Bioengineered endothelial constructs

Abstract

The present invention relates to bioengineered collagen constructs and methods for the preparation and use of said constructs, which may find application in, but are not limited to, providing replacement tissue and / or cells and / or delivering agents to biological targets, such as tissues and cells. The constructs and methods may also find application in, but are not limited to, replacing endothelial tissue.

Description

[Technical field]

[0001] Inclusion by cross reference This application claims priority to Australian Provisional Patent Application No. 2021 / 900795, filed on March 18, 2021, the entire contents of which are incorporated herein by cross-reference.

[0002] The present invention relates generally to the fields of biology and medicine. More specifically, the present invention relates to bioengineered collagen constructs and methods for the preparation and use of said constructs. The constructs and methods may find application in providing replacement tissues and / or cells and / or delivering drugs to biological targets such as tissues and cells. [Background technology]

[0003] When disease or injury results in significant damage to biological tissues, clinicians are often limited by the availability of suitable donor tissue for use in transplantation. Bioengineered constructs offer an exciting solution to this problem, but are limited by the requirements for strength and flexibility, as well as the need for synthetic materials to be compatible with biological tissues and / or cells.

[0004] One example of an area where there is a shortage of donor tissue available for transplantation is corneal disease and trauma. Currently, conventional eye banks represent storage and distribution centers for donated tissue. Ideally, these eye banks would also have the capacity to procure and culture corneal tissue and bioengineer additional tissue for therapeutic applications.

[0005] The cornea is the transparent, protective outer layer of the eye. It allows light to pass through the pupil and is the main refractive element of the eye's optical system. It acts as a barrier against bacteria, dirt, and other harmful substances. The cornea consists of five layers: the outer epithelium, Bowman's layer, stroma, Descemet's membrane, and the inner endothelium. Descemet's membrane is the basement membrane for the corneal endothelium and is a dense, thick, relatively transparent, cell-free matrix that separates the posterior corneal stroma from the underlying endothelium. The corneal stroma accounts for approximately 90% of the total thickness of the cornea and is composed primarily of collagen.

[0006] Corneal blindness is the second leading cause of blindness worldwide. Causes of corneal blindness include diseases such as trachoma, onchocerciasis, leprosy, ophthalmia neonatorum, and xerophthalmia, as well as other processes such as ocular trauma, corneal ulcers, and complications arising from the use of conventional ophthalmic medications.

[0007] Corneal injuries represent the most common eye emergency presentation in Australia, with approximately 75% of all cases resulting from the presence of a foreign body or abrasion within the cornea. These injuries alone are estimated to cost the Australian population over $155 million annually and, if not effectively treated, can lead to infection and scarring, potentially resulting in permanent vision loss.

[0008] In mild cases, the damaged cornea can regenerate through normal healing pathways, however, in other cases, the cornea's normal healing mechanisms are insufficient, resulting in corneal melting, corneal neovascularization, loss of transparency, infection, scarring, and the formation of non-healing defects that can lead to reduced vision to the point of blindness.

[0009] Corneal endothelial disease is one example of a condition affecting the cornea that would benefit from improved constructs for tissue replacement. Although the surgical treatment of corneal endothelial disease has evolved in recent years with the use of lamellar grafting, this surgery remains technically challenging and utilizes precious donor corneal tissue. Recognized complications include subluxation of the donor tissue.

[0010] Current treatments for corneal injuries include antibiotics, eye pads, sutures and surgical glues, which may be helpful for minor issues. However, they do not adequately address issues that arise in more advanced situations, including pain relief, infection, and / or scar tissue development. Infection represents a significant complication and often requires hospitalization. Scarring, common in severe corneal injuries, can result in permanent vision loss. In such cases, corneal transplantation is the only option for vision rehabilitation, but there is a shortage of donor corneas worldwide.

[0011] Many of the problems discussed above are not limited to corneal injury and disease, but also prevail in the event of injury and / or deterioration of other body tissues.

[0012] A need exists for improved constructs and methods for providing replacement tissues and / or cells and / or delivering agents to biological targets, such as tissues and cells. Summary of the Invention

[0013] The present invention alleviates at least one of the problems associated with current supplies and methods for replacing biological tissue and / or delivering drugs to biological targets, such as tissues and cells. The inventors have developed a construct comprising type IV collagen that is transparent, crosslinkable, strong, flexible and / or printable. Surprisingly, the construct can provide a unique method for culturing endothelial cells, such as human corneal endothelial cells.

[0014] Without being limited thereto, the constructs and methods described herein are generally useful for delivering agents (e.g., cells, drugs and / or other substances) to biological targets (e.g., tissues, membranes, cells) and may find application in endothelial tissue replacement, including, for example, human corneal endothelial tissue.

[0015] The invention is directed, at least in part, to the following embodiments.

