Method for constructing retinal model

Abstract

A method for constructing a retinal model, of the present invention, comprises the steps of: printing a tubular vascular structure by using a first bioink comprising a vascular-derived decellularized extracellular matrix (VdECM) and endothelial cells, and a second bioink comprising a VdECM and pericytes, such that the first bioink forms an inner layer and the second bioink forms an outer layer; printing an inner blood retinal barrier (BRB) structure around the tubular vascular structure such that a third bioink comprising a retina-derived decellularized extracellular matrix (RdECM) and retinal cells is filled; printing an outer BRB structure on a porous membrane such that a layer of a fourth bioink comprising an RdECM and not comprising retinal cells is formed, and a monolayer of retinal pigment epithelium (RPE) cells is formed thereon; and arranging the outer BRB structure on the inner BRB structure such that the porous membrane is in contact therewith.

Description

Method for manufacturing a retinal model

[0001] The present invention relates to a method for manufacturing a retinal model, and specifically, the present invention relates to a method for manufacturing a retinal model capable of accurately mimicking the retinal structure and, in particular, closely reflecting the progression of actual retinal vascular occlusion (RVO) lesions as well as the response to related drugs, and to a retinal model manufactured according to the same.

[0002] Due to changes in modern dietary habits, the prevalence of hyperlipidemia is increasing, leading to a rise in various retinal vascular diseases. In severe cases, these diseases can result in vision loss due to blood vessel blockage. A representative example of a retinal vascular disease is RVO. Since there is currently no complete cure for RVO, related research is necessary; however, despite efforts to develop in vitro retinal models for this purpose, reproducing the microenvironment of different retinal tissues remains a challenge, making the development of technologies to overcome this urgent need. In particular, to simulate the retina, the Blood-Retinal Barrier (BRB) must be fully replicated. The BRB consists of an inner BRB containing numerous microvessels and an outer BRB where retinal pigment epithelial (RPE) cells exist as a monolayer; currently, it is difficult to fully replicate this structure using existing in vitro retinal models. Furthermore, to simulate RVO in vitro, it is necessary to fully represent multilayered blood vessels with some parts narrowed; however, there are currently no attempts to create such an RVO model.

[0003] Accordingly, the inventors sought to solve these problems by developing a retinal model capable of accurately mimicking retinal structures, and in particular, a technology for manufacturing an RVO model that can closely reflect not only the progression of actual RVO lesions but also the response to related drugs.

[0004] [Prior Art Literature]

[0005] [Patent Literature]

[0006] Korean Registered Patent No. 10-1974716

[0007] Therefore, the main objective of the present invention is to provide a method for manufacturing a retinal model capable of mimicking an accurate retinal structure, in particular a retinal model capable of closely reflecting not only the progression of actual RVO lesions but also the response to related drugs.

[0008] Another objective of the present invention is to provide a retinal model capable of mimicking an accurate retinal structure, manufactured through the above-described method for manufacturing a retinal model, and in particular, a retinal model (RVO model) capable of closely reflecting not only the progression of actual RVO lesions but also the response to related drugs.

[0009] According to one aspect of the present invention, the present invention comprises the steps of: printing a tubular blood vessel structure using a first bioink comprising a vascular-derived decellularized extracellular matrix (VdECM) and endothelial cells and a second bioink comprising a vascular-derived decellularized extracellular matrix (VdECM) and perivascular cells, wherein the first bioink forms an inner layer and the second bioink forms an outer layer; printing an inner blood-retinal barrier (BRB) structure around the tubular blood vessel structure such that a third bioink comprising a retinal-derived decellularized extracellular matrix (RdECM) and retinal cells is filled; and printing an outer blood-retinal barrier (BRB) structure on a porous membrane such that a layer of a fourth bioink comprising a retinal-derived decellularized extracellular matrix (RdECM) and not retinal cells is formed, and a monolayer of retinal pigment epithelial (RPE) cells is formed thereon. A method for manufacturing a retinal model is provided, comprising the step of placing the outer blood retinal barrier structure so that the porous membrane contacts the inner blood retinal barrier structure.

[0010] In the method for manufacturing a retinal model according to the present invention, the step of printing the coronary blood vessel structure is preferably performed through a triple-coaxial nozzle comprising a first nozzle positioned at the center of the axis, a second nozzle surrounding the first nozzle, and a third nozzle surrounding the second nozzle, wherein the printing step is performed such that a support ink is ejected through the first nozzle, the first bioink is ejected through the second nozzle, and the second bioink is ejected through the third nozzle.

