Platelet lysate composition and use thereof

Abstract

The present invention relates to compositions, kits and methods for providing mechanical support and / or medicaments to biological targets. Specifically, the compositions include platelet lysates, fibrinogen and thrombin. The compositions can be used to treat wounds / injuries (e.g., cornea) and for other applications involving tissue cultures.

Description

[0001] Incorporated by cross reference

[0002] This application claims priority to Australian Provisional Application No. 2018902870 filed on August 7, 2018, the entire contents of which are incorporated herein by cross-reference. Technical Field

[0003] The present invention relates generally to the fields of biology and medicine, and more specifically to compositions and methods for providing mechanical support and / or agents to biological targets. Still more specifically, the present invention relates to compositions and methods that can be used to treat wounds / injuries and for other applications involving tissue culture. Background Art

[0004] The cornea is an integral component of the front surface of the eye that refracts light through the pupil and acts as a physical barrier to infection, UV light and mechanical trauma. Further, corneal injuries are the most common eye emergency visit in Australia, and approximately 75% of all cases are due to the presence of foreign objects or abrasions in the cornea. These injuries are estimated to cost the Australian population over AUD 155 million per year and, if not effectively treated, can lead to infection and scarring, permanently impairing vision. Additionally, corneal disease is the second most common cause of blindness in most developing countries, with an estimated 12% of the 39 million blind people worldwide suffering from bilateral corneal blindness, and an additional 23 million suffering from unilateral corneal blindness.

[0005] Corneal defects can result from trauma and other disease processes. In mild cases, the cornea is able to regenerate through normal wound healing pathways, and corneal injuries can be very painful due to the dense neuronal innervation of the cornea. However, in some cases, the normal wound healing mechanisms of the cornea may be insufficient. This results in the formation of non-healing defects, which can lead to corneal melting, corneal neovascularization, loss of transparency, infection, scarring, and decreased vision to the point of blindness.

[0006] Corneal wound healing occurs when the balance between cell replenishment and cell loss is disrupted, such as after an abrasion, infection, or refractive surgery. In mild cases, the cornea can upregulate physiological processes to restore this imbalance, however, in more severe cases, additional treatment is required. In cases where wound healing is ineffective, harmful consequences such as impaired vision and even corneal blindness may result.

[0007] Current medical treatments for corneal injuries include antibiotics, eye pads, sutures, and surgical glues, which may help minor wounds. However, they do not adequately address the problems that arise in more severe wounds, including pain relief, infection, and / or the development of scar tissue. Infection represents a serious complication and may require hospitalization. These problems are not limited to corneal injuries and are also prevalent in cases of wounds of other body tissues. Scarring, which is common in severe corneal injuries, may lead to permanent vision loss, with corneal transplantation being the only option for visual rehabilitation.

[0008] There is a need for improved compositions and methods for treating corneal injuries.The improved compositions and methods may also find application in the treatment of other tissue wounds. Summary of the invention

[0009] The present invention alleviates at least one of the problems associated with current compositions and / or methods for treating wounds (eg, corneal wounds).

[0010] The mechanical properties of platelet lysate are similar to water, so it is difficult to be applied to tissue in a structured form. For example, it is very challenging and infeasible in most cases to apply the original platelet lysate layer by layer to tissue to generate a two-dimensional or three-dimensional structure (for example, as required by technologies such as bioprinting). The inventor has developed a platelet lysate composition with mechanical and structural properties that promotes application to tissue in a structural form. Specifically, the composition of the present invention can be used to apply platelet lysate to tissue (for example, eyes) using two-dimensional or three-dimensional bioprinting technology. The composition can be prepared by initially separating components, which collectively include at least platelet lysate, fibrinogen and thrombin. When mixed, the separated components solidify into a gel-like material including platelet lysate. The solidification rate can be controlled by changing the timing and / or amount of various composition components, thereby providing flexibility during application. Without limitation, the compositions and methods described herein are generally useful for delivering agents (e.g., drugs and / or other substances) to biological targets (e.g., tissues, membranes, cells), and can be used, for example, to treat wounds, including wounds in corneal tissue (e.g., non-healing corneal ulcers or abrasions).

[0011] The present invention relates, at least in part, to the following embodiments:

[0012] Example 1 . A composition comprising 0.1-20 mg / ml fibrinogen, 2-20 U / mL thrombin and 1-40% (v / v) platelet lysate.

[0013] Example 2.The composition according to embodiment 1, further comprising any one or more of the following: ions, ion sources, amino acids, fibronectin, anesthetics, antibiotics, growth factors, tissue factor XIII, matrix proteins (e.g., collagen).

[0014] Example 3 .The composition according to Example 1 or Example 2, wherein the ions include calcium ions, and / or the growth factor includes human epidermal growth factor (hEGF).

[0015] Example 4 . The composition according to any one of embodiments 1 to 3, wherein the composition comprises a culture medium comprising the ions and the amino acids.

[0016] Example 5 . A composition according to any one of embodiments 1 to 4, wherein the ion is a component of an ionic salt solution contained in the composition.

[0017] Example 6 .The composition according to any one of embodiments 1 to 5, wherein the composition further comprises cells (e.g., mammalian cells, human cells).

[0018] Example 7 . A composition according to any one of embodiments 1 to 6, wherein the platelet lysate comprises or consists of human platelet lysate.

[0019] Example 8 .A composition according to any one of embodiments 1 to 7, wherein the platelet lysate is free of or substantially free of an anticoagulant (e.g., heparin) or comprises less than 10% (v / v), 9% (v / v), 8% (v / v), 7% (v / v), 6% (v / v), 5% (v / v), 4% (v / v), 3% (v / v), 2% (v / v), 1% (v / v), 0.5% (v / v).

[0020] Example 9 . A composition according to any one of embodiments 1 to 8, wherein the composition comprises:

[0021] (i) 0.1-15 mg / ml fibrinogen; 2-15 U / ml thrombin; and 5-40% (v / v) platelet lysate; or

[0022] (ii) 0.1-10 mg / ml fibrinogen; 2-10 U / ml thrombin; and 10-35% (v / v) platelet lysate; or

[0023] (iii) 0.2-5 mg / ml fibrinogen; 2-8 U / ml thrombin; and 15-30% (v / v) platelet lysate; or

[0024] (iv) 0.2-3 mg / ml fibrinogen; 2-4 U / ml thrombin; and 15-25% (v / v) platelet lysate.

[0025] Example 10 . A composition according to any one of embodiments 1 to 8, wherein the composition comprises:

[0026] (i) less than 1 mg / ml (e.g., about 0.2 mg / ml) fibrinogen; 2-20 U / ml (e.g., about 3 U / ml, about 5 U / ml, about 10 U / ml) thrombin; and 5-40% (v / v) (e.g., about 20-30% (v / v)) platelet lysate; or

[0027] (ii) 1-4 mg / ml (e.g., about 2 mg / ml) fibrinogen; 5-15 U / ml (e.g., about 7 U / ml, about 10 U / ml, about 12 U / ml) thrombin; and 10-40% (v / v) (e.g., about 15-30% (v / v)) platelet lysate; or

[0028] (iii) 3-8 mg / ml (e.g., about 5 mg / ml) fibrinogen; 2-15 U / ml (e.g., about 4 U / ml, about 8 U / ml, about 12 U / ml) thrombin; and 15-30% (v / v) (e.g., about 20-25% (v / v)) platelet lysate.

[0029] Embodiment 11 . A composition according to any one of embodiments 1 to 8, wherein the composition comprises:

[0030] (i) about 0.4 mg / ml, about 0.8 mg / ml or about 1 mg / ml fibrinogen, and

[0031] about 2 U / ml, about 10 U / ml, or about 20 U / ml thrombin, and

[0032] about 20% (v / v), about 25% (v / v), or about 30% (v / v) platelet lysate; or

[0033] (ii) about 2 mg / ml, about 4 mg / ml or about 8 mg / ml fibrinogen, and

[0034] about 2 U / ml, about 10 U / ml, or about 20 U / ml thrombin, and

[0035] about 10%, about 15% (v / v), or about 20% (v / v) platelet lysate; or

[0036] (iii) about 5 mg / ml, about 10 mg / ml or about 15 mg / ml fibrinogen, and

[0037] about 2 U / ml, about 10 U / ml, or about 20 U / ml thrombin, and

[0038] About 5% (v / v), about 15% (v / v), or about 25% (v / v) platelet lysate.

[0039] Example 12 . A composition according to any one of embodiments 1 to 8, wherein the composition comprises:

[0040] (i) at least the following: about 0.4 mg / ml fibrinogen, about 2 units / mL thrombin, and about 20% (v / v) platelet lysate; or

[0041] (ii) at least the following: about 20% (v / v) platelet lysate, about 4 mg / mL fibrinogen, and about 10 U / mL thrombin.

[0042] Embodiment 13 . A kit, package or device comprising:

[0043] - a first compartment comprising fibrinogen and a second compartment comprising thrombin, wherein the kit, the package or the device is configured to separate the fibrinogen of the first compartment from the thrombin of the second compartment during and after loading of the fibrinogen and the thrombin into the kit, the package or the device;

[0044] - Platelet lysate;

[0045] - means for promoting mixing of said fibrinogen of said first compartment with said thrombin and said platelet lysate of said second compartment.

[0046] Embodiment 14 .A kit, a package or a device according to Example 13, wherein the platelet lysate is positioned in a third compartment of the kit, the package or the device, and the kit, the package or the device is configured to separate the platelet lysate in the third compartment from the fibrinogen in the first compartment and the thrombin in the second compartment during and after loading the fibrinogen, the thrombin and the platelet lysate into the kit, the package or the device.

[0047] Embodiment 15. The kit, package or device of embodiment 13, wherein said first compartment comprises said fibrinogen and said platelet lysate, and said second compartment comprises said thrombin.

[0048] Example 16 . A kit, package or device according to any one of embodiments 13 to 15, wherein:

[0049] - any of the compartments further comprises any one or more of: ions, ion sources, amino acids, fibronectin, anesthetics, antibiotics, growth factors (e.g., hEGF), tissue factor XIII, matrix proteins (e.g., collagen); or

[0050] - the kit, the package or the device comprises an additional compartment, the additional compartment comprising any one or more of the following: ions, ion sources, amino acids, fibronectin, anesthetics, antibiotics, growth factors (e.g., hEGF), tissue factor XIII, matrix proteins (e.g., collagen); wherein the kit, the package or the device is configured to separate the contents of the additional compartment from the contents of other compartments during and after loading of the kit, the package or the device.

[0051] Embodiment 17 . The kit, package or device according to embodiment 16, wherein:

[0052] The ion is a calcium ion, or

[0053] The ion source is an ionic salt and / or includes calcium ions.

[0054] Embodiment 18 .A kit, package or device according to any one of Examples 13 to 17, wherein the device is configured to facilitate mixing of the fibrinogen, the thrombin and the platelet lysate outside of the kit, the package or the device.

[0055] Embodiment 19 . A kit, package or device according to any one of embodiments 13 to 18, wherein:

[0056] The device is configured to direct the flow stream of fibrinogen and the separate flow stream of thrombin to a point of convergence and thereby promote said mixing, and

[0057] At least one of the flow streams comprises the platelet lysate.

[0058] Embodiment 20. A kit, package or device according to any one of embodiments 13 to 19, wherein either or both of the first compartment and the second compartment and / or the third compartment comprises cells (e.g., mammalian cells, human cells).

[0059] Embodiment 21 . A kit, package or device according to any one of embodiments 13 to 20, wherein the platelet lysate comprises or consists of human platelet lysate.

[0060] Embodiment 22 .A kit, package or device according to any one of embodiments 13 to 21, wherein the platelet lysate is free of or substantially free of an anticoagulant (e.g., heparin) or comprises less than 10% (v / v), 9% (v / v), 8% (v / v), 7% (v / v), 6% (v / v), 5% (v / v), 4% (v / v), 3% (v / v), 2% (v / v), 1% (v / v), 0.5% (v / v).

[0061] Embodiment 23 . A kit, package or device according to any one of embodiments 13 to 22, wherein:

[0062] (i) the first compartment comprises 0.1-20 mg / ml fibrinogen; the second compartment comprises 1-40 U / ml thrombin; and the device comprises a total of 5-40% (v / v) platelet lysate; or

[0063] (ii) the first compartment comprises 0.1-15 mg / ml fibrinogen; the second compartment comprises 1-30 U / ml thrombin; and the device comprises a total of 5-40% (v / v) platelet lysate; or

[0064] (iii) the first compartment comprises 0.1-12 mg / ml fibrinogen; the second compartment comprises 1-25 U / ml thrombin; and the device comprises a total of 7-14% (v / v) platelet lysate; or

[0065] (iv) the first compartment comprises 0.5-10 mg / ml fibrinogen; the second compartment comprises 1-10 U / ml thrombin; and the device comprises a total of 0.5-20% (v / v) platelet lysate.

