WO2019211873A2

WO2019211873A2 - A liquid cornea composition

Info

Publication number: WO2019211873A2
Application number: PCT/IN2019/050352
Authority: WO
Inventors: Tuhin BHOWMICK; Arun CHANDRU; Tanmay GHARAT; Vivek Singh; Sayan BASU; Virender S SANGWAN
Original assignee: Pandorum Technologies Private Limited; Hyderabad Eye Research Foundation
Priority date: 2018-05-02
Filing date: 2019-05-02
Publication date: 2019-11-07
Also published as: WO2019211873A3

Abstract

The present disclosure discloses a Liquid Cornea composition comprising: at least one polymer; an antifibrinolytic agent; decellularized extracellular matrix; human corneal cell suspension; at least one extracellular matrix cross-linker; and calcium chloride (CaCl2). Also provided is a method for preparing the liquid cornea composition. The present disclosure also discloses a process for obtaining the decellularized extracellular matrix.

Description

A LIQUID CORNEA COMPOSITION

FIELD OF INVENTION

[001] The present disclosure relates to the field of biological hydrogels in general and corneal hydrogels in particular. There is provided a Liquid Cornea composition for biomedical application.

BACKGROUND OF INVENTION

[002] The organ eye in an organism represent the visual system and performs various photo sensory functions.

[003] The cornea is the outermost layer of the eye appearing as a transparent membrane- like tissue. The primary function of the cornea is to help focus vision and therefore it plays an important role in sight. Although it appears to possess a simplified tissue structure, this tissue is comprised of multiple layers.

[004] The layers of the cornea are sequentially: the epithelium, Bowman’s membrane, Stroma, Descemet's Membrane, and Endothelium. Each of the tissue layers comprise different cell types. The maintenance of this tissue relies on a regular supply of nutrients from tear fluid from the aqueous humour.

[005] The cornea can be affected by trauma, infection and several diseases such as, corneal abrasion, corneal dystrophy, corneal ulcer , corneal neovascularization , Fuchs' dystrophy, keratitis , keratoconus among others. These conditions can lead to temporary or complete blindness and are among the leading causes of blindness in the world.

[006] Some of the commonly used procedures for the treatment of corneal diseases include laser surgery, corneal transplant surgery, anterior lamellar keratoplasty, endothelial lamellar keratoplasty, and the use of artificial corneas. These treatments involve the replacement of a part or whole of the cornea. The healing of the cornea after these treatments is often compromised and thus, research is on-going to find better and effective alternatives.

SUMMARY OF INVENTION

[007] In an aspect of the present disclosure, there is provided a Liquid Cornea composition comprising: (a) at least one polymer; (b) an antifibrinolytic agent; (c) decellularized extracellular matrix; (d) human corneal cell suspension; (e) at least one extracellular matrix cross-linker; and (f) calcium chloride (CaCL).

[008] In an aspect of the present disclosure, there is provided a process for the preparation of a Liquid Cornea composition comprising: (i) at least one polymer; (ii) an antifibrinolytic agent; (iii) decellularized extracellular matrix; (iv) human corneal cell suspension; (v) at least one extracellular matrix cross-linker; and (vi) calcium chloride (CaCL), said process comprising: (a) obtaining the at least one polymer, the antifibrinolytic agent, decellularized extracellular matrix, the human corneal cell suspension, the at least one extracellular matrix cross-linker, and CaCL: and (b) contacting the polymers, the antifibrinolytic agent, the decellularized extracellular matrix, the human corneal cell suspension, the extracellular matrix cross-linkers, and CaCL to obtain a mixture; and (c) curing the mixture to obtain the Liquid Cornea composition.

[009] In an aspect of the present disclosure, there is provided a process to obtain a decellularized extracellular matrix (dECM) of at least one biological material, said process comprising: (a) processing at least one biological material, to obtain a processed biological material; (b) subjecting the processed biological material to cutting, to obtain pieces ranging in 1 to 10 mm diameter in size; (c) subjecting the pieces to freeze-milling for a time period in a range of 5-15 minutes, to obtain a mixture I; (d) subjecting the mixture-I to thawing to attain a temperature in a range of 25-30°C, to obtain a paste I; (e) repeating a combination of step (c) and (d) for at least two times, to obtain a paste-II; (f) subjecting the paste-II to a freeze-drying step to obtain a mass; and (g) subjecting the mass to cutting, followed by freeze-milling for a time period in a range of 5-15 minutes, to obtain the decellularized extracellular matrix of the at least one biological material.

[0010] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

[0011] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0012] Figure 1 depicts the validation of the decellularization procedure done by performing DAPI and H&E staining on sections of the decellularized tissues with untreated cornea tissues as a control, in accordance with an embodiment of the present disclosure.

[0013] Figure 2 depicts the Dynamic Light Scattering (DLS) graph for the human cornea decellularized extracellular matrix (CdECM) obtained by process PP1 and PP2, in accordance with an embodiment of the present disclosure.

[0014] Figure 3 depicts the Scanning Electron Micrograph (SEM) images for the human cornea decellularized extracellular matrix (CdECM) obtained by process PP1 (Figure 3A) and PP2 (Figure 3B), in accordance with an embodiment of the present disclosure.

[0015] Figure 4 depicts the SEM imaging at higher magnification confirming the presence of aligned collagen fibrils native to the corneal tissue in the CdECM powder particles obtained from physical processing (PP1), in accordance with an embodiment of the present disclosure.

[0016] Figure 5 depicts comparison of average size of CdECM particles obtained by PP1 and PP2 process, in accordance with an embodiment of the present disclosure.

[0017] Figure 6 depicts the SEM images for Cornea dECM processed with enzymatic digestion (extreme left) along with the elemental analysis performed on the CdECM particle to confirm the organic origin of the tissue microparticles and rule out the presence of salt/inorganic particles, in accordance with an embodiment of the present disclosure.

[0018] Figure 7 depicts transmittance studies conducted for 1 mg/ml suspension of CdECM powders prepared using physical process 1 , physical process 2 and enzymatic digestion (left) and for fibrin and Liquid Cornea hydrogels prepared using CdECM from the different processing steps (right), in accordance with an embodiment of the present disclosure.

[0019] Figures 8 and 9 show the process flow for forming the Liquid Cornea composition by contacting the extra cellular matrix powder with the extracellular matrix cross linkers and polymers, respectively, in accordance with an embodiment of the present disclosure.

[0020] Figures 10 and 11 show the process flow for forming the Liquid Cornea composition with a suspension of extra cellular matrix powder in buffer or saline solution, in accordance with an embodiment of the present disclosure.

