Method for generating organoids in hydrogel microparticles

Abstract

The present invention relates to the generation of organoids using particulate hydrogels, which are compacted assemblies of hydrogel microparticles.

Description

Technical Field

[0001] This invention relates to organoids, methods for generating organoids, and their uses. Background Technology

[0002] Physiologically relevant human tissue models are crucial for translational research, including the discovery and validation of new therapies.

[0003] The emergence of organoids as an alternative to human models has the potential to solve this problem. An organoid is an in vitro 3D organ-type culture that captures the structural, cellular, and molecular features of a target organ or tissue. Organoids are also known in the field as microphysiological systems or organ-on-a-chip. Organoids have demonstrated transformative potential in several different fields, including cardiovascular, neurological, and cancer research. However, in the field of bone and bone marrow-related diseases, progress has been limited in generating culture systems that faithfully capture the structural and cellular complexity of the bone and bone marrow microenvironment.

[0004] Creating organoids that capture the complexity of bone and bone marrow in vitro remains challenging due to the intricate arrangement of various cell types within their microenvironments. For example, the central myelogenic bone marrow is thought to consist primarily of sinusoidal endothelial cells and arteriolar endothelial cells that support the generation of myeloid lineage cells. Conversely, the endosteal niche, with its own specific vascular cells (e.g., H-type capillaries), is thought to maintain the generation of lymphoid progenitor cells and quiescent, long-lived hematopoietic stem cells (HSCs). Perivascular cells, adipocytes, and osteoblastic lineage cells derived from bone marrow mesenchymal stem cells (BMMSCs) are also key components of these distinct microenvironments. Bone structure is maintained through a dynamic homeostasis fostered by the balance of osteoblastic lineage cells (osteoclasts and osteoblasts).

[0005] WO 2023 / 156774 reports a method for generating bone marrow organoids involving embedding mesodermal aggregates in a bulk hydrogel to drive vascular budding and subsequently generate three-dimensional structures with vascular and hematopoietic structures. WO 2023 / 194370 reports a method for generating bone marrow organoids involving embedding mesodermal aggregates in a polymerized 3D type I collagen / Matrigel matrix. However, these methods are costly, labor-intensive, time-consuming, and cannot be automated. Reducing the use of matrix gel and manual steps would greatly benefit improvements in intra- and inter-experimental variability in size and composition. Excessive use of extracellular matrix can also be detrimental in the biological context of the cells within organoids, despite its practicality in supporting the development of complex vascular networks. Nevertheless, despite its limitations, bulk hydrogels have been the gold standard for generating self-organized vascular structures in vitro for decades.

[0006] Therefore, the object of the present invention is to provide a further and / or improved method for generating organoids, which significantly reduces costs and improves usability and scalability. Summary of the Invention

[0007] The inventors have identified a method for efficiently generating organoids that capture the physiological characteristics of natural human tissues using particulate hydrogels (i.e., compacted assemblies of hydrogel microparticles). Specifically, the method involves simply generating a single formulation comprising cells and particulate hydrogels. Organoids are generated after supplementation with various factors (e.g., growth factors, cytokines, and / or differentiation factors) over time. The resulting organoids can be used as models (e.g., in vitro, in vivo, or ex vivo) with a variety of applications, such as for studying tissue biology (e.g., development, homeostasis, or regeneration), regenerative medicine (production of cell therapies), and disease modeling (e.g., disease mechanisms, drug screening, or personalized medicine).

[0008] Particulate hydrogels are primarily used for 3D bioprinting due to their shear-thinning behavior and self-healing properties. They have not yet been used to generate complex self-organized vascularized organoid cultures because state-of-the-art techniques in the field utilize bulk hydrogels, in which cross-linked matrix components form scaffolds, and bulk hydrogels are considered necessary to drive angiogenic budding and angiogenesis.

[0009] Surprisingly, the inventors discovered that using granular hydrogels to generate organoids that capture the full physiological complexity of bone marrow, similar to the traditional "bulk" hydrogel approach, also brings several key advantages. Specifically, organoids generated using granular hydrogels contain a network of blood vessels forming a lumen and include various cell types, such as hematopoietic cells and stromal cells, as well as mineral deposits (see [link to article]). Figures 3 to 6Compared to previous methods, these organoids also exhibit significantly increased cell density, promoting biologically relevant cell-cell and cell-matrix interactions, thereby allowing for realistic simulation of cellular functions and signaling pathways occurring in vivo. The inventors have also demonstrated that bone marrow organoids are particularly useful for transplanting cells from other sources, such as adult donors (see [link to original text]). Figures 7 to 9 This includes cell types from patients that have been shown to be difficult to culture in vitro to model disease and evaluate therapy in relevant human tissue settings.

[0010] In addition to the higher throughput and lower cost of the particulate method, the inventors also demonstrated that, compared to the bulk hydrogel method, the particulate hydrogel-based method resulted in increased expression of native extracellular matrix (ECM) proteins in organoids while maintaining the same range of cell types (see [link to documentation]). Figure 10 and Figure 11 ).

[0011] Compared to the block approach, key ECM proteins known to be essential for the natural biology of bone marrow—namely, type I and type III collagen (COL1A1, COL3A1), fibronectin, and periostein—are significantly upregulated in relevant stromal cells (fibroblasts, MSCs, osteoblast lineage cells) generated in organoids derived from granular hydrogels. Figure 11 Therefore, organoids generated via a particulate hydrogel-based approach better mimic the natural expression of key ECM proteins (Bandyopadhyay, Shoviket al., (2024), Cell, Volume 187, Issue 12, 3120-3140.E29), promoting the observed improved mineralization. Figure 5 and Figure 17 ).

[0012] The inventors also demonstrated that bone marrow organoids derived from particulate hydrogels can be successfully transplanted from cells derived from patients (e.g., patients with hematologic malignancies such as multiple myeloma, etc.). Figures 12 to 15 (As illustrated) to model human diseases, and the practicality of generating blood cells (including immune cells) for example, for cell therapy (see [reference]). Figure 16 ).

[0013] The organoid generation method based on granular hydrogels is cost-effective, involves simple liquid handling steps, is highly reproducible, scalable, and automatable, and results in the consistent production of uniformly vascularized organoids. In particular, compared to prior art methods using bulk hydrogels (e.g., those disclosed in WO 2023 / 156774), the granular hydrogel-based method uses a significantly reduced gel volume per organoid. The granular hydrogel-based method is highly automatable due to its simple liquid handling steps and high reproducibility. The granular hydrogel-based method also offers significant scalability, as the average volume and diameter of the organoids are reduced without compromising complexity or cellularity, making the method suitable for high-throughput / automated pipetting techniques with strict size constraints.

[0014] Furthermore, particulate hydrogels are particularly effective for generating organoids because the hydrogel microparticles can be easily dispersed between cells and cell aggregates, thus providing good bioavailability. This material is more bioavailable than standard cross-linked bulk hydrogels, which tend to collapse and lose their structure, while particulate gels retain this structure when the particles are compressed. Particulate gels are also more tunable because their mechanical properties can be controlled and maintained through the degree of fragmentation and compaction methods. For example, rotating at higher centrifuge speeds will increase stiffness, while rotating at lower speeds will decrease stiffness. It is also easier to layer different gel compositions compared to standard bulk gels, and the stiffness of each layer can be adjusted.

[0015] The formulation of hydrogel microparticles can be customized to the organoids to be generated. For example, hydrogel microparticles contain extracellular matrix and factors that can effectively promote cell growth and differentiation. Furthermore, it is not necessary to extract the particulate hydrogel from the resulting organoids, so the organoids can be cultured in their encapsulated microenvironment throughout their entire life cycle, and they can integrate materials and be remodeled to better mimic physiological organs. In addition, the particulate hydrogel is prepared as a suspension, so it can be uniformly and reproducibly distributed throughout the culture vessel. The use of particulate hydrogels also leads to methodological simplicity, as it involves generating a single formulation containing cells, particulate hydrogel, and culture medium (see [link to documentation]). Figure 1 ).

[0016] Organoids generated by particulate hydrogel-based methods are superior to existing techniques using bulk hydrogels (e.g., those disclosed in WO 2023 / 156774). Organoids generated by bulk hydrogel-based methods exhibit cell-free spaces due to excess extracellular material. Conversely, organoids generated by particulate hydrogel methods show increased cell density (see [link to article]). Figures 2 to 4Furthermore, particulate hydrogel-based methods overcome the limitations of bulk hydrogel-based methods in terms of throughput and waste, while generating organoids that are preserved and enhance tissue modeling. For example, bulk hydrogel-based methods involve manually extracting buds from hydrogels (see...). Figure 1 This manual step is technically challenging and can impact downstream assays. It is also a source of inter- and intra-experimental variability, resulting from user variability and the technical challenges of isolating individual organoids from large, protein-rich bulk hydrogels. In contrast, particulate hydrogel-based methods have simpler liquid handling steps, requiring only a single formulation of cells and particulate hydrogels, and eliminating the need for manual bud extraction. Therefore, particulate hydrogel-based methods achieve consistent production of homogeneous, vascularized organoids due to the absence of an extraction step, compared to bulk hydrogel-based methods. Batch-to-batch variability is also significantly reduced because the particulate hydrogel structure is not driven by the kinetics of bulk hydrogel formation.

[0017] Therefore, the present invention provides a method for generating organoids, comprising culturing cells in a hydrogel microparticle suspension.

[0018] The present invention also provides a method for generating organoids, comprising culturing cells in a particulate hydrogel.

[0019] The present invention also provides a method for generating bone marrow organoids from pluripotent stem cells (e.g., iPSCs), the method comprising: (i) driving pluripotent stem cells (e.g., iPSCs) to form mesodermal aggregates; (ii) culturing the mesodermal aggregates from (i) into hematopoietic cells and vascular lineage cells in a culture medium containing hematopoietic and matrix-supporting cytokines (e.g., BMP4, FGF2, VEGFA, SCF, and FLT3L); and (iii) adding the mesodermal aggregates from (ii) to a hydrogel microparticle suspension; and (iv) culturing the mixture from (iii) to form bone marrow organoids.

[0020] The present invention also provides organoids that are obtained or obtainable by the method of the present invention, optionally wherein the organoids are bone marrow organoids.

[0021] The present invention further provides bone marrow organoids comprising: (a) a vascular network forming a lumen, optionally wherein the vascular network is composed of bone marrow-specific endothelial cell subsets (e.g., arterioles, sinusoids, H-type capillaries); (b) stromal cells comprising mesenchymal stem cells (MSCs) and osteoblast / adipocyte / CAR lineage cells, fibroblasts, and / or endothelial cells differentiated from MSCs; and (c) hematopoietic cells comprising, for example, hematopoietic stem and progenitor cells (HSPCs) and hematopoietic stem cells. (d) stem cells (HSCs) and myeloid and lymphoid lineage cells (including, but not limited to, T lineage progenitor cells, B lineage cells, natural killer cells, erythroid cells, eosinophils / basophils / mast cells, megakaryocytes, and myeloid mononuclear cells); and (e) osteoblasts; and optionally (e) mineral deposits; optionally wherein the bone marrow organoids are obtained by or derived from the methods of the present invention, and / or wherein the bone marrow organoids have an average vascularized area of ​​5% or more.

[0022] The present invention also provides the use of the bone marrow organoids described herein as a model for studying hematopoietic and stromal cell biology in healthy, aging, and diseased individuals, and for studying immune responses to infection and inflammation.

[0023] The present invention also provides a method for preparing a bone marrow organoid disease model, comprising seeding donor cells onto the bone marrow organoids of the present invention, optionally wherein the donor is an individual suffering from leukemia (e.g., leukemia). The present invention also provides disease models that are obtained or available through the method.

[0024] The present invention further provides a model for bone and / or bone marrow-related conditions (e.g., blood / bone marrow diseases, including primary and secondary cancers, and myelofibrosis), wherein the model comprises bone marrow organoids described herein that have been treated with reagents that induce the conditions.

[0025] The present invention also provides a method for producing cells for cell therapy, wherein the method includes bone marrow organoids generated by the method of the present invention, and wherein the method further includes collecting cells released from the bone marrow organoids. The present invention also provides cells for cell therapy that are obtained or available by said method.

[0026] This invention also provides the use of the models described herein for identifying agents capable of preventing or treating the diseases, including treating the models before, during, or after disease induction. This may include testing disease-modifying or preventative therapies involving targeted transplantation of cells into organoids. For example, organoids can be used to test anticancer immunotherapy agents by transplanting patient-derived cancer cells together with autologous or allogeneic immune cells into organoids to evaluate the therapy in relevant human tissue or tumor microenvironment.

[0027] The present invention also provides a method for identifying an agent for treating or preventing a disease, comprising: adding the agent to a disease model comprising an organoid of the present invention transplanted with diseased cells from a patient, wherein improvement in the disease state of the disease model indicates the effectiveness of the agent in treating or preventing the disease; optionally, the method further comprises adding the agent to a reference model comprising an organoid of the present invention transplanted with healthy cells, and comparing the effects of the agent on the reference model and the disease model.

[0028] The present invention also provides the use of a composition comprising a suspension of hydrogel microparticles for generating organoids, wherein the hydrogel microparticles are optionally compacted to form a particulate hydrogel, and optionally wherein the organoids are bone and / or bone marrow organoids.

