CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of SG provisional application No. 10201801025W, filed 6 February 2018, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of biomaterial and synthetic biosystems. In particular, the present invention relates to a modified agarose, a modified agarose membrane and methods to produce the modified agarose membrane.

BACKGROUND OF THE INVENTION

[0003] Comeal transplantation is a technology that has existed more than 110 years, and today, the cornea is the most commonly transplanted tissue. For example, more than 48,000 transplants were performed in 2015 in America alone. Globally, corneal dysfunction is a major problem and is ranked second as the main cause of blindness, after cataract. In many cases, keratoplasty represents the only hope of regaining vision.

[0004] The cornea comprises three main layers and two basal membranes. It is known that the outermost epithelium is 6-8 cell layers thick in human, and the cells in the outermost epithelium can replicate to repair any damage to the cornea. The stroma consists of multiple stacked arrays of collagen fibrils and is approximately 500 pm thick, which accounts for approximately 90% of tissue thickness. In order to maintain corneal transparency to allow light transmission, the fibrils must be kept precisely aligned. This is mainly achieved by the innermost endothelium, where a monolayer of amitotic cells is responsible for regulating homeostatic fluid pressure. Without a functional endothelium, the fibrils lose the precise alignment and become disordered, which causes the cornea to lose transparency and eventually leading to the vison loss.

[0005] In most cases, abnormalities to the comeal endothelium accounts for about half of all cornea transplantations undertaken. Penetrating keratoplasty is the standard method of care and most commonly used technique where the diseased cornea is replaced with a full-thickness cornea from a donor. Recently, other endothelial keratoplasty procedures such as Descemet’s stripping automated endothelial keratoplasty (DSAEK) has become popular. Descemet’s stripping automated endothelial keratoplasty (DSAEK) includes a small incision that is made to access only the endothelium, therefore leaving the other layers intact. Advantages of using Descemet’s stripping automated endothelial keratoplasty (DSAEK) include faster vision recovery with less astigmatism inpatients. However, Descemet’s stripping automated endothelial keratoplasty (DSAEK) is a complicated procedure and endothelial cell loss is more significant due to the extensive endothelium manipulation.

[0006] Since all variations of keratoplasty require donor corneas, it leads to limited availability of transplantation-grade donor corneas for all patients. Methods to create bioengineered corneal endothelium are therefore required to alleviate the global shortage of donor tissue. So far,“cell sheet engineering” has been explored where corneal endothelial cells (CEC) are first grown in two-dimensional (2D) cultures until it can be lifted as a sheet for further uses. However, reports have suggested that such cell-sheets are too fragile for surgery.

[0007] Various materials have been investigated as scaffolds for corneal endothelial cells, wherein collagen appears to be a popular choice. Evidence has shown that reconstruction of the different corneal layers is possible, for example, when different cell types were seeded onto collagen I vitrigel membranes. In another example, the development of corneal endothelium is possible when the human corneal endothelial cells (CEC) were seeded onto rat-tail collagen I membranes. Rat-tail collagen I was also used to co-culture limbal epithelial cells with stromal stem cells to study the epithelial- stromal interaction. In another example, porcine atelocollagen I can either be used as an acidified crosslinked gel for stromal replacement, or crosslinked with a glycopolymer to form an interpenetrating polymer network gel for use in lamellar keratoplasty.

[0008] Another material that is investigated as scaffolds for corneal endothelial cells is matrices prepared from de-cellularized porcine corneas and amniotic membranes of the placenta. These matrices can be further repopulated with corneal cells and evaluated as tissue replacements. Other examples of scaffolds also include porcine gelatine coated with atelocollgen and poly(ethylene glycol-caprolactone) hydrogel films.

[0009] With any choice of material, it is prudent to consider both its benefits and limitation . For instance, while collagen produces strong membranes that can support corneal endothelial cell (CEC) attachment, there are issues associated with batch variation, processing methods and high cost. Gelatine on the other hand, though significantly cheaper, more than 98% of its annual world output is bovine- or porcine- sourced, therefore gelatine -based material and the corneal endothelium grown on gelatine-based material might not be readily accepted for use by some religious groups. In addition, there is also the possibility of transmitting pathogens such as the Bovine Spongiform Encephalopathy. Ideally, synthetic scaffolds would be advantageous if they can be more defined in their composition and material properties. In addition, the production of the synthetic scaffolds should minimize the amount of chemical work-up needed and the use of toxic and/or expensive solvents or catalysts. These are important considerations for scalability and potential clinical translation.

[0010] Therefore, there is a need to create a biocompatible application that can be used as a scaffold for the attachment and growth of functional comeal endothelial cells (CEC) for use in a clinical setting. There is also a need to develop an alternative method to grow comeal endothelial cells (CEC) such that the cells can be used to form a functional bioengineered corneal endothelium.

SUMMARY

[0011] In one aspect, the present invention refers to a membrane comprising a modified agarose, wherein the water content of the membrane is less than 15% and wherein the modified agarose is an agarose covalently linked to a cell attachment signal.

[0012] In another aspect, the present invention refers to a membrane comprising a modified agarose, wherein the water content of the membrane is less than 15% and wherein the modified agarose is an agarose covalently linked to a cell attachment signal that is bound to a cell.

[0013] In yet another aspect, the present invention refers to a method of manufacturing a membrane comprising a modified agarose, wherein the water content of the membrane is less than 15%, wherein the method comprises:

a) covalently linking an agarose to a cell attachment signal to form a modified agarose;

b) dissolving the modified agarose in water to form a solution;

c) solidifying the solution to form a modified agarose hydrogel;

d) dehydrating the modified agarose hydrogel, thereby forming the membrane comprising the modified agarose.

[0014] In yet another aspect, the present invention refers to a membrane obtainable (or obtained) by the method as disclosed herein.

[0015] In a further aspect, the present invention refers to a corneal implant comprising a membrane as disclosed herein.

[0016] In yet another aspect, the present invention refers to a comeal implant comprising a membrane obtainable (or obtained) by the method as disclosed herein.

[0017] In yet another aspect, the present invention refers to a physical supporting structure comprising a membrane as disclosed herein.

[0018] In yet another aspect, the present invention refers to a physical supporting structure comprising a membrane obtainable (or obtained) by the method as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0020] Figure 1 shows an image of two schematics of modified agarose. (A) shows that the modified agarose comprises an agarose and a cell attachment signal that is linked by a covalent link. (B) shows the chemical structures of modification of agarose with various cell attachment signals. Agarose (A) was first activated with l,r-carbonyldiimidazole (CDI). Signals such as glycine-arginine-glycine-aspartic acid (GRGD) (resulting in product: AR), lysine (AK), poly lysine (AP) and fish-derived gelatine (AG) were then conjugated. Peptides glycine- arginine- glycine-aspartic acid (GRGD) and lysine (K) had their side chains protected to restrict the conjugation sites to their terminal amines. The protection groups were subsequently removed using catalytic hydrogenation and base. Thus, Figure 1 illustrates chemical structures of the modified agarose described herein.

[0021] Figure 2 shows an image with 2 chemical structures of agarose (A) and AR(N0 ₂ ). Fig. 2 also shows 4 line graphs representing data generated by 1H nuclear magnetic resonance (NMR) showing the conjugation of protected- glycine-arginine-glycine-aspartic acid (GRGD) to give AR(N0 ₂ ) as an intermediate. The protection groups were subsequently removed to result in agarose-glycine-arginine-glycine-aspartic acid (GRGD) (AR). The appearance and then disappearance of signals due to Obzl confirmed the success of conjugation and deprotection. The area integration of peaks due to Obzl relative to backbone agarose was used for the quantification of degree of conjugation. Figure 2 illustrates the success of covalently linking of agarose to a cell attachment signal agarose-glycine-arginine-glycine-aspartic acid (GRGD) in the modified agarose described herein.

[0022] Figure 3 shows an image with 2 chemical structures of agarose (A) and AK(Fmoc). Fig. 3 also shows 4 line graphs representing data generated by 1H NMR showing the conjugation of protected-K to give AK(Fmoc) as an intermediate. Fmoc was eventually removed to result in agarose-lysine (AK). The appearance and then disappearance of signals due to Fmoc confirmed the success of conjugation and deprotection. Figure 3 illustrates the success of covalently linking of agarose to a cell attachment signal lysine (K) in the modified agarose described herein.

[0023] Figure 4 shows 2 column charts representing data of quantification of degree of conjugation, in terms of weight percent (wt%). (A) shows the quantification of degree of conjugation of the three batches of agarose-polylysine (AP), AP1-3. (B) shows the quantification of degree of conjugation of the four batches of agarose-gelatine (AG), AG1-4. Figure 4 illustrates the success of covalently linking of agarose to a cell attachment signals of polylysine and gelatine in the modified agarose described herein.

[0024] Figure 5 shows 3 photos and 1 line graph that represent the gelation and membrane forming abilities of one of the agarose-gelatine (AG), AG3. (A) shows a photo of the bulk hydrogel placed over the star, wherein the bulk hydrogel is clear and transparent even at a thickness of ~8 mm. Bulk hydrogel is marked up with black dotted line. (B) shows a photo of the bulk hydrogel that was collapsed into an ultra-thin membrane through a process of controlled dehydration. The membrane is marked up with black dotted line and placed over the star. (C) shows a photo of the membrane that is transparent and strong enough to be handled with a pair of forceps. (D) shows a line graph that represents results from light transmittance studies of the dry membrane which allowed >90% of visible light (400-750 nm) to pass through. Thus, Figure 5 illustrates an example of a membrane that is optically transparent.

[0025] Figure 6 shows photos of cells grown on the four batches of agarose-gelatine (AG) series of membrane, AG1-4. (A) shows photos of cell adhesion of RK13 cells on Day 3. (B) shows photos of cell adhesion of human dermal fibroblast cell (NHDF) on Day 4. The cells were seeded on membranes prepared from AG 1-4 and observed to remain attached and viable, as evidenced by the positive calcein staining. Figure 6 illustrates the success of cell attachment and cell viability in the modified agarose described herein.

[0026] Figure 7 shows photos of rabbit comeal endothelial cells (RCEC) grown on the four batches of agarose-gelatine (AG) series of membrane, AG1 and AG3 for a longer term on AG1 and 3 membranes. (A) shows photos of rabbit corneal endothelial cells (RCEC) attached to AG1 and AG3 membranes on day 5, and remained viable on AG1 and AG3 membranes, as evidenced by the positive calcein staining. (B) shows photos of rabbit comeal endothelial cells (RCEC) attached to AG1 and AG3 membranes on Week 4, wherein the cells on AG1 and AG3 stayed confluent and viable. Figure 7 illustrates the cell attachment and cell viability across the batches of modified agarose described herein.

[0027] Figure 8 shows photos of rabbit corneal endothelial cells (RCEC) seeded on AG3 membranes that are labelled with different antibodies on the left panels, DAPI in the middle panels, and a merged photo on the right panel. The top row shows rabbit corneal endothelial cells (RCEC) labelled with CD 166, a functional cell surface marker. The middle row shows rabbit corneal endothelial cells (RCEC) labelled with ZO-l, a marker for the formation of tight cellular junction. The bottom row shows rabbit comeal endothelial cells (RCEC) labelled with Na ⁺ /K ⁺ ATPase, a regulator for the cellular pump function. Scale of the images are 20 pm. Figure 8 illustrates the rabbit corneal endothelial cells (RCEC) grown on the modified agarose described herein are functional.