[0066] Embodiment 24 . A kit, package or device according to any one of embodiments 13 to 22, wherein:

[0067] (i) the first compartment comprises less than 2 mg / ml (e.g., about 0.8 mg / ml) fibrinogen; the second compartment comprises 0.5-20 U / ml (e.g., about 1 U / ml, about 10 U / ml, about 20 U / ml) thrombin; and the device comprises a total of 5-40% (v / v) (e.g., about 20% (v / v)) platelet lysate; or

[0068] (ii) the first compartment comprises 1-16 mg / ml (e.g., about 8 mg / ml) fibrinogen; the second compartment comprises 0.5-20 U / ml (e.g., about 1 U / ml, about 10 U / ml, about 20 U / ml) thrombin; and the device comprises a total of 5-40% (v / v) (e.g., about 20% (v / v)) platelet lysate; or

[0069] (iii) the first compartment comprises 5-15 mg / ml (e.g., about 10 mg / ml); the second compartment comprises 0.5-20 U / ml (e.g., about 1 U / ml, about 10 U / ml, about 20 U / ml) thrombin; and the device comprises a total of 5-40% (v / v) (e.g., about 20% (v / v)) platelet lysate.

[0070] Embodiment 25 . A kit, package or device according to any one of embodiments 13 to 22, wherein:

[0071] (i) the first compartment comprises 0.8 mg / ml fibrinogen, and

[0072] The second compartment comprises about 2 U / ml, about 10 U / ml or about 20 U / ml thrombin, and

[0073] The device comprises a total of about 20% (v / v) platelet lysate; or

[0074] (ii) the first compartment comprises about 8 mg / ml fibrinogen, and

[0075] The second compartment comprises about 2 U / ml, about 10 U / ml or about 20 U / ml thrombin, and

[0076] The device comprises a total of about 20% (v / v) platelet lysate; or

[0077] (iii) the first compartment comprises about 20 mg / ml fibrinogen, and

[0078] The second compartment comprises about 2 U / ml, about 10 U / ml or about 20 U / ml thrombin, and

[0079] The device included a total of approximately 10% (v / v) platelet lysate.

[0080] Embodiment 26 The kit, package or device of any one of embodiments 13 to 22, wherein the formulation resulting from the mixing of said thrombin, said fibrinogen and said platelet lysate comprises:

[0081] (i) at least 0.2 mg / ml fibrinogen, at least 1 unit / mL thrombin, and about 10% (v / v) platelet lysate; or

[0082] (ii) about 20% (v / v) platelet lysate, about 2 mg / mL fibrinogen, and about 5 U / mL thrombin.

[0083] Embodiment 27 . A kit, package or device according to any one of embodiments 13 to 26, wherein the first compartment and the second compartment do not include the platelet lysate.

[0084] Embodiment 28 . A kit, package or device according to any one of embodiments 13 to 27, wherein the first compartment and the second compartment are equal volumes or less than the following different volumes of liquid formulation: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%.

[0085] Embodiment 29 . A method for preparing a composition, the method comprising:

[0086] (i) Provide the following:

[0087] a first formulation comprising fibrinogen and a second formulation comprising thrombin, wherein the first formulation is not in contact with the second formulation; and

[0088] (ii) mixing the first formulation and the second formulation together with platelet lysate to thereby provide the composition.

[0089] Embodiment 30 .The method according to embodiment 29, wherein prior to said mixing, said platelet lysate is combined with said first formulation comprising fibrinogen and is not combined with said second formulation comprising thrombin.

[0090] Embodiment 31 .The method according to embodiment 29, wherein prior to said mixing, said platelet lysate is not combined with said second formulation comprising thrombin.

[0091] Embodiment 32.The method according to embodiment 29, wherein prior to said mixing, said platelet lysate is not combined with said first formulation comprising fibrinogen or with said second formulation comprising thrombin.

[0092] Embodiment 33 .A method according to any one of embodiments 29 to 32, wherein the method further comprises mixing any one or more of the following with the first formulation and the second formulation and the platelet lysate: ions, ion sources, amino acids, fibronectin, anesthetics, antibiotics, growth factors (e.g., hEGF), tissue factor XIII, matrix proteins (e.g., collagen).

[0093] Embodiment 34 . The method of any one of embodiments 29 to 33, wherein:

[0094] The ion is a calcium ion, or

[0095] The ion source is an ionic salt and / or includes calcium ions.

[0096] Embodiment 35 .The method according to any one of embodiments 29 to 34, further comprising mixing cells (e.g., mammalian cells, human cells) with the first formulation and the second formulation.

[0097] Embodiment 36 .A method according to any one of embodiments 29 to 35, wherein the platelet lysate comprises or consists of human platelet lysate.

[0098] Embodiment 37 .A method according to any one of embodiments 29 to 36, wherein the platelet lysate contains no or substantially no anticoagulant (e.g., heparin) or comprises less than 10% (v / v), 9% (v / v), 8% (v / v), 7% (v / v), 6% (v / v), 5% (v / v), 4% (v / v), 3% (v / v), 2% (v / v), 1% (v / v), 0.5% (v / v).

[0099] Embodiment 38 . The method of any one of embodiments 29 to 37, wherein:

[0100] (i) the first formulation comprises 0.1-20 mg / ml fibrinogen; the second formulation comprises 1-40 U / ml thrombin; and the first formulation and / or the second formulation comprises a total of 5-40% (v / v) platelet lysate; or

[0101] (ii) the first formulation comprises 0.1-15 mg / ml fibrinogen; the second formulation comprises 1-30 U / ml thrombin; and the first formulation and / or the second formulation comprises a total of 5-20% (v / v) platelet lysate; or

[0102] (iii) the first formulation comprises 0.1-12 mg / ml fibrinogen; the second formulation comprises 1-25 U / ml thrombin; and the first formulation and / or the second formulation comprises a total of 7-14% (v / v) platelet lysate; or

[0103] (iv) the first formulation comprises 0.2-10 mg / ml fibrinogen; the second formulation comprises 2-20 U / ml thrombin; and the first formulation and / or the second formulation comprises a total of 8-12% (v / v) platelet lysate.

[0104] Embodiment 39 . The method of any one of embodiments 29 to 37, wherein:

[0105] (i) the first formulation comprises less than 1 mg / ml (e.g., about 0.4 mg / ml) fibrinogen; the second formulation comprises 0.5-20 U / ml (e.g., about 1 U / ml, about 10 U / ml, about 20 U / ml) thrombin; and the first formulation and / or the second formulation comprises a total of 5-40% (v / v) (e.g., about 20% (v / v)) platelet lysate; or

[0106] (ii) the first formulation comprises 1-8 mg / ml (e.g., about 4 mg / ml) fibrinogen; the second formulation comprises 0.5-20 U / ml (e.g., about 1 U / ml, about 10 U / ml, about 20 U / ml) thrombin; and the first formulation and / or the second formulation comprises a total of 5-40% (v / v) (e.g., about 20% (v / v)) platelet lysate; or

[0107] (iii) the first formulation comprises 5-15 mg / ml (e.g., about 10 mg / ml); the second formulation comprises 0.5-20 U / ml (e.g., about 1 U / ml, about 10 U / ml, about 20 U / ml) thrombin; and the first formulation and / or the second formulation comprises a total of 5-40% (v / v) (e.g., about 20% (v / v)) platelet lysate.

[0108] Embodiment 40 . The method of any one of embodiments 29 to 37, wherein:

[0109] (i) the first formulation comprises about 0.4 mg / ml fibrinogen, and

[0110] The second formulation comprises about 2 U / ml, about 10 U / ml or about 20 U / ml thrombin, and

[0111] The first formulation and / or the second formulation comprises a total of about 10% (v / v) platelet lysate; or

[0112] (ii) the first formulation comprises about 4 mg / ml fibrinogen, and

[0113] The second formulation comprises about 2 U / ml, about 10 U / ml or about 20 U / ml thrombin, and

[0114] The first formulation and / or the second formulation comprises a total of about 20% (v / v) platelet lysate; or

[0115] (iii) the first formulation comprises about 10 mg / ml fibrinogen, and

[0116] The second formulation comprises about 2 U / ml, about 10 U / ml or about 20 U / ml thrombin, and

[0117] The first formulation and / or the second formulation comprises a total of about 10% (v / v) platelet lysate.

[0118] Embodiment 41 The method of any one of embodiments 29 to 37, wherein the composition comprises:

[0119] (i) at least 0.2 mg / ml fibrinogen, at least 1 unit / mL thrombin, and about 20% (v / v) platelet lysate; or

[0120] (ii) about 20% (v / v) platelet lysate, about 2 mg / mL fibrinogen, and about 5 U / mL thrombin.

[0121] Embodiment 42 .A method according to any one of embodiments 29 to 41, wherein the first formulation and the second formulation are equal in volume or less than the following different volumes: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%.

[0122] Embodiment 43 The method of any one of embodiments 29 to 42, wherein said mixing said first formulation and said second formulation with said platelet lysate comprises:

[0123] generating a first flow stream comprising said fibrinogen, a second flow stream comprising said thrombin and optionally a third flow stream, wherein said flow streams are initially separated from each other, and

[0124] combining the flow streams together;

[0125] And wherein any one or more of the first flow stream, the second flow stream and / or the third flow stream comprises the platelet lysate.

[0126] Embodiment 44 . A composition obtained or obtainable by the method according to any one of embodiments 29 to 43.

[0127] Example 45. A use of the kit, package or device according to any one of embodiments 13 to 28 for applying a formulation comprising fibrinogen, thrombin and platelet lysate to a tissue of a subject.

[0128] Embodiment 46 . A method of treating a tissue of a subject, the method comprising applying to the tissue a composition according to any one of Examples 1 to 12 or according to Example 44.

[0129] Embodiment 47 . A composition according to any one of embodiments 1 to 12 or according to embodiment 44, for use in treating a tissue in a subject.

[0130] Embodiment 48 . Use of a first formulation comprising fibrinogen and a second formulation comprising thrombin for the preparation of a medicament for treating a tissue of a subject, wherein the medicament further comprises a platelet lysate.

[0131] Embodiment 49 .The use according to embodiment 48, wherein the drug further comprises any one or more of the following: ions, ion sources, amino acids, fibronectin, anesthetics, antibiotics, growth factors (e.g., hEGF), tissue factor XIII, matrix proteins (e.g., collagen).

[0132] Embodiment 50 .The use according to Example 48 or Example 49, wherein the drug further comprises cells (e.g., mammalian cells, human cells).

[0133] Embodiment 51.A first formulation comprising fibrinogen, a second formulation comprising thrombin and optionally a third formulation for simultaneous use in treating a tissue of a subject, wherein any one or more of the first formulation, the second formulation and / or the optional third formulation comprises a platelet lysate.

[0134] Embodiment 52 . The formulation of embodiment 51, wherein:

[0135] Any one or more of the first formulation, the second formulation, and / or the optional third formulation include any one or more of the following: ions, ion sources, amino acids, fibronectin, anesthetics, antibiotics, growth factors (e.g., hEGF), tissue factor XIII, matrix proteins (e.g., collagen).

[0136] Embodiment 53 . The formulation of embodiment 51 or embodiment 52, wherein:

[0137] Any one or more of the first formulation, the second formulation, and / or the optional third formulation comprises cells (eg, mammalian cells, human cells).

[0138] Embodiment 54 The use according to embodiment 49 or embodiment 50 or the formulation according to embodiments 52 to 53, wherein:

[0139] The ion is a calcium ion, or

[0140] The ion source is an ionic salt and / or includes calcium ions.

[0141] Embodiment 55 . The use according to any one of embodiments 48 to 50 or according to embodiment 54 or the formulation according to any one of embodiments 51 to 54, wherein:

[0142] The platelet lysate comprises or consists of human platelet lysate.

[0143] Embodiment 56 The use according to any one of embodiments 48 to 50, 54 or 55 or the formulation according to any one of embodiments 51 to 55, wherein:

[0144] The platelet lysate contains no or substantially no anticoagulant (e.g., heparin) or comprises less than 10% (v / v), 9% (v / v), 8% (v / v), 7% (v / v), 6% (v / v), 5% (v / v), 4% (v / v), 3% (v / v), 2% (v / v), 1% (v / v), 0.5% (v / v) of anticoagulant (e.g., heparin).

[0145] Embodiment 57 The use according to any one of embodiments 48 to 50 or 54 to 56 or the formulation according to any one of embodiments 51 to 56, wherein:

[0146] (i) the first formulation comprises 0.1-20 mg / ml fibrinogen; the second formulation comprises 1-40 U / ml thrombin; and the drug or the formulation comprises a total of 5-40% (v / v) platelet lysate; or

[0147] (ii) the first formulation comprises 0.1-15 mg / ml fibrinogen; the second formulation comprises 1-30 U / ml thrombin; and the drug or the formulation comprises a total of 5-15% (v / v) platelet lysate; or

[0148] (iii) the first formulation comprises 0.1-12 mg / ml fibrinogen; the second formulation comprises 1-25 U / ml thrombin; and the drug or the formulation comprises a total of 7-14% (v / v) platelet lysate; or

[0149] (iv) the first formulation comprises 0.2-10 mg / ml fibrinogen; the second formulation comprises 2-20 U / ml thrombin; and the medicament or the formulation comprises a total of 8-12% (v / v) platelet lysate.

