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WO2019156630A1 - Procédés de production d'une membrane d'agarose modifié - Google Patents

Procédés de production d'une membrane d'agarose modifié Download PDF

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Publication number
WO2019156630A1
WO2019156630A1 PCT/SG2019/050069 SG2019050069W WO2019156630A1 WO 2019156630 A1 WO2019156630 A1 WO 2019156630A1 SG 2019050069 W SG2019050069 W SG 2019050069W WO 2019156630 A1 WO2019156630 A1 WO 2019156630A1
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Prior art keywords
membrane
agarose
glycine
modified
cell
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Inventor
Wei Yang Seow
William Sun
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Agency for Science Technology and Research Singapore
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Agency for Science Technology and Research Singapore
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0036—Galactans; Derivatives thereof
    • C08B37/0039—Agar; Agarose, i.e. D-galactose, 3,6-anhydro-D-galactose, methylated, sulfated, e.g. from the red algae Gelidium and Gracilaria; Agaropectin; Derivatives thereof, e.g. Sepharose, i.e. crosslinked agarose
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/12—Agar or agar-agar, i.e. mixture of agarose and agaropectin; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • C08L89/04—Products derived from waste materials, e.g. horn, hoof or hair
    • C08L89/06—Products derived from waste materials, e.g. horn, hoof or hair derived from leather or skin, e.g. gelatin