[0011] In the method for manufacturing a retinal model according to the present invention, the step of printing the coronary blood vessel structure is a step of printing by temporarily increasing the ejection speed of the support ink, the first bioink, and the second bioink, and it is preferable that the retinal model is a retinal vascular occlusion (RVO) disease model.

[0012] In the method for manufacturing a retinal model according to the present invention, the third bioink is preferably a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen to which retinal cells are added.

[0013] In the method for manufacturing a retinal model according to the present invention, the mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen of the third bioink is preferably a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen in a weight ratio of 1:0.5 to 2.

[0014] In the method for manufacturing a retinal model of the present invention, the fourth bioink is preferably a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen.

[0015] In the method for manufacturing a retinal model according to the present invention, the mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen of the fourth bioink is preferably a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen in a weight ratio of 1:0.5 to 2.

[0016] According to another aspect of the present invention, the invention comprises a structure in which an outer blood-retinal barrier structure is disposed upon an inner blood-retinal barrier structure, wherein the inner blood-retinal barrier structure comprises a coronary vessel structure and a matrix structure formed around the coronary vessel structure, wherein the coronary vessel structure is printed such that a first bioink comprising a vascular-derived decellularized extracellular matrix (VdECM) and endothelial cells and a second bioink comprising a vascular-derived decellularized extracellular matrix (VdECM) and perivascular cells are printed such that the first bioink forms an inner layer and the second bioink forms an outer layer, and the matrix structure is printed such that a third bioink comprising a retinal-derived decellularized extracellular matrix (RdECM) and retinal cells is filled around the coronary vessel structure, and the outer blood-retinal barrier structure comprises a porous membrane, an extracellular matrix layer formed upon the porous membrane, and a monolayer of retinal pigment epithelial (RPE) cells formed upon the extracellular matrix layer. A retinal model is provided, wherein the extracellular matrix layer formed on the porous membrane comprises retinal-derived decellularized extracellular matrix (RdECM) and is printed with a fourth bioink that does not contain retinal cells, and the porous membrane of the outer blood-retinal barrier structure is positioned to be in contact with the inner blood-retinal barrier structure.

[0017] In the retinal model of the present invention, the tubular vascular structure is preferably formed in a tubular shape in a part that is thinner than other parts, and the retinal model is preferably a retinal vascular occlusion (RVO) disease model.

[0018] In the retinal model of the present invention, the third bioink is preferably a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen to which retinal cells are added.

[0019] In the retinal model of the present invention, the mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen of the third bioink is preferably a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen in a weight ratio of 1:0.5 to 2.

[0020] In the retinal model of the present invention, the fourth bioink is preferably a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen.

[0021] In the retinal model of the present invention, the mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen of the fourth bioink is preferably a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen in a weight ratio of 1:0.5 to 2.

[0022] According to the present invention, it is possible to manufacture a retinal model that mimics an accurate retinal structure by reproducing the tissue-specific microenvironment of the retina—namely, the medial BRB region and the lateral BRB region, which is the RPE region—very closely to reality. In particular, it is possible to manufacture a retinal model that not only reflects lesion characteristics such as those found in actual RVO very well but also reflects the response to related drugs exactly as they are. In particular, the manufacturing method of the present invention has significant advantages in that it allows for the easy and uniform production of such a retinal model by applying 3D printing technology. The retinal model provided according to the present invention will be of great help in studying the retinas of animals, particularly humans, and in developing treatments or therapeutic agents for retinal-related diseases, particularly RVO.

[0023] FIG. 1 schematically illustrates a method for manufacturing a retinal model according to one embodiment of the present invention. (A), a detailed structural schematic of an on-a-chip type retinal model mimicking the BRB structure of the retina; (B), a schematic diagram of a method for manufacturing an on-a-chip type retinal model; (C), a schematic diagram of (1) a normal model and (2) an RVO model.

[0024] FIG. 2 shows the experimental results of the effects of a bioink containing retinal-derived decellularized extracellular matrix (RdECM) according to one embodiment of the present invention. (A) Heatmap showing the types and content of proteins by category included in porcine RdECM; (B) VE-cadherin and CD31 immunofluorescence staining results on day 7 of endothelial cell culture using the bioink; (C) CCK analysis results on days 1, 7, and 14 of culture; (D) qRT-PCR results for VE-cadherin and CD31 markers on day 7 of culture; laminin, laminin treatment group; collagen, collagen treatment group; hybrid RdECM, RdECM-containing bioink of the present invention (RdECM + type 1 collagen (1:1)) treatment group.

