory response genes, for example, increased TNF, S100A4, and S100A9 expression in key haematopoietic cells (e.g. see

[0138] Figure 15). Furthermore, the organoids of the invention may respond to diseased cells e.g. by increasing B2M in stromal cells and decreasing expression of NFIA and NFIB transcription factors in osteolineage cells (e.g. see Figure 15). Hence, the invention also provides a system that can be used to model multiple myeloma.

[0139] The organoids of the invention may be engrafted with cells from a patient having a disease, such as a bone and / or bone marrow related condition, such as myelofibrosis, bone marrow fibrosis, or blood cancers, e.g. multiple myeloma, myeloma, solid tumour metastases, leukaemia, (e.g. acute or chronic lymphoblastic leukaemias, or acute or chronic myeloid leukaemias), myelodysplastic syndrome, myeloproliferative neoplasms, lymphomas, or mast cell neoplasms, or any disease described herein. Following seeding the organoids of the invention with patients cells, the organoids may undergo remodelling of the microenvironment, resulting in engrafted organoids having features of said disease. In the embodiment relating to a model of bone marrow fibrosis, the engrafted organoids may develop progressive scarring. In the embodiment relating to a model of multiple myeloma, the engrafted organoids may develop changes in stromal cell metabolism, cell fate, and inflammation (e.g. see Figures 12 to 15).

[0140] The organoids of the invention or a disease model of the invention may be used for screening for biomarkers associated with the disease state of the organoids, e.g. fibrosis or other bone marrow disorders. For example, the invention may provide a method comprising the monitoring of biomarkers released from the bone marrow organoids or cells engrafted therein, or biomarkers in tissue or cellular extracts of the bone marrow organoids.

[0141] A disease model comprising the organoids of the invention may be used to test a therapy for preventing or treating said disease. Hence, the invention also provides a method of identifying an agent for use in treating or preventing a disease, comprising adding an agent to a disease model comprising organoids of the invention engrafted with diseased cells of a patient, wherein an improvement in the disease state of the disease model is indicative of the effectiveness of the agent in treating or preventing said disease. The method may further comprise adding the agent to a reference model comprising organoids of the invention engrafted with healthy cells, and comparing the effects of the agent on the reference model and the disease model. The healthy cells may be from the same patient or a different donor. The method may further comprise adding further cells, such as immune cells, to the model, e.g. to enhance or facilitate cancer cell killing.

[0142] A disease state may develop, or be induced in the bone marrow organoid, whereby changes to the biomarker profile may be determined and linked to the disease state.

[0143] The biomarkers may comprise proteins, glycoproteins, glycans, peptides, nucleic acids, or any cellular product which may indicate a diseased state of the bone marrow organoid or engrafted cells in the bone marrow organoid. The biomarkers may be cell markers, such as surface proteins.

[0144] The organoids of the invention may be useful for supporting engraftment and survival of cells from patients having a condition, e.g. a range of blood malignancies, including cancer cell types which are difficult to keep alive ex vivo in standard liquid culture systems. The cancer cell types may include, but not be limited to, cells from patients with myeloid or lymphoid blood malignancies such as multiple myeloma, myeloma, acute or chronic lymphoblastic leukaemias, acute or chronic myeloid leukaemias, myelodysplastic syndrome, myeloproliferative neoplasms, lymphomas, and mast cell neoplasms.

[0145] Therefore, the organoids (e.g. bone marrow organoids) of the invention may be used for an engraftment and / or survival assay for cells from a patient with a blood malignancy.

[0146] The organoids (e.g. bone marrow organoids) may be seeded with cells from a donor and used for tracking the cells to assay one or more of survival, proliferation and isolation of engrafted cells, for example for downstream functional testing. The organoids (e.g. bone marrow organoids) may also be used to study cancer-associated pathogenic remodelling of the bone marrow niche, such as the fibrosis induced by a malignant clone in a proportion of patients with myeloproliferative neoplasms.

[0147] The cells may be tracked by fluorescent markers or tags. Tracking the cells may comprise the use of a fluorescent cell tracking system.

[0148] The cell donor may be an adult or child donor.

[0149] For example, methods or uses of the invention may relate to modelling fibrosis, comprising treating the bone marrow organoids of the invention with an agent for inducing fibrosis and / or collagen deposition. Hence, the invention also provides a model for bone and / or bone marrow related condition, such as bone marrow fibrosis, wherein the model comprises bone marrow organoids of the invention that have been treated with an agent to induce said condition. The model may be used to identify agents capable of preventing or treating the condition, comprising treating the model before, during or after induction of the condition and monitoring if the agent has an inhibitory or reductive effect in fibrosis development. The model may also be used to identify if the agent inhibits or reduces the expression of smooth muscle actin and / or collagen expression.

[0150] The agent may be used to induce fibrosis in the bone marrow organoids of the invention. The agent may be used to induce extracellular matrix deposition in the bone marrow organoids, which respond to fibrosis-promoting factors by increasing smooth muscle actin and collagen expression, resulting in fibrosis. The agent may comprise or consist of a growth factor or cytokine. The agent, such as a cytokine, may be TGFp. The bone marrow organoids may be treated with TGFP to emulate fibrosis. Fibrosis may be induced genetically, such as by genetic modification, genetic overexpression smooth muscle actin and / or collagen, or by siRNA silencing, or by treatment with other proteins or pharmacological agents. Fibrosis may be induced genetically, such as by overexpression of genes, such as TGFB1 or other fibrosis-promoting genes, that induce expression of aSMA / collagen. Fibrosis may be induced by using an iPSC line from a patient suffering from fibrosis, or iPSCs may be gene edited to harbour a known fibrosis causing gene.

[0151] The amount of agent and incubation time with the agent may be an amount and time sufficient to cause collagen deposition in the bone marrow organoid, such as at least 2 ng / ml TGFP, or such as at least lOng / ml, for at least 24 hours.

[0152] The amount and incubation time with TGFp may be at least 2ng / ml, such as at least 5ng / ml for at least 2 hours, such as at least 72 hours. The amount and incubation time with TGFp may be between about 2ng / ml and 500ng / ml, such as between about 5ng / ml and 500ng / ml, for a period of between about 2 and 96 hours or more, such as between about 24 and 96 hours or more. The amount and incubation time with TGFP may be between about 2ng / ml and 500ng / ml, such as between about 5ng / ml and 500ng / ml for a period of between about 2 and 72 hours, such as between about 24 and 72 hours. The amount and incubation time with TGFP may be between about 2ng / ml and 50ng / ml, such as between about 5ng / ml and 50ng / ml, for a period of between about 24 and 72 hours. The amount and incubation time with TGFp may be between about 2ng / ml and 50ng / ml, such as between about 5ng / ml and 50ng / ml for a period of between about 36 and 72 hours. The skilled person will appreciate that where a higher concentration is used the incubation time may be shorter, and vice versa.

