Multicellular spheroids and uses thereof

Abstract

This disclosure concerns multicellular spheroids containing endothelial cells, vascular smooth muscle cells and immune cells derived from a subject, and methods of preparation and uses thereof. Also disclosed are mixes and methods for isolating cells from tissue samples from the subject.

Description

[0001] Multicellular spheroids and uses thereof

[0002] Technical field

[0003] The present invention relates, in general terms, to spheroid cell cultures and more specifically to multicellular spheroids comprising endothelial cells, vascular smooth muscle cells and immune cells derived from a subject, and methods of preparation and uses thereof.

[0004] Background

[0005] Currently, there is a need for ways to stratify patients with cardiovascular disease (e.g., atherosclerosis, cardiomyopathy, etc.) based on their response to different treatments. Clinically, it is known that patients can respond differently to the standard care, with some patients developing resistance to particular therapies and others showing accelerated disease progression. Therefore, patient-specific models are needed for testing to help physicians tailor treatments to patients. 2D cell cultures, such as traditional plate-based monolayer cultures and engineered cell sheets, have been used as in vitro vascular models, but such culture systems may not fully recapitulate in vivo cellular interactions or drug uptake and distribution. Other culture systems such as cell-laden hydrogels, engineered or bioprinted vessels, and tissue-on-a-chip platforms require specialised devices or complex fabrication methods, and are difficult to adapt for high-throughput screening studies.

[0006] It would be desirable to overcome or ameliorate at least one of the above-described problems, or at least to provide a useful alternative.

[0007] Summary

[0008] Disclosed herein is a method of preparing a multicellular spheroid, the method comprising a) separately isolating endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and immune cells from one or more tissue samples that have been obtained from a subject, and b) culturing the ECs, VSMCs and immune cells under low adherence conditions to form a multicellular spheroid.

[0009] Also disclosed herein is a multicellular spheroid obtained by a method as defined herein. Disclosed herein is a multicellular spheroid comprising endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and macrophages.

[0010] Disclosed herein is a method of screening for a drug, comprising a) contacting a multicellular spheroid as defined herein with a drug library, and b) identifying a drug based on one or more biological responses from the multicellular spheroid.

[0011] Disclosed herein is the use of a multicellular spheroid as defined herein for drug testing.

[0012] Disclosed herein is a digestion mix for isolating cells from a tissue sample, comprising one or more collagenases, an elastase, a hyaluronidase, a DNase and a trypsin inhibitor.

[0013] Disclosed herein is a kit for isolating cells from a tissue sample, comprising collagenase Type II, collagenase type IV, collagenase type XI, pancreatic elastase, hyaluronidase Type II, DNase I and soybean trypsin inhibitor.

[0014] Disclosed herein is a method for isolating cells from a tissue sample, the method comprising: (a) contacting the tissue sample with a digestion mix as defined herein, under conditions for enzymatic digestion of the tissue; and (b) isolating cells released from the digested tissue.

[0015] Brief description of the drawings

[0016] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0017] Figure 1 is flowchart showing various options for downstream processing of cell suspensions obtained from an aortic punch using the methods of this disclosure.

[0018] Figure 2 shows (A) viability of cells isolated from different aortic punches; and (B) a comparison of the average number of live and dead cells in the samples.

[0019] Figure 3 shows the morphological differences in VSMCs and ECs from human aortic punch. Figure 4 shows (A) flow cytometry profile of isolated cell suspensions showing cell type heterogeneity: fibroblast (CD45- / CD90+), EC (CD45- / CD31+), and VSMC (CD31- / CD90). (B) Flow cytometry overlay histograms for side scatter. (C) and (D) Dot plots for CD31 and CD90 staining showing typical cell profiles for VSMC and EC.

[0020] Figure 5 shows examples of downstream analysis of cells derived from aortic punch. (A) Morphology of VSMC from different clinical groups (inverted microscope scale bar = 50 pm) and (B) corresponding quantification analysis of cell area. (C) EdU staining showing the proliferation ability of VSMC from two clinical groups and (D) corresponding quantification analysis. (E) Collagen gel contraction assay showing the contractile ability of VSMC from different clinical groups and (F) corresponding quantification analysis.

[0021] Figure 6 shows representative image slices of triple- and dual-culture spheroids. (A) Spheroids containing macrophages of different origins. Left: macrophages derived from PBMCs in blood; right: macrophages derived from commercial THP-1 cell line. (B) Spheroids containing SMCs and ECs in different ratios. The SMCs and ECs are commercially available patient-derived cells.

[0022] Figure 7 shows cell viability analysis of cultured 3D spheroids. (A) Livc / Dcad screening using flow cytometry. (B) Confocal imaging using live / dead stain (Calcein: green, representing live cells; AM-Red: red, representing dead cells; Blue: SMCs; Orange: Macrophages / MO) .

[0023] Figure 8 shows atherogenic stimulation of 3D spheroids using an atherogenic cocktail (IPCDilO) containing oxidised LDL conjugated to the lipophilic dye Dil (Dil-conjugated oxidised LDL). (A) Spheroids containing SMCs and macrophages treated with IPCDilO for 72 hrs. (B) Spheroids containing ECs, SMCs and macrophages treated with IPCDilO for 48 hrs (bottom panels) or not treated with the atherogenic cocktail (top panels).

[0024] Figure 9 shows transcriptomic analysis of SMCs in 2D and 3D culture. (A) Volcano plot showing an overview of the differential expression of all genes from RNAseq analysis comparing 3D-grown against 2D-grown SMCs. The threshold in the Volcano plot was - log! 0 adjusted P>2 and |log2 fold change| >2. The top 5 GO (gene ontology) identified using Gorilla arc listed in the table. Figure 10 shows gene expression of IL-ip, MCP-1, ADAM8, 1CAM-1, IL-8 and CXCR4 in 3D LSS and HSS spheroids.

[0025] Figure 11 shows (A) inflammatory status of 3D SMC spheroid after IL-ip treatment; and (B) higher uptake of Dil oxLDL in multi-cell spheroids compared to SMC-only spheroids.

[0026] Figure 12 shows KEGG analysis of gene signatures in multicellular spheroids compared to single cellular spheroids.

[0027] Detailed description

[0028] The present disclosure is predicated on the discovery that multicellular spheroids comprising vascular and immune cells may he produced which exhibit characteristics that mimic those of a vascular tissue. For example, the multicellular spheroid may exhibit a similar or identical behaviour to that of a vascular cell or tissue system with respect to cell composition, organisation, architecture, cell-cell interactions, cellular microenvironment, cell growth, viability, metabolic status, antigen profile, genetic profile, cytokine expression, protein secretion, extracellular matrix production or remodelling, oxidative stress response, shear stress response, molecular uptake, drug response and / or therapeutic resistance. The inventors have discovered that endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and immune cells (e.g., macrophages) derived from a subject can be mixed in various proportions to quickly generate three-dimensional subject-specific spheroid cultures. The presence of the immune cells are advantageous for providing a more representative vascular model for understanding disease development (such as the development of atherosclerosis) and for drug screening. For example, monocyte-derived macrophages and macrophage-like VSMCs in arteries are responsible for internalising and accumulating oxidised low density lipoproteins (ox-LDL), leading to formation of foam cells within atherosclerotic plaques. This progression can be modelled in spheroid cultures containing macrophages by exposing the spheroids to ox-LDL or other atherogenic stimuli to induce foam cell formation and other phenotypic changes characteristic of atheroma formation.