Definitions

  • the present invention relates generally to the field of biomaterial and synthetic biosystems.
  • the present invention relates to a modified agarose, a modified agarose membrane and methods to produce the modified agarose membrane.
  • 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.
  • 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.
  • 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.
  • Descemet stripping automated endothelial keratoplasty
  • DSAEK stripping automated endothelial keratoplasty
  • 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.
  • 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.
  • CEC comeal endothelial cells
  • 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.
  • 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.
  • 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:
  • the present invention refers to a membrane obtainable (or obtained) by the method as disclosed herein.
  • the present invention refers to a corneal implant comprising a membrane as disclosed herein.
  • the present invention refers to a comeal implant comprising a membrane obtainable (or obtained) by the method as disclosed herein.
  • the present invention refers to a physical supporting structure comprising a membrane as disclosed herein.
  • the present invention refers to a physical supporting structure comprising a membrane obtainable (or obtained) by the method as disclosed herein.
  • 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.
  • CDI l,r-carbonyldiimidazole
  • 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.
  • GRGD glycine-argin
  • Figure 1 illustrates chemical structures of the modified agarose described herein.
  • 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.
  • NMR nuclear magnetic resonance
  • 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
  • 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.
  • 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.
  • 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.
  • Figure 5 illustrates an example of a membrane that is optically transparent.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • RCEC rabbit comeal endothelial cells
  • 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.
  • 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.
  • 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.
  • agarose dissolves in hot water and yields a clear, rigid hydrogel upon cooling.
  • unaltered agarose does not natively support cell attachment nor cell adhesion.
  • agarose 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.
  • another ingredient e.g., bovine collagen I
  • 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.
  • photolabile groups 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the modified agarose comprises one or more compounds that can be an amino acid, a peptide, or a protein.
  • amino acid refers to an organic molecule that is made up of a basic amino group, an acidic carboxyl group and a side chain
  • 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.
  • 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.
  • the modified agarose comprises a cell attachment signal.
  • the modified agarose is an agarose covalently linked to a cell attachment signal.
  • the modified agarose is agarose that is modified by being covalently linked to a cell attachment signal.
  • the modified agarose is an agarose covalently linked to a cell attachment signal that is bound to a cell.
  • the modified agarose is agarose that is modified by being covalently linked to a cell attachment signal that is bound to a cell.
  • 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.
  • 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.
  • 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.
  • the short peptide comprises 4 amino acids.
  • the short peptide is glycine-arginine-glycine-aspartic acid (GRGD).
  • the cell attachment signal is selected from the group consisting of gelatine, glycine- arginine- glycine-aspartic acid (GRGD), lysine, poly-lysine, and mixture thereof.
  • 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.
  • 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.
  • the degree of conjugation comprises the cell attachment content in the modified agarose.
  • 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.
  • 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.
  • 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%.
  • 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%.
  • 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
  • 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%.
  • 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.
  • 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.
  • 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.
  • the glycine-arginine-glycine-aspartic acid (GRGD) to agarose ratio is or from 1:44 to 1:16.
  • 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.
  • 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.
  • GRGD glycine-arginine-glycine-aspartic acid
  • GRGD total weight of agarose-glycine-arginine-glycine-aspartic acid
  • AR total weight of agarose-glycine-arginine-glycine-aspartic acid
  • the weight of glycine-arginine-glycine-aspartic acid (GRGD) and a single repeat unit of glucose is about 402.4 and about 306 respectively.
  • 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%.
  • 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%.
  • the glycine-arginine-glycine-aspartic acid (GRGD) content in modified agarose is or from 2.90 wt% to 7.59 wt%.
  • 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%
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 1:889, or about 1:888, or about 1:313,
  • 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.
  • 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%, or from
  • 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%.
  • 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%.
  • 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 w
  • 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%.
  • the degree of conjugation of gelatine or gelatine content in the modified agarose is defined as the weight
  • 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%.
  • 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%.
  • 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%.
  • 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
  • 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%.
  • 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.
  • 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.
  • 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.
  • gelatine that is soluble in water or aqueous solution at room temperature can be used.
  • the gelatine is a gelatine that is soluble in water or aqueous solution at room temperature.
  • the gelatine is a fish-derived gelatine that is soluble in water or aqueous solution at room temperature.
  • 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.
  • 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.
  • the modified agarose is formed by covalently linking an agarose to a cell attachment signal.
  • Covalent linking can be accomplished via the use of one or more reagents.
  • the reagent can be a chemical.
  • 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 (CDI), carbodiimides, aminium salt, uranium salt, phosphonium salts, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) cyanogen bromide (CDI), carbodiimides, aminium salt, uranium salt, phosphonium salts, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) cyanogen bromide (CDI), car
  • the reagent to covalently link an agarose to a cell attachment signal to form a modified agarose is l, -carbonyldiimidazole (CDI).
  • the covalently linking an agarose to a cell attachment signal to form a modified agarose is by l,r-carbonyldiimidazole (CDI)-mediated coupling.
  • l,l’-carbonyldiimidazole (CDI) is advantageous as it is specific in its chemistry and will not result in unwanted reactions.
  • l,l’-carbonyldiimidazole 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.
  • the membrane comprises the modified agarose as disclosed 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.
  • 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%.
  • 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.
  • 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.
  • 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).
  • GRGD glycine-arginine-glycine- aspartic acid
  • 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.
  • 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.
  • 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.
  • the cell can be a prokaryote or eukaryote cell.
  • the cell can be adherent or free-floating.
  • 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.
  • 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.
  • the cell can be, but is not limited to, human dermal fibroblast cell (NHDF), comeal endothelial cell (CEC) or kidney cell.
  • the membrane can remain in a dehydrated form or in a hydrated form.
  • the membrane can be hydrated in an aqueous solution.
  • 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).
  • PBS phosphate buffered saline
  • TBS Tris buffered saline
  • TAE Tris-acetate-EDTA
  • TBE Tris-botate-EDTA
  • the membrane 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the membrane is optically transparent.