[0153] The organoids of the invention may provide a source of immune and blood cells in ex vivo culture systems, including body-on-a-chip approaches and organoid or microtissue co-cultures. For example, the invention provides a method for producing platelets and / or erythroid cells (RBCs), comprising the incubation of bone marrow organoids of the invention in vitro, and harvesting the platelets and / or erythroid cells (RBCs) produced from the bone marrow organoids. The platelets and / or erythroid cells (RBCs) may be produced naturally by the bone marrow organoids or induced, for example by the dosing of heparin, hirudin, and / or ROCKi to drive more proplatelet formation. The platelets and / or erythroid cells (RBCs) may be harvested by separating them from the cells of the bone marrow organoids, such as by FACS. The platelets and / or erythroid cells (RBCs) may be harvested by BSA gradient and / or centrifugation.

[0154] The organoids may also facilitate the maintenance and culture of said immune and blood cells.

[0155] Where maintenance and culture of blood cells is concerned, the methods and uses of the invention may also be used in a method for maintaining the viability of cells from a patient donor with blood cancer ex vivo.

[0156] The organoids of the invention may provide allogeneic or autologous cells for use in cell therapy (e.g. transplant or CAR-T therapies). The organoids may facilitate the maintenance and culture of said allogenic or autologous cells.

[0157] To produce cells (e.g. blood cells and / or immune cells) useful in cell therapy, the bone marrow organoids of the invention or a bone marrow organoid model of disease of the invention may be cultured, and cells released from the organoids may be collected. Cells may be released into the media of the culture. To facilitate release of cells from said the organoids, the organoids may be grown on a low attachment surface and cultured under agitation, e.g. on an incubation rocker at an appropriate speed (e.g. 15 degree oscillations over 5 second intervals). The released cells may be collected following differentiation, e.g. at day 28, 32 or 35 of differentiation. Hence, the invention also provides a method of producing cells for cell therapy comprising the steps described herein. The invention also provides cells obtained by said method.

[0158] The cells for use in cell therapy may be haemopoietic stem cells, histeocytes, macrophages, megakaryocytes, neutrophils, monocytes, myeloid progenitor cells, erythroid progenitor cells, mature erythrocytes, platelets, eosinophil progenitor cells, basophil progenitor cells, mast cell progenitor cells, fat cells, adipocytes, T cells, NK cells, B cells, dendritic cells (DCs), and / or NKT cells.

[0159] The cell therapy may be immunotherapy. The immunotherapy may be CAR-T cell therapy, TCR-T cell therapy, NK cell therapy, B cell therapy, DC cell therapy, CAR-NK cell therapy, TIL therapy and / or haematopoetic stem cell therapy.

[0160] The organoids (e.g. bone marrow organoids) of the invention may provide a method of producing hematopoietic and / or stromal cells. For example, the invention also provides a method comprising: a. driving induced pluripotent stem cells (iPSCs) to form mesodermal aggregates; b. inducing vascular and haematopoietic commitment of the mesodermal aggregates by further culturing the mesodermal aggregates in medium, supplemented with recombinant BMP4, FGF2, VEGFA, FLT3L and SCF; c. embedding the mesodermal aggregates in a hydrogel, and incubating the hydrogel in a sprouting medium comprising: a media suitable for stem cell maintenance supplemented with cytokines for differentiation, to form a mixture of hematopoietic and stromal cells.

[0161] The organoids of the invention may also facilitate the production of kits / reagents for the generation of the organoids (e.g. bone marrow organoids).

[0162] The organoids of the invention or disease models of the invention may be further treated, e.g. cryopreserved or frozen, such that they can be stored and distributed. Hence, the invention also provides a kit comprising organoids or disease models of the invention that are cryopreserved or frozen. The kit may comprise one or more, such as all of, recombinant BMP4, FGF2, VEGFA, FLT3L and SCF. The kit may comprise or consist of VEGFA and FGF2. The kit may comprise or consist of VEGFA, FGF2, BMP4 and VEGFC. The kit may comprise or consist of VEGFA, FGF2, SCF, TPO and FLT3L. The kit may comprise or consist of VEGFA, VEGFC, FGF2, SCF, TPO, BMP4 and FLT3L. The kit may comprise or consist of VEGFA, VEGFC, FGF2, SCF, TPO, EPO, BMP4, IL3, IL6 and FLT3L. Any kit may further comprise IL7 and / or calcium.

[0163] The kit may further comprise a set of instructions. The instructions will enable the reader to perform any method disclosed herein. The recombinant growth factors and / or cytokines may be provided in one solution in the kit, or two, three, four, five, six, seven or more separate solutions in the kit. In this way, one or more than one of the recombinant growth factors and / or cytokines may be provided in each solution, if required. The kit may also comprise a mesoderm-inducing medium, sprouting medium and / or hydrogel (fragmented or otherwise) required to carry out any method disclosed herein.

[0164] The organoids of the invention may be used as a model platform for target prioritization and validation of novel therapeutics. In line with identification of novel therapeutics, the organoids of the invention may be used for high throughput screening of drug candidates for disease, e.g. bone and / or bone marrow related conditions as described herein. The organoids may be monitored for their response to novel therapeutics, thereby identifying promising candidates for treatment of disease.

[0165] The organoids of the invention may be used as a pre-clinical model and alternative to animal systems. The organoids may provide a first model of a disease, with animal systems used only when promise is shown in the organoid model.

[0166] The organoids of the invention may be used for predicting outcome from stem cell transplantation or pharmacogenomic testing. The organoids may provide a model system to monitor organ / organ system response to foreign stem cell engraftment.

[0167] The organoids of the invention may be used as a model of a tumour microenvironment and / or to study metastatic disease, e.g. prostate metastasis to the bone.

[0168] The organoids of the invention may be used for providing a method of screening for agents capable of preventing or treating fibrosis, for example using the bone marrow organoid described herein, wherein the bone marrow organoid is genetically manipulated or treated with a potential agent before, during or after the bone marrow organoid is treated to induce fibrosis; and

[0169] A) determining if the potential agent or genetic target has any effect in inhibiting or preventing the development of fibrosis in the bone marrow organoid, or the reduction in fibrosis after it has developed in the bone marrow organoid;

[0170] B) determining if the potential agent or genetic target has any effect in inhibiting or reducing smooth muscle actin and / or collagen expression or other markers of fibrosis in the bone marrow organoid; and / or

[0171] C) determining if the potential agent or genetic target has any effect in inhibiting or reducing collagen deposition in the bone marrow organoid.

[0172] The bone marrow organoids of the invention may be used for one or more of:

[0173] A) determining if agent(s) or potential agent(s) have any effect in reducing the survival or proliferation of cancer cells from the patient;

[0174] B) determining if agent(s) or potential agent(s) have any effect on the pathogenicity of cancer cells from the patient;

[0175] C) determining if agent(s) or potential agent(s) have any effect on reducing the pathogenic remodelling of the organoid stroma or microenvironment / niche induced by cancer cells from the patient;

[0176] D) screening for potential biomarkers of cancer in the patient from whom engrafted cells are isolated;

[0177] E) studying clonal evolution of cancer in the organoids following seeding with cells from the patient, to predict future cancer progression in the patient donor;

[0178] F) studying treatment response of cancer cells seeded in the organoids to determine the optimal treatments for the donor patients;

[0179] G) studying the impact of a cancer on its microenvironment and / or niche; and

[0180] H) studying the role of the microenvironment and / or niche in initiating, or promoting cancer development and / or progression.

