Method and products for cell culture

Abstract

Structures for cell culture comprise a first layer that is not degradable and a second layer that is degradable. Also disclosed are structures for cell culture comprising: a) A first layer comprising (i) a co-polymer hydrogel comprising monomeric units of acrylamide, bisacrylamide and a coupling compound, and (ii) a first cell adhesion molecule, said first cell adhesion molecule being covalently coupled to coupling monomeric units of the co-polymer; and b) a second layer comprising (i) matrigel, synthetic hydrogel, such as polyethylene glycol hydrogel or polyacrylamide hydrogel, and / or agarose gel, and (ii) a second cell adhesion molecule. Also disclosed are systems including the structures and cells growing thereon, and methods of producing the structures and culturing the cells.

Description

[0001] Method and products for cell culture

[0002] Introduction

[0003] The present invention relates to structures, including layers of substrates for the culture of mammalian cells, such as stem cells and progenitor cells.

[0004] Background to the Invention

[0005] Historically, cells have been cultured in vitro under conditions that poorly replicate the natural cellular microenvironment, using simple plastic and glassware. More recently, substrates have been developed that better mimic that microenvironment, for example substrates, or biomimetic matrices, that replicate aspects of the extracellular matrix (ECM).

[0006] Important aspects of the ECM that may be replicated in substrates for cell culture include the presence of bioactive materials, with which the cultured cells may form attachment, and an appropriate stiffness of the material.

[0007] Such biomimetic matrices most commonly are made of hydrogel substrates, such as polyacrylamide (PA), hydroxyl-PA, alginate, hyaluronic acid and polyethylene glycol, with cross-linking chemistries used to allow attachment of the cells to the biomimetic matrix. Cell culture substrates are described in PCT / EP2018 / 085079 that comprise a co-polymer of monomeric units of acrylamide, bisacrylamide and a coupling compound, such as 6-acylamidohexanoic acid (6AHA: 6-(prop-2-enoylamino) hexanoic acid) (a PA-coupling compound hydrogel), with a cell adhesion molecule, such as a component of the ECM, covalently coupled to the coupling compound monomeric units of the hydrogel. However, there remains the need for improvements in cell culture substrates and systems to be used in the culturing of cells, such as mammalian cells.

[0008] Summary of the Invention

[0009] A first aspect of the invention provides a structure for cell culture comprising: a first layer comprising a first substrate - the first layer is suitably not degradable. A second layer can comprise a second substrate - wherein the second layer is suitably degradable.The first substrate may comprise a hydrogel and / or a first cell adhesion molecule. The hydrogel may be selected from the group consisting of polyacrylamide (PA), hydroxyl- polyacrylamide, polyacrylamide-coupling compound (PA-CC) co-polymer, alginate, hyaluronic acid and / or polyethylene glycol. The first substrate may comprise a hydrogel that comprises a co-polymer of monomeric units of acrylamide, bisacrylamide and a coupling compound, preferably wherein the coupling compound is 6-acylamidohexanoic acid (6AHA: 6-(prop-2-enoylamino) hexanoic acid).

[0010] The second substrate may comprise: matrigel, synthetic hydrogel, such as polyethylene glycol hydrogel or polyacrylamide hydrogel, and / or agarose gel; and / or a second cell adhesion molecule. The second substrate may comprise a fibrin gel and / or a fibrin-agarose gel.

[0011] A second, related, aspect of the invention provides a structure for cell culture comprising:

[0012] a) A first layer comprising a first substrate, wherein the first substrate comprises (i) a co-polymer hydrogel comprising monomeric units of acrylamide, bisacrylamide and a coupling compound, and (ii) a first cell adhesion molecule, said first cell adhesion molecule being covalently coupled to coupling monomeric units of the co-polymer; and b) a second layer comprising a second substrate, wherein the second substrate comprises (i) matrigel, synthetic hydrogel, such as polyethylene glycol hydrogel or polyacrylamide hydrogel, and / or agarose gel, and (ii) a second cell adhesion molecule.

[0013] The coupling compound may be 6-acylamidohexanoic acid (6AHA: 6-(prop-2-enoylamino) hexanoic acid). The second substrate may comprise a fibrin gel and / or a fibrin-agarose gel.

[0014] The first and second cell adhesion molecules of the first and second aspects may be independently selected from the group consisting of: integrins, cadherins, selectins, fibronectin, collagen, fibrin, laminin, tenascin, vitronectin, thrombospondin.

[0015] The structures of the invention may be for use in mammalian cell culture.

[0016] The invention also provides a system for cell culture comprising a structure according to the invention, and which comprises cells between the first and second layers, and cell culture medium.The cells may be mammalian, plant, or insect cells. The cells may be mammalian cells selected from human, mouse and rat cells. The mammalian cells may be selected from epithelial cells, endothelial cells, neural cells, fibroblasts, such as human dermal or tendon fibroblasts, stromal cells, such as bone marrow derived stromal cells and smooth muscle cells, cancer cells, progenitor and precursor cells, iPSCs, spheroid forming cells, colony forming cells, anchorage independent cells, and embryonic, foetal and adult stem cells.

[0017] The cells may be stem cells. The stem cells may be pluripotent, multipotent, oligopotent, unipotent, embryonic stem cells (ESCs), foetal stem cells, adult stem cells, amniotic stem cells, cord stem cells and / or induced pluripotent stem cells (iPSCs). The stem cells may be corneal (limbal) stem cells; oligodendrocyte progenitor cells (OPCs); embryonic stem cells; mesenchymal stem cells, adipose-derived stem cells, endothelial stem cells, dental pulp stem cells, skin epidermal stem cells; gut (intestinal) stem cells; orogenital stem cells; bronchial and other epithelial stem cells; muse cells, haematopoietic stem cells, amniotic stem cells bone marrow stromal stem cells; or growth plate stem cells.

[0018] The cells may be progenitor or precursor cells, for example oligodendrocyte progenitor cells.

[0019] The cells may form a 3D structure in the system.

[0020] The invention also provides methods of preparing a system of the invention for cell culture comprising: i) preparing the first layer comprising the first substrate; ii) applying the cells to the first layer; iii) applying culture medium to the first layer and the cells; and iv) applying the second layer to the cells on the first layer.

[0021] The invention also provides methods of culturing cells in vitro in a system of the invention, comprising: i) preparing the first layer comprising the first substrate; ii) applying cells to the first layer; iii) applying the second layer to the cells on the first layer; and iv) culturing the cells and layers in culture medium.

[0022] Methods of the invention may further comprise differentiating the cells. The differentiation of the cells may occur before the cells are applied to the first layer, after the cells are applied to the first layer, before the second layer is applied, and / or after the second layer is applied.In the methods and structures of the invention, the cells may form a 3D structure in the second layer or between the first and second layers.

[0023] Preferred features and / or characteristics of one aspect of the invention are applicable to another aspect mutatis mutandis.