[0025] [Correction pursuant to Rule 91 10.12.2025] FIG. 3 shows the experimental results of the effect of an RdECM-containing bioink according to one embodiment of the present invention. (A) Immunofluorescence staining results of GFAP and Nestin on culture days 1, 7, and 14; (B) qRT-PCR results of Nestin, GFAP, SOX-2, RHO, PDE6B, and CRX on culture days 1, 7, and 14. FIG. 4 is the experimental result of FIG. 3, showing (A) Immunofluorescence staining results of GFAP, Nestin, and RHO; (B) qRT-PCR results of GFAP, Nestin, SOX-2, RHO, PDE6B, and CRX.

[0026] [Correction pursuant to Rule 91 10.12.2025] FIG. 5 shows the results of the operation of a retinal model and the confirmation of RVO pathology according to one embodiment of the present invention. (A) Operation schedule of each retinal model; (B) VE-cadherin and CD68 immunofluorescence staining results of the vascular portion of the medial BRB of each retinal model on day 7 of operation (two photos on the left) and ICAM-1 immunofluorescence staining results on day 9 of operation (two photos on the right); (C) CD68 immunofluorescence staining results for macrophage localization in the RVO model on days 1, 7, and 9 of operation; (D) qRTPCR results for VE-cadherin on day 7 of operation and ICAM-1 markers on day 9 of operation; (E) ELISA results for TNF-alpha and IL-6 on day 7 of operation.

[0027] [Correction pursuant to Rule 91 10.12.2025] FIG. 6 shows the results of confirming the pathology of an RVO model according to one embodiment of the present invention. (A) Immunofluorescence staining results of Rhodopsin and GFAP in the medial BRB portion of each retinal model on day 9 of operation; (B) QRT-PCR results for Rhodopsin and GFAP markers in the medial BRB portion of each retinal model on day 9 of operation; (C) Immunofluorescence staining results of E-cadherin and RPE65 in the RPE portion of each retinal model on day 9 of operation; (D) QRT-PCR results for E-cadherin and RPE65 markers in the RPE portion of each retinal model on day 9 of operation.

[0028] [Correction pursuant to Rule 91 06.05.2026][Correction pursuant to Rule 91 10.12.2025] FIG. 7 is a drug test schedule for four types of experimental models for drug test results for a retinal model according to one embodiment of the present invention. FIG. 8 is the drug test results of FIG. 7, comprising: (A) ELISA results for TNF-alpha, IL-6, and VEGF-A on the 7th day of operation of each experimental model; and (B) qRT-PCR results for CD31, VE-cadherin, and VEGF-A on the 7th day of operation of each experimental model.

[0029] The method for manufacturing a retinal model according to the present invention comprises the steps of: printing a tubular blood vessel structure using a first bioink containing vascular-derived decellularized extracellular matrix (VdECM) and endothelial cells and a second bioink containing vascular-derived decellularized extracellular matrix (VdECM) and perivascular cells, wherein the first bioink forms an inner layer and the second bioink forms an outer layer; printing an inner blood-retinal barrier (BRB) structure around the tubular blood vessel structure such that a third bioink containing retinal-derived decellularized extracellular matrix (RdECM) and retinal cells is filled; and printing an outer blood-retinal barrier (BRB) structure on a porous membrane such that a layer of a fourth bioink containing retinal-derived decellularized extracellular matrix (RdECM) and not containing retinal cells is formed, and a monolayer of retinal pigment epithelial (RPE) cells is formed thereon. and the step of arranging the outer blood retinal barrier structure so that the porous membrane contacts the inner blood retinal barrier structure; characterized by including

[0030] The method for manufacturing a retinal model according to the present invention enables the manufacturing of a retinal model having a structure very similar to an actual retinal structure through a configuration in which an inner BRB portion and an outer BRB portion are separately manufactured and arranged, a configuration in which a coronary blood vessel structure is printed and the surrounding area is filled with bioink to form a structure that mimics the inner BRB, and a configuration in which an appropriate printing material is used for each corresponding tissue.

[0031] In particular, the method for manufacturing a retinal model according to the present invention makes it possible to manufacture a retinal model exhibiting characteristics very similar to RVO by simply controlling the printing speed when printing the coronary blood vessel structure.