[0029] The present invention also provides the use of particulate hydrogels for generating organoids, optionally wherein the organoids are bone marrow organoids.

[0030] The present invention also provides a kit comprising the composition described herein, a hydrogel microparticle suspension, and / or a culture medium. The kit may also include instructions for carrying out the methods described herein. Attached Figure Description

[0031] Figure 1 : A schematic diagram showing the present invention's method using particulate hydrogels and a prior art method (e.g., disclosed in WO 2023 / 156774) using bulk hydrogels to generate bone marrow organoids.

[0032] Figure 2 (a) Comparison of the average size of paraffin-embedded and sectioned organoids generated by the bulk hydrogel method and (b) the particulate hydrogel method. (c) Quantification of the diameter of individual organoids of paraffin-embedded organoids collected from two independent biological replicates. (d) Graphs of the minimum, maximum, and range sizes of organoids.

[0033] Figure 3(a) Comparison of cellularity (cell density) of embedded and sectioned organoids generated using a bulk hydrogel method and (b) a particulate hydrogel method. (c) Quantification of cell density (by counting cell nuclei in sections) across two biological replicates.

[0034] Figure 4 (a) CD34 staining of cross-sections of bone marrow organoids generated by (b) a bulk hydrogel-based method and (c) a particle-based hydrogel-based method. (d) Magnified images of organoids generated by the particle-based hydrogel-based method. Quantification of total CD34+ vascular area. Samples were analyzed in two independent biological replicates, within each individual organoid.

[0035] Figure 5 Von Kossa staining of paraffin-embedded sections of bone marrow organoids generated by (a) a bulk hydrogel method and (b) a particulate hydrogel method.

[0036] Figure 6 Characterization of organoids generated from bulk hydrogels and granular hydrogels. Organoids generated using either bulk or granular hydrogel methods were compared by flow cytometry on day 35 of culture. (A) Matrix cell group, identifying populations: endothelial cells, fibroblasts, BM MSCs, LEPR+BM MSCs, and osteoblast lineage cells. (B) Lymphoid cell group, identifying populations: natural killer (NK) cells, B lineage cells, and T cell progenitors (thymocyte population). (C) Myeloid cell group, identifying populations: erythroid cells, megakaryocytes, myeloid monocytes, and basophils and eosinophils. (D) Stem cell-specific group, identifying populations: hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), and myelo-erythroid progenitors (MEPs).

[0037] Figure 7 Image of donor cell transplantation. Fluorescent BAF3 cells migrate into bone marrow organoids (indicated by arrows).

[0038] Figure 8 Image of bone marrow organoids transplanted from CD34+ hematopoietic cells from a patient with myelofibrosis. The image shown is a Z-stack projection of a 150 μm region of the imaged organoid. Patient cells are marked in white and indicated by arrows. Background signal is type I collagen staining used to delineate the volume of the organoid.

[0039] Figure 9 Images of bone marrow organoids transplanted from CD34+ hematopoietic cells derived from healthy donors or patients with myelofibrosis. Patient cells were labeled with CellTrace violet, and the entire organoid was labeled with αSMA (a marker of fibroblast activation) and type I collagen.

[0040] Figure 10 (a) UMAP maps of cells from human bone marrow organoids generated using either a bulk hydrogel strategy or a particulate hydrogel strategy. The data shown were derived from day 35 organoids in four independent differentiations and generated using single-cell RNA sequencing (10X genomics, 3' v3.4 chemistry). Data were analyzed using Seurat (v5.1) and cell types were annotated based on the expression of typical genes, such as CD3 for T cells. (b) A comparison of cells generated using the bulk and particulate hydrogel methods reveals that the particulate hydrogel method achieves superior representativeness of key hematopoietic and stromal cell types present in human bone marrow, similar to organoids derived from bulk hydrogels.

[0041] Figure 11 Comparison of extracellular matrix (ECM) protein expression in stromal cell subtypes of organoids generated by bulk hydrogel-based and granular hydrogel-based methods. A violin plot shows a significant increase in ECM gene expression in stromal cells of organoids generated using granular hydrogels. This includes: COL1A1 (type I collagen) in fibroblasts, mesenchymal stromal cells (MSCs), and nestin (NES) + MSCs; COL3A1 (type III collagen) in fibroblasts, MSCs, NES + MSCs (nestin + mesenchymal stem cells), and osteoblast lineage cells; fibronectin (FN1) in endothelial cells, MSCs / fibroblasts, osteoblast lineage cells, and osteoblasts; and postosteoprotein (POSTN) in fibroblasts, NES + MSCs, and osteoblasts.

[0042] Figure 12(A) Schematic diagram of a protocol used for modeling diseases (e.g., myeloma) using particulate hydrogel-derived organoids. CD138+ plasma cells isolated from bone marrow aspirates from patients with myeloma (MM) or CD34+ blood stem / progenitor cells from peripheral blood of healthy mobilized donors were seeded onto bone marrow organoids on day 21. (B) Donor cells were labeled with a fluorescent dye (CellTrace Violet) to allow for tracking of transplantation and proliferation over time. Imaging of the entire organoid is shown on days 3, 7, and 14 post-transplantation (days 24, 28, and 35 of the entire protocol). Images show successful transplantation of donor cells into the organoid on day 3, and expansion of fluorescently labeled cells on day 14. (c and d) Images show (c) a centrifuged smear of cells from the dissociated organoid and (d) an H&E-stained histological section of the entire organoid 14 days after transplantation of primary cells. Red arrows indicate morphologically identifiable plasma cells.

[0043] Figure 13 UMAP plot of single-cell RNA sequencing data (10x Genomics) generated from organoids derived from particulate hydrogels transplanted with healthy donor control cells (n=2 donors) or myeloma patient cells (n=3 donors), as well as untransplanted organoids. Figure 12 As shown, organoids were dissected for scRNA-seq 14 days after transplantation of adult donor cells. Data analysis was performed using Seurat (v 5.1.0), and cell types were annotated based on typical gene expression. The UMAP plot represents 45,706 hematopoietic cells and 14,577 stromal cells. (a) UMAP of hematopoietic cells is shown, including myeloid cells (erythroids, megakaryocytes, eosinophils / basophils / mast cells, monocytes / macrophages, dendritic cells, and T lymphocyte progenitors). Organoid-derived plasma cells from transplanted myeloma samples were successfully captured (indicated by circles on the plot). (b) Captured stromal cells included endothelial cells, osteoblastic lineage cells, adipocyte lineage cells, fibroblasts, and mesenchymal stem cells.

[0044] Figure 14The heatmap shows a significant enrichment of gene sets and pathways (GSEA analysis) when comparing organoids derived from particulate hydrogels transplanted with myeloma patient cells to control organoids (transplanted with healthy donor cells). Black boxes highlight the cell populations (X-axis) and gene sets (Y-axis) with the most significant changes. This confirms increased interferon responses and inflammatory pathways in stromal cells (including CD271+ MSCs, osteoblast lineage cells, and early osteoblasts) as well as macrophages and dendritic cells, similar to findings observed in previous studies analyzing bone marrow from myeloma patients (de Jong, MME et al., (2021), Nature Immunology, 22, 769-780), demonstrating that organoids accurately model human disease.

[0045] Figure 15 Violin plot comparing the expression of inflammation-related genes in myeloma (MM) transplanted and control organoids. (a) The figure shows significantly increased expression of TNF, S1000A4, and S100A9 in macrophages, dendritic cells (cDC2), and neutrophils in myeloma transplanted organoids. (b) Significantly higher B2M expression was observed in stromal cells isolated from myeloma transplanted organoids, as previously shown in the bone marrow of myeloma patients. (c) Significantly lower expression of NFIA and NFIB in osteoblasts of myeloma transplanted organoids compared to osteoblasts of control organoids, indicating 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).

[0046] Figure 16 To investigate the ability of bone marrow organoids to generate hematopoiesis, flow was induced by agitation of the organoids in an ultra-low adhesion plate. (A) Schematic diagram of a simple flow system generated using a built-in agitation system in an incubator. (B) Bright-field image of bone marrow organoids after 7 days of flow / agitation. The large, dark 3D structure of the organoids is surrounded by a large number of cells extruded from the organoids due to flow dynamics. (C) Pore region showing the extruded cells. (D) Higher resolution bright-field image of cells extruded from the organoids using this system. (E) Extruded cells were collected, centrifuged, smeared onto glass slides, and stained with Giemsa. Numbers indicate morphologically defined cell types, including: macrophages: 1; megakaryocytes: 2; neutrophils: 3; monocytes: 4; myeloid progenitors: 5; erythroid progenitors: 6; adipocytes: 7.

[0047] Figure 17Mineralization / ossification was assessed over time. The mineral dyes Alizarin Red and Von Kossa were applied to paraffin-embedded sections of granular hydrogel-derived organoids collected at days 35 and 80. A significant increase in mineralization was observed between days 35 and 80, confirming the ability of osteoblast lineage cells to calcify organoids over time. Detailed Implementation

[0048] Methods for generating organoids using particulate hydrogels

[0049] This invention relates to the use of particulate hydrogels, i.e., compacted assemblies of hydrogel microparticles. Typically, particulate hydrogels are prepared by breaking up a bulk hydrogel (which comprises a solid or semi-solid network of cross-linked polymers) into a suspension of hydrogel microparticles, and then compacting the hydrogel microparticles to form a particulate hydrogel. Particulate hydrogels are a type of microgel, also known as fragmented particulate hydrogels or particulate microgels (see, for example, Muir et al., 2022, JoVE, e63867).

[0050] Therefore, the present invention provides a method for generating organoids, comprising culturing cells in a composition comprising a suspension of hydrogel microparticles (e.g., particulate hydrogel). The present invention also provides the use of a composition comprising a suspension of hydrogel microparticles (e.g., particulate hydrogel) for generating organoids.

[0051] The methods and uses of the present invention may include generating particulate hydrogels. For example, the methods and uses may include breaking up blocky hydrogels to produce hydrogel microparticles, and optionally compacting the hydrogel microparticles to produce particulate hydrogels. Alternatively, the methods and uses of the present invention may include using a suspension of hydrogel microparticles (e.g., particulate hydrogels) prepared as described herein.

[0052] Hydrogel microparticles can be prepared by any method that breaks down bulk hydrogels (e.g., chemical and / or mechanical fragmentation). The methods and uses of the present invention can utilize hydrogel microparticles (e.g., particulate hydrogels) that are obtained or available through the fragmentation and / or compaction methods described herein.

[0053] Chemical fragmentation can include hydrolytic degradation, co-aggregation, or enzymatic digestion (e.g., using proteases such as lipases).

[0054] Mechanical fragmentation can include extrusion techniques (e.g., by syringe, screen, filter, or sieve), mechanical abrasion (e.g., grinding with a mortar and pestle or using a mixer), or mechanical agitation (e.g., vortexing with or without broken beads).

[0055] For example, bulk hydrogels can be fragmented using batch emulsions, where mixing immiscible liquids leads to droplet formation, which then crosslinks to form hydrogel microparticles. A similar process can be performed in microfluidic devices and is referred to as a microfluidic emulsion.

[0056] For example, bulk hydrogels can be extruded and broken up, for instance, through a series of increasingly smaller needle sizes (e.g., from 18G (inner diameter 838 μm) to 30G (inner diameter 159 μm) or higher). For example, a bulk hydrogel can be extruded through a 23G needle, then through a 25G needle, and then through a 27G needle.

[0057] Bulk hydrogels can be electrohydrodynamic sprayed, in which the hydrogel is extruded through a syringe with a voltage applied to the needle tip.

[0058] Bulk hydrogels can have a compressive modulus of about 1 kPa to about 80 kPa, for example, about 10 kPa to about 70 kPa, or about 50 kPa to about 60 kPa. Hydrogels with a compressive modulus greater than 80 kPa may cause syringe clogging or increased overpressure during the fragmentation step. Bulk hydrogels with a compressive modulus less than 1 kPa may deform during the mechanical fragmentation step.

[0059] The hydrogel microparticles used in this invention can have regular or irregular shapes. They can be spherical. They can have serrated polygonal shapes. They can have uniform or polydisperse diameters. They can comprise mixtures of fibrillary structures and mixtures of regular or irregular spherical or polygonal shapes.

[0060] Hydrogel microparticles can have an average diameter of 10 μm to 500 μm, for example, an upper average diameter of ≤450 μm, ≤400 μm, ≤350 μm or ≤300 μm, and a lower average diameter of ≥20 μm, ≥40 μm, ≥60 μm, ≥80 μm or ≥100 μm.

[0061] The sphericity of hydrogel microparticles can range from 0.2 (non-circular) to close to 1 (perfectly circular).

[0062] The equivalent circle diameter of hydrogel microparticles can range from 10 μm to 300 μm.

[0063] The feret diameter of the hydrogel microparticles can range from about 20 μm to about 600 μm.

[0064] The aspect ratio of hydrogel microparticles can range from 1 to 3.

