Cell culture methods

Abstract

The present invention relates to novel cell culture and transfection methods using a wildtype Caf1 ("Caf1-WT") polymer or a variant thereof. Use of a Caf1-WT polymer or a variant thereof to increase cell viability, cell proliferation, cell migration and / or cell clustering; promote the formation of a 3D cell structure; and / or promote suspension cell culture of cells that are normally adherent are also provided herein.

Description

[0001] Cell culture methods

[0002] The present invention relates to novel cell culture and transfection methods using a wildtype Caf1 (“Caf1-WT”) polymer or a variant thereof. Use of a Caf1-WT polymer or a variant thereof to increase cell viability, cell proliferation, cell migration and / or cell clustering; promote the formation of a 3D cell structure; and / or promote suspension cell culture of cells that are normally adherent are also provided herein.

[0003] Background

[0004] Cell culture plays a considerable role in both basic and applied life science research. In traditional 2D cell culture, cells are grown in a flat monolayer on a growth substrate such as a cell culture plate. By contrast, 3D cell culture provides a cell culture environment that allows cells to grow and interact with a surrounding extracellular framework in three dimensions. 3D cell cultures can be grown with a supporting scaffold such as a hydrogel or inert matrix to allow growth in all directions. Alternatively, 3D cell cultures can be grown without a supporting scaffold, where the scaffold-free method relies on cells to self-assemble into clusters or spheroids. Several methods for cell culture are known in the art.

[0005] Brief summary of the disclosure

[0006] The wildtype Caf1 (“Caf1-WT”) polymer has a “non-stick” property that was previously considered to render the Caf1-WT polymer biologically inert (Roque et al., Adv Mater 2014 May;26(17):2704-9, 2616). In view of this property, Caf1-WT has been utilised as a scaffold into which cell adhesion motifs are introduced to facilitate specific cell interactions (WO2013186545). Caf1-WT polymers have also been used in hydrogels for cell culture, wherein hydrogels made up of Caf1-WT polymers are generated by crosslinking the Caf1- WT polymers using a chemical crosslinker (e.g. DTSSP, NHS-PEG-NHS, 4-arm NHS-PEG).

[0007] The invention is based in part on the surprising finding that when chemical crosslinking is replaced with a gel-forming polymer such as alginate or Matrigel ®, the presence of Caf1- WT in the hydrogel provides advantageous biological effects. The inventors have also shown that the presence of Caf1-WT polymer during cell culture provides a number of advantages including (i) increasing cell viability; (ii) increasing cell proliferation, cell migration and / or cell clustering; (iii) promoting the formation of a 3D cell structure, such as a spheroid or organoid; and / or (iv) promoting suspension cell culture of cells that are normally adherent. Advantageously, unlike other reagents that prevent cell clumping, the presence of Caf1-WT during cell culture does not adversely affect or prevent transfection of the cells. The effects observed herein when Caf1-WT polymer is used during cell culture in each of these contexts are surprising, as the Caf1-WT polymer was previously considered to be inert (Roque et al., Adv Mater 2014 May;26(17):2704-9, 2616). The inventors have demonstrated herein that Caf1-WT polymers can provide several benefits to cells in cell culture. In vitro or ex vivo methods and uses for Caf1-WT polymers in cell culture are therefore provided herein. The observed benefits of using Caf1-WT polymers in cell culture may also be extrapolated to its use in vivo, where the presence of Caf1-WT polymers (for example as part of a hydrogel that is transplanted into a subject) may provide a number of advantages including (i) increasing cell viability; (ii) increasing cell proliferation, cell migration and / or cell clustering; and / or (iii) promoting the formation of a 3D cell structure, such as a spheroid or organoid in vivo.

[0008] Accordingly, the invention provides use of a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof to increase cell viability during cell culture, wherein the variant has Caf1-WT activity.

[0009] The invention also provides use of a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof to promote suspension cell culture of cells that are normally adherent and to increase cell viability during the cell culture, wherein the variant has Caf1- WT activity.

[0010] Suitably, the Caf1-WT polymer or variant thereof may increase cell proliferation and / or cell migration.

[0011] The invention also provides use of a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof to promote the formation of a 3D cell structure during cell culture, wherein the variant has Caf1-WT activity, and wherein the Caf1-WT polymer or variant thereof increases cell viability during the cell culture.

[0012] Suitably, the 3D cell structure may be selected from the group consisting of: a spheroid, an organoid or cultivated meat, optionally wherein the organoid is an endothelial tube structure or a ciliated 3D cell structure.

[0013] The invention also provides use of a hydrogel comprising a gel-forming polymer and a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof as a 3D cell support scaffold during cell culture, wherein the variant has Caf1-WT activity, and wherein the Caf1-WT polymer or variant thereof increases cell viability during the cell culture.

[0014] Suitably, the Caf1-WT polymer or variant thereof may increase cell proliferation, cell migration and / or cell clustering.

[0015] The invention also provides a method of suspension cell culture of cells that are normally adherent comprising: (i) culturing the cells in a cell culture medium in the presence of a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof, wherein the variant has Caf1- WT activity, and wherein the Caf1-WT polymer or variant thereof increases cell viability during the cell culture.

[0016] The invention also provides a 3D cell culture method comprising:

[0017] (i) culturing cells on or in a hydrogel wherein the hydrogel comprises a gel-forming polymer and a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof, wherein the variant has Caf1-WT activity, and wherein the Caf1-WT polymer or variant thereof increases cell viability during cell culture.

[0018] Suitably, the method may further comprise (ii) transfecting the cells in the presence of the Caf1 polymer.

[0019] The invention also provides a cell culture method comprising:

[0020] (i) culturing cells in a cell culture medium in the presence of a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof, wherein the variant has Caf1-WT activity, and wherein the Caf1-WT polymer or variant thereof increases cell viability during the cell culture; and

[0021] (ii) transfecting the cells in the presence of the Caf1-WT polymer or variant thereof.

[0022] Suitably, the Caf1 polymer may be comprised in a hydrogel, wherein the hydrogel comprises a gel-forming polymer and at least about 0.025 % (w / v) of the Caf1 polymer.

[0023] Suitably, the Caf1 polymer may be present in the hydrogel at a concentration of from about 0.025 % (w / v) to about 0.4% (w / v).

[0024] Suitably, the Caf1 polymer may be present in the hydrogel at a concentration of about 0.2 % (w / v).

[0025] Suitably, the gel-forming polymer may be an alginate or a basement membrane extract.

[0026] Suitably, the gel-forming polymer may be Matrigel®.

[0027] Suitably, the gel-forming polymer may be an alginate, optionally wherein the alginate is present in the hydrogel at a concentration of from about 0.5 % (w / v) to about 10% (w / v), further optionally wherein the alginate is present in the hydrogel at a concentration of about 1 % (w / v).

[0028] Suitably, the gel-forming polymer and the Caf1 polymer may be present in the hydrogel in a ratio of between about 5:2 and about 40:1. Suitably, the gel-forming polymer and the Caf1 polymer may be present in the hydrogel in a ratio of about 5:1.

[0029] Suitably, the gel-forming polymer may be an alginate or a basement membrane extract, optionally wherein the gel forming polymer is an alginate.

[0030] Suitably, the cells may be on or attached to the hydrogel.

[0031] Suitably, the cells may be entrapped or encapsulated in the hydrogel.

[0032] Suitably, the cells may be mammalian cells.

[0033] Suitably, the cells may be myosatellite cells, induced pluripotent and embryonic stem cells, adipose-derived cells, hepatocytes, chondrocytes (such as MSC-derived chondrocytes), osteoblasts (such as MSC-derived osteoblasts), cancer cells, neurons, cardiomyocytes, keratinocytes, fibroblasts, epithelial cells, endothelial cells, macrophages, lymphoblasts, mesenchymal stem cells (hMSCs), embryonic cells, cell lines; or a combination thereof.

[0034] Suitably, the cells in (i) may be cultured on or in the hydrogel for at least 24 hours.

[0035] Suitably, the method may be for producing a 3D cell structure, optionally wherein the 3D cell structure is selected from the group consisting of: a spheroid, an organoid and cultivated meat; optionally wherein the organoid is an endothelial tube structure or a ciliated 3D cell structure.

[0036] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0037] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0038] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0039] Various aspects of the invention are described in further detail below.

[0040] Brief description of the Figures

[0041] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 shows that supplementing 0.2% Caf1-WT polymer into alginate hydrogel allows increased number of viable human mesenchymal stromal cells after (A) 10 and (B) 21 days of culture compared to alginate only gels. Cell viability was measured via MT assay which was prepared as per manufacturer specifications. The reagent was added 24 hours prior to reading the luminescence on an Omega plate reader. All conditions were run in triplicate with the graphs depicting the average and error bars showing the SEM.

[0042] Figure 2 shows that three independent repeats determine 0.025% (w / v) of Caf1-WT polymer is the minimum amount needed to enhance performance of a 1 % alginate hydrogel. HEK293 cells were seeded at 1x104cells per well in alginate hydrogels supplemented with increasing Caf1-WT. Cell viability was assessed after 2 days. All conditions were run in triplicate with the graphs depicting the average and error bars showing the SEM. The horizontal dashed line signifies the average readout for the alginate only gels. A) Presto blue reagent was added to wells and incubated for 2 hours prior to reading fluorescence. Increased fluorescence correlates to an increased number of viable cells. B and C) MT assay was added to the wells 24 hours prior to reading luminescence.

[0043] Figure 3 shows that the addition of Caf1-WT polymer to alginate hydrogel enhances migration of cancer cells. Alginate droplets were formed by mixing the alginate or alginate mixed with Caf1-WT polymer with 5 pL of 50mM CaChand incubating at 37°C for 45 minutes. Standard growth medium (10% FBS and 1 % L-glutamine supplemented DMEM) was gently added. Each condition was seeded in triplicate. Images were taken at 10X magnification. The images were used to calculate the percentage migrated using Imaged. The average of three repeats was plotted and the error bars represent the SEM.

[0044] Figure 4 shows tube Formation Assay Data for AngicyteTMcells. Caf1-WT polymer mixed with the Matrigel® leads to greater tube formation than Caf1-FGF (modified Caf1 displaying the sequence RSRKYSS-WYVALKR identified by Baird et al. Proc. Natl. Acad. Sci. II. S. A. 1988, 85 (7), 2324-8) or recombinant soluble FGF. The assay uses microscopy to determine the % area of cultured cells that form tube structures. Example micrographs are shown (right) for each of the four conditions. Values of % for each condition are plotted on the left with error bars.

[0045] Figure 5 shows the results of culturing primary nasal epithelial cells in Matrigel®, Alginate or Alginate hydrogels supplemented with CAF1-WT. Both Matrigel® and alginate + Caf1-WT polymer produce organoids with lumens and beating cilia.

[0046] Figure 6 shows HCT116 or HEK293 cells that were seeded at 1x104cells per 400 pl culture media (DMEM supplemented with 10% FBS). This was followed by the addition of 150 pl of 1 mg / mL Caf1-WT polymer or 150 pl of 1 mg / mL Caf1 -Fibronectin. In the negative control wells, no Caf1 products were added. Cells were left to grow for 3 days prior to taking images via bright field microscopy. Magnifications are specified in figure.

[0047] Figure 7 shows the effects of coating with Caf1-WT. Non-tissue culture treated 24-well plate was left uncoated, or coated with Caf1-WT. The coating solution (300pL of a 1 in 5 dilution of 1 mg / mL Caf1-WT polymer in PBS) was applied to each well and incubated for 1 hour at room temperature. Prior to seeding cells in culture medium, the coating solution was aspirated. Images were taken at 10X magnification via brightfield microscopy.

[0048] Figure 8 shows that HEK293 suspension cultures grown in the presence of Caf1-WT are transfectable. HEK293 cells were grown in single cell suspension in Freestyle F17 media supplemented with 250pg / mL Caf1-WT. Half of the cell population were transfected with the pEGFP-C2 plasmid encoding GFP protein for 24 hours and grown for a further 24 hours. The other half were left untransfected and grown for 48 hours. Fluorescent images were taken using an Incucyte S7 at 10X magnification. Both phase and green channels were used to produce the images. Black arrows show areas of successful transfection. A) Transfected cells. B) Untransfected controls. C) Quantification of the number of green cells in the transfected and untransfected samples. Scale bar is 400 pm.

[0049] Figure 9 shows that Caf1-WT enhances cellular proliferation in 1% alginate gels. Using 96 well plates HEK293 cells were grown in 1% alginate mixed with 0.2% Caf1-WT polymer. During initial seeding, 104cells / well (25pl) were added 75pl of alginate solution which was added to 75 pl of 25 mM calcium solution in each well. It was crosslinked for 30 min at 37°C before adding 100 pl of media (DM EM supplemented with either 2% or 10% FBS and 1% L- glutamine) to the gel surface. Cell viability was measured using a Presto Blue assay according to manufacturer’s instructions and each data point is the result of 3 independent repeats of the experiment and the error bars indicate the standard error. Three wells were used for each set of readings and media was changed each day a Presto Blue assay was performed.

[0050] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0051] Various aspects of the invention are described in further detail below.

[0052] Detailed Description The inventors have surprisingly identified that use of a Caf1-WT polymer during cell culture affects cell behavior and / or cell growth. The observed effects are particularly relevant to 3D cell culture (wherein cells grow and interact in three dimensions), but have also been observed in suspension cell culture (e.g. wherein single cells are grown in suspension). The observed benefits of using Caf1-WT polymers in cell culture may also be extrapolated to its use in vivo. They may also be extrapolated to Caf1-WT polymer variants which exhibit the same “non-stick” characteristics. More details on each of these aspects are provided below.

[0053] Capsular F1 Antigen (Caf1) polymer is formed from individual subunits (monomers) that adopt an immunoglobulin-like fold (Zavialov AV. Structure and Biogenesis of the Capsular F1 Antigen from Yersinia pestis: Preserved Folding Energy Drives Fiber Formation. Cell. 2003; 113(5): 587-96) that is shared with fibronectin which associate non-covalently to form long, flexible polymers. Caf1 polymer derives from Yersinia pestis bacteria, where its function is to protect the bacteria from phagocytosis by denying macrophages the opportunity to bind to the bacterial surface. The polymers can be expressed recombinantly in E. coli.

[0054] As used herein, “Caf1 polymer” refers to polymeric protein comprising a plurality of non- covalently associated Caf1 monomers. A Caf1 polymer may also be referred to as a polymeric pilus, or a polymeric protein fibre. A Caf1 polymer comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 Caf1 monomers. Typically, a Caf1 polymer comprises at least 10 Caf1 monomers.

[0055] For example, a Caf1 polymer may comprise at least 20 Caf1 monomers. It may comprise at least 30 Caf1 monomers, for example, it may comprise at least 40 Caf1 monomers.