[0181] The organoids (e.g. bone marrow organoids) of the invention may also be used for providing a method for maintaining the viability of cells from a patient donor with blood cancer ex vivo, to enable mechanistic studies or screening of agents or potential agents consisting of one or more of:

[0182] A) determining if agent(s) or potential agent(s) have any effect in reducing the survival or proliferation of cancer cells from the patient;

[0183] B) determining if agent(s) or potential agent(s) have any effect on the pathogenicity of cancer cells from the patient;

[0184] C) determining if agent(s) or potential agent(s) have any effect on reducing the pathogenic remodelling of the organoid stroma induced by cancer cells from the patient;

[0185] D) screening for potential biomarkers of cancer in the patient from whom engrafted cells are isolated;

[0186] E) studying clonal evolution of cancer in the organoids following seeding with cells from the patient, to predict future cancer progression in the patient donor; and

[0187] F) studying treatment response of cancer cells seeded in the organoids to determine the optimal treatments for the donor patients.

[0188] The organoids (e.g. bone marrow organoids) of the invention also provide a model for bone marrow related conditions, including blood cancers, wherein the model comprises bone marrow organoids cultured with primary patient or donor cells which then reproduce features of disease within the organoid.

[0189] The agent to be screened or investigated may be dosed at a physiological relevant amount. The agent to be screened or investigated may be dosed at a therapeutically relevant amount. Combinations of agents may be investigated.

[0190] The determination may be relative to an untreated bone marrow organoid (i.e. not treated with the potential agent) and / or relative to a control or reference value.

[0191] The agent to be investigated may be a small molecule (e.g. less than 900Da), nucleic acid, antibody therapy, cellular therapy, drug compound, metabolite or peptide. The agent to be investigated may be a small molecule (e.g. less than 900Da), nucleic acid or peptide. The peptide may comprise or consist of an antibody. The agent to be investigated may be a genetic manipulation agent, such as siRNA, shRNA, CRISPR- CAS9, lentiviral or retroviral vectors, for example for over expression. Other

[0192] It is to be understood that different applications of the disclosed methods, organoids or compositions of the invention may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0193] In addition as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise.

[0194] Reference to the size of a cell aggregate herein is understood to be the average of the largest diameter of the aggregate. Aggregates are defined by clear edges and boundaries.

[0195] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0196] The following examples illustrate the invention. with reduced extracellular matrix i and increased to those bulk

[0197] The inventors compared the effects of using granular hydrogel or bulk hydrogel in a method of generating bone marrow organoids. The protocols of the two methods are provided in Example 6, and summarised in Figure 1.

[0198] Bone marrow organoids generated using bulk hydrogel or granular hydrogel were fixed, embedded in paraffin, sectioned and subsequently imaged. The granular hydrogelbased method consistently produced organoids that were round, smaller and more evenly formed than the bulk hydrogel method (Figures 2A and 2B). When quantitatively analysed, the bone marrow organoids generated by the granular hydrogel method exhibited more consistent diameter and structure (Figures 2C and 2D), which are key features for reproducible, scalable biological models. The smaller diameter of organoids generated from the granular hydrogel-based method will enable automated liquid handing, critical for large scale applications.

[0199] It was also observed that organoids generated from the bulk hydrogel-based method contained an excess of internalised hydrogel, spreading cells over the surface area and generating inconsistent architectures (Figure 3A). In comparison, organoids generated from the granular hydrogel -based method are consistently more round, with less incorporated ECM and thus an increased cellular density (Figure 3B). It was also observed that organoids generated from the granular hydrogel-based method has a significant increase in cellularity per organoid section compared with the organoids generated by the bulk hydrogel method (Figure 3C). This architecture and cellular organization will favor improved cell-cell communication and interaction, and allows for improved scalability due to the smaller size of the resulting, cell rich organoids.

[0200] Therefore, this Example shows that bone marrow organoids generated using granular hydrogel are of higher consistency, cellularity and are of a size amenable to automation, which greatly increases the utility and application potential as compared to the bulk hydrogel method.

[0201] Example 2 - Bone marrow organoids generated from the granular hydrogel method exhibit similar vascularity but enhanced ossification compared to those generated using bulk hydrogel

[0202] The architectural complexity of the bone marrow organoids generated by the bulk hydrogel method and the granular hydrogel method were assessed.

[0203] The bone marrow organoids were stained for CD34, which is a marker of progenitor cells of blood vessels and stromal tissues. Histological cross sections of the organoids were imaged.

[0204] Organoids generated from the bulk hydrogel -based method produced clear, CD34+ lumen forming vasculature into which cells egress, and the large vessels aligned with the fibers of the bulk hydrogel (Figure 4A). The organoids generated from the granular hydrogel method also generated CD34+ lumen forming vessels, with cells interacting with and egressing into the vascular space (Figures 4B and 4C). Interestingly, the vessels of the organoids generated from the granular hydrogel method were typically smaller. Quantification of the total CD34+ vessel area across organoids generated from the bulk hydrogel method and the granular hydrogel method revealed that there was no loss of vascularity in the organoids generated from the granular hydrogel method (Figure 4D).

[0205] The bone marrow organoids were also stained for Von Kossa, which is a specific, calcification stain used to highlight areas of calcium / mineral deposition, indicating successful mineral deposition and the development of an ossified niche. Dark, positive Von Kossa staining was completely negative in organoids generated by the bulk hydrogel method (Figure 5A) whereas this staining was observed in organoids generated by the granular hydrogel method (Figure 5B).

[0206] Generating a bone rich or endosteal, ossified niche ex vivo has been a significant challenge and a limitation of the methods in the art. The observed positive Von Kossa staining in organoids generated from the granular hydrogel method provides the first evidence of mineralization in complex, vascularized hematopoietic organoids and is a significant advance towards creating a scalable, biomimetic environment for the study of this organ, and its application as an ex vivo model. The use of granular hydrogel is particularly effective because (a) microparticles ensure that materials are bioavailable (and it has been shown that fibrin and collagen I are required for ossification in vitro) and (b) granular hydrogel generated organoids are smaller and more cellular, facilitating more cellcell interactions and driving niche changes.

[0207] Example 3 - Bone marrow organoids generated from the granular hydrogel method comprise key haematopoietic and stromal lineages and display strong lineage complexity

[0208] Having established that granular hydrogel can be used to generate vascularised 3D cultures of a consistent size, shape, and cellularity using a simplified one step method, with evidence of mineral deposition that supports the development of an ossified niche, the cultures were then differentiated to establish lineage complexity of the organoids.