[0150] Embodiment 58 The use according to any one of embodiments 48 to 50 or 54 to 56 or the formulation according to any one of embodiments 51 to 56, wherein:

[0151] (i) the first formulation comprises less than 1 mg / ml (e.g., about 0.4 mg / ml) fibrinogen; the second formulation comprises 0.5-20 U / ml (e.g., about 1 U / ml, about 10 U / ml, about 20 U / ml) thrombin; and the drug or the formulation comprises a total of 5-40% (v / v) (e.g., about 20% (v / v)) platelet lysate; or

[0152] (ii) the first formulation comprises 1-8 mg / ml (e.g., about 4 mg / ml) fibrinogen; the second formulation comprises 0.5-20 U / ml (e.g., about 1 U / ml, about 10 U / ml, about 20 U / ml) thrombin; and the drug or the formulation comprises a total of 5-40% (v / v) (e.g., about 20% (v / v)) platelet lysate; or

[0153] (iii) the first formulation comprises 5-15 mg / ml (e.g., about 10 mg / ml); the second formulation comprises 0.5-20 U / ml (e.g., about 1 U / ml, about 10 U / ml, about 20 U / ml) thrombin; and the drug or the formulation comprises a total of 5-40% (v / v) (e.g., about 20% (v / v)) platelet lysate.

[0154] Embodiment 59 The use according to any one of embodiments 48 to 50 or 54 to 56 or the formulation according to any one of embodiments 51 to 56, wherein:

[0155] (i) the first formulation comprises about 0.4 mg / ml fibrinogen, and

[0156] The second formulation comprises about 2 U / ml, about 10 U / ml or about 20 U / ml thrombin, and

[0157] and the medicament or the formulation comprises a total of about 20% (v / v) platelet lysate; or

[0158] (ii) the first formulation comprises about 4 mg / ml fibrinogen, and

[0159] The second formulation comprises about 2 U / ml, about 10 U / ml or about 20 U / ml thrombin, and

[0160] and the medicament or the formulation comprises a total of about 20% (v / v) platelet lysate; or

[0161] (iii) the first formulation comprises about 10 mg / ml fibrinogen, and

[0162] The second formulation comprises about 2 U / ml, about 10 U / ml or about 20 U / ml thrombin, and

[0163] And the medicament or the formulation comprises a total of about 20% (v / v) platelet lysate.

[0164] Embodiment 60 The use according to any one of embodiments 48 to 50 or 54 to 56 or the formulation according to any one of embodiments 51 to 56, wherein said medicament or said formulation comprises:

[0165] (i) at least 0.2 mg / ml fibrinogen, at least 1 unit / mL thrombin, and about 10% (v / v) platelet lysate; or

[0166] (ii) about 10% (v / v) platelet lysate, about 2 mg / mL fibrinogen, and about 5 U / mL thrombin.

[0167] Embodiment 61 The use according to any one of embodiments 48 to 50 or 54 to 56 or the formulation according to any one of embodiments 46 to 51, wherein:

[0168] The first formulation and the second formulation are equal in volume or less than the following different volumes: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%.

[0169] Embodiment 62 .The use according to any one of embodiments 45, 48 to 50 or 54 to 61, the formulation according to any one of embodiments 51 to 61, the method according to embodiment 46 or the composition according to embodiment 47, wherein the tissue is ocular tissue (e.g., corneal tissue).

[0170] Embodiment 63 .The use according to any one of embodiments 45, 48 to 50 or 54 to 61, the formulation according to any one of embodiments 51 to 61, the method according to embodiment 46 or the composition according to embodiment 47, wherein the tissue comprises a wound or is ocular tissue comprising a wound (e.g., corneal tissue comprising a wound).

[0171] Embodiment 64 .A method according to any one of embodiments 45 or 54 to 62, wherein the formulation is applied to the tissue of the subject using three-dimensional (3D) bioprinting.

[0172] Embodiment 65 .A method according to any one of embodiments 48 to 50 or 54 to 62, wherein the drug used to treat the tissue of the subject is produced using three-dimensional (3D) bioprinting.

[0173] Embodiment 66 .A method according to Example 46, wherein the composition is applied to the tissue using three-dimensional (3D) bioprinting.

[0174] Example 67. The composition of embodiment 47, wherein said treating said tissue utilizes three-dimensional (3D) bioprinting.

[0175] Example 68. Embodiment 6. The formulation of any one of embodiments 51-62, wherein said treating said tissue utilizes three-dimensional (3D) bioprinting.

[0176] definition

[0177] As used in this application, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "component" also includes plural components.

[0178] As used herein, the term "comprising" means "including". Variations of the word "comprising" ("comprise" and "comprises") have correspondingly different meanings. Thus, for example, a composition "comprising" component "A" may consist solely of component A or may include one or more additional components (e.g., component "B" and / or component "C").

[0179] As used herein, the term "subject" includes any animal of economic, social or research importance, including bovine, equine, ovine, primate, avian and rodent species. Thus, a "subject" can be a mammal, such as a human or non-human mammal.

[0180] As used herein, the term "tissue" will be understood to encompass both cells as one or more components of the tissue and one or more organs formed from the tissue.

[0181] As used herein, the term "kit" refers to any delivery system for delivering materials. Such delivery systems include support materials (e.g., buffers, written instructions, etc., for performing assays, etc.) that allow storage, transportation or delivery of reaction reagents (e.g., labels, reference samples, support materials, etc. in appropriate containers) and / or from one location to another. For example, a test kit may include one or more shells, such as boxes, containing relevant reaction reagents and / or support materials. The term "kit" includes both fragmentation test kits and combined test kits. "Fragmentation test kit" refers to a delivery system comprising two or more separate containers of sub-parts each containing all test kit components. The container can be delivered to the intended recipient together or individually. Any delivery system comprising two or more separate containers of sub-parts each containing all test kit components is included in the meaning of the term "fragmentation test kit." Combination test kit" refers to a delivery system containing all components of a reaction assay in a single container (e.g., in a single box containing each component in a desired component).

[0182] As used herein, the term "about" when used in reference to a recited value includes the recited value and values ​​within plus or minus ten percent of the recited value.

[0183] Any description in this document of a prior art document or statements in this document derived from or based on these documents does not constitute an admission that the document or the derived statements are part of the common general knowledge in the relevant art.

[0184] For descriptive purposes, all documents cited herein are incorporated by reference in their entirety unless otherwise stated. BRIEF DESCRIPTION OF THE DRAWINGS

[0185] Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which

[0186] Figure 1 is a series of graphs showing the storage (G') and loss (G") moduli of compositions according to embodiments of the present invention at 34°C under 1 Hz time sweep oscillation for 10 minutes for (A) Bioink 1, (B) Bioink 2, (C) Bioink 3, (D) Bioink 4, (E) Bioink 5, (F) Bioink 6, (G) Bioink 7, (H) Bioink 8, and (I) Bioink 9 (see Table 3).

[0187] Figure 1 legend:

[0188]

[0189]

[0190] Figure 2 A graph is provided showing the viscosity of compositions according to embodiments of the present invention for bio-ink formulations 1-9 (see Table 3), as measured at a fixed temperature of 34° C. between 0.1 s -1 With 100s -1 The shear rate between ramps is measured in Pascal seconds.

[0191] Figure 2 legend:

[0192] 10% hPL + 2mg / mL FI + 5U / mLT

[0193] ..........10%hPL+0.2mg / mLFI+1U / mLT 10% hPL + 2mg / mL FI + 10U / mLT

[0194] 10%hPL+0.2mg / mLFI+5U / mLT..........10%hPL+5mg / mLFI+1U / mLT

[0195] 10% hPL + 0.2 mg / mL FI + 10 U / mLT 10% hPL + 5mg / mL FI + 5U / mLT

[0196] 10%hPL+2mg / mLFI+1U / mLT--10%hPL+5mg / mLFI+10U / mLT

[0197] Figure 3 Shown is a graph indicating the average percent transmittance of compositions according to embodiments of the present invention over wavelengths in the visible spectrum (400-700 nm) for bio-ink formulations 1-9 (see Table 3).

[0198] Figure 3 legend:

[0199] 10% hPL + 2mg / mL FI + 5U / mLT

[0200] ..........10%hPL+0.2mg / mLFI+1U / mLT 10% hPL + 2mg / mL FI + 10U / mLT

[0201] 10%hPL+0.2mg / mLFI+5U / mLT..........10%hPL+5mg / mLFI+1U / mLT

[0202] 10% hPL + 0.2 mg / mL FI + 10 U / mLT 10% hPL + 5mg / mL FI + 5U / mLT

[0203] 10%hPL+2mg / mLFI+1U / mLT --10%hPL+5mg / mLFI+10U / mLT

[0204] Figure 4 is an image of a composition of an example of Bio-ink 5 (see Table 3) according to the invention adhered to a glass slide.

[0205] Figure 5 A graph showing the mean confluence of HCE-T cells over time (n=6 for each condition) as measured by Inuccyte Zoom for Bioinks 1-9 (see Table 3) is provided.

[0206] Figure 5 legend:

[0207] ...0.2_1

[0208] 0.2_5

[0209] ...0.2_10

[0210] 2_1

[0211] 2_5

[0212] 2_10

[0213] 5_1

[0214] 5_5

[0215] ...5_10

[0216] Figure 6 A series of images showing primary human corneal cells cultured and grown in Bio-Ink 5 (Table 3) are provided. (A) shows corneal epithelial cells proliferating from corneal explants. (B) shows keratinocyte growth. (C) shows corneal neuron growth. (D) shows corneal endothelial cell growth.

[0217] Figure 7 Images showing successful growth of SHSY-5Y in a bio-ink formulation according to an example of bio-ink 5 (Table 3) of the present invention are provided. (A) shows cell growth in the ink. (B) shows cells reaching full confluence in the ink.

[0218] Figure 8 Images of spheroid aggregates cultured in a bioink formulation according to an embodiment of the invention are shown (A and B). (C) Corneal keratinocytes grown from spheroid aggregates are shown.

[0219] Fig. 9 Images of absorption and degradation of the composition of Example 5 of Bioink (Table 3) according to the present invention over 2 to 7 days of absorption are provided.

[0220] Fig.10Images (A) and graphs (B) showing the results of scratch assays of HCE-T cells (n=3 per condition) in 5% human platelet lysate (hPL), 5% fetal bovine serum (FBS), and DMEM alone are provided. No wound closure was observed under DMEM alone. (A) shows a representation of wound closure at 0 hours, 5 hours, and 24 hours (top row 0 hours; middle row 5 hours; bottom row 24 hours; left column 5% hPL; middle column 5% FBS; right column DMEM alone). (B) shows the scratch healing rate ± SEM observed under 5% hPL conditions (0.19 ± 0.01 microns / hour) and 5% FBS conditions (0.18 ± 0.03). There was no significant difference in the healing rate between 5% FBS and 5% hPL (p>0.05).

[0221] Fig.11 Images showing re-epithelialization of an ex vivo ulcerated human cornea previously rejected for transplantation purposes are provided. Bioink was applied on days 1 and 4 and fixed, cryosectioned and stained with H&E on day 7. (A) shows the initial ulcer, with the ulcerated area highlighted in red. (B) shows the size of the surface without epithelium after wound debridement. (C) H&E staining of the central portion of the cornea is shown and shows complete re-epithelialization with thickness and morphology comparable to healthy human corneas. (D) H&E staining of the central portion of the de-epithelialized cornea is shown for comparison. (i): epithelium; (ii): stroma; (iii): epithelial loss; (iv): stroma

[0222] Fig.12 Images showing the creation and successful sealing of a perforation (1 cm in diameter) on a post-mortem porcine cornea are provided. (A) Depicts the creation of a perforation. (B) Depicts a leaking cornea. (C) Depicts the application of bio-ink using an applicator device. (D) Depicts the sealed perforation 2 minutes after application of the bio-ink.

[0223] Fig.13 Images demonstrating layered printing of bio-inks of the present invention are provided.

[0224] Fig.14 Images of printed bioinks of the present invention are shown, including P18 HCET cells stained with Hoescht (blue) or propidium iodide (red). A and B: 2 hours after extrusion; C and D: 72 hours after extrusion.

[0225] Fig.15 The transparency of the printed bio-ink composition according to an embodiment of the present invention is shown.

[0226] Fig.16The images of the bio-ink according to the present invention on the slide after the immersion test are shown. According to Table 5, A. shows composition 1, B. shows composition 2, C. shows composition 3, D. shows composition 4, E. shows composition 5, F. shows composition 6, and E. shows composition 7.

[0227] Fig.17 Graph showing the level of force that can prevent leakage in a human corneal wound (1.5 mm) treated with the bio-ink of the present invention: A: 1.63 N, i.e., 112 mmHg; B. 1.33 N, i.e., 105 mmHG; C: -.71 N, i.e., 56 mmHG.

[0228] Fig.18 is a graph showing the average burst pressure (mmHg±SEM) that the bio-ink of the present invention can withstand through a 1.5 mm diameter perforation sealed with the bio-ink.

[0229] Fig.19 The viscosity of the bio-ink of the present invention ± SEM is shown at a fixed shear rate of 1 s -1 (measured in Pascal seconds (Pa.s)) versus time (in seconds, n=3). Component B was added at t=60 seconds. The setting time is defined as the time required for the viscosity to reach its peak value after addition and is represented by the pink dashed line. -1 The setting time was determined to be 25 seconds at a fixed shear rate.