[0056] In one example, a Caf1 polymer comprises at least 50 Caf1 monomers. It may comprise at least 60 Caf1 monomers, for example, it may comprise at least 70 Caf1 monomers.

[0057] In one example, a Caf1 polymer comprises at least 80 Caf1 monomers. It may comprise at least 90 Caf1 monomers, for example, it may comprise at least 100 Caf1 monomers.

[0058] In general, a Caf1 polymer may comprise one or more distinct types of Caf1 monomer. The Caf1 monomers may be wildtype Caf1 monomers (also referred to as “Caf1-WT” or “naturally occurring Caf1 monomer” herein). Alternatively, they may be variants of wildtype Caf1 monomers, which retain the “non-stick” properties of Caf1-WT (also referred to as “Caf1-WT variants” herein, which retain Caf1-WT activity). For example, they may include one or more conservative amino acid substitutions. In the context of the invention, Caf1-WT variants do not comprise any exogenous bioactive sequences as they would then lose the Caf1-WT activity (i.e. the “non-stick properties of Caf1-WT). Alternatively, Caf1 monomers may be modified Caf1 monomers (e.g. functionally modified Caf1 monomers, such as those that comprise an exogenous bioactive sequence). The term “Caf1 monomer” therefore encompasses wildtype Caf1 monomers, wildtype Caf1 monomer variants (that retain Caf1- WT activity) and modified Caf1 monomers.

[0059] As used herein, “wildtype Caf1 monomer” or “Caf1-WT monomer” refers to an unmodified / naturally occurring Caf1 monomer. The wildtype Caf1 monomer sequence is shown in SEQ ID NO: 1 (mature wildtype Caf1 monomer, without the signal sequence) or SEQ ID NO: 3 (with signal sequence). The terms “wildtype”, “unmodified” and “naturally occurring” are used interchangeably herein. As used herein, “naturally occurring” refers to a polypeptide sequence that occurs in nature or to a nucleic acid molecule, e.g. a RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). The terms “naturally-occurring” and “wildtype” are used interchangeably herein.

[0060] A Caf1-WT polymer (or “wildtype Caf1 polymer”) is made up of Caf1-WT monomers (or “wildtype Caf1 monomers”) only. It does not comprise any modified Caf1 monomers that comprise an exogenous bioactive sequence. Such modified Caf1 monomers (and portions thereof) are defined elsewhere herein. In addition, it does not comprise any Caf1-WT monomer variants (that retain Caf1-WT activity). Such variants are also defined elsewhere herein. Accordingly, a Caf1-WT polymer as described herein is a polymeric protein comprising a plurality of non-covalently associated Caf1-WT monomers. A Caf1-WT polymer comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 Caf1- WT monomers. Typically, a Caf1-WT polymer comprises at least 10 Caf1-WT monomers.

[0061] For example, a Caf1-WT polymer may comprise at least 20 Caf1-WT monomers. It may comprise at least 30 Caf1-WT monomers, for example, it may comprise at least 40 Caf1- WT monomers.

[0062] In one example, a Caf1-WT polymer comprises at least 50 Caf1-WT monomers. It may comprise at least 60 Caf1-WT monomers, for example, it may comprise at least 70 Caf1- WT monomers.

[0063] In one example, a Caf1 polymer comprises at least 80 Caf1-WT monomers. It may comprise at least 90 Caf1-WT monomers, for example, it may comprise at least 100 Caf1-WT monomers. Methods for making Caf1-WT polymers are well known in the art, see for example Miller et al., FEMS Immunology & Medical Microbiology, Volume 21 , Issue 3, July 1998, Pages 213- 221.

[0064] Caf1-WT polymers are shown herein to affect cell behavior and / or cell growth. Several uses for Caf1-WT polymers are therefore provided herein.

[0065] As used herein, “wildtype Caf1 monomer variant” or “Caf1-WT monomer variant”, “Caf1 monomer variant” or “variant” refers to Caf1 monomer that is a variant of the Caf1 monomer sequence shown in SEQ ID NO: 1 (mature wildtype Caf1 monomer, without the signal sequence) or SEQ ID NO: 3 (with signal sequence). In the context of the invention, a Caf1 monomer variant retains the functional properties of SEQ ID NO:1 (particularly the “nonstick” properties described in example 7 below). In the context of the invention, the Caf1 monomer variants described herein do not comprise an exogenous bioactive sequence. Suitable variants may readily be identified by a person of skill in the art using the assay provided in example 7 below. The wildtype Caf1 monomer variants described herein may therefore be described as wildtype Caf1 monomer variants having Caf1-WT activity.

[0066] A “wildtype Caf1 monomer variant” may be a functional variant (or functional fragment) of SEQ ID NO:1. Such variants may be naturally occurring (e.g. allelic), synthetic, or synthetically improved functional variants of SEQ ID NO:1. The term “variant” also encompasses homologues.

[0067] Functional variants may contain only conservative substitutions of one or more amino acids of SEQ ID NO:1 , or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the protein. A functional variant of SEQ ID NO:1 may therefore be a conservative amino acid sequence variant of SEQ ID NO:1 , wherein the variant has Caf1- WT activity (particularly the “non-stick” properties described in example 7 below). Caf1-WT activity can therefore be measured using the assay provided in example 7.

[0068] Non-functional variants are amino acid sequence variants of SEQ ID NO:1 that do not have Caf1-WT activity (particularly the “non-stick” properties described in example 7 below). Nonfunctional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:1 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants (e.g. functional and non-functional allelic variants) are well known to a person of ordinary skill in the art.

[0069] A person of skill in the art would readily be able to identify amino acids that may be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants, of SEQ ID NO:1. Homologues of SEQ ID NO:1 can also readily be identified using standard sequence alignment programmes by a person of ordinary skill in the art.

[0070] A wildtype Caf1 monomer variant may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:1 , or portions or fragments thereof, wherein the variant retains Caf1-WT activity. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:1), or portions or fragments thereof.

[0071] As another example, the wildtype Caf1 monomer variant may comprise an amino acid sequence with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions compared to SEQ ID NO: 1 , wherein the variant retains Caf1-WT activity. It may have no more than 5, no more than 4, no more than 3, for example no more than 2, or no more than 1 amino acid substitutions, insertions or deletions compared to SEQ ID NO: 1 , wherein the variant retains Caf1-WT activity. In some examples, the wildtype Caf1 monomer variant comprises 0 to 10 (or 0 to 5) conservative amino acid substitutions compared to SEQ ID NO: 1 , wherein the variant retains Caf1-WT activity. In some examples, the wildtype Caf1 monomer variant has no more than 5, no more than 4, no more than 3, for example no more than 2, or no more than 1 conservative amino acid substitutions, wherein the variant retains Caf1-WT activity.

[0072] A “non-essential” or “non-critical” amino acid residue is a residue that can be altered from the wild-type sequence of (e.g., the sequence of SEQ ID NO:1) without abolishing or, more preferably, without substantially altering Caf1-WT activity, whereas an “essential” amino acid residue results in such a change.

[0073] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of coding sequences, such as by saturation mutagenesis, and the resultant mutants can be screened for Caf1-WT activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:1 , the encoded proteins can be expressed recombinantly and the biological activity of the protein can be determined.

[0074] Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences are performed as follows.

[0075] To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0076] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a BLOSLIM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSLIM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Alternatively, the percent identity between two amino acid sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0077] The protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the N BLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-410). BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402). When using BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See <http: / / www.ncbi.nlm.nih.gov>.

[0078] The wildtype Caf1 monomer variants described herein can have amino acid sequences sufficiently or substantially identical to the amino acid sequence of SEQ ID NO:1 , wherein the variant retains Caf1-WT activity. The terms “sufficiently identical” or “substantially identical” are used herein to refer to a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent (e.g. with a similar side chain) amino acid residues to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently or substantially identical.

[0079] The Caf1-WT monomer variants provided herein do not comprise an exogenous bioactive sequence (i.e. they do not have a bioactive sequence that is not present in SEQ ID NO:1 or SEQ ID NO:3 as this would abolish or reduce the variant’s Caf1-WT activity). A person of skill in the art would know how to identify Caf1-WT monomer variants as provided herein. They could compare the sequence of the variant to the sequence of SEQ ID NO:1 and SEQ ID NO:3 and identify any differences. Using the definitions provided herein, they would know that such differences are considered “exogenous” sequences. Using their common general knowledge, they could then derive whether these sequences represent a bioactive sequence. They could also perform the assay outlined in example 7 to confirm whether the monomer retains the Caf1-WT monomer functionality (and thus does not comprise an exogenous bioactive sequence). Suitable variants may therefore readily be identified by a person of skill in the art using the assay provided in example 7 below. A Caf1-WT polymer variant (or “wildtype Caf1 polymer variant”) is made up of Caf1-WT monomer variants. It may also include one or more Caf1-WT monomers. It does not comprise any modified Caf1 monomers that comprise an exogenous bioactive sequence. A Caf1-WT polymer variant comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 Caf1-WT monomer variants. Typically, a Caf1-WT polymer variant comprises at least 10 Caf1-WT monomer variants. As demonstrated in the examples section below, Caf1-WT polymer may be used to increase cell viability, cell proliferation, cell migration and / or cell clustering. In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used to increase cell viability, cell proliferation, cell migration and / or cell clustering during cell culture (such that the use is in vitro or ex vivo).

[0080] In one example, a Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is used to increase cell viability {in vitro, ex vivo, or in vivo). In one example, a Caf1- WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is used to increase cell viability during cell culture (such that the use is in vitro or ex vivo). In this example the cell culture may be 3D cell culture, for example, wherein the cells are cultured on or in a scaffold, such as on or in a hydrogel. For example the cell culture may be 3D cell culture, wherein the cells are cultured on or in an alginate or Matrigel® hydrogel, wherein the hydrogel comprises Caf1-WT polymer (e.g. at least 0.025 % (w / v) Caf1-WT polymer), or a variant thereof, wherein the variant has Caf1-WT activity (e.g. at least 0.025 % (w / v) of the variant) For example the cell culture may be 3D cell culture, wherein the cells are cultured on or in an alginate or Matrigel® hydrogel, wherein the hydrogel comprises a maximum ratio of about 40:1 of the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT (or variant) polymer).

[0081] In one example, a Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is used to increase cell proliferation in vitro, ex vivo, or in vivo). In one example, a Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is used to increase cell proliferation during cell culture (such that the use is in vitro or ex vivo). In this example the cell culture may be 3D cell culture, for example, wherein the cells are cultured on or in a scaffold, such as on or in a hydrogel. For example the cell culture may be 3D cell culture, wherein the cells are cultured on or in an alginate or Matrigel® hydrogel, wherein the hydrogel comprises Caf1-WT polymer (e.g. at least 0.025 % (w / v) Caf1-WT polymer), or a variant thereof, wherein the variant has Caf1-WT activity (e.g. at least 0.025 % (w / v) of the variant) For example the cell culture may be 3D cell culture, wherein the cells are cultured on or in an alginate or Matrigel® hydrogel, wherein the hydrogel comprises a maximum ratio of about 40:1 of the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT (or variant) polymer).

[0082] Increase in cell proliferation may also be referred to as increased cell growth rates and / or increased cell yields herein.

[0083] In one example, a Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is used to increase cell migration {in vitro, ex vivo, or in vivo). In one example, a Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is used to increase cell migration during cell culture (such that the use is in vitro or ex vivo). In this example the cell culture may be 3D cell culture, for example, wherein the cells are cultured on or in a scaffold, such as on or in a hydrogel. In this example, the cells may be cultured in a hydrogel droplet such as an alginate hydrogel droplet or a Matrigel® hydrogel droplet. For example, the cells may be cultured in a hydrogel, wherein the hydrogel comprises Caf1-WT polymer (e.g. at least 0.025 % (w / v) Caf1-WT polymer), or a variant thereof, wherein the variant has Caf1-WT activity (e.g. at least 0.025 % (w / v) of the variant). For example, the cells may be cultured in a hydrogel, wherein the hydrogel comprises a maximum ratio of about 40:1 of the gel forming polymer (e.g. alginate or Matrigel®) to Caf1-WT (or variant) polymer.

[0084] In one example, a Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is used to increase cell clustering in vitro, ex vivo, or in vivo). In one example, a Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is used to increase cell clustering during cell culture (such that the use is in vitro or ex vivo). In this example the cell culture may be 3D cell culture, for example, wherein the cells are cultured on or in a scaffold, such as on or in a hydrogel. Advantageously, Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used to increase cell clustering during cell culture, wherein the cells are cultured in the absence of a scaffold (e.g. where the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is coated onto the surface of the cell culture vessel that is being used for cell culture). The Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) increases cell clustering thereby promoting the formation of 3D cell structures such as spheroids.

[0085] As is also demonstrated in the examples section below, Caf1-WT polymer may be used to promote the formation of a 3D cell structure {in vitro, ex vivo, or in vivo). In one example, Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used to promote the formation of a 3D cell structure in cell culture (such that the use is in vitro or ex vivo). In this example, the cell culture is 3D cell culture. Examples of suitable 3D cell structures that may be formed include a spheroid, an organoid (such as an endothelial tube structure or a ciliated 3D cell structure), or cultivated meat. In this example the cells may be cultured on or in a scaffold, such as on or in a hydrogel. For example, the cells may be cultured in a hydrogel such as an alginate hydrogel or a Matrigel® hydrogel, wherein the hydrogel comprises Caf1-WT polymer (e.g. at least 0.025 % (w / v) Caf1-WT polymer), or a variant thereof, wherein the variant has Caf1-WT activity (e.g. at least 0.025 % (w / v) of the variant). For example, the cells may be cultured in a hydrogel such as an alginate hydrogel or a Matrigel® hydrogel, wherein the hydrogel comprises a maximum ratio of about 40:1 of the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT (or variant)_polymer. Alternatively, they may be cultured without a scaffold (where the method relies on cells to self-assemble into the 3D cell structure).

[0086] Details of suitable scaffolds (such as hydrogels) are provided elsewhere herein. The terms “scaffold”, “support scaffold” and “supporting scaffold” are used interchangeably herein.

[0087] In examples where the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1- WT activity) is used to promote the formation of a 3D cell structure (such as a spheroid, an organoid (such as an endothelial tube structure or a ciliated 3D cell structure) or cultivated meat) in the absence of a scaffold, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be present via any appropriate means. For example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used to promote the formation of a 3D cell structure (such as a spheroid, an organoid (such as an endothelial tube structure or a ciliated 3D cell structure), or cultivated meat) in the absence of a scaffold, e.g. the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be coated onto the surface of the cell culture vessel that is being used for cell culture. In another example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used to promote the formation of a 3D cell structure (such as a spheroid, an organoid (such as an endothelial tube structure or a ciliated 3D cell structure), or cultivated meat) in the presence of a scaffold, e.g. a hydrogel. Alternatively, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be present in solution, e.g. within cell culture media that is being used for cell culture. Methods for coating the surface of the cell culture vessel are well known in the art. Methods for introducing polymers such as Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) into scaffolds or cell culture media are also well known.