[0029] Advantageously, spheroid culture readily allows the integration of multiple cell types, and the spheroids can self-assemble within 24—48 hours. Unlike 2D culture systems, spheroid cultures are more representative of in vivo 3D tissue organisation and may be better suited for modelling drug uptake and metabolism in tissues. Furthermore, spheroid culture docs not require complex microfluidic or bioprinting systems, fabrication of hydrogel constructs, or preparation of induced pluripotent stem cells from patients. The spheroid model is relatively simple, efficient and low cost to set up, and is amenable to high throughout culture formats (such as multiwell plates) for high-throughput screening or analysis. The inventors have found that biochemical stimuli can be applied during spheroid culture to produce models that mimic atherosclerosis and other vascular conditions. The subject-specific spheroids can be used in a wide variety of research, diagnostic, drug screening and testing, therapeutic and other industrial applications. They can be particularly useful for discovering and developing subject-specific therapies, especially for cardiovascular diseases.

[0030] The inventors have also developed a tissue dissociation protocol that allows cell isolation from tissues with a substantial amount of extracellular matrix (ECM) material, such as arterial tissue. The protocol uses an enzyme mix to efficiently digest ECM to release cells. There are currently no established methods for isolating cells from arteries. Many of the current methods require long incubation times with enzymes or chemicals which can affect the viability of primary cells in the tissue and result in a loss of cell type heterogeneity. The inventors showed that the disclosed method enables the isolation of a highly viable heterogeneous cell population containing endothelial cells, vascular smooth muscle cells and fibroblasts from ECM-rich arterial tissue.

[0031] Accordingly, disclosed herein is a method of preparing a multicellular spheroid, the method comprising a) separately isolating endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and immune cells from one or more tissue samples that have been obtained from a subject, and b) culturing the ECs, VSMCs and immune cells under low adherence conditions to form a multicellular spheroid. Also disclosed herein is a multicellular spheroid obtained by a method as defined herein.

[0032] Disclosed herein is a multicellular spheroid comprising endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and macrophages. The ECs, VSMCs and macrophages may be isolated from one or more tissue samples obtained from a subject.

[0033] Disclosed herein is a digestion mix for isolating cells from a tissue sample, comprising one or more collagenases, an elastase, a hyaluronidase, a DNase and a trypsin inhibitor. The digestion mix may comprise, collagenase Type 11, collagenase type IV, collagenase type XI, pancreatic elastase, hyaluronidase Type II, DNase I and soybean trypsin inhibitor.

[0034] Disclosed herein is a method for isolating cells from a tissue sample, the method comprising: (a) contacting the tissue sample with a digestion mix as defined herein, under conditions for enzymatic digestion of the tissue; and (b) harvesting the isolated cells.

[0035] As used herein, the term “spheroid” refers to an aggregate, cluster or assembly of cells cultured to allow three-dimensional growth, in contrast to the two-dimensional growth of cells in a monolayer or on a two-dimensional substrate, or the growth of cells in a suspension wherein the potential for cell aggregation is limited. The spheroid may be highly organised with a well-defined morphology or it may be a mass of cells that have clustered or adhered together with little organisation. A “heterocellular” or “heterotypic” spheroid is one which contains more than one cell type.

[0036] Cells in the spheroid may be primary cells, cell lines or derivatives thereof. As used herein, a “primary cell” refers to a cell that is obtained by direct removal from an organism, organ, tissue or biological sample. Most primary cells exhibit only a limited life span in cell culture. A “cell line” herein refers to cells which are able to grow continuously in cell culture under suitable culture conditions. Such cells arc also called immortalised cells. Cell derivatives may be differentiated from primary cells or cell lines (e.g., from stem cells, progenitor cells, or cells earlier in a differentiation lineage), or modified from those cells (e.g., through genome or epigenetic editing). The cells may be healthy or diseased.

[0037] An “immune cell” herein refers to an immune cell of myeloid or lymphoid lineage, nonlimiting examples of which include lymphoid and myeloid progenitor cells, monocytes, macrophages (e.g., Ml and M2 macrophages), dendritic cells (e.g., myeloid and plasmacytoid dendritic cells), basophils, neutrophils, eosinophils, mast cells, natural killer cells, natural killer T cells, T cells (e.g., CD4+ and CD8+ T cells), B cells and plasma cells.

[0038] The terms “subject”, “patient”, “host” or “individual”, used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrate animals that fall within the scope of the invention include, but are not limited to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such as from the genus Macaca (e.g., cynomolgus monkeys such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatto) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish. In one embodiment, the subject is a human subject.

[0039] A “tissue sample” herein may be any solid, semi-solid or fluid sample (such as a biopsy sample) from a healthy or diseased tissue. The sample may be from a solid biopsy (e.g., from a blood vessel) or a liquid biopsy (e.g., a blood, serum, plasma or lymph sample). The sample may also include materials derived from a cell or tissue culture, e.g., a culture from a biopsy. Any suitable methods for obtaining a biological sample can be employed. Exemplary methods include phlebotomy, fine needle aspiration, incisional biopsy, excisional biopsy and surgical resection. The sample may be pooled from multiple aliquots. The sample may also be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., cold storage, enzyme treatment, filtration, centrifugation, etc.) prior to cell isolation.

[0040] As used herein, a “non-adherent substrate” refers to a surface or material which does not promote cell adhesion. The substrate may be made of a non-adherent material or coated with a non-adherent or ultra-low attachment material to prevent cell adhesion. Examples of non- adherent or ultra-low attachment materials include perfluorinated polymers, alkenes or similar polymers or mixtures thereof. Other examples include agarose, non-ionic hydrogels such as polyacrylamide, polyethers such as polyethylene oxide, polyols such as polyvinyl alcohol, polysiloxanes such as polydimethylsiloxane, or similar materials or mixtures thereof. The substrate may also be shaped into particular surface geometries that promote spheroid formation, for example, a rounded or curved surface that would allow cells to pool and cluster. The substrate can form a part of a cell culture apparatus or device. For example, the substrate can form a part of a multiwcll plate, a flask, a dish, a tube (e.g., a microtluidic channel), a multi-layer cell culture flask, a bioreactor, or any other laboratory container intended to grow cells or spheroids.

[0041] Tissue digestion, cell isolation

[0042] Cells for spheroid culture may be isolated from one or more tissues samples from a subject. In some embodiments, immune cells are isolated from a blood sample from the subject for spheroid culture. In some embodiments, endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and optionally fibroblasts are isolated from a vascular tissue sample (e.g., an arterial tissue sample) from the subject for spheroid culture.

[0043] Cells may be isolated from a tissue sample in step (a) using any known method which retains cell viability and native phenotype. Generally, for isolation from solid or semi-solid tissues, cells in a sample are first dissociated or separated from each other before forming a cell suspension. Dissociation of cells may be accomplished, e.g.. by chemical treatment or by mechanical disruption (e.g., using a scalpel or scissors or by using a machine such as a homogeniser).

[0044] The inventors have devised a cell isolation protocol capable of isolating a highly viable heterogeneous cell population from solid or semi-solid tissue samples, especially ECM-rich tissues which generally do not give good cell yields. The protocol uses an digestion mix containing various ECM-degrading enzymes to efficiently digest ECM material within a few hours. The short digestion time minimises phenotypic changes and impact on cell viability. The cell isolation protocol is especially useful for extracting cells (including endothelial cells, vascular smooth muscle cells and fibroblasts) from vascular tissues, such as arterial and aortic tissue.