[0016] Embodiment 1. A construct comprising a composition, the composition comprising: -Type IV collagen, one or more crosslinkers, endothelial cells, - a platelet lysate and / or a component thereof. Embodiment 2. A construct comprising a composition, the composition comprising: -Type IV collagen, one or more crosslinkers, - an endothelial cell; Embodiment 3. The construct of embodiment 1 or 2, wherein the endothelial cell is a corneal endothelial cell. Embodiment 4. A construct according to any one of embodiments 1 to 3, wherein the endothelial cells are human endothelial cells. Embodiment 5. The construct of any one of embodiments 1, 3 or 4, wherein the platelet lysate is human platelet lysate. Embodiment 6. The construct according to any one of embodiments 1 or 3 to 5, wherein the component of the platelet lysate is any one or more of the following: fibrinogen, basic fibroblast growth factor (bFGF), transforming growth factor (TGF-β), insulin-like growth factor (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), IgG, albumin. Embodiment 7. A construct according to any one of embodiments 1 or 3 to 6, wherein the component of the platelet lysate is fibrinogen. Embodiment 8 The construct of embodiment 7, wherein the fibrinogen is human fibrinogen. Embodiment 9. A construct according to any one of embodiments 1 to 8, wherein the construct and / or composition further comprises sodium ions and / or calcium ions. Embodiment 10. A construct according to any one of embodiments 1 to 9, wherein one or more crosslinkers can be activated by UV light, blue light, green light, or white light. Embodiment 11. A construct according to any one of embodiments 1 to 10, wherein the one or more cross-linking agents comprises riboflavin. Embodiment 12. A construct comprising at least one layer comprising type IV collagen and one or more crosslinkers, each layer comprising: -6 to 24 mg / ml of type IV collagen; 12. The construct according to any one of the preceding embodiments, which is produced by crosslinking a solution comprising: 0.04 to 0.15 M sodium ions, and / or 0.008 to 0.4 M calcium ions. Embodiment 13. The solution is (i) 6 to 24 mg / ml of type IV collagen, 0.06 to 0.1 M of sodium ion, and 0.01 to 0.04 M of calcium ion, or (ii) 3 to 15 mg / ml of type IV collagen, 0.06 to 0.08 M of sodium ion, and 0.015 to 0.03 M of calcium ion, or (iii) The construct according to embodiment 12, comprising 4 to 12 mg / ml type IV collagen, 0.06 to 0.07 M sodium ions, and 0.018 to 0.02 M calcium ions. Embodiment 14. The solution is (i) less than 24 mg / ml type IV collagen; (ii) greater than 0.04M sodium ions; (iii) greater than 0.008M calcium ions. Embodiment 15. A construct according to any one of embodiments 12 to 14, wherein the solution contains 0.01 to 0.1 mg of riboflavin. Embodiment 16. A construct according to any one of embodiments 1 to 15, wherein the construct comprises two or more layers comprising type IV collagen and one or more crosslinking agents, each of the two or more layers being crosslinked to at least one other layer. Embodiment 17. A construct comprising at least one additional layer comprising type I collagen and one or more crosslinkers, the additional layer comprising: -3 to 15 mg / ml of type I collagen; 17. The construct according to any one of embodiments 12 to 16, which is produced by crosslinking a solution comprising: 0.135 to 0.5 M sodium ions and / or 0.008 to 0.4 M calcium ions. Embodiment 18. The construct of embodiment 17, wherein each layer is crosslinked to at least one other layer. Embodiment 19. A construct described in any one of embodiments 1 to 18, wherein the construct comprises a first layer comprising collagen and one or more crosslinking agents, the first layer being crosslinked separately prior to crosslinking to one or more other layers. Embodiment 20. A construct comprising at least one layer comprising type IV collagen, type I collagen, and one or more crosslinkers, each layer comprising: -6 to 24 mg / ml of type IV collagen; 12. The construct according to any one of the preceding embodiments, which is produced by crosslinking a solution comprising: - 3 to 15 mg / ml of type I collagen. Embodiment 21. The construct of embodiment 20, wherein the solution contains 0.01 to 0.1 mg of riboflavin. Embodiment 22. A construct according to embodiment 20 or 21, wherein the construct comprises two or more layers comprising type IV collagen, type I collagen, and one or more crosslinkers, each of the two or more layers being crosslinked to at least one other layer. Embodiment 23. A construct described in any one of embodiments 20 to 22, wherein the construct comprises a first layer comprising collagen and one or more crosslinking agents, the first layer being crosslinked separately prior to crosslinking to one or more other layers. Embodiment 24. A construct according to any one of embodiments 1 to 23, wherein the construct and / or composition further comprises a mammalian cell. Embodiment 25. The method of embodiment 24, wherein the mammalian cells comprise or consist of human cells. Embodiment 26. A construct according to any one of embodiments 1 to 25, wherein the construct and / or composition further comprises any one or more of the following: culture medium, growth factors, hormones, matrix proteins, glycoproteins, vitamins, ions, ion sources, fibronectin, amino acids, antibiotics, anesthetics, Factor XIII, Bovine Serum (FBS), Calf Serum (FCS), human serum, platelet lysate, human platelet lysate, therapeutic agent. Embodiment 27. The construct of embodiment 26, wherein the construct and / or composition comprises a culture medium comprising ions and amino acids. 28. (i) the growth factors include VEGF and / or FGF; and / or (ii) the vitamins include riboflavin, and / or (iii) the matrix protein comprises type I collagen; and / or (iv) The construct of embodiment 26 or 27, wherein the matrix protein comprises laminin. Embodiment 29. A construct according to any one of embodiments 1 to 28, wherein the construct and / or composition comprises an ion, and the ion is a component of an ionic salt contained in the construct and / or composition. Embodiment 30. A construct according to any one of embodiments 1 to 29, wherein the type IV collagen is neutralized. Embodiment 31. A construct according to any one of embodiments 1 to 30, wherein the construct and / or composition further comprises one or more of type VIII collagen, laminin, nidogen, and perlecan. Embodiment 32. A method for preparing a construct, comprising: (i) providing a composition, the composition comprising: -Type IV collagen, - providing a solution comprising one or more crosslinking agents; (ii) endothelial cells, - adding a platelet lysate and / or components thereof. Embodiment 33. A method for preparing a construct, comprising: (i) providing a composition, the composition comprising: -Type IV collagen, - providing a solution comprising one or more crosslinking agents; (ii) adding endothelial cells. Embodiment 34. A method for culturing endothelial cells, comprising: (i) providing a composition, the composition comprising: -Type IV collagen, - providing a solution comprising one or more crosslinking agents; (ii) endothelial cells, - adding a platelet lysate and / or components thereof. Embodiment 35. A method for culturing endothelial cells, comprising: (i) providing a composition, the composition comprising: -Type IV collagen, - providing a solution comprising one or more crosslinking agents; (ii) adding endothelial cells. Embodiment 36. The composition further comprises, prior to (ii): -Type I collagen, - one or more crosslinking agents. Embodiment 37. The method of any one of embodiments 32 to 35, wherein the solution further comprises type I collagen. Embodiment 38. The method of any one of embodiments 32 to 37, wherein the endothelial cells are corneal endothelial cells. Embodiment 39. The method of any one of embodiments 32, 34, or 36-38, wherein the platelet lysate is human platelet lysate. Embodiment 40. The method of any one of embodiments 32, 34, or 36-39, wherein the component of the platelet lysate is any one or more of the following: fibrinogen, bFGF, TGF-β, IGF-1, BDNF, VEGF, EGF, HGF, PDGF, IgG, albumin. Embodiment 41. The method of any one of embodiments 32, 34, or 36-40, wherein the component of the platelet lysate is fibrinogen. Embodiment 42 The method of embodiment 41, wherein the fibrinogen is human fibrinogen. Embodiment 43. The method of any one of embodiments 32 to 42, further comprising adding sodium ions and / or calcium ions in step (i) and / or step (ii). Embodiment 44. The method of any one of embodiments 32 to 43, wherein the one or more crosslinkers can be activated by UV light, blue light, green light, or white light. Embodiment 45. The method of any one of embodiments 32 to 44, wherein the one or more cross-linking agents comprises riboflavin. Embodiment 46. The composition comprises at least one layer comprising a crosslinking solution, the solution comprising: -6 to 24 mg / ml of type IV collagen; - 0.04 to 0.15 M sodium ions, and / or 0.008 to 0.4 M calcium ions. Embodiment 47. The solution is (i) 6 to 24 mg / ml of type IV collagen, 0.06 to 0.1 M of sodium ion, and 0.01 to 0.04 M of calcium ion, or (ii) 3 to 15 mg / ml of type IV collagen, 0.06 to 0.08 M of sodium ion, and 0.015 to 0.03 M of calcium ion, or (iii) The method according to embodiment 46, comprising 4 to 12 mg / ml type IV collagen, 0.06 to 0.07 M sodium ions, and 0.018 to 0.02 M calcium ions. Embodiment 48. The solution is (i) less than 24 mg / ml type IV collagen; (ii) greater than 0.04M sodium ions; 48. The method of embodiment 46 or 47, comprising: (iii) greater than 0.008M calcium ions. Embodiment 49. The method of any one of embodiments 45 to 48, wherein the solution comprises 0.01 to 0.1 mg of riboflavin. Embodiment 50. The method of any one of embodiments 32 to 49, wherein the composition comprises two or more layers comprising collagen and one or more crosslinking agents, and each of the two or more layers is crosslinked to at least one other layer. Embodiment 51. The method of any one of embodiments 32 to 50, wherein the composition comprises a first layer comprising collagen and one or more crosslinking agents, the first layer being crosslinked separately prior to crosslinking to one or more other layers. Embodiment 52. The method of embodiment 50 or 51, wherein at least one layer of the composition is crosslinked on the hydrophobic material. Embodiment 53. The method of embodiment 52, wherein the hydrophobic material is silicone or parafilm. Embodiment 54. The method of embodiment 50 or 51, wherein at least one layer of the composition is crosslinked on a low temperature material. Embodiment 55. The method of embodiment 54, wherein the low temperature material is a low temperature metal. Embodiment 56. The method of any one of embodiments 32 to 55, further comprising adding mammalian cells in step (i) and / or step (ii). Embodiment 57. The method of embodiment 56, wherein the mammalian cells comprise or consist of human cells. Embodiment 58. The method according to any one of embodiments 32 to 57, further comprising adding in step (i) and / or step (ii) any one or more of the following: culture medium, growth factors, hormones, matrix proteins, glycoproteins, vitamins, ions, ion sources, fibronectin, amino acids, antibiotics, anesthetic agents, factor XIII, FBS, FCS, human serum, platelet lysate, human platelet lysate, therapeutic agent. Embodiment 59. The method of embodiment 58, wherein ions and / or amino acids are provided in the culture medium. 60. (i) the growth factors include VEGF and / or FGF; and / or (ii) the vitamins include riboflavin, and / or (iii) the matrix protein comprises type I collagen; and / or (iv) The method of embodiment 58 or 59, wherein the matrix protein comprises laminin. Embodiment 61. The method of any one of embodiments 32 to 60, further comprising adding ions as components of an ionic salt in step (i) and / or step (ii). Embodiment 62. The method of any one of embodiments 32 to 61, wherein the type IV collagen is neutralized. Embodiment 63. The method according to any one of embodiments 32 to 62, further comprising adding any one or more of type VIII collagen, laminin, nidogen, and perlecan in step (i) and / or step (ii). Embodiment 64. A construct obtained or obtainable by the method according to any one of embodiments 32, 33 or 36 to 63. Embodiment 65. A method for replacing endothelial tissue, the method comprising applying a construct according to any one of embodiments 1 to 31 or 64. Embodiment 66 The method of embodiment 65, wherein the endothelial tissue comprises or consists of corneal endothelium. Embodiment 67 The method of embodiment 65 or 66, wherein the endothelial tissue is human endothelial tissue. Embodiment 68. The method of any one of embodiments 65 to 67, wherein the endothelial tissue comprises or consists of Descemet's membrane. Embodiment 69. A method for treating corneal damage or disease, the method comprising applying a construct described in any one of embodiments 1 to 31 or 64. Embodiment 70. A method of delivering a drug to a tissue, the method comprising applying to the tissue a composition described in any one of embodiments 1 to 31 or 64. Embodiment 71. A construct according to any one of embodiments 1 to 31 or 64 for use in replacing endothelial tissue. Embodiment 72. The use according to embodiment 71, wherein the endothelial tissue comprises or consists of corneal endothelium. Embodiment 73. The use according to embodiment 71 or 72, wherein the endothelial tissue is human endothelial tissue. Embodiment 74. The use according to any one of embodiments 71 to 73, wherein the tissue comprises or consists of Descemet's membrane. Embodiment 75. A construct according to any one of embodiments 1 to 31 or 64 for use in treating corneal injury or disease. Embodiment 76. A construct according to any one of embodiments 1 to 31 or 64 for use in delivering a drug to a tissue. Embodiment 77. A kit, package or device for producing a construct comprising a composition, the composition comprising: -Type IV collagen, one or more cross-linking agents, The kit, package, or device comprises: endothelial cells, - a platelet lysate and / or components thereof; Embodiment 78. A kit, package or device for producing a construct comprising a composition, the composition comprising: -Type IV collagen, one or more cross-linking agents, The kit, package or device further comprises endothelial cells. Embodiment 79. A kit, package or device according to embodiment 77 or 78, wherein the composition further comprises type I collagen. Embodiment 80. The kit, package or device is for producing a further composition, the composition comprising: -Type IV collagen, -Type I collagen, - one or more cross-linking agents. Embodiment 81. Use of a kit, package or device for preparing a construct comprising a composition, the composition comprising: -Type IV collagen, one or more cross-linking agents, The kit package or device comprises: endothelial cells, - A platelet lysate and / or components thereof. Embodiment 82. Use of a kit, package or device for preparing a construct comprising a composition, the composition comprising: -Type IV collagen, one or more cross-linking agents, The kit package or device comprises: - The use further comprising endothelial cells. Embodiment 83. The use according to embodiment 81 or 82, wherein the composition further comprises type I collagen. Embodiment 84. The use is for producing a further composition, the composition comprising: -Type IV collagen, -Type I collagen, - one or more cross-linking agents. Embodiment 85. A kit, package or device described in any one of embodiments 77 to 80, or a use described in any one of embodiments 80 to 84, wherein the endothelial cells are corneal endothelial cells. Embodiment 86. A kit, package or device according to any one of embodiments 77 to 80 or 85, or a use according to any one of embodiments 80 to 85, wherein the endothelial cells are human endothelial cells. Embodiment 87. A kit, package or device according to any one of embodiments 77, 79, 80, 85 or 86, or a use according to any one of embodiments 80, 81 or 83 to 86, wherein the platelet lysate is a human platelet lysate. Embodiment 88. A kit, package or device according to any one of embodiments 77, 79, 80 or 85 to 87, or a use according to any one of embodiments 80, 81 or 83 to 87, wherein the components of the platelet lysate are any one or more of fibrinogen, bFGF, TGF-β, IGF-1, BDNF, VEGF, EGF, HGF, PDGF, IgG, albumin. Embodiment 89. A kit, package or device according to any one of embodiments 77, 79, 80 or 85 to 88, or a use according to any one of embodiments 80, 81 or 83 to 88, wherein the component of the platelet lysate is fibrinogen. Embodiment 90. The kit, package or device, or use according to embodiment 89, wherein the fibrinogen is human fibrinogen. Embodiment 91. A kit, package or device according to any one of embodiments 77 to 80 or 85 to 90, or a use according to any one of embodiments 80 to 90, wherein the construct and / or composition further comprises sodium ions and / or calcium ions. Embodiment 92. A kit, package or device according to any one of embodiments 77 to 80 or 85 to 91, or a use according to any one of embodiments 80 to 91, wherein one or more crosslinkers can be activated by UV light, blue light, green light, or white light. Embodiment 93. A kit, package or device according to any one of embodiments 77 to 80 or 85 to 92, or a use according to any one of embodiments 80 to 92, wherein the one or more cross-linking agents comprises riboflavon. Embodiment 94. A construct comprising at least one layer comprising type IV collagen and one or more crosslinkers, each layer comprising: -6 to 24 mg / ml of type IV collagen; A kit, package or device according to any one of embodiments 77 to 80 or 85 to 93, or a use according to any one of embodiments 80 to 93, which is produced by crosslinking a solution comprising: 0.04 to 0.15 M sodium ions and / or 0.008 to 0.4 M calcium ions. Embodiment 95. The solution comprises: (i) 6 to 24 mg / ml of type IV collagen, 0.06 to 0.1 M of sodium ion, and 0.01 to 0.04 M of calcium ion, or (ii) 3 to 15 mg / ml of type IV collagen, 0.06 to 0.08 M of sodium ion, and 0.015 to 0.03 M of calcium ion, or (iii) The kit, package or device, or use according to embodiment 94, comprising 4 to 12 mg / ml type IV collagen, 0.06 to 0.07 M sodium ions, and 0.018 to 0.02 M calcium ions. Embodiment 96. The solution is (i) less than 24 mg / ml type IV collagen; (ii) greater than 0.04M sodium ions; (iii) more than 0.008M calcium ions. Embodiment 97. A kit, package or device, or use according to any one of embodiments 94 to 96, wherein the solution contains 0.01 to 0.1 mg of riboflavin. Embodiment 98. A kit, package or device described in any one of embodiments 77 to 80 or 85 to 97, or a use described in any one of embodiments 80 to 97, wherein the construct comprises two or more layers comprising type IV collagen and one or more crosslinking agents, each of the two or more layers being crosslinked to at least one other layer. Embodiment 99. A kit, package or device described in any one of embodiments 77 to 80 or 85 to 98, or a use described in any one of embodiments 80 to 98, wherein the construct comprises a first layer comprising type IV collagen and one or more crosslinking agents, the first layer being crosslinked separately before being crosslinked to one or more other layers. Embodiment 100. A kit, package or device according to any one of embodiments 77 to 80 or 85 to 99, or a use according to any one of embodiments 80 to 99, wherein the construct and / or composition further comprises mammalian cells. Embodiment 101. A kit, package or device or use according to embodiment 100, wherein the mammalian cells comprise or consist of human cells. Embodiment 102. A kit, package or device according to any one of embodiments 77 to 80 or 85 to 101, or a use according to any one of embodiments 80 to 101, wherein the construct and / or composition further comprises any one or more of the following: culture medium, growth factors, hormones, matrix proteins, glycoproteins, vitamins, ions other than sodium ions or calcium ions, ion sources, fibronectin, amino acids, antibiotics, anesthetics, factor XIII, FBS, FCS, human serum, platelet lysate, human platelet lysate, therapeutic agent.