[0088] As is also demonstrated in the examples section below, Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used to promote suspension cell culture of cells that are normally adherent. In this example, the cells are cultured without a scaffold, as the cells remain in suspension during cell culture (i.e. they do not adhere to a surface or scaffold during cell culture). This effect of the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is particularly surprising, as it enables cells that are normally adherent to be cultured in suspension, whilst retaining cell viability even though it has been shown not to interact with cells (Roque, et al., Adv Mater 2014 May;26(17):2704-9, 2616; Peters et al., PLoS Pathog. 2022 Mar 31 ;18(3):e1010447).

[0089] In examples where the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1- WT activity) is used to promote suspension cell culture of cells that are normally adherent, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be present in solution, within the cell culture media that is being used for cell culture (in other words, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be soluble in the cell culture media). Methods for introducing polymers such as Caf1- WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) into cell culture media are also well known. Suitably, the cells may be cultured in the presence of soluble Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) for a suitable time period such as at least 24 hours, at least 48 hours, or at least 72 hours. Other suitable time periods are provided below. Suitably, the cells may be cultured in the presence of soluble Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) for a suitable time period (such as at least 24 hours, at least 48 hours, or at least 72 hours) without agitation. In other words, the cell culture may be static. Suspension cell culture of normally adherent cells is advantageous as it allows for high cell densities and / or high cell yields to be achieved. Static suspension cell culture of normally adherent cells is particularly advantageous as it allows for high cell densities and / or high cell yields to be achieved at low cost.

[0090] As is also demonstrated in the examples section below, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may part of a 3D cell support scaffold, such as a hydrogel. Hydrogels comprising wildtype Caf1 (Caf1-WT) polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may therefore be used as a 3D cell support scaffold. This use may be in vitro, ex vivo or in vivo. The presence of Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) in the 3D cell support scaffold can affect cell behaviour and / or cell growth as described in detail elsewhere herein.

[0091] The term “increase” or “promote” is used herein to generally mean an increase by a statistically significant amount. The increase may be of at least 5% as compared to a threshold value or range, for example an increase of at least 10% as compared to a threshold value or range, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a threshold value or range, or at least about a 0.1 -fold, at least about a 0.25-fold, at least about a 0.5-fold, or at least about a 1.0-fold, or at least about a 1.2-fold, or at least about a 1.5-fold, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 1.0-fold and 10-fold or greater as compared to a threshold value or range. A person of skill in the art would readily understand how to determine the appropriate threshold value or range for determining whether an increase is observed. As a non-limiting example, where it is stated that Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used to increase cell proliferation during cell culture, the skilled person would understand that the increase in cell proliferation is the difference in cell proliferation that occurs in the presence of Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) compared to that which occurs under the equivalent conditions (but in the absence of Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity)). Similarly, where it is stated that Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used to promote suspension cell culture of cells that are normally adherent, the skilled person would understand that there is an increase in the success rate of suspension cell culture of normally adherent cells in the presence of Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) compared to that which occurs under the equivalent conditions (but in the absence of Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity)). Equally, where it is stated that Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used to promote the formation of a 3D cell structure, the skilled person would understand that there is an increase in the success rate of forming a 3D structure (or an increase in the size of the resultant 3D structure) in the presence of Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) compared to that which forms under the equivalent conditions (but in the absence of Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity)).

[0092] As used herein, “cell viability” refers to the number of live, functional cells in a sample. The overall cell viability of a sample may be increased by increasing the longevity or maintaining the health of cells in a sample and / or by increasing the proliferation of cells in a sample. Cell viability assays detect the number of viable cells by measuring the physical and physiological health of cells. Cell viability assays contain specially designed reagents that determine cell viability based on cell viability markers. Several cell viability markers are known in the art, including cellular membrane integrity, cellular function such as enzymatic activity, or metabolic activity. Further non-limiting examples of cell viability markers include cell adherence, adenosine triphosphate (ATP) production, co-enzyme production, and nucleotide uptake activity. Cell viability assays can be used across detection platforms that are well known in the art, including dye exclusion assays, colorimetric assays, fluorometric assays, luminometric assays, and flow cytometric assays.

[0093] As used herein, “cell proliferation” refers to an increase in the number of cells as a result of cell growth and cell division. Cell proliferation assays detect changes in the number of cells in a division or changes in a cell population. Several cell proliferation assays are known in the art, including metabolic activity assays, cell proliferation marker assays, ATP concentration assays, and DNA synthesis assays. The method you choose depends on the type of cell you are studying and the type of information you are looking to gain from the cell proliferation assay. DNA synthesis assays are generally the most accurate and reliable way to detect cell proliferation in the laboratory. Traditionally, radiolabeled 3H-thymine is incubated with cells for several hours or overnight. The newly proliferated DNA cells will incorporate the radiolabels and can be detected by a scintillation counter after elution. The advantages are that thymidine (3H-TdR) is accurate and reliable, highly sensitive and highly replicable. Alternatively, you could use the non-radioactive bromodeoxyuridine (Brdll) to label the DNA cells, and then analyze the cells using colorimetry, chemiluminescence or fluorescence detection. Cell proliferation of BrDU-labeled cells can be measured using anti- Brdll monoclonal antibodies and ICC staining after incorporating the Brdll using in vivo injection or cell culture. Other methods may also be used, such as the Prestoblue and MT assays described in the examples section below.

[0094] As used herein, “cell migration” refers to cell movement towards a chemical concentration gradient (chemotaxis) or ECM protein gradient (haptotaxis) over time. Several methods for measuring cell migration are known in the art, including the Boyden Chamber assay, which is a classic transwell migration assay system uses a hollow plastic chamber, sealed at one end with a porous membrane. This chamber is suspended over a larger well which may contain medium and / or chemoattractants. Cells are placed inside the Chamber and allowed to migrate through the pores, to the other side of the membrane. Migratory cells are then stained and counted. Alternatively bright field microscopy may be used to view cell migration (see example 2 below).

[0095] Cell aggregation encompasses cell clustering and cell clumping. As used herein, “cell cluster” refers to the clustering and adhesion of initially spaced cells to form a cell mass. The term “cell clustering” as used herein includes the association of cells based on cellular similarity. Clustering typically occurs naturally between viable cells and therefore is distinct from cell clumping, the undesirable or uncontrolled aggregation of cells, that can be observed in the presence of free DNA and cell debris in cell culture medium, which often occurs following cell lysis. The morphology of the cluster promotes the re-establishment of cell-to-cell contacts which is found in the tissues, thus functionality and viability of cells are enhanced in the cellular cluster. Methods for measuring cell clustering are well known. For example, cell clustering may be measured with a particle size counter; this provides information on the particle volume distribution curve as a function of the particle diameter. Alternatively bright field microscopy may be used.

[0096] Cell clusters may also be referred to as 3D cell structures. The term “3D cell structure” as used herein includes any cell cluster, including a spheroid, an organoid (such as an endothelial tube structure (also referred to as an endothelial tubule herein) or a ciliated 3D cell structure), or cultivated meat.

[0097] Spheroids and organoids are 3D structures composed of multiple cells. Spheroids are simple clusters of broad-ranging cells, such as from tumor tissue, embryoid bodies, hepatocytes, nervous tissue, or mammary glands. They don't require scaffolding to form 3D cultures; they do so by simply sticking to each other. However, they can't self-assemble or regenerate and, thus, aren't as advanced as organoids. Organoids are complex clusters of organ-specific cells, such as those from the stomach, liver, or bladder. They're made of stem cells or progenitor cells and self-assemble when cultured in the presence of an appropriate scaffold.

[0098] Cultivated meat may also be referred to as cultured meat, healthy meat, slaughter-free meat, in vitro meat, lab-grown meat, cell-based meat, cellular meat, clean meat, artificial meat, and / or synthetic meat.

[0099] An endothelial tubule is a capillary-like structure with a lumen. In vitro generation of such 3D structures are described for example by Andree et al, Scientific Reports volume 9, Article number: 5437 (2019).

[0100] The term “cell culture” as used herein refers to keeping the cells in an artificial environment under conditions favouring growth, differentiation, and / or continued viability. Culturing may be on a substrate or in suspension. As it will be appreciated by a person of skill in the art, whether culturing is on a substrate or in suspension may depend upon the type of cells being cultured. Cell culture may be referred to as in vitro cell culture or ex vivo cell culture.

[0101] As used herein, “3D cell culture” is a culture environment that allows cells to grow and interact with surrounding extracellular framework in three dimensions. This is in contrast with traditional 2D cell cultures in which cells are grown in a flat monolayer on a plate. 3D cell cultures can be grown with or without a supporting scaffold.

[0102] In 3D cell culture, the cells may be cultured within a scaffold (also referred to as a supporting scaffold herein) to allow growth in all directions. The term “scaffold”, as used herein, refers to any material that allows attachment of cells and subsequent proliferation and differentiation. “Attachment”, “attach” or “attaches” as used herein, refers to cells that adhere directly or indirectly to a substrate as well as to cells that adhere to other cells.

[0103] Suitable scaffolds are typically polymeric biomaterials that provide the structural support for cell attachment and subsequent 3D cell structure development. Popular types of scaffold include hydrogels and inert matrices.

[0104] A hydrogel is a polymeric material containing a network of crosslinked polymer chains that can absorb and retain water. Hydrogels can be generated using polymers derived from animals (such as Matrigel® or collagen) or plants (such as alginate / agarose). Alternatively, the may be generated using synthetic polymers (e.g. QGel® Matrix). Further details on hydrogels are provided elsewhere herein.

[0105] Inert matrices are sponge-like membranes made of polystyrene which contain pores for cells to proliferate and grow. Examples of such scaffolds include spun fibres and porous plastic scaffolds. The scaffolds described herein are of particular use in cell-based assays and tissue culture systems.

[0106] In some examples of 3D cell culture, the cells are cultured without a scaffold (also referred to herein as scaffold-free 3D cell culture). Scaffold-free methods rely on cells to selfassemble into clusters or spheroids. Popular scaffold-free methods include hanging drop culture, where cells are placed in a suspended drop of medium, allowing cells to cluster and form spheroids at the bottom of the droplet.

[0107] As used herein, “suspension cell culture” refers to a type of cell culture in which single cells or small clusters of cells are allowed to function and multiply in suspension. Suspension culture is one of the two classical types of cell culture, the other being adherent culture. Suspension cell culture is commonly used to culture non-adhesive (or non-adherent) cell lines like hematopoietic cells, plant cells, and insect cells. While some cell lines are cultured in suspension, the majority of commercially available mammalian cell lines are naturally adherent. Suspension cell cultures are typically agitated to maintain cells in suspension, and may require specialized equipment (e.g. magnetic stir plate, orbital shakers, incubators) and flasks (e.g. culture flasks, spinner flasks, shaker flasks). Suspension cell cultures may also be cultured statically (without agitation) for a number of days (referred to as “static suspension cell culture” herein).

[0108] Cells that are “normally adherent” are cells that naturally adhere to an artificial substrate or cell culture vessel surface during cell culture. These cell types have to be detached from the surface before they can be sub-cultured. For subculture cells may be detached by one of several methods including trypsin treatment to break down the proteins responsible for surface adherence, chelating calcium ions with EDTA which disrupts some protein adherence mechanisms, or mechanical methods like repeated washing or use of a cell scraper. The detached cells are then resuspended in fresh growth medium and allowed to settle back onto their growth surface.

[0109] The majority of the cells derived from vertebrates, with the exception of hematopoietic cell lines and a few others, are anchorage-dependent and have to be cultured on a suitable substrate that is specifically treated to allow cell adhesion and spreading. Such cells are referred to as “normally adherent” herein. Suprisingly, in the presence of Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity), such cells may successfully be cultured in suspension. Accordingly, in such situations, the Caf 1 -WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be considered to “promote suspension cell culture of cells that are normally adherent”.

[0110] Examples of cells that are normally adherent include fibroblasts, epithelial cells (such as primary nasal airway cells), endothelial cells, macrophages, lymphoblasts, mesenchymal stem cells (hMSCs) and embryonic cells. Examples of cell lines that are normally adherent include HEK293, 3T6, A549, A9, BALB / 3T3, BHK-21 , BHL-100, BT, Caco-2, Chang, CHO- K1 , Clone 9, Clone, M-3, COS-1 , COS-3, COS-7, CRFK, CV-1 , D-17, GH1 , GH3, HCT-15, HeLa, HT-1080, HT-29, HUVEC, 1-10, JEG-3, Jensen, L2, LLC-WRC 256, McCoy, MCF7, WI-38, WISH, XC, Y-1 , HCT116, PANC1 , NIH3T3, A431 , 3T3L1 , U2OS, MDAMB231 , NTERA2, RAW 264.7, T47D, PC3, C2C12, NCCIT, SKNAS, L6, GTL16, B16F10, C8D1A, HAEC, F9, HSkMs, SKOV3, LNCAP, A375, H1975, SW480, SKBR3, RD, SCC15, HMEC- 1 , CAKI-1 , RSC96, OVCAR3, NMUMG, AML12, SKMEL-31 , MEF-IM, BEWO, 769P, MCF10A, LOVO, H1299, A673, BT-549, H4, HACAT, Htert-RPE1 , RT4, U118MG, SW620, 786-0, and MJ cell lines.

[0111] The Caf1-WT polymers (or a variant thereof, wherein the variant has Caf1-WT activity) described herein may be used in a cell culture method. A cell culture method is therefore provided herein, wherein the method comprises the step of culturing cells in the presence of Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity).

[0112] The Caf1-WT polymers (or a variant thereof, wherein the variant has Caf1-WT activity) described herein are particularly useful when used in a 3D cell culture method. A 3D cell culture method is therefore provided herein, wherein the method comprises the step of culturing cells on or in a scaffold, wherein the scaffold comprises Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity).

[0113] In one example, a 3D cell culture method is provided, wherein the method comprises the step of culturing cells on or in a hydrogel, wherein the hydrogel comprises Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity). In this example, the hydrogel may comprise at least about 0.025 % (w / v) wildtype Caf1 polymer (or variant). In this example, the hydrogel comprises a maximum ratio of about 40:1 of the gel forming polymer (e.g. alginate or Matrigel®) to Caf1-WT polymer (or variant).