[0021] Figure 12 shows the biodegradation profile for hydrogels fabricated from corneal stromal stem cell (CSSCs) encapsulated in hydrogels containing Fibrin alone and Liquid Cornea hydrogels

[0022] Figure 13 shows the Live-Dead staining performed on CSSCs encapsulated in Fibrin hydrogel (A) and Liquid Cornea hydrogel (B). Scale Bar - 275 pm, in accordance with an embodiment of the present disclosure. [0023] Figure 14 shows the IF staining for four markers namely CD73, CD90 (red) and a-SMA, Collagen type 1 (green) performed for CSSCs encapsulated in hydrogels containing fibrin and fibrin with CdECM and cultured for three days. Nuclear staining was performed with DAPI. Samples were recovered day 1 and 3 post encapsulation. Scale - 100 pm, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0024] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions

[0025] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0026] The articles“a”,“an” and“the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0027] The terms“comprise” and“comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0028] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as“comprises” and“comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0029] The term “including” is used to mean “including but not limited to”. “Including” and“including but not limited to” are used interchangeably.

[0030] The term“freeze-milling” is defined as an act of cooling a material and then reducing the material to small size. The cooling can be effectuated by liquid nitrogen, dry ice, or any other well-known means. The term“freeze-drying” is synonymous to lyophilisation. The process involves freezing the product to low temperatures and removing the ice by sublimation. The term“thaw” or“thawing” refers to the step of providing heat to any frozen substance so that it attains a desired state. The term “cornea” has been used to refer to the cornea obtained from cadaver. The term“room temperature” refers to the temperature in a range of 22-30°C. The term“curing” refers to a chemical process that leads to cross-linking of polymer chains to obtain a final polymerized product. The term“human corneal cell suspension” is intended to comprise human corneal cells, it is contemplated to include corneal stem cell also. One of the human corneal cell suspension is corneal stromal stem cell. The term“an antifibrinolytic agent” is intended to refer to at least one antifibrinolytic agent, or combinations of antifibrinolytic agent.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0032] Partial or complete corneal implants are one of the most successful therapies for the treatment of corneal diseases. However, in situations where a complete corneal replacement is not required, several compositions from natural and synthetic origins are used to fill the holes in the cornea. These compositions are collectively known as ocular sealants. Cyanoacrylate, collagen and fibrinogen-based gels are few of the most widely used compositions. The constituting components of both of these compositions are not naturally present in the cornea; both the compositions do not assimilate with the cornea and hence degrade/dissolve in longer term till the keratocytes present in the cornea secrete enough extracellular material to fill the holes.

[0033] The present disclosure provides a solution to the aforementioned problem in the form a Liquid Cornea composition which utilizes a unique composition of at least one polymer, antifibrinolytic agent, human cornea decellularized extracellular matrix (CdECM), human corneal cell suspension, at least one extracellular matrix cross-linker and CaCh, that facilitates the growth and regeneration of the cornea using biologically active ingredients. Amongst others, the composition can comprise a solution I and solution II. In one of the embodiments of the present disclosure, solution I consists of fibrinogen and aprotinin and solution II consists of thrombin, corneal cell suspension and calcium chloride. The CdECM powder or suspension can be mixed with either the solution I or solution II. Next, the solution I and the solution II is mixed in a 1: 1 ratio to obtain the Liquid Cornea composition. The composition can be cast as disc on flat surface or concave surface to mimic the curvature of the eye. The composition can be cast into any controlled shape and can be employed to fill void or a defect on the surface or inside of the cornea.

[0034] The following paragraphs depict the embodiments of the claimed Liquid Cornea composition. Additionally, the processes for preparing the composition is also depicted. However, a person skilled in the art can employ conditions as per his need and prepare the composition based on the representative examples, and such processes would fall within the scope of the present invention.

[0035] The Liquid Cornea composition as disclosed herein, comprises polymers, antifibrinolytic agent, decellularized extracellular matrix, human corneal cell suspension, extracellular matrix cross-linkers and CaCb.

[0036] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0037] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.

[0038] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition comprising: (a) polymers; (b) an antifibrinolytic agent; (c) decellularized extracellular matrix; (d) human corneal cell suspension; (e) extracellular matrix cross linkers; and (f) calcium chloride (CaCb). In another embodiment of the present disclosure, the decellularized extracellular matrix is a powder. In yet another embodiment of the present disclosure, the decellularized extracellular matrix is a suspension prepared in an isotonic buffer or saline solution.

[0039] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition comprising: (a) at least one polymer; (b) an antifibrinolytic agent; (c) decellularized extracellular matrix; (d) human corneal cell suspension; (e) at least one extracellular matrix cross-linker; and (f) calcium chloride (CaCb). In another embodiment of the present disclosure, the decellularized extracellular matrix is a powder. In yet another embodiment of the present disclosure, the decellularized extracellular matrix is a suspension prepared in an isotonic buffer or saline solution.

[0040] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition comprising: (a) at least one polymer; (b) an antifibrinolytic agent; (c) human corneal decellularized extracellular matrix; (d) human corneal cell suspension; (e) at least one extracellular matrix cross-linker; and (f) calcium chloride (CaCb).

[0041] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the at least one polymer has a concentration in a range of 17 to 110 mg/ml with respect to the composition. In another embodiment of the present disclosure, the at least one polymer has a concentration in a range of 25 to 100 mg/ml with respect to the composition. In yet another embodiment of the present disclosure, the at least one polymer has a concentration in a range of 35 to 95 mg/ml with respect to the composition. In one another embodiment of the present disclosure, the at least one polymer has a concentration in a range of 40 to 93 mg/ml with respect to the composition. In an alternate embodiment of the present disclosure, the at least one polymer has a concentration in a range of 42 to 92 mg/ml with respect to the composition.

[0042] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the at least one polymer is selected from the group consisting of fibrinogen, collagen and silk. In another embodiment of the present disclosure, the polymer is fibrinogen. In yet another embodiment, the polymer is collagen. In an alternate embodiment, the polymer is silk.

[0043] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the antifibrinolytic agent has a concentration in a range of 600 to 4000 KlU/ml with respect to the composition. In another embodiment of the present disclosure, the antifibrinolytic agent has a concentration in a range of 800 to 3800 KlU/ml with respect to the composition. In yet another embodiment of the present disclosure, the antifibrinolytic agent has a concentration in a range of 1000 to 3700 KlU/ml with respect to the composition. In an alternate embodiment of the present disclosure, the antifibrinolytic agent has a concentration in a range of 1200 to 3500 KlU/ml with respect to the composition. In one another embodiment of the present disclosure, the antifibrinolytic agent has a concentration in a range of 1300 to 3500 KlU/ml with respect to the composition.

[0044] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the antifibrinolytic agent is selected from a group consisting of aprotinin, e-aminocaproic acid, tranexamic acid, and combinations thereof. [0045] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the antifibrinolytic agent is aprotinin.