[0017] definition 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 "a component" also includes a plural component.

[0018] As used herein, the term "comprising" means "including." Variations of the word "comprising," such as "comprise" and "comprises," have correspondingly altered meanings. Thus, for example, a composition that "comprises" component "A" may be exclusively component "A" or may include one or more additional components (e.g., component "B" and / or component "C").

[0019] 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, for example, a mammal, such as a human, or a non-human mammal.

[0020] As used herein, the term "tissue" will be understood to encompass both cells which are components of tissues and organs formed from tissues.

[0021] As used herein, the term "kit" refers to any delivery system for delivering materials. Such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., labels in appropriate containers, reference samples, supporting materials, etc.) and / or supporting materials (e.g., buffers, written instructions for performing an assay, etc.) from one location to another. For example, a kit may include one or more containers, such as a box, that contain the relevant reaction reagents and / or supporting materials.

[0022] As used herein, the term "about" when used in reference to a cited numerical value includes the cited numerical value as well as numerical values ​​within plus or minus ten percent of the cited numerical value.

[0023] As used herein, the term "plurality" means two or more. In certain aspects or embodiments, plurality can mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or more, and any numerical values ​​derivable therein, and any ranges derivable therein.

[0024] As used herein, when used in reference to a range of numerical values, the term "between" includes the numerical values ​​at each endpoint of the range. For example, calcium ion having a concentration between 0.008M and 0.4M includes calcium ion having a concentration of 0.008M and calcium ion having a concentration of 0.4M.

[0025] As used herein, the term "more than," when used in reference to a numerical value, will be understood to mean "more than or equal to." For example, greater than 0.018M calcium ion includes concentrations of 0.018M calcium ion and all concentrations of calcium ion greater than 0.018M.

[0026] As used herein, the term "less than" when used in relation to a numerical value will be understood to mean "less than or equal to." For example, less than 15 mg / ml of type IV collagen includes type IV collagen at a concentration of 15 mg / ml and all concentrations of type IV collagen less than 15 mg / ml.

[0027] As used herein, the term "neutralized" when used to describe type IV collagen will be understood to mean that the collagen solution has a pH of 6.7 to 7.6. For example, "neutralized" type IV collagen can have a pH of 6.8 to 7.5, or 6.9 to 7.4, or 7.0 to 7.3, or 6.9 to 7.2, etc.

[0028] As used herein, the term "crosslinking," when used in reference to collagen, will be understood to mean an increase in intra- and intermolecular covalent bonds. Variations of the word "crosslinking," such as "crosslinked" and "crosslinking," have correspondingly altered meanings. In some instances, "crosslinking" collagen will be understood to mean an increase in intra- and interfibrillar covalent bonds in collagen fibrils.

[0029] Any description in this specification of a prior art document, or any statement in this specification derived from or based on such documents, is not an admission that the document or the statement derived is part of the general knowledge in the relevant art.

[0030] For purposes of description, all documents referenced herein are incorporated by reference in their entirety unless otherwise noted. [Brief description of the drawings]

[0031] Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0032] [Figure 1] Representative images of the cell line corneal endothelial cells growing on bilayer and triple layer col-4, respectively (Figure 1A and Figure 1B). Upon exposure to 5% hPL, both bilayer and triple layer col-4 membranes began to detach from the coverslips in the wells by day 3. Figure 1C provides a representative image of the cell borders of corneal endothelial cells on a bilayer of col-4 that remained attached after mechanical disruption. Figures 1A and 1B were taken at 10x magnification. Figure 1C was taken at 20x magnification. [Diagram 2] Representative images of bilayer col-4 membranes in culture on day 5 are provided. In Figure 2A, the membrane was cultured for 5 days in 5% hPL medium and a col-4 corneal endothelial sheet was now detached from the coverslip in the well. In Figure 2B, the membrane was cultured for 5 days in 5% FCS medium and shows no signs of detachment as the sheet is confluent. Figure 2C provides a representative image of the detached sheet detached from the well, which is clear and flat. [Diagram 3] Representative images of cell recovery after mechanical disruption are provided. A comparison is shown between recovery when col-4 was left to potentially expand on the cells and when the col-4 film was torn. All images were taken at 10x magnification. [Figure 4]We provide representative images of col-4 membranes marked with either a simple dot that does not fade after washing with PBS (Figure 4A), or a more complex shape such as an "F" (Figure 4B), an asymmetric shape often used to mark Descemet's membrane in surgery to allow for eye orientation. [Diagram 5] Representative images are provided showing the volume of col-4 membrane (specifically the bilayer membrane shown) aspirated into a Stryker syringe (Figure 5A) and stained with VisionBlue (Figure 5B). The dashed line in Figure 5B represents the area remaining following trephine treatment of the membrane with an 8.5 mm trephine. [Figure 6] We provide a representative image of a bilayer col-4 membrane with corneal endothelial cells (shown in the image as mottled surfaces present in the cornea) adhered to the stromal surface within a human cornea after 1 minute when minimal liquid is present. [Figure 7] Representative images of the assembly of the molding platform are provided. A: Support material - silicone. B: Middle layer - polyester film with holes. C: Top layer - polyester film. D: Assembled platform. E: The support material can also be paraffin. F: A low-temperature metal plate was the support material with a middle layer on top. The bioink is filled into the middle layer and covered by the top layer. [Figure 8] Images of immunostaining for laminin (red) on collagen membranes are provided. A: col-4 membrane containing laminin. B: col-4 only membrane. The line in the upper right corner of each image indicates the membrane border. [Figure 9] Phase contrast images of four endothelial constructs are provided. All showed high cytocompatibility with confluent primary corneal endothelial cells. A: col-4 at 10.8mg / mL. B: col-1 and col-4 mixed. C: col-1 layer with col-4 layer on top. [Figure 10] 1 provides images of cells bearing collagen membranes that have self-detached from culture dishes. [Figure 11]Images taken after immunostaining are provided. Corneal primary cells cultured on representative membranes showed higher expression of Na / K-ATPase compared to the control. A: 10.8 mg / mL col-4 membrane, B: col-1 layer with col-4 layer on top, C: control. [Figure 12] Provides images of membranes made with three support materials: A: silicone, B: paraffin, and C: low temperature metal plate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The inventors have developed constructs that include type IV collagen that are transparent, crosslinkable, strong, flexible, and / or printable. The constructs of the present invention can provide a unique method of culturing endothelial cells, e.g., corneal endothelial cells, and can provide a means of delivering drugs to biological targets (e.g., organs, tissues, cells). The constructs can be used as carriers for cells, e.g., endothelial cells. The constructs described herein can be capable of mimicking Descemet's membrane in the cornea. The constructs can provide a replacement membrane and / or allow endothelial healing. Although suitable for application to the cornea, the constructs provide a platform for numerous applications in the fields of endothelial replacement and / or repair, and drug delivery, for example, by providing structural support, transparency, flexibility, viable cells, and other factors.