[0114] As used herein, the term “hydrogel” refers to a water-swellable polymeric matrix, with a network of macromolecules held together by covalent crosslinks. The matrix can absorb a substantial amount of water to form an elastic gel. The hydrogel swelling can be affected by conditions in which the hydrogel is placed, such as by pH, temperature, and the local ion concentration and type. In a non-limiting example, the hydrogel is insoluble.

[0115] The swollen state of a hydrogel may be characterised by several parameters, including the swelling ratio under changing conditions, the permeability coefficient of certain solutes, and the mechanical behaviour of the hydrogel under conditions of its intended use.

[0116] In a non-limiting example, the monomers and / or polymers of the hydrogel are crosslinked so as to provide structure and physical integrity to the matrix. The cross linking may be due to chemical, physical, or radiation crosslinking.

[0117] In the case of physical crosslinking, the linking may result from hydrogen bonding, van der Waals interactions, ionic bonding, or combinations thereof. Physical cross linking may be initiated by mixing two precursors that are physically separated until combined in situ or as a consequence of a prevalent condition in the physiological environment, including temperature, pH, ionic strength, combinations thereof.

[0118] Chemical (covalent) crosslinking may be accomplished by any of a number of mechanisms including, but not limited to, free radical polymerisation, condensation polymerisation, anionic or cationic polymerisation, step growth polymerisation, electrophile-nucleophile reactions or combinations thereof.

[0119] Radiation crosslinking may be achieved by any number of mechanisms including, but not limited to, exposing the hydrogel article to at least one of visible light radiation, infrared radiation, ultraviolet radiation, electron beam radiation, gamma radiation, or x-ray radiation.

[0120] Typically, the polymer constituents are crosslinked via chemical or physical processes such that they form a “mesh-like” insoluble polymer network.

[0121] The hydrogels described herein comprise at least one gel-forming polymer. The terms “gelforming polymer” and “hydrogel-forming polymer” are used interchangeably herein. A “gelforming polymer” is a polymer that is capable of forming a hydrogel (in the presence or absence of Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity)). The term "hydrogel-forming polymer" refers to a polymer which is capable of forming a cross-linked or network structure or matrix under appropriate conditions, wherein an interstitial liquid and an extracellular nucleic acid may be retained within such a structure or matrix. The hydrogel may comprise internal pores.

[0122] The gel-forming polymer may be a natural polymer or a synthetic polymer. Hydrogel-forming natural polymers include proteins such as collagen and gelatine and polysaccharides such as starch, alginate, and agarose. Synthetic polymers that form hydrogels are traditionally prepared using chemical polymerization methods. The gel-forming polymer provides the capability to generate a hydrogel, and the Caf1-WT polymer is introduced into the hydrogel (e.g. by simple mixing in the liquid state) to impart the beneficial properties described above to the resultant hydrogel (e.g. the ability to (i) increase cell viability; (ii) increase cell proliferation, cell migration and / or cell clustering; and / or (iii) promote the formation of a 3D cell structure). The presence of the Caf1-WT polymer in the hydrogel therefore provides specific beneficial properties to the hydrogel, thereby reducing the costs and / or increasing the efficiency of the process.

[0123] The hydrogels described herein may be formed using any appropriate gel-forming polymer. The polymer will in general be a hydrophilic polymer. It will be capable of swelling in an aqueous liquid. Some examples of appropriate gel-forming polymers include collagen, fibrin, alginate, polyacylamide, polyethylene glycol (PEG), hyaluronic acid and / or agarose. The hydrogel may be a collagen hydrogel, fibrin hydrogel, alginate hydrogel, polyacrylamide hydrogel, polyethylene glycol (PEG) hydrogel, hyaluronic acid hydrogel, agarose hydrogel, Matrigel® hydrogel, or a Pura Matrix™ (Corning®) hydrogel. Other appropriate hydrogels and gel-forming polymers may be readily identified by a person of skill in the art (see for example, Caliari, S., Burdick, J. A practical guide to hydrogels for cell culture. Nat Methods 13, 405-414 (2016), incorporated herein in its entirety).

[0124] In one example, the hydrogel-forming polymer is collagen. In this example, the collagen hydrogel comprises a matrix of collagen fibrils which form a continuous scaffold around an interstitial liquid and the extracellular nucleic acid. Dissolved collagen may be induced to polymerise by the addition of dilute alkali to form a gelled network of cross-linked collagen fibrils. The gelled network of fibrils supports the original volume of the dissolved collagen fibres, retaining the interstitial liquid. General methods for the production of such collagen gels are well known in the art (e.g. W02006 / 003442, W02007 / 060459 and WG2009 / 004351).

[0125] The collagen which is used in the collagen gel may be any fibril-forming collagen.

[0126] Examples of fibril-forming collagens are Types I, II, III, V, VI, IX and XI. The gel may comprise all one type of collagen or a mixture of different types of collagen. The gel may comprise or consist of Type I collagen. In some examples, the gel is formed exclusively or substantially from collagen fibrils, i.e. collagen fibrils are the only or substantially the only polymers in the gel. In other examples, the collagen gel may additionally comprise other naturally-occurring polymers, e.g. silk, fibronectin, elastin, chitin and / or cellulose. Generally, the amounts of the non- collagen naturally-occurring polymers will be less than 5%, preferably less than 4%, 3%, 2% or 1 % of the gel (wt / wt). Similar amounts of non-natural polymers may also be present in the gel, e.g. peptide amphiphiles, polylactone, polylactide, polyglycone, polycaprolactone and / or phosphate glass.

[0127] A non-limiting example of a suitable gel-forming polymer is a basement membrane extract. In one example, the basement membrane extract (BME) is solubilized extracellular matrices (ECM) purified from the Englebreth-Holm-Swarm (EHS) murine tumor. In this example, the BME is rich in extracellular matrix proteins, such as laminin, collagen IV, entactin, and heparan sulfate proteoglycans. In one example, the hydrogel is a Matrigel® hydrogel (which is a collagen containing hydrogel, see examples section below). Matrigel® hydrogels and methods of making the same are well known in the art (Passaniti et al., J Cell Commun Signal. 2021 Aug 31 ;16(4):621-626). Other equivalent examples of BME include Mogengel® (AcroBiosystems), Geltrex® (Thermo Fisher), and Cultrex® (R&D Systems). In one example, the BME is Mogengel® (AcroBiosystems). In one example, the BME is Geltrex® (Thermo Fisher). In one example, the BME is Cultrex® (R&D Systems). As is well known to a person of ordinary skill in the art, BME products, such as Matrigel®, show significant batch-to-batch variability. A person of ordinary skill in the art would understand that differences between batches can include variations in protein content, growth factors, and structural proteins, such as the presence of different levels of laminin (Hughes, C.S; Postovit, L.M and Lajoie, G.A. Proteomics, (2010) 10; 1886-1890).

[0128] Another non-limiting example of a suitable gel-forming polymer is alginate. In one example the hydrogel is an alginate hydrogel (see examples section below). Alginate hydrogels and methods of making the same are well known in the art (Lee et al., Prog Polym Sci. 2012 Jan;37(1):106-126.) In one example, the alginate hydrogel is set with calcium chloride. In some examples, the hydrogel-forming polymer is alginic acid or an alginate salt of a metal ion. In one example, the metal is a Group 1 metal (e.g. lithium, sodium, or potassium alginate) or a Group 2 metal (e.g. calcium, magnesium, barium or strontium alginate). For example, the polymer may be calcium alginate or sodium alginate or strontium alginate.

[0129] In examples where the hydrogel is an alginate hydrogel, the alginate may be present in the hydrogel in any appropriate amount, for example it may be present in the hydrogel at a concentration of from about 0.5 % (w / v) to about 10% (w / v). See for example Hoesli et al., J Vis Exp. 2017; (124): 55280, where a concentration of about 10% (w / v) is used. In one example, the hydrogel is an alginate hydrogel and the alginate is present in the hydrogel at a concentration of from about 0.5 % (w / v) to about 5% (w / v), or about 0.5 % (w / v) to about 2% (w / v). In one example, the alginate is present in the hydrogel at a concentration of about 1 % (w / v).

[0130] In the context of hydrogels, as used herein, “w / v” refers to the weight (w) of the recited reagent (in the paragraph above as an example, the alginate) that is added to the total liquid volume (v) used to generate the hydrogel. The weight (w) and volume (v) are in equivalent units (i.e. mg (w) and mL (v)). An example of how to calculate this is shown under “General methods used for mixing Caf1 polymer with other polymers e.g. alginate” in the examples section below. For the avoidance of doubt, the default units for the w / v provided herein in is mg (w; for the weight of the polymer e.g. alginate in the paragraph above) and mL (v; for the total liquid volume used to generate the hydrogel). For the avoidance of doubt, in these calculations the “total liquid volume (v) used to generate the hydrogel” does not include any additional cell-containing liquid (e.g. cell culture media comprising cells) that is added to the hydrogel solution before gelation has occurred. This is because the cells and corresponding cell culture medium are not part of the hydrogel per se, but are added to the hydrogel components. Similarly, the “total liquid volume (v) used to generate the hydrogel” also does not include any volume attributed to the crosslinker that is used to induce gelation of the hydrogel.

[0131] The hydrogels described herein comprise Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity). Preferably, the hydrogels described herein comprise at least about 0.025 % (w / v) Caf1-WT polymer (or variant) (e.g. in the context of an about 1% (w / v) alginate or Matrigel® hydrogel). In one example, the hydrogels comprise at least about 0.05 % (w / v) Caf1-WT polymer (or variant)(e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel).

[0132] In one example, the hydrogels comprise at least about 0.1 % (w / v) Caf1-WT polymer (or variant) (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel). In one example, the hydrogels comprise at least about 0.2 % (w / v) Caf1-WT polymer (or variant) (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel).

[0133] In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1- WT activity) is present in the hydrogel at a concentration of from about 0.025 % (w / v) to about 0.5% (w / v); or from about 0.05 % (w / v) to about 0.5 % (w / v) (e.g. in the context of an about 1% (w / v) alginate or Matrigel® hydrogel). In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is present in the hydrogel at a concentration of from about 0.1 % (w / v) to about 0.5% (w / v) (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel). In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1- WT activity) is present in the hydrogel at a concentration of from about 0.025% (w / v) to about 0.4% (w / v); or from about 0.05% (w / v) to about 0.4% (w / v) (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel). In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is present in the hydrogel at a concentration of from about 0.1 % (w / v) to about 0.4% (w / v) (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel).

[0134] For example the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be present in the hydrogel at a concentration of from about 0.025% (w / v) to about 0.3% (w / v); or from about 0.05% (w / v) to about 0.3% (w / v) (e.g. in the context of an about 1% (w / v) alginate or Matrigel® hydrogel). In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is present in the hydrogel at a concentration of from about 0.1 % (w / v) to about 0.3% (w / v) (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel).

[0135] In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1- WT activity) is present in the hydrogel at a concentration of about 0.2% (w / v) (e.g. in the context of an about 1% (w / v) alginate or Matrigel® hydrogel).

[0136] In the context of hydrogels, in line with the explanation provided elsewhere herein, as used herein, “w / v” refers to the weight (w) of the recited reagent (in the paragraph above as an example, the Caf1-WT polymer) that is added to the total liquid volume (v) used to generate the hydrogel. The weight (w) and volume (v) are in equivalent units (i.e. mg (w) and mL (v)). An example of how to calculate this is shown under “General methods used for mixing Caf1 polymer with other polymers e.g. alginate” in the examples section below. For the avoidance of doubt, the default units for the w / v provided herein in is mg (w; for the weight of the polymer e.g. Caf1-WT polymer in the paragraph above) and mL (v; for the total liquid volume used to generate the hydrogel). As stated above, for the avoidance of doubt, in these calculations the “total liquid volume (v) used to generate the hydrogel” does not include any additional cell-containing liquid (e.g. cell culture media comprising cells) that is added to the hydrogel solution before gelation has occurred. This is because the cells and corresponding cell culture medium are not part of the hydrogel per se, but are added to the hydrogel components. Similarly, the “total liquid volume (v) used to generate the hydrogel” also does not include any volume attributed to the crosslinker that is used to induce gelation of the hydrogel. The equivalent explanation applies to the Caf1-WT polymer variants described herein. An alternative means for describing the hydrogels provided herein is by the ratio of gelforming polymer to Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) within the hydrogel. Preferably, the hydrogels described herein comprise maximum ratio of about 40:1 of the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer(or variant)). In this context, it will be clear to a person of skill in the art that a “maximum ratio” refers to a maximal difference in the relative proportions of the two recited components (in this case gel-forming polymer and Caf1-WT polymer (or variant)) within the hydrogel. By defining a maximum ratio, it is clear that smaller ratios (i.e. ratios with smaller differences between the relative proportions of the two recited components) are also encompassed.

[0137] In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is a maximum ratio of about 40:1 (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel). In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 40:1 to about 5:2 (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel).

[0138] In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is about 5: 1 (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel).

[0139] In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 40:1 to about 20:1 (e.g. in the context of an about 1% (w / v) alginate or Matrigel® hydrogel).

[0140] In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 40:1 to about 10:1 (e.g. in the context of an about 1% (w / v) alginate or Matrigel® hydrogel).

[0141] In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 40:1 to about 5:1 (e.g. in the context of an about 1% (w / v) alginate or Matrigel® hydrogel).

[0142] In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 40:1 to about 2:1 (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel). In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 20: 1 to about 2:1 (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel). In one example, the gel forming polymer (e.g. the alginate or 1 Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 10:1 to about 2:1 (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel).

[0143] In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 40:1 to about 5:2 (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel). In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 20: 1 to about 5:2 (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel). In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 10:1 to about 5:2 (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel).

[0144] In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 40:1 to about 3:1 (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel). In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 20: 1 to about 3: 1 (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel). In one example, the gel forming polymer (e.g. the alginate or Matrigel®) to Caf1-WT polymer (or variant) ratio in the hydrogel is in a range of from about 10:1 to about 3:1 (e.g. in the context of an about 1 % (w / v) alginate or Matrigel® hydrogel).

[0145] Any discussion of the amount of Caf1-WT polymer (or variant) may be in the context of an about 1% (w / v) alginate or Matrigel® hydrogel) unless the context does not allow it.

[0146] The hydrogel may be in any suitable format, such as a hydrogel droplet, hydrogel bead, hydrogel disc or hydrogel sheet. The hydrogel disc may for example, have a diameter of 5- 50 mm or 10-50 mm, for example 10-30 mm, 15-25 mm, or about 19 mm. The thickness of the hydrogel may be generally 0.1 - 5mm, such as 0.5-2.0 mm, e.g. about 1.0 or 1.5 mm, or about 1 , 2, 3, 4 or 5 mm. In some examples, the final volume of hydrogel is about 200 pl to 1 ml, e.g. 200-600 pl, 300-500 pl, or 400-450 pl.