[0230] Fig. 20 Images of wound healing over time are provided: (i) eyes of rabbits with corneal epithelial wounds treated with the bioink of the present invention (column B); and (ii) eyes of control rabbits with corneal epithelial wounds treated with histoacryl (column A). At t=52 hours, wound healing was achieved in both control and treated rabbits. - (before wound initiation); Number (hours after wound initiation).

[0231] Fig.21 is depicted as measured over the time until wound healing was achieved (t = 52 hours) Fig. 20 Figure 3. Pain scores of control rabbits. The time point at which additional analgesia was administered was recorded. 1: administration of ketamine (rescue analgesia); 2: administration of methadone (rescue analgesia); 3: administration of buprenorphine (IM); 4. administration of meloxicam.

[0232] Fig. 22 The measured time until wound healing was achieved (t = 52 hours) is depicted Fig. 20 Figure 3. Pain scores of rabbits treated with bio-ink. The time point at which additional analgesia was administered was recorded. 1: administration of buprenorphine (IM); 2: administration of meloxicam.

[0233] Fig.23 Images of wound healing over time are provided, wherein: (i) two rabbit eyes each with corneal perforations treated with the bioink of the present invention (columns A and B); and (ii) the eye of a control rabbit with a corneal perforation treated with cyanoacrylate (column C). When corneal perforation wound healing is achieved, the pink border is marked. When secondary ulcer formation is healed, the green border is marked. Corneal perforation wound healing in the control rabbit was observed at t=80 hours, and corneal perforation wound healing in the treated rabbits was observed at t=28 hours and t=32 hours, respectively. A: Rabbit 2 treated with bioink; B: Rabbit 2 treated with bioink; C: Control rabbit. Numbers (hours of wound initiation).

[0234] Fig.24 is depicted as measured over time until complete wound healing was achieved (t = 80 hours). Fig.23 Figure 3 is a graph of pain scores of control rabbits (column C) with 100 mg / kg shampoos. The time points at which additional analgesia was administered were recorded. A: administration of buprenorphine (IM); B: administration of meloxicam; C: cyanide-free acrylate; D: secondary ulceration.

[0235] Fig.25 is depicted as measured over time until complete wound healing was achieved (t = 52 hours). Fig.23 Figure 3. Pain scores of rabbits treated with bio-inks (column A). The time point at which additional analgesia was administered was recorded. A: secondary ulceration; B: buprenorphine (IM) administration; meloxicam administration.

[0236] Fig.26 is depicted as measured over time until complete wound healing was achieved (t = 48 hours). Fig.23 Figure 3. Pain scores of rabbits treated with bio-inks (column B). The time point at which additional analgesia was administered was recorded. A: secondary ulceration; B: buprenorphine (IM) administration; meloxicam administration.

[0237] Fig. 27Images of wound healing over time are provided, wherein: (i) rabbit eyes with modified corneal perforations treated with the bioink of the present invention (column A); and (ii) control rabbit eyes with modified corneal perforations treated with cyanoacrylate (column C). When modified corneal perforation wound healing is achieved, the pink border is marked. When secondary ulcer formation is healed, the green border is marked. Healing of the modified corneal perforation in the control rabbit was observed at t=48 hours, and healing of the corneal perforation in the treated rabbit was observed at t=8 hours. Column A: Rabbit 2 treated with bioink; Column B: Control rabbit. Number (hours of wound initiation).

[0238] Fig.28 is a graph depicting the following pain scores: (i) as measured over the time taken before complete wound healing was achieved (t = 48 hours); Fig. 27 (ii) the time taken before complete wound healing was achieved (t = 8 hours) Fig. 27 Figure 2. Rabbits treated with bio-ink (lighter line) (column A). The time point at which additional analgesia was administered was recorded. No additional analgesics were administered to rabbits treated with bio-ink. A: Buprenorphine (IM) was administered; B: methadone was administered (rescue analgesia); C: Meloxicam was administered; D: No cyanoacrylate and secondary ulcer formation. Fig.28 legend:

[0239] Control

[0240] ink

[0241] Fig.29 Shown are (A) images of bioink samples freeze-dried at -40 degrees Celsius; (B) reconstitution of the freeze-dried samples of (A) with 50 uL of water per tube to clarify the liquid; and (C) the adhesive and clear gel formed after reconstitution. DETAILED DESCRIPTION

[0242] The inventor has developed a platelet lysate composition with mechanical and structural properties that promotes application to tissues in a structural form. Specifically, the composition of the present invention can be used to apply platelet lysate to tissues (e.g., eyes) using two-dimensional or three-dimensional bioprinting technology. The composition provides a device for delivering a medicament to a biological target (e.g., an organ, tissue, cell). Although suitable for use in treating corneal injuries, the composition described herein provides a platform for multiple treatments in the field of wound healing by providing, for example, structural support, living cells and other factors that promote the wound healing process. Effective blood-derived treatments for wounds such as ocular surface wounds are needed in the art. The composition described herein is based on platelet lysate, which not only lubricates the ocular surface to serve as a tear substitute in the context of eye treatment, but also provides a source of multiple growth factors and cytokines that promote wound healing. The composition described herein can utilize a biomaterial that mimics in vivo tissue and serves as a scaffold filled with cells, and / or encourages cells to regenerate their surrounding matrix by the manipulation of conditions. In the context of its suitability for application to eye tissue, the inventors, for example, have solved the difficulty of producing a matrix that can be embodied in the structural integrity of the tissue (e.g., cornea) under treatment while maintaining transparency and still being porous and biocompatible to allow infiltration, migration and / or proliferation of corneal cells and the growth factors required to promote healing. The balance between providing the nutritional needs of damaged tissue and simultaneously meeting the structural, mechanical and physical requirements of damaged tissue (e.g., eye tissue such as cornea) is a problem that exists when the present invention appears. The present invention provides improved compositions and methods for delivering agents to various biological targets. Without being limited to any particular application, the compositions can be used to treat wounds and other forms of tissue damage.

[0243] Compositions for delivery of biological agents

[0244] The present invention provides compositions suitable for delivering pharmaceutical agents to biological targets such as tissues and cells.

[0245] The composition utilizes a base scaffold material to provide structural support when applied to a biological target (eg, tissue, membrane, cell, organ) to facilitate delivery of an agent to the biological target and / or promote regeneration of damaged tissue and / or cells within the biological target.

[0246] There is no particular limitation regarding the specific material or materials used to create the scaffold.

[0247] For example, the support can be a fibrin support. These can be produced, for example, by using fibrinogen and thrombin in the composition. As known to those skilled in the art, the conversion of fibrinogen to fibrin usually occurs in three stages. Thrombin can catalyze the first stage, which is a limited proteolysis to release fibrin peptides (FpA and FpB) from fibrinogen, and thus provide fibrin monomers. In the second stage, the fibrin monomers can form intermediate polymers by non-covalent interactions. In the third stage, the intermediate polymers can be aggregated to form a three-dimensional fibrin matrix. Fibrin formation and its use in bioscaffolds are described in, for example, Ahmed et al. (" Tissue Engineering Part B Review (Tissue Eng Part B Rev.) ", June 2008; 14 (2): 199-215).

[0248] The compositions of the present invention may further optionally include ions and / or ion sources. Non-limiting examples of suitable ions include calcium ions. Non-limiting examples of suitable ion sources include compounds including calcium (e.g., calcium chloride).

[0249] The composition of the present invention may include platelet lysates. Platelet lysates may be, for example, mammal platelet lysates (e.g., platelet lysates produced using human, dog, cat, cattle, pig, horse, caprine, hircine, murine, hare, cricetine or mustelid platelets or any combination thereof). The platelet source used to generate platelet lysates generally depends on the specific purpose of the composition to be used. As known to those of ordinary skill in the art, platelet lysates are generated by separating platelets, cracking the platelets and removing cell debris. The composition and application of platelet lysates have been well analyzed (see, for example, Burnouf et al., Biomaterials. January 2016; 76: 371-87).

[0250] The inventors have identified the optimal relative concentrations of fibrinogen, thrombin and platelet lysate of the compositions of the present invention, some of which are described in the examples and claims of this application. It should be understood that the relative concentrations of fibrinogen, thrombin and platelet lysate disclosed are exemplary only.

[0251] The composition according to the invention may comprise cells. The cells may be, for example, mammalian cells (e.g., human cells, canine cells, cat cells, bovine cells, porcine cells, horse cells, caprine cells, hircine cells, murine cells, rabbit cells, hamster cells, or mustelid cells, or any combination thereof). The type of cell utilized will generally depend on the specific purpose for which the composition is to be used. For example, the cell can be the same type as the tissue to which the composition is to be applied (e.g., ocular surface cells, including cells of the central and / or peripheral corneal epithelium, medullary and / or conjunctival epithelium, conjunctival stroma, and / or eyelid margin; skin cells, including but not limited to keratinocytes, melanocytes, Merkel cells, and Langerhans cells; and neural tissue cells, including but not limited to neurons and glial cells. Other examples include epithelial cells, keratinocytes, neuronal cells, and endothelial cells. In some embodiments, the cell can be a hematopoietic stem cell, a bone marrow stem cell, a neural stem cell, an epithelial stem cell, a skin stem cell, a muscle stem cell, an adipose stem cell, a pluripotent stem cell, an induced pluripotent stem cell, an embryonic stem cell, a mesenchymal stem cell, or any combination thereof. In some embodiments, the cell can be a neuronal cell.

[0252] The platelet lysate and / or cells of the composition can be autologous (ie, self-derived from a given subject intended to receive the composition or (ie, donor-derived)).

[0253] The compositions of the present invention may include essential and / or non-essential amino acids. Non-limiting examples of suitable essential amino acids include isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, cysteine, tyrosine, histidine and arginine.

[0254] The compositions of the present invention may include additional components (e.g., one or more agents) including, but not limited to, fibronectin, anesthetics, antibiotics, growth factors (e.g., human epidermal growth factor (hEGF), platelet-derived growth factor, vascular endothelial growth factor, fibroblast growth factor, epithelial growth factor, transforming growth factor [including β] and connective tissue growth factor), fibrin stabilizing factors (e.g., tissue factor XIII), one or more matrix proteins (e.g., collagen, laminin, integrin), and any combination thereof.

[0255] The composition of the present invention may include other suitable ingredients, including water and / or culture medium (e.g., DMEM, DMEM / F-12, MEM, CnT-PR). The culture medium may include, for example, glycine, L-alanine, L-arginine hydrochloride, L-asparagine-H2O, L-aspartic acid, L-cysteine ​​hydrochloride-H2O, L-cystine 2HCl, L-glutamic acid, L-glutamine, L-histidine hydrochloride-H2O, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine disodium salt dihydrate, L-valine, vitamins, biotin, choline chloride, D-calcium pantothenate, folic acid, nicotinamide pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, i-inositol, inorganic salts , calcium chloride (CaCl2) (anhydrous), copper sulfate (CuSO4-5H2O), ferric nitrate (Fe(NO3)3"9H2O), ferric sulfate (FeSO4-7H2O), magnesium chloride (anhydrous), magnesium sulfate (MgSO4) (anhydrous), potassium chloride (KCl), sodium bicarbonate (NaHCO3), sodium chloride (NaCl), anhydrous disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4-, zinc sulfate (ZnSO4-7H2O), other components, D-glucose (dextrose), hypoxanthine Na, linoleic acid, lipoic acid, putrescine 2HCl, sodium pyruvate, thymidine or any combination thereof.

[0256] In some embodiments, the composition does not include an anticoagulant or is substantially free of an anticoagulant that may only be present in trace amounts. Non-limiting examples of these anticoagulants include heparin, vitamin K antagonists (e.g., warfarin, coumarin), rivaroxaban, edoxaban, apixaban, dabigatran, etc.

[0257] Non-limiting properties of the composition include one or more of the following:

[0258] - Non-Newtonian shear-thinning fluid properties, whereby the viscosity of the composition may decrease with increasing shear rate. In some embodiments, the viscosity of the composition may be in the range of 0.01 and 1000 Pa.s at room temperature.

[0259] - Optical clarity without obstructing or substantially obstructing vision resulting from the transmission of light, for example, greater than 90% in the visual color range of 400-700 nm.

[0260] - Suitability for printing (e.g., bioprinting / extrusion printing) with the ability to maintain or substantially maintain shape / structure after printing.

[0261] - Suitability for printing while maintaining cell viability in the composition during the printing process.

[0262] - Capability to be provided in two-dimensional or three-dimensional structures with or without the inclusion of living cells.

[0263] - Ability to maintain and / or promote cell growth (for example, maintain and / or promote the proliferation and growth of primary human cells such as epithelial cells, keratinocytes, neuronal cells and endothelial cells).

[0264] - Ability to promote spheroid organoid formation.

[0265] - The ability to be degraded by cells over time (e.g., 2-7 days).

[0266] - Wound healing capacity (eg corneal ulcer) over time (eg 7 days).

[0267] -Maintain cell viability over time (e.g., 7 days at 34°C).

