Thymic organoids

Abstract

The present invention provides thymic organoids, methods for their production, hydrogel scaffolds useful in producing thymic organoids, and uses of the thymic organoids, in particular their use in producing human T cells and lymphoid progenitor cells, as well as their use in methods of treating immune disorders and drug screening.

Description

THYMIC ORGANOIDSFIELD OF THE INVENTION

[0001] The present invention relates to thymic organoids, methods for their production, hydrogel scaffolds useful in producing thymic organoids, and uses of the thymic organoids, in particular their use in producing human T cells and lymphoid progenitor cells, as well as their use in methods of treating immune disorders and drug screening.BACKGROUND

[0002] There is a clinical need for restoring T cell immunity in patients with life-threatening combined immunodeficiencies, such as athymic (complete) DiGeorge syndrome (cDiGS), FOXN1 -deficiency and other congenital conditions. These patients suffer from severe infections and, in absence of treatment by allogeneic thymus transplantation that restores minimal T cell numbers, this condition is fatal in the first or second year of life.

[0003] Transplantation (Tx) of allogeneic thymic tissue slices in paediatric cDiGS patients, though partly efficacious, manifests clinical complications and requires immunosuppression. This treatment provides only limited T cell reconstitution and tissue minimal processing does not remove donor thymocytes that can react against and damage the patient’s tissues. Additionally, the heterogeneity of tissue slices affects quality and variability of donors affects the reproducibility and efficacy of the treatment. Autoimmunity (e.g. cytopenia) occurs in as many as 70%, and Graft versus Host Disease (GvHD) is a serious risk. Matched donor blood is not possible in most cases owing to the urgency of the treatment, thus increasing the risk for these complications. Finally, transplanting tissue intrinsically lacks wider scalability and flexibility to meet medical needs.

[0004] Alternative strategies to establish T cell immunity for cDiGS is a priority. Restoring human thymus-specific stromal epithelial, mesenchyme and vascularization to an acellular scaffold as a potential bio-engineered thymus is an alternative to the current clinical solution and we have engaged with clinicians at GOSH (Prof Graham Davies) and an SME (Videregen) to develop a cure based on bioengineered thymic constructs. Decellularised scaffolds processed from unrelated human donors are clinically compliant. A tissue-engineered thymus is a potentially curative therapy for children who have a fatally deficient immune system. However, the process of developing this approach into an Advanced Therapy Medicinal Product (ATMP) is challenging especially for the complexity of the GMP manufacture which includes a donor matrix component. Organ-on-a-chip devices have significant advantages compared to tissue slices, as they are potentially highly reproducible, scalable and show minimal variability.

[0005] Therefore, an alternative approach that may represent a therapeutic solution in the short-term and that holds the potential to become a therapeutic providing long-term benefit, is represented by the use of Adoptive Cell Therapy (ACT) to directly restore T cell immunity in these patients. ACT is an approach used - in particular for autologous T cells - but it is currently restricted to cancer therapies as personalised medicine which target specific antigens. Instead, athymic patients are in need of T cells able to respond to pathogens that the body has not encountered yet (naive T cells). Naive T cells develop only in the thymus. Donor-derived naive T cells are not an option as they would attack also own patients’ healthy cells causing GvHD.

[0006] Accordingly, there is a need in the art for approaches which can provide naive T cells for adoptive cell transfer in individuals with athymia and related conditions, in order for them to maintain immunity.

[0007] The thymus is a primary lymphoid organ that controls, supports and regulates the composition of a broad, self-tolerant T cell repertoire necessary for our immune system to fight infections, tumours and avoid autoimmunity. The development of mature T cells is called thymopoiesis and depends on serial steps that occur in specific compartments of the thymus, namely the cortex and the medulla. Haematopoietic precursors cells (HSPC) migrate from the bone marrow (BM) and enter the thymus where they become committed to the T-cell lineage (thymocytes). According to the stage of their development, thymocytes upregulate specific surface molecules including CD3, CD4 and CD8. In early stages the precursors are triple negative (TN, CD3-CD4-CD8-), then they start rearranging their T-cell receptor (TCR) genes and upregulate CD3 but also CD4 and CD8 (double positive, DP). DP thymocytes undergo positive and negative selection resulting in the elimination of non-reactive and self-reactive T cells respectively. Successful thymopoiesis occurs thanks to the lympho-stromal crosstalk between the developing thymocytes and professional antigen-presenting cells (APC) that in the thymus are represented by thymic epithelial cells (TEC). Tissue Restricted (self) Antigens (TRA) promiscuously generated in TEC are presented to the developing thymocytes. T cells recognising TRA are either deleted or develop down the pathway of regulatory CD4+ T cells (Treg). Once mature, single positive (SP) naive T cells emigrate to the periphery as SP CD4+ or CD8+ cells where they coordinate the function of our adaptive immune system.

[0008] Approaches for engineering microenvironments able to support conventional T cell maturation ex vivo would be of great relevance in a number of clinical conditions where T cell immunity is deficient or abnormal and therefore several attempts have been pursued. These include co-culture systems with Notch ligand-expressing murine bone marrow (BM) stromal cells - called artificial thymic organoids (ATOs) such as those in documents [1 ,2,3], the contents of which are incorporated herein by reference. These systems are currently constrained by limited capacity for lineage differentiation due to the following issues:1. Absence of human thymic epithelial cells (TEC) and thus of professional antigen presenting cells (APCs) expressing human MHC-molecules. MHC-restricted antigen presentation is necessary for proper T cell selection via MHC-TCR interaction.2. Bias towards CD8+ unconventional, non-MHC-restricted T cells and absence of CD4+ T cells owing to the absence of MHC-class Il-dependent positive selection.3. Absence of negative selection mechanisms owing to the lack of specialised APCs (i.e. medullary TEC, mTEC).4. Lack of TEC organization and compartmentalization (cortex and medulla, cTEC and mTEC), resulting in less diverse T cell repertoire.5. Absence of thymic mesenchymal cells named thymic interstitial cells (TIC). Thymus 3D organisation relies on interaction between TEC and TIC which also produce essential cytokines for T cell development (e.g., CXCL12).6. Notch-ligands are necessary to induce lymphoid lineage of differentiating HSPC. ATOs use murine BM stromal cell lines overexpressing DLL1 or DLL4 with consequent variable expression levels; additionally, murine cells may pose translational issues.7. ATOs, which are of very small size (200K cells / aggregate), have low T cell output and are non- scalable.

[0009] Thus, there is a need in the art for systems capable of producing human T cells which overcome the above issues.SUMMARY OF THE INVENTION

[0010] In a first aspect, the invention provides a thymic organoid comprising a hydrogel scaffold and a plurality of thymic stromal cells.

[0011] In a second aspect, the invention provides a method for producing a thymic organoid, comprising: (a) providing a hydrogel scaffold; (b) seeding the hydrogel scaffold with at least one thymic stromal cell, wherein the at least one thymic stromal cell comprises at least one isolated thymic epithelial stem cell, at least one isolated thymic interstitial cell, or a combination thereof; and (c) culturing the at least one thymic stromal cell.

[0012] In a third aspect, the invention provides a hydrogel scaffold comprising at least one thymic extracellular matrix protein or fragment thereof.

[0013] In a fourth aspect, the invention provides use of a hydrogel scaffold for producing a thymic organoid.

[0014] In a fifth aspect, the invention provides a method for producing a human T cell, comprising: (a) providing a thymic organoid as described herein, (b) seeding the thymic organoid with at least onelymphoid progenitor cell, at least one haematopoietic stem cell, or a combination thereof, and (c) culturing the thymic organoid under conditions suitable for T cell development.

[0015] In a sixth aspect, the invention provides a method for producing a lymphoid progenitor cell, comprising (a) providing a thymic organoid as described herein, (b) seeding the thymic organoid with at least one haematopoietic stem cell, wherein the haematopoietic stem cell is CD34+, and (c) culturing the thymic organoid under conditions suitable for differentiation of the at least one haematopoietic stem cell into a lymphoid progenitor cell.

[0016] In a seventh aspect, the invention provides a human T cell obtained or obtainable by the methods disclosed herein.

[0017] In an eighth aspect, the invention provides a human lymphoid progenitor cell obtained or obtainable by the methods disclosed herein.

[0018] In a ninth aspect, the invention provides a method of treating an immune disorder in a subject, the method comprising administering a human T cell obtained or obtainable by the methods disclosed herein or a human lymphoid progenitor cell obtained or obtainable by the methods disclosed herein to the subject.

[0019] In a tenth aspect, the invention provides a human T cell obtained or obtainable by the methods described herein or a human lymphoid progenitor cell obtained or obtainable by the methods described herein for use in a method of treating an immune disorder in a subject.

[0020] In an eleventh aspect, the invention provides a method of treating an immune disorder in a subject, the method comprising implanting a thymic organoid as disclosed herein in the subject.

[0021] In a twelfth aspect, the invention provides the use of a thymic organoid as disclosed herein as a thymus implant.

[0022] In a thirteenth aspect, the invention provides a method of screening for agents that modulate T cell development, the method comprising: (a) seeding a thymic organoid as disclosed herein with at least one lymphoid progenitor cell, at least one haematopoietic stem cell, or a combination thereof, (b) adding a candidate agent to the thymic organoid, (c) culturing the thymic organoid under conditions suitable for T cell development, and (d) determining the effect of said agent on T cell development.BRIEF DESCRIPTION OF THE DRAWINGSFigure 1 : Human thymic epithelial and interstitial cells seeded onto high (HMW) (A) and low (LMW) (B) molecular weight alginate-RGD polymer after 7 days of expansion. Z-stack 3D image showing thatthymic stromal cells are re-organising within the matrix with different patterns. Epithelial cells are stained mostly as double positive for KRRT5 / 14 and KRT8 / 18 and are also positive for Vimentin . Single Vimentin positive cells indicate interstitial cells.Figure 2: Human thymic epithelial and interstitial cells seeded onto high (HMW) (A) and low (LMW) (B) molecular weight alginate-RGD polymer after 7 days of expansion and differentiation in air liquid interphase. Z-stack confocal image showing that thymic epithelial cells differentiate into single positive KRT5 / 14 medullary and or LY75 cortical cells within the matrix in both conditions. Some sparse epithelial cells stain for KRT10, a marker of medullary Hassall’s Bodies.Figure 3: Human thymic epithelial and interstitial cells seeded onto high (HMW) (A) and low (LMW) (B) molecular weight alginate-RGD polymer after 7 days of expansion have been cocultured for an additional 7 days with total thymocytes. Z-stack confocal image showing that thymic epithelial cells positive for KRT5 / 14 interacts with human CD45 positive T cells within the 3D matrix. CD3 positive T cells are more abundant in LMW condition. Some cells also stain for MHC class II molecule HLADR-A.Figure 4: Cytokines in 3D Engineered Thymic Organoids (ETOs) were analysed utilising the GeniePlex Multiplex Immunoassays, employing a similar principle of a sandwich assay, where fluorescent beads conjugated with a specific antibody trap the cytokine of interest and recorded with a Fortessa Flow Cytometry analyser. The following cytokines were analysed: CCL19 (A), CXCL12 (B), IL-6 (C), IL-7 (D) in cFAD media (first columns), Pneumacult (second columns), or co-culture (third columns).Figure 5: Exemplary functionalisation methodologies.Figure 6: Human thymic epithelial and interstitial cells embedded into the hydrogel (alginate with ECM ligands) with (A) and without (B) Matrigel®, after 7 days of expansion. Confocal images showing that thymic epithelial cells positive for KRT5 / 14 demonstrate proliferation (nuclear mKi67, white arrows) and remodeling of the matrix with thymic interstitial cells (TE7) in the presence and absence of Matrigel®.Figure 7: Human thymic epithelial and interstitial cells embedded into hydrogel with increasing alginate- ECM ligand content, after 7 days of expansion. Confocal 20x images showing that thymic epithelial cells positive for KRT5 / 14 and thymic interstitial cells can migrate and remodel the 3D matrix with a minimum composition of 50% ECM ligand. Scale bar: 50 / zm.Figure 8: Human thymic epithelial and interstitial cells embedded into hydrogel 3D matrix with increasing viscoelastic properties, after 7 days in differentiation medium. Confocal 2x2 tiles showing that thymic epithelial cells positive for KRT5 / 14 reorganize into differentiated structures, particularly in the LMW stiff sample. Scale bar: 50 / zm.Figure 9: Infiltration and long-term survival of human thymocytes co-cultured with human thymic stromal cells in 3D hydrogel matrices. (A) Representative flow cytometry plots depicting the profile of human total thymocytes seeded in the 3D-ETOs. (B) Confocal 20x image (left) and Z-stack (right) depicting infiltration of thymocytes (small nuclei) within all planes of the 3D matrix. (C) Representative flow cytometry plot depicting profile of CD45+ thymocyte precursors seeded onto 3D-ETOs and cultured for 28 days giving rise to CD3+ T cells.Thymic organoid