[0034] The constructs described herein may utilize biomaterials that mimic in vivo tissues and act as scaffolds for cells to colonize and / or through manipulation of conditions to encourage cells to regenerate themselves into the surrounding matrix. In the context of their suitability for application to ocular tissues, the inventors have addressed the challenge of creating constructs that can embody the structural integrity of the tissue being treated (e.g., Descemet's membrane in the cornea) while remaining transparent, yet porous, and sufficiently biocompatible to allow infiltration, migration, and / or proliferation of corneal endothelial cells and growth factors.

[0035] Constructs The present invention provides constructs suitable for endothelial tissue replacement, for example, corneal endothelial tissue replacement. The constructs may also be used to deliver agents to biological targets, such as tissues and cells.

[0036] The construct can include a composition comprising type IV collagen and one or more crosslinking agents. The construct of the present invention can include a composition comprising type IV collagen, type I collagen, and one or more crosslinking agents. In some embodiments, the construct further comprises endothelial cells. The construct can include a platelet lysate and / or components thereof. The platelet lysate can be human platelet lysate. Non-limiting examples of components of the platelet lysate include fibrinogen, basic fibroblast growth factor (bFGF), transforming growth factor (TGF-β), insulin-like growth factor (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), IgG, and albumin. In some embodiments, the component of the platelet lysate can be fibrinogen, which can be human fibrinogen. The construct can include one or more layers of the composition. The composition can include type IV collagen. The collagen used may be unmodified compared to its naturally occurring counterpart. Modified type IV collagen may also be used. In some embodiments, the modification is at the end of the collagen fiber.

[0037] The construct may comprise one or more layers of a composition comprising type IV collagen. Some constructs of the present invention comprise one or more layers of a composition comprising type IV collagen and type I collagen. Additionally or alternatively, the construct may comprise one or more layers of a composition comprising type IV collagen crosslinked to one or more layers of a composition comprising type I collagen. The layers of a composition comprising type IV collagen and the layers of a composition comprising type I collagen may alternate. In some embodiments of the present invention, the construct comprises two layers, one layer comprises a composition comprising type I collagen and the other layer comprises a composition comprising type IV collagen.

[0038] The composition used to generate the construct of the present invention may optionally further comprise ions and / or one or more ion sources. Non-limiting examples of suitable ions include calcium ions and sodium ions. Non-limiting examples of suitable ion sources include compounds containing calcium (e.g., calcium chloride) and sodium (e.g., sodium chloride). Calcium ions and sodium ions may be present together or individually in the composition. The ions may be present in the construct and / or in the composition.

[0039] The construct and / or composition may include endothelial cells, e.g., corneal endothelial cells. The endothelial cells may be human endothelial cells. In some embodiments, the construct and / or composition further includes fetal calf serum (FCS) and / or platelet lysate. The platelet lysate may be human platelet lysate. In further embodiments, the construct and / or composition includes fibrinogen, which may be human fibrinogen. The construct and / or composition may also include laminin.

[0040] The inventors have identified optimal relative concentrations of type IV collagen, type I collagen, sodium ions and / or calcium ions for the compositions used to generate the constructs of the present invention, some of which are described in the examples and claims of this application. It will be understood that the disclosed relative concentrations of type IV collagen, type I collagen, sodium ions and / or calcium ions are merely exemplary.

[0041] The composition may comprise 6-24 mg / ml type IV collagen, 0.04-0.15 M sodium ion, and / or 0.008-0.4 M calcium ion. The composition may comprise 1-20 mg / ml type IV collagen, 0.07-0.5 M sodium ion, and / or 0.008-0.4 M calcium ion. In some embodiments, the composition may comprise 1-20 mg / ml type IV collagen, 0.06-0.25 M sodium ion, and / or 0.008-0.1 M calcium ion. The composition may comprise 3-15 mg / ml type IV collagen, 0.06-0.25 M sodium ion, and / or 0.008-0.4 M calcium ion. Other possible ranges for the components of the composition include 3-15 mg / ml type IV collagen, 0.06-0.1 M sodium ion, and / or 0.01-0.05 M calcium ion. In some embodiments, the composition may comprise 4-12 mg / ml type IV collagen, 0.06-0.08 M sodium ions, and / or 0.015-0.03 M calcium ions. Alternatively, the composition may comprise 5-10 mg / ml type IV collagen, 0.06-0.07 M sodium ions, and / or 0.018-0.02 M calcium ions. In some embodiments, the composition may comprise less than 15 mg / ml type IV collagen and greater than 0.06 M sodium ions and / or greater than 0.018 M calcium ions.

[0042] The compositions of the present invention may further comprise one or more crosslinking agents. One non-limiting example of a suitable crosslinking agent is riboflavin. Riboflavin may be present at a concentration of 0.01-0.5% (w / v). Riboflavin may be present in an amount of about 0.01-0.1 mg. In some embodiments, the amount of riboflavin present is 0.01, 0.1 mg, or any amount between these values. Light, such as UV light or blue light, may be used to activate the riboflavin and crosslink the composition. Those skilled in the art will recognize other suitable crosslinking agents and / or light sources, such as rose bengal dye and green light, both of which are approved for some applications to the cornea. In some embodiments, rose bengal is used as a photocrosslinking agent and is activated by green light. Alternatively, rose bengal is used as a photocrosslinking agent and is activated by white light. In some embodiments, 0.01-0.5% (w / v) of rose bengal is used for photocrosslinking. In further embodiments, the provided compositions will be colored by crosslinking the collagen composition with Rose Bengal and a suitable light source. In some embodiments, the color will be pink. These colored compositions can be used to monitor collagen metabolism in tissues or for other applications that require tracking of collagen activity. The constructs of the present invention may be provided in their crosslinked form.

[0043] The construct may comprise a single layer of collagen composition which may be crosslinked, or may comprise two or more layers of collagen composition. Each layer may be crosslinked to the existing construct prior to the addition of the next layer. This may include the first layer of the construct which may be crosslinked separately prior to the addition of any other layers.

[0044] The construct may be produced using a molding platform. A suitable molding platform may include a support surface, an intermediate material, and a top material. The composition may be crosslinked on a support surface made of a hydrophobic material. Non-limiting examples of suitable hydrophobic materials include silicone and parafilm. Crosslinking may also be performed on a cold surface. The cold surface may be a metal surface. In some embodiments of the present invention, an intermediate material may be used that may have a hollow center. The hollow center of the intermediate material may be used to transfer the composition onto the support surface. One non-limiting example of a suitable material for the intermediate layer is polyester. The top material may be added before compressing with a weight. One non-limiting example of a suitable material for the top layer is polyester.

[0045] The construct and / or composition may further comprise cells. The cells may be, for example, mammalian cells (e.g., human cells, canine cells, feline cells, bovine cells, porcine cells, equine cells, caprine cells, hircine cells, murine cells, rabbit cells, cricetine cells, weasel cells, or any combination thereof). The type of cells utilized will generally depend on the particular purpose for which the composition is used. For example, the cells may be of the same type as the tissue to which the composition is administered. Non-limiting examples of suitable cell types include ocular cells, including those of central and / or peripheral corneal epithelium, bulbar and / or palpebral conjunctival epithelium, palpebral conjunctival stroma, corneal endothelium, 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, keratocytes, neural cells, and endothelial cells. The endothelial cells may be primary endothelial cells. In some embodiments, the cells may be hematopoietic stem cells, bone marrow stem cells, neural stem cells, epithelial stem cells, skin stem cells, muscle stem cells, adipose stem cells, pluripotent stem cells, induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, or any combination thereof. In some embodiments, the cells may be neural cells. A non-limiting example of a suitable epithelial cell is a lens epithelial cell. A non-limiting example of a suitable endothelial cell is a corneal endothelial cell. The cells of the construct and / or composition may be autologous (i.e., autologous from a given subject intended to receive the composition), or allogeneic (i.e., donor-derived).