[0045] Disclosed herein is a digestion mix for isolating cells from a tissue sample, comprising one or more collagenases, an elastase, a hyaluronidase, a DNase and a trypsin inhibitor.

[0046] In some embodiments, the tissue sample is a solid or semi-solid tissue, such as vascular, muscle, cartilage, skin, lung, oral, stomach, intestinal, bladder, pancreatic, liver, adipose or lymphoid tissue. In one embodiment, the tissue sample is an arterial tissue. Suitable collagenases include but are not limited to collagenase Type I, Type TT, Type ITT, Type IV, Type V, Type VI, Type Vll, Type Vlll and Type XI. In some embodiments, the digestion mix comprises at least three collagenases. In some embodiments, the digestion mix comprises collagenase Type 11, Type IV and / or Type XI. In some embodiments, the digestion mix comprises collagenase Type II, Type IV and Type XI.

[0047] In one embodiment, the digestion mix comprises collagenase Type II at a concentration of about 1.5 mg / ml to about 2.5 mg / ml, such as about 1.5 mg / ml, about 1.6 mg / ml, about 1.7 mg / ml, about 1.8 mg / ml, about 1.9 mg / ml, about 2 mg / ml, about 2.1 mg / ml, about 2.2 mg / ml, about 2.3 mg / ml, about 2.4 mg / ml, or about 2.5 mg / ml. In one embodiment, the digestion mix comprises collagenase Type II at a concentration of about 2 mg / ml.

[0048] In one embodiment, the digestion mix comprises collagenase Type IV at a concentration of about 0.5 mg / ml to about 1.5 mg / ml, such as about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml. about 0.9 mg / ml, about 1 mg / ml, about 1.1 mg / ml. about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, or about 1.5 mg / ml. In one embodiment, the digestion mix comprises collagenase Type IV at a concentration of about 1 mg / ml.

[0049] In one embodiment, the digestion mix comprises collagenase Type XI at a concentration of about 0.05 mg / ml to about 0.15 mg / ml, such as about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml, about 0.11 mg / ml, about 0.12 mg / ml, about 0.13 mg / ml, about 0.14 mg / ml, or about 0.15 mg / ml. In one embodiment, the digestion mix comprises collagenase Type XI at a concentration of about 0.1 mg / ml.

[0050] Suitable elastases include but are not limited to pancreatic elastase and neutrophil elastase. In some embodiments, the digestion mix comprises pancreatic elastase. In one embodiment, the digestion mix comprises pancreatic elastase at a concentration of about 0.15 mg / ml to about 0.25 mg / ml, such as about 0.15 mg / ml, about 0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.2 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, or about 0.25 mg / ml. In one embodiment, the digestion mix comprises pancreatic elastase at a concentration of about 0.19 mg / ml.

[0051] Suitable hyaluronidases include but are not limited to hyaluronidase Type 1 and Type II. In some embodiments, the digestion mix comprises hyaluronidase Type I. In one embodiment, the digestion mix comprises hyaluronidase Type 1 at a concentration of about 0.2 mg / ml to about 0.3 mg / ml, such as about 0.2 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, about 0.26 mg / ml, about 0.27 mg / ml, about 0.28 mg / ml, about 0.29 mg / ml, or about 0.3 mg / ml. In one embodiment, the digestion mix comprises hyaluronidase Type I at a concentration of about 0.24 mg / ml.

[0052] Suitable DNases include but are not limited to DNase I and DNase II. In some embodiments, the digestion mix comprises DNase I. In one embodiment, the digestion mix comprises DNase I at a concentration of hyaluronidase Type I about 0.5 mg / ml to about 1.5 mg / ml, such as about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 1.1 mg / ml, about 1.2 mg / ml, about 1.3 mg / ml, about 1.4 mg / ml, or about 1.5 mg / ml. In one embodiment, the digestion mix comprises DNase I at a concentration of about 1 mg / ml.

[0053] Suitable trypsin inhibitors include but are not limited to soybean trypsin inhibitor, Kunitz soybean trypsin inhibitor, Bowman-Birk inhibitor, pancreatic secretory trypsin inhibitor and aprotinin. In some embodiments, the digestion mix comprises soybean trypsin inhibitor. In one embodiment, the digestion mix comprises soybean trypsin inhibitor at a concentration of about 0.2 mg / ml to about 0.3 mg / ml, such as about 0.2 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, about 0.26 mg / ml, about 0.27 mg / ml, about 0.28 mg / ml, about 0.29 mg / ml, or about 0.3 mg / ml. In one embodiment, the digestion mix comprises soybean trypsin inhibitor at a concentration of about 0.25 mg / ml.

[0054] In some embodiments, the digestion mix comprises collagenase Type II, collagenase type IV, collagenase type XI, pancreatic elastase, hyaluronidase Type I, DNase I and soybean trypsin inhibitor.

[0055] In some embodiments, the digestion mix further comprises a calcium salt, such as CaCh, Ca( 03)2 or CaSO4. In some embodiments, the digestion mix further comprises a magnesium salt, such as MgCh, Mg(NOa)2 or MgSO4.

[0056] The digestion mix may comprise enzymes and salts in a buffer. Any suitable physiological buffer with an effective pH range of 6 to 8 may be used, non-limiting examples of which include buffers prepared from inorganic salts, such as mono- or dibasic phosphate, bicarbonate or carbonate, and buffers prepared from organic salts, such as HEPES, MOPS, MES or TES. In some embodiments, the buffer comprises HEPES. In some embodiments, the digestion mix further comprises dextrose. In some embodiments, the digestion mix comprises Hanks’ balanced salt solution (HBSS).

[0057] Disclosed herein is a kit for isolating cells from a tissue sample, comprising one or more collagenases, an elastase, a hyaluronidase, a DNase and a trypsin inhibitor. In some embodiments, the kit comprises collagenase Type II, collagenase type IV, collagenase type XI, pancreatic elastase, hyaluronidase Type II, DNase I and soybean trypsin inhibitor.

[0058] Some or all of the components of the kit may be provided together as a mix in dried form (e.g., a powder), to be reconstituted with a liquid prior to use. The reagents may be dried through lyophilisation or other methods known in the art. The kit may further comprise an aqueous buffer for reconstituting the dried components. The buffer may comprise a calcium or magnesium salt (e.g., CaCh, Ca(NOs)2, CaSCE, MgCh, Mg(NOs)2 or MgSCE) and / or an organic solvent, such as dimethyl sulfoxide.

[0059] Disclosed herein is a method for isolating cells from a tissue sample, the method comprising: (a) contacting the tissue sample with a digestion mix as defined herein, under conditions for enzymatic digestion of the tissue; and (b) isolating cells released from the digested tissue.

[0060] In some embodiments, the tissue sample for cell isolation is a vascular tissue sample. A vascular tissue sample herein is not limited to any particular vessel and may be obtained from, for example, lymphatic vessels, arteries, arterioles, veins, venules or capillaries. A vascular tissue sample may be a vessel segment or a section of a vessel wall (full-thickness or a specific part of the vessel wall).