[0032] In the present invention, VdECM may be obtained from vascular tissue through a conventional tissue decellularization method. For example, it may be obtained through a VdECM production process as described in Korean Registered Patent No. 10-1974716 (the entire contents of which are incorporated herein by reference). For example, VdECM may be obtained by applying physical, chemical, and / or enzymatic treatments to the vascular tissue of an animal, for example, a pig, such as the aorta, thereby preserving the extracellular matrix of the vascular tissue, such as collagen, GAG, and elastin, while removing the genes. The vascular tissue used in the production of such VdECM may be vascular tissue derived from various animals and may be appropriately selected depending on the subject being mimicked. For example, if the subject being mimicked is the retina of a pig, pig-derived vascular tissue may be used. When the subject to be simulated is the human retina, human-derived vascular tissue, for example, vascular tissue cultured through isolation and tissue culture from humans, may be used; however, if this is difficult, vascular tissue derived from other animals, for example, pig-derived vascular tissue, may be used.

[0033] In the present invention, the RdECM may be obtained from retinal tissue through a conventional tissue decellularization method. For example, the RdECM may be obtained by separating an eyeball from an animal, removing surrounding tissue including muscles attached to the separated retina, removing the cornea and vitreous humor, peeling off the retina from the remaining eyeball tissue, washing the collected retina to remove blood within the retina, treating it with an SDS solution (e.g., 0.1% SDS solution), treating it with a Triton-X / EDTA solution (e.g., 1% PBS solution containing 2% Triton-X / 25mM EDTA), treating it with a DNase solution (e.g., 100 U / mL DNase solution), treating it with peracetic acid (e.g., 0.1% peracetic acid) and an ethanol solution (e.g., 4% ethanol), and then washing and freeze-drying. The retinal tissue used for such RdECM preparation may also be retinal tissue of various animal origin and may be appropriately selected depending on the target being simulated. For example, if the subject to be modeled is a pig's retina, pig-derived retinal tissue can be used. If the subject to be modeled is a human retina, human-derived retinal tissue, such as retinal tissue isolated from and cultured from humans, can be used; however, if this is difficult, retinal tissue derived from other animals, such as pig-derived tissue, can be used.

[0034] In the present invention, the first bioink is characterized by comprising VdECM and endothelial cells. Here, the endothelial cells may be endothelial cells derived from various animals and may be appropriately selected depending on the target being mimicked. For example, if the target being mimicked is a pig retina, pig-derived endothelial cells may be used. If the target being mimicked is a human retina, human-derived endothelial cells, for example, endothelial cells cultured from humans through isolation and tissue culture, such as HUVEC, may be used; however, if this is difficult, endothelial cells derived from other animals, for example, pig-derived endothelial cells, may be used. Furthermore, for the expected effects of the present invention, the concentration of endothelial cells is preferably 5 × 10⁻⁶ based on the total volume of the first bioink. 5 to 10 7 It is cells / mL. For the expected effects of the present invention, more preferably, the concentration of endothelial cells is 7×10 based on the total volume of the first bioink. 5 Up to 5×10 6 It is cells / mL, and more preferably 8×10 based on the total volume of the first bioink. 5 Up to 2×10 6 It is cell / mL. In addition, for the expected effects of the present invention, the concentration of VdECM is preferably 10 to 30 mg / mL based on the total volume of the first bioink. For the expected effects of the present invention, more preferably, the concentration of VdECM is 15 to 25 mg / mL based on the total volume of the first bioink, and more preferably 18 to 22 mg / mL based on the total volume of the first bioink.

[0035] In the present invention, the second bioink is characterized by comprising VdECM and pericytes. Here, the pericytes may be pericytes derived from various animals and may be appropriately selected depending on the target being mimicked. For example, if the target being mimicked is a porcine retina, porcine-derived pericytes may be used. If the target being mimicked is a human retina, human-derived pericytes, for example, pericytes cultured through isolation and tissue culture from humans, may be used; however, if this is difficult, pericytes derived from other animals, for example, porcine-derived pericytes may be used. Furthermore, for the expected effects of the present invention, the concentration of pericytes is preferably 5 × 10⁻⁶ based on the total volume of the second bioink. 4 to 10 6 It is cells / mL. For the expected effects of the present invention, more preferably, the concentration of perivascular cells is 7×10 based on the total volume of the second bioink. 4 Up to 5×10 5 It is cells / mL, and more preferably 8×10 based on the total volume of the second bioink. 4 Up to 2×10 5 It is cell / mL. In addition, for the expected effects of the present invention, the concentration of VdECM is preferably 10 to 30 mg / mL based on the total volume of the second bioink. For the expected effects of the present invention, more preferably, the concentration of VdECM is 15 to 25 mg / mL based on the total volume of the second bioink, and more preferably 18 to 22 mg / mL based on the total volume of the second bioink.