[0268] -The ability to adhere to various surfaces including tissues, organs, membranes (e.g., mammalian and human tissues, organs, membranes).

[0269] Preparation of wound healing compositions

[0270] The compositions of the present invention include platelet lysates (e.g., mammalian platelet lysates, human platelet lysates). Platelet lysates can be prepared by any suitable method (e.g., by freeze / thaw lysis; see, e.g., Chou and Burnouf, ISBT Science Series, Vol. 12, No. 1, February 2017, pp. 168-175).

[0271] In addition, the inventor has observed that the use of anticoagulants during platelet lysate preparation (e.g., when cultivating) can have an impact on the ability of platelet lysate to form compositions according to the present invention. Therefore, in some embodiments of the present invention, in some or all stages of preparation method, the platelet lysate utilized is prepared without using anticoagulants (e.g., heparin).

[0272] Typically, the compositions of the invention can be prepared by combining a variety of different formulations. In some embodiments, the formulation comprising thrombin can be maintained separately from the formulation comprising fibrinogen, and the two formulations are combined before or during application of the composition.

[0273] For example, the first preparation including fibrinogen and platelet lysate can be mixed with the second preparation including thrombin to provide compositions of the present invention.Alternately, the first preparation including fibrinogen can be mixed with the second preparation including thrombin and platelet lysate to provide compositions of the present invention.Other components (for example, ions, ion sources, amino acids, cells, antibiotics, growth factors, fibrin stabilizing factors, anesthetics, etc.) can be incorporated into compositions at any suitable time (for example, before the mixing stage) in any suitable manner.In certain embodiments, some or all of the components in other components (that is, components except fibrinogen and thrombin) can be included in the preparation including thrombin and / or the preparation including fibrinogen.In other embodiments, some or all of the components in other components (that is, components except fibrinogen and thrombin) can be provided in one or more preparations separated from those preparations including thrombin or fibrinogen. In still other embodiments, some of the additional components (i.e., components other than fibrinogen and thrombin) may be included in a preparation comprising thrombin and / or a preparation comprising fibrinogen, and some of the additional components (i.e., components other than fibrinogen and thrombin) may be included in one or more preparations separate from those preparations comprising thrombin or fibrinogen.

[0274] In some embodiments, the composition can be prepared by establishing separate flow streams of separated components. These streams can be maintained in a state of continuous flow for a suitable period of time and directed to mix with each other at a given point to thereby provide additional flows of mixed components deposited on the biological target. Alternatively, the streams can be directed to mix with each other at or on the surface of the biological target to which the composition is applied.

[0275] In certain embodiments, the present invention provides and promotes to separate and form the different preparations required for the present composition until the device and / or the test kit used.Usually, the device and the test kit can include at least two physically separated compartments, the first including the thrombin preparation, and the second including the fibrinogen preparation.In certain embodiments, one or two compartments can include the other components (for example, platelet lysate, ion, amino acid, cell, antibiotic, growth factor, fibrin stabilizing factor, anesthetic etc.) to be used in the generation composition.

[0276] Devices and kits may further include components that provide a means of promoting mixing of two separated preparations, for example, by removing a barrier separating the first compartment and the second compartment, and / or by piercing a seal or wall of one or both compartments. It will be readily appreciated by the skilled person that various arrangements may be made for this purpose.

[0277] Additionally or alternatively, the device and kit may be configured in a manner to ensure mixing of the two separate formulations during or after release of the formulations from the device or kit.

[0278] In some embodiments, the devices and kits may include additional compartments that include additional components to be used in generating the composition (e.g., platelet lysate, ions, amino acids, cells, antibiotics, growth factors, fibrin stabilizing factors, anesthetics, etc.). The device or kit may be configured in such a way as to facilitate mixing of these additional components with each other and / or with one or more preparations of fibrinogen and / or thrombin during use of the device or kit.

[0279] The devices and kits may facilitate mixing of the separate components before, during, or immediately after expulsion of the components from the device or kit.

[0280] In certain embodiments, device and test kit can provide the device of the independent stream of the component of separation.These streams can maintain the state of continuous flow during the use of device or test kit, and can be directed to mix with each other at a given point.The mixing point can be located in the device or test kit, at the site where the stream leaves the device or test kit, or outside the device or test kit (for example, at the surface of the biological target to be applied by the composition or before).In certain embodiments, the composition is a bio-ink, and the device is a three-dimensional (3D) printer (for example, an extrusion printer).

[0281] Application of the composition

[0282] The inventors have developed a composition for delivering one or more agents to target tissues and cells, characterized in that it is highly suitable for bioprinting. In addition to using the composition in therapeutic applications in wound healing, the composition of the present invention can also be used in therapeutic applications and / or tissue culture methods, in which it is necessary to apply delivery agents (e.g., natural growth factors, drugs, nanoparticles and / or cells) and / or to fix a single biological surface.

[0283] In some embodiments, the composition can act as a wound sealant and / or fixative for biological structures. It can provide structural and / or nutritional support for the wound. Additionally or alternatively, the composition can promote the growth of one or more target cell types, including those that can be provided as a component of the composition and / or cells present in the wound area.

[0284] Although there is no limitation on the type of tissue to which the composition can be applied, the inventors have demonstrated that the composition is highly effective in treating ocular wounds.

[0285] For example, it is demonstrated herein that the composition is effective in treating corneal wounds. In these embodiments, the composition can be used to promote the proliferation and / or migration of corneal epithelial cells. The composition can, for example, support the multidirectional growth and / or stratification of corneal epithelial cells, which can partially or completely biodegrade the composition once a cell monolayer is formed.

[0286] Thus, the present invention provides methods of treating wounds, and medicaments made for such treatment methods. The wound may, for example, be located in or around ocular tissue, including central and / or peripheral corneal epithelium, medullary and / or palpebral conjunctival epithelium, palpebral conjunctival stroma, and / or eyelid margin.

[0287] It will be appreciated by those skilled in the art that, without departing from the spirit or scope of the invention as broadly described, various changes and / or modifications may be made to the invention as disclosed in the specific embodiments. Therefore, the present embodiments are to be considered in all aspects as illustrative and not restrictive.

[0288] Examples

[0289] The invention will now be described with reference to specific examples, which should not be construed as limiting in any way.

[0290] Example 1: Preparation and characterization of bio-ink compositions

[0291] -Materials and methods

[0292] (i) Preparation of platelet lysate

[0293] Pharmaceutical human platelet lysate was prepared by the Australian Red Cross Blood Service using the following two methods:

[0294] 1. Apheresis

[0295] a. Platelets are obtained by apheresis.

[0296] b. Store the platelets at 4 degrees Celsius for 5 days.

[0297] c. Platelet lysate concentration is higher due to the apheresis process, but contains

[0298] d. Platelet lysates were prepared by multiple freeze-thaw cycles, centrifuged and cleared of debris by opti-press in each round, and then stored at -80°C.

[0299] 2. Pooling

[0300] a. Hang the blood bag and separate the red blood cells by in-line filtration.

[0301] b. The plasma was spun in the bag and then separated by opti-press to a plasma / platelet concentration of 30%.

[0302] c. The platelet product remaining in the bag is collected in a closed system by a tube welder.

[0303] d. Store the pooled platelets at 4 degrees Celsius for 5 days.

[0304] e. Pooled platelet units were pooled into 2-liter batches (50-70 donations).

[0305] f. The pooled platelet units were then centrifuged and the supernatant removed to obtain approximately 2 × 10 11 Platelet concentration.

[0306] g. Platelet lysates were prepared by multiple freeze-thaw cycles, centrifuged and cleared of debris by opti-press, and then stored at -80°C.

[0307] Notice:

[0308] • All hPLs prepared are stable at -80°C for up to 12 months.

[0309] • Pooled platelets do not undergo fibrinogen reduction due to low plasma concentrations of less than 30%.

[0310] • It is prepared in a closed system by final centrifugation and sterile filtration to remove pathogens.

[0311] ●All platelets are gamma irradiated prior to storage, and no pathogen inactivation technology is used.

[0312] Lysates obtained from the Red Cross Blood Bank were thawed overnight at 4°C and then placed in a 37°C water bath for 2 hours. Thereafter, if used immediately, they were stored at 4°C or -80°C until needed.

[0313] (ii) Bio-ink preparation

[0314] Pharmaceutical compositions ("bio-ink compositions") including platelet lysate, thrombin, and fibrinogen were manufactured. In some cases, the bio-ink compositions include additional ingredients such as ions (e.g., calcium), pharmaceutically acceptable essential amino acids (e.g., isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, cysteine, tyrosine, histidine, and / or arginine), fibronectin, human endothelial growth factor, tissue factor XIII, anesthetics, antibiotics, other growth factors (including but not limited to platelet-derived growth factor, vascular endothelial growth factor, fibroblast growth factor, epithelial growth factor, transforming growth factor (including β) and / or connective tissue growth factor), and / or other ingredients / excipients (see Table 1).

[0315] Table 1: Composition of bio-inks

[0316]

[0317] The composition was made by first preparing two separate solutions A and B (see Table 2), which were mixed together before applying the bio-ink. Complete mixing was not required, and partial mixing was sufficient to induce the formation of the bio-ink.

[0318] Table 2: Two solutions mixed to form bio-ink

[0319]

[0320] -Results and discussion

[0321] The bio-ink was produced by forming a transparent and colorless gel upon mixing solutions A and B. The following properties of the bio-ink were noted.

[0322] - Bio-inks are non-Newtonian shear-thinning fluids, whereby their viscosity decreases with increasing shear rate. The viscosity of bio-inks at room temperature ranges between 0.01 and 1000 Pa.s.

[0323] - With a transmittance of more than 90% in the visual color range of 400-700nm, the bioink is optically transparent and does not obstruct vision.

[0324] - The bioink can maintain its printability through extrusion printing and retain its shape once extruded, as determined by rheology.

[0325] -Bioinks can adhere to tissues.

[0326] - The bioink can sustain and promote the growth of cells in tissue culture in the absence of a secondary support medium.

[0327] - The bioink can maintain and promote the expansion and growth of human primary cells (such as epithelial cells, corneal cells, neuronal cells and endothelial cells).

[0328] -Bioinks promote the formation of spheroid organoids.

[0329] - The cells can degrade the bioink over a period of 2 to 7 days and reabsorb the bioink matrix.

[0330] -The bioink can heal wounds, such as corneal ulcers, over the course of seven days.

[0331] - The bioink could maintain the cell viability within the bioink for up to 7 days when stored at 34°C.

[0332] -Bio-inks enable 3D cell printing by suspending cells in a first component.

[0333] - Bioinks provide both structural support (scaffold) and nutritional support, thereby promoting cell growth and healing.

[0334] -Bioinks promote the growth of multiple cell types.

[0335] The bioink is able to promote the growth of multiple cell types in the cornea, allowing it to act as a wound healing treatment extending through multiple layers of the cornea and / or promote neuronal growth in human corneal explants. SHSY5Y as a non-corneal derived neuronal cell line has been successfully cultured in the bioink, allowing for applications across the cornea.

[0336] The bioink can be used in tissue culture to produce spheroidal aggregates of cells. The aggregates can be made in a simpler way than known methods and spheroids can be generated without enzymatic digestion of tissue or the use of microwells.

[0337] Bioinks are versatile and customizable, and can be tailored to the needs of individual patients, covering a wider range of applications that can extend across more pathologies and more tissue types. Autologous platelet lysates or lysates from donors can be included in the bioink, depending on what is most beneficial to the patient. The concentration and type of antibiotics and anesthetics / analgesics can be customized to target specific infections / diseases and address potential patient allergies. Preliminary data using components such as collagen and other proteins in bioinks, as well as various factors at different concentrations, have allowed their mechanical and nutritional properties to be modified and optimized for a variety of applications.

[0338] The bioink can be used to fill ocular defects at high resolution, is liquid, and once mixed can solidify in matter within minutes and is not affected by temperature at the wound site. The bioink is able to effectively fill defects and maintain shape to potentially accelerate wound healing and / or reduce the amount of time required in the clinic. The shear-thinning properties of the bioink can be provided within a range that makes it compatible with extrusion and droplet-based bioprinting.

[0339] The bio-ink is essentially transparent, a feature that is beneficial for preserving vision in addition to promoting wound healing and cell growth in the cornea.

[0340] Thus, bioinks can provide both structural and trophic support to, for example, corneal cells, have a wide range of potential applications, possess favorable physical properties, and have been shown to generate neuronal outgrowth and spheroid aggregates in primary culture.

[0341] Example 2: Optimization and exemplary properties of bioink formulations

[0342] -Materials and methods

[0343] (i) Reagents and antibiotics

[0344] Dulbecco's modified Eagle's medium / F12GlutaMax without phenol red (DMEM / F12), phosphate buffered saline (PBS), TrypLE, and penicillin / streptomycin were purchased from Gibco TM Human platelet lysate was obtained from the Red Cross Blood Bank. Fetal bovine serum (FBS) was purchased from Corning (USA). Human epidermal growth factor (hEGF), penicillin-streptomycin [(PS), 10,000 U / mL] were purchased from Life Technology (USA). Fibrinogen and thrombin were purchased from Merck-Millipore (USA). Hematoxylin and eosin were purchased from Sigma-Aldrich (USA).