[0061] In one embodiment, the vascular tissue sample is an arterial tissue sample. The arterial tissue sample may be obtained from any artery or arteriole, non-limiting examples of which include the ascending aorta, descending aorta, pulmonary artery, coronary arteries, carotid arteries, subclavian arteries, thoracic arteries, intercostal arteries, vertebral arteries, radial arteries, ulnar arteries, brachial arteries, femoral arteries, iliac arteries, tibial arteries, popliteal arteries, hepatic artery, splenic artery, celiac artery, mesenteric arteries, gastric arteries, renal arteries, and arterioles branching off from said arteries.

[0062] In some embodiments, step (a) is performed at a temperature of about 35°C to about 40°C, such as about 35°C, about 36°C, about 37°C, about 38°C, about 39°C or about 40°C.

[0063] In some embodiments, step (a) is performed for at least about 60 min. For example, step (a) may be performed for about 1 hr, about 1.5 hrs, about 2 hrs, about 2.5 hrs, about 3 hrs, about 3.5 hrs, about 4 hrs, or for longer than 4 hrs but less than 12 hrs. In one embodiment, step (a) is performed for about 2 hours. The inventors have found that a 2-hour incubation time produces sufficient tissue dissociation while minimising cell stress and the risk of cell death due to lack of nutrients.

[0064] The method may be suitably adapted to digest tissues of different types and sizes, and in particular different types of vascular tissue. By way of non-limiting example, a larger volume of the digestion mix may be used to digest larger tissue samples, while keeping the incubation time and enzyme concentration constant. The particular enzyme mix can produce higher cell yields and higher viability for the isolated cells as a result of more efficient tissue digestion.

[0065] Cell yields generally depend on the type and size of the tissue sample and on the incubation time, and may range from about 102cells to about 104cclls / mg tissue. For example, enzymatic dissociation of small tissues (e.g., an arterial punch) may yield between about 50 cells to about 500 cells / mg tissue, while larger tissues (e.g., full arterial resections) may yield about 1000 to about 10,000 cells / mg.

[0066] In some embodiments, the isolated cells are at least about 80% viable, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% viable. In some embodiments, the isolated cells are at least about 85% viable. Cell viability may be quantified using methods well known in the art, such as assays based on cell metabolism (e.g., MTT, MTS, resazurin or Alamar Blue assays), and assays based on changes in cell membrane permeability (e.g., Trypan blue, lactate dehydrogenase or live / dead fluorescent assays). In some embodiments, the method comprises isolating endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and / or fibroblasts.

[0067] Preferably, the harvested cells are treated to remove dead and / or dying cells and / or cell debris. The removal of such dead and / or dying cells is accomplished by any conventional means known to those skilled in the art, for example using filtration, centrifugation, bead extraction and / or antibody methods. It is known, for example, that phosphatidylserine is redistributed from the inner to outer plasma membrane leaflet in apoptotic or dead cells. The use of Annexin V-B iotin binding followed by binding of the biotin to streptavidin magnetic beads enables separation of apoptotic cells from living cells.

[0068] Suitable vascular or immune cells may be identified by antibody- or aptamer-based tagging of specific cell surface protein markers, and separated by chromatography (e.g., affinity chromatography) or flow cytometry using the same antibody- or aptamer-based tagging. Cells isolated in this way are known as primary cells. Primary cells may undergo further differentiation or modification before being used for spheroid culture. For example, isolated immune cells may be differentiated into specific immune cell lineages for incorporation into spheroids.

[0069] Spheroid culture

[0070] A skilled person may culture the different cell types in any suitable proportion to produce spheroids with desirable properties. For instance, cell ratios may be selected to recapitulate ratios in subject tissues. As an example, a EC:VSMC ratio of 1:5 may be chosen to reflect the cell proportion in non-diseased aorta, and a EC : VSMC ratio of between 1 : 1 and 1 : 2 may be chosen to reflect the cell proportion in diseased carotid arteries. In general, spheroids form more readily when the proportion of VSMCs is higher than the proportion of other cell types.

[0071] In some embodiments, the culture comprises a ratio of ECs to VSMCs of about 10:1 to about 1:10. The culture may comprise a ratio of ECs to VSMCs of about 10:1, about 9:1, about 8:1 , about 7:1 , about 6:1 , about 5:1 , about 4:1 , about 3:1 , about 2:1 , about 1 :1 , about 1 :2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10. In some embodiments, the culture comprises immune cells at a proportion of about 2% to about 20% of the total cell population. The culture may comprise immune cells at a proportion of about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% of the total cell population.

[0072] In some embodiments, the immune cell is a macrophage or a T cell (e.g., a CD4+ or CD+ T cell). In one embodiment, the immune cell is a macrophage.

[0073] In one embodiment, the culture comprises ECs, VSMCs and immune cells at a proportion of about 16%, about 72% and about 12% of the total cell population respectively.

[0074] In one embodiment, the ECs are CD45-, CD31+ and / or CD90- ECs. In one embodiment, the VSMCs are CD45-. CD31- and / or CD90- VSMCs. The presence or absence of these cell markers can be used to distinguish and / or separate ECs and VSMCs from other cells in a cell isolate.

[0075] The “CD” or “cluster of differentiation” designation refers to proteins found on the surface of cells, each unique surface protein having been assigned its own unique number identifier. Surface expression of a particular CD molecule and especially a particular combination of CD molecules is useful for the characterisation of cell phenotypes. ECs and VSMCs with suitable CD profiles may be identified using any means known in the art, e.g., using labelled antibodies or aptamers against specific CD molecules, using gene expression profiling (e.g., RNA-seq or other transcriptomic studies), or using single-cell proteomic analysis.

[0076] The spheroids may be cultured in any suitable culture medium. However, the inventors have discovered that a mixture of cell media may be used to support growth of the different cell types in the spheroid. Unlike conventional media formulations in which considerable effort may be expended to tailor a culture medium to a specific cell composition, an advantage of the mixed medium of this disclosure is that it can be readily customised for different spheroid compositions without the need for extensive reformulation. Eor example, the various media can be mixed in proportions corresponding to the cell composition of the spheroid. Accordingly, in some embodiments, step (b) of the method comprises culturing ECs, VSMCs and immune cells in a mixed medium comprising endothelial cell medium, smooth muscle cell medium and immune cell medium. In some embodiments, the proportion of the endothelial cell medium, smooth muscle cell medium and immune cell medium in the mixed medium is selected to correspond to the proportion of ECs, VSMCs and immune cells in the total cell population respectively. The skilled person will understand that the media proportion may be adjusted during the course of cell culture based on changes in the proportion of cells in the spheroid. The mixed medium may also contain additional media if additional cell types are present in the spheroid. For example, the mixed medium may additionally contain fibroblast medium if fibroblasts are used in spheroid culture.

[0077] The endothelial cell medium may be any medium that both supports the growth of and maintains the phenotype of endothelial cells from a subject. Endothelial cell medium will generally comprise a basal medium supplemented with defined concentrations of growth factors and other additives, non-limiting examples of which include epidermal growth factor (EGF), basic fibroblast growth factor (bFGF or FGF2), vascular endothelial growth factor (VEGF), hydrocortisone, L-glutamine, ascorbic acid, serum (e.g., fetal bovine serum) and other tissue extracts (e.g., bovine brain, pituitary or hypothalamic extract). Commercially available endothelial cell medium includes ATCC Microvascular EC Growth Kit-BBE, LifeLine Celltech’s VascuLife® EnGS-Mv LifeFactors Kit, Lonza EGM™-MV BullctKit™, Cell Applications Human EC Growth Medium Kit, PAN-Biotcch Endopan MV Kit, and Gibco Microvascular Growth Supplement.