[0147] In a specific embodiment, the hydrogel comprises a crosslinking agent. These crosslinking agents may comprise for example monoaldehydes, dialdehydes, sodium hypochlorite, diisocyanates, dicarboxylic acid halides and chlorinated epoxides.

[0148] In a specific embodiment, the crosslinking agent comprises Poly(ethylene glycol) (PEG). PEG is a chemical compound composed of repeating ethylene glycol units and is non- reactive, non-toxic, non-immunogenic, soluble and highly flexible to create insoluble networks, it requires end-functionalisation with cross-linking groups. A number of chemistries have been developed for the functionalisation of PEG including the addition of acrylate, thiol, amine, maleimide or vinyl sulfone reactive groups. As crosslinked networks, these materials are non-degradable under physiological conditions. Polyethylene glycol spacer arms have a defined structure and molecular weight which ensures reproducible protein-modification effects. Moreover, it provides high stability, reduced tendency toward clumping and immunogenicity even at high molecular weights.

[0149] In a specific embodiment, the crosslinking agent is a functionalised 4 arm PEG crosslinker.

[0150] In a specific embodiment, the crosslinking agent is a 4-arm PRG succinimidyl glutarate.

[0151] In a specific embodiment, the crosslinking agent is a 4-arm PEG succinimidyl carboxy methyl ester.

[0152] Other possible crosslinking agents include but are not limited to:

[0153] (i) the linear homobifunctional, short spacer arm, DTSSP (Sulfo-DSP) (3,3'- Dithiobis[sulfosuccinimidylpropionate]) with molecular weight of 608.51 and a spacer arm with approximately of 12.0 A. DTSSP is water-soluble and thiol-cleavable.

[0154] (ii) the linear homobifunctional, long spacer arm, NHS-PEG-NHS (O,O'-Bis[2-(N- Succinimidyl-succinylamino)ethyl]polyethylene glycol) with a molecular weight of 10000 and a spacer arm of approximately 197 A.

[0155] In a specific embodiment, the crosslinking agent is a biodegradable crosslinking agent. Advantageously such crosslinking agents make the hydrogel biodegradable or absorbable. As used herein, the term “biodegradable” refers to material or polymer that can be degraded, preferably adsorbed and degraded in a patient’s body. Alternatively, the cross linking agent is a non-degradable cross linking agent.

[0156] The skilled person would readily identify crosslinker to monomer / polymer ratios that are suitable in accordance with the present invention. By way of example, but not by way of limitation, ratios of 1 :120, 1 :5, 1 :3, or 1 :2 (monomer: crosslinker (w / w)) may be used.

[0157] The cells may be cultured in or on the hydrogel described herein. Cells may be cultured in the hydrogel if they are encapsulated in or entrapped by the hydrogel. Typically this occurs when the cells are mixed with the hydrogel components in the liquid state, before gelation occurs to form the hydrogel. It may also occur if the cells are seeded on the hydrogel surface (after gelation has generated the hydrogel), and they sequentially migrate into the hydrogel such that they become encapsulated in or entrapped by the hydrogel after the hydrogel has been generated. Cells may be cultured on the hydrogel if they are seeded on the hydrogel surface (after gelation has generated the hydrogel). In such circumstances, the cells may be the cells are on or attached to the hydrogel.

[0158] The cells may be any suitable cells. For example, they may be cells that are normally adherent. Examples of cells that are normally adherent are provided elsewhere herein. In some examples the cells are mammalian cells. In some examples the cells are mammalian cells that are normally adherent cells. In some examples, the cells are selected from the group consisting of: myosatellite cells, induced pluripotent and embryonic stem cells, adipose-derived cells, hepatocytes, chondrocytes (such as MSC-derived chondrocytes), osteoblasts (such as MSC-derived osteoblasts), cancer cells, neurons, cardiomyocytes, keratinocytes, fibroblasts, epithelial cells, endothelial cells, macrophages, lymphoblasts, mesenchymal stem cells (hMSCs), embryonic cells, and cell lines, such as: HEK293, 3T6, A549, A9, BALB / 3T3, BHK-21 , BHL-100, BT, Caco-2, Chang, CHO-K1 , Clone 9, Clone, M- 3, COS-1 , COS-3, COS-7, CRFK, CV-1 , D-17, GH1 , GH3, HCT-15, HeLa, HT-1080, HT-29, HUVEC, 1-10, JEG-3, Jensen, L2, LLC-WRC 256, McCoy, MCF7, WI-38, WISH, XC, Y-1 , HCT116, PANC1 , NIH3T3, A431 , 3T3L1 , U2OS, MDAMB231 , NTERA2, RAW 264.7, T47D, PC3, C2C12, NCCIT, SKNAS, L6, GTL16, B16F10, C8D1A, HAEC, F9, HSkMs, SKOV3, LNCAP, A375, H1975, SW480, SKBR3, RD, SCC15, HMEC-1 , CAKI-1 , RSC96, OVCAR3, NMUMG, AML12, SKMEL-31 , MEF-IM, BEWO, 769P, MCF10A, LOVO, H1299, A673, BT- 549, H4, HACAT, Htert-RPE1 , RT4, U118MG, SW620, 786-0, and MJ cell lines.

[0159] The step of culturing the cells may be performed for any appropriate length of time. For example, the cells may be cultured in the presence of Caf1-WT polymer or a variant thereof, wherein the variant has Caf1-WT activity) (e.g. wherein the Caf1-WT polymer (or variant) is comprised with a hydrogel as described herein and the cells are cultured on or in the hydrogel as described herein; or wherein the Caf1-WT polymer (or variant) is coated on a cell culture surface as described herein; or wherein the Caf1-WT polymer (or variant) is in solution in the cell culture medium as described herein) for at least 24 hours. In one example, the cells are cultured in the presence of Caf1-WT polymer (or variant) for at least 48 hours.

[0160] In one example, the cells may be cultured in the presence of Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) (e.g. wherein the Caf1-WT polymer (or variant) is comprised with a hydrogel as described herein and the cells are cultured on or in the hydrogel as described herein; or wherein the Caf1-WT polymer (or variant) is coated on a cell culture surface as described herein; or wherein the Caf1-WT polymer (or variant) is in solution in the cell culture medium as described herein) for at least 3 days or at least 4 days. In one example, the cells are cultured in the presence of Caf1-WT polymer (or variant) for at least 5 days.

[0161] In one example, the cells may be cultured in the presence of Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) (e.g. wherein the Caf1-WT polymer (or variant) is comprised with a hydrogel as described herein and the cells are cultured on or in the hydrogel as described herein; or wherein the Caf1-WT polymer (or variant) is coated on a cell culture surface as described herein; or wherein the Caf1-WT polymer (or variant) is in solution in the cell culture medium as described herein) for at least 7 days or at least 10 days. In one example, the cells are cultured in the presence of Caf1-WT polymer (or variant) for at least 14 days. In one example, the cells are cultured in the presence of Caf1-WT polymer (or variant) for at least 21 days.

[0162] Cells are typically cultured in a cell culture medium. The terms "cell culture medium" and "culture medium" (plural "media" in each case) refer to a nutritive solution for cultivating live cells and may be used interchangeably. The medium may be supplemented with differentiation factors. Such a medium may be referred to as a “differentiation medium”. However, it will be appreciated that a differentiation medium is a type of cell culture medium.

[0163] The cell culture medium may be a complete formulation, i.e., a cell culture medium that requires no supplementation to culture cells, or may be an incomplete formulation, i.e., a cell culture medium that requires supplementation or may be a medium that may supplement an incomplete formulation or in the case of a complete formulation, may improve culture or culture results. Various cell culture media will be known to those skilled in the art, who will also appreciate that the type of cells to be cultured may dictate the type of culture medium to be used.

[0164] Merely by way of example and not limitation, the culture medium may be selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Ham's F-12 (F-12), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI-1640, Ham's F-10, □Minimal Essential Medium (aMEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium(IMDM), or any combination thereof. Other media that are commercially available (e.g., from Thermo Fisher Scientific, Waltham, MA) or that are otherwise known in the art can be equivalently used in the context of this disclosure. Again, only by way of example, the media may be selected from the group consisting of 293 SFM, CD-CHO medium, VP SFM, BGJb medium, Brinster's BMOC-3 medium, cell culture freezing medium, CMRL media, EHAA medium, eRDF medium, Fischer's medium, Gamborg's B-5 medium, GLUTAMAX™ supplemented media, Grace's insect cell media, HEPES buffered media, Richter's modified MEM, I PL-41 insect cell medium, Leibovitz's L-15 media, McCoy's 5A media, MCDB 131 medium, Media 199, Modified Eagle's Medium (MEM), Medium NCTC-109, Schneider's Drosophila medium, TC-100 insect medium, Waymouth's MB 752 / 1 media, William's Media E, protein free hybridoma medium II (PFHM II), AIM V media, Keratinocyte SFM, defined Keratinocyte SFM, STEM PRO® SFM, STEM PRO® complete methylcellulose medium, HepatoZYME-SFM, Neurobasal™ medium, Neurobasal-A medium, Hibernate™ A medium, Hibernate E medium, Endothelial SFM, Human Endothelial SFM, Hybridoma SFM, PFHM II, Sf 900 medium, Sf 900 II SFM, EXPRESS FIVE® medium, CHO-S-SFM, AMINOMAX-II complete medium, AMINOMAX-C100 complete medium, AMINOMAX-C140 basal medium, PUB-MAX™ karyotyping medium, KARYOMAX bone marrow karyotyping medium, and KNOCKOUT D-MEM, or any combination thereof. Suitably, the combination may be of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 (F-12).

[0165] In some embodiments, the cell culture medium may comprise serum. In other embodiments, the culture medium may comprise low levels of serum. Such a medium may be referred to as cell culture medium with “reduced serum”. The term “reduced serum” is defined herein below. In other embodiments, the culture medium may be serum-free. When the cell culture medium has reduced serum, it may be said that the co-culture is performed in reduced- serum conditions. By the same token, when the cell culture medium is serum-free, it may be said that the co-culture is performed in serum-free conditions.

[0166] The term “reduced-serum” conditions as used herein is used to describe the presence of serum in the co-culture, but at a lower level than would usually be used for optimal co-culture of the cells of interest. For example, it is accepted in the field that muscle cells are typically cultured in a cell culture medium that comprises at least 10% (v / v) serum for cell growth, and at least 5% (v / v) serum for cell differentiation. Thus, a cell culture medium having no more than about 2% (v / v) serum may therefore be considered “reduced-serum” conditions. Reduced-serum conditions may comprise no more than about 2% (v / v) serum in the cell culture medium, no more than about 1.5% (v / v) serum in the cell culture medium, no more than about 1% (v / v) serum in the cell culture medium, no more than about 0.5% (v / v) serum in the cell culture medium, or less. Reduced-serum conditions may have (substantially) no serum. In this context, “substantially no serum” means that there are no more than trace amounts of serum. Trace amounts may be defined as a maximum of 0.1 % (v / v) serum. For example, the reduced-serum cell culture medium may have about 0.1 % to about 1.5% (v / v) serum. For example, the reduced-serum cell culture medium may have about 0.1% to about 1 % (v / v) serum. As another example, the reduced-serum cell culture medium may have about 0.1% to about 0.5% (v / v) serum.

[0167] Cell culture media wherein there is no detectable serum are referred to herein as “serum- free” cell culture media (SFM). Typically, for media that contains serum, the serum is added as a supplement at the start of, or during cell culture. The term “serum-free” cell culture medium therefore includes cell culture media which have not been supplemented with serum. The term “serum-free” is very well known in the art.

[0168] As would be clear to a person of skill in the art, “serum-free” media may comprise a number of additives and supplements, provided that it does not contain detectable levels of serum. The serum-free or reduced-serum cell culture media described herein are particularly advantageous as they provide a more chemically defined media for cell culture, using reagents that are more sustainable, and with a lower risk of contamination compared to equivalents that are reliant on serum.

[0169] Additional polymers may also be present during cell culture. For example, additional polymers may be present within the hydrogels described herein. For example, the hydrogels may comprise at least about 0.025 % (w / v) wildtype Caf1 (Caf1-WT) polymer (or variant) and also comprise additional polymers. In one example, the hydrogels may comprise a maximum ratio of about 40:1 of the gel forming polymer (e.g. alginate or Matrigel®) to Caf1- WT polymer (or variant), and also comprise additional polymers. An example of an additional polymer that may be present is a modified Caf1 polymer, wherein the modified Caf1 polymer comprises an exogenous bioactive sequence. Modified Caf1 polymers are described for example in EP2858675, which is incorporated herein in its entirety. Modified Caf1 polymers comprise at least one modified Caf1 monomer, wherein the modified Caf1 monomer retains the ability to form a Caf1 polymer but does not comprise the sequence shown in SEQ ID NO:3 because it comprises an exogenous bioactive sequence. In one example, the hydrogels described herein do not comprise any Caf1 polymer that is not Caf1-WT polymer. In other words, in this example, the hydrogels do not comprise any modified Caf1 polymers.

[0170] In the context of cell culture in cell culture medium (e.g. without the presence of a hydrogel), as used herein, “w / v” refers to the weight (w) of the recited reagent (e.g. the Caf1-WT polymer) that is added to the total liquid volume (v) of the cell culture medium used. The weight (w) and volume (v) are in equivalent units (i.e. mg (w) and mL (v)). For the avoidance of doubt, the default units for the w / v provided herein in is mg (w; for the weight of the polymer e.g. Caf1-WT) and mL (v; for the total liquid volume of the cell culture medium used).

[0171] During cell culture the cell culture medium may comprise Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity). Preferably, the cell culture medium may comprise at least about 0.005 % (w / v) Caf1-WT polymer (or variant). In one example, the cell culture medium may comprise at least about 0.01 % (w / v) Caf1-WT polymer (or variant). In one example, the cell culture medium may comprise at least about 0.015 % (w / v) Caf1- WT polymer (or variant). In one example, the cell culture medium may comprise at least about 0.02 % (w / v) Caf1-WT polymer (or variant). In this context, at least about 0.005 % (w / v) Caf1-WT polymer (or variant) equates to at least about 50 pg polymer Caf1-WT polymer (or variant) per ml of cell culture medium. Similarly, at least about 0.01 % (w / v) Caf1-WT polymer (or variant) equates to at least about 100 pg polymer Caf1-WT polymer (or variant) per ml of cell culture medium. As would be clear to a person of skill in the art, at least about 0.015 % (w / v) Caf1-WT polymer (or variant) equates to at least about 150 pg polymer Caf1-WT polymer (or variant) per ml of cell culture medium; and at least about 0.015 % (w / v) Caf1-WT polymer (or variant) equates to at least about 200 pg polymer Caf1-WT polymer (or variant) per ml of cell culture medium.

