FIELD OF INVENTION

The present invention relates to a method of producing supramolecular structures comprising a plurality of fused fibrils wherein each fibril comprises a plurality of cell adhesion motif lipopeptides. The invention also relates to supramolecular structures obtainable by the method of the invention.

BACKGROUND

There are a number of established and emerging commercial markets exploiting scientific advances in growing living cells to manufacture new products, including pharmaceutical drugs, stem cells, gene therapies or even cell-based meat. The goal of these products is to create an offering suitable to humanity’s future needs. Such products may be more sustainable and address issues associated with scarceness of food, global warming, or access to medical treatments.

Many of these applications rely on the use of adherent cells. However, currently there are a number of challenges hampering the development of these fields. These challenges include use of animal-derived components (such as serum), which are associated with a risk of contamination, high costs and batch-to-batch variability. Use of animal products is also not compliant with Good Manufacturing Practice and often ethically controversial. Whilst serum- free media alternatives exist, these are expensive and not available for all cell types.

Furthermore, the current methods of adhesive cell culture are associated with the limitations of traditional batch cell culture. Current batch bioprocessing techniques require large surface areas and significant amounts of growth media (contributing up to 70% of overall cost), which are both a limitation to scalability. As a result, companies are not able to meet the market demand, reducing the potential of their offering, and overall slowing the development of more sustainable, cheaper, and more environmentally friendly products.

The present invention aims to overcome or partially ameliorate the issues associated with the prior art by providing methods for making products for maintaining, culturing and bioprocessing cells (including adhesive cells) more efficiently and with less potential side effects.

SUMMARY OF INVENTION

The present invention is based on the inventors’ development of a new method for producing supramolecular structures comprising a plurality of fused fibrils, wherein each fibril comprises a plurality of cell adhesion motif lipopeptides. Specifically, the inventors have shown that by dissolving lyophilised lipopeptides in serum-free cell culture medium (SFM) the lipopeptides self-assemble to form fibrils that fuse together to generate a new supramolecular structure that is topologically distinct from lipopeptide structures defined in the art which are generated in water. The inventors believe that this new structure is formed as a result of the ionic strength of the solvent in which the lipopeptides self-assemble. The inventors hypothesise that the increased ionic strength of the solvent (compared to water) generates electrostatic attraction between the lipopeptides which changes how the lipopeptides assemble. Accordingly, whilst the method of the invention has been exemplified using SFM, it will be appreciated that the inventive concept also applies to other solvents having an ionic strength that is greater than the ionic strength of water.

Further, the inventors have demonstrated that this new supramolecular structure may be formed by self-assembly of a variety of different lipopeptides. Specifically, the inventors have demonstrated this through the use of three lipopeptides that comprise a cell adhesion motif such as RGD (SEQ ID NO: 1), KTTKS (SEQ ID NO: 2) or YEALRVANEVTLN (SEQ ID NO: 3).

The inventors have found that the supramolecular structures generated herein using solvents with high ionic strength have a higher fibril density than those generated in the art using water. It will therefore be appreciated that the methods provided herein generate supramolecular structures with a higher density of cell adhesion motifs, which may be advantageous.

Surprisingly, the inventors have also demonstrated that the denser supramolecular structures made in accordance with the method of the invention increase cell growth and/or cell collagen production. The inventors believe that the supramolecular structures described herein have a different bioactivity as compared to supramolecular structures self-assembled in water and can better mimic the function of the endogenous biomolecules from which part or all of the amino acid portion of the lipopeptide is obtained from.

In one aspect, provided herein is a method of producing a supramolecular structure comprising a plurality of fused fibrils wherein each fibril comprises a plurality of cell adhesion motif lipopeptides, the method comprising dissolving the plurality of cell adhesion motif lipopeptides in a solvent having an ionic strength that is greater than the ionic strength of distilled water to produce the supramolecular structure.

In an embodiment, the solvent has an ionic strength of at least 100 mM. In an embodiment, the fibrils are nanotapes.

In an embodiment, the plurality of cell adhesion motif lipopeptides are lyophilised lipopeptides prior to dissolving in the solvent.

In an embodiment, the dissolving comprises a step of mixing the plurality of cell adhesion motif lipopeptides in the solvent to obtain a substantially transparent solution.

In an embodiment, the solvent is serum-free.

In an embodiment, the solvent is a cell culture medium.

In an embodiment, the solvent is Dulbecco’s Modified Eagle Medium (DMEM), Ham’s F12, Leibovitz's L-15 medium, RPMI-1640, Mesencult™ Basal Medium, or DMEM-F12.

In an embodiment, the plurality of cell adhesion motif lipopeptides are C16-GGG-RGDS (SEQ ID NO: 4).

In an embodiment, the cell adhesion motif is an extracellular matrix protein sequence or a fragment or a variant thereof.

In an embodiment, the extracellular matrix protein is selected from the group consisting of fibronectin, collagen, lumican, decorin, laminin, vitronectin, fibrinogen, elastin, biglycan, heparin, tenascin and osteopontin.

In an embodiment, the cell adhesion motif is:

a) a fibronectin fragment comprising or consisting of an amino acid sequence selected from RGD (SEQ ID NO: 1), RGDS (SEQ ID NO: 5), PHSRN (SEQ ID NO: 6), LDVP (SEQ ID NO: 7), WQPPRARI (SEQ ID NO: 8), IGD (SEQ ID NO: 9), REDV (SEQ ID NO: 10), and IDAP (SEQ ID NO: 11) or a variant thereof;

b) a collagen fragment comprising or consisting of an amino acid sequence selected from KTTKS (SEQ ID NO:2), GTPGPQGIAGQRGW (SEQ ID NO: 12), GROGER (SEQ ID NO: 13), GLKGEN (SEQ ID NO: 14), GFOGER (SEQ ID NO: 15), and MNYYSNS (SEQ ID NO: 16) or a variant thereof; or

c) a lumican fragment comprising or consisting of an amino acid sequence selected from EVTLN (SEQ ID NO: 17), ELDLSYNKLK (SEQ ID NO: 18) and YEALRVANEVTLN (SEQ ID NO: 3); or d) a laminin fragment comprising or consisting of an amino acid sequence selected from the YIGSR (SEQ ID NO: 19), IKVAV (SEQ ID NO: 20), CCRRIKVAVWLC (SEQ ID NO: 21) and RGD.

In an embodiment, the plurality of cell adhesion motif lipopeptides comprise at least two different cell adhesion motif lipopeptides.

In an embodiment, the at least two different cell adhesion motif lipopeptides are:

a) a cell adhesion motif lipopeptide comprising or consisting of KTTKS (SEQ ID NO: 2); and b) a cell adhesion motif lipopeptide comprising or consisting of YEALRVANEVTLN (SEQ ID NO: 3).

In an embodiment, the lipopeptide comprises a lipid portion comprising a carbon chain of 6 to 24 carbon atoms.

In one aspect, provided herein is a supramolecular structure comprising a plurality of fused fibrils wherein each fibril comprises a plurality of cell adhesion motif lipopeptides, obtainable by the method described herein.

Except for where the context requires otherwise, the considerations set out in this disclosure should be considered to be applicable to the structure, aqueous medium and surface in accordance with the invention, and the uses thereof.

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.

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.

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

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 shows topographical characterisation of the single-type RGDS lipopeptide self- assembled either in water (left) or serum-free medium (SFM; right) by forward deflection scan analysis performed by atomic force microscopy (AFM) of RGDS lipopeptide. Scale bars: 1 pm. Insert size: 1 pm x 1 pm. The figure highlights the difference in the self-assembled structure derived from lipopeptide solubilisation in water versus SFM (the latter comprising fused fibrils as described herein).