[0078] The smooth muscle cell medium may be any medium that both supports the growth of and maintains the phenotype of vascular smooth muscle cells from a subject. Smooth muscle cell medium will generally comprise a basal medium supplemented with defined concentrations of growth factors and other additives, non-limiting examples of which include epidermal growth factor (EGF), basic fibroblast growth factor (bFGF or FGF2), insulin, L-glutamine, ascorbic acid and serum (e.g., human or fetal bovine serum). Commercially available smooth muscle cell medium includes ATCC Vascular Smooth Muscle Cell Growth Kit, LifeLine Celltech’s VascuLife® SMC Medium Complete Kit, Lonza SmGM™-2 BulletKit™, PromoCell Smooth Muscle Cell Growth Medium 2, Ceil Applications Smooth Muscle Cell Medium Kit, and ScienCell Smooth Muscle Cell Growth Supplement. The immune cell medium may be any medium that hoth supports the growth of and maintains the phenotype of specific immune cell types from a subject. Immune cell medium will generally comprise a basal medium supplemented with defined concentrations of growth factors and other additives specific for particular immune cells. For instance, scrum (e.g., human or fetal bovine serum), interleukin-4 (IL-4), macrophage colony-stimulating factor (M-CSF or CSF-1) and granulocyte macrophage colony-stimulating factor (GM-CSF or CSF-2) are common additives in macrophage culture, while insulin, transferrin, L- glutamine, IL-2, IL-7 and IL-15 may be used for T cell culture. Commercially available macrophage medium includes STEMCELL Technologies ImmunoCult™-SF Macrophage Medium, Lonza X-VIVO-15, ScienCell Macrophage Medium, and Gibco Macrophage - SFM. Commercially available T cell medium includes STEMCELL Technologies ImmunoCult™-XF T Cell Expansion Medium, Miltenyi Biotec TexMACS™ Medium, CellGenix GMP T Cell Medium, and Takara Bio LymphoONE T-Cell Expansion Xeno-Free Medium.

[0079] In some embodiments, step (b) of the method further comprises isolating fibroblasts from the one or more tissue samples from the subject and culturing the fibroblasts with the ECs, VSMCs and immune cells to form the multicellular spheroid. A skilled person may culture the cells in any suitable ratio to produce spheroids with desirable properties.

[0080] In some embodiments, the spheroid comprises ECs and SMCs isolated from a vascular tissue sample from a subject. In one embodiment, the spheroid comprises ECs and SMCs isolated from an arterial tissue sample.

[0081] In some embodiments, step (b) of the method comprises culturing the ECs, VSMCs and immune cells on a non-adherent substrate.

[0082] In some embodiments, the cells are grown in wells or microwells that have a concave surface, such as a hemi-spherical surface, a conical surface having a rounded bottom, and similar surface geometries or a combination thereof. The well and well bottom can ultimately terminate, end, or bottom-out in a spheroid-conducive rounded or curved surface, such as a dimple, a pit, and similar concave or frusto-conical relief surfaces, or combinations thereof. A combination of non-adherent wells, well geometry, and gravity can induce cells cultured within wells to sclf-asscmblc into spheroids, while the geometry of the wells impose a size limit to spheroid growth. Tn addition, the wells may he structured and arranged to provide for the movement of liquid into and out of the wells without trapping air between the substrate and liquid or liquid droplets that are introduced into the wells For example, well geometries may allow for the efficient displacement of air upon introduction of liquid to the well, or provide pathways for the flow of liquid into the well without blocking the escape of ah from the well.

[0083] Other examples of co-culture methods include pellet culture, hanging drop, liquid overlay, spinner culture, rotating wall vessels, liquid marble culture, magnetic levitation, gel embedding, microfluidic culture, and combinations thereof. Pellet culture uses centrifugal force to cluster cells, e.g., at the bottom of a rounded microwells. The hanging-drop method relies on surface tension and gravitational force for cell clustering; in a typical set-up, small droplets of cell suspension prepared in a culture medium are formed on the lid of a nonadherent culture dish and allowed to grow as pendant drops. In the liquid overlay method, cells are seeded on a non-adherent substrate and allowed to aggregate naturally into spheroidal clusters. In spinner bioreactors, cells in the culture medium are exposed to constant stirring, which provides shear stress that forms a 3D structure by hindering the cells from sticking to the bioreactor walls. Similarly, rotating bioreactors rely on constant circular rotation of the culture vessel to prevent cell adhesion to the walls while allowing for cellular self-organisation to form spheroids. With the liquid marble technique, hydrophobic coating material (e.g., polytetrafluoroethylene powders) is added to droplets of cell suspension to create a thin, porous, elastic hydrophobic outer shell which allows the entrapped cells to interact and self-assemble into spheroids over time. Magnetic levitation uses a magnetic force to levitate cell aggregates labelled with magnetic or diamagnetic beads. Positive magnetophoresis is based on directing cells labelled with magnetic beads, while negative magnetophoresis is based on controlling diamagnetic cells in a paramagnetic medium. In the gel-embedding technique, cells are encapsulated in or injected into a scaffold (typically an inert polymeric material which may or may not be crosslinked) which supports the formation of a spheroid therein. The scaffold or polymer may comprise natural polymers (e.g., collagen, fibrinogen, laminin, fibronectin, vitronectin, hyaluronic acid, fibrin, alginate, agarose, chitosan, dextran, Ficoll or Matrigel), decellularised natural polymeric matrices, or synthetic polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol, polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), or polyethylene oxide (PEO)). Microtluidic culture uses microfluidic flow to perfuse and support spheroid growth in microchannels. Spheroids may he grown in microwells or hanging drops in microfluidic channels. In addition, external forces such as acoustic actuation using acoustic wave patterning, or dielectrophoretic focusing using electrical forces applied to electrodes surrounding a culture tube or channel, can be used to structure spheroids during growth.

[0084] In some embodiments, the spheroids are cultured to a cell density of about 4000 to about 6000 cells per spheroid, such as a cell density of about 4000 cells, about 4500 cells, about 5000 cells, about 5500 cells, or about 6000 cells per spheroid. In one embodiment, the spheroids are cultured to a cell density of about 5000 cells per spheroid. The inventors have found that spheroids of this cell density can remain viable for the duration of most drug screening or testing studies while being amenable to imaging.

[0085] The method for spheroid production may be automated or semi-automated to allow high- throughput production of multicellular spheroids. For example, the spheroids may be produced in multi-well plates (such as 96, 384. 1536 well plates and the like), and robotic and / or computer controlled ejectors used to dispense the cell suspension and / or culture medium. Preferably, the multi-well plate is transparent which allows for imaging of the spheroids during or after growth.

[0086] Assaying spheroid formation is routine in the art and may be done, for example, by microscopy and / or image analysis. For example, the size of the spheroids may be monitored under microscopy. Cells may also be treated with fluorescent stains and analysed with epi- or confocal microscopy, or with brightfield microscopy, to determine cell viability or cell distribution.