[0024] The invention will now be described with reference to the accompanying drawings, which are not intended to be limiting, and in which:

[0025] Brief Description of the Drawings

[0026] Figure 1 provides an introduction to the production of functionalised gels for use according to the invention. Figure 1a depicts the synthesis of 0.7kPa polyacrylamide hydrogels, as specified in Labouesse, C. etal. Nature Communications, 2021, 12(1), 6132, incorporated herein by reference, on coverslips and with some minor modifications. Figure 1b depicts the functionalisation of the hydrogel with the target extracellular matrix protein, which is required before cells can adhere to the hydrogel.

[0027] Figure 1c describes the full chemical reaction for the 1 -ethyl-3-(-3-dimethylaminopropyl) carbodiimide N-hydroxysuccinimide (EDAC-NHS) cross-linker chemistry. ECM: Extracellular Matrix. AHA: Coupling compound 6-acylamidohexanoic acid (6AHA: 6-(prop-2-enoylamino) hexanoic acid).

[0028] Figure 2 provides an introduction to the 3D hydrogel scaffolds. It is a schematic focusing on methods for the production of hydrogels. A 2D polyacrylamide hydrogel with a non-degradable matrix is coated with an inert agarose gel, which is made biologically activated through the polymerization of fibrinogen to fibrin by thrombrin. Extracellular matrix components are added as desired to create a degradable matrix on top of the 2D gel, in which cells are encapsulated. ECM: Extracellular Matrix. PAAm: Polyacrylamide 2D hydrogel.

[0029] Figure 3 depicts a schematic overview of a method resulting in mature cholangiocyte formation in tubes, according to methods of the invention and using structures and systems of the invention. +: supplemented throughout. + / -: supplemented for one day and then removed. E6: Essential 6 media. CDM-PVA: CDM media with polyvinylalcohol. BMP4: bone morphogenetic protein 4. LY94002: phosphatidylinositol-3-OH kinase inhibitor. CHIR: Wnt inhibitor Chiron. HGF: hepatocyte growth factor. RA:retinoic acid. EGF: epidermal growth factor. FSK: forskolin. bFGF and FGF10: fibroblast growth factors 4 and 10 respectively.

[0030] Figure 4 shows that cholangiocyte differentiation is enhanced on 2D polyacrylamide hydrogels, as illustrated by cholangiocyte marker protein and gene expression. Figure 4a demonstrates that expression of cholangiocyte markers SOX9, keratin? and keratin19 is enhanced in the hydrogel conditions at the protein level. Hydrogels were coated with Collagen type I. Polyacrylamide: cells passaged onto a 2D hydrogel at day 10. Tissue culture plastic: cells passaged onto plastic at day 10. Non-passaged: cells remaining on plastic throughout differentiation as a negative control. Matrigel organoids: intrahepatic cholangiocyte organoids in Matrigel as a positive control.

[0031] Figure 4b shows expression of cholangiocyte markers SOX9 and keratin? is enhanced in the hydrogel conditions at the gene expression level. Results are provided from 3-4 biological replicates. Polyacrylamide: cells passaged onto a 2D hydrogel at day 10. TCP: cells passaged onto plastic at day 10. Non-passaged: cells remaining on plastic throughout differentiation as a negative control. ICOs: intrahepatic Cholangiocyte organoids in Matrigel as a positive control. Day 0: undifferentiated cells as a negative control.

[0032] Figure 5 shows cholangiocytes form tubes on 3D hydrogel scaffolds, as illustrated by cell morphology. Figure 5a shows cholangiocytes exhibit a 3D tubular morphology when sandwiched between polyacrylamide and agarose and a flat sheet morphology when between tissue culture plastic and agarose. The agarose hydrogels either contained the extracellular matrix protein Laminin or Fibronectin. Sandwich: cells covered in agarose-fibrin at day 17. Controls: cells not covered in agarose-fibrin at day 17. Fibronectin / Laminin: cells passaged onto polyacrylamide at day 10 and covered with agarose fibrin coating the respective protein at day 17. MTG: cells passaged onto polyacrylamide at day 10 and covered with Matrigel at day 17. TCP: cells passaged onto plastic at day 10. Non-passaged: cells remaining on plastic throughout differentiation. ICOs: intrahepatic cholangiocyte organoids in Matrigel. Figure 5b shows cholangiocyte morphology on 3D hydrogel scaffolds as measured by SEM. Cholangiocytes exhibit a 3D branching tubular morphology akin to bile ducts when sandwiched between hydrogels.

[0033] Figure 6 shows cholangiocytes form tubes in 3D hydrogel scaffolds, as illustrated by protein and gene expression. Figure 6a shows the tubes express cholangiocyteprotein markers SOX9, keratin19 AND keratin? when in hydrogel sandwiches containing either Laminin or Fibronectin. 0.7kPa: cells on a 0.7kPa polyacrylamide gel. Agarose + Fb: Polyacrylamide- agarose fibrin 3D gel containing fibronectin. Agarose + Lm: Polyacrylamide-agarose fibrin 3D gel containing laminin. - Agarose: Polyacrylamide 2D gel without agarose as a negative control. MTG: Intrahepatic cholangiocyte organoids in Matrigel as a positive control. Figure 6b shows cholangiocyte tubes express cholangiocyte marker keratin? when in hydrogel sandwiches containing either Laminin or Fibronectin. They also exhibit less expression of the hepatocyte marker Albumin (ALB), indicating less liver cell contamination. Polyacrylamide: cells on a 0.7kPa polyacrylamide gel without agarose as a negative control. Agarose + Fibronectin: Polyacrylamide-agarose fibrin 3D gel containing fibronectin. Agarose + Laminin: Polyacrylamide-agarose fibrin 3D gel containing laminin. Non-passaged (TCP): cells remaining on tissue culture plastic (TCP) throughout differentiation as a negative control. ICOs: Intrahepatic cholangiocyte organoids in Matrigel as a positive control.

[0034] Figure 7 shows cholangiocytes form tubes in 3D hydrogel scaffolds, as illustrated by luminal structures in orthogonal views of the cholangiocyte tubes and SEM images.

[0035] Figure 7a shows in orthogonal views that tubes exhibit a lumen, akin to the organization of bile ducts. Circled regions indicate the luminal structures. Figure 7b shows the luminal structures in SEM images. Tubes, particularly in the laminin condition, are hollow and exhibit a lumen. Therefore, indicating that they are bile duct organoid structures.

[0036] Detailed Description

[0037] The invention provides structures for use in the culturing of cells. The cells may be insect, plant, or mammalian cells. In preferred embodiments the cells are mammalian cells. Cell culture substrates can provide a solid or semi-solid surface, suitably on which cells immersed in a culture medium are able to grow and proliferate. The structures of the invention thus provide supports that may be used when growing cells, for example mammalian cells, in vitro or ex vivo.

[0038] The structure includes a first layer. The first layer includes a first substrate. In particularly preferred embodiments of the invention, the first substrate is not degradable, but is remodellable. The first substrate can provide biomimetic support tocells growing on the first layer.