  • optically transparent refers to the physical property of allowing light to pass through the material without being scattered.
  • 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.
  • the membrane that is optically transparent allows greater than 90% of visible light to pass through.
  • the membrane that is optically transparent allows greater than 96% of visible light to pass through.
  • the membrane 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • the tensile strength of the membrane is from 40 MPa to 60 MPa.
  • 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).
  • 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.
  • 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.
  • the Young’s modulus of the membrane is from 525 MPa to 709 MPa.
  • 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.
  • the membrane as described herein can be used for other purposes or for developing other applications.
  • 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.
  • 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.
  • 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.
  • the membrane can be used for supporting cell attachment and growth.
  • the membrane can be used to grow corneal endothelial cells.
  • 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.
  • 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.
  • the membrane can be used in a corneal implant.
  • the corneal implant comprises the membrane as disclosed herein.
  • the corneal implant comprises the membrane obtainable or obtained by the method as disclosed herein.
  • the membrane can be used as a physical supporting structure.
  • 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.
  • the physical supporting structure comprises the membrane as disclosed herein.
  • the physical supporting structure comprises the membrane obtainable or obtained by the method as disclosed herein.
  • 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.
  • the methods of manufacturing the membrane comprise multiple steps.
  • 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.
  • 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.
  • the membrane is obtainable (or obtained) by the method as disclosed herein.
  • 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.
  • 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.
  • the method after step a) and before step b) can further comprise a precipitation step al) to extract the modified agarose.
  • 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.
  • the modified agarose can be precipitated by alcohol or water.
  • the modified agarose can be precipitated using for example, ethanol or any other solution in the amount leading to the same effect.
  • 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.
  • 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.
  • the method after step a) and before step b) can further comprise step a3) dissolving the modified agarose in water to form a solution.
  • 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.
  • the method after step a) and before step b) can further comprise step a4) solidifying the modified agarose to form a modified agarose gel.
  • 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.
  • 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.
  • 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.
  • CDI l,r-carbonyldiimidazole
  • GRGD glycine-arginine-glycine- aspartic acid
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method of manufacturing a membrane comprising a modified agarose comprises b) dissolving the modified agarose to form a solution.
  • the modified agarose is dissolved in a liquid or aqueous solution.
  • the modified agarose is dissolved in a liquid or aqueous solution at a suitable temperature to form a solution.
  • 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).
  • PBS phosphate buffered saline
  • TBS Tris buffered saline
  • TBE Tris-acetate-EDTA
  • TBE Tris-botate-EDTA
  • the method of manufacturing a membrane comprising a modified agarose comprises b) dissolving the modified agarose in water to form a solution.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • gelation concentration refers to a concentration of a solution, above which, the solution is able to solidify and form a gel or a hydrogel.
  • 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
  • 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,
  • the method of manufacturing the membrane comprises the step of c) solidifying the solution to form a modified agarose hydrogel.
  • the term“hydrogel” refers to a macromolecular gel constructed of a network of crosslinked polymer chains.
  • 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.
  • the solidifying in c) is by cooling.
  • the solution is left to cool to solidify to form a modified agarose hydrogel.
  • 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.
  • 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.
  • the temperature can be room temperature, which is generally known in the art that room temperature can be from 20°C to 25°C.
  • 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.
  • the method of manufacturing the membrane comprises the step of d) dehydrating the modified agarose hydrogel, thereby forming the membrane comprising the modified agarose.
  • the membrane can then be formed by dehydrating the modified agarose hydrogel.
  • 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.
  • 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.
  • 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.
  • 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.
  • the temperature for dehydrating is about 60°C.
  • 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.
  • 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.
  • 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.
  • the length of time for dehydrating in d) is about 48 hours.
  • 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.
  • the singular form“a,”“an,” and“the” include plural references unless the context clearly dictates otherwise.
  • the term“a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof.
  • 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.
  • 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.
  • 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).
  • 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.
  • DMSO dimethyl sulfoxide
  • AK agarose-lysine
  • 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.
  • CDI l,l’-carbonyldiimidazole
  • DMSO dimethyl sulfoxide
  • 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.
  • 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).
  • agarose-gelatine 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.
  • 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.
  • 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).
  • 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.
  • Jeol JSM-7400F Tokyo, Japan
  • 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.
  • RCEC primary rabbit comeal endothelial cells
  • RCEC Rabbit comeal endothelial cells
  • NHDF normal human dermal fibroblasts
  • ATCC normal rabbit kidney cells
  • calcein-AM Invitrogen was added to identify live cells and images were obtained with an IX-83 inverted fluorescence microscope (Olympus, Japan).
  • 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).
  • DAPI 4',6-diamidino-2- phenylindole dihydrochloride
  • 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.
  • 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.
  • Agarose has an outstanding mechanical property and was therefore selected as a base material to ensure the structural integrity of ultra-thin membranes.
  • 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).
  • GRGD glycine-arginine-glycine-aspartic acid
  • AR glycine-arginine-glycine-aspartic acid
  • AK agarose-lysine
  • AP polylysine
  • fish-derived gelatine resulting in agarose-gelatine (AG) as a product
  • the modified agarose must be activated by, for example, carbonyldiimidazole (CDI) before conjugation.
  • CDI carbonyldiimidazole
  • the hydroxyl groups on agarose became susceptible to attacks by nucleophiles.
  • 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.
  • AR1, AR2, AR3 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.
  • GRGD conjugation degree
  • AR2 (1 glycine-arginine-glycine-aspartic acid (GRGD) : 28 repeat units of agarose)
  • AR3 (1 glycine-arginine-glycine-aspartic acid (GRGD) : 16 repeat units of agarose) in ascending order.
  • 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.
  • TFA trifluoroacetic acid
  • AK1, AK2, AK3, AK4 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).
  • 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.
  • 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).
  • Figure 10B the order-of-magnitude was not observed to drop in mechanical property post-conjugation, as reported by another group studying modified agarose.
  • 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.
  • 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).
  • GRGD glycine-arginine-glycine-aspartic acid
  • AR asine-glycine-aspartic acid
  • AK agarose-lysine
  • AP polylysine
  • fish-derived gelatine giving agarose-gelatine (AG) as product.
  • the advantages of fish-derived gelatine over mammal-derived sources were discussed. For optimization purposes,
  • 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.