[0023] As used herein, the term “thymic organoid” or “engineered thymic organoid” refers to the composition of thymic cells and hydrogel scaffold, in which the thymic cells are organised into a thymuslike structure. The thymic organoid can reproduce the chemical, physical, structural and / or cellular characteristics of thymus tissue. In particular, thymic organoids described herein comprise a 3D network resembling the histologically native thymus. Generally, thymic organoids have a cortical portion and a medullary portion, though this may not always be the case depending on the application. The thymic organoids described herein can be distinguished from artificial thymus organoids (ATOs) in the art in that they do not comprise DLL1 / DLL4-overexpressing mouse bone marrow stromal cells. ATOs also do not generally comprise a matrix or scaffold, and further do not comprise components of the thymus such as medullary or cortical portions. ATOs also do not include thymic epithelial or mesenchymal components.

[0024] A thymic organoid as described herein may comprise a hydrogel scaffold and a plurality of thymic stromal cells. The plurality of thymic stromal cells may comprise at least one thymic epithelial stem cell, at least one thymic interstitial cell, at least one cortical thymic epithelial cell, at least one medullary thymic epithelial cell, or a combination thereof. The exact composition of the cells may vary depending on the age or developmental stage of the thymic organoid. By way of example, in order to produce the thymic organoid, a hydrogel scaffold may be seeded with thymic epithelial stem cells and / or thymic interstitial cells. Over time, said cells will differentiate into cortical and medullary thymic epithelial cells to produce the thymic organoid. The plurality of thymic stromal cells may thus comprise at least one cortical thymic epithelial cell and at least one medullary thymic epithelial cell.

[0025] Thymic epithelial stem cells have been described in PCT / EP2023 / 056975, the contents of which is incorporated herein by reference. In particular, isolated thymic epithelial stem cells have been shown to be capable of differentiation into both cortical and medullary thymic epithelial cells, irrespective of whether the initial cell was a cortical thymic epithelial stem cell or a medullary thymic epithelial stem cell. By “differentiation”, it is meant the ability of a dividing cell (for example, an isolated thymic epithelial stem cell) to change its functional or phenotypical type, to give rise to a different cell type.

[0026] In general, isolated thymic epithelial stem cells are BCAMP°S, CD49FP°S, CD90P°Sand CD24nes Isolation of thymic epithelial stem cells using such markers can be achieved by methods known in theart. Thymic epithelial stem cells generally also express at least one cytokeratin gene, for example a cytokeratin gene selected from the group consisting of KRT5, KRT8, KRT13, KRT14, KRT15, KRT17, KRT18, and KRT19, or combinations thereof.

[0027] Isolated thymic epithelial stem cells generally express at least one gene selected from the group consisting of EPCAM, CD49F, FN1 , TIMP1 , IFITM3, VCAM1 , BCAM, LIFR, CEPBD, CLU, CCL19, CH25H, COL7A1 , CTGF, APOE, FGFR2, BOC, ITGA5, SOX17, YAP1 , PTGDS, CD34, VWF, SPARC, CAV-1 , EPAS-1 , TIMP3, COL4A2, COL5A1 , COL6A3, TP63 (for example ANTP63a) and cMYC, and combinations thereof.

[0028] The isolated thymic epithelial stem cells described herein are generally capable of differentiating into a medullary thymic epithelial cell. Exemplary medullary thymic epithelial cells (mTEC) include mTEC progenitors, neuroendocrine cells, myoid cells, ionocytes and Hassall body region cells. In some embodiments, mTECs express CLDN3, CLDN4 and / or ASCL1 , or combinations thereof.

[0029] The isolated thymic epithelial stem cells described herein are generally capable of differentiating into a cortical thymic epithelial cell. Cortical thymic epithelial cells include cells that express CD205, CTSV, FOXN1 , KCNIP13, CD274 (PDL-1) or combinations thereof.

[0030] In the thymic organoids as described herein, the at least one cortical thymic epithelial cell may be CD205pos / KRT5nes / KRT14nes. The at least one medullary thymic epithelial cell may be CD205ne9 / CK5P°s / KRT14pos.

[0031] The thymic organoids as described herein may comprise a cortical or cortical-like region, and / or a medullary or medullary-like region. By “cortical or cortical-like” region, it is meant a region of the thymic organoid which comprises cortical thymic epithelial cells and may also have a cellular organisation or architecture which resembles the cortex of the human thymus. By “medullary or medullary-like” region, it is meant a region of the thymic organoid which comprises medullary thymic epithelial cells and may also have a cellular organisation or architecture which resembles the medulla of the human thymus.

[0032] The plurality of thymic stromal cells may comprise thymic stromal cells which express at least one of CD205, MHC-1 , MHC-II, b5T, Foxnl , KRT10, KRT7, ASCL1 , AIRE1 , CK5 and KRT14.

[0033] The plurality of thymic stromal cells may comprise thymic stromal cells which are capable of antigen presentation, for example by acting as professional antigen presenting cells (APCs). MHC- restricted antigen presentation is necessary for proper T cell selection via MHC-TCR interaction. Typically, the thymic stromal cells which are capable of acting as APCs are differentiated medullary thymic epithelial cells.

[0034] The plurality of thymic stromal cells may comprise thymic stromal cells which produce at least one cytokine selected from the group consisting of SCF, IL7, FLT3-L, IL8 and CXCL12. The plurality of thymic stromal cells may comprise thymic stromal cells which produce at least one chemokine selected from the group consisting of CCL19, CCL21 and CCL25. The production of cytokines and / or chemokines may facilitate the development of naive T cells in the thymic organoid. In particular, the thymic stromal cells may produce cytokines necessary for T cell survival, and chemokines important for T cell migration through the thymus compartments.

[0035] In general, thymic organoids as described herein do not comprise bone marrow or bone marrow- derived stromal cells. Advantageously, Notch ligand-expressing bone marrow stromal cells can be dispensed with in the thymic organoids of the invention, since Notch ligands can be provided as part of the hydrogel scaffold, as described elsewhere herein.

[0036] The thymic organoids described herein may comprise thymic interstitial cells (TICs). TICS generally express one or more mesenchymal markers such as TE7, VIM, PDGFRp, Chondroitin Sulfate Proteoglycan 4 (NG2), Smooth Muscle Actin (aSMA), surface molecules such as PDGFRa, PDGFRp, CD90, CD146, alkaline phosphatase (ALP), CD73, PI16, or other markers consistent with a perivascular phenotype. The thymic organoids described herein may comprise thymic mesenchymal cells. TICs may be obtained via explant in Megacell complete medium, or by sorting CD45neg EpCAMnegCD205neg CD49fneg and CD90positive population and by culturing in Megacell complete medium.

[0037] In general, the cells described herein (including the cells of the thymic organoids, cells used to produce the thymic organoids, and T cells or other immune cells obtained via the thymic organoids) may be eukaryotic cells. In particular, the cells may be mammalian cells, for example human cells.DETAILED DESCRIPTION

[0038] The invention provides novel 3D-Engineered Thymic Organoids (3D-ETOs). In particular, the invention utilises hydrogel scaffolds to act as an extracellular matrix and recreate the thymic 3D microenvironment. When the hydrogel scaffolds are seeded with thymic epithelial stem cells having critical sternness properties, a thymic organoid can be produced. This thymic organoid recapitulates the 3D organisation, histology and morphology of thymus tissue, and can be used as a platform in which to produce a highly diverse repertoire of T cells, suitable for use in adoptive cell transfer.

[0039] In particular, the inventors have identified that hydrogel scaffolds provide a surprisingly advantageous growth support for thymic epithelial stem cells. This combination of scaffold and cell type provides numerous advantages over existing approaches, such as the use of artificial thymic organoids obtained from murine bone marrow stromal cells. In particular:1 . The human thymic epithelial cells used in the thymic organoids are capable of in vitro expansion and long-term storage (i.e. banking), as well as being compatible with clinical translation due to their human origin.2. T cells can be produced with physiologic CD4 / CD8 ratios, reflecting normal in vivo thymopoiesis. Tregs can also be produced, which are essential for self-tolerance induction and suppression of autoimmunity.3. The thymic organoids recapitulate the 3D thymic cell-cell organisation that requires thymic epithelial and thymic interstitial cell cooperation and interaction.4. The thymic organoids can reconstitute different thymic niches, mimicking the function of the natural thymus. In particular, the thymic organoids include the presence of medullary thymic epithelial cells, advantageous for T cell negative selection and Treg induction.5. Functionalisation of the hydrogel scaffolds with specific peptides affords controlled expression and standardisation of key molecular signalling molecules, such as the Notch ligands DLL1 and DLL4.6. T cell output can be increased compared to existing approaches such as ATOs, and is also scalable so is more amenable to clinical translation.

[0040] The thymic organoids described herein are able to reliably produce naive human T cells ex vivo with broad TCR repertoire and diverse T cell phenotypes. Such naive T cells can be used in T cell replacement therapy via ACT to maintain immunity in DiGS and related life-threatening disorders in children. The approaches described herein provide several advantages. One is the capacity to supply functional T cells (as somatic-cell therapy medicinal product, SCTMP) to support patients while waiting for the proper donor for transplant, thus eliminating the urgency of the transplant and allowing safer donor HLA-matching. A further advantage is that T cells obtained using the thymic organoids described herein can be used to support therapy in cases of transplant failure. Yet another advantage is that the thymic organoids can produce T cells with clinical translation capability that is faster than that of a bioengineered organ (otherwise known as a tissue-engineered product or TEP).Definitions

[0041] Below are provided certain definitions ofterms, technical means, and embodiments used herein.Hydrogel scaffold

[0042] The hydrogel scaffold may comprise a polysaccharide and / or a polyethylene glycol-based scaffold. The hydrogel scaffold may be selected from the group consisting of alginate, hyaluronic acid, chitosan and polyethylene glycol-based scaffolds. The hydrogel scaffold may be selected from the group consisting of alginate, hyaluronic acid and chitosan scaffolds. The hydrogel scaffold may be an alginate scaffold. Alginate is a polysaccharide that can form viscoelastic hydrogels which are nanoporous. The hydrogel scaffold may be an hyaluronic acid scaffold. The hydrogel scaffold may be an chitosan scaffold. The hydrogel scaffold may be an hyaluronic acid scaffold. The hydrogel scaffoldmay be an polyethylene glycol-based scaffold. It is anticipated that hydrogels comprising other polysaccharides (such as hyaluronic acid or chitosan), as well as polyethylene glycol-based hydrogels, would yield similar results. Alginate polymers are biocompatible and are resistant to degradation in mammalian cell culture systems. Hyaluronic acid polymers are biocompatible and are degradable in mammalian cell culture systems. Chitosan polymers are biocompatible and are resistant to degradation in non-mammalian cell culture systems. Polyethylene glycol-based polymers are biocompatible and are resistant to degradation in mammalian cell culture systems but can be chemically modified to become degradable.