[0065] Methods for measuring the equivalent circular diameter (μm), Ferrette diameter (μm), aspect ratio, and roundness are known in the art, for example, as disclosed by Muir et al. (JoVE, 2022, 183, e63867). For example, these parameters can be measured by first creating a diluted suspension of particles that can be imaged on epifluorescence or confocal microscopy, and then converting the image into a binary image (e.g., in ImageJ) to analyze the particles. The equivalent circular diameter (μm) of each particle can be determined from a scale of μm / pixel. The area of ​​the hydrogel particles can be determined from a scale of pixels. 2 Convert to μm 2 Area (μm) 2 The equivalent circle diameter can then be derived by taking the square root of the area, dividing by π, and then doubling the result. The μm / pixel scale can also be used to obtain the Freette diameter (μm) of the hydrogel particles. Various other parameters, namely roundness, aspect ratio, circularity, and solidity, can be derived directly from the output.

[0066] Hydrogel microparticles can be compacted to produce particulate hydrogels. Compaction techniques are well known in the art, and any compaction technique can be used. For example, compaction can be centrifugation. Hydrogel microparticle suspensions can be centrifuged at 10,000 xg to 18,000 xg (e.g., 10,000 xg, 12,000 xg, 14,000 xg, 16,000 xg, or 18,000 xg) for up to 10 min (e.g., 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min). For cell-containing suspensions, hydrogel microparticle suspensions can be centrifuged at 5 xg, 10 xg, or 1000 xg for between 30 seconds and 30 min. Hydrogel microparticle suspensions can also be centrifuged at 300 xg for 4 min.

[0067] Another compaction technique could be filtration, such as vacuum-driven filtration, which involves loading a suspension of hydrogel microparticles onto a membrane filter and reducing the amount of suspended media by filtration or vacuum suction.

[0068] The method of the present invention may include adding cells before compacting the hydrogel microparticles.

[0069] The method of the present invention may include adding cells after compacting hydrogel microparticles.

[0070] The method of the present invention may include creating multiple layers of a compacted mixture comprising hydrogel microparticles and cells by repeatedly adding cells to a suspension of hydrogel microparticles and then compacting the mixture. For example, the multiple layers of the compacted mixture may contain hydrogel microparticles with different formulations and / or different cell types.

[0071] The method of the present invention may include creating multiple layers of a compacted mixture comprising hydrogel microparticles and cells on multiple surfaces (e.g., functionalized surfaces (e.g., coated plates)), followed by alternative materials (e.g., ceramics) or in a microfluidic device.

[0072] The particulate hydrogel may contain about ≥0.1% w / v of hydrogel microparticles, such as about ≥0.5% w / v, ≥1% w / v, ≥10% w / v, ≥20% w / v, ≥30% w / v, ≥40% w / v, ≥50% w / v, or ≥60% w / v. For example, the particulate hydrogel may contain about 0.1% w / v to 60% w / v, such as about 0.5% w / v to about 1.5% w / v, or 0.1% w / v to 2% w / v of hydrogel microparticles. The hydrogel microparticle density of the particulate hydrogel may be about 0.5 or higher, such as about 0.6, about 0.7, about 0.8, or about 0.9.

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

[0074] The method of the present invention may include the step of removing the particulate hydrogel. However, this step is not necessary. In fact, it is beneficial to retain the particulate hydrogel within the organoids because the organoids will be cultured in their encapsulated microenvironment throughout their lifespan, and they are able to integrate the material and remodel to better mimic physiological organs. Therefore, in some embodiments of the present invention, the method and use do not include the removal of the particulate hydrogel, for example, by extracting organoids from the particulate hydrogel.

[0075] Hydrogel microparticles may contain natural polymers, such as collagen or fibroin. Hydrogels may also contain synthetic hydrogels, such as those comprising or composed of synthetic peptides or peptide / adhesion-functionalized polysaccharides. Hydrogel microparticles may contain polypeptide chains, polysaccharide chains, and / or extracellular matrix proteins.

[0076] Extracellular matrix proteins can be collagen, such as type I collagen and / or type IV collagen. Hydrogel microparticles can contain both type I and type IV collagen. The ratio of type I collagen to type IV collagen can range from 1:1 to 3:1, for example, 1:1. It has been found that a mixture of type I and type IV collagen in the hydrogel produces a high proportion of myeloid cells and a population of mesenchymal stromal cells, which is important for remodeling the bone marrow space.

[0077] Hydrogel microparticles may contain matrigol. Matrigol contains dissolved basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, such as laminin, type IV collagen, and nestin. Matrigol may also contain growth factors such as IGF and FGF.

[0078] Hydrogel microparticles can contain fibrin.

[0079] Hydrogel microparticles can contain matrix gel, fibrin, type I collagen, and type IV collagen.

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

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

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

[0083] The cell can be an iPSC.

[0084] The cell can be an ESC.

[0085] The cells can be CD34+ hematopoietic stem cells and progenitor cells.

[0086] The cells can be leukemia blasts, terminally differentiated cells (such as T cells and macrophages), or solid tumor cells to model bone marrow metastasis.

[0087] The cells can be immortalized cell lines, such as leukemia cell lines (e.g., HEL, MOLM-13).

[0088] The cells may be obtained from the subject. The subject may be a person. The subject may be healthy. The subject may have a disease or 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.

[0089] The methods and uses of this invention may include culturing one or more cell types (e.g., more than one cell type, such as 2, 3, or 4 cell types) in a hydrogel microparticle suspension. For example, the input cells may be iPSCs or cells derived therefrom (e.g., mesodermal aggregates), and more cells may then be added to the culture. The additional cells may be any cells described herein. More cells may be added at any time point after the iPSCs or cells derived therefrom (e.g., mesodermal aggregates) have been added to the hydrogel microparticles (e.g., from day 1 to day 21, such as on days 1, 7, 14, or 21). For example, more cells may be added when the iPSCs or cells derived therefrom (e.g., mesodermal aggregates) have sufficiently differentiated to establish a structure and / or some cell fate has been determined. For example, more cells, such as immortalized MSC cell lines (which secrete hematopoietic factors), may be added early in the protocol.

[0090] The methods and uses of this invention may include growing cells into aggregates and / or differentiated cells prior to culturing cells in a hydrogel microparticle suspension to generate organoids. The average diameter of the aggregates may be 50 μm to 700 μm.

[0091] In embodiments of the invention involving the use of pluripotent stem cells (e.g., iPSCs) as input cells, cells can be stimulated to undergo germ layer specialization (e.g., endoderm, mesoderm, or ectoderm) by culturing with appropriate stimulating factors, and then induced to differentiate and mature by culturing with specific growth factors and signaling factors to obtain an organized mixture of specific cell types that form the desired organ.

[0092] Therefore, the methods and uses of the present invention may include: (i) driving pluripotent stem cells (e.g., iPSCs) to form aggregates of germ layer-specific cells (e.g., endoderm, mesoderm, or ectoderm); (ii) culturing aggregates from (i) into fractionated lineages in a culture medium containing appropriate stimulating factors; (iii) adding aggregates from (ii) to a hydrogel microparticle suspension according to the methods described herein; and (iv) culturing a mixture from (iii), for example, according to the methods described herein, until organoids are produced.

[0093] 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.

[0094] In embodiments of the invention involving the use of adult stem cells (ASCs) as input cells, cells can be stimulated with appropriate growth factors to allow proliferation.

[0095] Supplemental factors can be added to the culture medium to drive differentiation and maturation. The supplemental factors and culture conditions will depend on the organoids to be generated. Those skilled in the art will be able to determine appropriate supplemental factors and culture conditions using common knowledge in the art, for example, 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 supplemental factors used to generate bone marrow organoids are described below.

[0096] Various differentiated cell types can be identified using markers and methods known in the art, such as those demonstrated in the examples. For instance, mesodermal differentiation markers include Brachyury (a typical marker), BMP family proteins, TWIST, TWIST2, and / or HAND1. Ectodermal differentiation markers include NES and / or OTX2. Endoderm differentiation markers include SOX17 and / or FOXA2. Hematopoietic differentiation markers include CD34 and CD35. Vascular differentiation markers include CD31 and / or CD144. Matrix differentiation markers include CD90, CD271, and / or LEPR.

[0097] The methods and uses of the present invention may not include separating cells from hydrogel microparticles. For example, the methods and uses of the present invention may not include extracting (e.g., manually extracting) cells (e.g., aggregates, developing organoids, or organoids) from hydrogel microparticles. The methods and uses of the present invention may include culturing cells (e.g., aggregates, developing organoids, or organoids) in a hydrogel microparticle suspension in the same container (e.g., a 96-well plate) throughout the culture period until organoids are formed.

[0098] The methods and uses of this invention can be used to generate any organoid, such as bone, bone marrow, bone and bone marrow, mammary gland, retina, kidney, stomach, intestine, liver, lung, prostate, bladder, endometrium, blood vessel or thyroid organoid.

[0099] This invention also provides organoids that are obtained or obtainable by the methods described herein. The organoids of this invention are in vitro or ex vivo organoids. The organoids of this invention are synthetically produced (i.e., in vitro) and do not contain natural tissue extracts.

[0100] The average size of the organoids of the present invention can be from about 100 μm to about 5 mm, for example, from about 500 μm to about 4 mm. The organoids of the present invention can have a narrow size distribution, for example, a standard deviation of 2 or lower.

[0101] The organoids described herein are 3D organoid cultures that possess the structural, cellular, and molecular characteristics of a target organ or tissue. For the avoidance of doubt, the organoids described herein (e.g., bone marrow organoids) differ from spheroids and other 3D cell aggregates, which may be multi-lineage but are not organized into structures (e.g., blood vessels, luminal spaces). The organoids described herein can capture a specific region (e.g., myelogenic bone marrow) or multiple regions (e.g., both lymphoid and myeloid bone marrow), or the entire target organ.

[0102] Mesodermal induced from pluripotent stem cells

[0103] Methods for forming mesodermal aggregates from pluripotent stem cells (e.g., iPSCs) are known in the art. For example, mesodermal aggregates can be formed by: incubating induced pluripotent stem cells (iPSCs) to induce iPSC aggregate formation; and culturing the iPSC aggregates in a culture medium for stem cell maintenance, supplemented with factors (e.g., BMP4, FGF2, and VEGFA). The iPSCs can be cultured with BMP4. The iPSCs can be cultured with BMP4 under hypoxic conditions (e.g., 1%-5%), with or without a WNT inhibitor (e.g., CHIR99021). For example, the iPSCs can be cultured with BMP4 and a WNT activator (e.g., CHIR99021) under 5% oxygen. The iPSCs can be cultured with BMP4, CHIR99021, VEGFA, and FGF2 under 5% oxygen.

[0104] Prior to the formation of aggregates of induced pluripotent stem cells (e.g., iPSCs), the pluripotent stem cells can be maintained and cultured according to methods commonly used in the art. The iPSCs can be passaged once or multiple times, for example at approximately 20%–90% confluence, or even approximately 70%–80% confluence. The pluripotent stem cells (e.g., iPSCs) can be isolated for passage and / or aggregate formation, for example using physical / mechanical separation and / or non-physical / mechanical separation, such as using EDTA or enzymatic separation. For the formation of aggregates of pluripotent stem cells (e.g., iPSCs), the isolated pluripotent stem cells (e.g., iPSCs) can be cultured in a stem cell differentiation medium for at least 5 hours or for at least 12 hours, for example, for approximately 5–24 hours. The stem cell differentiation medium can contain a basal medium (e.g., StemFlex™) supplemented with a ROCK inhibitor (e.g., RevitaCell™ (Thermo) or an equivalent thereof).

[0105] The pluripotent stem cell (e.g., iPSC) aggregates can be incubated under standard cell maintenance conditions (e.g., 37°C, 5% CO2) for a period of time, for example, 5-24 hours, and then added to a mesotherapy induction medium. The obtained pluripotent stem cell (e.g., iPSC) aggregates can be collected by gravity or centrifugation and resuspended in the mesotherapy induction medium. In one embodiment, the date on which the pluripotent stem cell (e.g., iPSC) aggregates are transferred for culture in the mesotherapy induction medium is designated as day 0. Culture of the pluripotent stem cell (e.g., iPSC) aggregates in the mesotherapy induction medium can be performed under suitable cell growth conditions, such as 5% O2, 5% CO2, and 37°C.

[0106] The early mesodermal induction step may include incubating the pluripotent stem cell (e.g., iPSC) aggregates for a period sufficient to form mesodermal cells in a cell population of ≥60% (e.g., ≥70%, ≥80%, ≥90%). The pluripotent stem cell (e.g., iPSC) aggregates may be cultured in the mesodermal induction medium for a period of approximately 2–7 days, for example, approximately 3–7 days or approximately 3–5 days. The pluripotent stem cell (e.g., iPSC) aggregates may be cultured in the mesodermal induction medium for a period of time until the aggregates reach an average size of approximately 200–250 μm. It should be understood that the size of the aggregates is the average of the maximum diameter of the pluripotent stem cell (e.g., iPSC) aggregates.

[0107] Technicians will understand that as many mesodermal cells as needed can be produced, and should understand that the number or percentage of mesodermal cells formed at this stage will affect the final yield of the resulting organoids. For example, a higher number of aggregates will produce a higher number of bone marrow organoids, but a higher number of mesodermal cells will lead to more efficient differentiation into bone marrow lineage cells.