[0028] Figure 9 shows 4 photos and 2 column charts that represent data for the characterization of membrane thickness. (A) shows a photo of the membrane mounted upright and thickness was measured using electron microscopy. The box with the dotted lines represents the magnified image of the photo, and thickness of the membrane is represented by the gap between the arrow heads. (B) shows a photo of the membrane mounted upright and thickness was measured using light microscopy. The box with the dotted lines represents the magnified image of the photo. (C) shows a column chart representing data of the representative thickness of the agarose-gelatine (AG) membranes, AG1-4. (D) shows a column chart representing data of the representative thickness of the agarose-gelatine (AG) membranes before and after immersion in PBS at 37°C for 14 days, wherein the membranes did not swell to any great extent. Figure 9 illustrates the thickness of the membrane described herein.

[0029] Figure 10 shows 2 column charts representing data of the mechanical properties of membranes prepared from the four batches of agarose-gelatine, AG1-4. (A) shows tensile testing to determine the tensile strength. (B) shows tensile testing to determine the Young’s modulus of membranes. Figure 10 illustrates the tensile strength and Young’s modulus of the membrane described herein.

[0030] Figure 11 shows a column chart representing data of the optimization of conditions used to prepare the ultra-thin membrane. (A) shows the data of the effect of the volume of solution : surface area of chamber on the tensile strength of membranes. (B) shows the data of the effect of concentration on the tensile strength of membranes. Figure 11 illustrates the optimized volume and concentration used to manufacture the modified agarose described herein.

[0031] Figure 12 shows 2 photos that represent rabbit comeal endothelial cells (RCEC) seeded onto membranes formed using unmodified agarose and imaged after 5 days. Both the bright field and fluorescence images showed the absence of adherent cells. The cells were most probably washed away during media change prior to imaging. Figure 12 illustrates that cells are unable to attach to unmodified agarose.

[0032] Figure 13 shows 1 photo that represents rabbit comeal endothelial cells (RCEC) seeded onto membranes formed using gelatine (5.7 wt%) simply mixed in (physical blend, no chemical conjugation) with agarose and imaged after 7 days. Bright field image showed the absence of adherent cells. Figure 13 illustrates that cells are unable to attach to a mixture of unmodified agarose and gelatine, wherein the gelatine is not covalently linked to the agarose. [0033] Figure 14 shows 1 photo that represents the transplantation of the membrane comprising the modified agarose into a rabbit’s cornea. The area circled by the white dotted lines represents the membrane that was implanted into the rabbit’s cornea. Figure 14 illustrates that the membrane can be used for in vivo corneal implant.

DETAILED DESCRIPTION

[0034] Agarose is a polysaccharide derived from plants, and has the advantage of being renewable and biocompatible. Agarose consists of alternate D-galactose and 3,6-anhydro-L- galactose disaccharide repeat units. Typically, agarose dissolves in hot water and yields a clear, rigid hydrogel upon cooling. However, unaltered agarose does not natively support cell attachment nor cell adhesion. In order for cell attachment or cell adhesion to occur, agarose must either be physically mixed with another ingredient (e.g., bovine collagen I) or must be chemically altered, for example, by conjugating cell attachment signals onto agarose. For instance, agarose can be altered with photolabile groups, which can be cleaved upon irradiation to expose free amines or thiols that permit further conjugation with other thiol-containing peptides, wherein micro-patterning can be achieved using the precision of the irradiation source. However, this is not a cost-effective method. A more economical method would be direct conjugation of cell attachment signals onto agarose, for example, conjugating gelatine and a laminin-derived peptide onto agarose to form altered agarose. However, such altered agarose can only be used when spatial control is not required, wherein cells are not required to grow and proliferate in a directed and organised manner. Such altered agarose and the applications derived from the altered agarose would not be useful for applications in a clinical setting, especially when the application is required to enable controlled and directed growth of cells for use, for example, but not limited to, to artificially culture a functional tissue, during transplantation, or during surgery. However, currently known altered agarose and the applications derived from the altered agarose are not able to enable controlled and directed growth of cells for use.

[0035] In addition, previous examples have only shown the use of the altered agarose as bulk gels, which have limited use and functionality in real applications, especially in the clinical setting. Bulk gels are too large in size for delicate types of clinical procedures, for example, during transplantation or during surgery. An example of such procedure is comeal transplantation. As mentioned earlier, as there is limited availability of transplantation-grade donor corneas for all patients, there is a need to create bioengineered corneal endothelium to alleviate the global shortage of donor tissue. However, as the size of the comeal is limited, there is yet a functional application that can be used to create a bioengineered corneal endothelium for corneal transplantation.

[0036] In view of the above problems, there is a need to provide functional applications for use in the clinics. Therefore, the inventors of the present disclosure have found different applications that comprise the modified agarose. More specifically, the inventors of the present disclosure have found a membrane comprising the modified agarose.

[0037] As used herein, the term“modified agarose” refers to the alteration of agarose by changing the chemical or physical structure of agarose. This can include linking one or more compounds to agarose.

[0038] The one or more compounds can be synthetic or naturally occurring. In one example, the modified agarose comprises one or more compounds that can be an amino acid, a peptide, or a protein. As used herein,“amino acid” refers to an organic molecule that is made up of a basic amino group, an acidic carboxyl group and a side chain, and“peptide” and“protein” are used interchangeably throughout and refer to a molecule comprising one or more amino acid residues joined to each other by peptide bonds. In another example, the modified agarose comprises one or more compounds that can bind to a cell, cell membrane, cell membrane proteins, cell adhesion molecules, cell receptor, protein channel, glycolipid, glycoprotein, integrin, fibronectin or any combinations thereof. In another example, the modified agarose comprises a cell attachment signal. In another example, the modified agarose is an agarose covalently linked to a cell attachment signal. In another example, the modified agarose is agarose that is modified by being covalently linked to a cell attachment signal. In another example, the modified agarose is an agarose covalently linked to a cell attachment signal that is bound to a cell. In another example, the modified agarose is agarose that is modified by being covalently linked to a cell attachment signal that is bound to a cell.

[0039] As used herein, the term“cell attachment signal” refers to an entity (such as a sequence of amino acids, chemical moieties, and the like) that cells recognize and interact with in order for the cells to adhere to a surface. In one example, the cell attachment signal includes, but are not limited to, gelatine, short peptide, lysine, poly-lysine (such as alpha-polylysine and epsilon-polylysine), mixture thereof, and the like. In one example, wherein when the cell attachment signal is a short peptide, the short peptide comprises 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30, or 31, or 32, or 33, or 34, or 35, or 36, or 37, or 38, or 39, or 40, or 41, or 42, or 43, or 44, or 45, or 46, or 47, or 48, or 49, or 50 amino acids.

In another example, wherein when the cell attachment signal is a short peptide, the short peptide comprises 4 amino acids. In another example, wherein when the cell attachment signal is a short peptide, the short peptide is glycine-arginine-glycine-aspartic acid (GRGD). In another example, the cell attachment signal is selected from the group consisting of gelatine, glycine- arginine- glycine-aspartic acid (GRGD), lysine, poly-lysine, and mixture thereof.

[0040] The levels of the cell attachment signals in the modified agarose are used to ensure the functionality of the modified agarose, wherein the level or amount or content of the cell attachment signals can be quantified by the degree of conjugation. As used herein, the term “degree of conjugation” refers to a measurement of the level or amount or content of cell attachment signal that has been covalently linked to agarose in the modified agarose. In one example, the degree of conjugation comprises the cell attachment content in the modified agarose. In another example, the degree of conjugation comprises, for example, but is not limited to, polylysine content in the modified agarose, glycine-arginine-glycine-aspartic acid (GRGD) content in the modified agarose, lysine content in the modified agarose, gelatine content in the modified agarose. In another example, the degree of conjugation can be measured as weight percent (wt%) of a cell attachment signal in the modified agarose, or as a ratio of a cell attachment signal and the repeat units of agarose. The weight percent (wt%) of a cell attachment signal in the modified agarose can be derived from the ratio of a cell attachment signal and agarose or the repeat units of agarose.

[0041] As used herein, the degree of conjugation of polylysine or polylysine content in the modified agarose is defined as the weight percent (wt%) of polylysine, wherein wt% of polylysine = - we ⁱ ght of poiyiysme - j _{n one} example, the polylysine

total weight of agarose-polylysine (AP)

content in the modified agarose is from 1 wt% to 3 wt%, or from 3 wt% to 6 wt%, or from 6 wt% to 9 wt%, or from 9 wt% to 12 wt%, or from 12 wt% to 15 wt%, or from 15 wt% to 18 wt%, or from 18 wt% to 21 wt%, or from 21 wt% to 24 wt%, or from 24 wt% to 27 wt%, or from 27 wt% to 30 wt%. In another example, the polylysine content in the modified agarose is from 1 wt% to 30 wt%, or from 2 wt% to 27 wt%, or from 2 wt% to 24 wt%, or from 3 wt% to 21 wt%, or from 3 wt% to 18 wt%, or from 4 wt% to 15 wt%, or from 4 wt% to 12 wt%, or from 5 wt% to 9 wt%, or from 5 wt% to 6.5 wt%, or from 1.6 wt% to 6.8 wt%. In another example, the polylysine content in the modified agarose is about 1.6 wt%, or about 1.7 wt%, or about 1.8 wt%, or about 1.9 wt%, or about 2.0 wt%, or about 2.1 wt%, or about 2.2 wt%, or about 2.3 wt%, or about 2.4 wt%, or about 2.5 wt%, or about 2.6 wt%, or about 2.7 wt%, or about 2.8 wt%, or about 2.9 wt%, or about 3.0 wt%, or about 3.1 wt%, or about 3.2 wt%, or about 3.3 wt%, or about 3.4 wt%, or about 3.5 wt%, or about 3.6 wt%, or about 3.7 wt%, or about 3.8 wt%, or about 3.9 wt%, or about 4.0 wt%, or about 4.1 wt%, or about 4.2 wt%, or about 4.3 wt%, or about 4.4 wt%, or about 4.5 wt%, or about 4.6 wt%, or about 4.7 wt%, or about 4.8 wt%, or about 4.9 wt%, or about 5.0 wt%, or about 5.1 wt%, or about 5.2 wt%, or about 5.3 wt%, or about 5.4 wt%, or about 5.5 wt%, or about 5.6 wt%, or about 5.7 wt%, or about 5.8 wt%, or about 5.9 wt%, or about 6.0 wt%, or about 6.1 wt%, or about 6.2 wt%, or about 6.3 wt%, or about 6.4 wt%, or about 6.5 wt%, or about 6.6 wt%, or about 6.7 wt%, or about 6.8 wt%. In another example, the polylysine content in the modified agarose is 1.7 ± 0.1 wt%, 3.1 ± 0.2 wt%, 6.5 ± 0.3 wt%, or 6.5 wt%. In another example, the polylysine content in the modified agarose is 6.5 wt%.