[0043] The hydrogel scaffold may be a polyethylene glycol (PEG) hydrogel or a PEG-containing hydrogel (e.g. PLGA-PEG-PLGA, PEG-MAL, PEG-DBCO). The hydrogel may be a hyaluronic acid hydrogel. In some embodiments, the hydrogel may comprise an animal-derived polymer. For example, the hydrogel may comprise Matrigel (Corning) or Cultrex BME (Bio-Techne), which include murine ECM proteins such as laminin and collagen. Accordingly, the hydrogel scaffold may comprise one or more ECM proteins. In some cases, the hydrogel may comprise one or more of collagen, laminin and fibronectin. These may be animal, in particular human, in origin. Conversely, the hydrogel may not comprise Matrigel® (Corning) or Cultrex BME (Bio-Techne). The hydrogel scaffold may not comprise one or more ECM proteins. The hydrogel may not comprise one or more of collagen, laminin and fibronectin. The hydrogel scaffold may not comprise decellularized ECM. The hydrogel scaffold may be two dimensional (2D). The hydrogel scaffold may be three dimensional (3D).

[0044] The chemical and mechanical properties of alginate scaffolds can also be precisely controlled to facilitate cell growth, maintenance and differentiation. Methods for preparing hydrogel scaffolds, in particular alginate scaffolds, are known in the art [4, 5, 10].

[0045] When alginate is used as the hydrogel in the hydrogel scaffold, the alginate may have a molecular weight of about 1000 kDa or less (for example, about 900kDa or less, about 800kDa or less, about 700kDa or less, about 600kDa or less, about 500kDa or less, about 450 kDa or less, about 400 kDa or less, about 350 kDa or less, about 300 kDa or less, about 250 kDa or less or about 200 kDa or less). In a preferred embodiment, the alginate may have a molecular weight of about 10kDa to about 400kDa (for example about 10kDa to about 300kDa, about 20kDa to about 300kDa, about 50kDa to about 300kDa, about 50kDa to about 200kDa, about 70kDa to about 200kDa, about 70kDa to about 150kDa or about 10OkDa to about 150kDa). Alginate from algae is typically up to 400kDa. Alginate made by bacteria can range from 10kDa to 10000kDa. The alginate may have an (average) molecular weight of about 86kDa. Molecular weight may be determined by methods known in the art such as sizeexclusion chromatography [6].

[0046] The hydrogel scaffolds described herein may be functionalised with proteins, peptides or peptide motifs in order to better mimic the extracellular matrix and chemical organisation of the thymus. For example, the hydrogel scaffold may comprise a thymic extracellular matrix protein or a fragmentthereof. The thymic extracellular matrix protein may be a cortical extracellular matrix protein or a medullary extracellular matrix protein, or the hydrogel scaffold may comprise a combination of both cortical and medullary extracellular matrix proteins. Examples of suitable extracellular matrix proteins include fibronectins such as fibronectin 1 (FN1). Accordingly, the hydrogel scaffold may comprise fibronectin, a fibronectin domain, or a fragment thereof.

[0047] Alginate scaffolds in general cannot be bound or degraded by cells. Hydrogel scaffolds (including alginate scaffolds) may be modified, for example so that cells are able to interact with the scaffold. Hydrogel scaffolds (including alginate scaffolds) described herein may be functionalised with adhesion mimetics. Accordingly, the hydrogel may comprise adhesion mimetics. The adhesion mimetic may comprise a peptide. The adhesion mimetic may comprise a protein.

[0048] Peptides that may be used as adhesion mimetics include those peptides that mimic the binding domains of proteins. Cells cultured on the scaffold may bind to these peptides via their integrins. One particularly useful peptide is the Arginine-Glycine-Aspartic acid (RGD) motif found on extracellular matrix proteins (such as fibronectin, fibrinogen, and vitronectin). If a hydrogel scaffold comprises or is functionalised with RGD, cellular integrins (for example, of thymic stromal cells) recognise and bind the RGD motif, enabling binding to the scaffold. The hydrogel scaffold may also comprise other peptides mimicking extracellular matrix components, for example the collagen I mimetic DGEA (SEQ ID NO: 1), and / or the laminin mimetics YIGSR (SEQ ID NO: 2) or IKVAV (SEQ ID NO: 3).

[0049] Thus, hydrogel scaffolds as described herein may comprise a peptide suitable for cell binding. A hydrogel scaffold (such as an alginate scaffold) may comprise an adhesion mimetic peptide, an RGD motif, a collagen I mimetic, a laminin mimetic, a DGEA motif, a YIGSR motif, a IKVAV motif, or any combination thereof. At least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the hydrogel scaffold may be cross-linked with the ligand suitable for cell binding.

[0050] Adhesion mimetics useful in the hydrogel scaffolds described herein also include proteins. These include extracellular matrix proteins such as laminin, growth factors or other ligands for cell binding. Thus, hydrogel scaffolds as described herein (including alginate scaffolds) may comprise an adhesion mimetic protein, a laminin, a collagen, a growth factor, a ligand for cell binding, or any combination thereof.

[0051] Generally, hydrogel scaffolds such as alginate scaffolds are resistant to degradation. In some cases, it may be advantageous to provide a hydrogel scaffold which can be degraded by cells (for example, which could be degraded by T cells). Thus, in some cases, the hydrogel scaffold may comprise a peptide, protein or other agent that permits degradation of the scaffold by cells. An example of such a peptide is the matrix metalloproteinase (MMP)-sensitive motif Pro-Val-Gly-Leu-lso-Gly (PVGLIG) (SEQ ID NO: 4).

[0052] In addition to the peptides and proteins described above, hydrogel scaffolds (including alginate scaffolds) may comprise a variety of other components. These include but are not limited to spacers, chemical groups allowing covalent crosslinking, chemical groups allowing photo-responsiveness, and fluorophores. Suitable spacers include polyethylene glycol (PEG) and the like. Spacers can act to increase the viscous properties of the hydrogel scaffold. Suitable chemical groups allowing covalent crosslinking include norbornene-tetrazine groups for click-chemistry reactions. Other suitable groups for crosslinking are known in the art and can be determined by the skilled person. Chemical groups allowing photo-responsiveness enable the hydrogel scaffold to be crosslinked in response to light. This can be particularly useful if a non-calcium based method of crosslinking is required. Suitable chemical groups allowing photo-responsiveness include methacrylate groups and the like. Suitable fluorophores may be used to enable visualisation of the hydrogel scaffold. Suitable fluorophores include rhodamine.

[0053] The hydrogel scaffold may comprise a Notch ligand or fragment thereof. In particular, the hydrogel scaffold may comprise the Notch ligands DLL1 or DLL4, or a combination thereof. Notch ligands are mainly produced by the endothelial cells of the vasculature, and are advantageous for inducing cells towards a lymphoid fate.

[0054] It will be appreciated that the hydrogel scaffolds can be functionalised with any number and combination of proteins, peptides, etc, in order to mimic the thymic environment and facilitate production of the thymic organoid.

[0055] Functionalisation may be performed by covalently coupling the adhesion peptide GGGGRGDSP (RGD, Peptide 2.0, SEQ ID NO: 5) to alginate utilizing carbodiimide chemistry (Sulfo- NHS, EDC) [7],

[0056] Exemplary functionalisation methodologies are shown in Figure 5A and 5B.

[0057] In addition to modification of the chemical properties of the hydrogel scaffold, the physical properties of the hydrogel scaffolds described herein can also be precisely controlled. Advantageously, the hydrogel scaffolds can be configured to comprise the mechanical properties of the human thymus or a portion thereof. In particular, properties such as stiffness, viscoelasticity and pore size can all be independently controlled to mimic the physical microenvironment of the human or animal thymus.

[0058] The hydrogel scaffolds described herein, or a portion thereof, may comprise a stiffness between about 10 Pa to about 500 kPa (for example about 10 Pa to about 400 kPa, about 10 Pa to about 300 kPa, about 10 Pa to about 200 kPa, about 10 Pa to about 100 kPa, about 100 Pa to about 100 kPa, about 200 Pa to about 100 kPa, about 500 Pa to about 100 kPa, about 1 kPa to about 100 kPa or about 10 kPa to about 100 kPa). The term “stiffness”, as used herein, refers to the mechanical property that characterizes the resistance of a hydrogel to deformation when subjected to an external force. It is a measure of how rigid or flexible the hydrogel material is and can be modified by altering the amount orconcentration of crosslinker. Generally, the amount of crosslinker is 100mM or less. Stiffness may be quantified by measuring the hydrogel's elastic modulus, which is a measure of its resistance to deformation under stress. Various techniques such as compression tests, atomic force microscopy, or rheological measurements can be used to determine the stiffness of hydrogels [8,9]. In some cases, the hydrogel scaffold or a portion thereof has an elastic modulus of about 100 Pa to 30 kPa.

[0059] The viscoelasticity of the hydrogel scaffolds described herein may also be varied or modified. The term “viscoelasticity”, as used herein, refers to a combination of two mechanical properties: viscosity and elasticity. It describes how the hydrogel behaves in response to applied forces over time, exhibiting both viscous and elastic characteristics. Viscosity is a measure of a material's resistance to flow. In the case of hydrogel scaffolds (including alginate scaffolds), it refers to their ability to dissipate energy and undergo deformation over time when subjected to an applied force. When a force is applied to a viscoelastic hydrogel scaffold, it deforms continuously and experiences stress relaxation, meaning the hydrogel scaffold gradually reduces the stress it bears over time. This characteristic is similar to how a viscous liquid, like honey, flows and deforms when a force is applied. Elasticity is a material's ability to regain its original shape after the applied force is removed. For hydrogel scaffolds, this property is related to the cross-linking of polymer chains within the three-dimensional network. When a force is applied to a viscoelastic hydrogel scaffold, it deforms elastically to some extent, and upon removing the force, it returns to its original shape. The extent of elastic response depends on the material’s crosslinking density and structure.

[0060] Viscoelasticity of hydrogel scaffolds can be modified to mimic the behaviour of soft tissues in the body. To understand and characterize the viscoelastic behaviour of hydrogel scaffolds, techniques like dynamic mechanical analysis (DMA) or rheological measurements are used. These methods involve subjecting the hydrogel scaffold to oscillatory or time-dependent forces and measuring its response to evaluate its viscoelastic properties, including storage (or elastic) modulus (a measure of elasticity) and loss modulus (a measure of viscosity).

[0061] Viscoelasticity can be measured by assessing the stress relaxation time of the hydrogel scaffold. Stress relaxation time may be assessed by dynamic mechanical analysis (DMA) or rheological measurements. The hydrogel scaffold or a portion thereof may have a stress relaxation time of 6000s or less, 5000s or less, 4000s or less, 3000s or less, 2500s or less, 2000s or less, 1500s or less, 1000s or less, or 500s or less.

[0062] One approach to modifying viscosity is to alter the molecular weight of the polymers used to prepare the hydrogel scaffold, such as by using a different molecular weight alginate. Elasticity can be modified by altering calcium crosslinker density / concentration. This approach is described in document

[0010] , the contents of which are incorporated herein by reference.