[0046] The constructs and / or compositions of the invention may comprise 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.

[0047] The constructs and / or compositions of the invention may include additional components (e.g., drugs) including, but not limited to, fibronectin, anesthetics, antibiotics, hormones (e.g., insulin), growth factors (e.g., human epidermal growth factor (hEGF), platelet-derived growth factor, vascular endothelial growth factor, fibroblast growth factor (FGF), epidermal growth factor, transforming growth factor [including beta], and connective tissue growth factor), fibrin stabilizing factors (e.g., factor XIII), matrix proteins (e.g., collagen [such as type I collagen], laminin, integrins), vitamins (e.g., vitamin C, riboflavin), glycoproteins (e.g., transferrin), bovine fetal serum (FBS), calf fetal serum (FCS), human serum, platelet lysate, human platelet lysate, therapeutic agents, and any combination thereof. In some embodiments, the compositions and / or constructs include a culture medium comprising ions and amino acids. Non-limiting examples of suitable growth factors include vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). The vitamin can be ascorbate (vitamin C, riboflavin). The matrix protein can include, but is not limited to, type I collagen, and / or laminin.

[0048] The constructs and / or compositions of the invention may include other suitable components, including water and / or culture medium (e.g., DMEM, DMEM / F-12, MEM, CnT-PR). The culture medium may contain, 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, niacinamide pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, i-inositol, inorganic salts, calcium chloride (CaCl2) (anhydrous), copper sulfate (C ions may include any one or more of the following: iron nitrate (Fe(NO3)3"9H2O), iron sulfate (FeSO4-7H2O), magnesium chloride (anhydrous), magnesium sulfate (MgSO4) (anhydrous), potassium chloride (KCl), sodium bicarbonate (NaHCO3), sodium chloride (NaCl), sodium phosphate dibasic anhydrous (Na2HPO4), sodium phosphate monobasic (NaH2PO4-, zinc sulfate (ZnSO4-7H2O), other ingredients, D-glucose (dextrose), sodium hypoxanthine, linoleic acid, lipoic acid, putrescine 2HCl, sodium pyruvate, thymidine, or any combination thereof. In some embodiments, the ions are provided as a component of an ionic salt included in the composition.

[0049] Non-limiting characteristics of the construct / composition include one or more of the following: - Due to non-Newtonian shear thinning fluid properties, the viscosity of the construct / composition may decrease as the shear rate increases. In some embodiments, the viscosity of the construct / composition may be in the range of 0.01 and 1000 Pa.s at room temperature. - Optical transparency without impeding or substantially without impeding the vision resulting from the transmission of light, e.g. greater than 90% in the visual color range of 400-700 nm. - Suitability for 2D and / or 3D printing (e.g. bioprinting / extrusion printing) with the ability to maintain or substantially maintain shape / structure after printing. - Suitability for printing while maintaining the viability of cells within the composition / construct during the printing process. - The ability to be provided in two- or three-dimensional structures, with or without living cells. - the ability to maintain and / or promote cell growth (e.g. the ability to maintain and / or promote the expansion and / or growth of primary human cells such as epithelial cells (e.g. lens epithelial cells), keratocytes, neuronal cells, and endothelial cells (e.g. corneal endothelial cells). -Ability to promote the formation of spheroid organoids. -Ability for degradation by cells over time (e.g., 2-7 days). -Maintenance of cell viability over time (e.g., 7 days at 34°C). -Ability to adhere to a variety of surfaces, including tissues, organs, and membranes (e.g., mammalian and human tissues, organs, and membranes).

[0050] Preparation of constructs The construct may be prepared by forming one or more layers of the composition. The construct may be crosslinked. The composition in one or more layers may be prepared by combining multiple different preparations. In some embodiments, lyophilized bovine type IV collagen may be used in the preparation of the composition. Additionally or alternatively, human collagen may be used. Type IV collagen may be neutralized before adding ions and other components of the composition. Those skilled in the art will recognize that various buffers may be used to keep collagen at physiological pH and maintain solubility. In some embodiments of the present invention, type IV collagen may be mixed in the preparation of the composition.

[0051] In some embodiments of the invention, a monolayer of the composition is applied to a surface to form a construct. The monolayer may be individually crosslinked. In further embodiments, one or more additional layers are added to the first layer. Each layer may be crosslinked to the existing construct before the addition of the next layer. There is no particular limit on the number of layers of the composition that may form the construct. The layers may include type IV collagen, type I collagen, or a mixture of the two. Alternating layers of compositions that include only type IV collagen and only type I collagen may be used to form the construct. In some embodiments, the construct has two layers, one layer includes a composition that includes only type IV collagen and the other layer includes a composition that includes only type I collagen.

[0052] The composition used to make the construct may include 6-24 mg / ml type IV collagen, 0.04-0.15 M sodium ion and / or 0.008-0.4 M calcium ion, or 1-20 mg / ml type IV collagen, 0.07-0.5 M sodium ion and / or 0.008-0.4 M calcium ion. In some embodiments, the composition may include 1-20 mg / ml type IV collagen, 0.06-0.25 M sodium ion and / or 0.008-0.1 M calcium ion. The composition may include 3-15 mg / ml type IV collagen, 0.06-0.25 M sodium ion and / or 0.008-0.4 M calcium ion. Other possible ranges for the components of the composition include 3-15 mg / ml type IV collagen, 0.06-0.1 M sodium ion and / or 0.01-0.05 M calcium ion. In some embodiments, the composition may comprise 4-12 mg / ml type IV collagen, 0.06-0.08 M sodium ions, and / or 0.015-0.03 M calcium ions. Alternatively, the composition may comprise 5-10 mg / ml type IV collagen, 0.06-0.07 M sodium ions, and / or 0.018-0.02 M calcium ions. In some embodiments, the composition may comprise less than 15 mg / ml type IV collagen and greater than 0.06 M sodium ions and / or greater than 0.018 M calcium ions.

[0053] The compositions used to make the constructs of the invention may further include one or more crosslinking agents. One non-limiting example of a suitable crosslinking agent is riboflavin. Riboflavin may be present in an amount of about 0.1 mg. Riboflavin may be present in an amount of 0.01-0.1 mg / ml. Riboflavin may be present in a concentration of 0.01-0.5% (w / v). Light, such as UV light or blue light, may be used to activate the riboflavin and crosslink the composition. Those skilled in the art will recognize other suitable crosslinking agents and / or light sources, such as rose bengal dye and green light, both of which are approved for some applications to the cornea. In some embodiments, rose bengal is used as a photocrosslinking agent and is activated by green light. Alternatively, rose bengal is used as a photocrosslinking agent and is activated by white light. In some embodiments, 0.01-0.5% (w / v) of rose bengal is used for photocrosslinking. The constructs and / or compositions may include endothelial cells, such as corneal endothelial cells. The endothelial cells may be human endothelial cells. In some embodiments, the construct and / or composition further comprises fetal calf serum (FCS) and / or platelet lysate.

[0054] In further embodiments, the construct and / or composition further comprises fibrinogen, which may be human fibrinogen. Human fibrinogen may be added to the construct at a concentration of 0.7-2 mg / ml. In some embodiments, platelet lysate and / or fibrinogen is added to the construct for use as a culture medium for culturing endothelial cells. Additionally or alternatively, components of a platelet lysate may be added. The platelet lysate may be human platelet lysate. Non-limiting examples of suitable components of a platelet lysate include fibrinogen, basic fibroblast growth factor (bFGF), transforming growth factor (TGF-β), insulin-like growth factor (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), IgG, and albumin.

[0055] In some embodiments, the invention provides devices and / or kits for the preparation of the constructs. The devices and / or kits may facilitate the separation of the different preparations required to form the constructs and / or compositions of the invention until use.

[0056] The devices and kits may further comprise components that provide a means for facilitating mixing of the two compartmentalized preparations, such as by removing a barrier separating the first and second compartments and / or puncturing a seal or wall of one or both compartments. Those skilled in the art will readily appreciate that a variety of configurations may be made for this purpose.

[0057] Additionally or alternatively, the devices and kits may be configured to ensure mixing of the two compartmentalized preparations during or after release of the preparations from the device or kit.

[0058] In some embodiments, the devices and kits may include additional compartments that contain additional components used in the production of the compositions and / or constructs (e.g., platelet lysates, ions, amino acids, cells, antibiotics, growth factors, fibrinogen stabilizing factors, anesthetics, etc.) The devices or kits may be configured to facilitate mixing of these additional components with each other and / or with other components of the composition.

[0059] The devices and kits can facilitate mixing of the separated components before, during, or immediately after release of the components from the device or kit.

[0060] In some embodiments, the composition is a bio-ink and the device is a three-dimensional (3D) printer (e.g., an extrusion printer).

[0061] In some embodiments of the invention, the cross-linking agent is riboflavin. In further embodiments, the riboflavin can be activated by UV light or blue light. A solution that can contain type IV collagen and sodium and / or calcium ions can be extruded in a line and UV light or blue light can be applied. Additional lines can be applied on top of the first line to form a structure that is cross-linked by the application of the cross-linking agent and light.

[0062] In some embodiments, crosslinking occurs in less than 15 minutes, less than 14 minutes, less than 13 minutes, less than 12 minutes, less than 11 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute. The light source used is 3 mW / cm 2 365nm UV, 10mW / cm 2 blue light, or tissue culture hood UV lamp.