[0036] In the present invention, the third bioink is characterized by comprising RdECM and retinal cells. Here, the retinal cells may be retinal cells derived from various animals and may be appropriately selected depending on the target being mimicked. For example, if the target being mimicked is a pig's retina, pig-derived retinal cells may be used. If the target being mimicked is a human retina, human-derived retinal cells, for example, retinal cells cultured from humans through isolation and tissue culture, for example, retinoblastoma, for example, Y79, may be used; however, if this is difficult, retinal cells derived from other animals, for example, pig-derived retinal cells, may be used. Furthermore, for the expected effects of the present invention, the concentration of retinal cells is preferably 10 based on the total volume of the third bioink. 6 to 10 7 It is cells / mL. For the expected effects of the present invention, more preferably, the concentration of retinal cells is 2×10 based on the total volume of the third bioink. 6 to 9×10 6 It is cells / mL, and more preferably 3×10 based on the total volume of the third bioink. 6 Up to 7×10 6 It is cells / mL. Additionally, for the expected effects of the present invention, the concentration of RdECM is preferably 5 to 30 mg / mL based on the total volume of the third bioink. For the expected effects of the present invention, more preferably, the concentration of RdECM is 5 to 25 mg / mL based on the total volume of the third bioink, more preferably 5 to 20 mg / mL based on the total volume of the third bioink, more preferably 5 to 15 mg / mL based on the total volume of the third bioink, and more preferably 8 to 12 mg / mL based on the total volume of the third bioink.

[0037] In the present invention, the fourth bioink is characterized by not containing retinal cells. Preferably, the fourth bioink does not contain retinal cells or any other living cells. Considering that cells may be unintentionally included due to insufficient decellularization during the RdECM manufacturing process, preferably 10 based on the total volume of the fourth bioink 3 Concentration of cells / mL or less, more preferably 10 2 Concentration of cells / mL or less, more preferably 10 1 It is included at a concentration of less than or equal to cells / mL. Additionally, for the expected effects of the present invention, the concentration of RdECM is preferably 5 to 30 mg / mL based on the total volume of the fourth bioink. For the expected effects of the present invention, more preferably, the concentration of RdECM is 5 to 25 mg / mL based on the total volume of the fourth bioink, more preferably 5 to 20 mg / mL based on the total volume of the fourth bioink, more preferably 5 to 15 mg / mL based on the total volume of the fourth bioink, and more preferably 8 to 12 mg / mL based on the total volume of the fourth bioink.

[0038] In the present invention, the step of printing a coronary blood vessel structure is characterized by using a first bioink and a second bioink to print such that the first bioink forms an inner layer and the second bioink forms an outer layer. Preferably, for the expected effects of the present invention, the step of printing a coronary blood vessel structure of the present invention is performed through a triple-coaxial nozzle comprising a first nozzle positioned at the center of an axis, a second nozzle surrounding the first nozzle, and a third nozzle surrounding the second nozzle, wherein the step involves printing such that a support ink is ejected through the first nozzle, the first bioink is ejected through the second nozzle, and the second bioink is ejected through the third nozzle. At this time, as the support ink, a support ink commonly used for printing in a tubular shape using bioink may be used, and for example, CPF-127 may be used as the support ink.

[0039] In the present invention, the step of printing a coronary blood vessel structure may be a step of printing by temporarily increasing the ejection speed of the support ink, the first bioink, and the second bioink. According to this, an RVO simulation model in which a part of the blood vessel is narrow can be efficiently manufactured. At this time, the temporarily high speed may be 2 to 10 times higher than the existing speed, for example, a speed of 300 to 1500 mm / min when the existing speed is 150 mm / min, or a speed 3 to 9 times higher, for example, a speed of 450 to 1350 mm / min when the existing speed is 150 mm / min, or a speed 4 to 8 times higher, for example, a speed of 600 to 1200 mm / min when the existing speed is 150 mm / min, or a speed 5 to 7 times higher, for example, a speed of 750 to 1050 mm / min when the existing speed is 150 mm / min.

[0040] In the present invention, the third bioink is preferably a mixture of RdECM and type 1 collagen with retinal cells added for the expected effects of the present invention. If composed solely of RdECM, there is a problem with difficulty in gelation, but if type 1 collagen is mixed, thermal gelation becomes possible. To this end, the ratio of RdECM to type 1 collagen is preferably 1:0.5 to 2 by weight, more preferably 1:0.6 to 1.8 by weight, more preferably 1:0.7 to 1.6 by weight, more preferably 1:0.8 to 1.4 by weight, and more preferably 1:0.9 to 1.2 by weight.