[0039] Of course, the skilled person will understand that the term “layer” is used in a broad way and refers to a thickness of material laying over or under something else. For example, the first layer may be a thickness of material laying under the second layer. The mammalian cells, for example, may be grown between the first and second layers. In some embodiments, the first layer will lie on a hard base, such as a plastic base. The second layer may be situated above the first layer. The (mammalian) cells maybe located or seeded in between the two layers.

[0040] The first layer comprises the first substrate. In some embodiments of the invention, the first layer may substantially consist of the first substrate. In some embodiments of the invention, the first layer may consist of the first substrate.

[0041] The first layer may be or comprise a hydrogel; that has a covalently, stably bound initial protein layer, such as ECM, that may act as a basement membrane for cells (applied to the first layer); and furthermore may not inhibit the addition of new proteins secreted by the cells (to be incorporated into the initial protein layer). In the sense that this layer can incorporate new proteins, but does not lose the initial protein layer -thus this layer may be referred to as remodellable but not degradable.

[0042] The structure also includes or comprises a second layer. The second layer includes a second substrate. In particularly preferred embodiments, the second substrate is remodellable and degradable. The second layer comprises the second substrate. In some embodiments of the invention, the second layer may substantially consist of the second substrate. In some embodiments of the invention, the second layer may consist of the second substrate.

[0043] The second layer may be added or located on top of the first layer, which may thus then act as a basement membrane (for this second layer). Thus, the second layer may be formed of a substrate that may be added on top of, or with, cells that are attached, or in the process of being attached, to the basement membrane comprising the first substrate. The second layer may have the property of being degradable but may also be able to incorporate new proteins secreted by the cells. In the sense that this second layer may be degraded and may also incorporate new proteins, this layermay be referred to as remodellable and degradable.

[0044] Remodelling is a natural activity that occurs when cells interact with the ECM. It plays an important part in the roles of the ECM, for example, allowing remodelling of tissue and regulating the behaviour and differentiation of cells. Remodelling can occur when the cells produce enzymes, such as metalloproteinases, that degrade proteins in the ECM, or enzymes that modify proteins in the ECM, for example cross-linking them. During cell culture, the cells can remodel biomimetic substrates in a similar way by ripping proteins from the surface of the substrate or cleaving or enzymatically degrading them. Remodelling can also occur when cells deposit molecules on the surface, for example molecules obtained from the surface and / or molecules adapted or produced by the cells.

[0045] Thus, cells can degrade, or modify, some remodellable substrates - because they can remove proteins, polysaccharides, or, generally, cell adhesion molecules, present in the remodellable substrate. However, cells may be unable to degrade or modify some other substrates, for example because any proteins, polysaccharides, and cell adhesion molecules are fixed within or to the substrate, such as in a way that renders them inaccessible. A substrate that is not degradable may, therefore, be considered stable in that cells adhering to the surface of the substrate are not able to significantly degrade the surface - and in particular, for example, are not able to significantly degrade the cell adhesion molecules through which the cells adhere to the surface. However, as used herein cells may still be able to deposit and build structures on a surface that is not degradable; the degradable nature can be determined by whether the cells can degrade the surface or not. The remodellable nature of a surface may encompass whether cells can deposit molecules on the surface or not and / or whether cells can degrade the surface or not. Thus, a surface may be remodellable but not degradable or may be remodellable and degradable.

[0046] Whether a surface is degradable or not can be determined, for example, by the method of Labouesse, C., et al. (Stem Bond hydrogels control the mechanical microenvironment for pluripotent stem cells. Nat Commun 12, 6132 (2021)), which is incorporated by reference herein in its entirety. In particular, whether a surface is degradable or not may be determined by plating an adherent cell type that secretes and remodels ECM, preferably a strongly adherent cell type such as mouse embryonic fibroblasts (MEFs), on the surface and monitoring for depletion of theECM (for example proteins, polysaccharides, and cell adhesion molecules). The surface may be considered degradable if there is significant depletion of the ECM after culturing the adherent cells on the surface. The surface may be considered to not be degradable if there is no significant depletion of the ECM after culturing the adherent cells on the surface. A significant depletion, for example, may mean a loss of 99%, 95%, 90%, 80%, 75%, 70%, 60%, 50%, 25%, or 10% of one or more, or all, ECM molecules. The depletion may be assessed for significance after culturing the cells for 1 day, 2 days, 3 days, 4 days, or 1 week. The surface may be considered degradable, for example, if there is depletion of 50% or more of the ECM on the surface after culturing of adherent cells on the surface for 3 days. The adherent cells may be of the cell type with which the surface is to be used in the structures and methods of the invention.

[0047] The inventors have surprisingly found that improvements in cell growth in vitro can be achieved when mammalian cells are grown on a first substrate that the cells do not degrade, and then a second substrate is applied to the growing cells, which second substrate the cells can and do remodel and degrade. For example, the improvements can be in the formation of 3D structures, such as tubules, that are more similar to those that would be formed by that cell type or cell lineage in vivo. Preferably the first substrate is a hydrogel. A hydrogel can comprise a semi-solid material comprising a matrix of polymeric fibres and an aqueous interstitial liquid. Hydrogels can be formed by the polymerisation and cross linking of matrix fibres to form a continuous network around the aqueous interstitial liquid which originally held the monomers. Different types of hydrogels include polyacrylamide (PA), hydroxyl-PA, alginate, hyaluronic acid and polyethylene glycol. Most hydrogels are inert and do not allow attachment, so suitable cell adhesion molecules should be added, for example via specific cross-linking chemistries, to allow use of the hydrogels in cell culture. A polyacrylamide-coupling compound (PA-CC) co-polymer is a hydrogel-forming polymer of cross-linked chains of acrylamide, bisacrylamide and coupling compound monomeric units.

[0048] Polyacrylamide (PA) is a hydrogel-forming polymer of cross-linked chains of acrylamide monomeric units.

[0049] Polyacrylamide (PA) gels are well-known in the art. Typically, chains of acrylamidemonomeric units in a PA gel are cross-linked using a cross-linking agent, such as bisacrylamide (N, N'-methylenebisacrylamide), which is mixed with acrylamide monomers before polymerisation. Suitable methods for the production of PA hydrogels are well-known in the art.

[0050] In some embodiments, the first substrate comprises: a hydrogel that comprises a co-polymer of monomeric units of acrylamide, bisacrylamide and a coupling compound, such as 6- acylamidohexanoic acid (6AHA: 6-(prop-2-enoylamino) hexanoic acid) (a PA-CC hydrogel); and a cell adhesion molecule, such as a component of the ECM, covalently coupled to the coupling compound monomeric units of the hydrogel. Such hydrogels are described in WO2019 / 115814 A1, which is incorporated by reference in its entirety, and may be referred to herein as a Stem Bond™ hydrogel.

[0051] The second substrate may be a matrigel, a mouse tumour-derived matrix that is the current gold standard for 3D cell culture. The second substrate may be a synthetic hydrogel, such as a polyethylene glycol hydrogel or a polyacrylamide hydrogel, which have been used as alternative substrates for 2D or 3D culture. The second substrate may be a fibrin gel, which have previously been used to encapsulate cells due to their biocompatibility and propensity to bind extracellular matrix proteins, therefore creating a biologically active scaffold which is capable of being remodelled and degraded by cells. The second substrate may be an agarose gel. The second substrate may be a fibrin-agarose gel.