[0108] Mesodermal tissue generated from aggregates of said pluripotent stem cells (e.g., iPSCs) can be identified by detecting the presence of mesodermal markers using any method known in the art. These mesodermal markers may include or consist of one or more of Brachyury, Snail, TBX6, and N-cadherin. Cellular markers can be detected by protein expression, such as using immunofluorescence or mRNA expression (e.g., using qRT-PCR).

[0109] The mesotherapy induction medium can be a chemically defined medium (CDM). The mesotherapy induction medium contains a basal medium, such as a stem cell differentiation medium. The mesotherapy induction medium may contain a stem cell differentiation medium, such as APEL2 or StemPro or equivalents. The mesotherapy induction medium may contain a ROCK inhibitor, such as Y-27632.

[0110] The mesodermal induction medium may contain appropriate amounts of supplementary factors to promote mesodermal settling and induce early endothelial / hematopoietic bipotent progenitor cells. The supplementary factors may be BMP4, FGF2, and VEGFA. BMP4 may be provided in the mesodermal induction medium at a concentration of about 10 ng / ml to about 100 ng / ml, for example, about 50 ng / ml. FGF2 may be provided in the mesodermal induction medium at a concentration of about 10 ng / ml to about 100 ng / ml, for example, about 50 ng / ml. VEGFA may be provided in the mesodermal induction medium at a concentration of about 10 ng / ml to about 100 ng / ml, for example, about 50 ng / ml. The supplementary factors may include the Wnt activator CHIR99021, activin A, and / or a TGFb inhibitor (SB).

[0111] bone marrow organoids

[0112] This invention relates to the production of bone marrow organoids that resemble natural human bone marrow. The bone marrow organoids described herein are 3D organoids or organoid cultures comprising stromal cells and hematopoietic cells. These bone marrow organoids may comprise multiple lineages of hematopoietic cells (including myeloid and lymphoid cells), stromal cell subsets (including, for example, mesenchymal stem cells, CAR cells, osteoblast lineage cells, and / or adipocytes), and a vascular system.

[0113] The methods and uses of the present invention may include generating bone marrow organoids from pluripotent stem cells (e.g., iPSCs, such as hiPSCs).

[0114] The methods and uses of the present invention may include: (i) driving iPSCs to form mesodermal aggregates; (ii) culturing the mesodermal aggregates from (i) into hematopoietic lineage cells and vascular lineage cells in a medium containing BMP4, FGF2, VEGFA, SCF and FLT3L; (iii) (e.g., according to the methods described herein) adding the mesodermal aggregates from (ii) to a hydrogel microparticle suspension; and (iv) (e.g., according to the methods described herein) culturing the mixture from (iii) until bone marrow organoids are produced.

[0115] The conditions applicable to mesodermal induction in step (i) are as described above.

[0116] Step (ii) may include culturing the mesodermal aggregates in a mesodermal induction medium for a period of time until the mesodermal aggregates have an average size of 100 μm-500 μm and / or exhibit markers of early endothelial and hematopoietic differentiation. Such markers may include one or more of CD144, CD31, RUNX1, and GATA1. The markers may be detected by any suitable method, such as by qRT-PCR.

[0117] This document describes a suitable mesodermal induction medium. The supplementary factors BMP4, FGF2, VEGFA, SCF, and FLT3L in the mesodermal induction medium of step (ii) can be provided in the medium at a concentration of about 10 ng / ml to about 100 ng / ml, for example, about 50 ng / ml. The medium may be further supplemented with one or more additional cytokines, such as IL7 or other interleukins, to induce lymphocyte production. These additional cytokines may include one or more of IL11, FLT3L, GM-CSF, M-CSF, G-CSF, EPO, IL1, IL12, IL13, IL33, TPO, IL3, IL6, IL2, IL10, and IL8. Those skilled in the art will be able to determine the appropriate amount of any supplementary factor based on common knowledge in the art. For example, in step (ii), the further supplementary factor may be provided in the medium at a concentration of about 10 ng / ml to about 100 ng / ml, for example, about 50 ng / ml.

[0118] The culture conditions in step (ii) can be standard cell culture conditions, such as 5% O2, 5% CO2, and 37°C. The culture conditions in step b can be normoxic conditions (e.g., about 20%-21% O2). The culture conditions can also be at hypoxic levels (e.g., 3%-10%, such as 5% O2). Culture conditions similar to physiologically relevant O2 stress (e.g., hypoxic levels, such as 3%-10%, such as 5% O2) promote bone marrow specialization and lymphoid cell generation.

[0119] Step (iii) may include culturing the mesodermal aggregates in a hydrogel microparticle suspension according to the method described herein.

[0120] Step (iv) may include culturing the mixture in a culture medium containing supplementary factors, such as a mixture of cytokines and / or growth factors for generating the desired hematopoietic lineage mixture. Supplementary factors that can be used in step (iv) may include one or more early hematopoietic factors (pan-hematopoietic factors), such as SCF, FLT3L, IL3, and / or IL6. Supplementary factors that can be used in step (iv) may include one or more factors that drive lymphoid lineage development, such as IL7. Supplementary factors that can be used in step (iv) may include one or more factors that drive lineage-specific development and diversify hematopoietic branches (such as EPO, TPO, and / or CSF) and interleukins. For example, supplementary factors that can be used in step (iv) may include small molecule enhancers of hematopoiesis or angiogenesis, such as Forskolin, UM171, PI3K inhibitors, TGFb inhibitors, and / or SR1. Supplementary factors that can be used in step (iv) may include one or more factors that drive sinusoidal space-specific endothelial vascular system development, such as VEGFC. VEGFC can be added at a later time, such as starting from d3 (from the overall differentiation timeline).

[0121] The supplementary factors that can be used in step (iv) may include VEGFA, VEGFC, FGF2, hSCF, FLT3L, IL3, IL6, IL7, TPO, EPO and BMP4.

[0122] The supplementary factors that can be used in step (iv) may include VEGFA, FGF2, BMP4, SCF, FLT3L, IL3, IL6, cGSF, EPO and / or TPO.

[0123] The supplementary factors that can be used in step (iv) may include VEGFA, FGF2, BMP4, SCF, FLT3L, IL3, IL6, cGSF, EPO, TPO, IL7 and / or VEGFC.

[0124] A skilled technician will be able to determine the appropriate amount of any supplemental factor based on common knowledge in the art. For example, the supplemental factor may be provided in the culture medium of step (iv) at a concentration of about 10 ng / ml to about 100 ng / ml, such as about 50 ng / ml.

[0125] The mesodermal aggregates in step (iv) can be cultured to an average size of about 100 μm to about 5 mm, for example about 500 μm to about 4 mm.

[0126] The mesodermal aggregates can be cultured until a vascular network is formed, and optionally cultured continuously as long as the vascular network is maintained.

[0127] The mesodermal aggregates can be cultured until hematopoietic cells are formed, such as CD45+ cells. The culture can be maintained in a basal medium, such as APEL2, StemPro-34, or a suitable alternative cell culture medium.

[0128] Technicians can adjust the concentration and / or composition of cytokines to alter the composition of the resulting bone marrow organoids. For example, when the budding stage is partially complete (e.g., about halfway through), the concentration of EPO can be increased to produce more erythrocytes, or the concentration of EPO can be decreased and the SCF / FLT3L content increased to produce more HSPCs. The increase or decrease can be sufficient to allow the desired cellular composition to be produced in the bone marrow organoids. A decrease in a given cytokine can be a reduction of about 10%, 30%, 50%, 80%, or 90%. An increase in a given cytokine can be an increase of about 10%, 30%, 50%, 80%, 100%, 150%, or 200%.

[0129] The present invention also provides bone marrow organoids. These bone marrow organoids can be obtained or derived by the methods described herein.

[0130] The composition of the bone marrow organoids of the present invention is generally similar to that of natural tissues, although the composition can be engineered to include other cell types, for example, for studying specific biological problems.

[0131] The bone marrow organoids of the present invention are in vitro or ex vivo organoids. The bone marrow organoids of the present invention are synthetically produced (i.e., in vitro) and do not contain natural bone and / or bone marrow tissue extracts.

[0132] The bone marrow organoids of the present invention are synthetically produced (i.e., in vitro) and do not contain bone marrow tissue extracts.

[0133] The bone marrow organoids of the present invention comprise cells from multiple lineages. The bone marrow organoids of the present invention may comprise stromal cells and / or hematopoietic cells, preferably comprising both stromal cells and hematopoietic cells. The stromal cells may comprise vascular cells, mesenchymal stem cells, adipocytes, and perivascular matrix. The hematopoietic cells may comprise myeloid cells and lymphoid cells and their stem cell progenitor cells. The bone marrow organoids of the present invention may comprise osteoblast lineage cells. The bone marrow organoids of the present invention may comprise mineral deposits.

[0134] For example, the bone marrow organoids of the present invention may comprise: (a) a network of vascular systems and / or sinusoids forming a lumen; (b) stromal cells comprising mesenchymal stem cells (MSCs), fibroblasts, and / or endothelial cells; (c) hematopoietic cells comprising hematopoietic stem-progenitor cells (HSPCs), erythroid cells, myeloid mononuclear cells, and / or megakaryocytes; and (d) osteoblastic lineage cells. The bone marrow organoids may further comprise mineral deposits.

[0135] The bone marrow organoids of the present invention are typically 3D cultures smaller than natural human tissue. The size can be adjusted based on the number of input cells. For example, the bone marrow organoids of the present invention can have an average size of about 100 μm to about 5 mm, or, for example, about 500 μm to about 4 mm. The bone marrow organoids of the present invention can have a narrow size distribution, for example, a standard deviation of 2 or less. The bone marrow organoids of the present invention can have an average vascularization area of ​​≥5%, for example, ≥10%, ≥20%, ≥40%, or ≥60%.

[0136] The bone marrow organoids of the present invention typically have long lifespans. For example, they can be maintained in culture for 30 days or longer, such as 50 days or more.

[0137] The bone marrow organoids of the present invention can have functions similar to natural tissues. For example, they can produce red blood cells, platelets, myeloid mononuclear cells, and lymphoid progenitor cells.

[0138] Applications / Uses

[0139] This invention relates to providing organoids that can be used as in vitro, ex vivo, or in vivo models for purposes such as studying tissue biology, regenerative medicine, or disease modeling. Therefore, this invention also provides methods and uses of said organoids for such purposes.

[0140] The organoids of this invention can be used to study tissue biology, such as development, homeostasis, or regeneration. The organoids of this invention can be used to model the bone and bone marrow environment for studying hematopoietic and stromal cell biology in healthy, aging, and diseased conditions. The organoids of this invention can be used to model immune responses to infection and inflammation.

[0141] The organoids of this invention can be used for disease modeling, such as disease mechanisms, drug screening, or personalized medicine. Therefore, this invention also provides a disease model comprising the organoids of this invention. Examples of personalized medicine include assessing individualized treatment responses, predicting treatment resistance (e.g., resistance to immunotherapy / cell therapy), stem cell transplantation outcomes, and pharmacogenomics testing, as well as high-throughput screening. The disease can be a bone and / or bone marrow-related condition, such as myelofibrosis, myelofibrosis, or blood cancers, such as multiple myeloma, myeloma, metastatic solid tumors, leukemia (e.g., acute or chronic lymphocytic leukemia, or acute or chronic myeloid leukemia), myelodysplastic syndromes, myeloproliferative neoplasms, lymphoma, or mast cell tumors.

[0142] For example, the organoids of the present invention can be used to model myelofibrosis. Extracellular matrix (ECM) proteins are known to be associated with myelofibrosis, and the organoids of the present invention can effectively express extracellular matrix (ECM) proteins (e.g., see...). Figure 11 Therefore, the present invention also provides a myelofibrosis model comprising the organoids of the present invention.

[0143] The organoids of this invention can be used to model multiple myeloma. The organoids of this invention can express inflammatory response genes, for example, increasing the expression of TNF, S100A4, and S100A9 in key hematopoietic cells (e.g., see...). Figure 15 Furthermore, the organoids of the present invention can respond to diseased cells, for example by increasing B2M in stromal cells and decreasing the expression of NFIA and NFIB transcription factors in osteoblast lineage cells (see, for example, [link to relevant documentation]). Figure 15 Therefore, the present invention also provides a system that can be used to model multiple myeloma.

[0144] The organoids of the present invention can be transplanted with cells from patients suffering from diseases such as bone and / or bone marrow-related conditions (e.g., myelofibrosis, myelofibrosis, or blood cancers such as multiple myeloma, myeloma, metastatic solid tumors, leukemia (e.g., acute or chronic lymphocytic leukemia, or acute or chronic myeloid leukemia), myelodysplastic syndromes, myeloproliferative neoplasms, lymphoma, or mast cell tumors) or any of the diseases described herein. After inoculation with patient cells, the organoids of the present invention may undergo microenvironmental remodeling, resulting in implanted organoids exhibiting characteristics of the disease. In embodiments involving myelofibrosis models, the implanted organoids may develop progressive scarring. In embodiments involving multiple myeloma models, the implanted organoids may undergo changes in stromal cell metabolism, cell fate, and inflammation (e.g., see...). Figures 12 to 15 ).

[0145] The organoids or disease models of this invention can be used to screen for biomarkers associated with disease states (e.g., fibrosis or other bone marrow diseases) in the organoids. For example, this invention can provide a method comprising monitoring biomarkers released from bone marrow organoids or from which cells have been transplanted, or biomarkers in tissue or cell extracts of bone marrow organoids.