[0209] Bone marrow organoids generated in parallel using either the bulk hydrogel method or the granular hydrogel method were compared by flow cytometry. Three independent differentiations were performed with organoid dissociated at day 35 for flow cytometry. Four flow cytometry panels were used to identify cells of the stromal and haematopoietic lineages, with three panels used to identify lymphoid, myeloid, and stem cell haematopoietic cells respectively.

[0210] The stromal cell panel revealed no significant differences in cell diversity at the stromal level, with key populations identified using both methods (Endothelial, Fibroblast, BM MSC, LEPR+ BM MSC, and Osteolineage cells) (Figure 6A). The lymphoid cell panel again showed the production of Natural Killer (NK), B Lineage, and T-Cell progenitor (thymocyte populations) using both methods (Figure 6B). The myeloid panel revealed erythroid cells, megakaryocytes, myelomonocytic cells as well as basophils and eosinophils, with a slight reduction in the percentage of megakaryocytes observed (Figure 6C). Finally, a dissection of stem cell populations using a stem cell specific panel revealed no significant differences in the fractions of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), Common Myeloid Progenitors (CMPs) and myelo -erythroid progenitors (MEPs) generated by each method (Figure 6D). Interestingly the variation across repeats in the HSC compartment is observably smaller in the granular hydrogel generated samples suggesting increased reproducibility in this compartment using the granular hydrogel approach.

[0211] Therefore, this Example shows that the granular hydrogel method successfully generates bone marrow organoids comprising key haematopoietic and stromal cell lineages.

[0212] Example 4 - Bone marrow organoids generated from the granular hydrogel method support the engraftment of donor cells

[0213] To test the utility of bone marrow organoids generated from the granular hydrogel method as a platform for personalised or precision medicine, the capacity of the organoids to support engraftment of donor cells was tested.

[0214] Bone marrow organoids were fabricated as described in Examples 1-3, and seeded with fluorescently labelled BAF3 cells, a B-lineage haematopoietic cell line. It was observed that, after 48 hours, donor cells homed into and populated the volume of the bone marrow organoids generated from the granular hydrogel method (Figure 7). This data show that this system creates an environment into which donor cells will migrate and survive.

[0215] Furthermore, CD34+ cells from a patient with myelofibrosis were labelled with a fluorescent dye (CellTrace Violet) and engrafted into bone marrow organoids generated from the granular hydrogel method. After incubation for 7 days (from day 21 to day 28 of culture) before fixation and subsequent imaging using a confocal microscope, it was clear that the adult donor derived cells had engrafted and proliferated in the organoids (Figure 8).

[0216] Therefore, this Example shows that bone marrow organoids generated from the granular hydrogel method can successfully support the engraftment of donor cells, e.g. for use as a platform for personalised or precision medicine.

[0217] Example 5 - Bone marrow organoids generated from the granular hydrogel method can be used to capture disease specific remodelling of the niche

[0218] The bone marrow organoid generated by the granular hydrogel method and the bulk hydrogel method were assessed for their usefulness as a tool for capturing disease specific remodelling of the niche.

[0219] Bone marrow organoids generated from the bulk hydrogel method and the granular hydrogel method were engrafted with healthy donor or myelofibrosis patient CD34+ cells that had been labelled with CellTrace violet at day 21 of culture and grown for a further 7 days before fixation and immunofluorescence imaging. The samples were imaged for CellTrace violet, aSMA (a marker of fibroblast activation, and hence fibrosis) and collagen type 1.

[0220] Figure 9 shows that there is an increase in aSMA staining in MF-engrafted organoids compared to healthy donor-engrafted organoids from granular hydrogels, which is at least as efficient as what was observed using organoids generated using the bulk hydrogel method (Figure 9A & 9B).

[0221] Therefore, this Example shows that bone marrow organoids generated from the granular hydrogel method are capable of supporting engraftment of primary cells from patients and of undergoing stromal activation, as indicated by aSMA expression, in response to cell engraftment.

[0222] Example 6 - Materials and methods

[0223] The following material and methods are applicable to Examples 1 to 6. iPSC Culture and. Maintenance

[0224] A human induced pluripotent stem cell line (iPSCs) bought from Gibco (Thermo) was maintained on GelTrex (Thermo) coated 6- well tissue culture plates (Corning). Cells were sub-cultured at approximately 70% confluence using an ethylenediaminetetraacetic acid EDTA (clump passaging) method of detachment. Briefly, cells were washed once with ImL phosphate buffered saline (PBS), and then once with EDTA before a 4-minute incubation at 37°C and 5% CO2. The EDTA was then aspirated, and cells are subsequently removed by pipetting StemFlex media against the bottom of the well. The resulting cell suspension is triturated before replating at the required concentration in freshly coated plates.

[0225] Differentiation Protocol for Granular Hydrogel Method

[0226] A directed differentiation approach was used to generate bone marrow organoids. First, iPSCs were grown to 70-80% confluence before EDTA detachment as described above. Cells were triturated twice before resuspension in StemFlex media supplemented with RevitaCell (Thermo) as per manufacturer’s recommendations. The resulting aggregates were then cultured in 6-well ultra low attachment (ULA) plates (Thermo - Nunc Sphera plates) and incubated overnight (12 hours) at 37°C and 5% CO2.

[0227] Aggregates were collected using a Pl 000 pipette, transferred to 15-mL conical tubes (Coming) by low speed (300G) centrifugation the following day (day 0). The supernatant was aspirated, and aggregates were resuspended in Phase I medium (APEL2 supplemented with 50ng / mL Bone Morphogenic Protein-4 (BMP4) (PeproTech), Fibroblast Growth Factor-2 (FGF2) (PeproTech) and Vascular Endothelial Growth Factor A (VEGFA-165) (PeproTech) and 4uM CHIR99201) and incubated for 72 hours (from day 0 to day 3) at 37°C and, 5 02%, 5% CO2. At 72 hours, mesodermal aggregates reached an average size of approximately 200-250um.

[0228] After 72 hours, cells were collected via gravitation in 15mL- conical tubes. Supernatant was aspirated before aggregates were resuspended in Phase II medium. Phase II media was comprised of APEL2 with 50ng each BMP4, FGF2, and VEGFA, supplemented with 25ng Fms Related Receptor Tyrosine Kinase-3 ligand (FLT3L) and stem cell factor (SCF) (25ng / mL) (PeproTech). Cells were cultured for a further 48 hours at 37°C and, 5% O2, 5% CO2or until they achieved an average size of 350-500um, under these conditions (typically d5).

[0229] The next stage of the protocol involved the preparation of granular hydrogel and embedding the iPSC aggregates within that structure.

[0230] First a hydrogel comprised of 40% (vol / vol) reduced growth factor Matrigel (Coming), 1.25mg / mL Fibrinogen (Thermo), 2mg / mL VitroCol (Collagen Type I, Advanced Biomatrix), and Img / mL Collagen Type IV (Advanced Biomatrix) was prepared on ice. IM NaOH was added to neutralise the resulting mixture, and 2U / ML thrombin (Thermo) was also added to drive fibrin formation. The hydrogel was carefully mixed on ice, before transfer to a sterile sealed syringe. The hydrogel was left to set at 37°C and 5% CO2for 90 minutes before further processing.