[0052] The second substrate may comprise a cell adhesion molecule.

[0053] A cell adhesion molecule is a molecule that binds to the surface of cells through cell adhesion.

[0054] Cell adhesion molecules may include ligands of cell surface receptors, such as integrins, cadherins and selectins. Suitable cell adhesion molecules for inclusion in hydrogels of the first substrate include components of the extracellular matrix. An extracellular matrix component is a protein, glycoprotein, oligosaccharide or proteoglycan that is present in the mammalian extracellular matrix. Suitable extracellular matrix components bind to mammalian cells and form a cell anchorage point in the extracellular matrix. For example, the extracellular matrix component maybe a ligand that binds a cell surface receptor, such as an integrin. Preferably, the extracellular matrix component is an amine containing molecule, such as a protein or peptide. In some embodiments, the extracellular matrix may comprise an RGD motif or other amino acid sequences (e.g. IKAV) which mediates cell attachment (Ruoslahti et al {1996) Ann Rev Cell Dev Biol 12 697-715). Suitable extracellular matrix components include fibronectin, collagen, fibrin laminin, tenascin, vitronectin and thrombospondin. In some embodiments, the cell adhesion molecule that is included in the first substrate, for example through coupling to the coupling compound, may be selected depending on the cell or tissue type understudy.

[0055] In some embodiments, the cell adhesion molecule may be a synthetic molecule, such as a peptide. The sequence of a peptide cell adhesion molecule may match an available cell surface receptor, such as an integrin, on the cell type to be cultured on the substrate. A substrate may thus be selective for cells that display a cell surface receptor that binds to the cell adhesion molecule.

[0056] In some embodiments, a mixture of two or more different cell adhesion molecules may be coupled to the first substrate, for example the hydrogel, or included in the second substrate, such as to produce the cell substrate. Thus, the first and / or second substrate may independently include one, two, or more of the following cell adhesion molecules: integrins, cadherins, selectins, fibronectin, collagen, fibrin, laminin, tenascin, vitronectin, thrombospondin. The first substrate may comprise collagen and / or the second substrate may comprise fibrin.

[0057] Cell adhesion molecules may be obtained from commercial suppliers or produced by recombinant or synthetic means using standard techniques.

[0058] Examples of suitable cell adhesion molecules include: collagen I from rat tail, Sigma; rhLaminin-521 , Thermo Scientific; human Fibronectin, Corning; Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane, Sigma-Aldrich.

[0059] In some embodiments, the second substrate is formed of a polysaccharide, such as agarose, combined with a cell adhesion molecule, such a fibrin. In particularly preferred embodiments, the second substrate comprises an agarose-fibrin gel.

[0060] Thus, in some aspects of the invention there is provided a structure for cell culture comprising:a) A first layer comprising a first substrate, wherein the first substrate comprises:

[0061] (i) a co-polymer hydrogel comprising monomeric units of acrylamide, bisacrylamide and a coupling compound, and (ii) a first cell adhesion molecule said first cell adhesion molecule being covalently coupled to coupling monomeric units of the co-polymer:

[0062] and

[0063] b)A second layer comprising a second substrate, wherein the second substrate comprises:

[0064] (i) matrigel, synthetic hydrogel, such as polyethylene glycol hydrogel or polyacrylamide hydrogel, and / or agarose gel, and (ii) a second cell adhesion molecule.

[0065] Preferably the coupling compound is 6-acylamidohexanoic acid (6AHA: 6-(prop-2-enoylamino) hexanoic acid). Preferably the second cell adhesion molecule is fibrin. Preferably the second layer comprises or substantively consists of or consists of fibrin gel or fibrin-agarose gel. Preferably the structure is for mammalian cell culture.

[0066] The first substrate of the first layer may be a polyacrylamide hydrogel, for example StemBondTM as described in WO 2019 / 115814 A1, which is incorporated by reference in its entirety. Details of how to make StemBondTM hydrogels are also described in WO 2019 / 115814 A1. The polyacrylamide hydrogel, for example StemBondTMmay include cross-linking of the cell adhesion molecules of the hydrogel, such that cells are unable to degrade or modify the cell adhesion molecules, i.e. so that the hydrogel is not degradable.

[0067] Cells, preferably mammalian cells, may be cultured between the first and second layers. Therefore, structures of the invention are also provided in which cells, for example mammalian cells, are present between the first and second layers of the structure.

[0068] In addition, a cell culture system is provided comprising a structure as describedherein, the cells between the first and second layers, and cell culture medium.

[0069] The cells may be of any type, for example mammalian, plant, or insect. Suitable mammalian cells include human cells and / or cells from laboratory models, such as mouse and / or rat. Suitable insect cells include Drosophila cells.

[0070] Suitable mammalian cells include epithelial cells, endothelial cells, neural cells, fibroblasts, such as human dermal and / or tendon fibroblasts, stromal cells, such as bone marrow derived stromal cells and smooth muscle cells, cancer cells, progenitor and precursor cells, iPSCs, spheroid forming cells, colony forming cells, anchorage independent cells, and embryonic, foetal and adult stem cells.

[0071] Stem cells are usually undifferentiated or partially differentiated cells that are capable of self-renewal and differentiation into one or more specialized cell-types. Stem cells may be pluripotent, multipotent, oligopotent or unipotent and may include embryonic stem cells (ESCs) and non- embryonic stem cells, for example foetal stem cells, adult stem cells, amniotic stem cells, cord stem cells and induced pluripotent stem cells (iPSCs). In some embodiments, the PSCs are not human embryonic stem cells. The stem cells may be obtained from mammals at any developmental stage, including foetal, neonatal, juvenile, mature or aged.

[0072] Suitable stem cells include corneal (limbal) stem cells; oligodendrocyte progenitor cells (OPCs); embryonic stem cells; mesenchymal stem cells, adipose-derived stem cells, endothelial stem cells, dental pulp stem cells, skin epidermal stem cells; gut (intestinal) stem cells; orogenital stem cells; bronchial and other epithelial stem cells; muse cells, haematopoietic stem cells, amniotic stem cells bone marrow stromal stem cells; growth plate stem cells and iPSCs.

[0073] Progenitor and precursor cells are partially differentiated cells that are capable of differentiation into one or more specialized cell-types. Suitable precursor cells include oligodendrocyte progenitor cells.

[0074] Preferably, the mammalian cells are of a type that forms a 3D structure, such as a tube, in the natural environment.

[0075] In some embodiments, the cells are cholangiocytes or progenitors of cholangiocytes.A cell culture medium is a nutritive solution that supports the growth and proliferation of mammalian cells. The substrates are not limited to any specific cell culture medium and any media may be used to culture cells on the substrate. Suitable cell culture media are well known in the art.