[0042] For modified agarose wherein the cell attachment signal is glycine- arginine-glycine- aspartic acid (GRGD), in one example, the degree of conjugation of glycine-arginine-glycine- aspartic acid (GRGD) or glycine-arginine-glycine-aspartic acid (GRGD) content in the modified agarose can be the glycine-arginine-glycine-aspartic acid (GRGD) to agarose ratio, wherein the glycine-arginine-glycine-aspartic acid (GRGD) to agarose ratio is the glycine-arginine-glycine- aspartic acid (GRGD) : repeat units of agarose in the modified agarose. In one example, the glycine-arginine-glycine-aspartic acid (GRGD) to agarose ratio is from 1:100 to 1:90, or from 1:90 to 1:80, or from 1:80 to 1:70, or from 1:70 to 1:60, or from 1:60 to 1:50, or from 1:50 to 1:40, or from 1:40 to 1:30, or from 1:30 to 1:20, or from 1:20 to 1:10, or from 1:10 to 1:1. In another example, the glycine-arginine-glycine-aspartic acid (GRGD) to agarose ratio is from 1:100 to 1:1, or from 1:90 to 1:2, or from 1:80 to 1:3, or from 1:70 to 1:4, or from 1:60 to 1:5, or from 1:50 to 1:5, or from 1:40 to 1:5. In another example, the glycine-arginine-glycine-aspartic acid (GRGD) to agarose ratio is or from 1:44 to 1:16. In another example, the glycine- arginine- glycine-aspartic acid (GRGD) to agarose ratio is about 1:44, or about 1:43, or about 1:42, or about 1:41, or about 1:40, or about 1:39, or about 1:38, or about 1:37, or about 1:36, or about 1:35, or about 1:34, or about 1:33, or about 1:32, or about 1:31, or about 1:30, or about 1:29, or about 1:28, or about 1:27, or about 1:26, or about 1:25, or about 1:24, or about 1:23, or about 1:22, or about 1:21, or about 1:20, or about 1:19, or about 1:18, or about 1:17, or about 1:16, or about 1:15, or about 1:14, or about 1:13, or about 1:12, or about 1:11, or about 1:10, or about 1:9, or about 1:8, or about 1:7, or about 1:6, or about 1:5. In another example, the glycine- arginine-glycine-aspartic acid (GRGD) to agarose ratio is 1:44, 1:28, or 1:16. In another example, the glycine-arginine-glycine-aspartic acid (GRGD) to agarose ratio is 1:16.

[0043] In another example, the degree of conjugation of glycine-arginine-glycine-aspartic acid (GRGD) or glycine-arginine-glycine-aspartic acid (GRGD) content in the modified agarose can be the weight percent (wt%) of glycine-arginine-glycine-aspartic acid (GRGD), wherein wt% of glycine— arginine— glycine— aspartic acid (GRGD) =

weight of glycine-arginine-glycine-aspartic acid (GRGD)

total weight of agarose-glycine-arginine-glycine-aspartic acid (GRGD)(aR)’ wherein the total weight of agarose-glycine-arginine-glycine-aspartic acid (GRGD) (AR) comprises the weight of the glycine-arginine-glycine-aspartic acid (GRGD) and repeat units of agarose. The weight of glycine-arginine-glycine-aspartic acid (GRGD) and a single repeat unit of glucose is about 402.4 and about 306 respectively. In one example, the glycine-arginine-glycine-aspartic acid (GRGD) content in the modified agarose is from 1.30 wt% to 1.44 wt%, or from 1.44 wt% to 1.62 wt%, or from 1.62 wt% to 1.84 wt%, or from 1.84 wt% to 2.14 wt%, or from 2.14 wt% to 2.56 wt%, or from 2.56 wt% to 3.18 wt%, or from 3.18 wt% to 4.20 wt%, or from 4.20 wt% to 6.17 wt%, or from 6.17 wt% to 11.62 wt%, or from 11.62 wt% to 56.80 wt%. In another example, the glycine- arginine-glycine-aspartic acid (GRGD) content in modified agarose is from 1.30 wt% to 56.80 wt%, or from 1.44 wt% to 39.67 wt%, or from 1.62 wt% to 30.48 wt%, or from 1.84 wt% to 24.74 wt%, or from 2.14 wt% to 20.82 wt%, or from 2.56 wt% to 20.82 wt%, or from 3.18 wt% to 20.82 wt%. In another example, the glycine-arginine-glycine-aspartic acid (GRGD) content in modified agarose is or from 2.90 wt% to 7.59 wt%. In another example, the glycine-arginine- glycine-aspartic acid (GRGD) content in the modified agarose is about 2.90 wt%, or about 2.97 wt%, or about 3.04 wt%, or about 3.11 wt%, or about 3.18 wt%, or about 3.26 wt%, or about 3.34 wt%, or about 3.43 wt%, or about 3.52 wt%, or about 3.62 wt%, or about 3.72 wt%, or about 3.83 wt%, or about 3.95 wt%, or about 4.07 wt%, or about 4.20 wt%, or about 4.34 wt%, or about 4.49 wt%, or about 4.64 wt%, or about 4.81 wt%, or about 5.00 wt%, or about 5.19 wt%, or about 5.41 wt%, or about 5.64 wt%, or about 5.89 wt%, or about 6.17 wt%, or about 6.47 wt%, or about 6.81 wt%, or about 7.18 wt%, or about 7.59 wt%, or about 8.06 wt%, or about 8.59 wt%, or about 9.19 wt%, or about 9.88 wt%, or about 10.68 wt%, or about 11.62 wt%, or about 12.75 wt%, or about 14.12 wt%, or about 15.82 wt%, or about 17.98 wt%, or about 20.82 wt%. In another example, the glycine-arginine-glycine-aspartic acid (GRGD) content in the modified agarose is 2.90 wt%, 4.49 wt%, or 7.59 wt%. In another example, the glycine-arginine-glycine-aspartic acid (GRGD) content in the modified agarose is 7.59 wt%.

[0044] For modified agarose wherein the cell attachment signal is lysine, in one example, the degree of conjugation of lysine or lysine content in the modified agarose can be the lysine to agarose ratio, wherein the lysine to agarose ratio is lysine : repeat units of agarose in the modified agarose. In one example, the lysine to agarose ratio is from 1:1500 to 1:1400, or from 1:1400 to 1:1300, or from 1:1300 to 1:1200, or from 1:1200 to 1:1100, or from 1:1100 to 1:1000, or from 1:1000 to 1:900, or from 1:900 to 1:800, or from 1:800 to 1:700, or from 1:700 to 1:600, or from 1:600 to 1:500, or from 1:500 to 1:400, or from 1:400 to 1:300, or from 1:300 to 1:200, or from 1:200 to 1:100, or from 1:100 to 1:75, or from 1:75 to 1:50. In another example, the lysine to agarose ratio is from 1:1500 to 1:5, or from 1:1400 to 1:10, or from 1:1300 to 1:15, or from 1:1200 to 1:20, or from 1:1100 to 1:20, or from 1:1000 to 1:25. In another example, the lysine to agarose ratio is from 1:924 to 1:54, or from 1:900 to 1:25, or from 1:800 to 1:30, or from 1:700 to 1:30, or from 1:600 to 1:35, or from 1: 500 to 1:35, or from 1:400 to 1:40, or from 1:300 to 1:40, or from 1:200 to 1:50, or from 1:100 to 1:50. In another example, the lysine to agarose ratio is about 1:924, or about 1:923, or about 1:922, or about 1:921, or about 1:920, or about 1:919, or about 1:918, or about 1:917, or about 1:916, or about 1:915, or about 1:914, or about 1:913, or about 1:912, or about 1:911, or about 1:910, or about 1:909, or about 1:908, or about 1:907, or about 1:906, or about 1:905, or about 1:904, or about 1:903, or about 1:902, or about 1:901, or about 1:900, or about 1:899, or about 1:898, or about 1:897, or about 1:896, or about 1:895, or about 1:894, or about 1:893, or about 1:892, or about 1:891, or about 1:890, or about 1:889, or about 1:888, or about 1:313, or about 1:312, or about 1:311, or about 1:310, or about 1:309, or about 1:308, or about 1:307, or about 1:306, or about 1:305, or about 1:304, or about 1:303, or about 1:302, or about 1:301, or about 1:100, or about 1:99, or about 1:98, or about 1:97, or about 1:96, or about 1:95, or about 1:94, or about 1:93, or about 1:92, or about 1:91, or about 1:90, or about 1:89, or about 1:88, or about 1:87, or about 1:86, or about 1:85, or about 1:84, or about 1:83, or about 1:82, or about 1:81, or about 1:80, or about 1:79, or about 1:78, or about 1:77, or about 1:76, or about 1:75, or about 1:74, or about 1:73, or about 1:72, or about 1:71, or about 1:70, or about 1:69, or about 1:68, or about 1:67, or about 1:66, or about 1:65, or about 1:64, or about 1:63, or about 1:62, or about 1:61, or about 1:60, or about 1:59, or about 1:58, or about 1:57, or about 1:56, or about 1:55, or about 1:54, or about 1:53, or about 1:52, or about 1:51, or about 1:50. In another example, the lysine to agarose ratio is 1:906+18, 1:307+6, 1:91+2, 1:56+2. In another example, the lysine to agarose ratio is 1:56.

[0045] In another example, the degree of conjugation of lysine or lysine content in the modified agarose can be the weight percent (wt%) of lysine, wherein wt% of lysine =

weight of lysine

total weight of agarose-lysine(AK ) , wherein the total weight of agarose-lysine (AK) comprises the weight of the lysine and repeat units of agarose. The weight of lysine and a single repeat unit of glucose is about 145 and about 306 respectively. In one example, the lysine content in the modified agarose is from 0.032 wt% to 0.034 wt%, or from 0.034 wt% to 0.036 wt%, or from 0.036 wt% to 0.039 wt%, or from 0.039 wt% to 0.043 wt%, or from 0.043 wt% to 0.047 wt%, or from 0.047 wt% to 0.053 wt%, or from 0.053 wt% to 0.059 wt%, or from 0.059 wt% to 0.068 wt%, or from 0.068 wt% to 0.079 wt%, or from 0.079 wt% to 0.095 wt%, or from 0.095 wt% to 0.118 wt%, or from 0.118 wt% to 0.158 wt%, or from 0.158 wt% to 0.236 wt%, or from 0.236 wt% to 0.472 wt%, or from 0.472 wt% to 0.628 wt%, or from 0.628 wt% to 0.939 wt%. In another example, the lysine content in the modified agarose is from 0.032 wt% to 8.567 wt%, or from 0.034 wt% to 4.524 wt%, or from 0.036 wt% to 3.062 wt%, or from 0.039 wt% to 2.314 wt%, or from 0.043 wt% to 2.314 wt%, or from 0.047 wt% to 1.860 wt%. In another example, the lysine content in the modified agarose is from 0.051 wt% to 0.870 wt%, or from 0.053 wt% to 1.860 wt%, or from 0.059 wt% to 1.555 wt%, or from 0.068 wt% to 1.555 wt%, or from 0.079 wt% to 1.336 wt%, or from 0.095 wt% to 1.336 wt%, or from 0.118 wt% to 1.171 wt%, or from 0.158 wt% to 1.171 wt%, or from 0.236 wt% to 0.939 wt%, or from 0.472 wt% to 0.939 wt%. In another example, the lysine content in the modified agarose is about 0.051 wt%, or about 0.052 wt%, or about 0.053 wt%, or about 0.151 wt%, or about 0.152 wt%, or about 0.153 wt%, or about 0.154 wt%, or about 0.155 wt%, or about 0.156 wt%, or about 0.157 wt%, or about 0.472 wt%, or about 0.476 wt%, or about 0.481 wt%, or about 0.486 wt%, or about 0.491 wt%, or about 0.496 wt%, or about 0.502 wt%, or about 0.507 wt%, or about 0.512 wt%, or about 0.518 wt%, or about 0.524 wt%, or about 0.530 wt%, or about 0.536 wt%, or about 0.542 wt%, or about 0.548 wt%, or about 0.554 wt%, or about 0.561 wt%, or about 0.568 wt%, or about 0.575 wt%, or about 0.582 wt%, or about 0.589 wt%, or about 0.596 wt%, or about 0.604 wt%, or about 0.612 wt%, or about 0.620 wt%, or about 0.628 wt%, or about 0.636 wt%, or about 0.645 wt%, or about 0.654 wt%, or about 0.663 wt%, or about 0.672 wt%, or about 0.682 wt%, or about 0.692 wt%, or about 0.702 wt%, or about 0.713 wt%, or about 0.724 wt%, or about 0.735 wt%, or about 0.747 wt%, or about 0.758 wt%, or about 0.771 wt%, or about 0.784 wt%, or about 0.797 wt%, or about 0.810 wt%, or about 0.824 wt%, or about 0.839 wt%, or about 0.854 wt%, or about 0.870 wt%, or about 0.886 wt%, or about 0.903 wt%, or about 0.921 wt%, or about 0.939 wt%. In another example, the lysine content in the modified agarose is 0.052+0.001 wt%, 0.154+0.003 wt%, 0.518+0.011 wt%, 0.839+0.030 wt%. In another example, the lysine content in the modified agarose is 0.839 wt%.