[0063] The hydrogel scaffolds as described herein are typically porous or nano-porous. As used herein, the term “pore size” refers to the distance between the two opposite sides of a pore. The pore size ofthe hydrogel scaffolds can also be varied or modified by altering the polymer (e.g. alginate) concentration. Typically, hydrogel scaffolds as described herein, or portions thereof, have a pore size between about 1 nm to 1000nm, for example 1 nm to 900nm, 3nm to 900nm, 5nm to 900nm, 5nm to 800nm, 5nm to 750nm, 8nm to 750nm, 8nm to 500nm, 10nm to 500nm, 10nm to 400nm, 10nm to 300nm, 10nm to 250nm, 10nm to 100nm, 10nm to 50nm, 10nm to 40nm, 5nm to 40nm, 5nm to 20nm or 2nm to 10nm. Porosity be measured by quantifying the diffusion of particles of known size out of the hydrogel scaffold. Porosity can also be qualitatively estimated by diffusion of big molecules (BSA, dextran). Other techniques include thermoporometry

[0011] .

[0064] Hydrogel scaffolds (including alginate scaffolds) can be prepared by ionic crosslinking, for example using a source of calcium ions. Methods to prepare hydrogel scaffolds, in particular alginate scaffolds, using calcium crosslinking, are known in the art. Various sources of calcium ion can be used based on the desired properties of the hydrogel scaffold. For example, the calcium source may comprise CaSO4, CaCOs and / or CaCh, among others.

[0065] The hydrogel scaffolds described herein may further comprise hyaluronic acid, collagen, agarose, polyethylene glycol (PEG), or combinations thereof.

[0066] When primarily comprised of a material such as alginate, which does not present intrinsic integrin adhesion ligands, the hydrogel scaffolds described herein may comprise peptides suitable for cell binding. Such peptides facilitate the binding of cells in culture to the scaffold, in order to support cell growth, maintenance and differentiation. An exemplary peptide suitable for cell binding is an Arg-Gly- Asp (RGD)-containing peptide.

[0067] The cells may be embedded in the hydrogel. The term “embedded” in such context refers to cells that are (initially) distributed homogeneously across the hydrogel. Embedded cells have an advantage in that they can move through the hydrogel and self-organise. Some hydrogels are merely designed to encapsulate cells using microgels, which acts like a container to keep the (aggregated) cells confined, which is different to the hydrogels of the present invention which are in the form of a scaffold.Methods for producing thymic organoids

[0068] Described herein are methods for producing thymic organoids. In general, the methods comprise providing a hydrogel scaffold as described herein, seeding the scaffold with thymic stromal cells, and culturing the thymic stromal cells until a thymic organoid is produced.

[0069] For example, a method for producing a thymic organoid may comprise: a) providing a hydrogel scaffold;b) seeding the hydrogel scaffold with at least one thymic stromal cell, wherein the at least one thymic stromal cell comprises at least one isolated thymic epithelial stem cell, at least one isolated thymic interstitial cell, or a combination thereof; and c) culturing the at least one thymic stromal cell.

[0070] The “seeding” step of the method may comprise seeding the hydrogel scaffold with at least one isolated thymic epithelial stem cell and at least one isolated thymic interstitial cell. The isolated thymic epithelial stem cell may be BCAMP°S, CD49FP°S, CD90P°Sand CD24nes. The isolated thymic interstitial cell may express one or more markers selected from the group consisting of TE7, VIM, PDGFRp, Chondroitin Sulfate Proteoglycan 4 (NG2), Smooth Muscle Actin (aSMA), surface molecules such as PDGFRa, PDGFRp, CD90, CD146, alkaline phosphatase (ALP), CD73, P116, or other markers consistent with a perivascular phenotype.

[0071] The seeding step of the method may comprise loading the thymic stromal cell or cells with the hydrogel in a bidirectional syringe, and subsequently mixing. The mixing is preferably performed in a way which avoids bubble formation. The seeding or mixing may be carried out in the presence of CaSO4 in DMEM high glucose. Once mixed, the thymic stromal cells and hydrogel may be deposited onto a surface, such as a plastic surface, to allow polymerisation.

[0072] After seeding, the thymic stromal cells are cultured with the hydrogel scaffold until a thymic organoid is produced. Generally, culturing includes growing the cells for 7days in expansion media cFAD medium composed by a mixture of 3:1 of DMEM1X (Gibco) and F-12 Nut Mix (Gibco), 10% Fetal Bovine Serum (SIGMA-ALDRICH), 1 % penicillin and streptomycin (100X, Sigma), Hydrocortisone (0.4 pg / ml, Calbiochem), Cholera Toxin (10-10 M, Sigma), Triodothyronine (T3) (2x10-9 M Sigma) and Insulin (5 pg / ml, SIGMA-ALDRICH) and kept in incubator at 37°C in a 6% CO2 atmosphere. Differentiation can be performed in Pneumacult plus supplement (STEMCELL Technologies). Differentiation can occur also in co-culture media, by removing growth factors and hormones from expansion medium e.g. hydrocortisone and cholera toxin. The expansion phase within alginate may range from 3-10 days depending on density; subsequent differentiation phase may range from 5 days to 15 days; in some cases it can be extended to 5-7 weeks to allow long term culture. Culturing may be performed for at least 1 day, at least 7 days, at least 14 days, at least 21 days or at least 28 days. Culturing may include the addition of signalling molecules to the culture medium to facilitate thymic organoid development, though this is not always necessary.

[0073] The properties of the thymic organoid can also be varied by changing the cell composition. For example, the thymic organoid can be seeded with different quantities of particular cell types, depending on the application or preferred use of the organoid. A single cell type can be used to seed the thymic organoid, or a combination of multiple cell types. The ratio of cell types can also be modified. For example, the ratio the ratio of thymic interstitial cells to thymic epithelial stem cells may be from about 10:1 to about 1 :10. For example, the ratio of thymic interstitial cells to thymic epithelial stem cells maybe 10:1 , 9:1 , 8:1 , 7:1 , 6:1 , 5:1 , 4:1 , 3:1 , 2:1 , 1 :1 , 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 :10. The number of cells seeded on the hydrogel scaffold may range from about 1 to about 5 million cells in total. For example, the number of thymic epithelial stem cells seeded on the hydrogel scaffold may be about 1 million, about 2 million, about 3 million, about 4 million or about 5 million cells. The number of thymic interstitial cells seeded on the hydrogel scaffold may be about 1 million, about 2 million, about 3 million, about 4 million or about 5 million cells. The total number of thymic epithelial stem cells and thymic interstitial cells seeded on the hydrogel scaffold may be about 1 million, about 2 million, about 3 million, about 4 million or about 5 million cells.

[0074] Thymic organoids can also be cultured using an air-liquid interface (ALI) culture system. Use of an ALI system can promote spatial organisation and differentiation of thymic stromal cells. In an ALI system, the hydrogel scaffold may be seeded with cells and maintained in culture medium to allow cell division and expansion. Culture media can then be partially removed from the culture vessel in order to expose the cells to air, which can promote differentiation. Thus, in some cases, the methods for producing thymic organoids described herein comprise a step of culturing the thymic stromal cells at an air-liquid interface. The methods described herein may comprise a step of exposing the thymic stromal cells to air.

[0075] Methods for producing thymic organoids as described herein may also comprise a step of applying shear stress. Shear stress may be applied by providing fluidic flow during culture. The fluidic flow mimics mechanical forces that thymic stromal cells may be exposed to in vivo, and so can promote shear-stress dependent cellular changes. Alternatively, thymic organoids may be cultured under static conditions (i.e. without the application of shear stress).

[0076] Methods for producing thymic organoids are generally carried out in vitro.Methods for producing a human T cell

[0077] The thymic organoids described herein provide a new platform by which to produce human T cells, in particular naive T cells. A method for producing a human T cell may comprise: a) providing a thymic organoid as described herein; b) seeding the thymic organoid with at least one lymphoid progenitor cell, at least one haematopoietic stem cell, or a combination thereof; and c) culturing the thymic organoid under conditions suitable for T cell development.

[0078] Step (b) may comprise seeding the thymic organoid with at least one lymphoid progenitor cell and at least one haematopoietic stem cell. Generally, an isolated plurality of each cell type will be used for seeding. The haematopoietic stem cells may be CD34+. The lymphoid progenitor cell may be athymocyte. In some cases, the lymphoid progenitor cell may be a “triple negative” cell, which does not express the surface molecules CD3, CD4 or CD8.

[0079] The thymic organoid may also be seeded with endothelial cells and / or neuronal progenitors. Thus, the thymic organoid may be seeded with at least one lymphoid progenitor cell and at least one endothelial cell. The thymic organoid may be seeded with at least one haematopoietic stem cell and at least one endothelial cell. The thymic organoid may be seeded with at least one lymphoid progenitor cell and at least one neuronal progenitor cell. The thymic organoid may be seeded with at least one haematopoietic stem cell and at least one neuronal progenitor cell. The thymic organoid may be seeded with at least one lymphoid progenitor cell, at least one haematopoietic stem cell, and at least one endothelial cell. The thymic organoid may be seeded with at least one lymphoid progenitor cell, at least one haematopoietic stem cell, and at least one neuronal progenitor cell. The thymic organoid may be seeded with at least one lymphoid progenitor cell, at least one haematopoietic stem cell, at least one endothelial cell, and at least one neuronal progenitor cell.

[0080] After seeding, the thymic organoid is cultured under conditions suitable for T cell development. Generally, culturing includes growing the organoid in a suitable medium. Suitable culture media may include co-culture media (co-culture medium complete (DMEM 1X (Gibco), 10% FBS (SIGMA- ALDRICH), 1 % penicillin and streptomycin (100X, SIGMA-ALDRICH), Triodothyronine (T3) (2x10-9 M SIGMA-ALDRICH), Insulin (5 pg / ml, SIGMA-ALDRICH) and Cytokines (lnterleukin-7, 5ng / mL (Invitrogen), Stem Cell Factor 5ng / mL (Cell Signalling) and FLT3-L (5ng / mL, CellGS)). This media has been adapted for culture with haematopoietic cells. Depending on the size of the hydrogel scaffold or organoid, oxygen tension may vary from 5% to 80%. High oxygen tension may be needed for large organoids to avoid internal hypoxia. For organoids below 1 cm3, oxygen tension may be 5-20%. Suitable oxygen tensions can be determined by the skilled person. Culturing may be performed for at least 1 day, at least 7 days, at least 14 days, at least 21 days or at least 28 days. Culturing may include the addition of signalling molecules to culture medium to facilitate T cell development, though this is not always necessary. Exemplary signalling molecules that may be used include IL2, IL7, IL15, stem cell factor (SCF), and Flt3-I.

[0081] In some cases, culturing step (c) comprises culturing the thymic organoid with at least one chemokine, at least one cytokine, at least one growth factor, at least one ubiquitin ligase, at least one peptide, or a combination thereof. Advantageously, these and other additives may be incorporated into the hydrogel scaffold. Alternatively, or additionally, additives may be added to the culture medium.

[0082] The method for producing a human T cell may further comprise a step of isolating the T cells from the thymic organoid. Isolating the T cells may comprise mechanical and / or enzymatic dissociation / digestion of the thymic organoid. For example, digestion may be performed using 10mM EDTA at 4°C. Digestion may also be performed using Collagenase I (2 mg / mL, SIGMA, C0130), Dispase II (1 U / mL, Roche) and DNAse I (80 pg / mL, Roche) in RPMI 1640 and 2% FBS. Followingdigestion, the cells may be isolated by passing the cell suspension through a cell strainer (100 pm). Antibody staining can subsequently be used for FACS sorting or analysis.