[0046] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the decellularized extracellular matrix has a concentration in a range of 7.5 to 60 mg/ml with respect to the composition. In another embodiment of the present disclosure, the decellularized extracellular matrix has a concentration in a range of 7.5 to 45.5 mg/ml with respect to the composition. In yet another embodiment of the present disclosure, the decellularized extracellular matrix has a concentration in a range of 7.5 to 55 mg/ml with respect to the composition. In an alternate embodiment of the present disclosure, the decellularized extracellular matrix has a concentration in a range of 10 to 45 mg/ml with respect to the composition. In one another embodiment of the present disclosure, the decellularized extracellular matrix has a concentration in a range of 12 to 35 mg/ml with respect to the composition.

[0047] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the decellularized extracellular matrix (dECM) has a concentration in a range of 8% to 57% dECM mass/total polymer mass of the composition. In another embodiment of the present disclosure, the decellularized extracellular matrix (dECM) has a concentration in a range of 10% to 50% dECM mass/total polymer mass of the composition. In yet another embodiment of the present disclosure, the decellularized extracellular matrix (dECM) has a concentration in a range of 12% to 45% dECM mass/total polymer mass of the composition. In an alternate embodiment of the present disclosure, the decellularized extracellular matrix (dECM) has a concentration in a range of 13% to 40% dECM mass/total polymer mass of the composition. In one other embodiment of the present disclosure, the decellularized extracellular matrix (dECM) has a concentration in a range of 13% to 35% dECM mass/total polymer mass of the composition.

[0048] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the decellularized extracellular matrix is a human corneal decellularized extracellular matrix. [0049] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human corneal cell suspension has a concentration in a range of 5x 10⁶ to lOx 10⁶ cells/ml with respect to the composition. In another embodiment of the present disclosure, the human corneal cell suspension has a concentration in a range of 5xl0⁶ to 9xl0⁶ cells/ml with respect to the composition. In yet another embodiment of the present disclosure, the human corneal cell suspension has a concentration in a range of 5xl0⁶ to 8xl0⁶ cells/ml with respect to the composition. In one another embodiment of the present disclosure, the human corneal cell suspension has a concentration in a range of 6xl0⁶ to 9xl0⁶ cells/ml with respect to the composition. In an alternate embodiment of the present disclosure, the human corneal cell suspension has a concentration in a range of 6xl0⁶ to 8xl0⁶ cells/ml with respect to the composition.

[0050] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human corneal cell suspension comprises human keratocytes.

[0051] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the extracellular matrix cross-linkers have a concentration in a range of 90 to 625 IU/ml with respect to the composition. In another embodiment of the present disclosure, the extracellular matrix cross-linkers have a concentration in a range of 125 to 600 IU/ml with respect to the composition. In yet another embodiment of the present disclosure, the extracellular matrix cross-linkers have a concentration in a range of 150 to 600 IU/ml with respect to the composition. In an alternate embodiment of the present disclosure, the extracellular matrix cross linkers have a concentration in a range of 175 to 550 IU/ml with respect to the composition. In one another embodiment of the present disclosure, the extracellular matrix cross-linkers have a concentration in a range of 200 to 550 IU/ml with respect to the composition

[0052] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the extracellular matrix cross-linkers is selected from a group consisting of thrombin, trilysine acetate, polyethylene amine, polyvianyl alcohol vinylamine, and combinations thereof. In another embodiment of the present disclosure, the extracellular matrix cross-linker is thrombin.

[0053] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein CaCb has a concentration in a range of 7.5 to 50 mM with respect to the composition. In another embodiment of the present disclosure, CaCb has a concentration in a range of 10 to 45 mM with respect to the composition. In yet another embodiment of the present disclosure, CaCL has a concentration in a range of 15 to 45 mM with respect to the composition. In an alternate embodiment of the present disclosure, CaCb has a concentration in a range of 17 to 42 mM with respect to the composition.

[0054] In an embodiment of the present disclosure, there is provided a process for the preparation of a Liquid Cornea composition comprising: (i) polymers; (ii) an antifibrinolytic agent; (iii) decellularized extracellular matrix; (iv) human corneal cell suspension; (v) extracellular matrix cross-linkers; and (vi) calcium chloride (CaCb), said process comprising: (a) obtaining the polymers, the antifibrinolytic agent, the decellularized extracellular matrix, the human corneal cell suspension, the extracellular matrix cross-linkers, and CaCb; and (b) contacting the polymers, the antifibrinolytic agent, the decellularized extracellular matrix, the human corneal cell suspension, the extracellular matrix cross-linkers, and CaCb to obtain a mixture; and (c) curing the mixture to obtain the Liquid Cornea composition.

[0055] In an embodiment of the present disclosure, there is provided a process for the preparation of a Liquid Cornea composition comprising: (i) at least one polymer; (ii) an antifibrinolytic agent; (iii) decellularized extracellular matrix; (iv) human corneal cell suspension; (v) at least one extracellular matrix cross-linker; and (vi) calcium chloride (CaCb), said process comprising: (a) obtaining the at least one polymer, the antifibrinolytic agent, the decellularized extracellular matrix, the human corneal cell suspension, the at least one extracellular matrix cross-linkers, and CaCb; and (b) contacting the at least one polymer, the antifibrinolytic agent, the decellularized extracellular matrix, the human corneal cell suspension, the extracellular matrix cross linkers, and CaCb to obtain a mixture; and (c) curing the mixture to obtain the Liquid Cornea composition.

[0056] In an embodiment of the present disclosure, there is provided a process for the preparation of a Liquid Cornea composition comprising: (i) at least one polymer; (ii) an antifibrinolytic agent; (iii) decellularized extracellular matrix; (iv) human corneal cell suspension; (v) at least one extracellular matrix cross-linker; and (vi) calcium chloride (CaCb), said process comprising: (a) obtaining a solution I comprising the at least one polymer, and the antifibrinolytic agent; (b) obtaining a solution II comprising the decellularized extracellular matrix, the at least one extracellular matrix cross-linker, the human corneal cell suspension and CaCb; (c) contacting the solution I and the solution II in a ratio range of 50: 1 to 1 : 50 to obtain a mixture; and (d) curing the mixture to obtain the Liquid Cornea composition. In another embodiment of the present disclosure, contacting the solution I and the solution II is done in a ratio range of 40: 1 to 1 :40 to obtain the mixture. In yet another embodiment of the present disclosure, contacting the solution I and the solution II is done in a ratio range of 30: 1 to 1 :30 to obtain the mixture. In an alternate embodiment of the present disclosure, contacting the solution I and the solution II is done in a ratio range of 20: 1 to 1 :20 to obtain the mixture. In one another embodiment of the present disclosure, contacting the solution I and the solution II is done in a ratio range of 10: 1 to 1: 10 to obtain the mixture. In a further embodiment, contacting the solution I and the solution II is done in a ratio of 1 : 1 to obtain the mixture.