[0146] Disease models comprising organoids of the present invention can be used to test therapies for the prevention or treatment of said diseases. Therefore, the present invention also provides a method for identifying agents for the treatment or prevention of diseases, comprising adding the agent to a disease model comprising an organoid of the present invention transplanted with diseased cells from a patient, wherein improvement in the disease state of the disease model indicates the effectiveness of the agent in treating or preventing said disease. The method may further comprise adding the agent to a reference model comprising an organoid of the present invention transplanted with healthy cells, and comparing the effect of the agent on the reference model and the disease model. The healthy cells may be from the same patient or different donors. The method may further comprise adding more cells (e.g., immune cells) to the model, for example to enhance or promote cancer cell killing.

[0147] Disease states can develop or be induced in bone marrow organoids, thereby allowing for the identification of changes in biomarker profiles and their correlation with disease states.

[0148] The biomarker may comprise proteins, glycoproteins, glycans, peptides, nucleic acids, or any cellular product that can indicate the disease state of bone marrow organoids or cells transplanted into bone marrow organoids. The biomarker may be a cellular marker, such as a surface protein.

[0149] The organoids of this invention can be used to support the transplantation and survival of cells from patients suffering from conditions such as a range of hematologic malignancies, including cancer cell types that are difficult to survive in vitro in standard liquid culture systems. The cancer cell types may include, but are not limited to, cells from patients suffering from myeloid or lymphoid hematologic malignancies, such as multiple myeloma, myeloma, acute or chronic lymphocytic leukemia, acute or chronic myeloid leukemia, myelodysplastic syndromes, myeloproliferative neoplasms, lymphoma, and mast cell tumors.

[0150] Therefore, the organoids of the present invention (e.g., bone marrow organoids) can be used for transplantation and / or survival assays of cells from patients with hematologic malignancies.

[0151] The organoids (e.g., bone marrow organoids) can be seeded with cells from a donor and used to track the cells to determine one or more of the following: survival, proliferation, and separation of transplanted cells, for example, for downstream functional testing. The organoids (e.g., bone marrow organoids) can also be used to study pathogenic remodeling of the bone marrow microenvironment associated with cancer, such as fibrosis induced by malignant clones in some patients with myeloproliferative neoplasms.

[0152] The cells can be tracked using fluorescent markers or tags. Tracking the cells may include using a fluorescent cell tracking system.

[0153] The cell donor can be an adult or a child donor.

[0154] For example, the methods or uses of the present invention may involve modeling fibrosis, including treating the bone marrow organoids of the present invention with agents that induce fibrosis and / or collagen deposition. Therefore, the present invention also provides a model for bone and / or bone marrow-related conditions (e.g., myelofibrosis), wherein the model comprises bone marrow organoids of the present invention that have been treated with agents that induce the condition. The model can be used to identify agents capable of preventing or treating the condition, including treating the model before, during, or after inducing the condition, and monitoring whether the agents have an inhibitory or mitigating effect on fibrosis development. The model can also be used to identify whether the agents inhibit or reduce the expression of smooth muscle actin and / or collagen.

[0155] The reagent can be used to induce fibrosis in bone marrow organoids of the present invention. The reagent can be used to induce extracellular matrix deposition in bone marrow organoids that respond to pro-fibrotic factors by increasing smooth muscle actin and collagen expression, leading to fibrosis. The reagent may contain or consist of growth factors or cytokines. The reagent (e.g., a cytokine) may be TGFβ. Bone marrow organoids can be treated with TGFβ to mimic fibrosis. Fibrosis can be induced genetically, for example by genetic modification, genetic overexpression of smooth muscle actin and / or collagen, or by siRNA silencing, or by treatment with other proteins or pharmacological agents. Fibrosis can be induced genetically, for example by overexpression of genes that induce αSMA / collagen expression, such as TGFB1 or other pro-fibrotic genes. Fibrosis can be induced by using iPSC lines from patients with fibrosis, or by gene editing of iPSCs to carry known pro-fibrotic genes.

[0156] The amount of reagent and the incubation time with the reagent can be sufficient to induce collagen deposition in bone marrow organoids, for example, at least 2 ng / ml TGFβ, or at least 10 ng / ml TGFβ, for at least 24 hours.

[0157] The amount and incubation time of TGFβ can be at least 2 ng / ml, for example, at least 5 ng / ml, for at least 2 hours, for example, at least 72 hours. The amount and incubation time of TGFβ can be between about 2 ng / ml and 500 ng / ml (for example, between about 5 ng / ml and 500 ng / ml), for a period of about 2 to 96 hours or longer (for example, about 24 to 96 hours or longer). The amount and incubation time of TGFβ can be between about 2 ng / ml and 500 ng / ml (for example, between about 5 ng / ml and 500 ng / ml), for a period of about 2 to 72 hours (for example, a period of about 24 to 72 hours). The amount and incubation time of TGFβ can be between about 2 ng / ml and 50 ng / ml (for example, between about 5 ng / ml and 50 ng / ml), for a period of about 24 to 72 hours. The amount and incubation time of TGFβ can be between approximately 2 ng / ml and 50 ng / ml (e.g., between approximately 5 ng / ml and 50 ng / ml), lasting for a period of approximately 36 to 72 hours. Technicians will understand that with higher concentrations, the incubation time can be shorter, and vice versa.

[0158] The organoids of the present invention can provide a source of immune cells and blood cells in in vitro culture systems, including body-on-a-chip approaches and organoid or microtissue co-culture. For example, the present invention provides a method for generating platelets and / or erythroid cells (RBCs), comprising: in vitro incubating the bone marrow organoids of the present invention; and harvesting platelets and / or erythroid cells (RBCs) generated from the bone marrow organoids. The platelets and / or erythroid cells (RBCs) can be naturally generated or induced from the bone marrow organoids, for example by administration of heparin, hirudin, and / or ROCKi to drive further proplatelet formation. The platelets and / or erythroid cells (RBCs) can be harvested by separating them from the cells of the bone marrow organoids (e.g., via FACS). The platelets and / or erythroid cells (RBCs) can be harvested by BSA gradient and / or centrifugation.

[0159] The organoids can also promote the maintenance and culture of the immune cells and blood cells.

[0160] In cases involving the maintenance and culture of blood cells, the methods and uses of the present invention can also be used as a method for maintaining the viability of cells from a patient donor suffering from leukemia in vitro.

[0161] The organoids of the present invention can provide allogeneic or autologous cells for cell therapy (e.g., transplantation or CAR-T therapy). The organoids can facilitate the maintenance and culture of the allogeneic or autologous cells.

[0162] To generate cells (e.g., blood cells and / or immune cells) suitable for cell therapy, bone marrow organoids of the present invention or disease-associated bone marrow organoid models of the present invention can be cultured, and cells released from the organoids can be collected. Cells can be released into a culture medium. To facilitate cell release from the organoids, the organoids can be grown on a low-attachment surface (e.g., on a culture shaker running at a suitable speed (e.g., 15-degree oscillation within 5-second intervals) and cultured under agitation. Released cells can be collected after differentiation (e.g., on day 28, 32, or 35). Therefore, the present invention also provides a method for generating cells for cell therapy, comprising the steps described herein. The present invention also provides cells obtained by said method.

[0163] Cells used in cell therapy can be hematopoietic stem cells, tissue cells, 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.

[0164] 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 hematopoietic stem cell therapy.

[0165] The organoids of the present invention (e.g., bone marrow organoids) can provide a method for generating hematopoietic cells and / or stromal cells. For example, the present invention also provides a method comprising:

[0166] a. Drive induced pluripotent stem cells (iPSCs) to form mesodermal aggregates;

[0167] b. The mesodermal aggregates were further cultured in a medium supplemented with recombinant BMP4, FGF2, VEGFA, FLT3L and SCF to induce vascular and hematopoietic characterization of the mesodermal aggregates;

[0168] c. The mesodermal aggregates are embedded in a hydrogel and the hydrogel is incubated in a budding medium to form a mixture of hematopoietic cells and stromal cells, the budding medium containing a culture medium supplemented with cytokines for differentiation suitable for stem cell maintenance.

[0169] The organoids of the present invention can also facilitate the production of kits / reagents for generating said organoids (e.g., bone marrow organoids).

[0170] The organoids or disease models of the present invention can be further processed (e.g., cryopreserved or frozen) so that they can be stored and distributed. Therefore, the present invention also provides a kit comprising cryopreserved or frozen organoids or disease models of the present invention.

[0171] The kit may contain one or more of the following: recombinant BMP4, FGF2, VEGFA, FLT3L, and SCF. The kit may contain or consist of VEGFA and FGF2. The kit may contain or consist of VEGFA, FGF2, BMP4, and VEGFC. The kit may contain or consist of VEGFA, FGF2, SCF, TPO, and FLT3L. The kit may contain or consist of VEGFA, VEGFC, FGF2, SCF, TPO, BMP4, and FLT3L. The kit may contain or consist of VEGFA, VEGFC, FGF2, SCF, TPO, EPO, BMP4, IL3, IL6, and FLT3L. Any kit may further contain IL7 and / or calcium.

[0172] The kit may further include a set of instructions. These instructions will enable the reader to perform any of the methods disclosed herein. Recombinant growth factors and / or cytokines may be provided in the kit as a single solution, or as two, three, four, five, six, seven, or more separate solutions. In this manner, one or more recombinant growth factors and / or cytokines may be provided in each solution, if desired. The kit may also contain mesodermal induction medium, budding medium, and / or hydrogel (crushed or otherwise) required to perform any of the methods disclosed herein.

[0173] The organoids of this invention can be used as a model platform for target prioritization and validation of novel therapies. To maintain consistency with the identification of novel therapies, the organoids of this invention can be used for high-throughput screening of candidate drugs for diseases, such as bone and / or bone marrow-related conditions as described herein. The responses of the organoids to novel therapies can be monitored to identify promising candidates for treating diseases.

[0174] The organoids of this invention can be used as an alternative to preclinical models and animal systems. The organoids can provide a first model of a disease, with animal systems only used if the organoid model shows promise.

[0175] The organoids of this invention can be used to predict the outcomes of stem cell transplantation or for pharmacogenomics testing. These organoids can provide a model system for monitoring the response of an organ / organ system to a foreign stem cell transplant.

[0176] The organoids of the present invention can be used as models of the tumor microenvironment and / or for studying metastatic diseases, such as prostate cancer metastasis to bone.

[0177] The organoids of the present invention can be used to provide a method for screening agents capable of preventing or treating fibrosis, for example using bone marrow organoids as described herein, wherein the bone marrow organoids have been genetically manipulated or treated with potential agents before, during, or after fibrosis-inducing treatment; and

[0178] A) Determine whether the potential reagent or genetic target has any effect on inhibiting or preventing the development of fibrosis in bone marrow organoids, or on reducing fibrosis after the development of fibrosis in bone marrow organoids;

[0179] B) Determine whether the potential reagent or genetic target has any effect in inhibiting or reducing smooth muscle actin and / or collagen expression or other fibrosis markers in bone marrow organoids; and / or

[0180] C) Determine whether the potential reagent or genetic target has any effect on inhibiting or reducing collagen deposition in bone marrow organoids.

[0181] The bone marrow organoids of the present invention can be used in one or more of the following:

[0182] A) Determine whether the reagent or potential reagent has any effect on reducing the survival or proliferation of cancer cells from the patient;

[0183] B) Determine whether the reagent or potential reagent has any effect on the pathogenicity of cancer cells from the patient;

[0184] C) Determine whether the agent or potential agent has any effect on reducing pathogenic remodeling of the organoid matrix or microenvironment / niche induced by cancer cells from the patient;

[0185] D) Screening for potential biomarkers of cancer in patients whose transplanted cells have been isolated;

[0186] E) After inoculation with cells from patients, study the clonal evolution of cancer in organoids to predict future cancer progression in patient donors;

[0187] F) Study the treatment response of cancer cells inoculated in organoids to determine the optimal treatment plan for donor patients;

[0188] G) Investigate the impact of cancer on its microenvironment and / or niche; and

[0189] H) Investigate the role of the microenvironment and / or niche in initiating or promoting cancer development and / or progression.

[0190] The organoids of the present invention (e.g., bone marrow organoids) can also be used to provide a method for maintaining cell viability in vitro from patient donors with leukemia, for mechanistic studies or screening of reagents or potential reagents, including one or more of the following:

[0191] A) Determine whether the reagent or potential reagent has any effect on reducing the survival or proliferation of cancer cells from the patient;

[0192] B) Determine whether the reagent or potential reagent has any effect on the pathogenicity of cancer cells from the patient;

[0193] C) Determine whether the reagent or potential reagent has any effect in reducing pathogenic remodeling of the organoid matrix induced by cancer cells from the patient;

[0194] D) Screening for potential biomarkers of cancer from patients whose implanted cells have been isolated;

[0195] E) Studying the clonal evolution of cancer in organoids after inoculation with cells from patients to predict future cancer progression in patient donors; and

[0196] F) Study the treatment response of cancer cells inoculated into organoids to determine the optimal treatment plan for donor patients.