[0172] Preferably, the cell culture medium may comprise at least about 0.025 % (w / v) Caf1-WT polymer (or variant). In one example, the cell culture medium may comprise at least about 0.05 % (w / v) Caf1-WT polymer (or variant). In this context, at least about 0.025 % (w / v) Caf1-WT polymer (or variant) equates to at least about 250 pg polymer Caf1-WT polymer (or variant) per ml of cell culture medium. Similarly, at least about 0.05 % (w / v) Caf1-WT polymer (or variant) equates to at least about 500 pg polymer Caf1-WT polymer (or variant) per ml of cell culture medium.

[0173] In one example, the cell culture medium comprise at least about 0.1 % (w / v) Caf1-WT polymer (or variant). In one example, the cell culture medium comprise at least about 0.2 % (w / v) Caf1-WT polymer (or variant). In this context, at least about 0.1 % (w / v) Caf1-WT polymer (or variant) equates to at least about 1 mg polymer Caf1-WT polymer (or variant) per ml of cell culture medium. Similarly, at least about 0.2 % (w / v) Caf1-WT polymer (or variant) equates to at least about 2 mg polymer Caf1-WT polymer (or variant) per ml of cell culture medium.

[0174] In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1- WT activity) is present in the cell culture medium at a concentration of from about 0.025 % (w / v) to about 0.5% (w / v); or from about 0.05 % (w / v) to about 0.5 % (w / v). In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is present in the cell culture medium at a concentration of from about 0.1 % (w / v) to about 0.5% (w / v).

[0175] In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1- WT activity) is present in the cell culture medium at a concentration of from about 0.025% (w / v) to about 0.4% (w / v); or from about 0.05% (w / v) to about 0.4% (w / v). In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is present in the cell culture medium at a concentration of from about 0.1 % (w / v) to about 0.4% (w / v).

[0176] For example the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be present in the cell culture medium at a concentration of from about 0.025% (w / v) to about 0.3% (w / v); or from about 0.05% (w / v) to about 0.3% (w / v). In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) is present in the cell culture medium at a concentration of from about 0.1 % (w / v) to about 0.3% (w / v). In one example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1- WT activity) is present in the cell culture medium at a concentration of about 0.2% (w / v).

[0177] The Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be present in the cell culture medium during transfection of the cells.

[0178] The term “cell transfection” as used herein is used to describe the process of artificially introducing nucleic acids into eukaryotic cells using non-viral methods. Such processes typically involve the use of a “transfection agent”. The term “transfection agent” is defined herein below. For example, the cell transfection method may be FuGENE®. As would be clear to a person of skill in the art, FuGENE® is representative of a common type of non- liposomal transfection method used in eukaryotic cell culture. FuGENE® is based upon a cationic polymer which forms a complex with the nucleic acid and then interacts with the cell membrane to deliver the genetic material into the cell. As would be clear to a person skilled in the art, other related methods use cationic lipids to achieve the same results. Conventional anti-clumping agents can interfere with cell transfection methods by disrupting mechanisms by which transfection reagents bind to and deliver genetic material into cells, particularly cationic transfection reagents. Anti-clumping agents are therefore removed when using these methods to achieve successful transfection. Anti-clumping agents also inhibit viral transfection by the same mechanism. In viral transfection, the virus attaches to or fuses with the cell membrane also via electrostatic / hydrophobic interactions, which are inhibited by many anti-clumping agents. As would be clear to a person skilled in the art, cell transfection methods all work using similar related mechanisms. It is surprising that the presence of Caf 1 - WT polymer does not inhibit the transfection process and that therefore transfection agents can successfully be used in the presence of Caf1-WT polymer.

[0179] The term “transfection agent” and / or “transfection reagent” as used herein is used to describe a substance that is used to introduce nucleic acids into eukaryotic cells. To introduce the nucleic acids into eukaryotic cells, the transfection agent or reagent must interact with the cell membrane, typically via electrostatic interactions. For example, the transfection agent or reagent can be cationic. A “cationic transfection agent” as used herein is used to describe a cationic molecule, which forms a complex with the nucleic acid and then interacts with the cell membrane to deliver the genetic material into the cell. As would be clear to a person of skill in the art, a cationic transfection agent is representative of a common type of transfection agent or reagent. As would be clear to a person of skill in the art, the transfection agent or reagent used herein may be cationic. As would be clear to a person of skill in the art, the transfection agent or reagent used herein may comprise a cationic polymer such as PEI (polyethylenimine),and Turbo293™. For example, the transfection agent or reagent may be FuGENE®. As would be clear to a person of skill in the art, the transfection agent or reagent used herein may comprise a cationic lipid such as Lipofectamine®. There are also liposomal methods using cationic and neutral lipids such as METAFECTENE® PRO or DOTAP.

[0180] The inventors have demonstrated herein that Caf1-WT polymers (or a variant thereof, wherein the variant has Caf1-WT activity) can provide several benefits to cells in cell culture. In vitro or ex vivo methods and uses for Caf1-WT polymers (or a variant thereof, wherein the variant has Caf1-WT activity) in cell culture are therefore provided herein. The observed benefits of using Caf1-WT polymers (or a variant thereof, wherein the variant has Caf1-WT activity) in cell culture may also be extrapolated to its use in vivo, where the presence of Caf1-WT polymers (or a variant thereof, wherein the variant has Caf1-WT activity) (for example as part of a hydrogel that is transplanted into a subject) may provide a number of advantages including (i) increasing cell viability; (ii) increasing cell proliferation, cell migration and / or cell clustering; and / or (iii) promoting the formation of a 3D cell structure, such as a spheroid or organoid in vivo.

[0181] Accordingly, the Caf1-WT polymers (or a variant thereof, wherein the variant has Caf1-WT activity) (or scaffolds such as hydrogels comprising the same) may have numerous biomedical applications. For example, the Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) and / or hydrogels comprising Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used as material for the treatment of wounds. In particular, scaffolds (such as hydrogels) comprising Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used to deliver cells to a tissue in need thereof. In one embodiment, scaffolds (such as hydrogels) comprising Caf 1 -WT polymer (or a variant thereof, wherein the variant has Caf1 -WT activity) are used to deliver cells to the eye of a mammalian subject. Alternatively, scaffolds (such as hydrogels) comprising Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used to deliver cells to a wound bed of a mammalian subject in need thereof. Accordingly, the invention provides the use of scaffolds (such as hydrogels) comprising Caf 1 -WT polymer (or a variant thereof, wherein the variant has Caf1 -WT activity) as a medicament.

[0182] The invention also provides the use of a Caf1-WT polymer (or a variant thereof, wherein the variant has Caf 1-WT activity) as a medicament. The Caf 1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) may be used in a method of treating a wound, for example in wound healing, in an equivalent manner as described below for the hydrogel of the invention. Accordingly, the invention provides a Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) of the invention for use in treating a wound, e.g. a skin wound. Also provided is a method of treating a skin wound comprising implanting a scaffold (such as hydrogel) comprising Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) into the skin, skin wound or skin wound bed of a mammalian subject in need thereof. The method is of particular use in re-epithel ial isation and of particular use in skin re-epithelialisation. The term “re-epithelialisation” relates to the repair, replacement, functional recovery and ultimate regeneration of damaged epithelium inside the body (including skin), or outside the body.

[0183] There is also provided scaffolds (such as hydrogels) comprising Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) for use in treating a skin wound.

[0184] As used herein the term “wound” relates to damaged tissues, preferably damaged skin, where the integrity of the skin or tissue is disrupted as a result from i.e. external force, bad health status, aging, exposure to sunlight, heat or chemical reaction or as a result from damage by internal physiological processes. Wounds where the epithelium such as the epidermis is damaged are considered to be an open wound. Wound healing is the process of regenerating the covering cell layers of a tissue, preferably by re-epithelialisation or reconstruction.

[0185] The introduction of a scaffold (such as hydrogel) comprising Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) capable of supporting normal skin cell attachment and migration and proliferation will help to accelerate wound healing by providing an immediate alternative substrate for unaffected skin cells at the wound margins to migrate across.

[0186] The invention provides a method of treating an ocular injury comprising implanting a scaffold (such as hydrogel) comprising Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) into the eye of a mammalian subject in need thereof.

[0187] There is also provided a scaffold (such as hydrogel) comprising Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1-WT activity) for use in treating an ocular injury.

[0188] As used herein, the term “ocular injury” refers to conditions resulting in an insufficient stromal micro-environment to support stem cell function, for example aniridia, keratitis, neurotrophic keratopathy, Keratoconus, Meesman’s dystrophy, Epithelial Basement Membrane Dystrophy and chronic limbitis; or conditions that destroy limbal stem cells such as Partial limbal stem cell deficiency, Total stem cell deficiency, Ocular herpes, chemical or thermal injuries, Stevens- Johnson syndrome, ocular cicatricial pemphigoid, contact lens wear, or microbial infection. There is also provided an ocular implant comprising a scaffolds (such as hydrogel) comprising Caf 1 -WT polymer (or a variant thereof, wherein the variant has Caf 1 -WT activity) .

[0189] In one embodiment the scaffold (e.g. hydrogel) is seeded with cells, such as corneal cells or stem cells prior to implantation. Alternatively, the scaffold (e.g. hydrogel) may be seeded with cells after implantation. Preferably, said cells are autologous, i.e. said cells are derived from the individual to be treated or alternatively the cells may be non-autologous.

[0190] The invention also provides a pharmaceutical composition comprising a scaffold (such as hydrogel) comprising Caf1-WT polymer (or a variant thereof, wherein the variant has Caf1- WT activity) together with a pharmaceutically acceptable excipient, diluent or carrier. In one embodiment the composition further comprises one or more of the following: growth factors, lipids, genes, etc., or compounds for altering the acidity / alkalinity (pH) of the wound, or compounds for altering the growth and performance of the transplanted cells and those at the margins of the wound I injury.

[0191] The term “pharmaceutically-acceptable carrier” as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human. When administered, the pharmaceutical compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, cytokines and optionally other therapeutic agents, preferably agents for use in wound healing such as growth factors, peptides, proteolytic inhibitors, extracellular matrix components, steroids and cytokines. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term “physiologically acceptable” refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. As used herein, a pharmaceutically acceptable carrier includes any conventional carrier, such as those described in Remington’s Pharmaceutical Sciences, by E. W Martin, Mack Publishing Co, Easton, PA, 15thEdition (1975).

[0192] In a further aspect there is provided a pharmaceutical composition in accordance with the invention for use as a medicament, for example, for use in treating ocular injury, corneal replacement or wound healing.

[0193] As used herein, the term “exogenous” refers to a heterologous amino acid sequence which is not present or naturally occurring within a Caf1-WT monomer sequence. It is therefore not present in SEQ ID NO:1 or SEQ ID NO:3. As used herein, “bioactive sequence” refers to a peptide sequence which has a specific biological function. Bioactive sequences are well known in the art and may be derived from any naturally occurring polypeptide including ECM components, cell adhesion molecules, cell surface receptors, growth factors, cytokines, chemokines, etc. For example, the bioactive sequence may mediate cell adhesion (or cell attachment), cell growth and / or cell differentiation (or induction of a cellular phenotype).

[0194] The bioactive sequence may be a cell adhesion recognition motif, a growth factor sequence motif or a protease site.

[0195] A cell adhesion recognition motif may be an extracellular matrix cell adhesion recognition motif, for example a motif derived from an extracellular matrix component such as collagen, elastin, fibronectin, laminin, osteopontin vitronectin or tenascin. The cell adhesion recognition motif may be derived from fibronenctin and may comprise the amino acid sequence RGD (Arg-Gly-Asp), more preferably RGDS (Arg-Gly-Asp-Ser). Alternatively, the cell adhesion recognition motif may be derived from fibronenctin and comprises the amino acid sequence PHSRN (Pro-His-Ser-Arg-Asn).

[0196] A cell adhesion recognition motif may be derived from Collagen I and may comprise the amino acid sequence GTPGPQGIAGQRGVV. Alternatively, the cell adhesion recognition motif may be derived from Collagen IV and may comprise the amino acid sequence MNYYSNS. Alternatively, the cell adhesion recognition motif may be derived from Laminin and may comprise the amino acid sequence YIGSR. Alternatively, the cell adhesion recognition motif may be derived from Laminin and may comprise the amino acid sequence IKVAV. Alternatively, the cell adhesion recognition motif may be derived from Fibronectin and may comprise the amino acid sequence FHRRIKA. Alternatively, the cell adhesion recognition motif may be derived from Fibronectin and may comprise the amino acid sequence LDVP. Alternatively, the cell adhesion recognition motif may be derived from Fibronectin and may comprise the amino acid sequence I DAP.

[0197] Alternatively, a bioactive sequence is a growth factor sequence motif derived from Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell line-derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nerve growth factor (NGF) and other neurotrophins, Platelet-derived growth factor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha (TGF-a), Transforming growth factor beta (TGF-P), Tumor_necrosis_factor-alpha (TNF-a), Vascular endothelial growth factor (VEGF), Wnt Signaling Pathway, placental growth factor (PIGF), Foetal Bovine Somatotrophin(FBS), IL- 1- Cofactor for IL-3 and IL-6 (activates T cells), IL-2- T-cell growth factor (stimulates IL-1 synthesis, activates B-cells and NK cells), IL-3 (stimulates production of all non-lymphoid cells); IL-4- Growth factor (for activated B cells, resting T cells, and mast cells), IL-5 (induces differentiation of activated B cells and eosinophils), IL-6 (stimulates Ig synthesis, growth factor for plasma cells), IL-7 (growth factor for pre-B cells), Neurone growth factor (NGF), Fibroblast growth factor (FGF) or Bone morphogenic protein 2 (BMP), e.g. a bioactive sequence comprising KIPKASSVPTELSAISTLYL.

[0198] Alternatively, a bioactive sequence is a protease site derived from known matrix metalloproteinase cleavage sites. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0199] Aspects of the invention are demonstrated by the following non-limiting examples.

[0200] Examples

[0201] General methods used for mixing Caf1 polymer with other polymers e.g. alginate

[0202] Materials needed:

[0203] All % are w / v unless otherwise defined

[0204] 2% Alginate in water (20 mg / mL) - sterile

[0205] Caf1-X in sterile water Freeze dried Caf1 may be used 100 mM sterile-filtered calcium chloride Protocol:

[0206] Dilute reconstituted Caf1-X to 8 mg / mL solution (0.8%) using sterile water

[0207] Perform serial dilution of Caf1-X to achieve 300 pL volumes of concentration series of Caf1-X / Alginate mixtures (where “Caf1-X” refers to any type of Caf1 , e.g. Caf1- WT polymer or Caf1 -Fibronectin).