[0046] For modified agarose wherein the cell attachment signal is gelatine, the degree of conjugation of gelatine or gelatine content in the modified agarose is defined as the weight

weight of gelatine

percent (wt%) of gelatine, wherein wt% of gelatine = total weight of agarose-gelatine (AG) . In one example, the gelatine content in the modified agarose is from 2 wt% to 5 wt%, or from 5 wt% to 10 wt%, or from 10 wt% to 15 wt%, or from 15 wt% to 20 wt%, or from 20 wt% to 25 wt%, or from 25 wt% to 30 wt%, or from 30 wt% to 35 wt%, or from 35 wt% to 40 wt%, or from 40 wt% to 45 wt%, or from 45 wt% to 50 wt%. In another example, gelatine content in the modified agarose is from 2 wt% to 50 wt%, or from 3 wt% to 40 wt%, or from 4 wt% to 30 wt%, or from 5 wt% to 25 wt%, or from 5 wt% to 20 wt%. In another example, gelatine content in the modified agarose is from 5.4 wt% to 21.5 wt%. In another example, gelatine content in the modified agarose is about 5.0 wt%, or about 6 wt%, or about 7 wt%, or about 8 wt%, or about 9 wt%, or about 10 wt%, or about 11 wt%, or about 12 wt%, or about 13 wt%, or about 14 wt%, or about 15 wt%, or about 16 wt%, or about 17 wt%, or about 18 wt%, or about 19 wt%, or about 20 wt%. In another example, gelatine content in the modified agarose is about 5.4 wt%, or about 5.5 wt%, or about 5.6 wt%, or about 5.7 wt%, or about 5.8 wt%, or about 5.9 wt%, or about 6.0 wt%, or about 8.0 wt%, or about 8.1 wt%, or about 8.2 wt%, or about 8.3 wt%, or about 8.4 wt%, or about 8.5 wt%, or about 8.6 wt%, or about 8.7 wt%, or about 8.8 wt%, or about 8.9 wt%, or about 9.0 wt%, or about 9.1 wt%, or about 9.2 wt%, or about 14.4 wt%, or about 14.5 wt%, or about 14.6 wt%, or about 14.7 wt%, or about 14.8 wt%, or about 14.9 wt%, or about 15.0 wt%, or about 15.1 wt%, or about 15.2 wt%, or about 15.3 wt%, or about 15.4 wt%, or about 15.5 wt%, or about 15.6 wt%, or about 20.9 wt%, or about 21.0 wt%, or about 21.1 wt%, or about 21.2 wt%, or about 21.3 wt%, or about 21.4 wt%, or about 21.5 wt%. In another example, gelatine content in the modified agarose is 5.7 ± 0.3 wt%, 8.6 ± 0.6 wt%, 15.0 ± 0.6 wt%, or 21.2 ± 0.3 wt%. In another example, gelatine content in the modified agarose is 15.0 ± 0.6 wt%.

[0047] Gelatine is useful as a cell attachment signal, however more than 98% of the world gelatine production annually is from bovine- or porcine-derived source. Bovine- or porcine- derived source is not accepted by some religions, and also carries the risk of transmitting mammal-borne pathogens to humans. Therefore, it is useful to use gelatine that is not from bovine or porcine. In one example, the gelatine is fish-derived gelatine. The use of fish-derived gelatine is advantageous as it is accepted by most major religion and has lower risk of zoonotic transmission to human when compared to the use of mammal-derived gelatine. In addition, chemical synthesis of fish-derived gelatine has been designed to be facile and green.

[0048] Bovine- or porcine-gelatine is also not very soluble in the room temperature, and requires heating to solubilize in water or aqueous solution, before it forms a gel upon cooling. This means that the extra time would be required to heat the water or aqueous solution to solubilize the bovine- or porcine-gelatine, which slows the production of modified agarose. In order to improve the efficiency of producing modified agarose, gelatine that is soluble in water or aqueous solution at room temperature can be used. In one example, the gelatine is a gelatine that is soluble in water or aqueous solution at room temperature. In another example, the gelatine is a fish-derived gelatine that is soluble in water or aqueous solution at room temperature.

[0049] In one example, room temperature is from 20°C to 25°C. In another example, room temperature is at about 20°C, or at about 2l°C, or at about 22°C, or at about 23°C, or at about 24°C, or at about 25°C.

[0050] The modified agarose is formed when any of the compounds as described herein binds to agarose by one or more chemical bonds, for example, covalent bond, ionic bond, hydrogen bond, van der Waals bond, or any combinations thereof between the one or more compounds to agarose. In one example, the modified agarose is formed by covalently linking an agarose to a cell attachment signal.

[0051] Covalent linking can be accomplished via the use of one or more reagents. The reagent can be a chemical. In one example, the reagent can include, but is not limited to 1,1’- carbonyldiimidazole (CDI), carbodiimides, aminium salt, uranium salt, phosphonium salts, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) cyanogen bromide (

pyrimidines

cyanate esters ( sulfonyl

chlorides ( ^R chloroformates ),

N-hydroxysuccinimide p-nitrophenyl derivatives or any combinations thereof. In another example, the reagent to covalently link an agarose to a cell attachment signal to form a modified agarose is l, -carbonyldiimidazole (CDI). In yet another example, the covalently linking an agarose to a cell attachment signal to form a modified agarose is by l,r-carbonyldiimidazole (CDI)-mediated coupling. Using l,l’-carbonyldiimidazole (CDI) is advantageous as it is specific in its chemistry and will not result in unwanted reactions. In addition, l,l’-carbonyldiimidazole (CDI) is safer and less toxic than, for example, cyanogen bromide. Additionally, unreacted l,l’-carbonyldiimidazole (CDI) can also be easily removed from the modified agarose with water.

[0052] As mentioned earlier, the membrane comprises the modified agarose as disclosed herein. As used herein, the term“membrane” is defined as a dehydrated gel comprising the modified agarose that mimic an extracellular matrix environment to support the adhesion, proliferation, and differentiation of living cells. The dehydration process occurs in a controlled environment, causing the gel to collapse into a membrane. This dehydration causes the loss of most of the water content in the gel, and leaving a membrane of lowered water content. In one example, the water content of the membrane is less than 20%, or less than 19%, or less than 18%, or less than 17%, or less than 16%, or less than 15%, or less than 14%, or less than 13%, or less than 12%, or less than 11%, or less than 10%. In another example, the water content of the membrane is less than 15%.

[0053] In one example, the membrane comprises a modified agarose, wherein the water content of the membrane is less than 15% and wherein the modified agarose is an agarose covalently linked to a cell attachment signal. In another example, the membrane comprises a modified agarose, wherein the water content of the membrane is less than 15% and wherein the modified agarose is an agarose covalently linked to gelatine. In another example, the membrane comprises a modified agarose, wherein the water content of the membrane is less than 15% and wherein the modified agarose is an agarose covalently linked to glycine-arginine-glycine- aspartic acid (GRGD). In another example, the membrane comprises a modified agarose, wherein the water content of the membrane is less than 15% and wherein the modified agarose is an agarose covalently linked to lysine. In another example, the membrane comprises a modified agarose, wherein the water content of the membrane is less than 15% and wherein the modified agarose is an agarose covalently linked to poly-lysine. In another example, the membrane comprises a modified agarose, wherein the water content of the membrane is less than 20% and wherein the modified agarose is an agarose covalently linked to gelatine. In another example, the membrane comprises a modified agarose, wherein the water content of the membrane is less than 10% and wherein the modified agarose is an agarose covalently linked to gelatine. [0054] In another example, the membrane comprises a modified agarose, wherein the water content of the membrane is less than 15% and wherein the modified agarose is an agarose covalently linked to a cell attachment signal that is bound to a cell. The cell as disclosed herein is at least one living cell. In one example, the cell can be a prokaryote or eukaryote cell. In another example, the cell can be adherent or free-floating. As used herein, the term“adherent cell” refers to anchorage-dependent cells or cells that have to attach or anchor to a surface in order to grow and/or proliferate. Non-limiting examples of adherent cells include, but are not limited to, fibroblast cell, epithelial cell, cancer cell (such as HepG2 cell, HeLa cell, and the like), endothelial cell (such as corneal endothelial cell), stem cell, and others generally known in the art. In another example, the cell can be, but is not limited to, human dermal fibroblast cell (NHDF), comeal endothelial cell (CEC) or kidney cell.

[0055] The membrane can remain in a dehydrated form or in a hydrated form. In one example, the membrane can be hydrated in an aqueous solution. In another example, the membrane can be hydrated in, for example, but is not limited to, phosphate buffered saline (PBS), water, Tris buffered saline (TBS), Tris-acetate-EDTA (TAE), or Tris-botate-EDTA (TBE).

[0056] One would appreciate that in order for the membrane to be useful for corneal endothelium transplantation, the membrane must be small and thin in order to fit into a cornea. In one example, the thickness of the membrane is from 1 pm to 100 pm, or from 2 pm to 90 pm, or from 3 pm to 80 pm, or from 4 pm to 70 pm, or from 5 pm to 60 pm, or from 6 pm to 50 pm, or from 8 pm to 40 pm, or from 10 pm to 30 pm. In another example, the thickness of the membrane is from 1 pm to 10 pm, or from 10 pm to 20 pm, or from 20 pm to 30 pm, or from 30 pm to 40 pm, or from 40 pm to 50 pm, or from 50 pm to 60 pm, or from 60 pm to 70 pm, or from 70 pm to 80 pm, or from 80 pm to 90 pm, or from 90 pm to 100 pm. In another example, the thickness of the membrane is from 1 pm to 5 pm, or from 5 pm to 10 pm, or from 10 pm to 15 pm, or from 15 pm to 20 pm, or from 20 pm to 25 pm, or from 25 pm to 30 pm, or from 30 pm to 35 pm, or from 35 pm to 40 pm, or from 40 pm to 45 pm, or from 45 pm to 50 pm, or from 50 pm to 55 pm, or from 55 pm to 60 pm, or from 60 pm to 65 pm, or from 65 pm to 70 pm, or from 70 pm to 75 pm, or from 75 pm to 80 pm, or from 80 pm to 85 pm, or from 85 pm to 90 pm, or from 90 pm to 95 pm, or from 95 pm to 100 pm. In another example, the thickness of the membrane is about 10 pm, or about 11 pm, or about 12 pm, or about 13 pm, or about 14 pm, or about 15 pm, or about 16 pm, or about 17 pm, or about 18 pm, or about 19 pm, or about 20 pm, or about 21 pm, or about 22 pm, or about 23 pm, or about 24 pm, or about 25 pm, or about 26 mih, or about 27 mih, or about 28 mih, or about 29 mih, or about 30 mih. In another example, the thickness of the membrane is about 15 mih.