[0076] In some embodiments, the cell culture medium may be an undefined medium. An undefined medium may contain one or more undefined components or constituents, such as feeder cells, stromal cells, serum, matrigel, serum albumin and complex extracellular matrices. In some embodiments, a cell culture medium may comprise serum and leukaemia inhibitory factor (LIF).

[0077] In some embodiments, the cell culture medium may be a defined medium. A defined medium contains only specified components, preferably components of known chemical structure and is devoid of undefined components or constituents, such as feeder cells, stromal cells, serum, matrigel, serum albumin and complex extracellular matrices. In some embodiments, the defined medium is humanised. A humanised defined medium is devoid of components or supplements derived or isolated from non-human animals, such as Foetal Bovine Serum (FBS) and Bovine Serum Albumin (BSA), and mouse feeder cells. Conditioned medium includes undefined components from cultured cells and is not defined.

[0078] A medium may comprise a defined basal medium supplemented with a serum-free media supplement and / or one or more additional components, for example transferrin, 1- thioglycerol, 2-mercaptoethanol, FGF2, defined lipids, L-GIn, non-essential amino acids, and optionally polyvinyl alcohol; polyvinyl alcohol and insulin; serum albumin; or serum albumin and insulin.

[0079] Suitable chemically defined basal medium, such as Advanced Dulbecco's modified eagle medium (DMEM) (Price et al Focus (2003) 25 3-6), Knockout Dulbecco's Modified Eagle's Medium (KO-DMEM), DMEM / F12, Iscove's Modified Dulbecco's medium (IMDM) and RPMI-1640 (Moore, G.E. and Woods L.K., (1976) Tissue Culture Association Manual. 3, 503-508) are known in the art and available from commercial sources (e.g. Sigma-Aldrich Ml USA; Life Technologies USA).

[0080] Serum-free media supplements, such as N2, B27 and N21, are well known inthe art and widely available commercially (e.g. Invitrogen; Sigma Aldrich Inc.). Suitable serum-free media supplements include B27 (Brewer et al Brain Res (1989) 49465-74; Brewer et al J. Neurosci Res 35567-576 (1993); Brewer et al Focus 16 1 6-9; Brewer et al (1995) J. Neurosci. Res. 42:674-683; Roth et al J Trace Elem Med Biol (2010) 24130-137), N2 (Lee et al (2000) Nat Biotechnol 18(6) 675-679; Lumelsky et al (2001 ) Science 292(5520): 1389-1394) and NS21 (Chen et al J. Neurosci Meths (2008) 171 239-247). In some embodiments, a suitable defined medium may comprise N2 and B27.

[0081] The medium may be a stem cell medium which supports the growth of stem cells without loss of potency. Examples of stem cell media include CDM-PVA (Johansson and Wiles (1995) Mol Cell Biol 15, 141-151), which comprises a basal medium supplemented with polyvinyl alcohol, insulin, transferrin and defined lipids. For example, a CDM-PVA medium may consist of: 50% Iscove's Modified Dulbecco's Medium (IMDM) plus 50% Ham's F12 with GlutaMAX-1TMor 50% F12 NUT-MIX (Gibco, supplemented with 1% chemically defined lipid concentrate, 450pM 1-thiolglycerol, 15pg / ml transferrin, 1 mg / ml polyvinyl alcohol, 7pg / ml Insulin. Other suitable chemically defined nutrient media include hESC maintenance medium (CDMA) which is identical to the CDM-PVA described above with the replacement of PVA with 5 mg / ml bovine serum albumin; and RPMI basal medium supplemented with B27 and Activin (for example at least 50ng / ml). Other suitable media are described in Vallier et al 2009 Stem Cells 27: 2655-2666, Touboul 2010 51: 1754-1765, Teo et al 2011 Genes & Dev. (2011) 25: 238-250 and Peterson & Loring Human Stem Cell Manual: A Laboratory Guide (2012) Academic Press.

[0082] In some embodiments, the medium may be a minimal defined medium.

[0083] Aspects of the invention provide methods for culturing cells, for example mammalian cells, using structures or systems provided and / or described herein. Suitable techniques for cell culture are well-known in the art (see, for example, Basic Cell Culture Protocols, C. Helgason, Humana Press Inc. U.S. (15 Oct 2004) ISBN: 1588295451; Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc., U.S. (9 Dec 2004) ISBN: 1588292223; Culture of Animal Cells: A Manual of Basic Technique, R. Freshney, John Wiley & Sons Inc (2 Aug 2005) ISBN: 0471453293, Ho WY et al J Immunol Methods. (2006) 310:40-52, Handbook of Stem Cells (ed. R. Lanza) ISBN: 0124366430) Basic CellCulture Protocols by J. Pollard and J. M. Walker (1997), 'Mammalian Cell Culture: Essential Techniques' by A. Doyle and J.B. Griffiths (1997), 'Human Embryonic Stem Cells' by A. Chiu and M. Rao (2003), Stem Cells: From Bench to Bedside' by A. Bongso (2005), Peterson & Loring (2012)Human Stem Cell Manual: A Laboratory Guide Academic Press and 'Human Embryonic Stem Cell Protocols' by K. Turksen (2006). Media and ingredients thereof may be obtained from commercial sources (e.g. Gibco, Roche, Sigma, Europa bioproducts, R&D Systems). Standard mammalian cell culture conditions may be employed for the above culture steps, for example 37°C, 21 % Oxygen, 5% Carbon Dioxide. Media is preferably changed every two days and cells allowed to settle by gravity.

[0084] In embodiments of the methods, the cultured cells present a 3D, for example tubular, morphology.

[0085] Thus, some aspects of the invention provide methods of preparing a cell culture system comprising:

[0086] i) Preparing a first layer comprising a first substrate;

[0087] ii) Applying cells to the first layer;

[0088] iii) Applying culture medium to the first layer and cells; and

[0089] iv) Applying a second layer comprising a second substrate to the cells on the first layer.

[0090] The cell culture system may be as described herein. Thus, the features of the method, including the first layer, first substrate, cells, culture medium, second layer, and second substrate, may be as described herein. As with the substrates and systems of the invention, preferably the cells are mammalian cells.

[0091] The methods may comprise differentiating the cells, for example the mammalian cells. In some embodiments of the invention, the cells applied to the first layer may have undergone, or be undergoing, differentiation. Thus, differentiation of the cells may begin before the cells are applied to the first layer. Differentiation of the cells may continue on the first layer, or the cells may be mature differentiated cells whenapplied to the first layer. Differentiation of the cells may continue when the second layer is applied, or the cells may be mature differentiated cells when the second layer is applied.

[0092] In some embodiments of the invention, the cells applied to the first layer may be differentiated after being applied to the first layer, and before, after and / or during application of the second layer.

[0093] Differentiation of the cells may be carried out according to methods known in the art, for example using the appropriate differentiation factors, such as growth factors.