[0208] NB Final concentration of alginate is 1%

[0209] To determine the “final %” of Caf1-WT polymer, the following explanation is provided. The 0.4% number comes from the mixture of the alginate and Caf1. Specifically, in this case, 150 pL of 8 mg / mL Caf1 is mixed with 150 pL 20 mg / mL alginate to make a mixture of 300 pL 4 mg / mL Caf1 and 10 mg / mL alginate. This equates to 0.004 g / mL and 0.01 g / mL or 0.4% and 1 % Caf1 and alginate respectively. These numbers are before the addition of the cell solution (100 pL) and the calcium chloride crosslinker solution (75 pL). The addition of these components makes the total gel volume 475 uL which then lowers as the gel shrinks during its formation. The percentages provided herein as (w / v) are defined before the addition of cells and crosslinker. This is in accordance with the standard way of working in the art.

[0210] Example 1 : A minimum level of WT- Caf1 is needed to improve 3D cell culture

[0211] Cells can be grown in 3D culture using polymeric materials such as alginate. Caf1 polymers can be easily mixed with these polymers and trapped by entanglement without the need for chemical modification. In addition to adding specific peptides such as RGDS, these experiments show that Caf1-WT polymer improves the environment for cell culture. Human mesenchymal stromal cells (hMSCs) were grown in 1% alginate, 1% alginate mixed with 0.2% Caf1-WT polymer or 1 % alginate mixed with 0.2% Caf1-RDGS. During initial seeding, 2x103cells / well were encapsulated in the gel with standard media conditions used (DM EM supplemented with 10% FBS and 1 % L-glutamine). After 10 or 21 days an MT cell viability assay was performed. The luminescent intensity relates to the number of viable cells present in the well. At day 10, supplementation of both Caf1-WT polymer and Caf1-RDGS polymer into alginate was beneficial to growth of hMSCs, indicated by increased number of viable cells. After 21 days, Caf1-WT polymer is seen to outperform Caf1-RDGS in enhancing the 3D culture of the hMSCs. See Figure 1.

[0212] HEK293 cells were encapsulated in increasing concentrations of Caf1-WT polymer mixed with 1% alginate. The cells were allowed to grow in StemPro MSC SFM (repeat 1) or complete DMEM supplemented with 2% FBS for 48 hours before the endpoint assay was performed to measure cell viability. Both presto blue and MT assays were used as per manufacturer specifications. Following 3 independent repeats of the experiment, it was determined the minimum concentration was 0.025% Caf1-WT, with a peak occurring at 0.2% Caf1-WT. See Figure 2.

[0213] Example 2. Addition of Caf1-WT polymer to alginate hydrogel enhances migration of cancer cells.

[0214] Migration of cancer cells is associated with metastatic cancer spread. Assays to imitate this are useful in various areas of cancer research. HCT116 colorectal cancer cells were encapsulated into either alginate only (1 % alginate hydrogel droplets) or Caf1-WT polymer supplemented 1% alginate hydrogel droplets. The droplets were formed using 50mM CaCh (compared to 25mM in previous experiments) to ensure structural integrity of the gel droplet. 2.5x104HCT116 cells were encapsulated in 7.5pL of alginate or alginate mixed with Caf1- WT. After 4 days, the droplets were imaged via bright field microscopy to view cell migration (representative images chosen). Imaged was used to calculate the % migration across an average of three wells. See Figure 3.

[0215] Example 3: WT-Caf1 mixed with Matrigel® induces endothelial cell tube formation in 3D cell culture

[0216] Endothelial cells can be grown in culture to create human vascular tissue for research use and regenerative medicine. This example uses basement cell extract (Matrigel®) as a 3D environment to produce the vascular tissue.

[0217] The tube formation assay measures the area of vascular tissue produced a percentage of the total cell area. The standard Matrigel® (Phenol Red Free, reduced growth factor Matrigel®, Corning Product# 356231) tube formation assay was used in combination with CaflWT and Caf1 FGF2. Angicyte™ cells were combined with a Matrigel® / medium mix at a ratio of 60:40. This suspension contained media with or without Caf1 in the following conditions.

[0218] 1. Matrigel® control (n=7)

[0219] 2. CAF control 20ng / ml (n=7)

[0220] 3. CAF FGF2 20ng / ml (n=7)

[0221] 4. Matrigel® + recombinant FGF2 (n=6)

[0222] The suspensions were then seeded in p-Angiogenesis slides (IBIDI #81506) and allowed to polymerise at 37°C in an incubator for 30 minutes. After polymerisation media was placed over the gels. After 48 hours, tube networks were stained with calcein and imaged. Quantification of the generated tubular networks total area percentage was carried out using Imaged. See Figure 4.

[0223] Example 4. Caf1-WT polymer mixed with Alginate induces human nasal airway epithelial cells to form ciliated 3D structures with lumen

[0224] Human primary nasal airway cells were collected and cultured. Using the standard protocol shown above, these were then mixed with either alginate or alginate plus Caf1-WT polymer to create 3D cultures that were stabilised by calcium ions which cross link the alginate. The aim is to create a model of the human airway epithelium where cells form organoids which display cilia and produce mucous. Such models can be used in models of cystic fibrosis for research and drug testing. The standard method uses basement membrane extract such as Matrigel® instead of alginate.

[0225] Method

[0226] The alginate hydrogels was prepared as above and contained either just 1% alginate or 1% alginate and 0.4% Caf1 , It was used as follows

[0227] 1 . Mix 75 pl hydrogel with 25 pl of cell solution

[0228] 2. Add 75 pl of calcium solution to a 96 well plate well

[0229] 3. Add 75 pl hydrogel / cell solution into the calcium solution

[0230] 4. Set for 60 min at 37 C in incubator.

[0231] 5. Add cell culture medium onto surface (max volume 75 pl).

[0232] 6. Alternatively Matrigel® was used as the 3D medium

[0233] Results

[0234] • Cells in alginate only; didn’t form a lumen

[0235] • Cells in alginate only after 24 hours were fewer than those seeded

[0236] • Cells in alginate + WT Caf1 formed a lumen where mucus was detected • Beating cilia observed only in cells kept in alginate + WT Caf1 or Matrigel® positive control.

[0237] See Figure 5.

[0238] Example 5: Adding soluble Caf1-WT polymer to cells in culture allows adherent cells to grow as a single cell suspension

[0239] HEK293 or HCT116 cells were seeded into wells of a 24-well non-tissue culture treated multi-well dish without Caf1 , with 150ug Caf1-WT polymer or 150ug Caf1 -Fibronectin. Cells were left to grow for 3 days prior to visualisation via bright field microscopy. See Figure 6.

[0240] Cells seeded without the addition of any Caf1 products were able to adhere to the surface of the dish. The addition of 150ug of Caf1-WT polymer encouraged the cells to grow in a single cell suspension. It is important to note the cells were left static during the three-day growth cycle. Finally, cell suspension supplemented with 150pg of Caf1 -Fibronectin caused the cells to evenly adhere to the surface of the well. No cells were seen to be floating.

[0241] Example 6. Caf1-WT polymer coated surfaces allow for the formation of spheroid structures.

[0242] HCT116 cancer cells were seeded into a non-tissue culture treated 24-well dish at 1x104cells per well in 10% FBS supplemented DMEM media. Prior to seeding, the wells were either left uncoated or coated with 250 pg / mL Caf1-WT polymer coating solution. The cell suspension was left to form spheroids for three days and visualised via brightfield microscopy.

[0243] In the uncoated non-TC plastic, some HCT116 cells have adhered to the surface in colonies. The well coated with Caf1-WT, however, has facilitated the formation of large cancer spheroids. No cells were seen to be attached to the surface of the well. See Figure 7.

[0244] Without wishing to be bound by a specific theory, the data provided herein suggests that Caf1-WT polymers affect the rheology of hydrogels, such as alginate hydrogels used herein. Alterations in alginate stiffness and stress relaxation have been shown to improve cell growth. The Caf1-WT polymers may also open up the gels and provide more space. This is supported by the presence of cell clusters in the CaflWT-alginate gels indicating that cells can migrate and associate more easily. This unexpected property of Caf1-WT polymers is of utility since it increases cell growth rates and yields in a range of 3D materials already used for cell culture in research and industry.

[0245] Example 7: method for identifying Caf1-WT polymer variants in accordance with the invention The wild type Yersinia pestis Caf1 monomer sequence is shown in SEQ ID NO:1. However, it is known that amino acid sequence variations occur in natural populations (Kopylov et al., PLoS One. 2016 Sep 8;11 (9):e0162308) and from laboratory research. These variants can retain the non-adherent and anti-phagocytic properties of the Caf1-WT sequence. This WT behaviour can be tested as shown by Roque et al (Roque et al., Adv. Mater. 2014;26:2704- 2709) by determining the ability of cells to adhere to Caf1 coated surfaces. In this example the WT behaviour is demonstrated by very low numbers of adhering fibroblast cells compared to positive control surfaces. This is referred to as Caf1-WT activity herein.

[0246] The method is as follows:

[0247] Cells (3T3 fibroblasts) were harvested using 0.05% trypsin- EDTA (1x) solution containing phenol red for detachment and centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in PBS pH 7.4 without calcium and magnesium and centrifuged at 1000 rpm for 5 min. This wash step was repeated once again in order to remove completely the serum. Cell suspension was diluted in cell culture medium to a final concentration of 5x105cells ml- 1. Glass round coverslips (12 mm diameter) were washed with 1% Hellmanex for 10 min and rinsed three times in water. The coverslips were placed into 4 well-plates (Nunc) and 500 pl of each protein solution (50 ug / ml) was applied to the corresponding well of the plate. The plates were placed on a rocking platform at 4°C, overnight. The solution was aspirated and 200 ul cells were added into the wells of the plates. After 24 h incubation, the medium was aspirated and the cell culture carefully washed once with PBS pH 7.4. The samples were fixed in 2% glutaraldehyde in PBS pH 7.4 at room temperature for 30 min. After fixation, the fixative was aspirated into a waste container for disposal. The samples were washed twice for 2 min in 1 ml PBS pH 7.4 to remove excess fixative, and the waste liquid was discarded. For the dehydration process, 1 ml of 25% ethanol was added into each well and incubated for 15 min at room temperature. The ethanol was aspirated and discarded. The last step was repeated using increasing concentrations of ethanol: 50 %, 75 %, 100 % and finally absolute ethanol. Once in absolute ethanol, the drying process was conducted using a Baltec Critical Point dryer. The sample was mounted on an aluminium stub with carbon discs. The samples were coated with 15 nm of gold using a Polaron SEM coating unit (Electron Microscopy Research Services). The samples were then ready for imaging using a conventional Cambridge Stereoscan 360 scanning electron microscope (SEM). The numbers of cells adhering to CaflWT surfaces was significantly less than the number that attached to untreated glass and the surfaces coated with fibronectin or Caf1-RDGS under the same conditions. See Roque et al (Roque et al., Adv. Mater. 2014;26:2704-2709) . Alternatively instead of drying, gold coating and SEM the fixed cells may be visualised using fluorescence microscopy by staining actin with selective fluorescent antibodies or Rhodamine-phalloidin. Also individual nuclei may be visualised and counted after DNA staining using DAPI, or 4' ,6-diamidino-2-phenylindol. Methods for these are well known in the field as are alternatives to glutaraldehyde fixing procedures such as those using formaldehyde. Thus, suitable procedures will enable accurate counting of numbers of adhered cells on identical surfaces coated with different proteins. In each case suitable controls will be used to compare the numbers bound and, in most cases, the positive control will be Caf1-RGDS and the negative control will be Caf1-WT. Positive Caf1 controls for cell lines other than fibroblasts may require adherent properties specific to those cells such as peptide motifs from laminin etc.

[0248] In each case a test of significance such as is used in Figure 2 of Roque et al (Roque et al., Adv. Mater. 2014;26:2704-2709 will establish whether the Caf1 used retains the WT ability to significantly reduce the number of adherent cells compared to control surfaces.

[0249] Example 8: HEK293 suspension cultures grown in the presence of Caf1-WT polymer are transfectable

[0250] HEK293 cells were grown in single cell suspension in Freestyle F17 media supplemented with 250pg / mL Caf1-WT. Half of the cell population were transfected with the pEGFP-C2 plasmid encoding GFP protein for 24 hours and grown for a further 24 hours. The other half were left untransfected and grown for 48 hours.

[0251] 6pl of FuGENE® 6 Transfection Reagent was added to a sterile tube containing 100pl OptiMEM transfection media, mixed and left to incubate at room temperature for 5 minutes. 1 pg of pEGFP-C2 plasmid DNA and was added to the mixture and incubated for a further 10 minutes. 50pL of the transfection mixture was added to wells of a 24-well non tissue culture treated multiwell plate, containing a single cell HEK293 culture (0.75x104cells in 400pL Freestyle F17 media). 100pL of OptiMEM media only was added to untransfected control wells. Cells were grown for a further 48 hours. Cell suspensions were agitated and 100pL samples transferred to a black walled 96-well plate. Transfection efficiency was measured by detecting fluorescent cells using an Incucyte S7 (phase and green channels) at 10X magnification. See Figure 8.

[0252] Example 9: WT Caf1 significantly improves 3D cell culture in alginate

[0253] Materials were prepared as in Example 1 by mixing WT Caf1 with alginate and crosslinking the gel with calcium. Using the optimum concentration of WT Caf1 (0.2% w / v) determined in Example 1 , this experiment confirms that Caf1-WT polymer can enhance cell viability of the 3D cell culture over an extended period. HEK293 cells were grown in 1% alginate mixed with 0.2% Caf1-WT polymer. During initial seeding, 1 x104cells / well were encapsulated in the gel with standard media conditions used (DM EM supplemented with either 2% or 10% FBS and 1% L-glutamine). The luminescent intensity of the Presto Blue assay relates to the number of viable cells present in the well and each data point is the result of 3 independent repeats of the experiment, From day 2, supplementation with Caf1-WT polymer into alginate was beneficial to growth of the HEK293 cells, indicated by increased numbers of viable cells.

[0254] Method

[0255] The alginate hydrogels was prepared as above and contained either just 1% alginate or 1% alginate and 0.2% Caf1. It was used as follows

[0256] 1 . Mix 75 pl hydrogel solution with 25 pl of cell solution containing 1 x104cells

[0257] 2. Add 75 pl of calcium solution to a 96 well plate well

[0258] 3. Add 100 pl hydrogel / cell solution into the calcium solution

[0259] 4. Set for 30 min at 37°C in incubator.

[0260] 5. Add cell culture medium onto surface (max volume 75 pl).

[0261] Results

[0262] • Cells in alginate + WT Caf1 showed significantly higher viability than in alginate alone.

[0263] • This was observed from Day 2 to 12

[0264] See Figure 9.