[0083] The human T cell may be a naive T cell, a CD4+ T cell, a CD8+ T cell, or a CD4+ / CD8+ T cell.

[0084] T cells obtained using thymic organoids may be assessed by methods known in the art to determine their cellular characteristics, for example profiling by flow cytometry to assess surface markers assessed by the T cells. In particular, T cells may be evaluated for CD3, CD4, CD8, TCRab, CD25 and Foxp3 expression. RNA and DNA sequencing may also be used to evaluate the TCR repertoire of obtained T cells. Isolated T cells obtained from the thymic organoids may also be tested on functional assays, such as response to anti-CD3 beads (to assess expansion capacity), PMA- ionomycin stimulation (to assess cytokine production) and mixed-lymphocyte reaction (MLR) (to assess allo-reactivity).

[0085] Also described herein are human T cells obtained or obtainable by the methods described above.Methods for producing a lymphoid progenitor cell

[0086] The thymic organoids described herein may also be used to produce lymphoid progenitor cells, in particular human lymphoid progenitor cells. A method for producing a human lymphoid progenitor cell may comprise: a) providing a thymic organoid as described herein; b) seeding the thymic organoid with at least one haematopoietic stem cell, wherein the haematopoietic stem cell is CD34+; and c) culturing the thymic organoid under conditions suitable for differentiation of the at least one haematopoietic stem cell into a lymphoid progenitor cell.

[0087] After seeding, the thymic organoid is cultured under conditions suitable for differentiation of the haematopoietic stem cells into lymphoid progenitor cells. Generally, culturing may include culturing the thymic organoid in a culture medium comprising reagents known in the art, such as DMEM 1X, FBS, antibacterial agents such as penicillin and streptomycin, triiodothyronine, insulin, and cytokines. Culturing may be performed for at least 1 day, at least 7 days, at least 14 days, at least 21 days or at least 28 days. Culturing may include the addition of signalling molecules to culture medium to facilitate T cell development, though this is not always necessary. Exemplary signalling molecules that may be used include IL2, IL7, IL15, stem cell factor (SCF), and Flt3-I.

[0088] In some cases, culturing step (c) comprises culturing the thymic organoid with at least one chemokine, at least one cytokine, at least one growth factor, at least one ubiquitin ligase, at least onepeptide, or a combination thereof. Advantageously, these additives may be incorporated into the hydrogel scaffold. Alternatively, or additionally, these additives may be added to the culture medium.

[0089] Following differentiation in culturing step (c), the lymphoid progenitor cell may express at least one selected from the group consisting of CD5, CD7, CD1 a, or a combination thereof. The lymphoid progenitor cell obtained by this method may be a thymocyte. The lymphoid progenitor cell may be a “triple negative” cell, which does not express the surface molecules CD3, CD4 or CD8.

[0090] The method may further comprise a step of isolating the lymphoid progenitor cells from the thymic organoid. The isolated lymphoid progenitor cells may be used in a subsequent method for producing T cells.

[0091] Also described herein are lymphoid progenitor cells obtained or obtainable by the methods described above.Methods of treating an immune disorder in a subject

[0092] As noted above, the thymic organoids described herein provide a new platform by which to generate T cells. Said T cells may subsequently be used to treat immune disorders or immune deficiencies by adoptive cell transfer.

[0093] Disclosed herein are methods of treating an immune disorder in a subject, the method comprising administering a T cell obtained by a method as described herein to the subject. The T cell may be a human T cell. The T cell may be a naive T cell, a CD4+ T cell, a CD8+ T cell, or a CD4+ / CD8+ T cell.

[0094] Also disclosed herein are methods of treating an immune disorder in a subject, the method comprising administering a human lymphoid progenitor cell obtained by a method as described herein to the subject.

[0095] Immune disorders which may be treated using the methods described herein include immunodeficiency, athymia, DiGeorge syndrome (DiGS), 22q11.2 deletion syndrome, FOXN1- deficiency, or an autoimmunity.

[0096] The thymic organoids described herein may also be useful in treating immune disorders. For example, a method of treating an immune disorder in a subject may comprise implanting a thymic organoid as described herein in the subject. Immune disorders which may be treated with such methods may include immunodeficiency, athymia, DiGeorge syndrome (DiGS), 22q11.2 deletion syndrome, FOXN1 -deficiency, or an autoimmunity. Accordingly, described herein is the use of a thymic organoid as a thymus implant.

[0097] Generally, where thymic organoids are used for treating immune disorders, production of the thymic organoid is carried out in vitro. However, it will be appreciated that further cell proliferation and / or differentiation of the organoid can occur after implantation in vivo. Thus, preferably the production of the organoid is carried out in vitro until an organoid is generated which is sufficiently populated with thymic epithelial cells that are sufficiently differentiated to allow successful implantation into a subject.

[0098] As used herein, the term “subject” generally refers to a mammal. The subject may be a human.

[0099] The cells used in the present methods (such as haematopoietic stem cells and / or lymphoid progenitor cells) will typically be autologous i.e. originate from or are derived from the intended recipient of the tissue or organ construct generated by the method of the invention. However, cells for use in the method may also be allogeneic, i.e. obtained or derived from a subject who is not the recipient of the tissue or organ construct to be generated. Further, xenogeneic cells may be used, i.e. cells derived from a different species to the recipient of the tissue / organ construct. The cells may also be produced from pluripotent stem cells or induced pluripotent stem cells.

[0100] As used herein, the term “administration” or “administering” refers to the administration of a composition to a subject. Administration to an animal subject (e.g., to a human) may be by any appropriate route. For example, in some embodiments, administration may be bronchial (including by bronchial instillation), buccal, enteral, intra-arterial, intra-dermal, intra-gastric, intra-medullary, intramuscular, intra-nasal, intra-peritoneal, intra-thecal, intra-venous, intra-ventricular, within a specific organ or tissue (e. g. intra-hepatic, intra-tumoral, peri-tumoral, etc), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intra-tracheal instillation), transdermal, vaginal and vitreal. The administration may involve intermittent dosing. Alternatively, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.Drug screening

[0101] The thymic organoids described herein enable the differentiation and growth of populations of T cells, as well as other cell types in the T cell lineage such as lymphoid progenitor cells. As discussed above, the hydrogel scaffolds also lend themselves to chemical and physical manipulation. Accordingly, the thymic organoids provide a useful model in which to screen for agents or physical properties which modulate T cell development.

[0102] Described herein are methods of screening for agents that modulate T cell development. Such a method may comprise: a) seeding a thymic organoid as described herein with at least one lymphoid progenitor cell, at least one haematopoietic stem cell, or a combination thereof; b) adding a candidate agent to the thymic organoid;c) culturing the thymic organoid under conditions suitable for T cell development; and d) determining the effect of said agent on T cell development.

[0103] The candidate agent may be added to the thymic organoid in various ways. For example, the candidate agent may be added to the hydrogel scaffold, to the culture medium, or both. The method may also employ gene editing to express the candidate agent in the at least one lymphoid progenitor cell, at least one haematopoietic stem cell, or a combination thereof. Any combination of said approaches is within the scope of this disclosure.

[0104] As will be appreciated, there are numerous effects on T cell development that could be assessed or screened for using the thymic organoids described herein. For example, step (d) may comprise comparing the number of T cells obtained by the method with addition of the candidate agent to the number of T cells obtained by the method without addition of the candidate agent. Step (d) may comprise comparing the gene expression profile of T cells obtained by the method with addition of the candidate agent to the gene expression profile of T cells obtained by the method without addition of the candidate agent. Step (d) may comprise comparing the protein expression profile of T cells obtained by the method with addition of the candidate agent to the protein expression profile of T cells obtained by the method without addition of the candidate agent.

[0105] While the above methods refer to modulation of T cell development, it will be appreciated that the methods may also be used to screen for agents which modulate the development of other cell types in the T cell lineage, for example lymphoid progenitor cells.Protein expression and surface markers

[0106] The term “surface markers” refers to a molecule, such as a protein, that is expressed on the surface of a cell. Patterns of protein expression and the presence of particular surface markers can be used to identify and isolate thymic epithelial stem cells, as well as other cells used in the thymic organoids and methods described herein, and to distinguish them from other cell populations. Protein expression and cell surface markers can be detected by any suitable method. For example, protein expression can be detected by immunohistochemistry (IHC), immunocytochemistry (ICC), magnetic- activated cell sorting (MACS), fluorescence activated cell sorting (FACS), mass cytometry or proteomics.

[0107] Expression of a surface marker or level of expressed protein may be described in positive or negative terms. A cell may be negative for a particular marker when the level of expression of that marker is equivalent or comparable to that of a negative control, for example a cell that is known not to express that particular marker. For example, a cell may be described as negative for a marker when the detectable level of that marker corresponds to a fluorescence intensity signal with a distribution comparable to the one of the negative control on a fluorescence log plot. The negative control may either be an unlabelled sample (e.g. a sample that does not comprise a detectable label) or the compensationcontrol, cells stained with all the fluorophores minus the one of the target protein, a.k.a. Fluorescence Minus One (FMO). A negative control may be used to set the PMT voltage values. A cell may be positive for a particular marker when the level of expression of that marker is increased compared to a control, e.g. a negative control. A cell may be positive for a particular marker when the level of expression of that marker is increased compared to a negative control by at least 10%, for example increased by at least 20%, increased by at least 30%, increased by at least 40%, increased by at least 50%, increased by at least 60%, increased by at least 70%, increased by at least 80%, increased by at least 90% or increased by at least 100% compared to a negative control. A cell may be positive for a particular marker when the level of expression of that marker is comparable to a positive control (i.e. a marker known to be expressed on the same cells).

[0108] The amount, degree or level of expression of a surface marker or expressed protein may also vary. For example, cells may exhibit a low level of expression of a surface marker. Cells may exhibit a high level of expression of a surface marker. Cells may exhibit an intermediate level of expression of a surface marker. The level of expression of a surface marker can be defined according to whether the surface marker is expressed to a level that is above or below a threshold value. The level of expression of a surface marker can be determined by any suitable method known in the art. For example, the level of expression of a surface marker may be determined by FACS.

[0109] In some embodiments, a cell has an intermediate level of expression of a surface marker if the cell expresses the marker at a level 1-2 logs (i.e. 10 to 100 times) higher compared to a negative control (e.g. a cell known not to express the surface marker). In some embodiments, a cell has a high level of expression of a surface marker if the cell expresses the marker at a level 1-2 logs (i.e. 10 to 100 times) higher than a cell that expresses the surface marker at an intermediate level (e.g. a cell as defined above), or 3-4 logs (i.e. 100 to 1000 times) higher compared to a negative control (e.g. a cell known not to express the surface marker). Methods of determining expression thresholds are known in the art.Gene expression

[0110] The expression of a gene or gene set may be used to detect, identify or distinguish cell types from one another. The inventors have surprisingly discovered a novel gene expression profile that can distinguish thymic epithelial stem cells from other cell types. This gene expression profile can be used to identify thymic epithelial stem cells as well as in methods of isolating and culturing thymic epithelial stem cells.

[0111] Gene expression may be detected or measured by any suitable method known in the art. For example, gene expression may be measured by polymerase chain reaction (PCR), fluorescent in situ hybridisation (FISH), single cell RNA sequencing (scRNA-seq), spatial transcriptomics, RNA-scope and HiPLEX.

[0112] Aspects and embodiments described herein with the term “comprising” may include other features or steps within the scope. It is also understood that aspects and embodiments described as “comprising” also describes aspect and embodiments wherein the term “comprising” is replaced by the term “consisting essentially of’ or “consisting of’.