[0087] In one embodiment, the method comprises culturing the ECs, VSMCs and immune cells in the presence of an atherogenic stimulus. As used herein, an “atherogenic stimulus” refers to a compound capable of promoting the development and / or progression of atherosclerosis. The atherogenic stimulus may be, for example, a peptide, protein, glycoprotein, lipoprotein, lipid, metabolite or small molecule endogenous or exogenous to a subject. The atherogenic stimulus may further comprise a detectable label (e.g., a fluorescent or colorimetric dye).

[0088] The atherogenic stimulus may he added to the culture medium at any time during cell culture. Cells may be exposed to the atherogenic stimulus for any length of time sufficient to induce a desired biological response. For example, the cells may be cultured in the presence of the atherogenic stimulus for at least about 12 hr, at least about 24 hr, at least about 36 hr, at least about 48 hr, at least about 60 hr, at least about 72 hr, at least about 84 hr, or at least about 96 hr. In some embodiments, the cells are cultured in the presence of the atherogenic stimulus for at least 48 hr.

[0089] The method may comprise detecting a biological response in the spheroids following exposure to the atherogenic stimulus. The biological response may be, for example, uptake of the atherogenic stimulus into one or more cell types, or a change in the survival, growth, proliferation, differentiation, migration, distribution, morphology, signalling, metabolic activity, gene expression, cell-cell interaction and / or expression of a cell surface marker in one or more cell types. The biological response may also be a change in the size or cell distribution of the spheroid.

[0090] The uptake of ox-LDL may provide an indication of the ability of a drug to penetrate the spheroids and the potential drug distribution in the cells of the spheroid.

[0091] In one embodiment, the atherogenic stimulus comprises a low-density lipoprotein (LDL), cholesterol, a cytokine or a combination thereof.

[0092] In some embodiments, the atherogenic stimulus comprises a cytokine. The cytokine may be a pro-inflammatory cytokine. In one embodiment, the cytokine is interleukin 1 beta (IL- Ip).

[0093] In some embodiments, the atherogenic stimulus comprises an LDL. In some embodiments, the atherogenic stimulus comprises an LDL and cholesterol. In some embodiments, the atherogenic stimulus comprises an LDL, cholesterol and a cytokine.

[0094] The LDL may be an oxidised LDL (ox-LDL) or a malondialdehyde (MDA)-modified LDL. In some embodiments, the atherogenic stimulus comprises ox-LDL at a concentration of about 10 pg / ml to about 100 ug / ml in the culture, such as a concentration of about 10 pg / ml, about 20 pg / ml, about 30 pg / ml, about 40 pg / rnl, about 50 ug / ml, about 60 pg / rnl. about 70 pg / ml, about 80 pg / ml, about 90 pg / ml, or about 100 pg / ml. In one embodiment, the atherogenic stimulus comprises ox-LDL at a concentration of about 50 g / ml. Disclosed herein is a multicellular spheroid obtained by a method as defined herein.

[0095] Disclosed herein is a multicellular spheroid comprising endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and macrophages. In some embodiments, the ECs, VSMCs and macrophages are isolated from one or more tissue samples that have been obtained from a subject.

[0096] Methods of use

[0097] The multicellular spheroids may find use in a wide variety of research, diagnostic, drug screening and testing, and other therapeutic applications. In particular, since the multicellular spheroids herein mimic in vivo vascular cell systems, these spheroids may be used for pharmacokinetic profiling, pharmacodynamic profiling, treatment efficacy studies, cytotoxicity studies, penetration studies of compounds, therapeutic resistance studies, personalised or tailored therapies, small molecule identification and / or testing, nucleic acid drug testing, biomarker identification, and the like.

[0098] Accordingly, disclosed herein is a method of screening for a ding, comprising a) contacting a multicellular spheroid as defined herein with a drug library, and b) identifying a drug based on one or more biological responses from the multicellular spheroid.

[0099] Also disclosed herein in the use of a multicellular spheroid as defined herein for drug testing.

[0100] The drug may be any biologically active agent, including polypeptides, peptides, nucleic acids, small molecules, natural products, and combinations and pharmaceutical compositions thereof. The candidate drug may be developed for, indicated for, or is being repurposed for treating or preventing a vascular or metabolic condition, e.g., atherosclerosis, vasculitis, pathogen-induced vasculopathy or diabetic vasculopathy. The candidate drug may target the expression of particular genes in VSMCs, or pathways associated with lipid accumulation and / or efflux from the spheroid cell types, or pathways associated with inflammation (e.g., the candidate drug may be colchicine or sirolimus or antibodies or inhibitors against pro-inflammatory cytokines such as IL-ip). The candidate drug may be a compound or a composition customised to a subject, particularly a subject from whom cells have been isolated to produce the multicellular spheroids. In some embodiments, the method of drug screening is a high throughput method. Assays which are capable of high throughput operation are particularly preferred, e.g., for screening a library of candidate drugs, or for testing a candidate drug on a library of subject-specific spheroids. The assays may be performed on multicellular spheroids cultured in multi-well plates as described above. Further, the assays may be automated or semi-automated. For instance, addition of the candidate drug to the spheroid may be automated.

[0101] The drug may be contacted with a spheroid at any point during spheroid culture. The drug may be contained within a delivery vehicle so as to control the rate at which the agent is released. Alternatively, the drug may be one (such as a polynucleotide) that is expressed in the cells of the multicellular spheroid.

[0102] Drug response may be assessed by examining one or more biological responses from the spheroid. Non-limiting examples of biological responses include changes to cell survival, growth, proliferation, differentiation, migration, morphology, distribution, signalling, metabolic activity, gene expression, cell-cell interaction, and expression of cell surface markers. Drag response may also be assessed by examining one or more properties of a spheroid, examples of which include spheroid size and cell distribution in the spheroid.

[0103] Assessment of biological response may be carried out using any suitable method known in the art. Spheroids may first be fixed by snap-freezing or chemical fixation techniques before observation. Any of cell survival, growth, proliferation, differentiation, migration and morphology may be assessed by microscopy or image analysis. Cell properties may also be detected using appropriate markers. For example, expression of detectably-labelled proteins, reporters and / or single-step labelling of cell components and markers can enable cell architecture, multicellular organisation and other readouts to be directly visualised, for example by fluorescence microscopy (e.g., E-cadherin staining to identify cell-cell contacts, detection of secreted inflammatory markers using labelled antibodies, detection of lipid accumulation or lipid efflux using suitably labelled lipids or lipoproteins, etc.). Gene expression may be assessed by functional genomic or transcriptomic (e.g., microarray) techniques, and so on. It is appreciated that the skilled person may select the appropriate technique to assess a given property. As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0104] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0105] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0106] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of' will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0107] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0108] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0109] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

[0110] Examples

[0111] Example 1: Isolation of patient-derived cells from vascular tissue

[0112] Methodology

[0113] Human aortic tissue was collected from the operating theatre, and mouse aortic tissue was collected from the dissection facility. The tissues were preserved in 4°C Custodiol solution (Dr. Franz Kohler Chemie GmbH) under sterile conditions immediately after collection, and transferred to the laboratory. The cut-off time was 30 minutes from collection to cell isolation, but an even shorter time can be better for tissue preservation. Under sterile conditions, the tissue was rinsed in 4°C Custodiol with penicillin-streptomycin solution (100 U / mL) (Thermo Fisher Scientific), followed by three rinses in ice-cold Dulbccco's phosphate -buffered saline (DPBS) (Thermo Fisher Scientific). Blood was removed as much as possible. The washed tissue was minced with a scalpel into 1 mm3pieces, while removing the non-aortic parts.