[0057] In an embodiment of the present disclosure, there is provided a process for the preparation of the Liquid Cornea composition comprising: (i) at least one polymer; (ii) an antifibrinolytic agent; (iii) decellularized extracellular matrix; (iv) human corneal cell suspension; (v) at least one extracellular matrix cross-linker; and (vi) calcium chloride (CaCb), said process comprising: (a) obtaining a solution I comprising the at least one polymer, the antifibrinolytic agent and the decellularized extracellular matrix; (b) obtaining a solution II comprising the at least one extracellular matrix cross-linker, the human corneal cell suspension and CaCh; (c) contacting the solution I and the solution II in a ratio range of 50: 1 to 1 : 50 to obtain a mixture; and (d) curing the mixture to obtain the Liquid Cornea composition. In another embodiment of the present disclosure, contacting the solution I and the solution II is done in a ratio range of 40: 1 to 1 :40 to obtain the mixture. In yet another embodiment of the present disclosure, contacting the solution I and the solution II is done in a ratio range of 30: 1 to 1 :30 to obtain the mixture. In an alternate embodiment of the present disclosure, contacting the solution I and the solution II is done in a ratio range of 20: 1 to 1:20 to obtain the mixture. In one another embodiment of the present disclosure, contacting the solution I and the solution II is done in a ratio range of 10: 1 to 1: 10 to obtain the mixture. In a further embodiment, contacting the solution I and the solution II is done in a ratio of 1 : 1 to obtain the mixture.

[0058] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the composition is used for treating corneal diseases.

[0059] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the composition is used for treating corneal diseases selected from a group consisting of corneal abrasion, corneal dystrophy, corneal ulcer, corneal neovascularization, Fuchs' dystrophy, keratitis, and keratoconus.

[0060] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the at least one polymer further contains a stabilizing factor.

[0061] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the at least one polymer further contains a stabilizing factor Human Factor XIII.

[0062] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human corneal cells are selected from the group consisting of corneal epithelial cells, corneal stromal cells, corneal endothelial cells, limbal epithelial cells, human keratocytes, mesenchymal stem cells and combinations thereof.

[0063] In an embodiment of the present disclosure, there is provided a process for the preparation of a Liquid Cornea composition as described herein, wherein curing is done for a time period in a range of 10 seconds to 60 seconds. In another embodiment, curing is done for a time period in a range of 20 seconds to 50 seconds. In yet another embodiment, curing is done for a time period in a range of 30 seconds to 60 seconds.

[0064] In an embodiment of the present disclosure, there is provided a process for the preparation of a Liquid Cornea composition as described herein, wherein curing the mixture is for up to a minute.

[0065] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition comprising: (a) at least one polymer; (b) an antifibrinolytic agent; (c) human corneal decellularized extracellular matrix; (d) human corneal cell suspension; (e) at least one extracellular matrix cross-linker; and (f) calcium chloride (CaCL), wherein the human corneal decellularized extracellular matrix is obtained by a process comprising: (i) processing cornea obtained from cadaver, to obtain a processed corneal sample; (ii) optionally subjecting the processed corneal sample to cutting, to obtain pieces ranging in 1 to 10 mm diameter in size; (iii) subjecting the pieces to freeze- milling for a time period in a range of 5-15 minutes, to obtain a mixture I; (iv) subjecting the mixture-I to thawing to attain a temperature in a range of 25-30°C, to obtain a paste I; (v) repeating a combination of step (c) and (d) for at least two times, to obtain a paste-II; (vi) subjecting the paste-II to a freeze-drying step to obtain a mass; and (vii) subjecting the mass to cutting, followed by freeze-milling for a time period in a range of 5-15 minutes, to obtain decellularized extracellular matrix of the at least one biological material. In another embodiment, processing comprises at least one step of: (a) saline wash; (b) DNase treatment; (c) PBS treatment; (d) antibiotic treatment; (e) washing; and (f) combinations thereof. In yet another embodiment, cutting yields pieces ranging in 2 to 5 mm diameter in size. In one alternate embodiment, cutting yields pieces ranging in 1 to 4 mm diameter in size. [0066] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human cornea decellularized extracellular matrix is processed using physical methods that includes first, freeze milling the decellularized corneal tissues with the inherent water content in the presence of liquid nitrogen followed by lyophilization to get a spongy, fibrous mass that is freeze milled again to get the decellularized extracellular matrix powder.

[0067] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using physical methods including freeze-drying and freeze milling in combination with enzymatic digestion and dialysis.

[0068] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using enzymatic digestion and the enzyme used is pepsin.

[0069] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using pepsin enzymatic digestion and the pepsin is immobilized on agarose beads.

[0070] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using pepsin enzymatic digestion and the solvent used for enzymatic digestion is acetic acid solution in water.

[0071] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using pepsin enzymatic digestion and the concentration of the acetic acid for enzymatic digestion is 8.5 M.

[0072] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using pepsin enzymatic digestion and the pH of the acetic acid solution for enzymatic digestion is 4.5. [0073] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using pepsin enzymatic digestion and the pH of the acetic acid for enzymatic digestion is 4.5 and concentration of the immobilized pepsin-bead slurry is 0.025 ml slurry per mg of the freeze-dried decellularized extracellular matrix powder.

[0074] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using pepsin enzymatic digestion and the enzymatic digestion is carried out for 72 hours at 37°C.

[0075] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using pepsin enzymatic digestion and the digested suspension is dialyzed using a cellulose ester (CE) membrane with MWCO 0.1 - 1 KDa.

[0076] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using pepsin enzymatic digestion and the digested suspension is dialyzed against distilled water for 48 hours at room temperature.

[0077] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using freeze milling.

[0078] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using physical methods including: freeze-drying in combination with freeze milling.

[0079] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is processed using treatments with salt solutions which include sodium chloride solution and detergents which include Triton-X, Sodium dodecyl sulphate, sodium lauryl sulphate, polyethylene glycol, and glyceryl laurate. [0080] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix has a particle size in the range of 3-10 pm.

[0081] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the polymers, human decellularized extracellular matrix and extracellular matrix cross-linkers are obtained in freeze-dried form and are reconstituted with a solvent for use in the method for the preparation of the Liquid Cornea composition.

[0082] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the Liquid Cornea composition optionally comprises a buffer solution. In yet another embodiment of the present disclosure, the buffer solution is Phosphate Buffer Saline (PBS). In one of the embodiment of the present disclosure, the buffer solution is Phosphate Buffer Saline having a concentration in the range of 0.4 to 0.6X.

[0083] In an embodiment of the present disclosure, there is provided a process for the preparation of a Liquid Cornea composition as described herein, wherein the decellularized extracellular matrix post-milling is sterilized using exposure to UV radiation for 30 minutes in a class II laminar hood.

[0084] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the Liquid Cornea composition is in the shape of circular disc or any other controlled shape that could be used to fill a void or defect on the surface or inside the cornea.

[0085] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition described herein, wherein the Liquid Cornea composition is in the shape of circular disc or any other controlled shape with a thickness of < 1 mm.

[0086] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition described herein, wherein the Liquid Cornea composition is in the shape of circular disc or any other controlled shape on a flat surface or a convex surface that mimics the curvature of the human cornea.