[0345] (ii) Sample Collection

[0346] Human corneas not suitable for transplantation were provided by Lions NSW Eye Bank with consent and ethical approval from the Local Health District Human Research Ethics Committee (HERC 14 / 275). All procedures were in accordance with the Declaration of Helsinki.

[0347] Frozen porcine eyes were obtained from the Vision Basic Training Laboratory at Sydney Eye Hospital.

[0348] (iii) Scratch test

[0349] SV40 immortalized human corneal epithelial cell line (HEC-T, Rilka, Japan) was thawed and cultured in 5% FBS medium (5% FBS, 10ng / mL hEGF, 5% PS, DMEM / F12 glutmax) until 80% confluence. The cells were then passaged into two T-75 flasks, one of which was cultured with 5% FBS medium and the other was grown in 5% hPL medium (5% hPL, 10ng / mL hEGF, 5% PS, DMEM / F12) until 80% confluence. HCE-T cells from 5% FBS were then inoculated into six wells of a 24-well plate (20,000 cells / well) and left until fully confluent. Similarly, HCE-T cells from 5% hPL were inoculated in three wells in the same 24-well plate at the same density and left until fully confluent. The culture medium was changed every 2 days. The day before scraping, three of the 6 wells cultured in 5% FBS were replaced with DMEM. After scraping, all wells were monitored and images were captured every 2 hours using an Olympus DP70 optical microscope (Olympus, the United States) with a 4x objective. All images were analyzed by Image J (v1.51). For the first 5 hours, the rate of wound closure was calculated as the reduction of wound size in 5 hours. This was calculated in Excel (Microsoft) and graphically and statistically analyzed in Prism (GraphPad Software Company (GraphPad), the United States).

[0350] (iv) Bio-ink formulation testing

[0351] Bio-ink preparation

[0352] The received hPL was thawed at 4 °C overnight and then incubated in a 37 °C water bath for 2 h. The hPL was then stored at 4 °C until further use.

[0353] Fibrinogen was dissolved in sterile water at 37°C and dispensed into 1 mL Eppendorf tubes at a concentration of 20 mg / mL. It was stored at -20°C and thawed at 37°C before use.

[0354] Thrombin was dissolved in DMEM / F12 and dispensed into 1 mL Eppendorf tubes at a concentration of 20 U / mL. It was stored at -20°C and thawed at 37°C before use.

[0355] The bio-inks were prepared by homogenously mixing two separate components: component one contained hPL and fibrinogen in DMEM / F12; and component two contained thrombin in DMEM / F12. Various concentrations of fibrinogen (0.2, 2, and 5 mg / mL) and thrombin (1, 5, 10 units / mL) were tested. All bio-inks contained an initial concentration of 20% hPL (Table 1).

[0356] Table 3. Nine bio-ink formulations were tested, with their individual component compositions listed. All concentrations tested refer to pre-mixed concentrations.

[0357]

[0358] Rheology

[0359] The rheological properties of the prepared inks were examined by an AR-G2 rheometer (TA Instruments, USA). A 2° / 40 mm cone-plate geometry was used in all tests. Time sweep tests were performed to determine the consistency of the ink at a constant frequency of 1 Hz and a strain of 1%. The temperature was also kept constant at 34°C to mimic the temperature at the corneal surface. The storage modulus (G') and loss modulus (G") of each bioink (n=3 for each bioink) were measured as a function of time over 10 minutes. The data were tabulated, graphed and analyzed using Prism (GraphPad, USA).

[0360] Shear thinning is a desirable property for extrusion-based bioprinting, therefore, the ink viscosity in response to applied shear stress was also measured by rheometer. For each bioink, the viscosity of the ink was measured at a fixed temperature of 34 °C over a period of 0.1 s -1 With 100s -1 Viscosity of shear rate ramp between 1 and 2 (n=3 for each bioink). Data were tabulated, graphed and analyzed using Prism (GraphPad, Inc., USA).

[0361] transparency

[0362] The average transmittance of light was measured over the visible spectrum (400-700 nm). The machine was zeroed on the slide, and then samples were placed on the slide (n=3 per sample) and the average transmittance was measured. A visual representation of the transparency of the ink could be created by writing "cornea" with ink on a glass slide and holding it up to the light.

[0363] Cytocompatibility

[0364] The bioink prepared as described above was also used for cytocompatibility testing. HCE-T cells (passage 23) were seeded in 96-well plates (6000 cells / well, n=6 for each condition) and cultured using Incucyte TM (ZOOM, Essen BioScience Inc) Confluence was measured every 2 hours over 7 days.

[0365] All subsequent experiments were performed using one formulation of Bioink 5 (Table 3, premix concentrations: 20% hPL, 4 mg / mL fibrinogen, 10 U / mL thrombin, DEME / F12).

[0366] Cultivation of corneal explants

[0367] Corneal explants were obtained from the Lyon NSW Eye Bank. The limbal and central regions were separated using 8 mm trephine. The limbal region was then cut into 8 equal-sized pieces with as much sclera removed as possible and placed in one well of a 6-well plate, and the central region was cut into 8 equal-sized pieces and placed in another well of a 6-well plate. The explants were then covered with 2 mL of bioink 5 (Table 3).

[0368] (v) In vitro wound healing assay

[0369] The epithelium of the ulcerated cornea was removed with a surgical spatula (received from the NSW Eye Bank in Lyon) and rinsed with PBS. Bio-ink 5 (Table 3) was applied immediately to the wound surface. The cornea was then suspended in a jar of organ culture medium. The ink was reapplied on the 4th day. On the 7th day, the cornea was fixed overnight with 4% paraformaldehyde, rinsed twice with PBS, and left in 30% sucrose overnight. The cornea was then cut in half, immersed in OCT compound (Tissue-Tek, USA), flash frozen in liquid nitrogen, and stored at -80°C until sectioning. The frozen cornea was cut into a thickness of 12 μm at -20°C using a Leica CM3050 S cryostat (Leica Biosystems, Germany, Germany) and transferred to a polylysine-coated slide, dried and stored at -20°C. For Mayer's hematoxylin and eosin staining, the slides were immersed in 95% ethanol for 15 seconds and then immersed in 4% paraformaldehyde for 10 immersions. The slides were then rinsed in distilled water for 10 immersions and suspended in hematoxylin for 30 seconds. The slides were then immersed in two changes of distilled water for 10 times, and then immersed in 95% ethanol for another 10 times. The slides were then counterstained in eosin for 15 seconds, and then dehydrated in 2 changes of 95% ethanol and 2 changes of 100% ethanol, each immersion 10 times. The slides were then immersed in 2 changes of xylene for 10 times, and mounted in DPX (Sigma Aldrich). The sections were examined with an Olympus DP70 optical microscope. Human donor corneas were used as negative controls, the epithelium was removed and immediately fixed, sliced ​​and stained using the same protocol.

[0370] (vi) In vitro perforation test

[0371] Corneas from frozen porcine eyes (n=3) were excised and then fixed in a Barron artificial anterior chamber. The cornea was expanded by injecting water through a syringe connected to the artificial anterior chamber. A perforation was made in each cornea with a metal needle (1 cm in diameter), and water leakage was observed to confirm that a full-thickness perforation had been made. Bio-ink 5 (Table 3) was then applied to the site of the perforation and allowed to coagulate for 2 minutes. The cornea was then re-expanded and any subsequent water leakage was observed.

[0372] (vii) Cultivation of non-corneal derived cell lines

[0373] SHSY-5Y cells were thawed and cultured in Bio-ink 5 (Table 3) in T-25 flasks. Cell morphology and confluence were observed by Olympus DP70. The cells were passaged when they reached 80% confluence, and the passage number was recorded.

[0374] Bio-ink production using calcium ion solution and MEM to replace the DMEM / F12 component of bio-ink

[0375] Calcium chloride solution (at a concentration of 200 mg / mL) and MEM were used to replace the DMEM / F12 component of the bioink, and gelation of the final product was observed.

[0376] (viii) Statistical analysis

[0377] All statistical analyses were performed in Prism (GraphPad, Inc., USA). T-tests were used to compare two separate conditions. In the case of three or more formulations used, one-

[0378] -Results and discussion

[0379] (i) Scratch measurement

[0380] At what rate and at what rate did the wells treated with 5% hPL and 5% FBS completely heal at 24 hours? Figure 1 No significant differences were observed (p>0.05). No wound closure was observed in the DMEM only condition after 24 hours.

[0381] (ii) Rheology

[0382] Time sweep experiments showed that bioink formulations 2-9 (Table 3) were able to gel because the storage modulus was greater than the loss modulus ( Figure 2 ), and the ink is consistent because both moduli are stable over time ( Figure 2 ). Time-dependent viscosity measurements showed that while the viscosity of all bioinks decreased with increasing shear rate ( Figure 3 ), all bioink formulations 1-9 (Table 3) exhibited non-Newtonian fluid and shear-thinning behavior.

[0383] (iii) Transparency

[0384] All variants of the ink (Table 3) showed more than 85% transparency for wavelengths in the visible spectrum ( Figure 4 Direct assessment of ink transparency enhances the printability of the ink, as it can be extruded and retain shape, and enhances transparency, as buildings can be easily seen through the ink ( Figure 5 ).

[0385] (iv) Cytocompatibility

[0386] With the exception of bioink formulations 6 and 9 (Table 3), cell confluence increased over time, indicating that bioink formulations 1-5 and 7-8 supported cell growth ( Figure 6). Cells incubated with premixed thrombin at a concentration of 20 U / mL and 4 and 10 mg / mL fibrinogen in the ink showed no increase in mean confluence. In addition, the mean confluence rate decreased with increasing fibrinogen concentration. Corneal epithelial cells, keratinocytes, endothelial cells, and neuronal cells were observed to be growing in the bio-ink ( Figure 7 Degradation of the bioink was also observed once cells began to migrate out of the explants ( Figure 7 a).

[0387] (v) In vitro wound healing

[0388] Compared with de-epithelialized corneas, multiple cell layers were observed in the ulcerated corneas treated with bioink ( Figure 8 ). This demonstrated that the bioink was able to promote complete re-epithelialization of ex vivo ulcerated human corneas.

[0389] (vi) Puncture test

[0390] All three perforated corneas showed complete wound sealing with ink application within 2 minutes of clotting time ( Fig. 9 ).

[0391] (vii) Compatibility with other cell types

[0392] SH-SY cells were able to reach confluence and were successfully cultured for 8 passages ( Fig.10 ).

[0393] -discuss

[0394] A transparent, printable, biocompatible and biodegradable platelet lysate-based ink was developed for wound healing on the ocular surface. 5% hPL promoted scratch healing of a corneal epithelial cell line at a rate comparable to fetal bovine serum (FBS), which is currently used in standard tissue culture protocols. By adding fibrinogen and thrombin to platelet lysate, it is possible to induce 3D fibrin matrix formation and manipulate properties such as biocompatibility, strength and printability.

[0395] To the best of the inventors' knowledge, human platelet lysate has not been used as a standalone bioprinting material. This is because platelet lysate alone is not a strong enough matrix to be printed on its own. The ability of platelet lysate to form a gel depends on its preparation method. For tissue culture, heparin is often added to platelet lysate to prevent clot formation. The clotting time, centrifugation, and dilution of blood products used across studies vary, however, these parameters have been shown to generally affect the efficacy and properties of blood products.

[0396] By developing two separate chambers (the first containing 20% ​​hPL, 4 mg / mL fibrinogen, DMEM / F12), and mixing the two components from both chambers to generate a transparent, adhesive, and bioprintable ink, a 3D matrix was achieved that was positioned directly at the wound site. Figure 2 ) showed that the minimum requirements for a printable ink consisted of ≥0.2 mg / ml fibrinogen, >1 unit / mL thrombin, and 10% hPL (all concentrations refer to the premix stage). The hPL must be prepared as described in the methods section to maintain its biological activity, and heparin needs to be avoided in the hPL preparation because it prevents the formation of the fibrin network. All ink compositions showed high transparency across wavelengths in the visible spectrum, making this product ideal for ophthalmic applications ( Figure 4 and 5 ). Cytocompatibility testing showed that the ink could not have ≥10U / mL thrombin ( Figure 6 ) to produce effective cell confluence.

[0397] Through a combination of cytocompatibility and rheological data, favorable premixed concentrations of fibrinogen and thrombin in the ink were observed to be 4 mg / mL and 10 U / mL, respectively (Bioink 5 in Table 3). These concentrations were used in subsequent ex vivo studies, which demonstrated that the bioink had the ability to promote complete re-epithelialization of ulcerated human corneas ( Figure 8 ) and the ability to seal a complete puncture with a diameter of 1 cm ( Fig.12 In addition, all four cell types were shown to be able to grow in the bioink ( Figure 7 ), which makes this product necessary for full-thickness intraocular application. All these results indicate that the bioink has the properties to treat corneal wound healing in a 3D printing manner.