[0197] The organoids of the present invention (e.g., bone marrow organoids) also provide models of bone marrow-related diseases (including leukemia), wherein the models comprise bone marrow organoids cultured with primary patient or donor cells, the cells subsequently reproducing disease features within the organoids.

[0198] The reagents to be screened or studied can be administered at physiologically relevant levels. The reagents to be screened or studied can be administered at therapeutically relevant levels. Combinations of reagents can be studied.

[0199] The determination can be relative to untreated bone marrow organoids (i.e., not treated with the potential reagent) and / or relative to a control or reference value.

[0200] The reagent to be studied can be a small molecule (e.g., less than 900 Da), nucleic acid, antibody therapy, cell therapy, pharmaceutical compound, metabolite, or peptide. The peptide may contain or be composed of an antibody. The reagent to be studied can be a genetic manipulation agent (e.g., siRNA, shRNA, CRISPR-CAS9, lentivirus, or retroviral vector), for example, for overexpression.

[0201] other

[0202] It should be understood that different applications of the methods, organoids, or compositions disclosed in this invention can be tailored to specific needs in the art. It should also be understood that the terminology used herein is for describing specific embodiments of the invention only and is not intended to be limiting.

[0203] Furthermore, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references, unless otherwise expressly provided.

[0204] In this article, references to cell aggregate size are understood to mean the average of the largest diameter of the aggregate. Aggregates are defined by clear edges and boundaries.

[0205] All publications, patents, and patent applications cited in this article, whether mentioned above or below, are incorporated herein by reference in their entirety.

[0206] The following examples illustrate the present invention.

[0207] Example 1 - A method using particulate hydrogels produced uniformly formed, consistent-shaped organoids, and Compared to organoids produced using bulk hydrogels, those with reduced extracellular matrix incorporation and increased density

[0208] The inventors compared the effects of using particulate hydrogels or bulk hydrogels in methods for producing bone marrow organoids. Schemes for both methods are provided in Example 6, and... Figure 1 Summary.

[0209] Bone marrow organoids generated using either bulk or granular hydrogels were fixed, embedded in paraffin, sectioned, and subsequently imaged. Granular hydrogel-based methods consistently produced organoids that were more rounded, smaller, and more uniformly formed than bulk hydrogel methods. Figure 2 A and 2B). When quantitatively analyzed, bone marrow organoids produced via the particulate hydrogel method exhibited more consistent diameter and structure. Figure 2 (C and 2D), which are key features of reproducible and scalable biological models. The smaller diameter of organoids generated from particulate hydrogel-based methods will enable automated liquid handling, which is crucial for large-scale applications.

[0210] It was also observed that organoids generated from bulk hydrogel-based methods contained excessive internalized hydrogels, which spread cells across the surface area and produced inconsistent structures. Figure 3 A). In contrast, organoids generated from particulate hydrogel-based methods are consistently more spherical, have less ECM incorporation, and therefore have higher cell density. Figure 3 B). It was also observed that the number of cells per organoid slice was significantly increased from organoids produced by the particulate hydrogel-based method compared to those produced via the bulk hydrogel method. Figure 3C). This structure and cellular organization will facilitate improved intercellular communication and interactions, and the smaller size of the resulting cell-rich organoids allows for improved scalability.

[0211] Therefore, this embodiment demonstrates that bone marrow organoids generated using particulate hydrogels have higher uniformity, cell number, and a size suitable for automation, which greatly increases their utility and application potential compared to bulk hydrogel methods.

[0212] Example 2 - Bone marrow organoids produced from the particulate hydrogel method exhibited similar vascular distribution, but compared with those using... Compared to those produced by bulk hydrogels, ossification enhancement

[0213] The structural complexity of bone marrow organoids generated by bulk hydrogel methods and particulate hydrogel methods was evaluated.

[0214] Bone marrow organoids were stained with CD34, a marker for vascular and stromal progenitor cells. Histological cross-sections of the organoids were then imaged.

[0215] Organoids generated from a bulk hydrogel-based approach produced a clear, CD34+ vascular system with luminal formation from which cells escaped, and large vessels aligned with the fibers of the bulk hydrogel. Figure 4 A). Organoids generated from the particulate hydrogel method also produced CD34+ luminous vessels, in which cells interacted with and escaped into the vascular space. Figure 4 B and Figure 4 C). Interestingly, the blood vessels in organoids derived from the particulate hydrogel method are generally smaller. Quantification of the total CD34+ blood vessel area in organoids derived from both the bulk and particulate hydrogel methods indicates that organoids derived from the particulate hydrogel method do not suffer from loss of blood vessel distribution (C). Figure 4 D).

[0216] Von Kossa staining (a specific calcification staining agent used to highlight areas of calcium / mineral deposition) was also performed on bone marrow organoids, indicating successful mineral deposition and development of ossification niches. In organoids produced via the bulk hydrogel method, dark, positive Von Kossa staining was completely negative. Figure 5 A), and this staining was observed in organoids produced by the particulate hydrogel method. Figure 5 B).

[0217] The in vitro generation of bone- or endosteal-rich, ossified niches has been a major challenge and limitation in this field. Positive Von Kossa staining observed in organoids generated from particulate hydrogel methods provides the first evidence of mineralization in complex, vascularized hematopoietic organoids and represents a significant step towards creating scalable, biomimetic environments for studying these organs and their applications as in vitro models. The use of particulate hydrogels is particularly effective because (a) the microparticles ensure the material is bioavailable (and fibrin and type I collagen have been shown to be essential for in vitro ossification), and (b) the smaller, more cellular organoids generated by particulate hydrogels promote greater intercellular interactions and drive niche changes.

[0218] Example 3 - Bone marrow organoids generated by the particulate hydrogel method contain key hematopoietic and stromal lineages and exhibit... It exhibits strong genealogical complexity.

[0219] After establishing that particulate hydrogels can be used to generate vascularized 3D cultures with consistent size, shape, and cellular composition through a simplified, one-step method, and after providing evidence of mineral deposition that supports ossification niche development, the cultures were differentiated to establish lineage complexity of organoids.

[0220] Bone marrow organoids generated in parallel using either bulk or particulate hydrogel methods were compared by flow cytometry. Three independent differentiations were performed, and the organoids were dissociated on day 35 for flow cytometry. Four flow cytometry panels were used to identify cells of the stromal and hematopoietic lineages, with three panels specifically used to identify lymphoid cells, myeloid cells, and stem cell hematopoietic cells, respectively.

[0221] The stromal cell group analysis showed no significant difference in cell diversity at the stromal level, and both methods identified key cell populations (endothelial cells, fibroblasts, BM MSCs, LEPR+BM MSCs, and osteoblast lineage cells). Figure 6 A). The lymphoid cell group again showed that both methods generated natural killer (NK) cells, B lineage cells, and T cell progenitor cells (thymocyte populations). Figure 6 B). The myeloid group revealed erythroid cells, megakaryocytes, myeloid mononuclear cells, as well as basophils and eosinophils, with a slightly decreased percentage of megakaryocytes observed. Figure 6C). Finally, stem cell populations were dissected using stem cell-specific assays, and the results showed no significant differences in the proportions of hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs), and myelo-erythroid progenitors (MEPs) generated by each method. Figure 6 D). Interestingly, in samples generated by particulate hydrogels, the variability between replicates in the HSC compartment was significantly smaller, indicating higher reproducibility in this compartment using the particulate hydrogel method.

[0222] Therefore, this embodiment demonstrates that the particulate hydrogel method successfully generates bone marrow organoids containing key hematopoietic cell lineages and stromal cell lineages.

[0223] Example 4 - Transplantation of bone marrow organoid support donor cells generated by particulate hydrogel method

[0224] To test the practicality of bone marrow organoids generated by the particulate hydrogel method as a platform for personalized or precision medicine, the ability of the organoids to support donor cell transplantation was tested.

[0225] Bone marrow organoids were prepared as described in Examples 1-3 and seeded with fluorescently labeled BAF3 cells (a B-lineage hematopoietic cell line). It was observed that after 48 hours, donor cells homed and filled the volume of the bone marrow organoids generated by the particulate hydrogel method. Figure 7 This data indicates that the system creates an environment in which donor cells can migrate and survive.

[0226] Furthermore, CD34+ cells from myelofibrosis patients were labeled with a fluorescent dye (CellTraceViolet) and implanted into bone marrow organoids generated using a particulate hydrogel method. After 7 days of incubation (from day 21 to day 28 of culture) before fixation and subsequent imaging using confocal microscopy, it was clear that the adult donor-derived cells had been transplanted and proliferated within the organoids. Figure 8 ).

[0227] Therefore, this embodiment demonstrates that bone marrow organoids generated by the particulate hydrogel method can successfully support the implantation of donor cells, for example, as a platform for personalized or precision medicine.

[0228] Example 5 - Bone marrow organoids generated by the particulate hydrogel method can be used to capture disease-specific microenvironments. Reshape

[0229] The usefulness of bone marrow organoids generated via particulate and bulk hydrogel methods as tools for capturing disease-specific microenvironment remodeling was evaluated.

[0230] Bone marrow organoids generated using both bulk and particulate hydrogel methods were implanted into CD34+ cells from healthy donors or myelofibrotic patients labeled with CellTrace Violet on day 21 of culture. After another 7 days of culture, the cells were fixed and subjected to immunofluorescence imaging. Imaging was performed on the samples for CellTrace Violet, αSMA (a marker of fibroblast activation, and therefore also a marker of fibrosis), and type I collagen.

[0231] Figure 9 The results showed that MF-transplanted organoids exhibited increased αSMA staining compared to organoids generated from particulate hydrogels and implanted with healthy donors, with an efficiency at least as high as that observed in organoids generated using the bulk hydrogel method. Figure 9 A and Figure 9 B).

[0232] Therefore, this embodiment demonstrates that bone marrow organoids generated by the particulate hydrogel method can support transplantation of primary cells from patients and can undergo matrix activation in response to cell transplantation, as indicated by αSMA expression.

[0233] Example 6 - Materials and Methods

[0234] The following materials and methods are applicable to Examples 1 to 6.

[0235] iPSC cultivation and maintenance

[0236] Human induced pluripotent stem cell lines (iPSCs) purchased from Gibco (Thermo) were maintained on GelTrex (Thermo) coated 6-well Corning culture plates. Cells were passaged at approximately 70% confluence using the ethylenediaminetetraacetic acid (EDTA) (clump passage) method. Briefly, cells were washed once with 1 mL of phosphate-buffered saline (PBS), then once with EDTA, and incubated at 37°C and 5% CO2 for 4 minutes. The EDTA was then aspirated, and cells were subsequently removed by pipetting StemFlex medium from the bottom of the wells. The resulting cell suspension was then re-seeded at the desired concentration onto freshly coated plates.

[0237] Differentiation scheme of particulate hydrogel method

[0238] Bone marrow organoids were generated using a directed differentiation approach. First, iPSCs were cultured to 70%-80% confluence, and then EDTA was removed as described above. After two pipetting cycles, the cells were resuspended in StemFlex medium supplemented with RevitaCell (Thermo) as recommended by the manufacturer. The resulting aggregates were then cultured in 6-well ultra-low adhesion (ULA) plates (Thermo-Nunc Sphera plates) and incubated overnight (12 hours) at 37°C and 5% CO2.

[0239] On the following day (Day 0), aggregates were collected using a P1000 pipette and transferred to 15 mL conical tubes by low-speed (300G) centrifugation. The supernatant was aspirated, and the aggregates were resuspended in Phase I medium (supplemented with 50 ng / mL bone morphogenetic protein-4 (BMP4) (PeproTech), fibroblast growth factor-2 (FGF2) (PeproTech), vascular endothelial growth factor A (VEGFA-165) (PeproTech), and 4 μM CHIR99201 APEL2) and incubated at 37°C, 5% O2, and 5% CO2 for 72 hours (from Day 0 to Day 3). After 72 hours, the mesodermal aggregates reached an average size of approximately 200-250 μm.

[0240] After 72 hours, cells were collected by gravity sedimentation in 15 mL conical tubes. The supernatant was aspirated, and the aggregates were then resuspended in phase II medium. The phase II medium consisted of APEL2 containing 50 ng each of BMP4, FGF2, and VEGFA, supplemented with 25 ng of Fms-associated receptor tyrosine kinase-3 ligand (FLT3L) and stem cell factor (SCF) (25 ng / mL) (PeproTech). Cells were cultured at 37°C, 5% O2, and 5% CO2 for another 48 hours, or under these conditions (usually day 5) until an average size of 350–500 μm was reached.

[0241] The next stage of the project involves preparing particulate hydrogels and embedding iPSC aggregates within the structure.

[0242] First, a hydrogel consisting of 40% (vol / vol) Corning growth factor matrix gel, 1.25 mg / mL fibrinogen (Thermo), 2 mg / mL VitroCol (Type I collagen, Advanced Biomatrix), and 1 mg / mL Type IV collagen (Advanced Biomatrix) was prepared on ice. The resulting mixture was neutralized with 1M NaOH, and 2 U / mL thrombin (Thermo) was added to drive fibrin formation. The hydrogel was carefully mixed on ice and then transferred to a sterile, sealed syringe. The hydrogel was allowed to solidify at 37°C and 5% CO2 for 90 minutes before further processing.