[0113] The phrase "selected from the group comprising" may be substituted with the phrase "selected from the group consisting of and vice versa, wherever they occur herein.

[0114] It is also understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and / or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

[0115] The invention will now be further described by way of the following Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention, with reference to the Figures.EXAMPLESMaterials & Methods

[0116] Alginate-based hydrogels are synthesised as follows: Alginate LF10 / 60 and 5Mrad RGD motifs are dissociated in DMEM high glucose (Gibco, 1 1965092) and Matrigel® (Corning), mixed for 1 hour at room temperature (RT) and then loaded at the tip of a bidirectional syringe.

[0117] TIC and TEC are cultivated as previously reported in Campinoti et al. (2020) Nature Communications 11 :6372, the contents of which is incorporated herein by reference, and seeded either alone or in combination at different ratios 1 :1 -10 in the range of 1 .5 to 5M of cells in total per condition.

[0118] Thymic stromal cells are mixed with the ionic crosslinker (e.g. calcium), alginate and DMEM High glucose. All components are mixed to allow polymerisation.

[0119] Repopulated alginate polymers are incubated at 37°C for at least 30 minutes.

[0120] cFAD medium is added 1 hour later and fresh medium replaced after 4h, 24h and every other day. The repopulated matrix is cultivated in cFAD for 5 to a maximum of 30 days and fixed in PFA 4% or dissociated (condition) for downstream analysis at different time points. After 5 days stromal cells are differentiated in various differentiation media by Stem Cell Technologies up to 21 days as described in Ragazzini R, (2023) Boeing S, Zanieri L, Green M, D’Agostino G, Bartolovic K, Agua-Doce A, Watson SA, Batsivari A, Ariza-McNaughton L, Gjinovci A, Greco M, Scoville D, Han A, Hayday AC, Bonnet D,and Bonfanti P. Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human postnatal thymus. Developmental Cell, in press.

[0121] Alternatively, after 4-5 days of expansion in cFAD medium, 1-5M millions of sorted lineage negative T cell precursors or haematopoietic stem cells purified from human cord blood (Stem Cells Technologies) are injected using Insulin Syringes (Thermo, 29.5G) in co-culture medium complete (DMEM 1X (Gibco), 10% FBS (SIGMA-ALDRICH), 1 % penicillin and streptomycin (100X, SIGMA- ALDRICH), Triodothyronine (T3) (2x10-9 M SIGMA-ALDRICH), Insulin (5 pg / ml, SIGMA-ALDRICH) and Cytokines (lnterleukin-7, 5ng / mL (Invitrogen), Stem Cell Factor 5ng / mL (Cell Signalling) and FLT3-L (5ng / mL, CellGS)). Co-culture of stromal and haematopoietic cells is maintained for up to 30 days and fixed in PFA 4% or dissociated (condition) for downstream analysis at different time points.

[0122] Cytokines produced by cells in 3D Engineered Thymic Organoids (ETOs) were analysed utilising the GeniePlex Multiplex Immunoassays, employing a similar principle of a sandwich assay, where fluorescent beads conjugated with a specific antibody trap the cytokine of interest. Cytokine production was quantified with a Fortessa Flow Cytometry analyser. The following cytokines were analysed: CCL19, CXCL12, IL-6, and IL-7.Example 1

[0123] Cell staining of the thymic organoids was performed to assess the organisation and differentiation of cells seeded onto the hydrogel scaffold. Both high and low molecular weight alginate- RGD polymers were used for this experiment. Figure 1 shows cellular organisation following 7 days of expansion on the scaffold. Thymic stromal cells re-organise within the matrix with different patterns. Figure 2 shows differentiation of thymic epithelial cells into single positive KRT5 / 14 medullary or LY75 cortical cells within the matrix, when grown on both high and low molecular weight alginate-RGD scaffolds. Some epithelial cells stain positive for KRT 10, a marker of medullary Hassall bodies. Figure 3 shows that thymic organoids can be used to support the development of CD45 positive T cells when co-cultured with thymocytes. CD45 positive T cells are shown in yellow, and were more abundant in the low molecular weight condition although were also present in the high molecular weight condition. Some cells also stained positive for MHC class II molecule HLADR-A.Example 2

[0124] Cytokine production was assessed to demonstrate that 3D-ETOs are capable of producing cytokines, without the need to include cytokines in the media. The 3D-ETOs were cultured for 5 days in cFAD (blue, first columns) media during the expansion / proliferation phase of the cells, followed by 7 days of cell differentiation in either Pneumacult (red, second columns) or Co-Culture (green, third columns) media. CCL19, CXCL12, IL-6, IL-7 were all expressed by 3D-ETOs. This exampledemonstrates that 3D-ETOs are capable of producing cytokines that are part of the endogenous thymic microenvironment, thus recapitulating functional aspects of thymus in vitro.

[0125] Together, the results of the experiments described herein indicate that thymic cells grown on hydrogel scaffolds as described herein surprisingly show spatial re-organisation, differentiation into medullary and cortical cell types, and functional cytokine production. The results demonstrate, for the first time, provision of thymic organoids which recapitulate both structural and functional markers of human thymus, and which can support the development of T cells.Example 3Thymic Stromal Cell expansion culture

[0126] Human Thymic Epithelial Cells (TECs) were thawed and expanded in T25 or T75 flasks over a feeder layer of lethally irradiated 3T3-J2 cells, with expansion medium (cFAD) and were cultured for several passages as described in [12,13].

[0127] Human Thymic Interstitial Cells (TICs) were thawed and expanded in T25 or T75 flasks as described in

[0013] .Embedding and culture of thymic stromal cells within hydrogels

[0128] Sodium alginate with molecular weight (MW) of maximum 400kDa, chemically conjugated or not to one or several ligands (e.g. ECM motifs) following the previously described chemical reaction

[0010] , was dissolved in DMEM (Gibco) with addition of other possible agents (e.g. Matrigel®, Corning) and loaded in a syringe. Thymic interstitial cells (TICs) and thymic epithelial cells (TECs) were embedded either alone or in combination at different ratios 1 :1-1 :10, in the range of 1-20 millions of cells / mL of hydrogel. Thymic stromal cells were mixed with the ionic crosslinker (e.g. calcium) and loaded to another syringe. The contents of this syringe and the one containing alginate were mixed using a syringe connector, dispensed and incubated at 37 C: for at least 30 minutes to polymerize. cFAD medium was added after polymerisation and replaced the following day, and then every other day. The cells in the alginate-based hydrogels were cultivated in cFAD for 5 to 30 days. After this, expansion was replaced with differentiation media of varying compositions (including, but not limited to, medium with and without retinoic acid; serum-free growth medium; Neurocult™ or Pneumacult™), up to an additional 30 days as described in

[0013] .

[0129] The samples were fixed in PFA 4% or dissociated for downstream analysis at different time points.Isolation of thymocytes and lymphoid precursors (LPs)

[0130] Thymic tissue, derived from patients undergoing cardiothoracic surgery, was mechanically dissociated to obtain small fragments, after which Phosphate Buffer Saline (PBS, Sigma) was added to collect the released thymocytes, which were filtered with a 40pm cell strainer into a larger container. These steps were repeated until the entire tissue was fully dissociated. After this, the thymocyte suspension was centrifuged and the pellet was used for subsequent steps.

[0131] LPs isolation was performed via magnetic sorting using biotinylated anti- CD3 / CD4 / CD8a / CD235ab antibodies (BioLegend) followed by Lineage-negative (BioLegend) sorting using streptavidin beads (Invitrogen). Negative-enriched cells underwent a FACS purity sorting using FACS Aria III machine (BD Bioscience). Purified cells were frozen in medium supplemented with 10% Dimethylsulfoxide.LPs seeding in alginate hydrogels repopulated with thymic stromal cells

[0132] Prior to thymocyte addition, hydrogel samples were lifted for air exposure. Total thymocytes or LPs, were centrifuged, and resuspended in RPMI (Gibco) with 10% Fetal Bovine Serum (Sigma) and counted. A range of 10-30 million TT cells per scaffold was used; in the case of LPs, a range of 15x104- 1x106 cells per scaffold was used. Cells were thus centrifuged and re-suspended in a small volume of medium completed with cytokines (including, but not limited to, interleukin 7 (IL7, Invitrogen), stem cell factor (SCF, Cell signalling), and FLT-L3 (CellGS)). The required volume of TT or LP suspension was added to the hydrogel via drop deposition, or direct injection inside the system. The next day, completed medium was added in the well. Medium was replaced every other day, and the culture was kept for a period of at least 3 - 28 days.Dissociation of 3D-ETOs:

[0133] 3D-ETOs containing thymic stromal and lymphoid cells were mechanically dissociated using sterile scissors, after which these were incubated in a 37°C water bath with an enzymatic solution containing Collagenase I (Sigma), Dispase II (Roche), and DNAse I (Roche), with varying concentrations of Alginate Lyase (Sigma) and EDTA. Reaction was stopped with 1 mL RPMI+FBS, and the cell suspensions were filtered through a 100 pm cell strainer onto a round bottom tube. The resulting cell suspension was used for RT-qPCR or Flow cytometry analyses.Immunostaining

[0134] 3D-ETOs from different expansion, differentiation, and lymphocyte infiltration timepoints were fixed with 4% paraformaldehyde (ThermoScientific) supplemented with Calcium. Samples were then washed, and incubated in a permeabilization & blocking solution (PB) containing normal donkey serum(Jacksonlmmuno) and Triton-X (Sigma). Samples were incubated in a solution containing the primary antibodies diluted in PB. 5 washes with PB were performed, after which the solution containing secondary antibodies in PB was added. Hydrogels were again washed 5 times, then mounted on slides and stored in humid boxes to avoid drying. The samples were imaged with a confocal microscope and 10x, 20x, 40x and 63x objectives.