[0398] The concept of using 3D printing in the cornea has been primarily associated with bioprinting whole or partial structures from the outside, however, the ability to print in vivo is advantageous for treating corneal wounds of various sizes that require prompt attention. Fibrin glue commercially used to seal wounds is opaque, has been shown to only seal wounds, and does not promote corneal cell growth due to its inhibition of corneal cell migration. This hinders the natural wound healing response and, therefore, overall wound healing. The resulting bioink incorporates a structurally supportive fibrin glue that may provide the additional benefit of allowing cell migration and proliferation, and may also include supplements that enhance corneal regeneration.

[0399] In addition to its printability, bio-inks provide both structural support and nutritional support that promote cell growth and healing. In addition, the properties of bio-ink products are versatile and customizable, and can be tailored to the needs of individual patients, with the potential for wide application covering more pathologies and more tissue types. Autologous platelet lysates or lysates from donors can be included in bio-inks, depending on what is most beneficial to the patient. The concentration and type of antibiotics and anesthetics / analgesics can be customized to target specific infections / diseases and to address potential patient allergies. Bio-inks can also be used as drug delivery tools to extend the half-life and accurately deliver drugs directly to the wound site. It can also be used to deliver other substances (such as nanoparticles) to targeted sites.

[0400] Spheroid formation is an indicator of 3D culture ( Fig.11 ). Over the past decade, there has been a growing interest in developing 3-dimensional cell aggregates for corneal applications. Compared to 2-dimensional monolayer cell cultures, 3-dimensional cell aggregates have been shown to promote tissue self-assembly at a higher rate. This cell culture method is particularly advantageous compared to single-cell primary cultures, not only because this configuration promotes more cell-to-cell adhesion, but also because it can reverse cells into younger offspring, whereby these cells have stronger progenitor potential than their primary culture equivalents, migrate and proliferate at a higher rate, and have enhanced telomerase activity and telomere length. This cell culture method shows great promise for corneal wound healing applications. Bioinks can be used as alternative matrices in 3D cultures and represent a simpler way to obtain spheroidal cells.

[0401] Finally, tissue culture results showed the ability of the bioink to support SHSY-5Y, a non-corneal derived neuronal cell line. Fig.10 ), suggesting that the range of potential future applications for this ink extends far beyond the cornea.

[0402] In summary, the inventors have developed a bioprintable, biodegradable, transparent bioink with properties that promote wound healing. It is also envisioned that the bioink has additional functions, such as a tool for 3D culture to generate spheroid cells, the ability to heal wounds in other tissues and serve as an effective drug / substance carrier for disease treatment.

[0403] Example 3: Bio-ink formulations 3D printed in layers

[0404] -method

[0405] P18 HCET cells were treated with trypsin and cultured at 1 × 10 6The final concentration of cells / mL was suspended in the bio-ink. The bio-ink was then extruded into a petri dish and allowed to coagulate for 2 minutes. Another layer of ink was then extruded orthogonally on top and allowed to coagulate. The two lines were observed under an optical microscope to determine if they were still distinct.

[0406] -result

[0407] like Fig.13 As demonstrated, the bioink can be printed in different layers.

[0408] Example 4: Survival of cells in printed bio-ink

[0409] -method

[0410] P18 HCET cells were treated with trypsin and cultured at 1 × 10 6 The final concentration of cells / mL was suspended in the bio-ink. The bio-ink was then extruded into a culture dish and allowed to coagulate for 2 minutes. Hoescht and propidium iodide (PI) live-dead staining was performed 2 hours and 72 hours after extrusion, and cell viability was calculated using Image J. Hoechst staining (blue) stains all cell nuclei, while PI staining (red) stains only dead cells.

[0411] -result

[0412] Fig.14 A and 14B show Hoechst and propidium iodide staining in the same area 2 hours after extrusion. Fig.14 C and 14D show Hoechst and propidium iodide staining in the same area 72 hours after extrusion.

[0413] In both cases, the cell viability determined by Image J analysis was over 90%, meaning that the products can be used as cell carriers for 3D printed cells.

[0414] Example 5: The role of calcium ions in the formation of bio-inks

[0415] -method

[0416] The conditions (n=3 for each condition) were tested as shown in Table 3.

[0417] Table 4: Utilization conditions

[0418]

[0419] -result

[0420] Transparent gels were obtained in all solutions containing hPL independent of calcium ions. Opaque gels were obtained without hPL but in the presence of calcium ions. The absence of hPL and the absence of calcium ions resulted in fibrin sheets floating on top of the transparent liquid and no gel was formed.

[0421] hPL is critical to the gelation and clarity of inks ( Fig.15 ). The bio-ink is able to form independently of calcium ions.

[0422] Example 6: Adding components to bio-ink.

[0423] -method

[0424] The different components were added to the bio-ink at various concentrations (Table 5) (n=3 per formulation) and extruded onto a glass slide in the form of 3 drops. The first drop was allowed to sit for 1 minute, the second for 2 minutes, and the third for 5 minutes. These time points were then subjected to an "immersion test", whereby they were immersed in water and then rinsed with 0.9% NaCl. The minimum sitting time was determined by the shortest time point at which the gel drop remained on the slide after the "immersion test".

[0425] Table 5. Various concentrations of different components tested

[0426]

[0427] -result

[0428] All compositions were able to coagulate at or below 5 minutes after mixing (Table 6). Fig.16 The images of the bio-ink on the glass slide after the immersion test are shown. According to Table 5, A. shows composition 1, B. shows composition 2, C. shows composition 3, D. shows composition 4, E. shows composition 5, F. shows composition 6, and E. shows composition 7.

[0429] Table 6. Minimum coagulation time for different compositions

[0430] Composition Coagulation time 1(Bio-ink only) 2 minutes 2(10μg / mL FXiii) 1 minute 3(1μg / mL FXiii) 1 minute 4(CnT-PR) 5 minutes 5 (endothelial cell-specific mediator) 5 minutes 6(3mg / mL collagen) 5 minutes 7(0.5mg / mL fibronectin) 2 minutes

[0431] Example 7: Use of bio-ink as ocular surface sealant

[0432] -method

[0433] The experiments in this example were all performed using one formulation of Bio-Ink 5 (Table 3, premixed concentrations: 20% hPL, 4 mg / mL fibrinogen, 10 U / mL thrombin, DEME / F12). A wound of approximately 1.5 mm in diameter was created in the center of a human cornea obtained from the Lyon NSW Tissue Bank. The cornea was then placed in a Hanna artificial anterior chamber (AAC). The Bio-Ink (n=3) was applied to the wound and subjected to pressure. The pressure at which the perforated cornea without sealant exhibited a stable leak was measured and taken as the baseline pressure inherent to the system. The cause of sealant failure was determined to be the observation of wound leakage and the burst pressure was calculated as:

[0434]

[0435] And recorded in mmHg.

[0436] -result

[0437] Results (see Fig.17 and 18 ) showed that bioinks can be used as sealants for small corneal perforations and as a biocompatible alternative to commercially available sealants such as cyanoacrylate glue. In perforations with a diameter of approximately 1.5 mm, the bioink was able to withstand an average pressure of 91 ± 27 mmHg. This far exceeds the average intraocular pressure (10-22 mmHg).

[0438] Example 8: Setting time versus fixed shear rate

[0439] -method

[0440] The experiments in this example were all conducted using a formulation of Bio-Ink 5 (Table 3, premixed concentrations: 20% hPL, 4 mg / mL fibrinogen, 10 U / mL thrombin, DEME / F12). -1 The setting time of the bio-inks was quantitatively measured at a fixed shear rate of 1 s. The couette cup and bob viscometer geometries were also used in an AR-G2 rheometer (ThermoAnalysis Instruments, USA) to determine the setting time of the bio-inks (n = 3) at a fixed shear rate of 1 s. -115mL component A (20% hPL, 4mg / mL hPL, DMEM / F12) was poured into the geometric shape, and the viscosity of component A was measured separately for 60 seconds. Then, Bob paused the rotation for 5 seconds, during which time 15mL component B (10U / mL thrombin, DMEM / F12) was added. Then Bob restarted the rotation, and the viscosity was measured for another 180 seconds. Prism (GraphPad, U.S.) was used to tabulate the data, plot graphs and analyze. Set up the coagulation time, and it is determined as the time spent to reach stable viscosity after adding component B.

[0441] -result

[0442] When 1s -1 When mixed at a fixed shear rate of 1.5 Å, the bio-ink coagulated within 25 seconds ( Fig.19 ). It has been previously determined that when components A and B are passively mixed on a benchtop, the coagulation time is 2-5 minutes. These results suggest that shear rate affects coagulation time, meaning that the resolution at which the bioink is extruded in a 3D printing device affects how quickly the bioink coagulates.

[0443] Example 9: In vivo animal testing:

[0444] -method

[0445] The experiments in this example were all conducted using one formulation of Bio-Ink 5 (Table 3, premix concentrations: 20% hPL, 4 mg / mL fibrinogen, 10 U / mL thrombin, DEME / F12).

[0446] Experiments were conducted to determine whether the bioink promoted / did not hinder wound healing in an in vivo corneal epithelial wound model and an in vivo corneal perforation model, and to determine whether it reduced the pain experienced associated with the wound healing process.

[0447] Subject: Female New Zealand albino rabbit

[0448] Epithelial wounds (rabbits 1 and 2)

[0449] 1. Rabbit subjects will be prepared for surgery using SOP Anesthesia and Analgesia Rabbit (Mixing Chamber) Surgery (SOP-ANA_04_LAS Anesthesia and Analgesia_Rabbit_20171130) and LAS SOP #38 Rabbit Handling and Restraint.

[0450] 2. Prior to surgery, a buprenorphine patch was placed on the rabbit's back.

[0451] 3. Prepare the eyelid margins by removing the fur from the periocular area. This is done by the veterinary assistant using veterinary scissors (#40 blade).

[0452] 4. The ocular surface and periocular skin surface area are prepared with 10% povidone-iodine wash.

[0453] 5. Place a sterile surgical drape over the rabbit exposing the periocular area.

[0454] 6. During the procedure, a pediatric speculum is placed in the eye to maintain access.

[0455] 7. The cornea is then rinsed with sterile saline to remove the 10% povidone-iodine wash.

[0456] 8. Gently apply the surgical spear to the cornea and allow the surface to dry.

[0457] 9. Prepare a 5 mm sterile corneal shield (sponge) with 3 drops (approximately 150 microliters) of 30% ethanol. The sponge is then placed on the central corneal surface for 20 seconds to promote the loosening of epithelial cells from the underlying corneal layers. Carefully observe the sponge for any residual fluid leaking from the sponge. This can be improved by using a surgical spear to absorb additional fluid (if necessary).

[0458] 10. The corneal shield / sponge is then removed using surgical forceps and discarded. A surgical spear is then used to absorb any ethanol liquid remaining on the cornea.

[0459] 11. Remove loose epithelium by passing a surgical spear through the central 5 mm area. If the epithelium cannot be removed by the surgical spear, the researcher will use a sterile hockey puck blade to gently remove the remaining cells.

[0460] 12. If the rabbit is considered a study rabbit, place the bioink over the central 5 mm defect. Start a timer for 120 seconds to ensure adequate adhesion to the exposed corneal surface.

[0461] 13. Antibiotic eye drops were placed in the operated eye and the speculum was removed. The rabbit was given 0.5 mg / kg meloxicam. Meloxicam was administered by a veterinary anesthesiologist.

[0462] 14. The investigator will externally inspect the wound and surrounding area to confirm that there are no intraoperative complications.

[0463] 15. Once the operation is complete, the rabbit is moved to the recovery place for monitoring. Before returning to personal housing, a drop of Celluvisc artificial tears is placed in both eyes. Before using antibiotic eye drops, Celluvisc is placed in the eye undergoing surgery, and for up to every 2 hours of wakefulness until the wound is closed. In the last inspection of the day, a gel lubricant without fresh preservatives is placed in the eye undergoing surgery to provide extended overnight coverage. This will continue until the wound is closed, or if the researcher believes that the rabbit subject shows surface irritation (e.g., chemical, dry or irregular corneal surface) under slit lamp examination.

[0464] 16. Score pain every 2 hours throughout the work hour according to the HT Composite Pain Score Rabbit Documentation.

[0465] 17. Routine inspections were performed 4 times daily until wound healing as demonstrated by slit lamp biomicroscopy (up to 7 days), then daily for 7 days after wound closure, and then weekly until euthanasia.

[0466] Corneal perforation (rabbits 3, 4, and 5)

[0467] 1. Rabbit subjects will be prepared for surgery using SOP Anesthesia and Analgesia Rabbit (Mixing Chamber) Surgery (SOP-ANA_04_LAS Anesthesia and Analgesia_Rabbit_20171130) and LAS SOP #38 Rabbit Handling and Restraint.

[0468] 2. Prior to surgery, a buprenorphine patch was placed on the rabbit's back.

[0469] 3. Prepare the eyelid margins by removing the fur from the periocular area. This is done by the veterinary assistant using veterinary scissors (#40 blade).

[0470] 4. The ocular surface and periocular skin surface area are prepared with 10% povidone-iodine wash.

[0471] 5. Place a sterile surgical drape over the rabbit exposing the periocular area.

[0472] 6. During the procedure, a pediatric speculum is placed in the eye to maintain access.