Figure 2 shows the topographical characterisation of single-type and composite lipopeptide systems self-assembled in serum-free media (SFM; top) or water (bottom). Forward deflection scans performed by atomic force microscopy (AFM) of RGDS, Lumican, Matrixyl and Lumican:Matrixyl lipopeptides (greyscale = 500 and 100 nm, respectively). Top images scale bars: 8 and 2 pm for the image and for the inset, respectively. Bottom images scale bars: 1 and 0.5 pm for the inset, respectively.

Figure 3 shows results of cellular biocompatibility and bioactivity assays performed on human stromal progenitor cells cultured with lipopeptides self-assembled in serum free media (SFM) or water. The graphs show human stromal progenitor cell proliferation at day 3 and 7 when using Lumican (A), Matrixyl (C) and RGDS lipopeptides (E) either solubilised in distilled water (H2O) or in SFM at different concentrations and the amount of collagen deposited by human stromal progenitor cells after 7 days of culture with Lumican (B), Matrixyl (D) and RGDS lipopeptides (F) previously solubilised either in water on in SFM. Mean ± S.D., n = 3 for all experiments; *, **, *** and **** referred to statistically significant differences compared to the control (0 SFM) and corresponded to p < 0.05, 0.01 , 0.001 and 0.0001 , respectively.

Figure 4 shows the effect the cell culture medium has on the proliferation of RGDS- supplemented human adipose-derived mesenchymal stem cells (hASCs). Proliferation of hASCs at day 3 and 7 in the presence of RGDS lipopeptide solubilised either in SFM or in distilled water (H2O) at 50 mM. Mean ± S.D., n = 3 for all experiments; **** referred to statistically significant differences compared to control in SFM and corresponded to p < 0.0001. Figure 5 shows the effect the cell culture medium has on the proliferation of Lumican or Matrixyl-supplemented hASCs. (A) Graph reporting hASCs proliferation at day 3 and 7 in presence of Lumican or Matrixyl lipopeptides solubilised either in serum-free medium (SFM) or in distilled water (H2O) at 50 mM using Alamar Blue assay. Graphs reporting the total amount of collagen deposited by hASCs after 7 days in culture with Lumican (B) or Matrixyl lipopeptides (C) solubilised either in SFM or in H2O at 50 or 25 mM. Mean ± S.D., n = 3 for all experiments.

Figure 6 shows how the cell culture medium in which the lipopeptide is self-assembled affects the biocompatibility and bioactivity on C2C12 cells. (A) Graph showing C2C12 proliferation at day 3 and 7 when using Lumican lipopeptide either solubilised in distilled water (H2O) or in serum-free medium (SFM) at different concentrations. (B) Graph showing the amount of collagen deposited by C2C12 after 7 days of culture with Lumican lipopeptide previously solubilised either in H2O on in SFM. Mean ± S.D., n = 1 for all experiments; *, **, *** and **** referred to statistically significant differences corresponded to p < 0.05, 0.01 , 0.001 and 0.0001 , respectively.

Figure 7 shows how the cell culture medium in which the lipopeptide is self-assembled affects the biocompatibility and bioactivity on C2C12 cells. (A) Graph showing C2C12 proliferation at day 3 and 7 when using Matrixyl lipopeptide either solubilised in distilled water (H2O) or in serum-free medium (SFM) at different concentrations. (B) Graph showing the amount of collagen deposited by C2C12 after 7 days of culture with Matrixyl lipopeptide previously solubilised either in H2O on in SFM. Mean ± S.D., n = 1 for all experiments; ; *, **, *** and **** referred to statistically significant differences corresponded to p < 0.05, 0.01 , 0.001 and 0.0001 , respectively.

Figure 8 shows how the cell culture medium in which the lipopeptide is self-assembled affects the biocompatibility and bioactivity on C2C12 cells. (A) Graph showing C2C12 proliferation at day 3 and 7 when using RGDS lipopeptide either solubilised in distilled water (H2O) or in serum-free medium (SFM) at different concentrations. (B) Graph showing the amount of collagen deposited by C2C12 after 7 days of culture with RGDS lipopeptide previously solubilised either in H2O on in SFM. Mean ± S.D., n = 1 for all experiments; ; *, **, *** and **** referred to statistically significant differences corresponded to p < 0.05, 0.01 , 0.001 and 0.0001 , respectively.

Figure 9 shows the topographical characterisation of the single-type RGDS lipopeptide self- assembled in (A) Leibovitz’s L-15 medium, (B) RPMI 1640 medium, (C) DMEM-F12 medium, (D) Mesencult™ Basal Medium; (E) water control. It can be seen that the lipopeptides assembled in cell culture media form a supramolecular structure comprising a plurality of fused fibrils. The scale bars correspond to 2 pm.

DETAILED DESCRIPTION

Provided herein is a method of producing a supramolecular structure comprising a plurality of fused fibrils. A supramolecular structure comprising a plurality of fused fibrils obtainable by a method described herein is also provided herein.

The terms“supramolecular structure”“fused fibrillar structure” or“the structure” are used herein interchangeably and refer to an aggregate composed of a plurality of fibrils. The fibrils are fused together within the supramolecular structure. Each fibril comprises a plurality of cell adhesion motif lipopeptides. The supramolecular structure may also be described as an arrangement of fibrils in which the fibrils are fused together.

It will be appreciated that fused fibrils can be identified using cryo-transmission electron microscopy (cryo-TEM), AFM, or small-angle X-ray scattering. Details of appropriate methods are provided elsewhere herein.

As used herein, the term“fibril” refers to a fibre-like structure made up of lipopeptides. The fibre-like structure (i.e. the fibril) may be a nanofiber, filament, tape, tube, twisted fibre, twisted filament, twisted tape, twisted tube, or network, or combination thereof. The structural characteristics of a fibril are well known in the art (see for example the following reviews by I. W. Hamley (Soft Matter, 2011 , 7: 4122) and Stupp et al. (Faraday Discussions, 2013, 166: 9- 30)).

Typically, a fibril may be in the region of about 40-290 nm wide and/or about 150-2500 nm long. The structure may be made up of uniformly and/or non-uniformly shaped fibrils. Within the structure, the fibrils may be of substantially the same size or of different size.

A plurality of fibrils present in the structure are fused together. For example, at least two, three, four, five, six, seven, eight, nine, ten, or more fibrils may be fused together to form the structure.

The fused fibrillar structure may form dense globular deposits. The globular deposits may have a diameter of at least 200 nm. For example, the globular deposits may have a diameter of at least 300, at least 400, at least 500, at least 600, at least 700, at least 800 etc nm. In one example, they have a diameter of from about 200 to about 800 nm wide.

The supramolecular structures generated herein using solvents with high ionic strength have a higher fibril density than those generated in the art using the same lipopeptides with water. It will be appreciated that density can be determined using cryo-transmission electron microscopy (cryo-TEM) or AFM, by analysing the total area occupied by structures formed in different conditions. Details of other appropriate methods are also well known in the art.

In one example, the supramolecular structures generated herein using solvents with high ionic strength have a density of fibrils that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50 % higher than the density of fibrils in a supramolecular structure generated using the same lipopeptides with water. For example, the supramolecular structures generated herein may have a density of fibrils that is at least 40% higher than the density of fibrils in a supramolecular structure generated using the same lipopeptides with water.

As will be appreciated by a person of skill in the art, in the context of this specification, when a comparison is made to a supramolecular structure generated using water as the solvent, the water that is meant is distilled water. Accordingly, any reference to“water” solvents herein refer to distilled water.