[0087] In an embodiment of the present disclosure, there is provided a Liquid Cornea composition as described herein, wherein the human decellularized extracellular matrix is prepared under sterile conditions prior to use including exposure to UV radiation for 30 minutes in a class II laminar hood.

[0088] In an embodiment of the present disclosure, there is provided a process to obtain a decellularized extracellular matrix (dECM) of at least one biological material, said process comprising: (a) processing at least one biological material, to obtain a processed biological material; (b) optionally subjecting the processed biological material to cutting, to obtain pieces ranging in 1 to 10 mm diameter in size; (c) subjecting the pieces to freeze-milling for a time period in a range of 5-15 minutes, to obtain a mixture I; (d) subjecting the mixture-I to thawing to attain a temperature in a range of 25-30°C, to obtain a paste I; (e) repeating a combination of step (c) and (d) for at least two times, to obtain a paste-II; (f) subjecting the paste-II to a freeze-drying step to obtain a mass; and (g) subjecting the mass to cutting, followed by freeze-milling for a time period in a range of 5-15 minutes, to obtain the decellularized extracellular matrix of the at least one biological material. In another embodiment of the present disclosure, cutting yields pieces ranging in 2 to 5 mm diameter in size. In one alternate embodiment, cutting yields pieces ranging in 1 to 4 mm diameter in size.

[0089] In an embodiment of the present disclosure, there is provided a process to obtain a decellularized extracellular matrix (dECM) of at least one biological material, said process comprising: (a) processing at least one biological material, to obtain a processed biological material; (c) subjecting the processed biological material to freeze-milling for a time period in a range of 5-15 minutes, to obtain a mixture I; (d) subjecting the mixture-I to thawing to attain a temperature in a range of 25-30°C, to obtain a paste I; (e) repeating a combination of step (c) and (d) for at least two times, to obtain a paste-II; (f) subjecting the paste-II to a freeze-drying step to obtain a mass; and (g) subjecting the mass to cutting, followed by freeze-milling for a time period in a range of 5-15 minutes, to obtain the decellularized extracellular matrix of the at least one biological material. In another embodiment of the present disclosure, repeating a combination of step (c) and (d) for 2-5 times. In yet another embodiment, repeating a combination of step (c) and (d) for 2-10 times.

[0090] In an embodiment of the present disclosure, there is provided a process to obtain a decellularized extracellular matrix (dECM) of at least one biological material as described herein, wherein the at least one biological material is selected from a group consisting of cornea, cartilage, liver, heart, skeletal muscle, lymph nodes and adipose tissue.

[0091] In an embodiment of the present disclosure, there is provided a process to obtain a decellularized extracellular matrix (dECM) of at least one biological material as described herein, wherein in step (c), subjecting the pieces to freeze-milling is done in presence of inherent or added water in the at least one biological material.

[0092] In an embodiment of the present disclosure, there is provided a decellularized extracellular matrix (dECM) of at least one biological material obtained by a process, said process comprising: (a) processing at least one biological material, to obtain a processed biological material; (b) subjecting the processed biological material to cutting, to obtain pieces ranging in 1 to 10 mm diameter in size; (c) subjecting the pieces to freeze-milling for a time period in a range of 5-15 minutes, to obtain a mixture I; (d) subjecting the mixture-I to thawing to attain a temperature in a range of 25-30°C, to obtain a paste I; (e) repeating a combination of step (c) and (d) for at least two times, to obtain a paste-II; (f) subjecting the paste-II to a freeze-drying step to obtain a mass; and (g) subjecting the mass to cutting, followed by freeze-milling for a time period in a range of 5-15 minutes, to obtain the decellularized extracellular matrix of the at least one biological material.

[0093] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.

EXAMPLES

[0094] The disclosure will now be illustrated with a working example, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

[0095] The paragraphs below illustrate the Liquid Cornea composition and the process of preparing the same using the components, fibrinogen, aprotinin, human corneal decellularized extracellular matrix, human keratocytes, thrombin and CaCK Working and non-working prototypes of Liquid Cornea hydrogel has also been depicted with different weight percentage of the components. Though the process for preparation of the Liquid Cornea composition has been illustrated by a process described herein, it is to be noted that composition can be prepared by adding the aforementioned components in any preferred sequence, and the process would fall within the scope of the present invention. A person skilled in the art will be able to employ any of the process and will be enabled to prepare the composition by way of the examples disclosed herein.

Example 1 [0096] One of the process flow used to obtain the Liquid Cornea composition is described in Figure 10. Briefly, the process involves mixing of solution I and solution II in a 1: 1 volume/volume ratio. The solution I comprises polymer (fibrinogen), antifibrinolytic agent (aprotinin solution) and decellularized extracellular matrix (human corneal decellularized extracellular matrix) powder and the second solution comprises corneal cells (human keratocytes), extracellular matrix cross-linkers (thrombin) and CaCL. The PBS used in solution I is in the range of 0.4X to 0.6X. The composition of IX PBS used in current study is 2.7mM potassium chloride, l37mM sodium chloride and l.76mM potassium phosphate.

[0097] Alternatively, other processes for preparing the Liquid Cornea composition are illustrated in Figure 8, Figure 9, and Figure 11.

[0098] As per Figure 8, the solution I comprises fibrinogen, aprotinin solution, and CdECM powder, whereas the solution II comprises corneal cell suspension, thrombin and CaCL. The solution I and the solution II are then mixed in 1: 1 ratio for the preparation of the Liquid Cornea composition.

[0099] In Figure 9, the solution I is obtained by mixing fibrinogen and aprotinin solution and the solution II is obtained by mixing thrombin, corneal cell suspension, CaCL, and CdECM powder. Similar to the afore -mentioned processes, the solution I and the solution II is mixed in a ratio of 1: 1 to obtain the final Liquid Cornea composition.

[00100] In an alternate process as described in the Figure 11 , the solution I is obtained by contacting fibrinogen and aprotinin solution, and the solution II is obtained by contacting thrombin, CaCL, corneal cell suspension, CdECM powder and 0.5X PBS. The solution I and solution II are then mixed in the ratio of 1 : 1 to obtain the Liquid Cornea composition.

[00101] It is to be appreciated that, all of the process as described herein above leads to the formation of Liquid Cornea composition which has desirable properties.

[00102] The basis of the regeneration of corneal tissue using the Liquid Cornea composition relies on the use of the human decellularized matrix (CdECM) obtained from the cornea. Therefore, the Liquid Cornea composition uses the same matrix naturally present in the human eye. However, the hydrogel obtained from the CdECM is not sustained on the cornea and is often washed away. To circumvent the problem, one or more active polymers along with the suitable cross-linkers are added in the hydrogel which also makes it easy to handle the composition during surgical procedures and sustain the regeneration of injured tissue.