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Abstract

La présente invention concerne une membrane ayant une teneur en eau de moins de 15 %, comprenant un agarose modifié qui est lié de manière covalente à un signal de fixation de cellule, tel qu'un acide aminé, un peptide et une fraction chimique, que les cellules reconnaissent et avec lequel elles interagissent afin que les cellules adhèrent à la membrane. Dans un mode de réalisation, le signal de fixation de cellule conjugué à l'agarose est choisi dans un groupe consistant en la gélatine issue de poisson, le peptide glycine-arginine-glycine-acide aspartique (GRGD), la lysine, la poly-lysine et un mélange de ceux-ci. L'invention concerne également des procédés de fabrication de la membrane comprenant : la liaison covalente d'un agarose à un signal de fixation de cellule ; la dissolution de l'agarose modifié dans de l'eau pour former une solution ; la solidification de la solution d'agarose modifié pour former un hydrogel ; et la déshydratation de l'hydrogel d'agarose modifié. L'invention concerne également des applications de la membrane en tant qu'échafaudage pour la fixation et la croissance de cellules telles que des cellules endothéliales cornéennes (CEC) pour une transplantation cornéenne.
PCT/SG2019/050069 2018-02-06 2019-02-07 Procédés de production d'une membrane d'agarose modifié Ceased WO2019156630A1 (fr)

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CN117815447A (zh) * 2023-12-04 2024-04-05 中国海洋大学 一种琼脂糖基人工角膜及其制备方法

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US20080286360A1 (en) * 2007-04-05 2008-11-20 Molly Shoichet Chemically patterned hydrogels, manufacture and use thereof
WO2013040559A1 (fr) * 2011-09-16 2013-03-21 Wake Forest University Health Sciences Fabrication de feuille d'hydrogel de gélatine pour transplantation d'endothélium cornéen
EP2735318A1 (fr) * 2012-11-26 2014-05-28 Albert-Ludwigs-Universität Freiburg Matrices comportant des polysaccharides modifiés et polysaccharides modifiés

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US20080286360A1 (en) * 2007-04-05 2008-11-20 Molly Shoichet Chemically patterned hydrogels, manufacture and use thereof
WO2013040559A1 (fr) * 2011-09-16 2013-03-21 Wake Forest University Health Sciences Fabrication de feuille d'hydrogel de gélatine pour transplantation d'endothélium cornéen
EP2735318A1 (fr) * 2012-11-26 2014-05-28 Albert-Ludwigs-Universität Freiburg Matrices comportant des polysaccharides modifiés et polysaccharides modifiés

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LUO Y. ET AL.: "A photolabile hydrogel for guided three- dimensional cell growth and migration", NAT MATER, vol. 3, no. 4, 21 March 2004 (2004-03-21), pages 249 - 253, XP055018575, [retrieved on 20190429], doi:10.1038/nmat1092 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117815447A (zh) * 2023-12-04 2024-04-05 中国海洋大学 一种琼脂糖基人工角膜及其制备方法
CN117815447B (zh) * 2023-12-04 2025-07-01 中国海洋大学 一种琼脂糖基人工角膜及其制备方法

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