[0231] At 90 minutes, the hydrogel was fully set and ready for fragmenting to a resuspended fragmented hydrogel. First, the hydrogel was extruded through the syringe into 2mL of APEL2 in a 6- well ULA plate. An 18G needle was affixed to the syringe, and the extruded hydrogel, resuspended in APEL2, was taken up into the syringe and extruded through the 18G needle three times. A fresh syringe was prepared with a 23G needle, and the hydrogel / media suspension again passed into and out of the syringe and 23G needle three times. The resuspended hydrogel was visibly broken down into a microgel or microparticle suspension. The above steps were repeated with a 25G and then 27G needle, before collecting the fragmented hydrogel in a 15-mL conical tube. The ULA well plate was washed with 3mL APEL2 to collect any residual microgel particles and this media collected in the same 15-mL tube containing the fragmented gel material. The resulting resuspended fragmented gel resided in a final volume of 5mL. The fragmented gel solution was supplemented with 5% Knock-Out Serum (Thermo), 5U / mL heparin (Merck), lOOng / mL VEGFA, 50ng / mL VEGFC, BMP4, FGF2, SCF, FLT3L and 20ng / mL EPO, TPO, IL3, and IL6.

[0232] Cell aggregates were collected in a 15-mL conical tube by gravitation. Aggregates were visible by eye. The supernatant was aspirated, and the cell mixture resuspended in the prepared fragmented hydrogel in APEL2. The resulting cell, cytokine, and fragmented gel suspension was placed in a reagent reservoir and, using a multi-channel pipette, 50uL of this suspension placed in each well of a 96-well ULA.

[0233] Once all wells were filled to maintain sterility and avoid accidental exposure and spillage, the plate was sealed with parafilm. The above process can be repeated to generate multiple well-plates. Using a plate centrifuge, the suspension was spun down to form compacted granular hydrogels by centrifugation at 300G for 4 minutes. The parafilm was subsequently removed.

[0234] The gels were checked to confirm compaction using a bench top brightfield microscope. In particular, it was checked that a solid structure was formed encapsulating the cell aggregate. The mixture was incubated at 37 °C and, 5% O2, 5% CO2 for 48 hours. After 48 hours, at day 7, a further 50uL / well of APEL2 supplemented with 5% KnockOut Serum, 5 U / mL Heparin, 50ng / mL VEGFA, 25ng / mL FGF2, BMP4, VEGFC, SCF, FLT3L, and lOng / mL EPO, TPO, IL3 and IL6 was added. Cells were then incubated at 37°C and, 5% O2, 5% CO2.

[0235] At day 10, myeloid factors were reduced and cultures spiked with IL7 to drive lymphoid development. APEL2 with 50ng / mL each of VEGFA, VEGFC, FGF2, hSCT, FLT3L, and IL7, as well as lOng / mL IL3 and IL6 were added at the 50:50 ratio previously described.

[0236] Cells were maintained until day 21 at 37 °C and, 5% O2, 5% CO2. Media changes were performed every 48-72 hours as 50 / 50 media changes (50uL aspirated, and 50uL added) from day 12 onwards in APEL2 completed with L-Ascrobic Acid (10 ug / mL final concentration), ImM Calcium Chloride, 2mM B-glycerolphosphate pentahydrate, 2% KnockOut Serum and 2% Chemically Defined Lipids (Thermo). Media supplementation with growth factors was performed as follows: Day 12: 20ng / mL each VEGFA, VEGFC, FGF2, SCF, FLT3L, IL7 and lOng / mL IL3, IL6.

[0237] Day 14, 17, 19 and 21: 20ng / mL SCF, FET3E, IE7, lOng / mE IE3, IE6, 5ng / mE mCSF and EPO, and finally Ing / mE TPO.

[0238] At day 21, samples were optionally moved to and maintained in normoxic 20% O2 conditions to drive cell proliferation. From day 21 onwards, cells were also cultured in supplemented StemPro-34 media (with added E-Glutamine (1%), E-Ascrobic Acid (10 ug / mE final concentration), ImM Calcium Chloride, 2mM B-glycerolphosphate pentahydrate, 2% KnockOut Serum and 2% Chemically Defined Lipids). On day 21 and 24 medium were supplemented with reduced growth factors (lOng / mL hSCF, FLT3L, IL7, 5ng / mL mCSF, IL3, IL6, EPO, and Ing / mL TPO).

[0239] From day 26 to day 33, modified StemPro-34 was supplemented with 5ng / mL EPO and IL7, and Ing / mL TPO. From day 33 onwards, only 5ng / mL EPO and Ing / mL TPO were supplemented to model growth physiological exposure of exogenous factors to the bone marrow.

[0240] Differentiation Protocol for Bulk Hydrogel Method

[0241] The iPSC differentiation protocol for generation of bulk hydrogel derived organoids mirrored that for the granular hydrogel derived organoids from dO to d5. At d5 in both protocols, cells were prepared for embedding in the hydrogel and the hydrogels themselves prepared. The protocol below follows the bulk hydrogel method from around d5, which differs to that for the granular hydrogel method.

[0242] Initially, different compositions of hydrogels were tested to determine the best conditions for the generation of myeloid and bone marrow specific lineages. Each matrix was comprised of 40% reduced growth factor Matrigel (Coming) and 60% either Collagen Type I or Collagen Type IV (Cell Systems), or a mixed Collagen I Collagen IV gel. All gels were prepared with Collagen at a concentration of Img / mL. Gel preparation began on d4 when Matrigel aliquots were thawed overnight at 4°C. Gel mixtures were prepared on ice, with Collagen mixes neutralised with IM NaOH prior to distribution in 12 well cell culture plates. Each hydrogel was allowed a minimum of 90 minutes to polymerise. An initial cell free layer was prepared before cells were collected by gravitation and resuspended in the remaining gel volume.

[0243] Once fully polymerised, 3D cultures were supplemented in Phase III sprouting medium which was comprised of APELII medium supplemented with 5% Foetal Bovine Serum (FBS), 5U / mL Heparin Sulfate, 50ng VEGFA, lOng each Interleukin 3 (IL3) and Interleukin 6 (IL6), as well as 25ng each of SCF, FLT3L, Thrombopoietin (TPO), Erythropoietin (EPO), Granulocyte Colony Stimulating Factor (G-CSF) (Stem Cell Technologies), FGF2, and BMP4. Cells were maintained in this media formulation until dl2, with media changes every 72 hours. From d5 onwards, media changes were performed as 60:40 splits fresh: conditioned medium. For experiments developing sinusoid specific endothelial vasculature Vascular Endothelial Growth Factor C (VEGFC, Stem Cell Technologies) was supplemented into d3 media.