In one example, the fibril is a nanotape. In other words, in this example, the supramolecular structure comprises a plurality of fused nanotapes.

Typically, the nanotape may be in the region of about 40-290 nm wide and/or about 150-2500 nm long. The structure may be made up of uniformly and/or non-uniformly shaped nanotapes. Within the structure, the nanotapes may be of substantially the same size or of different size.

A plurality of nanotapes present in the structure are fused together. For example, 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, or more nanotapes may be fused together to form the structure.

The method may comprise dissolving a plurality of lipopeptides in a solvent to obtain a solution. The term“dissolving” refers to incorporating the plurality of lipopeptides into a liquid solvent, so as to form a solution. The terms “dissolving” and “solubilising” (and variants thereof) are used interchangeably herein. The lipopeptides may be cell adhesion motif lipopeptides. The lipopeptides (for example cell adhesion motif lipopeptides) to be dissolved may be lyophilised. It will be appreciated that dissolving of the plurality of lipopeptides may be aided by mixing. Thus, in the context of the present disclosure, the step of dissolving may comprise the step of mixing. Mixing may comprise, for example, vortexing, sonicating, rotating, and/or irrigating the solvent comprising the lipopeptides. The step of mixing may be carried out until the lipopeptides are dissolved in the solvent so as to form a substantially transparent solution. By“substantially transparent” it is meant the lipopeptides have dissolved to the extent that they are no longer visible by eye (e.g. at a distance of 30 cm by a person with 20/20 vision). In other words, a substantially transparent solution is one that is optically clear.

Merely by way of example, the step of mixing may comprise vortexing, sonicating and/or rotating, or a combination thereof (e.g at least two of vortexing, sonicating and rotating, or all three of vortexing, sonicating and rotating).

Vortexing may last, for example, for at least about ten minutes, from about 10 minutes to about 120 minutes, from about 20 minutes to about 60 minutes, or from about 30 minutes to about 45 minutes. Vortexing may be, for example, carried out at a temperature from about 4°C to about 90°C, from about 10°C to about 50°C, or from about 18°C to about 28°C.

Sonicating may last, for example, for at least about 10 minutes, from about 10 minutes to about 60 minutes, from about 20 minutes to about 45 minutes, or for about 30 minutes. Sonicating may be, for example, carried out at a temperature from about 10°C to about 90°C, from about 20°C to about 80°C, from about 30°C to about 70°C, from about 40°C to about 60°C, or from about 50°C to about 55°C.

Rotating may last, for example, for at least about one hour, from about 1 hour to about 48 hours, orfrom about 12 to 24 hours. Rotating may be carried out, for example, at a temperature from about 2°C to about 25°C, from about 4°C to about 15°C, or at about 4°C to about 6°C.

By way of example, the step of mixing may comprise vortexing for 30 to 45 minutes at a temperature from about 18°C to about 28°C, sonicating for 30 minutes at 55°C, and/or rotating for about 10 hours at 4°C. The step of mixing may comprise vortexing for 30 to 45 minutes at a temperature from about 18°C to about 28°C. The step of mixing may comprise vortexing for 30 to 45 minutes at a temperature from about 18°C to about 28°C and sonicating for 30 minutes at 55°C. The step of mixing may comprise vortexing for 30 to 45 minutes at a temperature from about 18°C to about 28°C, sonicating for 30 minutes at 55°C and rotating for about 10 hours at 4°C. It will be appreciated that the step of mixing may be repeated until the solution is substantially transparent. It will be also appreciated that the step of mixing may be influenced by the desired concentration of lipopeptides in the solution.

Merely by way of example, the concentration of lipopeptides in the solution may be from about 0.5 mM to about 2 mM, or from about 1 mM to about 1.75 mM. For example, the concentration of lipopeptides in the solution may be from about 1.25 mM to about 1.55 mM.

A solvent is any liquid substance. The high-ionic strength solvent has an ionic strength that is greater than distilled water. For example the solvent has an ionic strength of at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM, at least 160 mM, at least 170 mM, at least 180 mM, at least 190 mM, at least 200 mM. For example, the solvent has an ionic strength from about 100 mM to about 200 mM. For example, the solvent has an ionic strength from about 125 mM to about 175 mM. For example, the solvent has an ionic strength from about 150 mM to about 170 mM.

The solvent may be serum-free.

The solvent may be selected from the group consisting of cell culture media, phosphate- buffered saline (PBS) or other saline solutions. For example, the cell culture media, phosphate-buffered saline (PBS) and/or saline solution may be serum free. The use of a serum-free cell culture medium may advantageously remove risks associated with contamination, batch-to-batch variability, as well as reduce cell culture costs, and diminish ethical considerations relating to the use of animal sources.

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 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. 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), Leibovitz's L-15 medium, RPMI-1640, Mesencult™ Basal Medium, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), Ham's F-10, aMinimal 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, 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, STEMPRO® SFM, STEMPRO® 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.

The cell culture medium may be serum-free. For example, the serum-free medium may be DMEM or F-12, or a combination thereof.

The culture medium (for example DMEM, F-12, Leibovitz's L-15 medium, RPMI-1640, Mesencult™ Basal Medium, or a combination thereof (for example DMEM-F12) may further comprise a supplement. A supplement may be for example ascorbic acid, insulin, transferrin and/or sodium selenite.

The ascorbic acid may be in the culture medium at a concentration of, for example, from about 0.1 mM to about 10 mM or from about 0.5 mM to about 5 mM. For example, the ascorbic acid may be at a concentration of about 1 mM. Insulin may be in the culture medium at a concentration of, for example, from about 1 mg/L to about 20 mg/L or from about 5 mg/L to about 15 mg/L. Insulin may be in the culture medium at a concentration of about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20 mg/L, or any range in between. For example, the insulin may be in the culture medium at about 10 mg/L.

Transferrin may be in the culture medium at a concentration of, for example, from about 1 mg/L to about 12 mg/L or from about 3 mg/L to about 10 mg/L. Transferrin may be in the culture medium at a concentration of about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or about 12 mg/L, or any range in between. For example, the transferrin may be in the culture medium at about 5.5 mg/L.

Sodium selenite may be in the culture medium at a concentration of, for example, from about 1 mg/L to about 16 mg/L or from about 4 mg/L to about 10 mg/L. Sodium selenite may be in the culture medium at a concentration of about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, or about 16 pg/L, or any range in between. For example, the sodium selenite may be in the culture medium at about 6.7 pg/L.

The solvent must allow lipopeptides to self-assemble in solution. Advantageously, use of a solvent having an ionic strength greater than water promotes self-assembly of the lipopeptides at a lower concentration than is required for self-assembly of these lipopeptides in water. Further advantageously, using a lower concentration of lipopeptides may be useful in the prevention of lipopeptide gelification.

The inventors have found that the critical aggregation concentration (c.a.c.) value of the lipopeptide may be at least 2.5-fold lower in a solvent having an ionic strength greater than water compared to its c.a.c. value in distilled water (13-18.2 MW/cm ² ). For example, the c.a.c. value of the lipopeptide may be at least 5-fold lower in the solvent compared to its c.a.c. value in distilled water (13-18.2 MW/cm ² ).

The lipopeptide may be dissolved in the solvent at a concentration of from about 15 mM to about 3000 pM. For example, the lipopeptide may be dissolved in the solvent at a concentration from about 50 pM to 2000 pM. For example, the lipopeptide may be dissolved in the solvent at a concentration from about 200 pM to 2000 pM. For example, the lipopeptide may be dissolved in the solvent at a concentration from about 200 pM to 1500 pM. For example, the lipopeptide may be dissolved in the solvent at a concentration from about 500 mM to 1500 mM.