[0057] Another key feature of the membrane for it to be useful for comeal endothelium transplantation is that the membrane is optically transparent. As used herein, the term optically transparent refers to the physical property of allowing light to pass through the material without being scattered. In one example, a membrane that is optically transparent allows greater than 90%, or greater than 91%, or greater than 92%, or greater than 93%, or greater than 94%, or greater than 95%, or greater than 96%, or greater than 97%, or greater than 98%, or greater than 99% of visible light to pass through, wherein the visible light as defined in the art is of wavelength 400 nm - 750 nm. In another example, the membrane that is optically transparent allows greater than 90% of visible light to pass through. In another example, the membrane that is optically transparent allows greater than 96% of visible light to pass through.

[0058] Since the membrane will be used during corneal endothelium transplantation, the membrane will undergo various forms of manipulation during surgery. Therefore, the membrane cannot be fragile and must have high tensile strength to withstand forms of manipulation. As used herein, the term“tensile strength” refers to a measure of the ultimate stress that a membrane can withstand before rupture. Tensile strength can be measured using any method known in the art. In one example, the tensile strength is measured using an Instron 5848 MicroTester (MA, USA) at room temperature (or about 25°C). In one example, the tensile strength of the membrane is from 10 MPa to 20 MPa, or from 20 MPa to 30 MPa, or from 30 MPa to 40 MPa, or from 30 MPa to 40 MPa, or from 40 MPa to 50 MPa, or from 50 MPa to 60 MPa, or from 60 MPa to 70 MPa, or from 70 MPa to 80 MPa, or from 80 MPa to 90 MPa, or from 90 MPa to 100 MPa. In another example, the tensile strength of the membrane is from 10 MPa to 100 MPa, or from 20 MPa to 90 MPa, or from 30 MPa to 80 MPa, or from 40 MPa to 70 MPa, or from 40 MPa to 60 MPa, or from 49 MPa to 60 MPa. In another example, the tensile strength of the membrane is about 40 MPa, or about 41 MPa, or about 42 MPa, or about 43 MPa, or about 44 MPa, or about 45 MPa, or about 46 MPa, or about 47 MPa, or about 48 MPa, or about 49 MPa, or about 50 MPa, or about 51 MPa, or about 52 MPa, or about 53 MPa, or about 54 MPa, or about 55 MPa, or about 56 MPa, or about 57 MPa, or about 58 MPa, or about 59 MPa, or about 60 MPa. In another example, the tensile strength of the membrane is from 40 MPa to 60 MPa.

[0059] In addition to tensile strength, another feature that the membrane must have in order to withstand forms of manipulation is a property that measures the stiffness of a material called Young’s modulus. As used herein, the term“Young’s modulus” refers to tensile stress divided by strain during the initial elastic deformation part of the stress-strain curve of a material. Young’s modulus can be measured using any method known in the art. In one example, the Young’s modulus is determined based on the tensile stress that is measured using an Instron 5848 MicroTester (MA, USA) at room temperature (or about 25°C). In one example, the Young’s modulus of the membrane is from 100 MPa to 200 MPa, or from 200 MPa to 300 MPa, or from 300 MPa to 400 MPa, or from 400 MPa to 500 MPa, or from 500 MPa to 600 MPa, or from 600 MPa to 700 MPa, or from 700 MPa to 800 MPa, or from 800 MPa to 900 MPa, or from 900 MPa to 1000 MPa. In another example, the Young’s modulus of the membrane is from 100 MPa to 1000 MPa, or from 200 MPa to 900 MPa, or from 300 MPa to 800 MPa, or from 400 MPa to 800 MPa. In another example, the Young’s modulus of the membrane is from 525 MPa to 709 MPa. In another example, the Young’s modulus of the membrane is about 400 MPa, or about 425 MPa, or about 450 MPa, or about 475 MPa, or about 500 MPa, or about 525 MPa, or about 550 MPa, or about 575 MPa, or about 600 MPa, or about 625 MPa, or about 650 MPa, or about 675 MPa, or about 700 MPa, or about 725 MPa, or about 750 MPa, or about 775 MPa, or about 800 MPa. In another example, the Young’s modulus of the membrane is from 400 MPa to 800 MPa.

[0060] The membrane as described herein can be used for other purposes or for developing other applications. In one example, the membrane can be used for other purposes including, but are not limited to, supporting cell adhesion, cell attachment, cell viability, cell proliferation, cell growth or any combinations thereof. As used herein, the term “cell adhesion” and“cell attachment” can be used interchangeably, and refer to the process by which cells interact and attach to neighbouring cells through specialised molecules of the cell surface. As used herein, the term“cell viability” refers to the ability of a cell to survive or live under certain conditions. The methods used for testing cell attachment or cell adhesion, and cell viability are methods that are generally known in the art. In one example, the membrane can be used for supporting cell attachment and growth.

[0061] In another example, the membrane can be used to grow corneal endothelial cells. In another example, the membrane can be used to support tissue growth and development. Examples of the types of tissue include, but are not limited to, cornea, kidney, muscle, liver, heart, pancreas, bladder, skin, stomach, and colon. In yet another example, the membrane can be used in an implant, for example, but not limited to, corneal, kidney, muscle, liver, heart, pancreas, bladder, skin, stomach, and colon. In another example, the membrane can be used in a corneal implant. In another example, the corneal implant comprises the membrane as disclosed herein. In another example, the corneal implant comprises the membrane obtainable or obtained by the method as disclosed herein. In yet another example, the membrane can be used as a physical supporting structure. As used herein, the term“physical supporting structure” refers to a device for holding, clasping, clutching, gripping, clenching, or maintaining the position of a glaucoma drainage device, which is a device that is useful for relieving the intra-ocular pressure following glaucoma surgery. In one example, the physical supporting structure comprises the membrane as disclosed herein. In another example, the physical supporting structure comprises the membrane obtainable or obtained by the method as disclosed herein. In one example, the physical supporting structure is placed on and/or over the glaucoma drainage device or the implant. The physical supporting structure is capable of holding the implant in place after the surgery and preventing rupture of the site of implantation. The membrane of the present invention is useful as a physical supporting structure because the physical supporting structure has to be relatively thin (for example, from lpm to lOOpm), strong, and cell compatible or biocompatible.

[0062] Also disclosed herein are the methods of manufacturing the membrane, wherein the methods comprise multiple steps. In one example, the method of manufacturing a membrane comprising a modified agarose, wherein the water content of the membrane is less than 15%, wherein the method comprises: a) covalently linking an agarose to a cell attachment signal to form a modified agarose; b) dissolving the modified agarose in water to form a solution; c) solidifying the solution to form a modified agarose hydrogel; d) dehydrating the modified agarose hydrogel, thereby forming the membrane comprising the modified agarose. In another example, the method comprises covalently linking an agarose to a cell attachment signal to form a modified agarose, wherein a) the covalent linking of an agarose to a cell attachment signal to form a modified agarose is described above. In another example, the membrane is obtainable (or obtained) by the method as disclosed herein.

[0063] The method of manufacturing the membrane can comprise further steps after step a) covalently linking an agarose to a cell attachment signal to form a modified agarose and before step b) dissolving the modified agarose in water to form a solution, wherein the further steps can be used to isolate and purify the modified agarose. In one example, the method can further comprise, after step a) and before step b): al) precipitating the modified agarose in a solvent suitable to precipitate the modified agarose; a2) isolating the modified agarose; a3) dissolving the modified agarose in water to form a solution; a4) solidifying the modified agarose to form a modified agarose gel; a5) submerging the modified agarose gel in water, and a6) lyophilising the modified agarose gel.

[0064] In one example, the method after step a) and before step b) can further comprise a precipitation step al) to extract the modified agarose. As used herein, the term“precipitation” refers to the extraction of the modified agarose in a solid form from an organic solution. The organic solution can be, but is not limited to, dimethyl sulfoxide (DMSO). Precipitation occurs when two or more solutions are mixed, thereby causing a reaction that creates a solid. Precipitation of the modified agarose can be done using a solvent suitable to precipitate the modified agarose. In one example, the modified agarose can be precipitated by alcohol or water. In another example, the modified agarose can be precipitated using for example, ethanol or any other solution in the amount leading to the same effect.

[0065] In one example, the method after step a) and before step b) can further comprise an isolation step a2) to purify the modified agarose. Isolating the modified agarose can be done by methods generally known in the art, for example, centrifugation, filtration, or any combination thereof.

[0066] In one example, the method after step a) and before step b) can further comprise a washing step between a2) and a3) to clean or wash the modified agarose. Washing the modified agarose can be done by using an aqueous solution generally known in the art, for example, using ethanol, water, buffers, or any combination thereof.

[0067] In one example, the method after step a) and before step b) can further comprise step a3) dissolving the modified agarose in water to form a solution. In another example the method after step a) and before step b) can further comprise step a3) dissolving the modified agarose in water at room temperature to form a solution.

[0068] In one example, the method after step a) and before step b) can further comprise step a4) solidifying the modified agarose to form a modified agarose gel. As used herein, the term “modified agarose gel” refers to a gel comprising a network of crosslinked polysaccharide conjugated with a cell attachment signal. Time is required for the solidification of the modified agarose to form an modified agarose gel. In one example, the time taken for the modified agarose to be solidified to the modified agarose gel is from 10 to 20 minutes, or from 20 to 30 minutes, or from 30 to 40 minutes, or from 40 to 50 minutes, or from 50 to 60 minutes, or from 10 minutes to 60 minutes. In another example, the time taken for the modified agarose to be solidified to the modified agarose gel is about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes. In yet another example, the time taken for the modified agarose to be solidified to the modified agarose gel is 30 minutes.

[0069] In one example, the method after step a) and before step b) can further comprise step a5) submerging the modified agarose gel in water or an aqueous solution. The modified agarose gel can be in an impure state, wherein impurities such as l,r-carbonyldiimidazole (CDI), unbound gelatine, unbound lysine, unbound polylysine, and unbound glycine-arginine-glycine- aspartic acid (GRGD) can be found in a mix with the modified agarose. Therefore, a further step can be included to purify the modified agarose gel to remove any impurities. In one example, the modified agarose gel can be submerged in water at room temperature from 1 to 2 days, or from 2 to 3 days, or from 3 to 4 days, or from 4 to 5 days, or from 1 to 5 days, or for 2 days. For example, the use of gelatine that is soluble in water or aqueous solution at room temperature as a cell attachment signal at room temperature can be helpful because the modified agarose can be easily isolated and purified. Because the gelatine used to prepare the product is a gelatine that is soluble in water or aqueous solution at room temperature, when the unpurified product is submerged in water, gelatine that are not covalently linked to agarose (or the unreacted starting material) can be removed. The isolation and purification method described above utilises a shorter time and does not utilise preparation of heated buffers.