[0063] The crosslinking agent can be Rose Bengal. In some embodiments, the Rose Bengal is activated by green light. In further embodiments, the Rose Bengal is activated by white light. In still further embodiments, the crosslinking occurs in less than 15 minutes, less than 14 minutes, less than 13 minutes, less than 12 minutes, less than 11 minutes, less than 10 minutes, less than 9 minutes, less than 8 minutes, less than 7 minutes, less than 6 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute. The light source used is 100 mw / cm 2 Those skilled in the art will recognize that the light source and parameters can vary according to the particular application.

[0064] The collagen constructs of the invention may have a variety of thicknesses depending on the application, for example the gel may be 50 μm to 3 mm thick, the constructs may be injectable and / or the mat may be handled with tweezers without breaking.

[0065] Application of constructs The inventors have developed constructs that may be useful, for example, as replacement endothelial tissue. Non-limiting examples of tissues that may be repaired / replaced using the constructs include corneal endothelium and Descemet's membrane. The constructs may be used to treat corneal injury or disease, for example, by repairing / replacing human corneal endothelium and human Descemet's membrane. In some embodiments, the constructs may be used as a replacement for donor corneal tissue in endothelial keratoplasty or as an alternative to keratoplasty in endokeratoplasty. The constructs may also be used to deliver drugs to target tissues and cells. The characteristics of the constructs make them highly suitable for bioprinting. The constructs of the present invention may be used in applications requiring delivery of drugs (e.g., natural growth factors, drugs, nanoparticles, and / or cells) and / or for fixation of individual biological surfaces and / or in tissue transplantation and / or in tissue culture methods.

[0066] In some embodiments, the constructs can function as tissue sealants and / or fixatives for biological structures. They can provide structural and / or nutritional support to tissues. Additionally or alternatively, the constructs can promote healing and / or promote the growth of target cell types, including those that may be provided as components of the constructs and / or cells present in the target tissue. The compositions can be useful for culturing cells, for example, human corneal endothelial cells.

[0067] While there are no limitations regarding the type of tissue to which the composition may be applied, the inventors have demonstrated that the composition is compatible with cells, such as endothelial cells, and / or may support the growth and / or proliferation of corneal endothelial cells.

[0068] For example, the compositions herein are demonstrated to be effective in supporting the growth and / or proliferation of corneal endothelial cells. In these embodiments, the compositions may be used to promote the proliferation and / or migration of corneal endothelial cells.

[0069] Thus, the present invention provides a method for delivering various drugs to biological targets.Target can be located in or around ocular tissue, including, for example, central and / or peripheral corneal epithelial tissue and / or Descemet's membrane.This method can also be used to repair and / or replace collagen IV-containing basal layer of other human tissue and / or deliver drugs to collagen IV-containing basal layer of other human tissue.

[0070] It will be appreciated by those skilled in the art that numerous variations and / or modifications may be made to the invention disclosed in the specific embodiments without departing from the spirit or scope of the invention as broadly described, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive. EXAMPLES

[0071] The present invention will now be described with reference to specific examples, which should not be construed as limiting in any way.

[0072] Example 1: Creation of col-4 membrane mimicking Descemet's membrane col-4 powder (minimum concentration of 6 mg / mL) was dissolved in 0.1 M acetic acid and then neutralized with 5 M NaOH and 27.4 mg / mL CaCl2 to a final pH of 6.7-7.4. These steps were performed at room temperature. Once the solution was neutralized, 0.1-0.2 mg of riboflavin was added for every 90 μL of col-4 originally used, and the col-4 / riboflavin solution was further dissolved by vortexing and centrifuging for 30 s and 1 min, respectively.

[0073] The addition of calcium ions produced a clear, neutralized col-4 solution. The resulting liquid can be photocrosslinked immediately or stored at -30°C for use after at least one week. The solution can be kept at room temperature for use after at least one hour. Neutralized col-4 without riboflavin can be stored at 30°C for use after at least one week without change in crosslinking properties.

[0074] Multiple morphologies of col-4 membranes were generated. a: A bilayer of col-4 (approximately 100 μm). This was generated by first pipetting 25 μL of col-4 / riboflavin solution onto a glass coverslip (13 mm diameter). The drop was spread over the coverslip area and placed under UV or blue light for 1 min. An additional 25 μL of solution was then pipetted onto the partially crosslinked solution and again spread over the area. The col-4 film was then crosslinked under UV or blue light for an additional 3 min to generate a bilayer crosslinked col-4 film. The layered nature of the generated film is intended to increase its strength compared to a monolayer of col-4 film.

[0075] b: col-4 triple layer (~200 μm). This was generated in a similar manner to the col-4 bilayer. 25 μL of col-4 / riboflavin solution was pipetted onto a glass coverslip and allowed to spread to cover the area. This layer was placed under UV or blue light for 1 min. An additional 25 μL was pipetted onto the partially crosslinked solution and allowed to spread to re-cover the area. The bilayer film was then allowed to crosslink for an additional minute before a final 25 μL was added and allowed to spread to re-fill the area, forming the triple layer structure.

[0076] The col-4 membranes were then placed into 35 mm petri dishes containing 1x PBS and washed 2x15 min until clear. These membranes could be stored in 1x PBS at 4°C until use. For culture experiments, the col-4 membranes were first sterilized under UV light in a cell culture hood for 15 min. Once sterilized, the membranes were placed into the wells of a 12-well plate.

[0077] Both bilayer and triple layer col-4 membranes produced were compatible with corneal endothelial cells when tested using cell lines (Figure 1A / B). In addition, the increased thickness seen between monolayer, bilayer, and triple layer col-4 membranes did not affect their ability to detach from the wells after 3-5 days of culture in 5% hPL medium. No difference in transparency between monolayer and bi / triple layer col-4 membranes was observed macroscopically or microscopically after washing. When bilayer and triple layer col-4 membranes were handled with tweezers, differences were observed with a greater number of layers crosslinked together, as the membrane structure was stronger. However, although triple layer col-4 was the strongest of those produced, this strength compromised some of its flexibility as it could not be folded into a scroll. Bilayer col-4 was stronger than monolayer and maintained an appropriate level of flexibility. Cells remained attached to all types of membranes even after mechanical disruption, and an example of this can be seen in Figure 1C. The ideal bioengineered endothelial membrane appears to be a bilayer membrane, although triple-layer membranes are also potential candidates.

[0078] Development of a unique culture system for corneal endothelial cells on col-4 membranes Both immortalized corneal endothelial cell line (B4G12) and primary corneal endothelial cells were used. B4G12 cells were thawed (after storage in liquid nitrogen) and expanded in coated tissue culture flasks until they reached 80% confluence. Cells were cultured using a fetal calf serum (FCS)-based growth medium of Nutrient Mixture Ham's F12 and Medium 199, 20 μg / mL ascorbic acid, 10 ng / mL bFGF and 5% FCS medium in a 1:1 mixture of 10,000 U / mL penicillin and 10,000 μg / mL streptomycin. Cells were then passaged and counted. 1×10 5The cell density was adjusted so that 1 cell was equivalent to 75–100 μL of cell-loading medium. This amount of medium was added to the generated col-4 membranes, and the membranes containing the cells were left in an incubator for 45 min to allow cell attachment. An additional 500 μL of FCS-based growth medium was added to the wells containing the membranes.

[0079] The next day, the FCS-based growth medium was replaced with 5% human platelet lysate (hPL) medium. This hPL medium had the same composition as the 5% FCS-based growth medium, except that FCS was replaced with hPL. The membranes were incubated in hPL medium for 3–5 days.

[0080] The cell lines reached full confluence after 5 days with an average density of 3784 cells / mm 2 The generation of corneal endothelial sheets during the culture of corneal endothelial cells on col-4 membranes required the addition of 5% hPL.

[0081] A total of 19 membranes; 3 trilayers and 16 bilayers col-4 were prepared and observed. All of these membranes were isolated after the cells reached confluence in 5% hPL medium (Figure 2A). All prepared triple-layer membranes were isolated 4 days after the initiation of 5% hPL culture. The average isolation time of the bilayer col-4 group, which produced more membranes for observation, ranged from 3 to 5 days of culture in 5% hPL medium (Table 1). In comparison, none of the col-4 corneal endothelial sheets cultured in 5% FCS medium could be detached (Figure 2B). [Table 1]

[0082] Wound healing test Bioengineered col-4 corneal endothelial sheets were subjected to mechanical disruption as surgeons manipulated them using tweezers. After mechanical trauma, including a scratch to the membrane, the sheets were fixed in a 35 mm Petri dish using tweezers and allowed to recover for one week in 5% FCS medium. Cell recovery across areas where col-4 remained intact and areas where no col-4 remained was compared. Some sheets were torn to completely remove col-4 (control condition), while in other sheets, cells were simply scratched off the col-4. The same area of ​​each sheet was imaged daily and the image analysis software Image J was used to determine the area of ​​recovered cells.

[0083] This test was commonly important during DMEK surgery, where 30% of cells are lost from the transplanted endothelium. After injury, endothelial cells were able to recover, forming a scar-free monolayer on the col-4 membrane after 7 days (Figure 3). However, if there was a tear in the col-4 layer itself, the cells were unable to recover because they could not expand the col-4.

[0084] Mock Clinical Trial Marking the bioengineered endothelium During surgery, it is essential that the Descemet's membrane can be marked to indicate orientation so that the surgeon can determine which side the endothelial cells are on so that they are correctly positioned for attachment to the patient's eye. A non-toxic pen was used to draw an asymmetric shape, such as an "F," on the col-4 membrane. The col-4 membrane with this marking was washed to determine if the marking remained visible after washing in various liquids and mechanical manipulation during surgery.