[0114] The tissue pieces were placed in a BSA-coated tube containing sufficient volume of a custom enzyme cocktail EM8. Incubation was performed in a water bath at 37°C, with gentle tube inverting every 10 minutes for a total of 60 minutes. The cell suspension was strained through MACS® Smarts trainers (30 pm) (Miltenyi Biotec) and washed with copious amounts of Hanks' balanced salt solution (HBSS) (Thermo Fisher Scientific). The cell suspension was centrifuged at 500 g for 5 minutes at 4°C and the supernatant was discarded. The cell pellet can be resuspended in an appropriate cell culture medium or in PBS. EM8 comprises 8 components (6 types of enzymes and trypsin inhibitor in buffer solution) (Tabic 1).

[0115] Table 1. EM8 Recipe

[0116] For QC check, 50 L of the cell suspension was added to 50 iiL of LIVE / DEAD™ Cell Viability / Cytotoxicity Kit for mammalian cells (Thermo Fisher Scientific) to determine cell viability and cell number. The mixture was mixed gently and transferred to a 96-well plate. The plate was wrapped with aluminium foil and incubated for 30 minutes. The cells were observed under a fluorescence microscope and live and dead cells were manually counted throughout the plate.

[0117] The cell suspension can be processed in different ways depending on the intended study (Figure 1 ). For example, the cell suspension can be used to develop cell cultures or it can undergo further sorting into specific cell populations or undergo enrichment for specific cell types.

[0118] For cell culture, either smooth muscle cell-enriched (SmGM) or endothelial cell-enriched (EGM) medium was used, depending on the cell type required. An additional amount of SmGM / EGM medium was used to dilute the cell suspension to the recommended seeding density, and an appropriate volume of cell suspension was transferred to new cell culture dishes, which were then placed in the incubator. The cell culture growth medium was changed once every 2 to 3 days to maintain the cell culture until it reached 70—80% confluence. The protocol can be varied depending on the amount of tissue available and the nature of the material available.

[0119] Results

[0120] Cells were isolated from human aortic biopsies (punch technique with a 3.00 mm device), human bulk aortic tissue and mouse aortic tissue using the EM8 enzyme cocktail and the described protocol. Around 10,000 cells could be obtained from the aortic biopsy with viability of 87% on average (Table 2, Figure 2). In general, an average cell yield of 149 cells per milligram of aortic punch tissue and 3798 cells per milligram of bulk aortic tissue, typically with cell viability of 90% or more, can be obtained using the present enzyme mix and dissociation protocol. The EM8 enzyme cocktail was also used to isolate cells from mouse tissue, and more than 80% viable single-cell suspensions were obtained from mouse aortic tissue samples, with an average cell yield of 257 cells per milligram. Therefore, it can be seen that this protocol enables the isolation of highly viable single cells from both human and animal tissue.

[0121] Table 2. Total cell numbers of dissociated aortic punches from 10 patients using enzymatic cocktail EM 8.

[0122] Cells which were morphologically short and spindle-shaped were identified as VSMCs and short rounded cells were identified as ECs (Figure 3). The cell characterisations were confirmed by flow cytometry profiling (CD31- / CD90- and CD31+) (Figure 4).

[0123] Cells were isolated from 12 patients: 6 samples from diabetic and 6 samples from nondiabetic patients. PARSE sequencing analysis were performed on the isolated cells, and QC analysis revealed that the dissociation protocol and enzymatic mix EM8 were suitable for single cell downstream processing. The resulting single-cell suspension was successfully used to generate patient-specific cell lines and single-cell transcriptomic analysis.

[0124] Example 2: Triple-cell spheroid culture and use in disease modelling

[0125] Methodology

[0126] The present study investigates the influence of 3D spheroid culture on cellular physiology and its potential for atherosclerosis modelling, using vascular smooth muscle cells (VSMCs), endothelial cells (ECs) and macrophages from primary arterial sources. A reproducible approach for generating patient-specific 3D spheroid culture is defined.

[0127] Patient-specific or commercial smooth muscle cells (SMCs), endothelial cells (ECs) and PBMC -derived macrophages were isolated for spheroid seeding at percentage of 72%, 16% and 12% respectively in each spheroid containing 5000 cells. Patient-derived VSMCs (CD45- / CD31- / CD90-) and ECs (CD45- / CD31+ / CD90-) were isolated via enzymatic dissociation of arterial samples and magnetic microbeads sorting based on the respective surface markers. Briefly, aortic biopsies were cut into small pieces and incubated with an enzymatic cocktail (EM8) to detach the cells and dissociate the tissue scaffolding. Detached ECs and SMCs were isolated and cultured in cell type-specific media and characterised using flow cytometry. Patient-derived macrophages were differentiated from CD14+ monocytes isolated from peripheral blood mononuclear cells (PBMC) using magnetic-activated cell sorting. The cells were seeded in an ultra-low protein binding round bottoms microplate (COSTAR #7007). The media used contained a mix of Smooth Muscle Cell Growth Mcdium-2 BulletKit™ (CC-3182), EGM™-2 Endothelial Cell Growth Medium-2 BulletKif™ (CC- 3162) and RPM1 supplemented with 10% EBS, at a ratio of 9:2:1.5. The microplatc was centrifuged at 600 g for 10 minutes to initiate cell congregation and subsequently incubated at 37°C and 5% CO?. Addition of 50 pl fresh media containing Smooth Muscle Cell Growth Medium-2 BulletKit (CC-3182), EGM™-2 Endothelial Cell Growth Medium-2 BulletKit™ (CC-3162) andRPMI supplemented with 10% FBS were required daily in each well. Abrupt, frequent, or unnecessary movements were avoided when handling the spheroids to avoid interference of spheroid formation through agitation during the first 24 hours.

[0128] Results

[0129] The effect of 3D spheroid culture on cellular physiology and in atherosclerosis modelling was monitored using spheroids containing vascular smooth muscle cells (VSMCs), endothelial cells (ECs) and macrophages from primary arterial sources.

[0130] It is shown that, in the 3D spheroids, ECs are generally localised within the spheroid core and macrophages are scattered throughout the spheroids when seeded (Figure 6). Moreover, in investigating the viability of 3D spheroids, it was observed that the majority of the cells in the spheroids remained viable up to at least Day 12, based on flow and confocal analysis (Figure 7), suggesting the possibility of using these spheroids for prolonged treatment in the future.

[0131] In studies of atherosclerosis, atherogenic stimuli such as low-density lipoprotein (LDL) may be used to activate VSMCs and ECs. It was observed in the present studies that prolonged treatment of SMC-macrophage co-culture spheroids with oxidised lipoprotein conjugated to the Dil dye (Dil-oxLDL) up to 72 hr gave rise to colocalisation of Dil-oxLDL with SMCs and macrophages (Figure 8), suggesting a uptake of the oxidised LDLs by both SMCs and macrophages and subsequent morphological changes. Uptake of Dil-oxLDL by SMCs and macrophages was also seen when EC-SMC-macrophage triple culture spheroids were treated with Dil-oxLDL for 48 hr. Lastly, transcriptomic analysis showed that 3D VSMCs in spheroids have gene expression profile that were enriched in gene ontology (GO) terms related to inflammatory response regulation, immune system regulation and aging compared to 2D-grown VSMCs (Figure 9).