[0244] Cultures were allowed to sprout until an optimal size of between 800um and 1.5mm was observed, typically between dlO-12. At this stage, sprouted bone marrow organoids were extracted from hydrogels and cultured individually in 96-well ultra-low attachment dishes. Organoids were harvested first by scraping with a sterile cell scraper, before pipetting in an excess of media into a 15mL Falcon (Coming). Samples were then spun down at 500G for 5 minutes to separate organoids from both the media and collagen. The free organoids were then resuspended in the desired volume of media and collected for individual culture in 96- well ultra-low attachment plates.

[0245] From dl2, bulk hydrogel derived organoids were maintained in parallel with granular hydrogel derived organoids.

[0246] Histology

[0247] Organoids were fixed in 4% paraformaldehyde (PFA) in a 15mL Falcon tube, washed three times with PBS, and then subject to serial dehydration (30%, 50%, 70%, 100%) in ethanol before immersion in Histoclear (Geneflow, Cat#A2-0101). Samples were then embedded in paraffin, and where necessary stained and mounted by C&C laboratories. Histological preparations were imaged using a Zeiss AxioScan.Zl slide scanner. Flow Cytometry

[0248] The organoids were prepared for flow cytometry Following digestion and dissociation, samples were dissociated using Collagenase Type B (Sigma) at 20mg / mL in sterile HEPES. Samples were collected by gravitation in a 15mL falcon tube before washing first in in 10 mL PBS. Once washed, samples were incubated in the prepared Collagenase solution at 37°C for 10 minutes before complete dissociation via tituration. Single cell suspensions were washed, spun at 500G and blocked in 2% fetal bovine serum (FBS) in PBS for 15 minutes (on ice) before labelling with flow cytometry antibodies.

[0249] Stromal cells were identified as CD45-, CD71- and CD235- cells using an optimised flow cytometry panels. Haematopoietic cells were identified within a CD45+ gate, with the exception of erythroid cells which were identified as CD71+ / CD235+.

[0250] Microscopy

[0251] Immunofluorescence was performed using a Zeiss LSM88O confocal microscope (lOx air objective). Samples were prepared first by fixation in 10% formalin, before 3x 5min successive washes with PBS. Samples were then blocked overnight in a detergent blocking solution comprised of 2% goat serum (Thermo), 1% Bovine Serum Albumin (BSA) in 50mL of PBS. 250pl of Triton X100 and Tween-20 (Sigma) were added, as well as 500pl sodium deoxycholate (Sigma) to permeabilise samples and encourage antibody preparation. Primary antibody incubations were performed overnight at 4°C, before 4x 5- minute successive washes in PBS. Samples were then incubated overnight once more at 4°C in secondary antibody mixes: DAPI for nuclear labelling, Alexa-488, Alexa-568, and Alexa-647 (Thermo) depending on the species mix of antibodies present. For example, this protocol was used to stain Collagen I and aSMA in whole organoid labelling.

[0252] Once labelled whole organoids were embedded in a small volume of 0.5% agarose in Ibidi 8-well slides (Ibidi). Once cooled, this gel was subject to a progressive dehydration by a series of Ethanol washes adjusted to pH 9. Finally, the sample was dehydrated completely in absolute Ethanol before clearance with Ethyl Cinnamate (SLS). At this stage samples were ready for confocal imaging. Seeding of organoids with primary cells from adults

[0253] CD34+ hematopoietic cells from healthy donors and myelofibrosis patient samples were used and labelled with a fluorescent dye (CellTrace Violet). Organoids were seeded with 5000 cells per wells and cultured for 8-14 days in StemPro. On collection day, organoids were fixed for imaging or digested for assessment by flow cytometry.

[0254] BAF3 cell engraftment

[0255] The BAF3 cell line used for engraftment experiments and were stably transduced with a fluorescent marker for easy identification.

[0256] CellTrace labelling for engraftment assays

[0257] Patient cells were labelled with CellTrace Violet as indicated by the manufacturer. Briefly, cells were washed IX with PBS and resuspended at IxlO6cells / mL in staining solution (CellTrace Violet 2pM in PBS). Cells were incubated in staining solution for 30min at 37°C. After incubation, CellTrace was quenched with 5 volumes of PBS with FBS (10%), spun down and resuspended in the appropriate media.

[0258] Example 7 - Single cell RNA sequencing and analysis of granular vs. bulk hydrogel derived bone / bone marrow organoids

[0259] Organoids generated using both bulk and granulated hydrogels were subject to single cell RNA sequencing on day 35 of the differentiation protocol as described in Example 6. Single cell annotation identified and confirmed cell types observed using flow cytometry (Figure 10) across both granular and bulk hydrogel derived organoid samples.

[0260] Surprisingly, while similar cell types were produced, a notable increase in the expression of extracellular matrix (ECM) proteins native to the bone marrow was observed across the stromal compartment (Figure 11). Figure 11 shows that key ECM proteins known to be essential to the native biology of the bone marrow, collagen types I and III (COL1A1, COL3A1), fibronectin and periostin are significantly upregulated in the relevant stromal cells (fibroblasts, MSCs, osteolineage cells) generated in granular hydrogel- derived organoids when compared to the bulk method. Hence, organoids generated by the granular hydrogel-based method better mimic the native expression of key ECM proteins (Bandyopadhyay, Shovik et al., (2024), Cell, Volume 187, Issue 12, 3120-3140. E29), facilitating the improved mineralisation observed (Figures 5 and 17).

[0261] ECM protein expression is subject to negative feedback loops, wherein excess ECM in the environment suppresses cellular expression of these protein, hence limiting their development and sensitivity to modelling changes in ECM expression as a consequence of disease (e.g. fibrosis). An increase in bone marrow ECM proteins in organoids grown in a granular hydrogel was observed, indicating that the excess hydrogel presented by the bulk hydrogel approach likely inhibits the cellular expression of ECM proteins.

[0262] Therefore, this Example shows that organoids generated in a granular hydrogel better express native ECM proteins better mimicking the physiological behaviour of these cells, and the organoid in general, when compared to the bulk hydrogel approach. Similarly, this is likely to enable better disease modelling where ECM proteins are involved (e.g. fibrosis).

[0263] Example 8 - Engraftment of organoids with primary multiple myeloma samples to model disease

[0264] Multiple myeloma is an exemplar blood cancer in which malignant plasma cells hijack the bone marrow to support the expansion of the diseased cells at the cost of normal haematopoiesis. The disease typically involves a loss of normal myeloid cell production as the plasma cells drive local inflammation, a remodelling of the bone rich niche (a loss of osteoblast formation in favour of bone resorbing osteoclasts), and an increase in bone marrow adiposity. This disease has been very difficult to model using conventional tools, with challenges including the lack of mouse models that capture disease heterogeneity, and the difficulty of culturing multiple myeloma cells ex vivo.