For example, the lipopeptide may be dissolved in the solvent at a concentration from about 15 pM to about 50 pM, from about 50 pM to about 100 pM, from about 100 pM to about 150 pM, from about 150 pM to about 200 pM, from about 200 pM to about 250 pM, from about 250 pM to about 300 pM, from about 300 pM to about 350 pM, from about 350 pM to about 400 pM, from about 400 pM to about 450 pM, from about 450 pM to about 500 pM. For example, the lipopeptide may be dissolved in the solvent at a concentration from about 200 pM to about 300 pM, from about 300 pM to about 400 pM, from about 400 pM to about 500 pM. For example, the lipopeptide may be dissolved in the solvent at a concentration from about 50 pM to about 100 pM, from about 100 pM to about 200 pM, from about 200 pM to about 300 pM, from about 300 pM to about 400 pM, from about 400 pM to about 500 pM, from about 500 pM to about 750 pM, from about 750 pM to about 1000 pM, from about 1000 pM to about 1250 pM, from about 1250 pM to about 1500 pM, from about 1500 pM to about 2000 pM, from about 2000 pM to about 2500 pM, from about 2500 pM to about 3000 pM. For example, the lipopeptide may be dissolved in the solvent at a concentration from about 500 pM to about 750 pM, from about 750 pM to about 1000 pM, from about 1000 pM to about 1250 pM, from about 1250 pM to about 1500 pM, or any range in between.

It will be appreciated that the supramolecular structures of the present disclosure may maintain their topology (i.e, their fused fibril arrangement) even if, after the structure has been formed in a solvent having an ionic strength greater than water, it is placed under conditions wherein the lipopeptide would not self-assemble into such a structure. Therefore, for example, once the structure (i.e, the fused fibril arrangement) has been produced, it may be placed in a different medium (e.g. a medium having a lower ionic strength than the solvent in which the structure self-assembled, such as water) and maintain its topology, even though the lipopeptides would not self-assemble into a structure as described herein under such conditions.

The term “lipopeptide” as used herein refers to an amphiphilic molecule comprising or consisting of a lipid portion and an amino acid portion. The terms“lipopeptide”,“amphiphilic molecule”,“peptide amphiphile” and“PA” are used interchangeably herein. The amphiphilic properties enable a plurality of lipopeptides to self-assemble into the supramolecular structure. Lipopeptides are well known and their self-assembly properties are well characterised in the art (see for example Cui H. et al., Biopolymers, 2010; 94(1): 1-18). Appropriate lipopeptides may therefore easily be identified by a person of skill in the art e.g. by testing their propensity to self-assemble under certain conditions and form supramolecular structures. Lipopeptide self-assembly and corresponding c.a.c. can be evaluated by the Thioflavin (ThT) and pyrene (Pyr) fluorescence spectroscopy methods. Fluorescence spectra are recorded with a Fluorescence Spectrometer. For the ThT assay, the spectra are typically recorded from 460 to 600 nm using an excitation wavelength A _ex = 440 nm and the lipopeptide dissolved in a 4-5 x 10 ^~3 % (w/v) ThT solution. For the Pyr assay, the spectra are typically recorded from 360 to 550 nm using an excitation wavelength A _ex = 339 nm. Pyr assays are performed using a 1-1.5 x 10 ^~5 % (w/v) Pyr solution as a diluent. The Florescence intensity is plotted against a log of the lipopeptide concentration. The inflection point for the data denotes a change of environment for the ThT/Pyr molecule and is used to identify the c.a.c.

A lipopeptide nanostructure can be evaluated by cryo-transmission electron microscopy (cryo- TEM) using a field-emission cryo-electron microscope (e.g. JEOL JEM-3200FSC), AFM, or small-angle X-ray scattering. For cryo-TEM, vitrified specimens are prepared onto holey carbon copper grids with 3.5 pm hole size. A lipopeptide solution is applied to the grid and then vitrified in a 1/1 mixture of liquid ethane and propane at -180 °C. The cryo-electron microscope is operated at -187 °C during the imaging. Lipopeptide solutions are heated from -187 °C to -60 °C at -10-5 Pa, before being imaged at -187 °C. The heating process from -187 to -60 °C, equivalent to a freeze drying process in the microscope, allows for the sublimation of the ice from the sample and removes the vitrified water. Images are taken using bright-field mode and zero-loss energy filtering (omega type) with a slit width 20 eV. Micrographs are recorded using a CCD camera (e.g. Gatan Ultrascan 4000).

The term“plurality”, as used herein, is defined as two or more than two. The two or more lipopeptides in the structure may be the same or they may be different.

The amino acid portion of the lipopeptide may be a natural or synthetic amino acid sequence. A natural amino acid sequence is one that exists in nature and encodes a protein or a fragment thereof. The natural amino acid sequence may encode a human, animal, plant, fungal, Protista, Archaea, and/or bacterial protein or fragment thereof. For example, the fragment may comprise from about 3 to about 40 amino acids, such as from about 3 to about 20, or from about 3 to about 10 amino acids.

The amino acid portion may comprise an extracellular matrix protein sequence (i.e. a sequence of an extracellular matrix protein; also referred to as a motif) that is involved in cell adhesion. The amino acid portion may alternatively comprise fragments or variants of such sequences, where the fragments or variants are also involved in cell adhesion. Such sequences (including fragments and variants thereof) are referred to herein as“cell adhesion motifs”. In other words, as used herein, the term “cell adhesion motif” encompasses extracellular matrix protein motifs, and fragments and variants thereof, which are involved in cell adhesion. The term “cell adhesion motif” therefore encompasses extracellular matrix protein cell adhesion motifs, or fragments or variants thereof, where the fragments or variants are also involved in cell adhesion.

As used herein, the term“cell adhesion motif lipopeptide” therefore describes a lipopeptide that comprises an amino acid portion that comprises or consists of an extracellular matrix protein motif, or a fragment or variant thereof, that is involved in cell adhesion.

As used herein,“involved in cell adhesion” refers to promoting cell adhesion to the lipopeptide, and/or directly adhering or binding to the cell e.g. by binding to cells via cell surface molecules, such as integrins, displayed on the surface of the cells. Cell adhesion motifs are typically capable of adhering to cells directly, for example, by binding to cells via cell surface molecules, such as integrins, displayed on the surface of the cells.

Many extracellular matrix proteins involved in cell adhesion are known in the art. By way of example, an extracellular matrix protein involved in cell adhesion may be selected from the group consisting of fibronectin, collagen (such as types I, II, III and V), lumican, decorin, laminin, vitronectin, fibrinogen, elastin, biglycan, heparin, tenascin and osteopontin. Additionally, the cell adhesion motifs may be any peptide derived from any of the aforementioned proteins, including derivatives or fragments containing the binding domains of the above-described molecules. Example motifs include integrin-binding motifs, such as the RGD (arginine-glycine-aspartate) motif, the YIGSR (tyrosine-isoleucine-glycine-serine- arginine) motif, and related peptides that are functional equivalents. For example, peptides containing RGD sequences (e.g., RGDS) and WQPPRARI sequences are known to direct spreading and migration properties of endothelial cells, and YIGSR peptide has been shown to promote epithelial cell attachment. Whether an amino acid sequence is a cell adhesion motif can be determined by screening peptide libraries for adhesion and selectivity to specific cell types. Cell adhesion motifs may also be developed empirically via Phage display technologies.