[0265] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0266] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0267] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0268] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0269] Sequences

[0270] SEQ ID NO :1 Wildtype Caf1 monomer (including signal sequence)

[0271] MKKISSVIAIALFGTIATANAADLTASTTATATLVEPARITLTYKEGAPITIMDNGNIDTELLVG

[0272] TLTLGGYKTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNG

[0273] ENLVGDDWLATGSQDFFVRSIGSKGGKLAAGKYTDAVTVTVSNQ

[0274] SEQ ID NO:2 Wildtype Caf1 monomer signal sequence

[0275] MKKISSVIAIALFGTIATANA

[0276] SEQ ID NO:3 Wildtype Caf1 monomer (without signal sequence)

[0277] ADLTASTTATATLVEPARITLTYKEGAPITIMDNGNIDTELLVGTLTLGGYKTGTTSTSVNFT

[0278] DAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDWLATGSQDFF

[0279] VRSIGSKGGKLAAGKYTDAVTVTVSNQ

[0280] SEQ ID NO: 4

[0281] RSRKYSS-WYVALKR

[0282] SEQ ID NO: 5 cell adhesion recognition motif derived from fibronectin

[0283] RGDS

[0284] SEQ ID NO: 6 cell adhesion recognition motif derived from fibronectin

[0285] PHSRN

[0286] SEQ ID NO: 7 cell adhesion recognition motif derived from Collagen I

[0287] GTPGPQGIAGQRGVV

[0288] SEQ ID NO: 8 cell adhesion recognition motif derived from Collagen IV

[0289] MNYYSNS

[0290] SEQ ID NO: 9 cell adhesion recognition motif derived from Laminin

[0291] YIGSR

[0292] SEQ ID NO: 10 cell adhesion recognition motif derived from Laminin

[0293] IKVAV SEQ ID NO: 11 cell adhesion recognition motif derived from Fibronectin FHRRIKA

[0294] SEQ ID NO: 12 cell adhesion recognition motif derived from Fibronectin

[0295] LDVP

[0296] SEQ ID NO: 13 cell adhesion recognition motif derived from Fibronectin

[0297] IDAP

[0298] SEQ ID NO: 14

[0299] KIPKASSVPTELSAISTLYL

[0300] NUMBERED PARAGRAPHS OF THE INVENTION

[0301] The invention will now be described with the following numbered paragraphs.

[0302] Para 1 . A 3D cell culture method comprising:

[0303] (i) culturing cells on or in a hydrogel wherein the hydrogel comprises a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof, wherein the variant has Caf1-WT activity.

[0304] Para 2. Use of a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof to increase cell proliferation, cell migration and / or cell aggregation, wherein the variant has Caf1-WT activity.

[0305] Para 3. Use of Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof to promote the formation of a 3D cell structure, wherein the variant has Caf1-WT activity, optionally wherein the 3D cell structure is selected from the group consisting of: a spheroid, an organoid or cultivated meat, optionally wherein the organoid is an endothelial tube structure or a ciliated 3D cell structure.

[0306] Para 4. Use of Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof to promote suspension cell culture of cells that are normally adherent, wherein the variant has Caf1-WT activity. Para 5. Use of a hydrogel comprising a Caf1 polymer selected from a wildtype Caf1 (Caf1- WT) polymer or a variant thereof as a 3D cell support scaffold, wherein the variant has Caf1- WT activity.

[0307] Para 6. The method or use according to any one of paragraphs 1 to 3 or paragraph 5, wherein the Caf1 polymer is comprised in a hydrogel, and wherein the hydrogel comprises at least about 0.025 % (w / v) of the Caf1 polymer.

[0308] Para 7. The method or use according to paragraph 6, wherein the Caf1 polymer is present in the hydrogel at a concentration of from about 0.025 % (w / v) to about 0.4% (w / v).

[0309] Para 8. The method or use according to paragraph 7, wherein the Caf1 polymer is present in the hydrogel at a concentration of about 0.2 % (w / v).

[0310] Para 9. The method or use according to any one of paragraphs 1 , 5, 6, 7 or 8, wherein the hydrogel comprises a gel-forming polymer.

[0311] Para 10. The method or use according to paragraph 9, wherein the gel-forming polymer is an alginate or a basement membrane extract.

[0312] Para 11. The method or use according to paragraph 10, wherein the hydrogel is a Matrigel® hydrogel.

[0313] Para 12. The method or use according to paragraph 10, wherein the hydrogel is an alginate hydrogel, optionally wherein the alginate is present in the hydrogel at a concentration of from about 0.5 % (w / v) to about 10% (w / v), further optionally wherein the alginate is present in the hydrogel at a concentration of about 1% (w / v).

[0314] Para 13. The method or use according to any one of paragraphs 9 to 12, wherein the gel forming polymer and the Caf1 polymer are present in the hydrogel in a ratio of between about 5:2 and about 40: 1.

[0315] Para 14. The method or use according to paragraph 13, wherein the gel forming polymer and the Caf1 polymer are present in the hydrogel in a ratio of about 5:1.

[0316] Para 15. The method or use according to paragraph 13 or 14, wherein the gel forming polymer is an alginate or a basement membrane extract, optionally wherein the gel forming polymer is an alginate.

[0317] Para 16. The method or use according to paragraph 1 , or any one of paragraphs 5 to 15, wherein the cells are on or attached to the hydrogel.

[0318] Para 17. The method or use according to paragraph 1 , or any one of paragraphs 5 to 16, wherein the cells are entrapped or encapsulated in the hydrogel. Para 18. The method or use according to any one of the preceding paragraphs, wherein the cells are mammalian cells.

[0319] Para 19. The method or use according to any one of the preceding paragraphs, wherein the cells are myosatellite cells, induced pluripotent and embryonic stem cells, adipose-derived cells, hepatocytes, chondrocytes (such as MSC-derived chondrocytes), osteoblasts (such as MSC-derived osteoblasts), cancer cells, neurons, cardiomyocytes, keratinocytes, fibroblasts, epithelial cells, endothelial cells, macrophages, lymphoblasts, mesenchymal stem cells (hMSCs), embryonic cells, cell lines; or a combination thereof.

[0320] Para 20. The method according to any one of the preceding paragraphs, wherein the cells in (i) are cultured on or in the hydrogel for at least 24 hours.

[0321] Para 21. The method according to any one of the preceding paragraphs, wherein the method is for producing a 3D cell structure, optionally wherein the 3D cell structure is selected from the group consisting of: a spheroid, an organoid and cultivated meat; optionally wherein the organoid is an endothelial tube structure or a ciliated 3D cell structure.

[0322] References

[0323] Baird et al. Proc. Natl. Acad. Sci. U. S. A. 1988, 85 (7), 2324-8.

[0324] Andree, B., Ichanti, H., Kalies, S. et al. Formation of three-dimensional tubular endothelial cell networks under defined serum-free cell culture conditions in human collagen hydrogels. Sci Rep 9, 5437 (2019). https: / / doi.org / 10.1038 / s41598-019-41985-6

[0325] Caliari, S., Burdick, J. A practical guide to hydrogels for cell culture. Nat Methods 13, 405- 414 (2016)

[0326] Hoesli CA, Kiang RLJ, Raghuram K, Pedroza RG, Markwick KE, Colantuoni AMR, Piret JM. Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel. J Vis Exp. 2017 Jun 29;(124):55280.

[0327] Zavialov AV. Structure and Biogenesis of the Capsular F1 Antigen from Yersinia pestis: Preserved Folding Energy Drives Fiber Formation. Cell. 2003; 113(5): 587-96

[0328] Ana I Roque , Andrei Soliakov, Mark A Birch, Sion R Philips, Deepan S H Shah, Jeremy H Lakey. Reversible non-stick behaviour of a bacterial protein polymer provides a tuneable molecular mimic for cell and tissue engineering. Adv Mater 2014 May;26(17):2704-9, 2616. Julie Miller, E. Diane Williamson, Jeremy H. Lakey, Martin J. Pearce, Steven M. Jones, Richard W Titball. Macromolecular organisation of recombinant Yersinia pestis F1 antigen and the effect of structure on immunogenicity. FEMS Immunology & Medical Microbiology, Volume 21 , Issue 3, July 1998, Pages 213-221 , Daniel T Peters , Antonio Reifs, Alvaro Alonso-Caballero, Azzeldin Madkour, Helen Waller, Brendan Kenny, Raul Perez-Jimenez, Jeremy H Lakey Unraveling the molecular determinants of the anti-phagocytic protein cloak of plague bacteria PLoS Pathog. 2022 Mar 31 ;18(3):e1010447. Antonino Passaniti, Hynda K Kleinman, George R Martin Matrigel: history / background, uses, and future applications. J Cell Commun Signal. 2021 Aug 31 ;16(4):621-626.

[0329] Kuen Yong Lee, David J Mooney. Alginate: properties and biomedical applications. Prog Polym Sci. 2012 Jan;37(1):106-126.

[0330] Pavel Kh Kopylov, Mikhail E Platonov, Vitaly G Ablamunits, Tat’yana I Kombarova, Sergey A Ivanov, Lidiya A Kadnikova, Aleksey N Somov, Svetlana V Dentovskaya, Vladimir N Uversky,

[0331] Andrey P Anisimov. Yersinia pestis Caf1 Protein: Effect of Sequence Polymorphism on Intrinsic Disorder Propensity, Serological Cross-Reactivity and Cross-Protectivity of Isoforms. PLoS One. 2016 Sep 8;11(9):e0162308

[0332] Roque A. I. SoliakovA. Birch M. A. Philips S. R. Shah D. S. Lakey J. H. Adv. Mater. 2014;26:2704-2709.

Claims

1. Claims1 . Use of a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof to increase cell viability during cell culture, wherein the variant has Caf1-WT activity.

2. The use of claim 1 , wherein the Caf1-WT polymer or variant thereof is used to promote suspension cell culture of cells that are normally adherent.

3. The use of claim 1 or claim 2, wherein the Caf1-WT polymer or variant thereof increases cell proliferation and / or cell migration.

4. The use of claim 1 , wherein the Caf1-WT polymer or variant thereof is used to promote the formation of a 3D cell structure during cell culture.

5. The use of claim 4, wherein the 3D cell structure is selected from the group consisting of: a spheroid, an organoid or cultivated meat, optionally wherein the organoid is an endothelial tube structure or a ciliated 3D cell structure.

6. Use of a hydrogel comprising a gel-forming polymer and a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof as a 3D cell support scaffold during cell culture, wherein the variant has Caf1-WT activity, and wherein the Caf1-WT polymer or variant thereof increases cell viability during the cell culture.

7. The use of any one of claims 4 to 6, wherein the Caf1-WT polymer or variant thereof increases cell proliferation, cell migration and / or cell clustering.

8. A method of suspension cell culture of cells that are normally adherent comprising: (i) culturing the cells in a cell culture medium in the presence of a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof, wherein the variant has Caf1- WT activity, and wherein the Caf1-WT polymer or variant thereof increases cell viability during the cell culture.

9. A 3D cell culture method comprising:(i) culturing cells on or in a hydrogel wherein the hydrogel comprises a gel-forming polymer and a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof, wherein the variant has Caf1-WT activity, and wherein the Caf1-WT polymer or variant thereof increases cell viability during cell culture.

10. The method of claims 8 or 9, wherein the method further comprises (ii) transfecting the cells in the presence of the Caf1 polymer.11 . A cell culture method comprising:(i) culturing cells in a cell culture medium in the presence of a Caf1 polymer selected from a wildtype Caf1 (Caf1-WT) polymer or a variant thereof, wherein the variant has Caf1-WT activity, and wherein the Caf1-WT polymer or variant thereof increases cell viability during the cell culture; and(ii) transfecting the cells in the presence of the Caf1-WT polymer or variant thereof.

12. The use of claim 1 or any one of claims 3 to 7; or the method of claims 9 to 11 , wherein the Caf1 polymer is comprised in a hydrogel, and wherein the hydrogel comprises a gel-forming polymer and at least about 0.025 % (w / v) of the Caf1 polymer.

13. The use or method of claim 12, wherein the Caf1 polymer is present in the hydrogel at a concentration of from about 0.025 % (w / v) to about 0.4% (w / v).

14. The use or method of claim 13, wherein the Caf1 polymer is present in the hydrogel at a concentration of about 0.2 % (w / v).

15. The use of claim 6; or the method of claim 9; or the use or method of any one of claims 12 to 14, wherein the gel-forming polymer is an alginate or a basement membrane extract.

16. The use of claim 6; or the method of claim 9; or the use or method of any one of claims 12 to 15, wherein the gel-forming polymer is Matrigel®.

17. The use of claim 6; or the method of claim 9; or the use or method of any one of claims 12 to 15, wherein the gel-forming polymer is an alginate, optionally wherein the alginate is present in the hydrogel at a concentration of from about 0.5 % (w / v) to about 10% (w / v), further optionally wherein the alginate is present in the hydrogel at a concentration of about 1% (w / v).

18. The use of claim 6; or the method of claim 9; or the use or method of any one of claims 12 to 17, wherein the gel-forming polymer and the Caf1 polymer are present in the hydrogel in a ratio of between about 5:2 and about 40:1.

19. The use or method of claim 18, wherein the gel-forming polymer and the Caf1 polymer are present in the hydrogel in a ratio of about 5:1.

20. The use or method of claim 18 or 19, wherein the gel-forming polymer is an alginate or a basement membrane extract, optionally wherein the gel-forming polymer is an alginate.

21. The use of claim 6; or the method of claim 9; or the use or method of any one of claims 12 to 20, wherein the cells are on or attached to the hydrogel.

22. The use of claim 6; or the method of claim 9; or the use or method of any one of claims 12 to 21 , wherein the cells are entrapped or encapsulated in the hydrogel.

23. The method of claim 9; or the method of any one of claims 12 to 22, wherein the cells in are cultured on or in the hydrogel for at least 24 hours.

24. The use or method of any one of the preceding claims, wherein the cells are mammalian cells.

25. The use or method of any one of the preceding claims, wherein the cells are myosatellite cells, induced pluripotent and embryonic stem cells, adipose-derived cells, hepatocytes, chondrocytes (such as MSC-derived chondrocytes), osteoblasts (such as MSC-derived osteoblasts), cancer cells, neurons, cardiomyocytes, keratinocytes, fibroblasts, epithelial cells, endothelial cells, macrophages, lymphoblasts, mesenchymal stem cells (hMSCs), embryonic cells, cell lines; or a combination thereof.

26. The method of any one of the preceding claims, wherein the method is for producing a 3D cell structure, optionally wherein the 3D cell structure is selected from the group consisting of: a spheroid, an organoid and cultivated meat; optionally wherein the organoid is an endothelial tube structure or a ciliated 3D cell structure.

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