[0243] At 90 minutes, the hydrogel completely solidified and was ready to break down into a resuspendable ruptured hydrogel. First, the hydrogel was extruded through a syringe into a 6-well ULA plate containing 2 mL of APEL2. An 18G needle was attached to the syringe, and the extruded hydrogel resuspended in APEL2 was drawn into the syringe and extruded three times through the 18G needle. A new syringe with a 23G needle was prepared, and the hydrogel / culture medium suspension was again extruded three times through the syringe and 23G needle. The resuspended hydrogel visibly broke down into microgels or particulate suspensions. The above steps were repeated with a 25G needle, then with a 27G needle, and the ruptured hydrogel was collected in a 15 mL conical tube. The ULA plate was washed with 3 mL of APEL2 to collect any remaining microgel particles, and this culture medium was collected in the same 15 mL tube containing the ruptured gel material. The final volume of the resulting resuspended ruptured gel was 5 mL.

[0244] The ruptured gel solution was supplemented with 5% knockout serum (Thermo), 5 U / mL heparin (Merck), 100 ng / mL VEGFA, 50 ng / mL VEGFC, BMP4, FGF2, SCF, FLT3L, and 20 ng / mL EPO, TPO, IL3, and IL6.

[0245] Cell aggregates were collected by gravity sedimentation in 15 mL conical tubes. The aggregates were visible to the naked eye. The supernatant was aspirated, and the cell mixture was resuspended in a ruptured hydrogel prepared in APEL2. The resulting cell, cytokine, and ruptured gel suspension were placed in a reagent tank, and 50 μL of the suspension was added to each well of a 96-well ULA plate using a multichannel pipette.

[0246] After all wells have been filled, seal the plate with sealing film to maintain sterility and prevent accidental exposure and spillage. This process can be repeated to create multiple well plates. Centrifuge the suspension at 300G for 4 minutes using a plate centrifuge to form a compacted granular hydrogel. Then remove the sealing film.

[0247] The gel was examined using a benchtop bright-field microscope to confirm compaction. Special attention was paid to checking for the formation of solid structures encapsulating cell aggregates. The mixture was incubated at 37°C, 5% O2, and 5% CO2 for 48 hours. After 48 hours, on day 7, 50 μL of APEL2 supplemented with 5% knockout serum, 5 U / mL heparin, 50 ng / mL VEGFA, 25 ng / mL FGF2, BMP4, VEGFC, SCF, FLT3L, and 10 ng / mL EPO, TPO, IL3, and IL6 was added to each well. The cells were then incubated again at 37°C, 5% O2, and 5% CO2.

[0248] On day 10, myeloid factor was reduced, and IL7 was added to the culture to drive lymphoid development. APEL2 containing 50 ng / mL each of VEGFA, VEGFC, FGF2, hSCF, FLT3L, and IL7, and 10 ng / mL of IL3 and IL6 was added in a 50:50 ratio as previously described.

[0249] Cells were maintained at 37°C, 5% O2, and 5% CO2 until day 21. From day 12 onwards, the 50 / 50 medium was replaced every 48–72 hours (50 μL aspirated and 50 μL added), using APEL2 supplemented with L-ascorbic acid (final concentration 10 μg / mL), 1 mM calcium chloride, 2 mM β-glycerophosphate pentahydrate, 2% knockout serum, and 2% chemically defined lipids (Thermo). Growth factor supplementation was performed as follows:

[0250] Day 12: 20 ng / mL VEGFA, VEGFC, FGF2, SCF, FLT3L, IL7 and 10 ng / mL IL3, IL6.

[0251] Days 14, 17, 19 and 21: 20 ng / mL SCF, FLT3L, IL7, 10 ng / mL IL3, IL6, 5 ng / mL mCSF and EPO, and finally 1 ng / mL TPO.

[0252] On day 21, samples were optionally transferred to normoxic 20% O2 conditions to drive cell proliferation. From day 21 onwards, cells were also cultured in supplemented StemPro-34 medium (fortified with L-glutamine (1%), L-ascorbic acid (final concentration 10 μg / mL), 1 mM calcium chloride, 2 mM β-glycerophosphate pentahydrate, 2% knockout serum, and 2% chemically determined lipids). On days 21 and 24, the medium was supplemented with reduced growth factors (10 ng / mL hSCF, FLT3L, IL7, 5 ng / mL mCSF, IL3, IL6, EPO, and 1 ng / mL TPO).

[0253] From day 26 to day 33, the modified StemPro-34 was supplemented with 5 ng / mL EPO and IL7 and 1 ng / mL TPO. From day 33 onwards, only 5 ng / mL EPO and 1 ng / mL TPO were supplemented to mimic the physiological exposure of bone marrow to exogenous factors.

[0254] Differentiation scheme for bulk hydrogel method

[0255] The iPSC differentiation protocol for generating bulk hydrogel-derived organoids is identical to the protocol for granular hydrogel-derived organoids during days 0 to 5. On day 5 in both protocols, preparation is made to embed cells in the hydrogel and to prepare the hydrogel itself. The following protocol follows the bulk hydrogel method, which differs from the granular hydrogel method, starting from approximately day 5.

[0256] First, hydrogels with different compositions were tested to determine the optimal conditions for generating myeloid and bone marrow-specific lineages. Each matrix consisted of 40% Corning growth factor matrix gel and 60% type I or type IV collagen (CellSystems), or a mixture of type I and type IV collagen. All gels were prepared with a collagen concentration of 1 mg / mL. Gel preparation began on day 4, when aliquots of matrix gel were thawed overnight at 4°C. Gel mixtures were prepared on ice, and the collagen mixtures were neutralized with 1 M NaOH before being partitioned into 12-well cell culture plates. Each hydrogel polymerized for at least 90 minutes. A cell-free sublayer was prepared before collecting cells by gravity sedimentation and resuspending them in the remaining gel volume.

[0257] Once fully polymerized, cells were supplemented with 3D culture in Phase III budding medium, which consisted of APELII medium supplemented with 5% fetal bovine serum (FBS), 5 U / mL heparin sulfate, 50 ng VEGFA, 10 ng each of interleukin-3 (IL3) and interleukin-6 (IL6), and 25 ng 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 medium formulation until day 12, with the medium changed every 72 hours. From day 5 onwards, the medium was changed at a ratio of fresh to conditioned medium of 60:40. For experiments developing sinusoidal specific endothelial vascular systems, vascular endothelial growth factor C (VEGFC, Stem Cell Technologies) was supplemented in the medium on day 3.

[0258] The culture was allowed to sprout until an optimal size between 800 μm and 1.5 mm was observed, typically between days 10 and 12. At this stage, the sprouted bone marrow organoids were removed from the hydrogel and cultured individually in 96-well ultra-low adhesion plates. The harvested organoids were first scraped off with a sterile cell scraper and then pipetteed in excess culture medium into 15 mL Falcon tubes (Corning). The samples were then centrifuged at 500 G for 5 min to separate the organoids from the culture medium and collagen. The free organoids were then resuspended in the desired volume of culture medium and collected for individual culture in 96-well ultra-low adhesion plates.

[0259] From day 12 onwards, organoids derived from bulk hydrogels and organoids derived from particulate hydrogels were maintained in parallel.

[0260] Histology

[0261] Organoids were fixed in 15 mL Falcon tubes with 4% paraformaldehyde (PFA), washed three times with PBS, and then serially dehydrated in ethanol (30%, 50%, 70%, 100%) before being immersed in Histoclear (Geneflow, Cat#A2-0101). The samples were then embedded in paraffin and stained and mounted by the C&C laboratory as needed. Histological preparations were imaged using a Zeiss AxioScan.Z1 slide scanner.

[0262] Flow cytometry

[0263] Organoids were prepared for flow cytometry. After digestion and dissociation, samples were dissociated using 20 mg / mL type IV collagenase (Sigma) in sterile HEPES. Samples were collected by gravity sedimentation in 15 mL Falcon tubes and washed first with 10 mL PBS. After washing, the samples were incubated in the prepared collagenase solution at 37°C for 10 min, and then completely dissociated by pipetting. The single-cell suspension was washed, centrifuged at 500 G, and blocked on ice with 2% fetal bovine serum (FBS) in PBS for 15 min, followed by flow cytometry antibody labeling.

[0264] Using an optimized flow cytometry setup, stromal cells were identified as CD45-, CD71-, and CD235- cells. Hematopoietic cells were identified within the CD45+ phylum, except for being recognized as CD71+ / CD235+ erythroid cells.

[0265] Microscopic examination

[0266] Immunofluorescence was performed using a Zeiss LSM880 confocal microscope (10x air objectives). Samples were first fixed in 10% formalin and then washed three times consecutively with PBS for 5 minutes each time. The samples were then blocked overnight in a blocking buffer consisting of 2% goat serum (Thermo) and 1% bovine serum albumin (BSA) in 50 mL PBS. 250 μl of Triton X100 and Tween-20 (Sigma) and 500 μl of sodium deoxycholate (Sigma) were added to permeate the samples and promote antibody penetration. Primary antibody incubation was performed overnight at 4°C, followed by four consecutive washes with PBS for 5 minutes each time. The samples were then incubated again overnight at 4°C in a secondary antibody mixture: DAPI for nuclear labeling, and Alexa-488, Alexa-568, and Alexa-647 (Thermo), depending on the combination of antibody species present. For example, this protocol was used for Collagen I and αSMA staining in whole organoid labeling.

[0267] After labeling, the entire organoid was embedded in a small amount of 0.5% agarose in an Ibidi 8-well slide (Ibidi). After cooling, the gel was progressively dehydrated by a series of ethanol washes adjusted to pH 9. Finally, the sample was completely dehydrated in anhydrous ethanol and then cleared with ethyl cinnamate (SLS). The sample was then ready for confocal imaging.

[0268] Using adult primary cells to graft various organs

[0269] CD34+ hematopoietic cells from healthy donors and myelofibrosis patients were used and labeled with a fluorescent dye (CellTrace Violet). 5000 cells were seeded into organoids per well and cultured in StemPro for 8–14 days. On the collection day, the organoids were fixed for imaging or digested for flow cytometry evaluation.

[0270] BAF3 cell implantation

[0271] The BAF3 cell line used in the implantation experiment was stably transduced with fluorescent markers for easy identification.

[0272] Cell tracer markers for implantation assays

[0273] Following the manufacturer's instructions, label the patient's cells with CellTrace Violet. In short, wash the cells once with PBS and label them with 1x10⁻⁶ cells. 6 Cells were resuspended at a concentration of 10 cells / mL in staining solution (2 μM CellTrace Violet in PBS). Cells were incubated in staining solution at 37°C for 30 minutes. After incubation, cell tracers were quenched with 5 volumes of PBS containing 10% FBS, centrifuged, and resuspended in appropriate culture medium.

[0274] Example 7 - Single-cell RNA sequencing and analysis of bone / bone marrow organoids derived from particulate and bulk hydrogels

[0275] As described in Example 6, single-cell RNA sequencing was performed on organoids generated using bulk and particulate hydrogels on day 35. Single-cell annotation identified and confirmed the cell types observed in organoid samples derived from both particulate and bulk hydrogels by flow cytometry. Figure 10 ).

[0276] Surprisingly, although similar cell types were generated, a significant increase in the expression of bone marrow-inherent extracellular matrix (ECM) proteins was observed in the stromal compartment. Figure 11 ). Figure 11 The results showed that, compared with the bulk approach, key ECM proteins known to be crucial to the natural biology of bone marrow—namely, type I and type III collagen (COL1A1, COL3A1), fibronectin, and periostrin—were significantly upregulated in the relevant stromal cells (fibroblasts, MSCs, and osteoblastic lineage cells) generated from organoids derived from granular hydrogels. Therefore, organoids generated based on the granular hydrogel approach better mimic the natural expression of key ECM proteins (Bandyopadhyay, Shovik et al., (2024), Cell, Volume 187, Issue 12, 3120-3140.E29), promoting the observed improved mineralization. Figure 5 and Figure 17 ).

[0277] ECM protein expression is influenced by a negative feedback loop in which excess ECM in the environment inhibits cellular expression of these proteins, thus limiting their sensitivity and development in modeling disease-induced changes in ECM expression, such as fibrosis. Increased bone marrow ECM proteins were observed in organoids grown in granular hydrogels, suggesting that excess hydrogel presented by bulk hydrogel methods may suppress cellular expression of ECM proteins.

[0278] Therefore, this embodiment demonstrates that, compared to bulk hydrogel methods, organoids generated in particulate hydrogels better express native ECM proteins and better mimic the physiological behavior of these cells and the organoids as a whole. Similarly, this may enable better modeling of diseases involving ECM proteins, such as fibrosis.

[0279] Example 8 - Implantation of organoids with primary multiple myeloma samples to simulate the disease

[0280] Multiple myeloma is a classic blood cancer in which malignant plasma cells hijack the bone marrow to support the proliferation of diseased cells, sacrificing normal hematopoietic function. The disease typically involves the loss of normal myeloid cell production (because plasma cells drive local inflammation), remodeling of the bone-rich microenvironment (reduced osteoblast formation, replaced by osteoclasts that support bone resorption), and increased bone marrow fat. Modeling this disease using conventional tools is very difficult, with challenges including the lack of mouse models that capture disease heterogeneity and the difficulty of culturing multiple myeloma cells in vitro.