The amino acid portion of the lipopeptide may comprise or consist of one or more cell adhesion motifs. For example, the amino acid portion of the lipopeptide may comprise or consist of 2, 3, 4, 5 or more cell adhesion motifs (which may be in tandem or may be spatially separated e.g. by other amino acids or linkers within the amino acid portion of the lipopeptide). In an example where the amino acid portion of the lipopeptide comprises more than one cell adhesion motif, some or all of the motifs may be same. Alternatively, some or all of the motifs may be different.

The cell adhesion motif may be an extracellular matrix protein or a fragment or variant thereof that is involved in cell adhesion (i.e. an extracellular matrix protein that is involved in cell adhesion; a variant of the extracellular matrix protein, wherein the variant is involved in cell adhesion; a fragment of the extracellular matrix protein, wherein the fragment is involved in cell adhesion; or a variant of a fragment of the extracellular matrix protein, wherein the variant of the fragment is involved in cell adhesion).

The cell adhesion motif may be a fibronectin fragment that comprises or consists of an amino acid sequence selected from the group consisting of RGD, RGDS, PHSRN, LDVP, WQPPRARI, IGD, REDV, and IDAP or a variant thereof. The variant may be a conservative amino acid substitution variant e.g. having one, two or three conservative amino acid substitutions compared to an amino acid sequence selected from the group consisting of RGD, RGDS, PHSRN, LDVP, WQPPRARI, IGD, REDV, and IDAP.

Alternatively, the cell adhesion motif may be a collagen fragment that comprises or consists of an amino acid sequence selected from the group consisting of KTTKS, GTPGPQGIAGQRGVV, GROGER, GLKGEN, GFOGER, and MNYYSNS or a variant thereof. The variant may be a conservative amino acid substitution variant e.g. having one, two or three conservative amino acid substitutions compared to an amino acid sequence selected from the group consisting of KTTKS, GTPGPQGIAGQRGVV, GROGER, GLKGEN, GFOGER, and MNYYSNS.

Alternatively, the cell adhesion motif may be a lumican fragment that comprises or consists of an amino acid sequence selected from the group consisting of EVTLN, ELDLSYNKLK and YEALRVANEVTLN or a variant thereof. The variant may be a conservative amino acid substitution variant e.g. having one, two or three conservative amino acid substitutions compared to an amino acid sequence selected from the group consisting of EVTLN, ELDLSYNKLK and YEALRVANEVTLN.

Alternatively, the cell adhesion motif may be a laminin fragment that comprises or consists of an amino acid sequence selected from the group consisting of YIGSR, IKVAV, CCRRIKVAVWLC and RGD or a variant thereof. The variant may be a conservative amino acid substitution variant e.g. having one, two or three conservative amino acid substitutions compared to an amino acid sequence selected from the group consisting of YIGSR, IKVAV, CC R R I KVAVWLC and RGD.

As mentioned, amino acid portions of the lipopeptides may be synthetic. By“synthetic” it is meant that they comprise amino acid sequences that do not exist in nature. A synthetic amino acid portion may resemble an amino acid sequence, for example a peptide, that occurs in nature. Merely by way of example, a synthetic cell adhesion motif may be selected from the group consisting of V ₂ A ₂ E _2, HSNGLPLGGGSEEEAAAWV (SEQ ID NO: 22), HSNGLPLGGGSEEEAAAVVV(K) (SEQ ID NO: 23) and HSNGLPLGGGSEEEAAAVWK (SEQ ID NO: 24) or a variant thereof.

The amino acid portion may comprise an enzyme-cleavable sequence.

In one example, the amino acid portion may comprise or consist of an enzyme-cleavable sequence and a cell adhesion motif. In such an example, the enzyme-cleavable sequence may be at the N-terminal of the amino acid portion, whereas the cell adhesion motif may be at the C-terminal of the amino acid portion.

The term“enzyme-cleavable sequence” refers to an amino acid sequence that can be cleaved by an enzyme. The enzyme may be, for example, a protease. It will be appreciated that an enzyme-cleavable sequence that can be cleaved by a protease may also be referred to as a protease-cleavable sequence. Examples of proteases and sequences cleaved by proteases will be well known to those skilled in the art. Merely by way of example, a protease may be selected from the group consisting of a metalloprotease, serine protease, cysteine protease, threonine protease, aspartic protease, glutamic protease and asparagine peptide lyases. A metalloprotease may be, for example, MMP1 or MMP2. TPGPQGIAGQ (SEQ ID NO: 25) is an example of an enzyme-cleavable sequence that may be cleaved by MMP1 or MMP2.

The presence of an enzyme-cleavable sequence may be useful for enabling self-directed release of cells maintained and/or cultured on the fused fibrillar supramolecular structure disclosed herein. Cells released from the supramolecular structure by cleavage of the enzyme- cleavable sequence may be carrier-free, structurally, and/or phenotypically equivalent to their natural counterparts. Such cells may be advantageous for example in the context of autologous transplantation. By“carrier-free” it is meant that the cells are free from remnants of the supramolecular structure on which they were maintained and/or cultured. The term“fragment” as used herein refers to a peptide that is a truncation of the corresponding wild type amino acid sequence. A fragment of the peptide may share 100% identity with the portion of the wild type amino acid sequence that it corresponds to.

As used herein, the term "variant" refers to a peptide in which one or more amino acid have been replaced by different amino acids as compared to the corresponding wild type amino acid sequence. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the peptide (conservative substitutions). Generally, the substitutions which are likely to produce the greatest changes in a peptide's properties are those in which (a) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g. Leu, lie, Phe or Val); (b) a cysteine or proline is substituted for, or by, any other residue; (c) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp) or (d) a residue having a bulky side chain (e.g., Phe or Trp) is substituted for, or by, one having a smaller side chain (e.g. , Ala, Ser) or no side chain (e.g., Gly).

The fragment or variant of a cell adhesion protein may substantially retain the biological function of the corresponding wild type peptide. The term“biological function” as used herein may refer to the ability to promote cell binding. By "substantially retains" biological function, it is meant that the fragment or variant retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological function of the wild type peptide to, for example, promote cell binding. Indeed, the fragment or variant may have a higher biological function than the wild type peptide. The fragment or variant may have 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, of the biological function of the wild type peptide to, for example, promote cell binding.

The lipid portion of the lipopeptide may be linear, branched or cyclic. For example, the lipid portion may be linear.

The lipid portion may comprise a hydrophobic carbon chain of 6 to 24 carbon atoms. The lipid portion may therefore comprise a carbon chain of 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more carbon atoms. For example, the lipid portion will comprise a carbon chain of 16 or 18 carbon atoms. It will be appreciated that, when a lipid portion is referred to as, for example, C16 or C18, it means that the lipid portion comprises carbon chain of 16 or 18 carbon atoms, respectively. By way of example and not limitation, the lipid portion may comprise or consist of dodecanoic acid (lauric acid), tetradecanoic acid (myristic acid), hexadecanoic acid (palmitic acid), octadecenoic acid (stearic acid), oleic acid, linoleic acid, and linolenic acid.

The lipid portion may be saturated or unsaturated.

The lipid portion and amino acid portion of the lipopeptide may be attached directly or indirectly. By attached directly, it is meant that the lipid and peptide portions are not separated by a linker. For example, the lipid and amino acid portion may be covalently coupled. By attached indirectly it meant that the lipid and peptide portions are separated by a linker.