[00103] In order for use in corneal applications, the processing of the CdECM is important to determine the properties of the Liquid Cornea. In the present disclosure, the following methods were used to process the CdECM. There are two methods which can be used for physical processing of CdECM. Apart from these two methods, one more method can be used for a combination of physical and enzymatic method for processing cornea. The two methods Physical Process 1 (PP1) and Physical Process 2 PP2 are discussed below followed by a detailed protocol for all the three methods for processing cornea to obtain CdECM.

Study on Method 2 and a comparison with Method 1 for physical

processing to obtain CdECM.

[00104] Corneas extracted from human cadaver were decellularized using 1.5M NaCl and DNase treatment. The decellularization process was validated by performing DAPI and H&E staining on sections of the decellularized tissues with untreated cornea tissues as a control (Figure 1). The absence of any nuclear staining confirmed successful decellularization of the tissues.

[00105] The decellularized corneas were stored in IX Phosphate Buffer Saline (PBS) + 1% antibiotic at 4°C until further use.

[00106] To prevent any interference of the PBS and antibiotic with the hydrogel formation process, all the decellularized corneas were washed three times with deionized water for 30 minutes for each wash at room temperature. [00107] Post-washing, the decellularized corneas (with inherent water content or added water) were cut into small pieces and transferred to a micro vial set in a freeze- miller.

[00108] The cornea pieces were allowed to pre-cool in liquid nitrogen for five minutes followed by freeze-milling in liquid nitrogen for 10 minutes. Then the pre-cooled cornea pieces were thawed to room temperature to obtain a CdECM paste. This step of wet-freeze milling followed by thawing, was repeated twice or more times to obtain a finely milled CdECM paste (thick suspension of CdECM particles in water).

[00109] The CdECM paste from the previous step was freeze-dried to obtain a spongy, fibrous mass of CdECM that was again cut into small pieces.

[00110] The cut pieces were allowed to pre-cool in liquid nitrogen for five minutes followed by freeze-milling in liquid nitrogen for 10 minutes.

[00111] Prior studies performed in-house and other research groups related to processing of decellularized tissues includes corneal (Yin, H. et al. Acta Biomater. 2018; Kim, H. et al. J. Tissue Eng. 10, 1-12 2019), adipose (Christman, K. L. decellularized and delipidzed extracellular matrix and methods of use. 1, (2012); Shridhar, A. et al. Methods Mol. Biol. (2017)), cartilage tissue (Shridhar, A. et al. Methods Mol. Biol. (2017) involved freeze-drying (lyophilisation) of the decellularized tissue followed by freeze-milling to get the final dECM powder. This method of freeze - drying followed by freeze-milling is represented as PP 1.

[00112] However, the modified PP2 method disclosed herein involves multiple cycles of wet freeze milling and thawing of CdECM, followed the regular process of PP1 (freeze drying followed by freeze-milling) of the decellularized tissue.

[00113] Firstly, the decellularized tissue is freeze-milled with the inherent water content (or added water). This is wet freeze milling, in the presence of water. The ice crystals resulting from snap freezing in liquid nitrogen appear to aid in breaking down the CdECM particles, and reduce the final particle size. Next, the wet milled CdECM paste is subject to thawing at room temperature, i.e., converting the ice crystals into water. After thawing, the CdECM paste is again snap frozen in liquid nitrogen, and freeze milled with water content (wet freeze milling). The milled paste is again thawed to room temperature. This process of: la) wet freeze milling and lb) thawing can be done multiple times, to obtain a finely milled CdECM paste (thick suspension of CdECM particles in water).

[00114] Secondly, the wet paste obtained from previous step (multiple cycles of wet freeze milling and thawing), is subjected to the same process as previously reported in literature i.e., PP1. The wet paste is freeze dried (lyophilisation) and freeze milled (under dry conditions) to obtain a finely milled dry CdECM powder.

Study of particles in CdECM obtained by PP1 and PP2

[00115] The reduction in particle size (and polydispersity) was tested in-house using dynamic light scattering and scanning electron microscopy methods. 1 mg/ml suspension of the CdECM powder processed using both the methods was used.

[00116] As can be seen via Dynamic Light Scattering (DLS), average particle size (hydrodynamic diameter) of PP2 is about half of PP1 process (with narrower size distribution) (Figure 2).

[00117] Scanning Electron Micrograph (SEM) shows that the average particle size of PP2 is about 3 to 4 times lesser than PP1 (Figure 3 A and 3B). SEM imaging at higher magnification confirmed the presence of aligned collagen fibrils native to the corneal tissue in the CdECM powder particles obtained from physical processing (Figure 4). Figure 5 depicts a comparison of average particle size obtained from PP 1 and PP2.

Method 1 - Physical processing of CdECM (PP1):

[00118] Corneas extracted from human cadaver were decellularized. The decellularized corneas were stored in IX Phosphate Buffer Saline (PBS) + 1% antibiotic at 4 °C until further use. To prevent any interference of the PBS and antibiotic with the hydrogel formation process, all the decellularized corneas were washed three times with deionized water for 30 minutes for each wash at room temperature. Post washing, the corneas were frozen at -80 °C and lyophilized in a freeze dryer at a pressure of 5 mT and -104.5 °C for 7 hours. Post lyophilization, the corneas were weighed for their dry weight and stored at -80 °C until further use.

[00119] The lyophilized corneas were taken out of the freezer and allowed to warm up to room temperature. Further, they were cut into small pieces and transferred to a micro vial set in a freeze-miller. The cornea pieces were allowed to pre-cool in liquid nitrogen for five minutes followed by freeze-milling in liquid nitrogen for 10 minutes. This cycle was repeated twice to obtain a finely milled CdECM powder.

Method 2 - Physical processing of CdECM :

[00120] Corneas extracted from human cadaver were decellularized. The decellularized corneas were stored in IX Phosphate Buffer Saline (PBS) + 1% antibiotic at 4 °C until further use. To prevent any interference of the PBS and antibiotic with the hydrogel formation process, all the decellularized corneas were washed three times with deionized water for 30 minutes for each wash at room temperature. Post washing, the corneas were cut into small pieces and transferred to a micro vial set in a freeze-miller. The cornea pieces were allowed to pre-cool in liquid nitrogen for five minutes followed by freeze-milling in liquid nitrogen for 10 minutes. This cycle was repeated twice to obtain a finely milled CdECM paste (thick suspension of CdECM particles in water). The freeze-milled CdECM paste was freeze-dried to obtain a spongy, fibrous mass that was again cut into small pieces and freeze-milled to obtain the finely milled CdECM powder.