[0132] The protocol described for generating the 3D spheroid culture is defined and reproducible. Moreover, the cells in the spheroids were shown to behave differently from single VSMC spheroids, i.e., the cells in the spheroids have different gene expression profiles and exhibit more enrichment in gene ontology pathways such as inflammatory response regulation. The spheroids may be used to study vascular diseases driven by inflammation, such as atherosclerosis.

[0133] Example 3: 3D spheroid cultures as patient-specific disease models

[0134] It was previously shown that aortic tissue obtained from patients with low syntax score (LSS) exhibited higher tissue expression of IL- 1(3 and upregulation of differentially-expressed genes (DEGs) associated with atherosclerosis signalling, immune pathways and inflammation-associated pathways involving genes IL- 1 (3, CCL2, CXCL8, CXCR4, 1CAM1, ADAM8, MMP9, PCYOX1 , compared to patients with high syntax score (HSS) (Dorajoo, R. ct al., Atherosclerosis. 2023;365:15-24).

[0135] Using SMCs derived from patients with LSS and HSS, single-cell spheroids were generated and analysed for gene expression and DEGs. It was observed that 3D spheroids derived from LSS and HSS patients exhibited gene signatures that were similar to the previously reported data (Figure 10). This suggests the potential of triple-cell spheroids as patient-specific models.

[0136] Stimulation with the inflammatory cytokine IL- 1(3 is known to cause a phenotypic switch towards a distinct inflammatory state. Using patient-specific single-cell SMC spheroids, it was observed that treatment with IL- 1(3 resulted in increased inflammatory status of the spheroids (Figure 11 A). KEGG analysis of cells within multicellular spheroids also showed enrichment in several pathways related to inflammation (e.g., cytokine-cytokine receptor interaction, IL-17, TNF and NF-kappa B signalling) (Figure 12). Thus, it can be seen that the spheroids retained patient-specific features during culture, and are potentially useful as patient-specific models for studying vascular diseases such as atherosclerosis which are driven by inflammation, and for drug screening for personalised therapy.

[0137] This suggests the potential of spheroids as a means of drug screening in responding to external drug uptake whereby multi-cell spheroids would likely be more responsive towards drugs with greater uptake of oxLDL compared to single -cell SMC spheroids (Figure 1 IB).

[0138] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

Claims

CLAIMS1. A method of preparing a multicellular spheroid, the method comprising a) separately isolating endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and immune cells from one or more tissue samples that have been obtained from a subject, and b) culturing the ECs, VSMCs and immune cells under low adherence conditions to form a multicellular spheroid.

2. The method of claim 1, wherein the immune cell is a macrophage or a T cell.

3. The method of claim 1 or 2, wherein the culture comprises a ratio of ECs to VSMCs of about 10:1 to about 1:10.

4. The method of any one of claims 1 to 3, wherein the culture comprises immune cells at a proportion of about 2% to about 20% of the total cell population.

5. The method of claim 3 or 4, wherein the culture comprises ECs, VSMCs and immune cells at a proportion of about 16%, about 72% and about 12% of the total cell population respectively.

6. The method of any one of claims 1 to 5, wherein step (b) comprises culturing the ECs, VSMCs and immune cells in a mixed medium comprising endothelial cell medium, smooth muscle cell medium and immune cell medium.

7. The method of claim 6, wherein the proportion of the endothelial cell medium, smooth muscle cell medium and immune cell medium in the mixed medium is selected to correspond to the proportion of ECs, VSMCs and immune cells in the total cell population respectively.

8. The method of any one of claims 1 to 7, wherein the method further comprises isolating fibroblasts from the one or more tissue samples from the subject and culturing the fibroblasts with the ECs, VSMCs and immune cells to form the multicellular spheroid.

9. The method of any one of claims 1 to 8, wherein the ECs and VSMCs are isolated from an arterial tissue sample from the subject.

10. The method of claim 9, wherein cell isolation comprises contacting the arterial tissue sample with a digestion mix comprising one or more collagenases, an elastase, a hyaluronidase, a DNase and a trypsin inhibitor, under conditions for enzymatic digestion of the tissue.1 1. The method of claim 10, wherein the digestion mix comprises collagenase Type 11, collagenase type IV, collagenase type XI, pancreatic elastase, hyaluronidase Type II, DNase I and soybean trypsin inhibitor.

12. The method of any one of claims 1 to 11, wherein the immune cells are isolated from a blood sample from the subject.1 . The method of any one of claims 1 to 12, wherein step (b) comprises culturing the ECs, VSMCs and immune cells on a non-adherent substrate.

14. The method of any one of claims 1 to 13, wherein the method comprises culturing the cells in the presence of an atherogenic stimulus.

15. The method of claim 14, wherein the cells are cultured in the presence of the atherogenic stimulus for at least 48 hours.

16. The method of claim 14 or 15, wherein the atherogenic stimulus comprises a low- density lipoprotein (LDL), cholesterol, a cytokine, or a combination thereof.

17. The method of claim 16, wherein the cytokine is interleukin 1 beta (IL- Ip).

18. The method of claim 16 or 17, wherein the LDL is an oxidised LDL (ox-LDL).

19. The method of any one of claims 16 to 18, wherein the atherogenic stimulus comprises ox-LDL at a concentration of about 10 pg / ml to about 100 pg / ml in the culture.

20. The method of claim 19, wherein the concentration of ox-LDL in the culture is about 50 pg / ml.

21. A multicellular spheroid obtained by a method according to any one of claims 1 to 20.

22. A multicellular spheroid comprising endothelial cells (ECs), vascular smooth muscle cells (VSMCs), and macrophages.

23. The multicellular spheroid of claim 22, wherein the ECs, VSMCs and macrophages are isolated from one or more tissue samples that have been obtained from a subject.

24. A method of screening for a drug, comprising a) contacting a multicellular spheroid according to any one of claims 21 to 23 with a drug library, and b) identifying a drug based on one or more biological responses from the multicellular spheroid.

25. Use of a multicellular spheroid according to any one of claims 21 to 23 for drug testing.

26. A digestion mix for isolating cells from a tissue sample, comprising one or more collagenases, an elastase, a hyaluronidase, a DNase and a trypsin inhibitor.

27. The digestion mix of claim 26, wherein the digestion mix comprises collagenase Type II, collagenase type IV, collagenase type XI, pancreatic elastase, hyaluronidase Type II, DNase I and soybean trypsin inhibitor.

28. A kit for isolating cells from a tissue sample, comprising collagenase Type II, collagenase type IV, collagenase type XI, pancreatic elastase, hyaluronidase Type II, DNase I and soybean trypsin inhibitor.

29. A method for isolating cells from a tissue sample, the method comprising: (a) contacting the tissue sample with a digestion mix of claim 26 or 27, under conditions for enzymatic digestion of the tissue; and (b) isolating cells released from the digested tissue.

30. The method of claim 29, wherein the tissue sample is an arterial tissue sample.

31. The method of claim 29 or 30, wherein the cells comprise endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and / or fibroblasts.

32. The method of any one of claims 29 to 31, wherein step (a) is performed at about 37°C for at least about 60 min.

33. The method of claim 32, wherein step (a) is performed for about 2 hours.

34. The method of any one of claims 29 to 33, wherein the isolated cells are at least about 80% viable.

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