[0473] 7. The cornea is then rinsed with sterile saline to remove the 10% povidone-iodine wash.

[0474] 8. Gently apply the surgical spear to the cornea and allow the surface to dry.

[0475] 9. Use a blade to create a complete perforation approximately 2 mm in length in the peripheral region of the cornea.

[0476] 10. If the rabbit is considered a study rabbit, place the bioink over the perforation. Start a timer for 120 seconds to ensure adequate adhesion to the exposed corneal surface.

[0477] 11. If the rabbits were to be considered control rabbits, commercially available cyanoacrylate was placed over the perforations.

[0478] 12. Antibiotic eye drops were placed in the operated eye and the speculum was removed. The rabbit was given 0.5 mg / kg meloxicam. Meloxicam was administered by a veterinary anesthesiologist.

[0479] 13. The investigator will externally inspect the wound and surrounding area to confirm that there are no intraoperative complications.

[0480] 14. Once the operation is complete, the rabbit is moved to the recovery place for monitoring. Before returning to personal housing, a drop of Xiaolaiwei artificial tears is placed in both eyes. Before using antibiotic eye drops, Xiaolaiwei is placed in the eye undergoing surgery, and up to every 2 awake hours until the wound is closed. In the last inspection of the day, a gel lubricant without fresh preservatives is placed in the eye undergoing surgery to provide extended overnight coverage. This will continue until the wound is closed, or, if the researcher believes that the rabbit subject shows surface irritation (e.g., chemical, dry or irregular corneal surface) under slit lamp examination.

[0481] 15. Score pain every 2 hours throughout the work hour according to the HT Composite Pain Score Rabbit Documentation.

[0482] 16. Routine inspections were performed 4 times daily until wound healing as demonstrated by slit lamp biomicroscopy (up to 7 days), then daily for 7 days after wound closure, and then weekly until euthanasia.

[0483] Modified corneal perforation (rabbits 6 and 7)

[0484] 1. Rabbit subjects will be prepared for surgery using SOP Anesthesia and Analgesia Rabbit (Mixing Chamber) Surgery (SOP-ANA_04_LAS Anesthesia and Analgesia_Rabbit_20171130) and LAS SOP #38 Rabbit Handling and Restraint.

[0485] 2. Prior to surgery, a buprenorphine patch was placed on the rabbit's back.

[0486] 3. Prepare the eyelid margins by removing the fur from the periocular area. This is done by the veterinary assistant using veterinary scissors (#40 blade).

[0487] 4. The ocular surface and periocular skin surface area are prepared with 0.2% povidone-iodine wash.

[0488] 5. Place a sterile surgical drape over the rabbit exposing the periocular area.

[0489] 6. During the procedure, a pediatric speculum is placed in the eye to maintain access.

[0490] 7. The cornea is then rinsed with sterile saline to remove the 0.2% povidone-iodine wash.

[0491] 8. Gently apply the surgical spear to the cornea and allow the surface to dry.

[0492] 9. Use a blade to create a complete perforation approximately 2 mm in length in the peripheral region of the cornea.

[0493] 10. If the rabbit is considered a study rabbit, place the bioink over the perforation. Start a timer for 120 seconds to ensure adequate adhesion to the exposed corneal surface.

[0494] 11. If the rabbits were to be considered control rabbits, commercially available cyanoacrylate was placed over the perforations.

[0495] 12. Antibiotic eye drops were placed in the operated eye and the speculum was removed. The rabbit was given 0.5 mg / kg meloxicam. Meloxicam was administered by a veterinary anesthesiologist.

[0496] 13. The investigator will externally inspect the wound and surrounding area to confirm that there are no intraoperative complications.

[0497] 14. Once the operation is complete, the rabbit is moved to the recovery place for monitoring. Before returning to personal housing, a drop of Xiaolaiwei artificial tears is placed in both eyes. Before using antibiotic eye drops, Xiaolaiwei is placed in the eye undergoing surgery, and up to every 2 awake hours until the wound is closed. In the last inspection of the day, a gel lubricant without fresh preservatives is placed in the eye undergoing surgery to provide extended overnight coverage. This will continue until the wound is closed, or, if the researcher believes that the rabbit subject shows surface irritation (e.g., chemical, dry or irregular corneal surface) under slit lamp examination.

[0498] 15. Score pain every 2 hours throughout the work hour according to the HT Composite Pain Score Rabbit Documentation.

[0499] 16. Routine inspections were performed 4 times daily until wound healing as demonstrated by slit lamp biomicroscopy (up to 7 days), then daily for 7 days after wound closure, and then weekly until euthanasia.

[0500] -result

[0501] Overview of each rabbit

[0502] Surgery Day 1, August 20, 2018

[0503] Rabbit 1 - ID number: 233665 , arrived on July 11, 2018 (Group 1 - 1 of 2).

[0504] Perform debridement surgery with 30% ethanol

[0505] Tx.-Bio-ink

[0506] Result - moderate conjunctival inflammation, the wound healed only after 48 hours.

[0507] Rabbit 2-ID number: 233666 , arrived on July 11, 2018 (Group 1 - 2 of 2).

[0508] Debridement surgery with ethanol

[0509] Tx.-Control (antibiotic drops only)

[0510] Result - severe conjunctival inflammation and chemosis, the lesion doubled in size, the pain was initially uncontrollable but improved greatly on the second day. The wound healed only after 48 hours.

[0511] Surgery Day 2, September 3, 2018

[0512] Rabbit 3 - ID number: 239686 , arrived on August 1, 2018 (Group 2 - 1 of 5).

[0513] Perforation surgery

[0514] Tx.-Bio-ink

[0515] Results - Moderate conjunctival inflammation and secondary ulceration, the perforation healed in 32 hours, and the secondary ulcer healed in 52 hours.

[0516] Rabbit 4-ID number: 239685 , arrived on August 1, 2018 (Group 2 - 2 out of 5).

[0517] Perforation surgery

[0518] Tx.-Bio-ink

[0519] Results - Moderate conjunctival inflammation and secondary ulceration, the perforation healed in 28 hours, and the secondary ulcer healed in 48 hours.

[0520] Rabbit 5-ID number: 239684 , arrived on August 1, 2018 (Group 2 - 3 out of 5).

[0521] Perforation surgery

[0522] Tx.-Control (Cyanoacrylate Glue)

[0523] Results - Moderate conjunctival inflammation and chemosis. The rabbit was very uncomfortable and with appropriate pain management strategies, the rabbit is now less uncomfortable. Secondary ulceration was observed at 56 hours, but this has now healed at 72 hours. The perforation wound healed at 80 hours.

[0524] Surgical Day 3, September 4, 2018 - implemented a reduction in povidone-iodine concentration from 5% to 0.2% and added lubricating eye drops

[0525] Rabbit 6-ID number: 239687 , arrived on August 1, 2018 (Group 2 - 4 out of 5).

[0526] Perforation surgery

[0527] Tx.-Bio-ink

[0528] Results - 8 hour healing with no complications. No need to get additional analgesia from the first buprenorphine patch.

[0529] Rabbit 7-ID number: 239688 , arrived on August 1, 2018 (Group 2 - 5 of 5).

[0530] Perforation surgery

[0531] Tx.-Control (Cyanoacrylate Glue)

[0532] Results - The rabbit was initially in significant pain with moderate conjunctivitis and significant chemosis, which appeared to be related to the location of the cyanoacrylate glue. Euthanasia was considered, however, it seemed to respond to the administration of methadone and began eating that evening. Upon examination this morning (Day 2), the rabbit seemed to be more comfortable with the pain that was successfully managed - it was still uncomfortable but not in pain. The wound had healed in 48 hours and the rabbit is now very comfortable.

[0533] In the epithelial defect wound model (rabbits 1 and 2), wound healing was achieved at the same time points in control and treated rabbits ( Fig. 20 ). Significantly less postoperative analgesia was administered to the treated rabbits, and overall pain scores were lower than those experienced by the control rabbits ( Fig.21 and 22 ). Pain scores were based on the assessment of indicators including excessive blinking, blepharospasm, eye rubbing, blepharospasm, and tearing and eye rubbing.

[0534] In the corneal perforation model (rabbits 3, 4, and 5), wound healing occurred significantly faster in treated rabbits (t=28 and t=32 hours) compared to control rabbits (t=80 hours) ( Fig.23 ). Compared with control rabbits ( Fig.24 ) were also given less postoperative analgesia overall to treated rabbits ( Fig.25 and 26 ).

[0535] In the modified corneal perforation model (rabbits 6 and 7), wound healing occurred significantly faster in treated rabbits (t = 8 hours) compared to controls (t = 48 hours) ( Fig. 27 No postoperative analgesia was administered to the treated rabbits, and the pain scores recorded were significantly lower than those of the control rabbits ( Fig.28 ).

[0536] The bioink was biocompatible and did not impede corneal wound healing. These results suggest that the bioink reduces pain associated with corneal injuries and may also accelerate wound healing of corneal perforations compared to conventional treatment options.

[0537] Example 10: Freeze-drying and reconstitution of bio-ink

[0538] -method

[0539] ● 50uL of bio-ink components A and B were prepared.

[0540] Component A contained 20% hPL, 4 mg / mL fibrinogen in DMEM / F12, and component B contained 10 U / mL thrombin in DMEM / F12. The final volume of both components was 50 uL. Once mixed, it provided the same formulation as Bio-Ink 5 (Table 3, premix concentrations: 20% hPL, 4 mg / mL fibrinogen, 10 U / mL thrombin, DMEM / F12).

[0541] ● Freeze-dry the sample at -40 degrees Fig.29 A)

[0542] ●Reconstitute in seconds with 50uL of water per tube ( Fig.29 B)

[0543] ●Mix and form adhesive and clear gel ( Fig.29 C)

[0544] -result

[0545] Visual assessment showed that the bioink could still cure within 2 minutes after reconstitution. It was observed to be viscous (connected to Fig.29 C) and is transparent. Fig.29 C)。

Claims

1. A composition comprising 0.2-5 mg / ml fibrinogen, 5-10 U / mL thrombin and 5-10 % (v / v) platelet lysate.

2. The composition according to claim 1, further comprising any one or more of the following: ions, amino acids, fibronectin, anesthetics, antibiotics, growth factors, tissue factor XIII, matrix proteins.

3. The composition of claim 2, wherein the ions comprise calcium ions.

4. The composition of claim 2, wherein the growth factor comprises human epidermal growth factor (hEGF).

5. The composition of claim 2, wherein the matrix protein comprises collagen.

6. The composition according to any one of claims 1 to 5, wherein the composition comprises a culture medium comprising the ions and the amino acids.

7. The composition according to any one of claims 1 to 5, wherein the ions are components of an ionic salt solution contained in the composition.

8. The composition according to any one of claims 1 to 5, wherein the composition further comprises cells.

9. The composition of claim 8, wherein the cell is a mammalian cell.

10. The composition of claim 8, wherein the cells are human cells.

11. The composition of any one of claims 1 to 5, wherein the platelet lysate comprises or consists of human platelet lysate.

12. A kit, package or device comprising: - a first compartment comprising fibrinogen and a second compartment comprising thrombin, wherein the kit, the package or the device is configured to separate the fibrinogen of the first compartment from the thrombin of the second compartment during and after loading of the fibrinogen and the thrombin into the kit, the package or the device; - Platelet lysate; - means for promoting mixing of said fibrinogen of said first compartment with said thrombin and said platelet lysate of said second compartment: in: The first compartment comprises 0.2-5 mg / ml fibrinogen; the second compartment comprises 5-10 U / mL thrombin; and the device comprises a total of 5-10% (v / v) platelet lysate.

13. The kit, package or device of claim 12, wherein the first compartment comprises the fibrinogen and the platelet lysate, and the second compartment comprises the thrombin.

14. The kit, package or device according to claim 12 or 13, wherein: - any of the compartments further comprises any one or more of the following: ions, amino acids, fibronectin, anesthetics, antibiotics, growth factors, tissue factor XIII, matrix proteins; or - the kit, the package or the device comprises an additional compartment, the additional compartment comprising any one or more of the following: ions, amino acids, fibronectin, anesthetics, antibiotics, growth factors, tissue factor XIII, matrix proteins; wherein the kit, the package or the device is configured to separate the contents of the additional compartment from the contents of other compartments during and after loading of the kit, the package or the device.

15. The kit, package or device of claim 14, wherein the growth factor comprises hEGF.

16. The kit, package or device of claim 14, wherein the matrix protein comprises collagen.

17. The kit, package or device of claim 14, wherein: The ion is a calcium ion.

18. The kit, package or device of claim 12, wherein: (i) either or both of the first compartment and the second compartment and / or the third compartment comprises cells; and / or (ii) wherein the platelet lysate comprises or consists of human platelet lysate.

19. The kit, package or device of claim 18, wherein the cells are mammalian cells.

20. The kit, package or device of claim 18, wherein the cells are human cells.

21. Use of a composition according to any one of claims 1 to 5 in the preparation of a medicament for treating a wound in a subject, wherein the wound is located in the cornea of ​​the subject.

22. The use according to claim 21, wherein the drug is applied to the wound using three-dimensional (3D) bioprinting.

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