[0094] Other aspects of the invention provide methods, reagents and kits for producing substrates as described herein for cell culture. A kit may comprise;

[0095] (i) a substrate comprising a polyacrylamide-coupling (PA-CC) compound co-polymer hydrogel and a cell adhesion molecule, said cell adhesion molecule being covalently coupled to coupling compound monomeric units of the hydrogel;

[0096] (ii) a PA-CC compound co-polymer hydrogel; a cell adhesion molecule;

[0097] and one or more activating agents, optionally EDAC and NHS; or

[0098] (iii) coupling compound; acrylamide; a cross-linking agent, optionally bisacrylamide; one or more polymerisation initiators, optionally APS and TEMED; a cell adhesion molecule; and one or more coupling agents, optionally EDAC and NHS.

[0099] A kit may further comprise a mould for casting a PA-CC co-polymer hydrogel.

[0100] A kit may further comprise reagents for forming a second layer comprising

[0101] a second substrate, as described herein.

[0102] A kit may further comprise one or more cell culture media. The one or more culture media in the kit may be formulated in deionized, distilled water. The one or more media will typically be sterilized prior to use to prevent contamination, e.g. by ultraviolet light, heating, irradiation or filtration. The one or more media may be frozen (e.g. at -20°C or -80°C) for storage or transport. The one or more media maycontain one or more antibiotics to prevent contamination.

[0103] The one or more media may be a 1x formulation or a more concentrated formulation, e.g. a 2x to 250x concentrated medium formulation. In a 1x formulation each ingredient in the medium is at the concentration intended for cell culture, for example a concentration set out above. In a concentrated formulation one or more of the ingredients is present at a higher concentration than intended for cell culture. Concentrated culture media are well known in the art. Culture media can be concentrated using known methods e.g. salt precipitation or selective filtration. A concentrated medium may be diluted for use with water (preferably deionized and distilled) or any appropriate solution, e.g. an aqueous saline solution, an aqueous buffer or a culture medium.

[0104] The reagents in the kit may be contained in hermetically-sealed vessels. Hermetically-sealed vessels may be preferred for transport or storage of the reagents to prevent contamination. The vessel may be any suitable vessel, such as a flask, a plate, a bottle, a jar, a vial or a bag. The kits may include instructions for producing substrates and / or systems of the invention, and / or for carrying out methods of the invention.

[0105] Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term "comprising" replaced by the term "consisting of' and the aspects and embodiments described above with the term "comprising" replaced by the term "consisting essentially of'.

[0106] It is to be 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.

[0107] Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention. Equally, and to the extent allowed by local law, it is intended that the features of the invention will encompass equivalents. All documents mentioned in this specification are incorporated herein by referencein their entirety for all purposes.

[0108] "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0109] Examples

[0110] Example 1: Novel 2D Polyacrylamide Hydrogel Fabrication.

[0111] A) Synthesis

[0112] 0.7kPa polyacrylamide hydrogels are synthesised as specified in Labouesse, C. et al. Nature Communications, 2021, 12(1), 6132, incorporated herein by reference, with some minor modifications (Figure 1a).

[0113] Before preparing the 2D hydrogels, glass coverslips were washed in 70% ethanol for 10 minutes and then rinsed in deionised water. Next, coverslips were treated with 0.2M sodium hydroxide (NaOH) for 20 minutes to facilitate the binding of hydroxyl groups (-OH) to the coverslip surface. This is required for BindSilane (PlusOneTM GE Healthcare) functionalisation. The coverslips were dipped in MiliQ water to remove salt residues and then dried using lint-free tissues (KimWipes, Midland Scientific) and placed in Petri dishes lined with hydrophobic parafilm. One side of each coverslip was coated with 5% BindSilane dissolved in 10% acetic acid in ethanol (130pl and 85pl for 20mm and 14mm coverslips respectively) incubated at room temperature covered for 2 hours and then uncovered for half an hour to allow the BindSilane to evaporate. This enables the presentation of heterobifunctional linkers which allow polyacrylamide to covalently bind to the glass coverslips. After incubation, the coverslips were washed 3 times in absolute ethanol and wiped dry with KimWipes. Polyacrylamide hydrogel solutions of various stiffness were prepared according to Table 1 and subsequently desiccated for 2 and a half hours to prevent delamination. Polymerisation was initiated upon the addition of 0.5% N,N,NN - Tetramethylethylenediamine (TEMED, Sigma) and 0.1% Ammonium persulfate in MiliQ (APS, Sigma). The polymerising hydrogel solution was pipetted onto the BindSilane coated coverslips to enable droplet formation and hydrophobic top coverslips were placed onto the droplets to produce aflat surface. Gel mixture volumes of 155pl, 80pl and 25pl were used for 32mm, 20mm and 14mm coverslips respectively to produce hydrogels of a height >180pm. This is above the threshold at which cells can sense the underlying stiffness of the glass coverslip. After 15 minutes the gels were fully polymerised, and the coverslips were removed from the IPS. The gels were then stored in phosphate buffered saline (PBS) + penicillin / streptomycin (pen / strep) at 4°C until activation. The acrylamide to bisacrylamide ratio was chosen to achieve the target stiffness and the concentration of 80mM 6-acylamidohexanoic acid (AHA) was picked to enable maximum extracellular matrix protein binding. As an alternative to top coverslips, hydrophobic polymers called induced polymer stamps (IPS) can be used in the polymerisation process.

[0114]

[0115] Table 1: Gel mix composition for novel polyacrylamide hydrogels. Volumes shown are per 500pl aliquot. The 2M stock of 6-acylamidohexanoic acid (AHA) was prepared in methanol. TEMED: tetramethylethylenediamine, APS: ammonium persulfate.

[0116] B) Functionalisation

[0117] Before cells can adhere to the hydrogel, it needs to be functionalised with the target extracellular matrix protein (Examples include Collagen I from rat tail, Sigma; rhLaminin- 521, Thermo Scientific; human Fibronectin, Corning; Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane, Sigma-Aldrich) as depicted in Figure 1b.

[0118] This functionalisation was performed using 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide N-hydroxysuccinimide (EDAC-NHS) cross-linker chemistry (Figure 1b). To enable this, hydrogels were first equilibrated with 2-ethanesulfonic acid (MES) buffer (0.1 M MES hydrate, 0.5 M sodium chloride, pH 6.1). The buffer was thenremoved, and gels were covered with activation solution (0.5M NHS, 0.2M EDAC in MES buffer) and incubated at room temperature for 30 minutes. EDAC reacts with carboxyl (-COOH) groups on the hydrogel to form unstable intermediates, which are subsequently stabilised by NHS to form an amine- reactive intermediate. This enables activation of the terminal carboxyl groups of 6- acylamidohexanoic acid (AHA). The activation solution was subsequently removed, and gels were washed with pre-chilled phosphate buffered saline and 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES) buffer (pH 8.5) before being coated with the target protein diluted in HEPES buffer to the desired concentration. Gels were then incubated at 4°C overnight or for 2 hours at room temperature. Primary amines on the protein displace the active intermediate, facilitating the formation of strong covalent bonds between the activated carboxyl groups and primary amines. The full chemical reaction is described in Figure 1c.