[0085] The col-4 membrane created can be marked with the same marker used in surgery to create an asymmetric mark that allows the surgeon to distinguish which side the endothelial cells are on. Marks created on the non-endothelial side of the membrane could not be washed away and remained distinguishable (Figure 4A). More complex shapes, including the letter "F," can also be created using dots to form the letter, as drawing this shape directly on the membrane can cause tearing (Figure 4B).

[0086] Mock DMEK (Descemet's Membrane Endothelial Keratoplasty) Surgery Test The surgical test was performed in a surgical wet lab using an artificial eye. The entire process followed the steps of a regular DMEK surgery, where a donor Descemet's membrane is transplanted with its attached endothelium. In this test, all three types of membranes with cells were used: monolayer, bilayer, and trilayer. The membranes were first trephine using an 8.5 mm trephine and stained with VisionBlue to allow visualization during the injection process. The membranes were then aspirated into a Stryker syringe for insertion into the artificial eye. After insertion, an air bubble was placed on top of the sheet in the eye to allow for shrinkage and mimic the part of the DMEK surgery where the transplanted sheet can be attached to the patient's eye.

[0087] Both bilayer and triple layer col-4 corneal endothelial sheets were injectable and could be expelled from the Stryker syringe into the artificial eye (shown in Figure 5A). However, due to its increased strength and decreased flexibility, the triple layer membrane required more force. They could also be properly stained with VisionBlue dye (shown in Figure 5B), which is used in routine surgery for visualization of the eye during injection. Both col-4 corneal endothelial bilayer and triple layer sheets could be properly trephine to produce the correct size sheet (this size is shown in dashed line in Figure 5B). This trephine produced a smooth circular layer by removing uneven edges. They were also compatible with the big bubble technique and could be properly expelled into the artificial eye and porcine eye.

[0088] Ex vivo testing This test was performed to determine the adhesion of col-4 corneal endothelium to whole corneas lacking Descemet's membrane. The sheet was placed in the eye and flattened with the cell side facing up to mimic the normal structure and orientation of the corneal layers. At five different time points, 1 min, 5 min, 10 min, and 15 min, the whole cornea was picked up and the adhesion of the layer was determined by examining the movement of the layer from its original position.

[0089] After placement on a human cornea lacking Descemet's membrane, the bioengineered endothelial layer was able to adhere instantly (within 1 min) to the stroma when there was minimal fluid in the eye (Figure 6). This adhesion was maintained at all following time points checked. However, even after 1 h of adhesion time with minimal fluid, the addition of a certain amount of liquid caused the col-4 sheet to dissociate from the cornea and float to the surface. After overnight incubation in a humidified chamber, the addition of liquid allowed the col-4 sheet to remain adhered to the corneal stroma. This adhesion was found to be permanent as the sheets remained adhered after 2 weeks of culture in organ storage medium. The sheets also remained transparent within the cornea both initially and over the course of their culture.

[0090] Example 2: Preparation and testing of additional endothelial constructs In this example, additional endothelial constructs were prepared and tested, including constructs that use collagen I (col-1) and laminin as the major components of the membrane. Primary endothelial cells were used to provide further evidence and examples of additional constructs.

[0091] Materials and Methods Preparation of collagen I (col-1) solution Col-1 powder (Bovine skin, Sigma-Aldrich) was prepared according to the manufacturer's protocol. The powder was dissolved in 0.1 M acetic acid solution (pH=2.72) under continuous stirring at a speed of 800 rpm for 16 hours. The mixture was then visually observed to ensure that the collagen powder was completely dissolved. The collagen solution was then transferred to a 1.5 ml Eppendorf tube and stored in the refrigerator.

[0092] 90 parts collagen solution was neutralized with 2.7 parts 5M NaOH and then mixed with 7.3 parts CaCl2. 0.1% and 0.2% (w / v) solutions of riboflavin were made by adding 0.5 and 1 mg of riboflavin powder separately to 500 μL of 2 mg / ml CaCl2 solution. The tubes were vortexed for 2 minutes and checked for precipitates by centrifuging the tubes for 1 minute in a mini centrifuge. Riboflavin powder (Sigma-Aldrich) was added to the freshly made neutralized collagen solution with ions added to a final concentration of 1 mg / ml. The col-1 solutions used in this example had col-1 concentrations of 3-12 mg / mL.

[0093] Preparation of collagen 4 (col-4) solution col-4 powder (minimum concentration of 6 mg / mL) was dissolved in 0.1 M acetic acid and then neutralized with 5 M NaOH and 27.4 mg / mL CaCl2 to a final pH of 6.7-7.4. These steps were performed at room temperature. Once the solution was neutralized, 0.1-0.2 mg of riboflavin was added for every 90 μL of col-4 originally used, and the col-4 / riboflavin solution was further dissolved by vortexing and centrifuging for 30 s and 1 min, respectively.

[0094] The addition of calcium ions produced a clear, neutralized col-4 solution. The resulting liquid can be photocrosslinked immediately or stored at -30°C for use after at least one week. The solution can be kept at room temperature for use after at least one hour. Neutralized col-4 without riboflavin can be stored at 30°C for use after at least one week without change in crosslinking properties.

[0095] Preparation of Collagen I and / or IV Constructs Four collagen constructs were prepared. 1a: col-4 at 10.8 mg / mL 1b: col-1 and col-4 were mixed with 4 mg / mL col-1 and 7 mg / mL col-4 1c: col-1 layer (10.5 mg / mL) at the bottom, col-4 layer (10.5 mg / mL) at the top 1d: col-4 10mg / mL and laminin 10μg / mL All constructs were prepared in two layers. Briefly, 20 μL of collagen bioink (col-4 only, col-1 only, or a mixture of col-1 and col-4) was dropped onto a 22 mm diameter coverslip and spread using a p100 pipette to fill the surface. The hydrogel was crosslinked with 480 nm light from a distance of 3 cm for a minimum of 1 min. Following this, a second layer of 20 μL of collagen bioink was applied on top of the first. The completed membrane was crosslinked for a minimum of another 4 min. The membrane thickness was measured by placing it vertically under a microscope. The membrane was washed three times with sterile 1× PBS for 5 min each on a shaker and left overnight at 4°C until clear. The membrane was used immediately or stored in a refrigerator until use. Sterilization was performed by removing the membrane from PBS and placing it under UV for 15 min in a class II biosafety cabinet.

[0096] Transparency Test After crosslinking, all hydrogels were washed 3 times for 10 min with 1x PBS. The membranes were then placed flat in a 24-well plate, filling at least 90% of the wells. The optical density of each well from 400 to 1000 nm was measured using a plate reader (Tecan Safire II), and the % transmittance was obtained by 1-mean OD.

[0097] Immunostaining of collagen IV using laminin membrane Membranes were fixed with 4% paraformaldehyde for 15 min and rinsed with PBS. Samples were blocked with 5% BSA / PBS for 30 min and incubated with anti-laminin beta 1 antibody (1 out of 100 dilution; Abcam ab256380) at 4°C overnight. After incubation, membranes were rinsed with PBS and incubated with donkey anti-rabbit 594 secondary antibody for 2 h at room temperature (RT). Immunostaining was visualized and imaged using a Zeiss LSM700 scanning laser confocal microscope.

[0098] Primary corneal endothelial cell culture Primary corneal endothelial cells were cultured. Descemet's membrane was peeled from donor corneas and digested in collagenase A (2 mg / mL, Sigma) in tubes for 3–5.5 h at 37 °C. The digestion tubes were centrifuged at 190 g for 5 min at room temperature (RT). The supernatant was removed and TrypLE select 1× (ThermoFisher) was added for further digestion for 5 min. M5 medium was supplemented with 5% FBS, human endothelial serum-free medium (SFM, ThermoFisher), 10 μM Y-27632 (Sigma) and 1% antibiotic-antimycotic (ThermoFisher) to inactivate TrypLE, and the tubes were centrifuged again. The cell pellet was resuspended in fresh M5 medium. 50 μL of cell suspension (1.5×10^5 cells) was added to the center of each membrane already placed in 6-well or col-1 pre-coated petri dishes with 9 mm diameter spacers as control. Plates and dishes were placed in the incubator for 45 min, followed by the addition of 1.5 mL of M5 medium. The next day, M5 was replaced with growth medium M4 (Ham's F12:M199, 1:1, 5% FBS, 1% ITS, 10 μM Y-27632, 10 ng / ml hFGFb, and 1% antibiotic-antimycotic). Medium was changed 3 times a week and cultures were examined by phase contrast light microscopy.

[0099] Immunostaining of bioengineered endothelium After incubation, membranes and controls were fixed with cold methanol for 2 min and rinsed with PBS. Samples were blocked with 5% BSA / PBS for 30 min and incubated with mouse Na / K-ATPase (Abcam) overnight at 4°C. Samples were rinsed with PBS and incubated with goat anti-mouse 488 secondary antibody and Hoechst 33342 for 2 h at room temperature. Immunostaining was visualized and imaged using a Zeiss LSM700 scanning laser confocal microscope.

[0100] An alternative method to fabricate collagen constructs using a molding platform The molding platform was assembled with three layers: a support layer made of either a hydrophobic material (e.g., silicone, Parafilm) or a cold surface (e.g., cold metal), a middle layer that was a 50 μm or thicker polyester film with a hollow center, and the top layer was another polyester film (Figure 7).

[0101] Briefly, the support layer and middle layer were first assembled, and 30 μL of collagen bioink (col-4 only, col-1 only, or a mixture of col-1 and col-4) was pipetted into the hollow center of the middle layer. The top layer was then placed on top of the device. The entire mold was compressed by weight, and the molded hydrogel membrane was crosslinked with 480 nm light from a distance of 3 cm for 2 min.