[0281] The inventors have demonstrated the utility of bone marrow organoids derived from particulate hydrogels in supporting the growth of multiple myeloma cells and capturing the complexities of bone marrow microenvironment remodeling caused by the disease.

[0282] CD138+ plasma cells from three myeloma patients were labeled with CellTrace Violet, and 20,000 cells were added to each organoid on day 21 of differentiation. In parallel, CD34+ stem / progenitor cells from healthy donors were cultured in organoids for 14 days. Figure 12 a) Take images regularly to ensure successful cell implantation into the organoid. Figure 12 b). On day 14 post-implantation, organoids were dissociated and collected for single-cell RNA sequencing, cell smears, and histology. Cell smears of dissociated organoid samples ( Figure 12 c) Plasma cells were confirmed in all multiple myeloma implanted samples, but not in healthy donor controls. Similarly, histology of organoids on day 35 ( Figure 12 d) The presence of myeloma cells in the organoids implanted with myeloma samples was confirmed.

[0283] Single-cell RNA sequencing was performed on organoids on day 35 of differentiation. Unimplanted and healthy donor-implanted organoids served as controls, and parallel generation was performed with multiple myeloma-implanted organoids (from 3 myeloma patients). Analysis was performed using the Seurat package, identifying and naming cell types based on classical gene expression. A total of 45,706 hematopoietic cells and 14,577 stromal cells were identified from 6 samples. Hematopoietic cells included a complete myeloid lineage (erythroids, megakaryocytes, eosinophils / basophils / mast cells, monocytes / macrophages, dendritic cells) and lymphoid compartments (T lineage cells, patient-derived plasma cells, lymphoid cells). This included patient-derived plasma cell clusters, indicated by red circles in the figure (…). Figure 13 a). Stromal cells include endothelial cells, osteoblastic lineage cells, adipose-derived cell lines, fibroblasts, and mesenchymal stem cells ( Figure 13 ).

[0284] Gene set enrichment analysis (GSEA) comparing organoid implantation in multiple myeloma patients with controls showed increased levels of inflammatory response genes (interferon-α, interferon-γ, TNFα / NFkB, IL2 / IL6) in key stromal cell types (CD271+ MSCs, osteoblast lineage cells, osteoblasts) and inflammatory hematopoietic cells (macrophages and dendritic cells). Figure 14 ).

[0285] Violin plots of specific genes known to be closely related to the pathogenesis of multiple myeloma show a typical increase in TNF, S100A4, S100A8, and S100A9 in key hematopoietic cells (macrophages, dendritic cells, and neutrophils). Figure 15 a). Similarly, patient cells drove typical changes in matrix behavior, reproducing the complexity of human disease. Multiple myeloma implanted organoids showed increased B2M (a factor closely associated with myeloma), although the specific cellular origin remains unclear. Finally, osteoblasts in multiple myeloma implanted organoids showed significantly reduced expression of NFIA and NFIB transcription factors, which are crucial for osteoblastogenesis. Figure 15 b).

[0286] Therefore, this embodiment demonstrates the extensive and significant remodeling of the bone marrow microenvironment following multiple myeloma cell implantation. Notably, the observed changes in stromal cell metabolism and cell fate, along with significant inflammation, faithfully capture the multi-lineage and highly complex nature of this disease. This data is first-in-class and provides a viable preclinical model for complex, multi-lineage diseases like multiple myeloma.

[0287] Example 9 - Generation of blood cells from bone marrow organoids

[0288] The primary function of bone marrow is to produce the large amounts of blood and immune cells necessary for healthy hematopoiesis. The inventors' bone marrow system is capable of producing an unprecedented variety of different hematopoietic cell types. To determine whether this method can reliably produce cells for therapeutic purposes (such as cell therapy), the inventors designed an experimental workflow in which bone marrow organoids are placed on a shaker ( Figure 16 a, simulating flow through single-phase oscillations, to induce fluid flow through the organoid's volume.

[0289] This agitation is sufficient to drive the outflow of round hematopoietic cells over the next 14 days (days 21-35). Figure 16 b、 Figure 16 c and Figure 16 d). Collect the released cells ( Figure 16 d) Used for cell centrifugation smears and annotation ( Figure 16 e). Therefore, this embodiment demonstrates that bone marrow organoids prepared by the methods described herein are particularly suitable for producing blood and immune cells for cell therapy.

[0290] Example 10 - Materials and Methods

[0291] The following materials and methods are applicable to Examples 7-9.

[0292] Single-cell RNA sequencing and analysis

[0293] Cells derived from healthy donor hiPSC stem cell lines (Gibco, Life Technologies) were used for bone marrow organoid differentiation using both bulk and granular hydrogel protocols, as described in Example 6. On day 35, cells were dissociated using type IV collagenase and then single-cell sorted into phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA). Cells were collected from a total of four independent differentiations and cryopreserved prior to processing. 100,000 viable cells were sorted, as confirmed by 7AAD staining. 35,000 viable cells were then used for GEM preparation using a 3' high-throughput (3' HT, v3.4 Chemistry 10X Genomics) kit according to the manufacturer's protocol. The resulting emulsion was then used for library preparation and sequencing.

[0294] Data analysis was performed using a standard 10x genomics workflow. In short, demultiplexed sequencing data were analyzed using CellRanger (v7.1), followed by preprocessing with CellBender (v0.3.2) to denoise the resulting count matrix. Further analysis was performed using Seurat Pipeline (v5) for integration, clustering, annotation, and differential gene expression analysis.

[0295] Organoid implantation experiments (e.g., simulating multiple myeloma)

[0296] Organoids were generated using the described particulate hydrogel method. On day 21 of the differentiation protocol described in Example 6, cryopreserved primary CD138+ enriched cells and CD34+ healthy donor cells were thawed and resuspended at 20,000 and 5,000 cells / 50 μL, respectively. Then, 50 μL of each cell solution was added to each individual organoid in a 96-well culture plate.

[0297] After the addition of exogenous cells, the culture medium was changed regularly at 50:50 intervals every 72 hours to maintain the plate. The culture medium consisted of StemPro-34 (ThermoScientific) supplemented with penicillin / streptomycin (1%), IL7 10 ng / mL, EPO 5 ng / mL, and TPO 1 ng / mL. On day 14 post-implantation, 12 organoids were fixed for paraffin embedding and histology, and approximately 20 organoids were dissociated for cell centrifugation smears and single-cell RNA sequencing, as described in the previous section.

[0298] Cell agitation and collection: To determine whether the organoid system could serve as a source of released blood cells, individual organoids were cultured in ultra-low adhesion plates (6 wells, NUNC Sphera) and placed on incubator shakers (Mimetas) set to oscillate at 15-degree intervals for 5 seconds. Cells were periodically collected from the “release” from each well and subjected to Giesma staining and cell centrifugation smears.

Claims

1. A method for generating organoids, comprising culturing cells in a hydrogel microparticle suspension.

2. The method according to claim 1, wherein, The hydrogel microparticles have an average diameter of 10 μm-500 μm.

3. The method according to claim 1 or 2, wherein, The hydrogel microparticles are obtained by fracturing the blocky hydrogel, optionally by chemical and / or mechanical fracturing.

4. The method according to any one of the preceding claims further includes optionally compacting the hydrogel microparticles by centrifugation or vacuum-driven filtration to generate particulate hydrogels.

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

6. The method according to claim 4 or 5, wherein, The cells are added after the hydrogel microparticles are compacted.

7. The method according to any one of the preceding claims, comprising producing a multilayer compacted mixture comprising hydrogel microparticles and cells by repeating the steps described in claims 4 and 5.

8. The method according to any one of claims 4 to 7, wherein, The compacted mixture contains about ≥0.1% w / v, for example, from 0.1% w / v to 60% w / v hydrogel microparticles.

9. The method according to any one of the preceding claims, wherein, The hydrogel microparticles contain polypeptide chains, polysaccharide chains, and / or extracellular matrix proteins.

10. The method according to any one of the preceding claims, wherein, The hydrogel microparticles comprise: (a) a mixture of collagens, such as type I collagen and / or type IV collagen; and / or (b) fibrin, such as matrix collagen, fibrin, type I collagen and type IV collagen.

11. The method according to any one of the preceding claims, wherein, The cells are induced pluripotent stem cells (iPSCs) or cells differentiated from them, such as mesodermal aggregates.

12. The method according to any one of the preceding claims, wherein, The organoid in question is a bone marrow organoid.

13. A method for generating bone marrow organoids from pluripotent stem cells, such as iPSCs, the method comprising: (i) Driving the pluripotent stem cells, such as iPSCs, to form mesodermal aggregates; (ii) The mesodermal aggregates from (i) are cultured into hematopoietic and vascular lineages in a medium containing hematopoietic and matrix-supporting cytokines such as BMP4, FGF2, VEGFA, SCF, and FLT3L; and (iii) Adding the mesodermal aggregates from (ii) to the hydrogel microparticle suspension; and (iv) Cultivate the mixture from (iii) to form bone marrow organoids.

14. The method according to claim 13, wherein, Step (i) includes culturing the iPSCs to induce the formation of iPSC aggregates, and culturing the iPSC aggregates in a stem cell maintenance medium supplemented with BMP4 to induce the formation of mesodermal aggregates.

15. The method according to claim 13 or 14, wherein, Step (ii) includes culturing the mesodermal aggregates for a period of time until the mesodermal aggregates have an average size of about 100 μm to 500 μm and / or have markers of early endothelial and hematopoietic differentiation.

16. The method according to any one of claims 13 to 15, wherein, Step (iii) includes culturing the mesodermal aggregates in a hydrogel microparticle suspension according to any one of claims 1 to 12.

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

18. The method according to any one of claims 13 to 17, further comprising: (v) Add more cells to the bone marrow organoid from step (iv), optionally wherein the more cells are obtained from a subject, such as a subject with a bone marrow pathology.

19. An organoid that can be obtained or acquired by the method of any one of the preceding claims, optionally wherein, The organoid in question is a bone marrow organoid.

20. The organoid according to claim 19, wherein, The bone marrow organoids include: (a) A network of blood vessels that form the lumen; (b) Stromal cells, including mesenchymal stem cells (MSCs), fibroblasts, and / or endothelial cells; (c) Hematopoietic cells, including hematopoietic stem and progenitor cells (HSPCs), erythroid cells, myeloid mononuclear cells, and / or megakaryocytes; and (d) Osteoblastic lineage cells; And optionally (e) mineral deposits.

21. A bone marrow organoid comprising: (a) A vascular network forming a lumen, optionally wherein the vascular network is composed of bone marrow-specific endothelial cell subsets (e.g., arterioles, sinusoids, H-type capillaries); (b) Stromal cells, comprising mesenchymal stem cells (MSCs) and bone / fat / CAR lineage cells differentiated from MSCs, fibroblasts and / or endothelial cells; (c) Hematopoietic cells, including hematopoietic stem and progenitor cells (HSPCs), hematopoietic stem cells (HSCs), and myeloid and lymphoid lineage cells, including but not limited to T lineage progenitor cells, B lineage cells, natural killer cells, erythroid cells, eosinophils / basophils / mast cells, megakaryocytes, and myeloid mononuclear cells; and (d) Osteoblastic lineage cells; and optionally (e) mineral deposits; And choose any one of them, The bone marrow organoids can be obtained or acquired by the method according to any one of claims 1 to 17, and / or wherein the bone marrow organoids have an average vascularized area of ​​5% or higher.

22. The use of a bone marrow organoid according to any one of claims 19 to 21 as a model for studying hematopoietic and stromal cell biology in healthy, aging and diseased individuals, and for studying immune responses to infection and inflammation.

23. A method for preparing a bone marrow organoid disease model, comprising seeding donor cells onto bone marrow organoids according to any one of claims 19 to 21, optionally wherein, The donor is an individual suffering from cancer, such as leukemia.

24. A disease model that can be obtained or acquired by the method of claim 23.

25. A model for bone and / or bone marrow-related conditions such as myelofibrosis, wherein, The model comprises bone marrow organoids according to any one of claims 19 to 21 that have been treated with a reagent that induces the disease.

26. The use of the model of claim 25 for identifying an agent capable of preventing or treating the disease, comprising treating the model before, during or after inducing the disease.

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

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

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

30. A cell for cell therapy that can be obtained or acquired by the method of claim 28 or 29.

31. A method for identifying an agent used to treat or prevent a disease, comprising: Adding a reagent to a disease model comprising an organoid transplanted with diseased cells from a patient, as described in any one of claims 19 to 21, wherein improvement in the disease state of the disease model indicates the effectiveness of the reagent in treating or preventing the disease; Optionally, the method further includes adding the reagent to a reference model and comparing the effects of the reagent on the reference model and the disease model, the reference model comprising an organoid of any one of claims 19 to 21 transplanted with healthy cells.

32. The method according to claim 31, wherein, The disease is a bone and / or bone marrow-related condition, such as myelofibroma, myelofibrosis, or blood cancer, such as multiple myeloma, myeloma, metastatic solid tumor, leukemia (e.g., acute or chronic lymphocytic leukemia, or acute or chronic myeloid leukemia), myelodysplastic syndrome, myeloproliferative neoplasm, lymphoma, or mast cell tumor.

Images