[0119] Afterwards, the protein was removed and blocking solution (0.5 M ethanolamine in HEPES buffer) was added to the gels to block any unbound activated groups. Gels were incubated for 30 minutes at room temperature, then washed and stored in phosphate buffered saline + penicillin / streptomycin at 4°C until required for cell culture. Before seeding hydrogels were equilibrated in the desired media (HepatoZYME supplemented with hepatocyte growth factor and oncostatin M) overnight at 37°C. The extracellular matrix proteins incorporated in the agarose fibrin hydrogels used for bile duct organoid culture are described in table.

[0120] Example 2: Novel 3D Polyacrylamide-Agarose Hydrogel Sandwich Fabrication

[0121] A) Synthesis

[0122] Agarose Fibrin hydrogels are synthesised using a modified version of the matrix screening protocol specified in Mulas, C et al. Lab Chip, 2020, 20, 2580 — 2591, (Figure 1d). In brief, 0.02g of low melt agarose was diluted in 500ul (4% agarose) of MiliQ water and then incubated at 80°C for a minimum of 1 hour to allow the agarose to dissolve. Once the agarose was dissolved it was transferred to 37°C and kept there until use. In sterile conditions the agarose gel media mix (Table 2) was combined with the 500ul of dissolved agarose and 500ul of gel mix was placed on top of the polyacrylamide gel to form a 3D sandwich containing the cell type of choice. Once the gel had begun to polymerise then 40ul of thrombin was pipettedon top of the gel to cross link the fibrinogen. The gel was then allowed to polymerise at 4°Cfor 15 minutes. The extracellular matrix proteins incorporated in the agarose fibrin hydrogels used for bile duct organoid culture are described in table 4.

[0123] B) Optimisation

[0124] The synthesis of agarose fibrin hydrogels described above differs from that in the Mulas et al. matrix screening protocol by three factors; the concentrations of agarose and thrombin and the timing of the addition of thrombin. The protocol from Mulas et al. described the embedding of cells within agarose fibrin where thrombin was added directly to a 2% agarose mixture prior to polymerisation. We initially adapted this protocol for use within the StemBond-agarose sandwich culture as described above. However, the agarose fibrin gels dissolved once the cell media was added. This led us to double the agarose concentration used to create gels which proved resistant to dissolving upon media addition. During subsequent cell culture it was demonstrated that if 2X the amount of thrombin was incorporated into the gel after the agarose had begun to polymerise bile duct tube formation was observed. Therefore, we chose to proceed with this protocol during further experiments.

[0125]

[0126] Table 2: Gel mix composition for agarose fibrin hydrogels. Volumes shown are per 1ml aliquot. ECM: extracellular matrix.

[0127] Example 3: Bile Duct Cell Culture using Hydrogel Sandwich

[0128] Human progenitor cells were seeded onto the novel hydrogels and subsequently differentiated to cholangiocytes (bile duct / biliary cells) following a modified version of the protocol discussed in Sampaziotis, F. etal. Nature Protocols, 2017, 12, 814-827', see Figure 1e and summary in Table 3:

[0129]

[0130] >

[0131]

[0132] Table 3: Details of the full differentiation protocol corresponding to Figure 1e.

[0133] Briefly, at day -1 of differentiation human pluripotent stem cells were seeded onto tissue culture plastic using rho-associated protein kinase (ROCK) inhibitor at a densityof 50,000 cells / cm2 and left in Essential 8 media supplemented with fibroblast growth factor 2 and transforming growth factor (31 for one subsequent day to enable cell growth. The cells were incubated at 37°C and 5% CO2. Differentiation was initiated at day 1 and the plate was transferred to a high oxygen incubator (5% CO2, 5% 02, 37°C). Cells were differentiated into definitive endoderm, foregut endoderm and eventually hepatic progenitors using specific combinations of cell media and growth factors which mimic embryonic development. At day 10 of differentiation hepatic progenitor cells were washed with phosphate buffered saline and then dissociated with the enzyme TryplE (Gibco) for 5 minutes to produce a single cell suspension which was plated onto polyacrylamide hydrogels coated with the desired extracellular matrix protein at a density of 500,000-750,000 cells per cm2. At day 17 of differentiation immature cholangiocytes were coated with 500ul of the agarose fibrin hydrogel containing the extracellular matrix protein of choice, synthesised as described in Example 2, and placed at 4°C for 15 minutes to enable gel polymerisation. This volume was determined through optimisation experiments to be the minimum amount required to fully coat the hydrogel and cells. Room temperature cell medium was then placed on top of the hydrogels and cells were maintained in the incubator up until day 26, when they are predicted to have reached full maturity.

[0134]

[0135] Table 4: Extracellular matrix proteins used in the culture of bile duct cells (cholangiocytes) within Agarose-Polyacrylamide hydrogel sandwiches. EHS: Engelbreth-Holm-Swarm sarcoma.

[0136] Discussion of Alternative Substrates

[0137] Current substrates used in the culture of cells outside of the body (in vitro) are listed below alongside several caveats associated with each substrate:) Matrigel, a mouse tumour-derived matrix is the current gold standard for 3D cell culture.

[0138] i. Matrigel is non-good manufacturing process compilable due to its batch-to-batch variability in properties, such as protein composition and stiffness. This means that cell culture experiments performed using this substrate lack reproducibility and make it difficult to distinguish between biological effects caused by experimental conditions and Matrigel itself.

[0139] ii. The substrate is unable to be biochemically or physically manipulated. Hence, you cannot alter its physical (e.g. elasticity, stiffness) and chemical (e.g. protein composition) properties experimentally.

[0140] iii. Matrigel is not optimal for human cell culture as it is derived from another species (mouse).

[0141] iv. As an animal-derived product, Matrigel is likely to contain viral contaminants. Therefore, limiting its applicability in downstream cell therapy-based applications.

[0142] ) Tissue culture plastic is widely used in the 2D culture of cells.

[0143] i. Tissue culture plastic is much stiffer than the organ itself, with its stiffness being measured at 3,000,000kPa, and is therefore a bad representation of the environment that cells exist in within the body. For comparison, a healthy liver is approximately 2kPa in stiffness.

[0144] ii. This substrate also exhibits difficulty with cell growth and attachment when using serum-free media.

[0145] iii. Cells cultured on plastic exhibit increased proliferation and decreased differentiation in comparison to cells inside the body, indicating that plastic alters the properties to the cells and is therefore an incorrect model for cell

[0146] growth inside the organ.

[0147] iv. When grown on tissue culture plastic cells are less reactive tohormones and supplemental growth factors

[0148] ) Synthetic hydrogels, such as polyethylene glycol or polyacrylamide, have been used as alternative substrates for 2D or 3D culture which more closely represent the target organ stiffness.

[0149] i. Polyethylene glycol

[0150] a. Polyethylene glycol is the most extensively studied polymer for use in tissue engineering applications. b. Polyethylene glycol hydrogels have a low stiffness and are relatively soft. However, their stiffness cannot be tuned exactly to the desired value as the stiffness of polyacrylamide hydrogels can.