The term“linker” as used herein refers to a moiety which is located between the lipid portion and the amino acid portion. Choosing a suitable linker is within the capabilities of those having ordinary skill in the art. For example, where a rigid linker is desired, it may be a rigid polyunsaturated alkyl or an aryl, biaryl, heteroaryl, and the like. When a flexible linker is desired, it may be a flexible peptide, such as Gly-Gly-Gly, or a flexible saturated alkanyl or heteroalkanyl. Hydrophilic linkers or spacers may be, for example, polyalcohols or polyethers, such as polyalkyleneglycols. Hydrophobic linkers or may be, for example, alkyls or aryls.

For example, the linker may be a flexible peptide, such as Gly-Gly-Gly.

In one example, the cell adhesion motif lipopeptide may comprise a C16 lipid portion and an amino acid portion comprising or consisting of the sequence RGDS. In this example, the lipopeptide may comprise a linker (e.g. a Gly-Gly-Gly linker) between the lipid and the RGDS sequence.

In another example, the cell adhesion motif lipopeptide may comprise a C16 lipid portion and an amino acid portion comprising or consisting of the sequence RGDS and an enzyme- cleavable sequence. In this example, the enzyme cleavable-sequence may be TPGPQGIAGQ. The enzyme-cleavable sequence may be at the N-terminal of the amino acid portion and the cell adhesion motif may be at the C-terminal of the amino acid portion.

In another example, the cell adhesion motif lipopeptide may comprise a C16 lipid portion and an amino acid portion comprising or consisting of the sequence YEALRVANEVTLN. In this example, a linker between the lipid portion and the amino acid portion may not be needed e.g. the lipid portion and the YEALRVANEVTLN amino acid sequence may be attached directly. In another example, the cell adhesion motif lipopeptide may comprise a C16 lipid portion and an amino acid portion comprising or consisting of the sequence KTTKS. In this example, a linker between the lipid portion and the amino acid portion may not be needed e.g. the lipid portion and the KTTKS amino acid sequence may be attached directly.

The supramolecular structure may be single-type or composite.

A structure is composite when it is made up of non-identical molecules, wherein some or all of the molecules are lipopeptides and wherein at least one of the lipopeptides is a cell adhesion motif lipopeptide.

In one example, the composite structure comprises or consists of at least two different lipopeptides, wherein at least one of the lipopeptides is a cell adhesion motif lipopeptide. The composite structure may further comprise at least one non-cell adhesion motif lipopeptide. A non-cell adhesion motif lipopeptide is one that does not comprise an amino acid sequence that promotes cell binding. Composite structures may be comprised by different lipopeptides evenly interspersed, or separated in well-defined, discrete domains of the structure.

In one example, the at least one cell adhesion motif lipopeptide may comprise a C16 lipid portion and an amino acid portion comprising or consisting of the sequence YEALRVANEVTLN. The adhesion motif lipopeptide may also further comprise a Gly-Gly-Gly linker between the lipid portion and the amino acid portion (e.g. there may be a Gly-Gly-Gly linker between the lipid portion and the YEALRVANEVTLN sequence).

The at least one non-cell adhesion motif lipopeptide may be used to dilute cell adhesion motif lipopeptides that form the supramolecular structure, for reduced steric hindrance.

In one example, the composite structure may comprise or consist of at least two different lipopeptides, wherein the at least two lipopeptides are both (distinct) cell adhesion motif lipopeptides. For example, one of the at least two cell adhesion motif lipopeptides may comprise a C16 lipid portion and an amino acid portion comprising or consisting of the sequence YEALRVANEVTLN. Such a lipopeptide may be referred to as “Cie- YEALRVANEVTLN”. The adhesion motif lipopeptide may further comprise a Gly-Gly-Gly linker between the lipid portion and the amino acid portion. Such a lipopeptide may be referred to as “C16-GGG-YEALRVANEVTLN”. The second of the at least two cell adhesion motif lipopeptides may comprise a C16 lipid portion and an amino acid portion comprising or consisting of the sequence KTTKS. Such a lipopeptide may be referred to as“C16- KTTKS”. It will be appreciated that in a composite structure, the ratio of the different lipopeptides may vary. For example, where the structure comprises two different types of lipopeptides, the molar ratio of the first lipopeptide to the second lipopeptide may be from about 1 : 10 to about 10:1 , from about 1 :9 to about 9:1 , from about 1 :8 to about 8: 1 , from about 1 :7 to about 7:1 , from about 1 :6 to about 6: 1 , from about 1 :5 to about 5: 1 , from about 1 :4 to about 4: 1 , from about 1 :3 to about 3: 1 , from about 1 :2 to about 2: 1 , or about 1 : 1. For example, the ratio of the first lipopeptide to the second lipopeptide may be from about 1 :7 to about 7: 1 , from about 1 :6 to about 6: 1 , or from about 1 :5 to about 5: 1.

A structure is single-type when it is made up of identical lipopeptide molecules.

In an embodiment, where the structure is single-type, the structure may comprise a cell adhesion motif lipopeptide comprising a C16 lipid portion and an amino acid portion comprising a cell adhesion motif selected from the group consisting of RGDS,

EALRVANEVTLN and KTTKS. In one example, when the amino acid sequence is RGDS the cell adhesion motif lipopeptide may further comprise a Gly-Gly-Gly linker between the lipid portion and the RGDS sequence.

In an embodiment, where the structure is single-type, the structure may comprise a cell adhesion motif lipopeptide comprising a C16 lipid portion and an amino acid portion comprising a cell adhesion motif selected from the group consisting of RGDS,

EALRVANEVTLN and KTTKS. In one example, when the amino acid sequence is RGDS the amino acid portion may further comprise an enzyme-cleavable sequence (for example TPGPQGIAGQ). The enzyme-cleavable sequence may be at the N-terminal of the amino acid portion and the cell adhesion motif may be at the C-terminal of the amino acid portion.

The fused fibrillar supramolecular structure obtainable by the method described herein may be immobilised in or on the surface or provided in an aqueous medium. The aqueous medium may be the same as the solvent in which the lipopeptides were dissolved or different. This example gives rise to further aspects of the disclosure.

Accordingly, provided herein is an aqueous medium comprising a supramolecular structure obtainable by a method described herein. Provided herein is also a surface comprising a fused fibrillar supramolecular structure obtainable by a method described herein. The surface may be for maintenance, culture and/or bioprocessing of a cell. The term“aqueous medium” as used herein refers to any liquid medium containing water. The aqueous medium may be cell culture medium, or may in fact be water. However, it will be appreciated that the term "aqueous medium" does not imply that water should always be the major constituent of the medium. The aqueous medium may be serum-free.

The term“surface” as used herein refers to an area on which cells may be grown. The surface can be 2-dimensional (2D) or 3-dimensional (3D). An example of a 2D surface is a cover slip, or a surface of a culture vessel, such as a tube, a flask, a dish or a plate comprising a plurality of wells. The culture vessel may be a glass, plastic, or metal container that can provide an aseptic environment for culturing cells. An example of a 3D surface is a scaffold, such as a polystyrene scaffold (e.g. Alvetex™) or a gel scaffold (e.g. hydrogel).

The fused fibrillar structure may be immobilised on the surface. In such an example, it can be said that the surface is coated with the fused fibrillar structure. The surface may be coated partially or completely. Methods of coating surfaces with supramolecular structures are generally known in the art. By way of example, a surface may be coated by drop-spotting on the surface and homogenously distributing a solution comprising the lipopeptides, followed by drying the surface to form a thin film of self-assembled fused fibrillar supramolecular structure. In an example, the structure may be immobilised on a 2D surface, such as a cover slip, or a surface of a culture vessel such as a tube, a flask, a dish or a plate comprising a plurality of wells.