[0102] After crosslinking, the top layer was removed and 20 μL of collagen bioink was applied to the crosslinked membrane. The bioink was then covered with the top layer and crosslinked again. The bilayer membrane was then removed from the mounting apparatus and the membrane thickness was measured by placing the membrane vertically under a microscope.

[0103] The membranes were washed three times with sterile 1x PBS for 5 minutes each on a shaker and left overnight at 4°C until clear. Membranes were either used immediately or stored in the refrigerator until use. Sterilization was performed by removing the membranes from the PBS and placing them under UV for 15 minutes in a class II biosafety cabinet.

[0104] result Preparation of Collagen I and / or IV Constructs All four membranes were successfully constructed with an average thickness of 250 μm and a transparency of >90% (Table 2). [Table 2]

[0105] Immunostaining of collagen IV using laminin membrane Fluorescence images of collagen membranes with laminin showed stronger staining than the control (Figure 8), suggesting that laminin (red fluorescence) was incorporated across the membrane.

[0106] Primary corneal endothelial cell culture Primary corneal endothelial cells reached confluence for all membranes tested (Figure 9). At approximately 10-14 days of culture, the membranes were able to self-detach from the coverslips on which they were prepared (Figure 10). The detached membranes were then picked up with tweezers and transferred to a separate dish for immunostaining.

[0107] Immunostaining of bioengineered endothelium Primary human corneal endothelial cells grown on col-1 alone as well as col-1 I and col-4 layered membranes had higher intensity of the key marker Na / K-ATPase on the cell borders compared to controls (Petri dishes precoated with col-1) (Figure 11).

[0108] An alternative method to fabricate collagen constructs using a molding platform Two layered collagen membranes could be successfully fabricated by the molding platform (Figure 12). When the cold metal plate was used, the membrane adhered to the top layer rather than to the support material. However, the adhesion was weak and the membrane could be easily removed by rinsing with PBS. The molding platform method allowed for consistent membrane production. The top layer ensured a smooth surface of the membrane and allowed the membrane to be easily removed from the support material.

[0109] conclusion The data presented in this example show that col-4 membranes can incorporate structural proteins such as col-1 and laminin without affecting the transparency of the membrane. In addition, two layered constructs can be made using one layer of pure col-1 and another layer of col-4 via cross-linking methods. Primary corneal endothelial cells were tested and were able to self-detach the membranes from the discs in which they were generated after the cells reached confluence (usually around day 10 of culture). There is no need to add human platelet lysate when culturing primary endothelial cells. A new method of producing collagen constructs was also developed that produces a smooth surface on the membrane, allowing for easy removal and handling of the membrane.

Claims

1. A construct comprising a composition, the composition comprising: - Type IV collagen, - one or more cross-linking agents, - an endothelial cell.

2. (i) The composition further comprises a platelet lysate, preferably a human platelet lysate, and / or a component thereof, preferably the component being any one or more of fibrinogen, basic fibroblast growth factor (bFGF), transforming growth factor (TGF-β), insulin-like growth factor (IGF-1), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), IgG, albumin, preferably the component being fibrinogen; and / or (ii) the endothelial cells are corneal endothelial cells, preferably human endothelial cells; and / or (iii) the construct and / or the composition further comprises sodium ions and / or calcium ions; and / or (iv) the one or more cross-linking agents are capable of being activated by UV light, blue light, green light, or white light; and / or (v) the one or more cross-linking agents comprise riboflavin; and / or (vi) the construct and / or the composition further comprises a mammalian cell, preferably the mammalian cell comprises or consists of a human cell; and / or (vii) the construct and / or composition further comprises any one or more of a culture medium, a growth factor, a hormone, a matrix protein, a glycoprotein, a vitamin, an ion, an ion source, fibronectin, an amino acid, an antibiotic, an anesthetic, Factor XIII, Fetal Bovine Serum (FBS), Fetal Calf Serum (FCS), human serum, platelet lysate, human platelet lysate, a therapeutic agent; and / or (viii) the construct and / or the composition comprises a culture medium containing ions and amino acids; and / or (ix) the construct and / or the composition comprises an ion, the ion being a component of an ionic salt contained in the construct and / or the composition; and / or (x) the type IV collagen is neutralized; and / or (xi) the construct and / or the composition further comprises any one or more of type VIII collagen, laminin, nidogen, and perlecan; and / or (xii) the construct comprises at least one layer comprising the type IV collagen and the one or more crosslinkers, each layer comprising: -6 to 24 mg / ml type IV collagen; - 0.04 to 0.15 M sodium ions and / or 0.008 to 0.4 M calcium ions, preferably comprising: (a) 6 to 24 mg / ml type IV collagen, 0.06 to 0.1 M sodium ions, and 0.01 to 0.04 M calcium ions, or (b) 3-15 mg / ml type IV collagen, 0.06-0.08 M sodium ions, and 0.015-0.03 M calcium ions, or (c) comprising 4 to 12 mg / ml type IV collagen, 0.06 to 0.07 M sodium ions, and 0.018 to 0.02 M calcium ions; The construct of claim 1.

3. The solution is (i) less than 24 mg / ml type IV collagen, more than 0.04 M sodium ions and more than 0.008 M calcium ions; and / or (ii) 0.01 to 0.1 mg of riboflavin The construct of claim 2 , comprising:

4. 4. The construct of any one of claims 1 to 3, wherein the construct comprises two or more layers comprising the type IV collagen and the one or more crosslinking agents, each of the two or more layers being crosslinked to at least one other layer.

5. The construct comprises at least one additional layer comprising type I collagen and one or more crosslinkers, the additional layer comprising: - 3 to 15 mg / ml of type I collagen; - 0.135 to 0.5 M sodium ions and / or 0.008 to 0.4 M calcium ions, Preferably, each layer is crosslinked to at least one other layer. A construct according to any one of claims 2 to 4.

6. 6. The construct of any one of claims 1 to 5, wherein the construct comprises a first layer comprising the collagen and the one or more crosslinking agents, the first layer being crosslinked separately prior to crosslinking to one or more other layers.

7. The construct, (i) at least one layer comprising type IV collagen, type I collagen, and said one or more crosslinkers, each layer comprising: -6 to 24 mg / ml type IV collagen; - 3 to 15 mg / ml type I collagen, preferably said solution containing 0.01 to 0.1 mg riboflavin; and / or (ii) comprising two or more layers comprising the type IV collagen, type I collagen, and the one or more crosslinking agents, each of the two or more layers being crosslinked to at least one other layer; A construct according to any one of claims 1 to 6.

8. 1. A method for preparing a construct, the method comprising: (i) providing a composition, the composition comprising: - Type IV collagen, - one or more crosslinking agents; (ii) adding endothelial cells.

9. 1. A method for culturing endothelial cells, the method comprising: (i) providing a composition, the composition comprising: - Type IV collagen, - one or more crosslinking agents; (ii) adding endothelial cells.

10. The method of claim 8 or 9, further comprising adding in step (ii) a platelet lysate, preferably human platelet lysate, and / or a component thereof, preferably said component being any one or more of fibrinogen, bFGF, TGF-β, IGF-1, BDNF, VEGF, EGF, HGF, PDGF, IgG, albumin, preferably said component being fibrinogen. (a) the composition, prior to (ii), - type I collagen, - one or more cross-linking agents; and / or (b) the solution further comprises type I collagen; and / or (c) the endothelial cells are corneal endothelial cells; and / or (d) adding sodium ions and / or calcium ions in step (i) and / or step (ii); and / or (e) the one or more cross-linking agents are capable of being activated by UV light, blue light, green light, or white light; and / or (f) the one or more cross-linking agents comprise riboflavin; and / or (g) the composition comprises at least one layer comprising a crosslinking solution, the solution comprising: -6 to 24 mg / ml type IV collagen; - 0.04 to 0.15 M sodium ions and / or 0.008 to 0.4 M calcium ions; and / or (h) the composition comprises two or more layers comprising the collagen and the one or more crosslinking agents, each of the two or more layers being crosslinked to at least one other layer; and / or (i) the composition comprises a first layer comprising the collagen and the one or more crosslinking agents, the first layer being crosslinked separately prior to crosslinking to one or more other layers; and / or (j) adding mammalian cells in step (i) and / or step (ii), preferably said mammalian cells comprise or consist of human cells; and / or (k) adding in step (i) and / or step (ii) any one or more of a culture medium, a growth factor, a hormone, a matrix protein, a glycoprotein, a vitamin, an ion, an ion source, fibronectin, an amino acid, an antibiotic, an anesthetic agent, factor XIII, FBS, FCS, human serum, platelet lysate, human platelet lysate, a therapeutic agent; and / or (l) adding ions as components of an ionic salt in step (i) and / or step (ii); and / or (m) the type IV collagen is neutralized; and / or (n) in step (i) and / or step (ii), adding any one or more of type VIII collagen, laminin, nidogen, and perlecan; The method according to any one of claims 8 to 10.

12. (i) at least one layer of the composition is crosslinked onto a hydrophobic material, preferably the hydrophobic material being silicone or parafilm; or (ii) at least one layer of said composition is crosslinked on a low temperature material, preferably said low temperature material is a low temperature metal; The method of claim 11.

13. A construct obtained or obtainable by the method according to any one of claims 8 or 10 to 12.

14. A construct according to any one of claims 1 to 7 or 13 for replacing endothelial tissue, preferably said endothelial tissue comprises or consists of said corneal endothelium, more preferably said endothelial tissue is human endothelial tissue.

15. A construct according to any one of claims 1 to 7 or 13 for treating corneal damage or disease.

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