[0151] c. This means that polyethylene glycol hydrogels exhibit a smaller stiffness range than polyacrylamide hydrogels, which can easily be tuned to replicate the optimal stiffness required to culture various cell types, and therefore a large range of stiffnesses.

[0152] ii. Polyacrylamide

[0153] a. Without the incorporation of our novel carboxyl- terminating co- polymer, as discussed above in Example 1 , extracellular matrix proteins are typically conjugated to polyacrylamide hydrogels via a UV- activated method known as Sulfo-SANPAH.

[0154] b. When functionalised using this method, proteins are amenable to detaching from the hydrogel during cell culture, leading to cellular detachment over time. c. The introduction of our co-polymerisation technique enables stable protein, and therefore, cell tethering on a non-re-modellable matrix.

[0155] d. This enables long term cell culture, which is notachievable on Sulfo-SANPAH conjugated polyacrylamide hydrogels.

[0156] e. Furthermore, in Sulfo-SANPAH conjugated polyacrylamide hydrogels stiffness cannot be controlled independently of protein tethering. A caveat which our co-polymer method overcomes, enabling each property to be specified independently by polymer and co-polymer concentrations respectively.

[0157] 4) Fibrin gels have previously been used to encapsulate cells due to their biocompatibility and propensity to bind extracellular matrix proteins, therefore creating a biologically active scaffold which is capable of being remodelled and digested by cells.

[0158] i. However, without the addition of agarose, as discussed above in Example 2, fibrin gels exhibit low stability and cellular remodelling can result in gel disintegration over time. Therefore, leading to loss of the 3D support available to cells. ii. The addition of agarose confers increased stability to fibrin gels.

[0159] Enabling gels to retain their biologically active and remodellable properties without being suspectable to degradation over time. In bile duct organoid studies agarose fibrin gels were shown to remain intact over 9 consecutive days in culture.

Claims

- 28 -Claims1. A structure for cell culture comprising:a first layer comprising a first substrate, wherein the first layer is not degradable; anda second layer comprising a second substrate, wherein the second layer is degradable.

2. A structure according to claim 1 wherein the first substrate comprises a hydrogel and a first cell adhesion molecule.

3. A structure according to claim 1 or 2 wherein the hydrogel comprises polyacrylamide (PA), hydroxyl- polyacrylamide, polyacrylamide-coupling compound (PA-CC) co-polymer, alginate, hyaluronic acid and / or polyethylene glycol.

4. A structure according to any preceding claim wherein the first substrate comprises: a hydrogel that comprises a co-polymer of monomeric units of acrylamide, bisacrylamide and a coupling compound, preferably wherein the coupling compound is 6- acylamidohexanoic acid (6AHA: 6-(prop-2-enoylamino) hexanoic acid).

5. A structure according to any preceding claim wherein the second substrate comprises:matrigel, synthetic hydrogel, such as polyethylene glycol hydrogel or polyacrylamide hydrogel, and / or agarose gel; anda second cell adhesion molecule.

6. A structure according to any preceding claim wherein the second substrate comprises a fibrin gel and / or a fibrin-agarose gel.

7. A structure for cell culture comprising:a. A first layer comprising a first substrate, wherein the first substrate comprises:i. a co-polymer hydrogel comprising monomeric units of acrylamide, bisacrylamide and acoupling compound; andii. (ii) a first cell adhesion molecule, said first cell adhesion molecule being covalently coupled to coupling monomeric units of the co-polymer;andb. A second layer comprising a second substrate, wherein the second substrate comprises:i. matrigel, synthetic hydrogel, such as polyethylene glycol hydrogel or polyacrylamide hydrogel, and / or agarose gel; and(ii) a second cell adhesion molecule.

8. A structure according to any preceding claim wherein the first and second cell adhesion molecules are independently selected from the group consisting of: integrins, cadherins, selectins, fibronectin, collagen, fibrin, laminin, tenascin, vitronectin, thrombospondin.

9. A structure according to claim 7 or claim 8 wherein the coupling compound is 6-acylamidohexanoic acid (6AHA: 6-(prop-2- enoylamino) hexanoic acid).

10. A structure according to any of claims 7 to 9 wherein the second substrate comprises a fibrin gel and / or a fibrin-agarose gel.

11. A structure according to any preceding claim wherein the structure is for mammalian cell culture.

12. A system for cell culture comprising:a. a structure according to any preceding claim;b. cells between the first and second layers; andc. cell culture medium.

13. A system according to claim 12 wherein the cells comprise mammalian, plant, or insect cells.

14. A system according to claim 12 or 13 wherein the cells comprisemammalian cells selected from human, mouse and / or rat cells.

15. A system according to any of claims 12 to 14 wherein the cells comprise epithelial cells, endothelial cells, neural cells, fibroblasts, such as human dermal or tendon fibroblasts, stromal cells, such as bone marrow derived stromal cells and smooth muscle cells, cancer cells, progenitor and precursor cells, iPSCs, spheroid forming cells, colony forming cells, anchorage independent cells, and embryonic, foetal and adult stem cells.

16. A system according to any of claims 12 to 15 wherein the cells comprise stem cells.

17. A system according to claim 16 wherein the stem cells comprise pluripotent, multipotent, oligopotent, unipotent, embryonic stem cells (ESCs), foetal stem cells, adult stem cells, amniotic stem cells, cord stem cells and / or induced pluripotent stem cells (iPSCs).

18. A system according to claim 16 wherein the stem cells comprise corneal (limbal) stem cells; oligodendrocyte progenitor cells (OPCs); embryonic stem cells; mesenchymal stem cells, adipose-derived stem cells, endothelial stem cells, dental pulp stem cells, skin epidermal stem cells; gut (intestinal) stem cells; orogenital stem cells; bronchial and other epithelial stem cells; muse cells, haematopoietic stem cells, amniotic stem cells bone marrow stromal stem cells; or growth plate stem cells.

19. A system according to any of claims 12 to 15 wherein the cells comprise progenitor or precursor cells, for example oligodendrocyte progenitor cells.

20. A system according to any of claims 12 to 19 wherein the cells form a 3D structure in the system.

21. A method of preparing a system according to any of claims 12 to 20 for cell culture comprising:i) Preparing the first layer comprising the first substrate;ii) Applying the cells to the first layer;iii) Applying culture medium to the first layer and the cells; and iv) Applying the second layer to the cells on the first layer.

22. A method of culturing cells in vitro in a system according to any of claims 12 to 20 comprising:i) Preparing the first layer comprising the first substrate;ii) Applying cells to the first layer;iii) Applying the second layer to the cells on the first layer; and iv) Culturing the cells and layers in culture medium.

23. A method according to claim 21 or claim 22 further comprising differentiating the cells.

24. A method according to claim 23 wherein the differentiation of the cells occurs before the cells are applied to the first layer, after the cells are applied to the first layer, before the second layer is applied, and / or after the second layer is applied.

25. A method according to any of claims 21 to 24 wherein the cells form a 3D structure in the second layer or between the first and second layers.

Images