[0070] In one example, the method after step a) and before step b) can further comprise step a6) lyophilising the modified agarose gel. Lyophilising the modified agarose gel can result in a more purified form of the modified agarose. As used herein, the term “lyophilise” or “lyophilising” refer to the process of removing water from a sample of the modified agarose gel. Lyophilising can be accomplished by first freezing the sample and subsequently drying the sample to give rise to a lyophilised modified agarose gel that comprises the modified agarose. This lyophilised modified agarose gel can be used as the modified agarose for step b) in the method of manufacturing a membrane. Lyophilising the modified agarose gel can be done by methods generally known in the art, for example, freeze-drying.

[0071] In another example, the method of manufacturing a membrane comprising a modified agarose comprises b) dissolving the modified agarose to form a solution. In one example, the modified agarose is dissolved in a liquid or aqueous solution. In one example, the modified agarose is dissolved in a liquid or aqueous solution at a suitable temperature to form a solution. In another example, the modified agarose is dissolved in water or buffer at a suitable temperature to form a solution, wherein the buffer can be, but is not limited to, phosphate buffered saline (PBS), Tris buffered saline (TBS), Tris-acetate-EDTA (TAE), or Tris-botate-EDTA (TBE). In another example, the method of manufacturing a membrane comprising a modified agarose comprises b) dissolving the modified agarose in water to form a solution. In another example, the method of manufacturing a membrane comprising a modified agarose comprises b) dissolving the modified agarose in water at a suitable temperature to form a solution.

[0072] As used herein, the term“suitable temperature” refers to the temperature of the liquid or aqueous solution used to dissolve the modified agarose or the lyophilised modified agarose gel derived from step a6). For example, the lyophilised modified agarose gel derived from step a6) is insoluble in water at room temperature, but is soluble in water at higher temperatures. Since the degree of solubility of the modified agarose or lyophilised modified agarose gel is dependent on the temperature, it is therefore useful to regulate the temperature so that most, if not all, of the modified agarose or lyophilised modified agarose gel can be dissolved in the liquid or aqueous solution. In one example, the suitable temperature to form a solution can be at about 70°C and above. In another example, the suitable temperature to form a solution can be above about 70°C, above about 75°C, above about 80°C, above about 85°C, above about 90°C, above about 95°C, above about l00°C or above about l05°C. In another example, the suitable temperature to form a solution can be from about 70°C to about 80°C, from about 80°C to about 90°C, from about 90°C to about l00°C, from about l00°C to about H0°C. In yet another example, the suitable temperature to form a solution can be about 95.0°C, about 96.0°C, about 97.0°C, about 98.0°C, about 99.0°C, about l00.0°C, about 101.0°C, about l02.0°C, about l03.0°C, about l04.0°C, or about l05.0°C.

[0073] Another useful feature for dissolving the modified agarose is the gelation concentration. As used herein, the term“gelation concentration” refers to a concentration of a solution, above which, the solution is able to solidify and form a gel or a hydrogel. In one example, the concentration of the solution is from 2 mg/mL to 5 mg/mL, or from 5 mg/mL to 10 mg/mL, or from 10 mg/mL to 15 mg/mL, or from 15 mg/mL to 20 mg/mL, or from 20 mg/mL to 25 mg/mL, or from 25 mg/mL to 30 mg/mL, or from 30 mg/mL to 35 mg/mL, or from 35 mg/mL to 40 mg/mL, or from 40 mg/mL to 45 mg/mL, or from 45 mg/mL to 50 mg/mL, or from 50 mg/mL to 55 mg/mL, or from 55 mg/mL to 60 mg/mL, or from 60 mg/mL to 65 mg/mL, or from 65 mg/mL to 70 mg/mL, or from 70 mg/mL to 75 mg/mL, or from 75 mg/mL to 80 mg/mL, or from 80 mg/mL to 85 mg/mL, or from 85 mg/mL to 90 mg/mL, or from 90 mg/mL to 95 mg/mL, or from 95 mg/mL to 100 mg/mL, or from 2 mg/mL to 100 mg/mL, or from 2 mg/mL to 90 mg/mL, or from 3 mg/mL to 80 mg/mL, or from 3 mg/mL to 70 mg/mL, or from 4 mg/mL to 60 mg/mL, or from 4 mg/mL to 50 mg/mL, or from 5 mg/mL to 40 mg/mL, or from 5 mg/mL to 30 mg/mL, or from 5 mg/mL to 20 mg/mL. In another example, the concentration of the solution is about 5 mg/mL, or about 5.5 mg/mL, or about 6 mg/mL, or about 6.5 mg/mL, or about 7 mg/mL, or about 7.5 mg/mL, or about 8 mg/mL, or about 8.5 mg/mL, or about 9 mg/mL, or about 9.5 mg/mL, or about 10 mg/mL, or about 10.5 mg/mL, or about 11 mg/mL, or about 11.5 mg/mL, or about 12 mg/mL, or about 12.5 mg/mL, or about 13 mg/mL, or about 13.5 mg/mL, or about 14 mg/mL, or about 14.5 mg/mL, or about 15 mg/mL, or about 15.5 mg/mL, or about 16 mg/mL, or about 16.5 mg/mL, or about 17 mg/mL, or about 17.5 mg/mL, or about 18 mg/mL, or about 18.5 mg/mL, or about 19 mg/mL, or about 19.5 mg/mL, or about 20 mg/mL. In another example, the concentration of the solution is about 10 mg/mL.

[0074] The method of manufacturing the membrane comprises the step of c) solidifying the solution to form a modified agarose hydrogel. As used herein, the term“hydrogel” refers to a macromolecular gel constructed of a network of crosslinked polymer chains. As used herein, the term“modified agarose hydrogel” refers to a macromolecular gel constructed of a network of crosslinked polysaccharides conjugated with a cell attachment signal that is formed prior to the dehydration step to obtain a membrane comprising the modified agarose. In one example, the solidifying in c) is by cooling. In another example, the solution is left to cool to solidify to form a modified agarose hydrogel. In another example, the solution is left to cool to a temperature of below about 20°C, below about 25°C, below about 30°C, below about 35°C, below about 40°C, below about 45°C, below about 50°C or below about 55°C. In another example, the temperature can be from about 20°C to about 30°C, from about 30°C to about 40°C, from about 40°C to about 50°C, from about 50°C to about 60°C. In yet another example, the temperature can be room temperature, which is generally known in the art that room temperature can be from 20°C to 25°C. In another example, room temperature is at about 20°C, or at about 2l°C, or at about 22°C, or at about 23°C, or at about 24°C, or at about 25°C.

[0075] The method of manufacturing the membrane comprises the step of d) dehydrating the modified agarose hydrogel, thereby forming the membrane comprising the modified agarose. In one example, after the solution has been solidified into a hydrogel, the membrane can then be formed by dehydrating the modified agarose hydrogel. As used herein, the terms“dehydrate” or “dehydrating” refers to the use of heat to remove water from the hydrogel. This can be accomplished by general methods known in the art for dehydrating hydrogels, for example, but is not limited to, heating or vacuuming. In another example, when the dehydrating in d) is by heating, the temperature for dehydrating is from 25°C to 30°C, or from 30°C to 35°C, from 35°C to 40°C, from 40°C to 45°C, from 45°C to 50°C, from 50°C to 55°C, from 55°C to 60°C, from 60°C to 65°C, from 65°C to 70°C, from 70°C to 75°C, from 75°C to 80°C, from 80°C to 85°C, from 85°C to 90°C, from 90°C to 95°C, from 95°C to l00°C. In another example, when the dehydrating in d) is by heating, the temperature for dehydrating is from 25°C to l00°C, from 30°C to 95°C, from 35°C to 90°C, from 40°C to 85°C, from 45°C to 80°C, from 50°C to 75°C, from 50°C to 70°C. In another example, when the dehydrating in d) is by heating, the temperature for dehydrating is from about 50°C, or about 5l°C, or about 52°C, or about 53°C, or about 54°C, or about 55°C, or about 56°C, or about 57°C, or about 58°C, or about 59°C, or about 60°C, or about 6l°C, or about 62°C, or about 63°C, or about 64°C, or about 65°C, or about 66°C, or about 67°C, or about 68°C, or about 69°C, or about 70°C. In another example, when the dehydrating in d) is by heating, the temperature for dehydrating is about 60°C.

[0076] For the modified agarose hydrogel to become a membrane, time is required for the dehydration process. In one example, dehydration can be slow or fast. In another example, the length of time for dehydrating in d) is from 24 hours to 36 hours, or from 36 hours to 48 hours, or from 48 hours to 60 hours, or from 60 hours to 72 hours, or from 72 hours to 84 hours, or from 84 hours to 96 hours, or from 96 hours to 108 hours, or from 108 hours to 120 hours. In another example, the length of time for dehydrating in d) is from 24 hours to 120 hours, or from 24 hours to 108 hours, or from 24 hours to 96 hours, or from 24 hours to 84 hours, or from 24 hours to 72 hours. In another example, the length of time for dehydrating in d) is from about 24 hours, or about 36 hours, or about 48 hours, or about 60 hours, or about 72 hours. In another example, the length of time for dehydrating in d) is about 48 hours.

[0077] The method of manufacturing a membrane of the present invention is an alternative to the method known in the art such as direct casting of modified agarose, wherein the membrane obtained from the method of the present invention is stronger than the membrane obtained from direct casting. The membrane that is prepared using direct casting method is too weak to be properly handled and thus its properties (such as the thickness, the tensile strength, and the Young’s modulus) cannot be measured.

[0078] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0079] As used in this application, the singular form“a,”“an,” and“the” include plural references unless the context clearly dictates otherwise. For example, the term“a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof. [0080] As used herein, the term“about”, in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

[0081] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0082] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0083] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0084] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION

[0085] Materials

[0086] C-terminal amidated, side-chain protected peptides NH ₂ -G{R(N0 ₂ )}G{D(0bzl)}- CONH ₂ and NH ₂ -{K(Fmoc)]-CONH ₂ were purchased at >95% purity from GenScript (NJ, USA). Agarose- 1000 was purchased from Invitrogen (Singapore). Poly-L-lysine (4-15 kDa by viscosity), gelatine (cold water fish skin), l,r-carbonyldiimidazole (CDI) (98%), piperidine (99%), dimethyl sulfoxide (DMSO) (anhydrous), Pd/C (10 wt%) and celite were all purchased from Sigma (Singapore). All other solvents were from Fisher Scientific (UK).

[0087] Methods

[0088] Agarose-gelatine (AG) synthesis

[0089] Agarose was activated with l,l’-carbonyldiimidazole (CDI) typically as follows. 306 mg of agarose was completely dissolved in 15 mL of anhydrous dimethyl sulfoxide (DMSO) at l30°C under N ₂ and cooled to room temperature. 2-6 mg of l,l’-carbonyldiimidazole (CDI) in 0.5 mL of dimethyl sulfoxide (DMSO) was added and stirred for 2 hours.

[0090] For conjugation, appropriate amount of gelatine (~l mg l,l’-carbonyldiimidazole (CDI) per 17.5 mg gelatine) dissolved in 1.5 mL of dimethyl sulfoxide (DMSO) was added to the activated agarose and stirred for 20 hours. Agarose-gelatine (AG) was precipitated dropwise into 300 mL of ethanol, isolated by centrifugation and washed 3 more times with ethanol. After the last wash, 5 mL of water was added to the agarose-gelatine (AG) pellet. At this stage, agarose-gelatine (AG) was soluble at 25°C under vortex mixing to give a clear solution. Upon standing for 30 mins, a modified agarose gel (now insoluble in water at room temperature) was formed, which was then submerged in 3 L of water and purified for 2 days with twice daily water change before lyophilisation.