[0135] The invention can further be described by reference to the following numbered embodiments:1 . A thymic organoid comprising a hydrogel scaffold and a plurality of thymic stromal cells.2. The thymic organoid according to embodiment 1 , wherein the plurality of thymic stromal cells comprises at least one thymic epithelial stem cell, at least one thymic interstitial cell, at least one cortical thymic epithelial cell, at least one medullary thymic epithelial cell, or a combination thereof.3. The thymic organoid according to embodiment 1 or 2, wherein the plurality of thymic stromal cells comprises at least two different types of thymic stromal cells4. The thymic organoid according to any preceding embodiment, wherein the plurality of thymic stromal cells comprises at least one cortical thymic epithelial cell and at least one medullary thymic epithelial cell.5. The thymic organoid according to any preceding embodiment, wherein the plurality of thymic stromal cells comprises at least one thymic epithelial stem cell and at least one thymic interstitial cell.6. The thymic organoid according to any preceding embodiment, wherein the at least one cortical thymic epithelial cell is CD205pos / KRT5nes / KRT14nes.7. The thymic organoid according to any preceding embodiment, wherein the at least one medullary thymic epithelial cell is CD205ne9 / CK5P°s / KRT14P°s.8. The thymic organoid according to any preceding embodiment, wherein the plurality of thymic stromal cells comprises thymic stromal cells which express at least one of CD205, MHC-1 , MHC-II, b5T, and Foxnl .9. The thymic organoid according to any preceding embodiment, wherein the plurality of thymic stromal cells comprises thymic stromal cells which express at least one of KRT10, KRT7, ASCL1 , AIRE1 , CK5 and KRT14.10. The thymic organoid according to any preceding embodiment, wherein the plurality of thymic stromal cells comprises thymic stromal cells which are capable of antigen presentation.11. The thymic organoid according to any preceding embodiment, wherein the plurality of thymic stromal cells comprises thymic stromal cells which produce at least one cytokine selected from the group consisting of SCF, IL7, FLT3-L, IL8 and CXCL12.12. The thymic organoid according to any preceding embodiment, wherein the plurality of thymic stromal cells comprises thymic stromal cells which produce at least one chemokine selected from the group consisting of CCL19, CCL21 and CCL25.13. The thymic organoid according to any preceding embodiment, comprising a cortical or cortical- like region and / or a medullary or medullary-like region.14. The thymic organoid according to embodiment 13, wherein the cortical or cortical-like region comprises cortical thymic epithelial cells.15. Thymic organoid according to embodiment 14, wherein the cortical thymic epithelial cells express at least one of CD205, MHC-1 , MHC-II, b5T, and Foxnl .16. The thymic organoid according to any one of embodiments 13-15, wherein the medullary or medullary-like region comprises medullary thymic epithelial cells.17. The thymic organoid according to embodiment 16, wherein the medullary thymic epithelial cells express at least one of KRT10, KRT7, ASCL1 , AIRE1 , CK5 and KRT14.18. The thymic organoid according to any preceding embodiment, wherein the thymic organoid does not comprise murine bone marrow stromal cells.19. A method for producing a thymic organoid, comprising: a. providing a hydrogel scaffold; b. seeding the hydrogel scaffold with at least one thymic stromal cell, wherein the at least one thymic stromal cell comprises at least one isolated thymic epithelial stem cell, at least one isolated thymic interstitial cell, or a combination thereof; and c. culturing the at least one thymic stromal cell.20. The method for producing a thymic organoid according to embodiment 19, wherein step (b) comprises seeding the hydrogel scaffold with at least one isolated thymic epithelial stem cell and at least one isolated thymic interstitial cell.21. The method for producing a thymic organoid according to embodiment 19 or 20, wherein the isolated thymic epithelial stem cell is BCAMpos, CD49Fpos, CD90posand CD24nes.22. The method for producing a thymic organoid according to any one of embodiments 19-21 , wherein the isolated thymic interstitial cell expresses one or more markers selected from the group consisting of: a mesenchymal marker, TE7, VIM, PDGFRp, Chondroitin Sulfate Proteoglycan 4 (NG2), Smooth Muscle Actin (aSMA), PDGFRa, PDGFRp, CD90, CD146, alkaline phosphatase (ALP), CD73, and P116, or a combination thereof.23. The method for producing a thymic organoid according to any one of embodiments 19-22, wherein step (c) is performed for at least 1 day, at least 7 days, at least 14 days, at least 21 days or at least 28 days.24. A hydrogel scaffold comprising at least one thymic extracellular matrix protein or fragment thereof.25. Use of a hydrogel scaffold for producing a thymic organoid.26. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the hydrogel scaffold is an alginate scaffold.27. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the hydrogel scaffold comprises a thymic extracellular matrix protein or a fragment thereof.28. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the thymic extracellular matrix protein is a medullary extracellular matrix protein.29. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the thymic extracellular matrix protein is a cortical extracellular matrix protein.30. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the hydrogel scaffold comprises fibronectin, a fibronectin domain, or a fragment thereof.31. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the hydrogel scaffold comprises a Notch ligand or fragment thereof, optionally wherein the Notch ligand is DLL1 or DLL4.32. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the hydrogel scaffold or a portion thereof is configured to comprise the mechanical properties of the human thymus or a portion thereof.33. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the hydrogel scaffold or a portion thereof has a stiffness of about 100 Pa to 30 kPa.34. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the hydrogel scaffold or a portion thereof has a stress relaxation half time of 3000s or less.35. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the hydrogel scaffold or a portion thereof has a pore size of about 1 nm to 2000nm.36. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the hydrogel scaffold comprises alginate at a molecular weight of 300 kDa or less.37. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the hydrogel scaffold further comprises hyaluronic acid, collagen, agarose and / or polyethylene glycol (PEG).38. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to embodiment 37, wherein alginate has a molecular weight of from about 50kDa to about 200kDa.39. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the hydrogel scaffold comprises a peptide suitable for cell binding.40. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the peptide suitable for cell binding is an Arg-Gly-Asp (RGD)-containing peptide.41. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding embodiment, wherein the thymic stromal cells are human.42. A method for producing a human T cell, comprising: a. providing a thymic organoid according to any one of embodiments 1 -18 or 26-41 ; b. seeding the thymic organoid with at least one lymphoid progenitor cell, at least one haematopoietic stem cell, or a combination thereof; and c. culturing the thymic organoid under conditions suitable for T cell development.43. The method for producing a human T cell according to embodiment 42, wherein the haematopoietic stem cell is CD34+.44. The method for producing a human T cell according to embodiment 42 or 43, wherein the lymphoid progenitor is a thymocyte.45. The method for producing a human T cell according to any one of embodiments 42-44, wherein step (b) further comprises seeding the thymic organoid with at least one endothelial cell, at least one neuronal progenitor cell, or a combination thereof.46. The method for producing a human T cell according to any one of embodiments 42-45, wherein the T cell is a naive T cell, a CD4+ T cell, a CD8+ T cell, a CD4+ / CD8+ T cell, or a Treg.47. A method for producing a human lymphoid progenitor cell, comprising: a. providing a thymic organoid according to any one of embodiments 1 -18 or 26-41 ; b. seeding the thymic organoid with at least one haematopoietic stem cell, wherein the haematopoietic stem cell is CD34+; and c. culturing the thymic organoid under conditions suitable for differentiation of the at least one haematopoietic stem cell into a lymphoid progenitor cell.48. The method for producing a human lymphoid progenitor cell according to embodiment 47, wherein following step (c) the lymphoid progenitor cell expresses at least one selected from the group consisting of CD5, CD7, CD1a, or a combination thereof.49. The method for producing a human lymphoid progenitor cell according to embodiment 47 or 48, wherein step (c) comprises culturing the thymic organoid with at least one chemokine, cytokine, growth factor, ubiquitin ligase, peptide, or combination thereof.50. The method for producing a human T cell according to any one of embodiments 42-46, or method for producing a human lymphoid progenitor cell according to any one of embodiments 47-49, wherein step (c) comprises culturing the thymic organoid with at least one component selected from the group consisting of IL2, IL7, IL15, SCF, FLT3-L, or a combination thereof.51. The method for producing a human T cell according to any one of embodiments 42-46 or 50, or method for producing a human lymphoid progenitor cell according to any one of embodiments 47-50, wherein culturing is performed for at least 1 day, at least 7 days, at least 14 days, at least 21 days or at least 28 days.52. A human T cell obtained or obtainable by the method according to any one of embodiments 42-46, 50 or 51 .53. A human lymphoid progenitor cell obtained or obtainable by the method according to any one of embodiments 47-51 .54. A method of treating an immune disorder in a subject, the method comprising administering the human T cell according to embodiment 52 to the subject.55. A human T cell according to embodiment 52 for use in a method of treating an immune disorder in a subject.56. A method of treating an immune disorder in a subject, the method comprising administering a human lymphoid progenitor cell according to embodiment 53 to the subject.57. A human lymphoid progenitor cell according to embodiment 53 for use in a method of treating an immune disorder in a subject.58. The method according to embodiment 54 or 56, the human T cell for use according to embodiment 55 or the human lymphoid progenitor cell for use according to embodiment 57, wherein the immune disorder is an immunodeficiency, athymia, DiGeorge syndrome, 22q11 .2 deletion syndrome, FOXN1 -deficiency, or an autoimmunity.59. A method of treating an immune disorder in a subject, the method comprising implanting a thymic organoid according to any one of embodiments 1-18 or 26-41 in the subject.60. The thymic organoid according to any one of embodiments 1-18 or 26-41 for use in a method of treating an immune disorder in a subject, the method comprising implanting the thymic organoid in the subject.Use of the thymic organoid according to any one of embodiments 1 -18 or 26-41 as a thymus implant. A method of screening for agents that modulate T cell development, the method comprising: a. seeding a thymic organoid according to any one of embodiments 1-18 or 26-41 with at least one lymphoid progenitor cell, at least one haematopoietic stem cell, or a combination thereof; b. adding a candidate agent to the thymic organoid; c. culturing the thymic organoid under conditions suitable for T cell development; and d. determining the effect of said agent on T cell development. The method of screening according to embodiment 62, wherein step (b) comprises adding the candidate agent to the hydrogel scaffold, adding the candidate agent to a culture medium, and / or using gene editing to express the candidate agent in the at least one lymphoid progenitor, haematopoietic stem cell, or combination thereof. The method of screening according to embodiment 62 or 63, wherein step (d) comprises comparing the number of T cells obtained by the method with addition of the candidate agent to the number of T cells obtained by the method without addition of the candidate agent. The method of screening according to any one of embodiments 62-64, wherein step (d) comprises comparing the gene expression profile of T cells obtained by the method with addition of the candidate agent to the gene expression profile of T cells obtained by the method without addition of the candidate agent. The method of screening according to any of embodiments 62-65, wherein step (d) comprises comparing the protein expression profile of T cells obtained by the method with addition of the candidate agent to the protein expression profile of T cells obtained by the method without addition of the candidate agent. The thymic organoid, method for producing a thymic organoid, method of treating an immune disorder, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding embodiment, wherein the thymic stromal cell are human cells. The thymic organoid, method for producing a thymic organoid, method of treating an immune disorder, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding embodiment, wherein the thymic organoids comprise a 3D network.69. The thymic organoid, method for producing a thymic organoid, method of treating an immune disorder, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding embodiment, wherein the thymic stromal cell produce one or more cytokines selected from the group consisting of: CCL19, CXCL12, IL-6 and IL-7.70. The thymic organoid, method for producing a thymic organoid, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of the thymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding embodiment, wherein the thymic stromal cells are embedded in the hydrogel.71. The thymic organoid, method for producing a thymic organoid, the hydrogel scaffold, use of a hydrogel scaffold, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of the thymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding embodiment, wherein the hydrogel scaffold does not comprise decellularized ECM.72. The thymic organoid, method for producing a thymic organoid, the hydrogel scaffold, use of a hydrogel scaffold, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of the thymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding embodiment, wherein the hydrogel scaffold is cross-linked with a ligand suitable for cell binding.73. The thymic organoid, method for producing a thymic organoid, the hydrogel scaffold, use of a hydrogel scaffold, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of the thymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to embodiment 72, wherein the ligand is an (RGD)-containing peptide.74. The thymic organoid, method for producing a thymic organoid, the hydrogel scaffold, use of a hydrogel scaffold, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of thethymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to embodiment 72 or 73, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the hydrogel scaffold is cross-linked with the ligand suitable for cell binding.75. The thymic organoid, method for producing a thymic organoid, the hydrogel scaffold, use of a hydrogel scaffold, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of the thymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding embodiment, wherein the hydrogel scaffold, or a portion thereof, comprises a stiffness between about 1 kPa to about 100 kPa.76. The thymic organoid, method for producing a thymic organoid, the hydrogel scaffold, use of a hydrogel scaffold, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of the thymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to embodiment 75, wherein the hydrogel scaffold, or a portion thereof, comprises a stiffness between about 1 kPa and 10kPA.EQUIVALENTS AND SCOPE

[0136] Those skilled in the art will appreciate that the present invention is defined by the appended claims and not by the Examples or other description of certain embodiments included herein.

[0137] Similarly, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0138] Unless defined otherwise above, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, genetics and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art, or according to manufacturer’s specifications.