Method 3 - Physical processing and Enzymatic Digestion of CdECM (ED):

Corneas extracted from human cadaver were decellularized. The decellularized corneas were stored in IX Phosphate Buffer Saline (PBS) + 1% antibiotic at 4 °C until further use. To prevent any interference of the PBS and antibiotic with the hydrogel formation process, all the decellularized corneas were washed three times with deionized water for 30 minutes for each wash at room temperature. Post-washing, the corneas were freeze-dried and cut into small pieces and transferred to a micro vial set in a freeze- miller. The cornea pieces were allowed to pre-cool in liquid nitrogen for five minutes followed by freeze-milling in liquid nitrogen for 10 minutes. This cycle was repeated twice to obtain a finely milled CdECM powder. The freeze-milled CdECM powder was weighed and suspended in 8.5 M acetic acid solution with pH 4.5. A slurry of immobilized pepsin on agarose beads was also included in the suspension at a concentration of 0.025 ml slurry/mg of the CdECM powder. The CdECM particles and agarose beads were ensured to be in suspension using a magnetic stirrer and enzymatic digestion was carried out at 37°C for 72 hours.

[00121] Post 72 hours, the beads were separated from the CdECM suspension using centrifugation at lOOg for 5 mins and the CdECM suspension was dialyzed against distilled water using a CE membrane with 1 kDa MWCO cutoff for 48 hours.

[00122] Post dialysis, the CdECM suspension is freeze-dried to get the CdECM powder.

[00123] Figure 6 depicts the SEM images for Cornea dECM processed with enzymatic digestion (extreme left). The distinct change in particle size and surface morphology can be observed when this image is compared with particles of the physically processed powder (PP1 and PP2) from Figure 3 A and 3B. The other two images depict the elemental analysis performed on the CdECM particle to confirm the organic origin of the tissue microparticles and rule out the presence of salt/inorganic particles.

[00124] The CdECM powders obtained from the different methods (PP2 and ED) were also subjected to Dynamic Light Scattering (DLS) testing at 37 °C with a 1 mg/ml suspension in IX PBS. The results of the DLS testing are shown in in Table 1. The physically-processed freeze-milled powder has a high polydispersity index of 80% with average particle diameter of 950 nm. The enzymatically-digested freeze-milled powder has a polydispersity index of 23% with average particle diameter of 904 nm. Table 1:

[00125] The CdECM so obtained was then used in the process to form the Liquid Cornea hydrogel or composition as described in the process flow in Figures 8, 9, 10 and 11 and also in detail in the preceding paragraphs, except the corneal cell suspension component. Alongside the Liquid Cornea hydrogel, a control Fibrin hydrogel was also prepared. The results obtained are described in Table 2 below.

[00126] Post polymerization, the Liquid Cornea hydrogel turns opaque similar to the fibrin glue. The initial weights of the hydrogels were measured and the hydrogels were soaked in IX PBS overnight at room temperature. The final weight was measured and the % change in initial and final weights was noted. Due to the sticky nature of the hydrogels, it was difficult to handle the hydrogels without disrupting the 3D network. However, the swelling ratios of the hydrogels indicated that fibrin gels do not swell significantly. Moreover, addition of the CdECM powder to fibrin does not change the swelling characteristics of the resultant Liquid Cornea hydrogels.

[00127] Rheological testing was performed on the hydrogels to find their storage modulus using a frequency sweep at a constant shear strain rate. Parallel plate geometry was used along with 0.2% strain rate, 0.9 mm gap size and frequency sweep range from 0.1-10 hertz. Storage modulus were recorded at ~l hertz. The results obtained are described in Table 3. There was no significant change in stiffness observed with the addition of CdECM to the fibrin network for the tested composition. Table 2:

Table 3:

[00128] Transmittance studies were conducted for both suspensions prepared from the CdECM powder in IX PBS at 1 mg/ml concentration as well as for fibrin and Liquid Cornea hydrogels prepared using CdECM powder obtained using the different processing methods. Absorption spectrum was measured using a spectrophotometer between 350 - 900 nm wavelength range and the % transmittance was calculated using the formula

% T = lO^{(2 A)}

where T is transmittance and A is the absorbance value at the given wavelength.

% Transmittance was plotted against wavelength to get the transmittance profile (Figure 7). Enzymatically digested CdECM powder in suspension showed significant increase in % transmittance compared to physically milled (PP1 and PP2) CdECM samples. However, overall % transmittance values were less than 10% for the final hydrogel formulations.

[00129] For testing biodegradation characteristics, change in dry weight and was studied for the fibrin and Liquid Cornea hydrogels. Corneal stromal stem cells (CSSCs) encapsulated in hydrogels were prepared and cultured in cell culture medium at 37°C with 5% C0₂ environment with medium change every 48 hours. Hydrogels were collected at different time points for a period of 8 days. Biodegradation studies revealed that in the presence of CSSCs, both Liquid Cornea (as described in the present disclosure and obtained using a process PP1) and control fibrin hydrogels lost only 20% of their mass by the end of day 6 (Figure 12). However, both groups significantly lost >80% mass by the end of day 8. Moreover, no significant differences in percentage mass loss were observed between the two groups at any time point during the 8 -day study period.

[00130] 12 mΐ hydrogels for both fibrin and Liquid Cornea of the present disclosure (comprising CdECM obtained using process PP1) that encapsulated CSSCs were prepared between cover slips to get thin samples. The thin hydrogel samples were immersed in 0.2 pg/ml calcein acetoxymethylester (calcein-AM) and 2.5 pg/ml propidium iodide (PI) for 15 min in supplemented DMEM at 37°C to stain live cells green and dead cells red, respectively, when visualized using fluorescence microscopy. Results from live-dead assay demonstrated that the CdECM microparticles displayed very good cytocompatibility, comparable to fibrin-only controls (refer to liquid cornea composition of the present disclosure but devoid of CdECM and corneal cells), with cell viability exceeding >95% by the end of day 5 (Figure 13).

[00131] 12 mΐ hydrogels for both fibrin and Liquid Cornea that encapsulated CSSCs were prepared between cover slips to get thin samples. The thin hydrogel samples were recovered at different time points and washed with PBS. Post-washing step, the hydrogel samples were fixed with 3.7% formalin solution in IX PBS for 10 mins at RT and washed with IX PBS again. IHC (fluorescence) was performed to visualize the expression of scarless-wound healing related markers. Primary anti-CD73, anti-CD90, anti-alpha smooth muscle actin (aSMA) and anti-collagen type-l antibody were procured from Abeam. Goat anti-mouse and anti-rat antibodies (conjugated with Alexa Fluor 594 and Alexa Fluor 488; Thermo Fisher Scientific) were used as secondary antibodies. The sections were imaged with Evos FL Auto 2 (Thermo Scientific). Final images shown in Figure 13 were combined from several microscopic images, showing the relative effect of Liquid Cornea hydrogel on scarless wound healing in vitro.

[00132] Results from immunofluorescence studies showed that CSSCs cultured in Liquid Cornea hydrogels (CdECM obtained using PP1 process) exhibited better expression of CD73 and CD90 compared to fibrin controls over a 72-hour culture period. In addition, fibrocytic marker expression, a-SMA and Collagen type 1, was downregulated in cells cultured in Liquid Cornea hydrogels compared to fibrin gels (Figure 14). This data gives clear evidence that Liquid Cornea hydrogel inhibit the differentiation of CSSCs to myofibroblasts which play a major role in fibrotic ECM deposition that lead to corneal opacity. These data demonstrate that the presence of CdECM support and maintain CSSC phenotypic characteristics that are beneficial for corneal scar-less wound healing in vivo.