[0265] The inventors demonstrate the utility of the granular hydrogel derived bone marrow organoids to both support the growth of multiple myeloma cells and capture the complexity of the remodelling of the bone marrow niche that is a consequence of this disease. CD 138+ plasma cells from 3 patients with myeloma were labelled with CellTrace Violet and 20,000 cells were added to each organoid on day 21 of differentiation. In parallel, healthy donor CD34+ derived stem / progenitor cells were cultured within the organoids for 14 days (Figure 12a), with images taken at regular intervals to ensure that cells successfully engrafted the organoid (Figure 12b). At day 14 post-engraftment, organoids were dissociated and collected for single cell RNA sequencing, cytospin and histology. Cytospin of dissociated organoid samples (Figure 12c) confirmed the presence of plasma cells in all multiple myeloma engrafted samples, but not in healthy donor controls. Similarly, histology of organoids at day 35 (Figure 12d) confirmed myeloma cells within organoids engrafted with myeloma samples.

[0266] Single cell RNA sequencing was performed on organoids at day 35 of differentiation. Unengrafted and healthy donor engrafted organoids were used as controls and generated in parallel to multiple myeloma engrafted organoids (3 myeloma patients). Analysis was performed using the Seurat package to identify and call cell types on the basis of canonical gene expression. In total, 45,706 haematopoietic cells and 14,577 stromal cells were identified from the 6 samples. Haematopoietic cells included a full myeloid series (erythroid, MK, Eo / Baso / Mast, mono / mac, dendritic cells) and lymphoid compartment (T lineage, patient derived plasma cells, lymphoid cells). These included a patient derived plasma cell cluster as indicated by a red circle on the plot (Figure 13a). Stromal cells included endothelial, osteolineage, adipolineage, fibroblast and mesenchymal stem cells (Figure 13).

[0267] Gene set enrichment analysis (GSEA) comparing multiple myeloma engrafted organoids to controls demonstrated an increase in inflammatory response genes (interferon alpha, interferon gamma, TNF alpha / NFkB, IL2 / IL6) in key stromal cell types (CD271+ MSC, osteolineage cells, osteoblasts) and inflammatory haematopoietic cells (macrophages and dendritic cells) (Figure 14).

[0268] Violin plots of specific genes known to be heavily involved in the pathogenesis of multiple myeloma demonstrated a canonical increase in TNF, S100A4, S100A8, and S100A9 in key haematopoietic cells (macrophages, dendritic cells, and neutrophils (Figure 15a). Similarly, patient cells drove canonical changes in stromal behavior that reconstitute the complexity of the human disease. Multiple myeloma engrafted organoids demonstrated an increase in B2M a factor critically linked to myeloma, though the specific cells of origin remain unknown. Finally, osteoblasts in multiple myeloma engrafted organoids demonstrated a significant reduction in the expression of NF1A and NFIB transcription factors that are essential to osteoblastogenesis (Figure 15b).

[0269] Therefore, this Example shows an extensive and remarkable remodelling of the bone marrow microenvironment as a consequence of engraftment with multiple myeloma cells. Remarkably, changes in stromal cell metabolism, cell fate, and notable inflammation are observed faithfully capturing the multilineage and highly complex features of the disease. This data is the first of its kind and offers, for the first time, a viable preclinical model of a complex multilineage disease like multiple myeloma.

[0270] Example 9 - Generation of blood cells from bone marrow organoids

[0271] The primary function of the bone marrow is the production of the huge range of blood and immune cells required for healthy haematopoiesis. The inventors’ bone marrow system is capable of producing an unprecedented range of different haematopoietic cell types. To determine whether this approach could feasibly produce cells for therapeutic use e.g. cell therapy, the inventors devised an experimental workflow whereby bone marrow organoids were mounted on a rocker (Figure 16a, modelling flow through a single phase of oscillation) to induce fluid flow through the volume of the organoid.

[0272] This agitation was enough to drive the egress of rounded haematopoietic cells over the course of the following 14 days (days 21-35) (Figure 16b, 16c & 16d). Released cells (Figure 16d) were collected for cytospin and annotated (Figure 16e). Therefore, this Example shows that the bone marrow organoids prepared by the methods described herein are particularly useful for producing blood and immune cells for cell therapy.

[0273] Example 10 - Materials and methods

[0274] The following material and methods are applicable to Examples 7-9.

[0275] Single cell RNA sequencing and analysis Cells generated using a healthy donor-derived hiPSC stem cell line (Gibco, Life technologies) were subject to both the bulk and granular hydrogel bone marrow organoid differentiation protocol as described in Example 6. On day 35, cells were dissociated using collagenase IV before single cell sorting into 0.1% bovine serum albumin (BSA) in phosphate buffer saline (PBS). In total cells were collected from 4x independent differentiations and cryopreserved prior to processing. 100,000 live cells as determined by 7AAD staining were sorted. 35,000 live cells were then taken and subject to GEM preparation using a 3’ High Throughput (3’ HT, v3.4 chemistry 10X genomics) kit as described by the manufacturer’s protocol. The resulting emulsion was then subject to library preparation and sequencing.

[0276] Data analysis was performed using the standard lOx genomics pipeline. Briefly, demultiplexed sequencing data was analysed using CellRanger (v7.1) before preprocessing using CellBender (vO.3.2) to denoise the resulting count matrices. Further analysis was applied using the Seurat Pipeline (v 5) to integrate, cluster, annotate and perform differential gene expression analysis.

[0277] Organoid engraftment experiments (e.g. to model multiple myeloma)

[0278] Organoids were generated using a granular hydrogel approach as described. At day 21 of the differentiation protocol as described in Example 6, cryopreserved primary CD138+ enriched and CD34+ healthy donors were thawed and resuspended at 20,000 and 5000 cells / 50uL volume each. 50uL of each cell solution was then added to each individual organoid in a 96-well culture plate.

[0279] After the addition of exogenous cells, plates were maintained with regular 50:50 media changes at 72 hour intervals. Media was comprised of StemPro-34 (Thermo Scientific) supplemented with pen / strep (1%), IL7 lOng / mL, EPO 5ng / mL and TPO Ing / mL. At day 14 post engraftment, 12 organoids were fixed for paraffin embedding and histology, and approximately 20 organoids were dissociated for cytospin and single cell RNA sequencing as described in the previous section.

[0280] Cell agitation and collection: To establish whether the organoid system could function as a source of released blood cells, individual organoids were cultured in an ultra- low attachment plate (6-well, NUNC Sphera) and placed on an incubator rocker (Mimetas) set to 15 degree oscillations over 5 second intervals. Cells were collected from the ‘releasate’ in each well at regular intervals and subject to Giesma staining and cytospin.

Claims

Claims1. A method of generating organoids comprising culturing cells in a suspension of hydrogel microparticles.

2. The method of claim 1, wherein the hydrogel microparticles have a mean diameter of 10-500 pm.

3. The method of claim 1 or claim 2, wherein the hydrogel microparticles are obtained by fragmenting bulk hydrogel, optionally by chemical and / or mechanical fragmentation.

4. The method of any one of the preceding claims, further comprising compacting the hydrogel microparticles to generate a granular hydrogel, optionally by centrifugation or vacuum-driven filtration.