[0139] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.References[1] Edgar, J.M., Michaels, Y.S. & Zandstra, P.W. Multi-objective optimization reveals time- and dosedependent inflammatory cytokine-mediated regulation of human stem cell derived T-cell development, npj Regen Med 7, 11 (2022)[2] Montel-Hagen, A., Seet, C.S., Li, S., Chick, B., Zhu, Y., Chang, P., Tsai, S., Sun, V., Lopez, S., Chen, H.C. and He, C., 2019. Organoid-induced differentiation of conventional T cells from human pluripotent stem cells. Cell stem cell, 24(3), pp.376-389.[3] Seet, C.S., He, C., Bethune, M.T., Li, S., Chick, B., Gschweng, E.H., Zhu, Y., Kim, K., Kohn, D.B., Baltimore, D. and Crooks, G.M., 2017. Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids. Nature methods, 14(5), pp.521-530.[4] Jon A. Rowley, Gerard Madlambayan, David J. Mooney, Alginate hydrogels as synthetic extracellular matrix materials, Biomaterials, Volume 20, Issue 1 , 1999, Pages 45-53.[5] Chaudhuri, O., Gu, L., Klumpers, D. et al. Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nature Mater 15, 326-334 (2016).[6] Wang, Y., Moradali, M., Goudarztalejerdi, A. et al. Biological function of a polysaccharide degrading enzyme in the periplasm. Sci Rep 6, 31249 (2016).[7] https: / / www.thermofisher.com / uk / en / home / life-science / protein-biology / protein-biology-learning- center / protein-biology-resource-library / pierce-protein-methods / carbodiimide-crosslinker-chemistry.html[8] Vining, K.H., Marneth, A.E., Adu-Berchie, K. et al. Mechanical checkpoint regulates monocyte differentiation in fibrotic niches. Nat. Mater. 21 , 939-950 (2022).[9] Chaudhuri, O., Koshy, S., Branco da Cunha, C. et al. Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium. Nature Mater 13, 970- 978 (2014).

[0010] Elosegui-Artola, A., Gupta, A., Najibi, A.J., Seo, B.R., Garry, R., Tringides, C.M., de Lazaro, I., Darnell, M., Gu, W., Zhou, Q. and Weitz, D.A., 2023. Matrix viscoelasticity controls spatiotemporal tissue organization. Nature Materials, 22(1 ), pp.117-127.

[0011] Tanyarut Boontheekul, Hyun-Joon Kong, David J. Mooney, Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution, Biomaterials, Volume 26, Issue 15, 2005, Pages 2455-2465.

[0012] Campinoti S, Gjinovci A, Ragazzini R, Zanieri L, Ariza-McNaughton L, Catucci M, Boeing S, Park JE, Hutchinson JC, Munoz-Ruiz M, Manti PG, Vozza G, Villa CE, Phylactopoulos DE, Maurer C, Testa G, Stauss HJ, Teichmann SA, Sebire NJ, Hayday AC, Bonnet D, Bonfanti P, 2020. Reconstitution of a functional human thymus by postnatal stromal progenitor cells and natural whole-organ scaffolds. Nature Communications, 11(1 ).

[0013] Ragazzini R, Boeing S, Zanieri L, Green M, DAgostino G, Bartolovic K, Agua-Doce A, Greco M, Watson SA, Batsivari A, Ariza-McNaughton L, Gjinovci A, Scoville D, Nam A, Hayday AC, Bonnet D, Bonfanti P, 2023. Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human thymus. Developmental Cell. 58(22), pp. 2428-2446.

Claims

CLAIMS1 . A thymic organoid comprising a hydrogel scaffold and a plurality of thymic stromal cells.

2. The thymic organoid according to claim 1 , wherein the plurality of thymic stromal cells comprises at least one thymic epithelial stem cell, at least one thymic interstitial cell, at least one cortical thymic epithelial cell, at least one medullary thymic epithelial cell, or a combination thereof.

3. The thymic organoid according to claim 1 or 2, wherein the plurality of thymic stromal cells comprises at least two different types of thymic stromal cells.

4. The thymic organoid according to any preceding claim, wherein the plurality of thymic stromal cells comprises at least one cortical thymic epithelial cell and at least one medullary thymic epithelial cell.

5. The thymic organoid according to any preceding claim, wherein the plurality of thymic stromal cells comprises at least one thymic epithelial stem cell and at least one thymic interstitial cell.

6. The thymic organoid according to any preceding claim, wherein the at least one cortical thymic epithelial cell is CD205pos / KRT5nes / KRT14nes.

7. The thymic organoid according to any preceding claim, wherein the at least one medullary thymic epithelial cell is CD205ne9 / CK5P°s / KRT14P°s.

8. The thymic organoid according to any preceding claim, wherein the thymic organoid does not comprise murine bone marrow stromal cells.

9. A method for producing a thymic organoid, comprising: a. providing a hydrogel scaffold; b. seeding the hydrogel scaffold with at least one thymic stromal cell, wherein the at least one thymic stromal cell comprises at least one isolated thymic epithelial stem cell, at least one isolated thymic interstitial cell, or a combination thereof; and c. culturing the at least one thymic stromal cell.

10. The method for producing a thymic organoid according to claim 9, wherein step (b) comprises seeding the hydrogel scaffold with at least one isolated thymic epithelial stem cell and at least one isolated thymic interstitial cell.

11. The method for producing a thymic organoid according to claim 9 or 10, wherein the isolated thymic epithelial stem cell is BCAMP°S, CD49FP°S, CD90P°Sand CD24nes.

12. The method for producing a thymic organoid according to any one of claims 9-11 , wherein the isolated thymic interstitial cell expresses one or more markers selected from the group consisting of: a mesenchymal marker, TE7, VIM, PDGFRp, Chondroitin Sulfate Proteoglycan 4 (NG2), Smooth Muscle Actin (aSMA), PDGFRa, PDGFRp, CD90, CD146, alkaline phosphatase (ALP), CD73, and P116, or a combination thereof.

13. A hydrogel scaffold comprising at least one thymic extracellular matrix protein or fragment thereof.

14. Use of a hydrogel scaffold for producing a thymic organoid.

15. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding claim, wherein the hydrogel scaffold is an alginate scaffold.

16. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding claim, wherein the hydrogel scaffold comprises a thymic extracellular matrix protein or a fragment thereof.

17. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding claim, wherein the hydrogel scaffold comprises a Notch ligand or fragment thereof, optionally wherein the Notch ligand is DLL1 or DLL4.

18. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding claim, wherein the hydrogel scaffold comprises alginate at a molecular weight of 300 kDa or less.

19. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to claim 18, wherein alginate has a molecular weight of from about 50kDa to about 200kDa.

20. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding claim, wherein the hydrogel scaffold comprises hyaluronic acid, collagen, agarose and / or polyethylene glycol (PEG).

21. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to any preceding claim, wherein the hydrogel scaffold comprises a peptide suitable for cell binding.

22. The thymic organoid, method for producing a thymic organoid, hydrogel scaffold or use according to claim 21 , wherein the peptide suitable for cell binding is an Arg-Gly-Asp (RGD)- containing peptide.

23. A method for producing a human T cell, comprising: a. providing a thymic organoid according to any one of claims 1 -18 or 15-22; b. seeding the thymic organoid with at least one lymphoid progenitor cell, at least one haematopoietic stem cell, or a combination thereof; and c. culturing the thymic organoid under conditions suitable for T cell development.

24. A method for producing a human lymphoid progenitor cell, comprising: a. providing a thymic organoid according to any one of claims 1 -8 or 15-22; b. seeding the thymic organoid with at least one haematopoietic stem cell, wherein the haematopoietic stem cell is CD34+; and c. culturing the thymic organoid under conditions suitable for differentiation of the at least one haematopoietic stem cell into a lymphoid progenitor cell.

25. A human T cell obtained or obtainable by the method according to claim 23.

26. A human lymphoid progenitor cell obtained or obtainable by the method according to claim 24.

27. A method of treating an immune disorder in a subject, the method comprising administering a human T cell according to claim 25 or a human lymphoid progenitor cell according to claim 26 to the subject.

28. A human T cell according to claim 25 or a human lymphoid progenitor cell according to claim 26 for use in a method of treating an immune disorder in a subject.

29. The method according to claim 27, the human T cell for use according to claim 28 or the human lymphoid progenitor cell for use according to claim 28, wherein the immune disorder is an immunodeficiency, athymia, DiGeorge syndrome, 22q11 .2 deletion syndrome, FOXN1- deficiency, or an autoimmunity.

30. A method of treating an immune disorder in a subject, the method comprising implanting a thymic organoid according to any one of claims 1-8 or 15-22 in the subject.31 . The thymic organoid according to any one of claims 1 -8 or 15-22 for use in a method of treating an immune disorder in a subject, the method comprising implanting the thymic organoid in the subject.

32. Use of the thymic organoid according to any one of claims 1-8 or 15-22 as a thymus implant.

33. A method of screening for agents that modulate T cell development, the method comprising: a. seeding a thymic organoid according to any one of claims 1-8 or 15-22 with at least one lymphoid progenitor cell, at least one haematopoietic stem cell, or a combination thereof; b. adding a candidate agent to the thymic organoid; c. culturing the thymic organoid under conditions suitable for T cell development; and d. determining the effect of said agent on T cell development.

34. The thymic organoid, method for producing a thymic organoid, method of treating an immune disorder, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding claim, wherein the thymic stromal cell are human cells.

35. The thymic organoid, method for producing a thymic organoid, method of treating an immune disorder, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding claim, wherein the thymic organoids comprise a 3D network.

36. The thymic organoid, method for producing a thymic organoid, method of treating an immune disorder, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding claim, wherein the thymic stromal cell produce one or more cytokines selected from the group consisting of: CCL19, CXCL12, IL-6 and IL-7.

37. The thymic organoid, method for producing a thymic organoid, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of the thymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding claim, wherein the thymic stromal cells are embedded in the hydrogel.

38. The thymic organoid, method for producing a thymic organoid, the hydrogel scaffold, use of a hydrogel scaffold, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of thethymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding claim, wherein the hydrogel scaffold does not comprise decellularized ECM.

39. The thymic organoid, method for producing a thymic organoid, the hydrogel scaffold, use of a hydrogel scaffold, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of the thymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding claim, wherein the hydrogel scaffold is crosslinked with a ligand suitable for cell binding.

40. The thymic organoid, method for producing a thymic organoid, the hydrogel scaffold, use of a hydrogel scaffold, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of the thymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to claim 39, wherein the ligand is an (RGD)-containing peptide.

41. The thymic organoid, method for producing a thymic organoid, the hydrogel scaffold, use of a hydrogel scaffold, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of the thymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to claims 39 or 40, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the hydrogel scaffold is cross-linked with the ligand suitable for cell binding.

42. The thymic organoid, method for producing a thymic organoid, the hydrogel scaffold, use of a hydrogel scaffold, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of the thymic organoid, use of the thymic organoid or method of screening for agents that modulate T cell development according to any preceding claim, wherein the hydrogel scaffold, or a portion thereof, comprises a stiffness between about 1 kPa to about 100 kPa.

43. The thymic organoid, method for producing a thymic organoid, the hydrogel scaffold, use of a hydrogel scaffold, method for producing a human T cell, method for producing a human lymphoid progenitor cell, human T cell, human lymphoid progenitor cell, human T cell for use, human lymphoid progenitor cell for use, method of treating an immune disorder, use of the thymic organoid, use of the thymic organoid or method of screening for agents that modulateT cell development according to claim 42, wherein the hydrogel scaffold, or a portion thereof, comprises a stiffness between about 1 kPa and 10kPA.

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