[0091] Agarose-lysine (AK) synthesis

[0092] Agarose was activated as described above with l,l’-carbonyldiimidazole (CDI), before appropriate amount of fluorenylmethyloxycarbonyl (Fmoc)-protected lysine in 2.5 mL of dimethyl sulfoxide (DMSO) was added. After stirring for 20 hours, 20% (by volume) of the reaction solution was aspirated. This aliquot contained agarose conjugated with fluorenylmethyloxycarbonyl (Fmoc)-protected lysine, AK(Fmoc). To the remaining solution, a volume of piperidine (similar to what was removed earlier) was added and stirred for 20 mins to cleave the fluorenylmethyloxycarbonyl (Fmoc), resulting in agarose-lysine (AK). Both AK(Fmoc) and agarose-lysine (AK) were precipitated in ethanol, washed, purified and lyophilized as above.

[0093] Agarose-glycine-arginine-glycine-aspartic acid (GRGD) (AR) synthesis

[0094] Agarose was activated as described above with l,l’-carbonyldiimidazole (CDI), before appropriate amount of protected glycine-arginine-glycine-aspartic acid (GRGD) in 2.5 mL of dimethyl sulfoxide (DMSO) was added. After stirring for 20 hours, agarose conjugated with N0 ₂ protected glycine-arginine-glycine-aspartic acid (GRGD) [AR(N0 ₂ )] was precipitated in ethanol, washed, purified and lyophilized as above.

[0095] Catalytic hydrogenation was next used to remove the protection groups. 200 mg of AR(N0 ₂ ) was dissolved in 80 mL of dimethylformamide (DMF) at l50°C and cooled to room temperature. 100 mg of Pd/C was added and the vessel was charged with H ₂ to 90 psi. After stirring for 24 hours, the solution was centrifuged to remove most of the catalyst, before being passed through a column of dimethylformamide (DMF)-wetted celite. Agarose-glycine-arginine- glycine-aspartic acid (GRGD) (AR) was then precipitated in ether and isolated by centrifugation. The agarose-glycine-arginine-glycine-aspartic acid (GRGD) (AR) pellet was washed 3 more times in ether and dried overnight under vacuum. The pellet dissolved in water at 90°C and formed a hydrogel upon cooling. The hydrogel was then purified and lyophilized, as above, to give purified agarose-glycine-arginine-glycine-aspartic acid (GRGD) (AR).

[0096] Agarose-polylysine (AP) synthesis

[0097] Agarose was activated as described above with l,l’-carbonyldiimidazole (CDI), before appropriate amount of polylysine in 2.5 mL of dimethyl sulfoxide (DMSO) was added. After stirring for 20 hours, agarose-polylysine (AP) was precipitated in ethanol, washed, purified and lyophilized as above.

[0098] Degree of conjugation

[0099] The quantification method for degree of conjugation varied for each compound.

[00100] For agarose-gelatine (AG), the Pierce BCA protein assay kit (Thermo Scientific, Singapore) was used. Agarose-gelatine (AG) was dissolved in water at 2 mg/mL (below its gelation concentration) and 25 pL of sample was incubated with 200 pL of working reagent at 60°C for 30 mins. Absorbance was read at 560 nm and calibrated against standard gelatine solutions (R >0.99). Degree of conjugation was expressed as weight percent (wt%) of gelatine ± standard deviation (sd) of triplicates.

[00101] For agarose-lysine (AK) and agarose-polylysine (AP), the Pierce quantitative fluorometric peptide assay kit (Thermo Scientific) was used. This kit contains a fluorescent dye that specifically labels primary amines. Samples (2 mg/mL) and the respective standards (R >0.99) were prepared in water and 10 pL was added to 70 pL of buffer, followed by 20 pL of fluorescent dye. After 30 mins, fluorescence (Ex/Em 390/475 nm) were read. Degree of conjugation for agarose-lysine (AK) was expressed as number of agarose repeat units per lysine ± standard deviation (sd) of triplicates. For agarose-polylysine (AP), it was expressed as weight percent (wt%) of polylysine ± standard deviation (sd) of triplicates.

[00102] For AR, 1H nuclear magnetic resonance (NMR) was used (Bruker 400 MHz Ultrashield Plus, Singapore). Samples were dissolved in DMSO-d ₆ (Cambridge Isotope Laboratories, MA, USA) and chemical shifts were obtained in parts per million (ppm). Quantification was based on area integration of the characteristic peaks of Obzl on AR(N0 ₂ ), relative to backbone agarose. Degree of conjugation was expressed as number of agarose repeat units per glycine-arginine-glycine-aspartic acid (GRGD).

[00103] Gel permeation chromatography (GPC)

[00104] Samples were prepared in dimethylformamide (DMF) and syringe filtered (0.45 pm). Molecular weight was analysed with a Waters e2695 separation module fitted with a 2414 refractive index detector, relative to polystyrene standards.

[00105] Preparation of ultra-thin membrane

[00106] The lyophilised agarose-gelatine (AG), agarose-lysine (AK), agarose-glycine- arginine-glycine-aspartic acid (GRGD) (AR), or agarose-polylysine (AP) samples were dissolved in water (l00°C) at 10 mg/mL. Appropriate volumes of hot solution were dispensed into 1-, 2- or 4-well chamber slides with removable chamber tops (SPL Life Sciences, Korea) and left to gel upon cooling to room temperature. Membranes were subsequently formed by slowly dehydrating the gel in a 60°C oven over two days. The ultra-thin membrane was then carefully peeled off the slide after removing the chamber tops.

[00107] Light transmittance

[00108] Membranes were formed and mounted on a cuvette. The % transmittance of light in the visible spectrum was measured with a U-2800 spectrophotometer (Hitachi, Japan).

[00109] Membrane thickness

[00110] Membranes were mounted upright onto a carbon tape and sputtered with platinum, before being observed with a Jeol JSM-7400F (Tokyo, Japan) field-emission scanning electron microscope. For routine measurements, a light microscope with a calibrated scale bar was used instead.

[00111] T ensile properties of membrane [00112] Rectangular membranes were prepared with a length of 1.5 cm, width of 6 mm and thickness as measured above. The membranes were subjected to tensile tests with an Instron 5848 MicroTester (MA, USA). The starting gap was 5 mm and pulling rate was 0.2 mm/min.

[00113] Isolation of primary rabbit corneal endothelial cells (RCEC)

[00114] Isolation of primary rabbit comeal endothelial cells (RCEC) was performed using methods generally known, but with slight modifications. The Descemet’s membrane was peeled off from New Zealand White rabbit corneas and exposed to collagenase I (2 mg/mL in Endothelial-SFM medium) for 2-4 hours to dislodge the rabbit corneal endothelial cells (RCEC). This resulted in cell clusters, which were further mechanically dissociated into smaller clumps/single cells. Rabbit corneal endothelial cells (RCEC) was then maintained on tissue culture plates coated with FNC (Athena Environmental Sciences, USA) and cultured using dual media until needed.

[00115] For passaging or seeding onto experimental membranes, cells were lifted off with collagenase I (2 mg/mL in Endothelial-serum free medium (SFM)) for 1-2 hours and washed twice with medium before use. Unused cells were discarded after passage three.

[00116] Cell attachment study

[00117] Rabbit comeal endothelial cells (RCEC), normal human dermal fibroblasts (NHDF) or RK-13 normal rabbit kidney cells (ATCC, USA) were seeded onto the respective membranes and maintained at 37°C, 5% C0 ₂ . Media was changed every 2-3 days. At appropriated time points, calcein-AM (Invitrogen) was added to identify live cells and images were obtained with an IX-83 inverted fluorescence microscope (Olympus, Japan).

[00118] Immunostaining

[00119] Immunostaining was conducted by methods generally known in the art, using primary antibodies for CD166 and Na ⁺ /K ⁺ ATPase were obtained from Abeam (Cambridge, UK). Tight junction protein (ZO-l) antibody and Alexa- Fluor conjugated secondary antibodies were obtained from Life Technologies (Singapore). Nucleus was stained with 4',6-diamidino-2- phenylindole dihydrochloride (DAPI) (Merck Millipore, Singapore).

[00120] Optimization of the effect of the volume of solution : surface area of chamber on the tensile strength of the membrane [00121] AK2 was first dissolved in water (10 mg/mL, or 1%) at l00°C. 300, 400 or 500 pL was then dispensed into 4-well chambered slides with removable chamber tops (SPL Life Sciences, Korea). Therefore, only volume was varied, while concentration and surface area were fixed. Membranes were formed as described previously and subjected to tensile testing. Tensile strength increased with volume of solution used and 500 pL was selected to obtain membranes with greater reproducibility and ease-of-handling.

[00122] Optimization of the effect of concentration of modified agarose on the tensile strength of the membrane

[00123] AK2 was dissolved at different concentrations (0.5%, 0.75% and 1%) and 500 pL was dispensed into the 4-well chamber slides. Here, concentration was varied, while volume and surface area were maintained. Tensile testing revealed that membranes formed with 0.5% solution were too weak to be even tested. Membranes formed with 0.75% solution were strong enough to be tested but required careful handling. Eventually, conditions of 500 pL of a 1% solution were determined to be optimal for a 4-well chamber slide for future experiments. Conditions were scaled accordingly if a larger sheet of membrane was required.

[00124] Experimental Results

[00125] Agarose has an outstanding mechanical property and was therefore selected as a base material to ensure the structural integrity of ultra-thin membranes. However, agarose natively lacks the appropriate chemical groups to allow cell to attach or adhere, and must be conjugated with suitable signals for cell adhesion. Agarose (A) was therefore conjugated with signals such as: the glycine-arginine-glycine-aspartic acid (GRGD) integrin -binding sequence (resulting in agarose-glycine-arginine-glycine-aspartic acid (GRGD) (AR) as the product), lysine as a single amino acid (resulting in agarose-lysine (AK) as a product), polylysine (resulting in agarose- polylysine (AP) as a product) and fish-derived gelatine (resulting in agarose-gelatine (AG) as a product) (Figure 1A and 1B). Coupling was facilitated by 1,1’ -carbonyldiimidazole (CDI) and synthesis was designed to be facile. Indeed, with the exception of agarose-glycine-arginine- glycine-aspartic acid (GRGD) (AR), all the other modified agarose could be synthesised in single-pot reactions. This makes the production of the modified agarose amendable to scaling.

[00126] The modified agarose must be activated by, for example, carbonyldiimidazole (CDI) before conjugation. After 1,1’ -carbonyldiimidazole (CDI) activation, the hydroxyl groups on agarose became susceptible to attacks by nucleophiles. Hence, to restrict the site of conjugation to its N-terminal amine, the side chains of glycine-arginine-glycine-aspartic acid (GRGD) were initially protected with N0 ₂ and Obzl to eliminate competition for binding. The protected peptide was then used for conjugation, producing AR(N0 ₂ ) as an intermediate. Subsequently, the protection groups were removed by catalytic hydrogenation to result in AR. The success of conjugation was evidenced by the appearance of proton nuclear magnetic resonance (NMR) peaks due to Obzl, followed by their disappearance after deprotection (Figure 2). Peaks due to the backbone agarose and Obzl were identified and integrated to determine the conjugation ratio. Three batches (AR1, AR2, AR3) were prepared, with AR1 having the lowest conjugation degree (1 glycine-arginine-glycine-aspartic acid (GRGD) : 44 repeat units of agarose), followed by AR2 (1 glycine-arginine-glycine-aspartic acid (GRGD) : 28 repeat units of agarose) and AR3 (1 glycine-arginine-glycine-aspartic acid (GRGD) : 16 repeat units of agarose) in ascending order.