The fused fibrillar supramolecular structure may be in the surface for cell maintenance, cell culture and/or cell bioprocessing. By “in the surface” is meant that the supramolecular structure is incorporated into the surface, such that it is partially or fully encapsulated by the surface. Methods for incorporating a supramolecular structure into a surface are also known in the art. For example, the lipopeptides which form the supramolecular structure may be added to the solution from which the surface (such as a 3D scaffold) is made.

It will be appreciated that in some examples, the structure described herein may itself be in fact the surface for cell maintenance, cell culture and/or cell bioprocessing. In such an embodiment, the structure may be in an aqueous medium or may be immobilised in or on the surface. An embodiment in which the aqueous medium comprises the structure and wherein the structure is the surface may be particularly advantageous in the context of adherent cells, where the surface area may be a limiting factor for cell growth. The fused fibrillar supramolecular structures self-assembled in the solution in dense arrangements may provide a greater surface area for cell growth than, for example, the surfaces of the cell culture vessel containing the aqueous medium or coated with fibrillar structures of the art such as those self- assembled in water. This may have advantages such as reduction of cost associated with cell culture and/or improved bioprocessing. The structure may be a 3D surface.

The term“cell maintenance” as used herein refers to keeping the cells alive in an artificial (e.g., an in vitro) environment without substantially increasing the cell number. 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 of the cells. In the context of the present disclosure, the cell can be an individual or a population of cells, or a tissues, organ or organ system. The cell may be eukaryotic (e.g., animal, plant and fungal cells) or prokaryotic (e.g., bacterial cells). The cell may be an animal cell. For example, the cell is mammalian (for example human or mouse). Merely by way of example, a human cell may be a human stromal progenitor cell or a human adipose derived mesenchymal stem cell. Merely by way of example, a mouse cell may be immortalised mouse myoblast cell.

The aqueous medium, the surface, or indeed the structure described herein may promote cell growth. Cell growth may be promoted for example if cell number and/or cell viability is increased as compared to a suitable control.

The term“cell bioprocessing” as used herein refers to producing a molecule of biological origin. The aqueous medium, the surface, or indeed the fused fibrillar supramolecular structure described herein may improve bioprocessing of cells. By improve cell bioprocessing it is meant that cells maintained or cultured in the presence of the fused fibrillar supramolecular structure described herein may produce more of the molecule of biological origin as compared to a suitable control. The molecule of biological origin may be, for example, collagen.

A suitable control may be for example cells grown without the presence of the fused fibrillar supramolecular structure described herein. The control cells may be grown in the presence of a fibrillar supramolecular structure of the art such as a structure that has self-assembled in water.

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

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

EXAMPLES

1. Materials and methods

1.1 Preparation of lipopeptide solutions in serum free medium

Lyophilized lipopeptides were weighed with static eliminator turned on. Lyophilized lipopeptides are highly electrostatic thus the powder transfer is compromised without static eliminator. This is an essential requirement to not loose lipopeptide powder and precisely weight the required amount of lipopeptides.

Lipopeptides were then solubilized in serum-free medium at the desired concentration. The serum-free medium is DMEM-F12, Leibovitz’s L-15 medium, RPMI 1640 medium, or Mesencult™ Basal Medium supplemented with 1 mM ascorbic acid and 1 :100 insulin- transferrin-selenium. Stock solutions must be prepared to have the c.a.c. value greater than the c.a.c. value of lipopeptide used. C.a.c. value varies depending on the lipopeptide sequence and is determined via pyrene fluorescence measurements.

Exemplary stock: 1.52 mM for RGDS lipopeptide and 1.25 mM for LumicamMatrixyl lipopeptides (the latter consists of mixing together the 2 powders at the ratio 15:85 to obtain a final concentration of 1.25 mM). Solution vortexed for 30-45 minutes at room temperature then sonicate at 55 ^° C for 30 min. Solution should be rotated overnight at 4 ^° C. If the lipopeptides are still not well solubilized, repeat steps vortexing and sonication. The final stock solution needs to be substantially, preferably completely transparent and without lipopeptide aggregates. Stock should be stored at 4 ^° C.

1.2 Preparation of lipopeptide solutions in water

1) Weight the lipopeptides.

2) Dissolve the lipopeptides in aqueous solution at the desired concentration, above the critical aggregation concentration (c.a.c.) 3) Vortex the solution for at least 15 minutes

4) Sonicate the solution for at least 30 min and not above 55 ^° C

5) Rotate the solution overnight in a cool environment.

6) Store the final stock solution in a cool environment.

1.3 Cell proliferation

Cell proliferation can be evaluated via Alamar Blue assays, with cell number extrapolated from standard calibration curves using known number of cells for each experiment. Cells are incubated at 37 °C with resazurin reagent at 50 mM (Sigma Aldrich), prepared in 1 : 10 dilution using fresh culture medium, for 5-3 h, after which 100 pi of culture supernatants (in triplicate) are sampled for fluorescence emission analysis at 590 nm using either a Fluoroskan Ascent fluorescent spectrophotometer or a Varioskan™ Lux (both from Thermo Scientific). The incubation time differ depending on the experiment and on the cell type.

1.4 Collagen deposit assay

The amount of collagen deposited by the cells is investigated using the Sirius Red assay (Jimenez et al. , 1985). In specific, cells are fixed in 70% ice-cold ethanol and transferred to - 80 °C for at least 10 min to ensure the complete fixation of the material. Subsequently, the ethanol solution is removed and each well was gently washed with distilled water. Cells are treated with Sirius-red/picric acid solution (Sigma Aldrich) and incubated overnight at 4 °C with gentle agitation. The following day, any unbound dye is removed and cells were gently rinsed with distilled water. Cells are then treated with 500 mI of 1 M NaOH (Fisher Scientific) at room temperature for 10 min under agitation to disperse the solution and 100 pL aliquots of each sample were then transferred in triplicate to a 96 well plate. The total collagen is calculated by comparing the absorbance of the resulting samples at 490 nm, read using either a Multiskan Ascent™ or a Varioskan™ Lux (both from Thermo Fisher Scientific), to that of known standard concentrations of collagen. Each tested condition is performed in triplicate.

1.5 Atomic Force Microscopy (AFM)

Analysis of materials surface topography are performed using a Nanosurf Easyscan 2- controlled atomic force microscope equipped with ContAI-G soft contact mode cantilevers (BudgetSensors; Bulgaria) with a resonant frequency of 13 kHz and nominal spring constant of 0.2 N/m. Briefly, the different samples are mounted on parafilm-covered glass slides (Bemis; USA and Thermo Fisher Scientific, respectively) to minimise sample displacement and drift. Surface topography is analysed from three separate regions in each sample, with 512* two- direction lines scanned at 10 pm/s, at 1 nV, and with P- and l-gains of 1. Topographic data is processed for line wise and tilt correction using the Scanning Probe Image Processor software package (Image Metrology A/S). Data is analysed using the Orientation J plugin from ImageJ (open source Java application NIH, USA) v1.46 for measuring dimensions and distribution of specimens. All experiments were performed on four individual areas in each sample (n = 3).

2. Results

2.1 Self-assembling of lipopeptides prepared via solubilisation in culture medium

When the lipopeptides (lipidated peptides) are solubilised in serum-free culture medium (SFM) rather than double distilled water (as they normally are), their self-assembly occurs assuming a different supramolecular nanostructure (Figure 2 and Figure 9).