Advantages of the present disclosure

[00133] The present disclosure discloses a liquid cornea composition which comprises: at least one polymer; an antifibrinolytic agent; decellularized extracellular matrix; human corneal cell suspension; at least one extracellular matrix cross-linker; and calcium chloride (CaCb). The liquid cornea as disclosed herein provides a hydrogel which comprises decellularized corneal extracellular matrix and maintain corneal cell characteristics which is turn promotes scar-less wound healing in natural conditions in the eye. The liquid cornea composition as disclosed herein comprises decellularized corneal extracellular matrix which when prepared using the disclosed physical processing method provides ultra-fine particles which provides a beneficial effect in the final liquid cornea composition.

Claims

I/We Claim:

1. A Liquid Cornea composition comprising:

(a) at least one polymer;

(b) an antifibrinolytic agent;

(c) decellularized extracellular matrix;

(d) human corneal cell suspension;

(e) at least one extracellular matrix cross-linker; and

(f) calcium chloride (CaCh).

2. The Liquid Cornea composition as claimed in claim 1, wherein the at least one polymer has a concentration in a range of 17 to 110 mg/ml with respect to the composition.

3. The Liquid Cornea composition as claimed claim 1, wherein the at least one polymer is selected from the group consisting of fibrinogen, collagen, silk, and combinations thereof.

4. The Liquid Cornea composition as claimed in claim 3, wherein the at least one polymer is fibrinogen.

5. The Liquid Cornea composition as claimed in claim 1, wherein the antifibrinolytic agent has a concentration in a range of 600 to 4000 KlU/ml with respect to the composition.

6. The Liquid Cornea composition as claimed in claim 5, wherein the antifibrinolytic agent is aprotinin.

7. The Liquid Cornea composition as claimed in claim 1, wherein the decellularized extracellular matrix has a concentration in a range of 7.5 to 60 mg/ml with respect to the composition.

8. The Liquid Cornea composition as claimed in claim 7, wherein the decellularized extracellular matrix (dECM) has a concentration in a range of 8% to 57% dECM mass/total polymer mass of the composition.

9. The Liquid Cornea composition as claimed in claim 8, wherein the decellularized extracellular matrix is a human corneal decellularized extracellular matrix.

10. The Liquid Cornea composition as claimed in claim 1, wherein the human corneal cell suspension has a concentration in a range of 5xl0⁶ to lOxlO⁶ cells/ml with respect to the composition.

11. The Liquid Cornea composition as claimed in claim 10, wherein the human corneal cell suspension comprises human keratocytes.

12. The Liquid Cornea composition as claimed in claim 1, wherein the at least one extracellular matrix cross-linker has a concentration in a range of 90 to 625 IU/ml with respect to the composition.

13. The Liquid Cornea composition as claimed in claim 12, wherein the at least one extracellular matrix cross-linker is selected from a group consisting of thrombin, trilysine acetate, polyethylene amine, polyvinyl alcohol vinylamine, and combinations thereof.

14. The Liquid Cornea composition as claimed in claim 13, wherein the at least one extracellular matrix cross-linker is thrombin.

15. The Liquid Cornea composition as claimed in claim 1, wherein calcium chloride (CaCL) has a concentration in a range of 7.5 to 50 mM with respect to the composition.

16. A process for the preparation of the Liquid Cornea composition as claimed in claim 1, said process comprising: (a) obtaining at least one polymer, an antifibrinolytic agent, decellularized extracellular matrix, human corneal cell suspension, at least one extracellular matrix cross-linker, and CaCL: and (b) contacting the at least one polymer, the antifibrinolytic agent, the decellularized extracellular matrix, the human corneal cell suspension, the at least one extracellular matrix cross-linkers, and CaCk; and (c) curing the mixture to obtain the Liquid Cornea composition.

17. A process for the preparation of the Liquid Cornea composition as claimed in claim 1, said process comprising:

(a) obtaining a solution I comprising the at least one polymer and the antifibrinolytic agent;

(b) obtaining a solution II comprising the decellularized extracellular matrix, the at least one extracellular matrix cross-linker, the human corneal cell suspension and CaCk;

(c) contacting the solution I and the solution II in a ratio range of 50: 1 to 1 : 50 to obtain a mixture; and

(d) curing the mixture to obtain the Liquid Cornea composition.

18. A process for the preparation of the Liquid Cornea composition as claimed in claim 1, said process comprising:

(a) obtaining a solution I comprising the at least one polymer, the antifibrinolytic agent and the decellularized extracellular matrix;

(b) obtaining a solution II comprising the at least one extracellular matrix cross-linker, the human corneal cell suspension and CaCk; (c) contacting the solution I and the solution II in a ratio range of 50: 1 to 1 : 50 to obtain a mixture; and

(d) curing the mixture to obtain the Liquid Cornea composition.

19. The process for the preparation of the Liquid Cornea composition as claimed in any one of the claims 17 or 18, wherein contacting the solution I and the solution II is done in a ratio of 1 : 1 , to obtain the mixture.

20. The Liquid Cornea composition as claimed in any one of the claims 1-15 for use in treating corneal diseases.

21. The Liquid Cornea composition as claimed in claim 20, wherein the corneal diseases is selected from a group consisting of corneal abrasion, corneal dystrophy, corneal ulcer, corneal neovascularization, Fuchs' dystrophy, keratitis, and keratoconus.

22. A process to obtain a decellularized extracellular matrix (dECM) of at least one biological material, said process comprising: a) processing at least one biological material, to obtain a processed biological material;

b) optionally subjecting the processed biological material to cutting, to obtain pieces ranging in 1 to 10 mm diameter in size;

c) subjecting the pieces to freeze-milling for a time period in a range of 5-15 minutes, to obtain a mixture I;

d) subjecting the mixture-I to thawing to attain a temperature in a range of 25- 30°C, to obtain a paste I;

e) repeating a combination of step (c) and (d) for at least two times, to obtain a paste-II;

f) subjecting the paste-II to a freeze-drying step to obtain a mass; and

g) subjecting the mass to cutting, followed by freeze-milling for a time period in a range of 5-15 minutes, to obtain the decellularized extracellular matrix of the at least one biological material.

23. The process as claimed in claim 22, wherein the at least one biological material is selected from a group consisting of cornea, cartilage, heart, liver, skeletal muscle, lymph node and adipose tissue.

24. The process as claimed in claim 22, wherein in step (c), subjecting the pieces to freeze-milling is done in presence of inherent or added water in the at least one biological material.

25. A decellularized extracellular matrix (dECM) of at least one biological material obtained by a process as claimed in claim 22.