[00127] N0 ₂ /Obzl was selected in favour of the more common Pbf/OtBu protection groups because the former could be removed by catalytic hydrogenation. This avoided the need for acid treatment to remove the latter, which was undesirable as agarose is susceptible to acid hydrolysis. Indeed, under conditions typically used for acid deprotection (95% trifluoroacetic acid (TFA), 25°C, 2 hours), agarose was degraded into oligosaccharides and single disaccharide repeat units (-306 Da), as revealed by mass spectrometry.

[00128] The side chain of the lysine amino acid was protected with fluorenylmethyloxycarbonyl (Fmoc) to limit conjugation to its terminal amine. The success of conjugation and deprotection was confirmed by the appearance, and then disappearance, of fluorenylmethyloxycarbonyl (Fmoc) nuclear magnetic resonance (NMR) peaks in AK(Fmoc) and agarose-lysine (AK), respectively (Figure 3). In this case, the area integration method was not sensitive enough to the variation in conjugation ratio. Instead, an amine-specific fluorescent dye was used to quantify the amount of lysine side chain present in agarose-lysine (AK). This then allowed for the calculation of conjugation degree. Four batches (AK1, AK2, AK3, AK4) were prepared, with AK1 having the lowest conjugation degree of 0.052+0.001 wt% (1 lysine : 906+18 repeat units of agarose), followed by AK2 (1 lysine : 307+6 repeat units of agarose), AK3(l lysine : 91+2 repeat units of agarose) and AK4 (1 lysine : 56+2 repeat units of agarose).

[00129] The conjugation of K(Fmoc) was less efficient than GR(N0 ₂ )GD(Obzl). For instance, the feed ratio for AK1-4 was -30-40 times of what was achieved practically. In comparison, the feed ratio for AP1-3 was only -1.5 -1.9 times of the achieved ratio. A likely explanation is steric hindrance caused by the proximity of the bulky Fmoc group to the conjugation site. In contrast, the protection groups used for glycine-arginine-glycine-aspartic acid (GRGD) were less bulky and further away from the terminal amine. [00130] In light of the above, the protection of poly-lysine appears unfeasible. Therefore, poly lysine was unprotected, with the implications being that the specific site of conjugation cannot be controlled and multiple conjugations per poly-lysine chain are possible. Using the amine- specific fluorescent dye, the success of conjugation was confirmed by the detection of free amines in purified agarose-polylysine (AP). Conjugation degree was then quantified and expressed as weight percent (wt%) of poly-lysine. Three batches (AP1, AP2, AP3) were prepared, with AP1 having the lowest conjugation degree (1.7+0.1 wt%), followed by AP2 (3.1+0.2 wt%) and AP3 (6.5+0.3 wt%) (Figure 4A). As expected, the conjugation of poly-lysine was more efficient than K(Fmoc) as the feed ratio was only -1.2 times of what was achieved.

[00131] Fish-derived gelatine was next used for conjugation and the BCA protein assay kit was used to quantify the gelatine content in purified agarose-gelatine (AG) preparations. Degree of conjugation was expressed in terms of wt%. Four batches (AG1, AG2, AG3, AG4) were prepared, with AG1 having the lowest conjugation degree (5.7+0.3 wt%), followed by AG2 (8.6+0.6 wt%), AG3 (15.0+0.6 wt%) and AG4 (21.2+0.3 wt%) (Figure 4B). The reaction was also relatively efficient, with the feed being -1.0- 1.2 times the achieved ratio.

[00132] This study deliberately uses gelatine from cold water fish skin, which offers advantages such as being generally well accepted by the major religions. The risk of transmitting mammal-borne pathogens is also avoided. Moreover, the physical properties of gelatine from cold water fish skin differ usefully from its mammal-derived counterparts. In contrast, cold water fish-gelatine is naturally soluble in water (>40 mg/mL) and dimethyl sulfoxide (DMSO) at room temperature and does not form a gel even after standing overnight. This facilitates both synthesis and the efficient removal of unreacted gelatine during purification. With mammal-gelatine, purification is hindered because of precipitation and trapping within the gel matrix.

[00133] The gelation and membrane-forming abilities of the modified agarose preparations were next evaluated. Initially, bulk hydrogels were formed by dissolving samples in water (10 mg/mL or 1%) at l00°C and filling into ring moulds. A higher-than-required temperature was used for dissolution so as to sterilize the solution at the same time. This was in preparation for future cell culture experiments. A representative bulk gel formed with AG3 was clear and transparent (Figure 5A). Following conditions that had previously been optimized (Figure 11), the bulk gel was then dehydrated in a controlled environment and collapsed into an ultra-thin membrane. As can be seen, the membrane was transparent (Figure 5B) and robust enough to be easily handled with a pair of forceps (Figure 5C). Transmittance studies demonstrated that the dry membrane was >90% transparent to visible light (400-750 nm) (Figure 5D), suggesting that the membranes exhibited excellent optical clarity. [00134] Freshly-isolated primary rabbit comeal endothelial cells (RCEC), human dermal fibroblast cell (NHDF) and RK13 cells were seeded onto the various ultra-thin membranes to screen for cell adhesion. The live/dead cell assay kit based on calcein AM was then added at appropriate time points to identify and visualize viable cells. As expected, cells did not attach to membranes of unmodified agarose (Figure 12). Cells, however, attached well to membranes prepared from any one of the AG1-4, as shown at a relatively early time point for RK13 (Figure 6A), normal human dermal fibroblasts (NHDF) (Figure 6B) and rabbit corneal endothelial cells (RCEC) (Figure 7A).

[00135] Rabbit corneal endothelial cells (RCEC) were confluent on AG1 and AG3 membranes at an earlier time point (Figure 7A), wherein the cells were viable as shown by the positive calcein staining. In view of future applications, the long-term viability of rabbit comeal endothelial cells (RCEC) was assessed. By week 4, rabbit comeal endothelial cells (RCEC) also remained viable and confluent on AG1 and AG3 membranes (Figure 7B). This suggests that AG1 and AG3 are potential candidates for evaluation as scaffolds for CEC transplantation.

[00136] Immunofluorescence staining was next performed on rabbit corneal endothelial cells (RCEC) cultured on AG3 membranes (Figure 8). As can be seen, cells stained positive for CD 166 (also known as ALCAM), which belongs to the immunoglobulin superfamily. CD 166 forms part of the adhesive complex at intercellular junctions which help to maintain tissue architecture. Recently, CD 166 has been reported to be a functional cell surface marker for the non-fibroblastic phenotype of corneal endothelial cells (CEC). The positive ZO-l staining demonstrates that rabbit corneal endothelial cells (RCEC) formed tight cellular junctions, reminiscent of cells growing natively in rabbit corneas. Positive staining for Na ⁺ /K ⁺ ATPase, a regulator of pump function, further indicates that pumping actions were intact.

[00137] To demonstrate that chemical conjugation of gelatine to agarose was necessary, another set of membranes was formed where gelatine (5.7 wt%) was merely mixed physically with agarose rabbit comeal endothelial cells (RCEC) were then seeded and observed to be non adherent on day 7 (Figure 13). This is in contrast to the chemically conjugated AG1 (5.7+0.3 wt%) membranes where RCEC were already attached by day 5 (Figure 7A). This is likely due to the washing away of gelatine during media change, resulting in the loss of cell- attachment signals. This highlights the importance of chemical conjugation and also offers functional evidence that gelatine had been successfully conjugated with agarose in the agarose-gelatine (AG) series of membranes.

[00138] Gel permeation chromatography (GPC) analysis, relative to polystyrene standards, revealed that the number- (M _n ) and weight-average (M _w ) molecular weights of AG 1-4 were broadly comparable at the level of resolution offered by this technique. In general, M _n ranged between 200-206 kDa and M _w ranged between 291-298 kDa for AG1-4. The polydispersity index (PD I) was -1.4-1.5. This was a slight increase from unmodified agarose, which had M _n =l8l kDa, M _w =284 kDa and PDI=l.6. MALDI-TOF mass spectrometry was shown to work well in the determination of molecular weights of agarose oligosaccharides , however it is less useful with agarose polysaccharide samples.

[00139] Membranes were mounted upright and imaged using either electron (Figure 9A) or light (Figure 9B) microscopy to quantify thickness of the membranes. As seen, the thickness of AG1-4 membranes is typically -15 pm (Figure 9C). Furthermore, upon immersion in PBS (37°C, 2 weeks), the membrane did not swell to any large extent (Figure 9D).

[00140] The agarose-gelatine (AG) series of membrane was then subjected to tensile testing. The tensile strength is a measure of the ultimate stress that the membrane can withstand before rupture. From Figure 10A, membranes of AG1-4 had comparable tensile strength, which ranged between 49-60 MPa. Young’s modulus, which is a measure of stiffness, ranged between 525-709 MPa (Figure 10B). Usefully, the order-of-magnitude was not observed to drop in mechanical property post-conjugation, as reported by another group studying modified agarose. For reference, the tensile strength of native rabbit and human corneas has been quoted to be approximately 4 and 3.3 MPa, respectively. The membranes developed here are therefore significantly stronger than the native tissues.

[00141] Finally, to determine if the membrane could indeed withstand surgical manipulations during transplantation, corneas of New Zealand White rabbits were obtained and AG3 membranes (trephined into 6 mm circular discs) were prepared for a mock endothelial keratoplasty procedure. Briefly, a incision was made in the cornea and the endothelium was removed by scraping. The membrane was then folded in half and inserted into the anterior chamber (Figure 14). The membrane spread open easily and was held in place with a small bubble. This suggests that the membrane can be useful in a surgical setting. As part of future work, the transplantation of ultra-thin membranes bearing rabbit corneal endothelial cells (RCEC) will be evaluated for the restoration of corneal transparency in rabbits with damaged endothelia.

[00142] Agarose (A), which natively does not permit cell adhesion, was modified with several types of attachment signals such as: glycine-arginine-glycine-aspartic acid (GRGD) (giving agarose-glycine-arginine-glycine-aspartic acid (GRGD) (AR) as product), single lysine amino acid (giving agarose-lysine (AK) as product), polylysine (giving agarose-polylysine (AP) as product) and fish-derived gelatine (giving agarose-gelatine (AG) as product). The advantages of fish-derived gelatine over mammal-derived sources were discussed. For optimization purposes, samples with a range of conjugation ratios were prepared for each family. All products formed bulk hydrogels, which were then collapsed into ultra-thin membranes in a controlled environment, following conditions optimized previously. Membranes were evaluated for their ability to support the attachment of various cell types. Especially relevant is the primary rabbit corneal endothelial cells (RCEC), which remained confluent and viable (as evidenced by calcein staining) for at least 4 weeks. The cells also stained positive for CD166, ZO-l and Na ⁺ /K ⁺ ATPase, indicative of function. The hydrated agarose-gelatine (AG) membranes allowed >96% of visible light to be transmitted. The membranes were typically -15 pm thick and did not swell greatly after immersion in PBS for 14 days. Tensile strength of the agarose-gelatine (AG) series of membrane ranged between 49-60 MPa, while young’s modulus varied between 525-709 MPa. The outstanding mechanical property of the agarose-gelatine (AG) membrane was vindicated when it was successfully transplanted in a mock endothelial keratoplasty procedure. This membrane thus offers great promise as a scaffold for CEC during endothelial keratoplasty or other biomedical applications.