Results show that single-type RGDS lipopeptide self-assembled in medium quite differently compared to water. In water, RGDS lipopeptide forms networks of individual nanotapes /twisted nanotapes (Gouveia et al., 2013). On the other hand, in SFM such lipopeptide form bigger and denser arrangements comprising fused nanotapes. Similarly, single-type Lumican and Matrixyl lipopeptides self-assembled in SFM quite differently compared to water (Figure 2). In water, Lumican lipopeptide assembled in borderline structures between lipid-like and amyloid peptide-like self-assembly (i.e., between nanotape and fibril structures) (Hamley et al., 2015), whereas once self-assembled Matrixyl presented wide nanotapes (Castelletto et al., 2010). In contrast, Lumican and Matrixyl lipopeptides form fused fibrillar structures in SFM, arranged in dense aggregates. Finally, the composite systems Lum ⁸⁵ :Matrixyl ¹⁵ and Lum ¹⁵ :Matrixyl ⁸⁵ formed structures resembling more the single-type Lumican lipopeptide than the single-type Matrixyl lipopeptide. Altogether, these results show the presence of supramolecular self-assembled structures of the single-type and composite lipopeptides in medium and a striking difference in structure when compared to structures assembled in water.

2.2 Effect of the cell culture medium on lipopeptides biocompatibility and bioactivity

The effect of the different nanostructure on biocompatibility and bioactivity was investigated next. For this study, the lipopeptides were used as a media supplement, rather than coating. The bioactivity of the lipopeptides has been evaluated for three different cell types, namely human stromal progenitor cells, human adipose-derived mesenchymal stem cells (hASCs) and immortalized mouse myoblast cell line (C2C12).

2.2. 1 Effect on human stromal progenitor cells

The inventors hypothesised that the effect exerted by the same lipopeptide molecule can be different depending on the solvent in which the self-assembly is initiated. Lumican, Matrixyl and RGDS lipopeptides were investigated as supplements to culture media using stock solutions which were previously self-assembled either in water or in medium. Specifically, cells were cultured for 7 days and proliferation was assessed at day 3 and 7, whereas collagen deposition was analysed at day 7.

Results showed that Lumican lipopeptide significantly increased cell proliferation when self- assembled in water and supplemented at 25 mM (25 pM H2O) and 12 pM (12 pM H2O) compared to non-supplemented serum free medium (SFM) both at day 3 (p < 0.0001) and 7 (p = 0.001 and p = 0.0005 respectively) (Figure 3A).

On the other hand, Lumican lipopeptide self-assembled in medium and supplemented at the same concentrations, slightly increased cell proliferation only up to day 3 (p = 0.003 and p = 0.0012 for 12 and 25 pM, respectively) (Figure 3A).

Similarly, the amount of collagen deposited by human stromal progenitor cells cultured for 7 days with Lumican lipopeptide self-assembled in water was ^« 75% and 15% higher at 25 pM and 12 pM respectively, compared to their corresponding concentrations of PA self-assembled in medium (Figure 3B).

Nonetheless, the presence of the lipopeptide significantly increased collagen deposition compared to the control (SFM) independently of the solvent in which the self-assembly occurred. In contrast, Matrixyl lipopeptide self-assembled in SFM was found to have: i) dramatically decreased cell viability at day 7 in both conditions (p < 0.0001) compared to the control (Figure 3C) and ii) significantly increased the amount of deposited collagen at day 7 compared to the control (p < 0.0001) and was ^« 55% higher compared to Matrixyl lipopeptide self-assembled in water (Figure 3D).

Furthermore, RGDS lipopeptide self-assembled in water was found to greatly impair cell proliferation compared to RGDS lipopeptide self-assembled in medium at both concentrations (50 and 500 mM), with both conditions and concentrations significantly lower to the control (p < 0.0001) at day 3 and 7 (Figure 3E). However, both conditions and concentrations significantly increased the amount of deposited collagen compared to the control, with RGDS lipopeptide self-assembled in water increasing the amount of collagen of 65% and 35% at 500 pM and 50 pM respectively, compared to their corresponding concentrations of lipopeptide self-assembled in medium (Figure 3F). Altogether, results reported in Figure 3 indicate that the solvent in which the self-assembly initially occurs has a lasting effect on the biocompatibility and bioactivity of the lipopeptide on human stromal progenitor cell. 2.2.3 Effect on human adipose-derived mesenchymal stem cells (hASCs)

The hASCs were also cultured with RGDS lipopeptide previously solubilised above its c.a.c. in water and then diluted in SFM at the final concentration of 50 mM (Figure 4). This was to understand if the different self-assembled structures affected hASCs behaviour as found for human stromal progenitor cell. Results showed that RGDS self-assembled in water had indeed a statistically significant (p < 0.0001 since day 3) toxic effect on cell proliferation that RGDS lipopeptide self-assembled in medium did not display (comparable to Figure 3E).

Moreover, hASCs were also cultured with Lumican or Matrixyl lipopeptides previously solubilised above their c.a.c. in water and then diluted in SFM at the final concentration of 50 pM (Figure 5). Although not significant, results showed that both lipopeptides slightly increase cell proliferation when solubilised in medium rather than water (Figure 5A). Significant differences in collagen deposition were not present, however, Lumican lipopeptide (Figure 5B) in water and Matrixyl lipopeptide (Figure 5C) in SFM slightly increased the collagen deposited by hASCs after 7 days in culture.

2.2.3 Effect on immortalized mouse myoblast cell line (C2C12)

C2C12 were cultured up to 7 days in serum-free medium (SFM) supplemented either with Lumican or Matrixyl or RGDS lipopeptides. Each lipopeptide stock was prepared above its c.a.c. either in serum-free medium (SFM) or in water (H2O). C2C12 proliferation was evaluated using Alamar Blue assay at day 3 and 7. The amount of deposited collagen was quantified using the Sirius Red assay at day 7.

Lumican lipopeptide was tested at 25 and 50 mM and results showed that when solubilised in medium significantly increased cell proliferation since day 3 (Figure 6). Moreover, the collagen deposited significantly increased when such lipopeptide was solubilised in SFM rather than water (Figure 6).

Similarly, Matrixyl lipopeptide was tested at 25 and 50 pM and both cell proliferation and collagen deposition significantly increased when the lipopeptide was solubilised in medium (Figure 7).

Finally, RGDS lipopeptide was tested at 25, 50 and 500 pM and results showed as such lipopeptide has a quite strong toxic effect on C2C12 when solubilised in water (Figure 8). On the other hand, when solubilised in SFM it significantly reduced its toxic effect and increased collagen deposition (Figure 8). REFERENCES

Castelletto, V., Hamley, I.W., Perez, J., Abezgauz, L. and Danino, D. (2010) 'Fibrillar superstructure from extended nanotapes formed by a collagen-stimulating peptide', Chemical Communications, 46(48), pp. 9185-9187. Gouveia, R.M., Castelletto, V., Alcock, S.G., Hamley, I.W. and Connon, C.J. (2013) 'Bioactive films produced from self-assembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions', Journal of Materials Chemistry B, 1 (44), pp. 6157-6169

Jimenez, W., Pares, A., Caballeria, J., Heredia, D., Bruguera, M., Torres, M., Rojkind, M. and Rodes, J. (1985) 'Measurement of fibrosis in needle liver biopsies: evaluation of a colorimetric method', Hepatology, 5(5), pp. 815-8.

Hamley, I.W., Dehsorkhi, A., Castelletto, V., Walter, M.N.M., Connon, C.J., Reza, M. and Ruokolainen, J. (2015) 'Self-Assembly and Collagen-Stimulating Activity of a Peptide Amphiphile Incorporating a Peptide Sequence from Lumican', Langmuir, 31 (15), pp. 4490- 4495.