5. The method of claim 4, wherein the cells are added before compacting the hydrogel microparticles.

6. The method of claim 4 or claim 5, wherein the cells are added after compacting the hydrogel microparticles.

7. The method of any one of the preceding claims, comprising creating multiple layers of compacted mixture comprising hydrogel microparticles and cells by repeating the steps recited in claims 4 and 5.

8. The method of any one of claims 4 to 7, wherein the compacted mixture comprises about >0.1% w / v, e.g. 0.1 % to 60 % w / v, hydrogel microparticles.

9. The method of any one of the preceding claims, wherein the hydrogel microparticles comprise polypeptide chains, polysaccharide chains, and / or extracellular10. The method of any one of the preceding claims, wherein the hydrogel microparticles comprise: (a) a mixture of collagen, such as collagen I and / or collagen IV, and / or (b) fibrin, for example, Matrigel, fibrin, collagen type I and collagen type IV.

11. The method of any one of the preceding claims, wherein the cells are induced pluripotent stem cells (iPSCs) or cells differentiated therefrom such as mesoderm aggregates.

12. The method of any one of the preceding claims, wherein the organoids are bone marrow organoids.

13. A method of generating bone marrow organoids from pluripotent stem cells, e.g. iPSCs, and the method comprises:(i) driving the pluripotent stem cells, e.g. iPSCs, to form mesodermal aggregates;(ii) culturing the mesodermal aggregates from (i) into hematopoietic and vascular lineages in a medium comprising haematopoietic and stromal support cytokines, such as BMP4, FGF2, VEGFA, SCF and FLT3L; and(iii) adding the mesodermal aggregates from (ii) to a suspension of hydrogel microparticles; and(iv) culturing the mixture from (iii) to form bone marrow organoids.

14. The method of claim 13, wherein step (i) comprises culturing the iPSCs to induce formation of iPSC aggregates, and culturing the iPSC aggregates in a media for stem cell maintenance supplemented with BMP4 to induce formation of mesodermal aggregates.

15. The method of claim 13 or claim 14, wherein step (ii) comprises culturing the mesodermal aggregates for a period until the mesodermal aggregates have an average size of about 100-500 pm and / or the presence of markers of early endothelial and haematopoietic differentiation.

16. The method of any one of claims 13 to 15, wherein step (iii) comprises culturing the mesodermal aggregates in a suspension of hydrogel microparticles as described in the method of any one of claims 1 to 12.

17. The method of any one of claims 13 to 16, wherein step (iv) comprises culturing the mixture in a medium comprising VEGFA, FGF2, BMP4, SCF, FLT3L, IL3, IL6, cGSF, EPO and / or TPO.

18. The method of any one of claims 13 to 17, further comprising: (v) adding further cells to the bone marrow organoids from step (iv), optionally wherein the further cells are cells obtained from a subject, e.g. a subject with a bone marrow pathology.

19. Organoids obtainable or obtained by the method of any of the preceding claims, optionally wherein the organoids are bone marrow organoids.

20. Organoids of claim 19, wherein the bone marrow organoids comprise:(a) a lumen forming vasculature network;(b) stromal cells comprising mesenchymal stem cells (MSCs), fibroblast, and / or endothelial cells;(c) haematopoietic cells comprising haematopoietic stem and progenitor cells (HSPC), erythroid cells myelomonocytic cells, and / or megakaryocytic cells; and(d) osteolineage cells; and optionally (e) mineral deposits.

21. Bone marrow organoids comprising:(a) a lumen forming vasculature network, optionally wherein the vasculature network is comprised of subsets of endothelial cells specific to the bone marrow (e.g. arteriolar, sinusoidal, Type H capillaries);(b) stromal cells comprising mesenchymal stem cells (MSCs) and osteo / adipo / CAR lineage cells differentiated from MSCs, fibroblast, and / or endothelial cells;(c) haematopoietic cells comprising haematopoietic stem and progenitor cells (HSPC), haematopoietic stem cells (HSCs), and cells of the myeloid and lymphoid lineages (including but not limited to T-lineage progenitors, B-lineage cells, natural killer cells, erythroid, eo / baso / mast, megakaryocytic, and myelomonocytic cells); and(d) osteolineage cells; and optionally (e) mineral deposits; and optionally wherein the bone marrow organoids are obtainable or obtained by the method according to any of claims 1 to 17, and / or wherein the bone marrow organoids have a mean vascularised area of 5% or more.

22. Use of the bone marrow organoids of any one of claims 19 to 21 as a model for studies into healthy, ageing and diseased haematopoietic and stromal cell biology, for studies of immune response to infection and inflammation.

23. A method of making a bone marrow organoid model of disease, comprising seeding donor cells onto the bone marrow organoids of any one of claims 19 to 21, optionally wherein the donor is an individual with cancer, e.g. blood cancer.

24. A disease model obtained or obtainable by the method of claim 23.

25. A model for bone and / or bone marrow related condition, such as bone marrow fibrosis, wherein the model comprises bone marrow organoids of any one of claims 19 to 21 that have been treated with an agent to induce said condition.

26. Use of the model of claim 25 to identify agents capable of preventing or treating the disease, comprising treating the model before, during or after induction of the disease.

27. Use of a composition comprising a suspension of hydrogel microparticles to generate organoids, wherein the hydrogel microparticles are optionally compacted to form a granular hydrogel, and optionally wherein the organoids are bone and / or bone marrow organoids.

28. A method of producing cells for cell therapy, wherein the method comprises bone marrow organoids generated by the method of any one of claims 1 to 18 or claim 23, and wherein the method further comprises collecting cells released from the bone marrow organoids.

29. The method of claim 28, wherein the released cells are blood and / or immune cells, optionally wherein the blood and / or immune cells are haemopoietic stem cells, histeocytes, macrophages, megakaryocytes, neutrophils, monocytes, myeloid progenitor cells, erythroid progenitor cells, mature erythrocytes, platelets, eosinophil progenitor cells, basophil progenitor cells, mast cell progenitor cells, fat cells, adipocytes, T cells, NK cells, B cells, dendritic cells (DCs), and / or NKT cells.

30. Cells for cell therapy obtained or obtainable by the method of claim 28 or claim 29.

31. A method of identifying an agent for use in treating or preventing a disease, comprising: adding an agent to a disease model comprising organoids of any of claims 19 to 21 engrafted with diseased cells of a patient, wherein an improvement in the disease state of the disease model is indicative of the effectiveness of the agent in treating or preventing said disease; optionally wherein the method further comprises adding the agent to a reference model comprising organoids of any of claims 19 to 21 engrafted with healthy cells, and comparing the effects of the agent on the reference model and the disease model.

32. The method of claim 31, wherein the disease is a bone and / or bone marrow related condition, such as myelofibrosis, bone marrow fibrosis, or blood cancers, e.g. multiplemyeloma, myeloma, solid tumour metastases, leukaemia, (e.g. acute or chronic lymphoblastic leukaemias, or acute or chronic myeloid leukaemias), myelodysplastic syndrome, myeloproliferative neoplasms, lymphomas, or mast cell neoplasms.