[0041] For the same reason, the fourth bioink in the present invention is also preferably a mixture of RdECM and type 1 collagen for the expected effects of the present invention. At this time, the ratio of RdECM to type 1 collagen is preferably 1:0.5 to 2 by weight, more preferably 1:0.6 to 1.8 by weight, more preferably 1:0.7 to 1.6 by weight, more preferably 1:0.8 to 1.4 by weight, and more preferably 1:0.9 to 1.2 by weight.

[0042] In the method for manufacturing a retinal model according to the present invention, each bioink, namely the first bioink, the second bioink, the third bioink, and the fourth bioink, may further include auxiliary components in addition to the corresponding components mentioned above, such as components that help facilitate printing and / or components that help with cell survival and / or maintenance. Additionally, in the method for manufacturing a retinal model according to the present invention, each bioink may consist only of the corresponding components mentioned above.

[0043] The retinal model of the present invention is a structure in which an outer blood-retinal barrier structure is disposed upon an inner blood-retinal barrier structure, wherein the inner blood-retinal barrier structure comprises a tubular vessel structure and a matrix structure formed around the tubular vessel structure, wherein the tubular vessel structure is printed such that a first bioink comprising vascular-derived decellularized extracellular matrix (VdECM) and endothelial cells and a second bioink comprising vascular-derived decellularized extracellular matrix (VdECM) and perivascular cells are printed such that the first bioink forms an inner layer and the second bioink forms an outer layer, and wherein the matrix structure is printed such that a third bioink comprising retinal-derived decellularized extracellular matrix (RdECM) and retinal cells is filled around the tubular vessel structure, and the outer blood-retinal barrier structure comprises a porous membrane, an extracellular matrix layer formed on the porous membrane, and a monolayer of retinal pigment epithelial (RPE) cells formed on the extracellular matrix layer, and wherein the porous membrane The extracellular matrix layer formed above comprises retinal-derived decellularized extracellular matrix (RdECM) and is printed with a fourth bioink that does not contain retinal cells, and is characterized by a structure in which the porous membrane of the outer blood-retinal barrier structure is arranged to be in contact with the inner blood-retinal barrier structure.

[0044] The retinal model of the present invention may be manufactured using the method for manufacturing a retinal model of the present invention.

[0045] Details regarding each component, such as VdECM, RdECM, and their respective bioinks, in the retinal model of the present invention may be the same as those described above in relation to the method for manufacturing the retinal model of the present invention.

[0046] In the present invention, the tubular blood vessel structure may form a tube shape in which a part is thinner than another part. In this case, it can serve as a model mimicking an RVO in which a part of the blood vessel is narrow.

[0047] The present invention will be described in more detail below through examples. These examples are merely illustrative of the present invention, and therefore the scope of the present invention should not be interpreted as being limited by these examples.

[0048] [Example]

[0049] Example 1. Preparation of a retinal model

[0050] An on-a-chip retinal model was manufactured using 3D bioprinting.

[0051] As shown in (A) of Fig. 1, the retina includes an inner blood retina barrier (Inner BRB) portion and an outer blood retina barrier (Outer BRB) portion, which is a retinal pigment epithelium (RPE) portion. Accordingly, the retina model of this embodiment was also fabricated to consist largely of an inner blood retina barrier portion and an outer blood retina barrier portion (represented as the RPE part in Fig. 1).

[0052] First, as shown in (B) of FIG. 1, a first bioink containing human umbilical vein endothelial cells (HUVEC) in a vascular-derived decellularized extracellular matrix (VdECM) bioink (the concentration of endothelial cells is approximately 10 based on the total volume of the first bioink). 6cells / mL), a second bioink containing human pericytes in VdECM bioink (the concentration of human pericytes is approximately 10 based on the total volume of the second bioink). 5 After preparing cells / mL) and CPF-127 as a support ink, a double-layer structure of blood vessels in the inner blood-retinal barrier region (inner: endothelial cells, outer: pericytes) was created using a triple-coaxial printing technique, and a third bioink containing retinal cells (human retinoblastoma (Y79)) was prepared around these vessels using retinal-derived decellularized extracellular matrix (RdECM) bioink (the concentration of retinal cells is approximately 5×10⁶ based on the total volume of the third bioink). 6 The entire inner blood-retinal barrier was formed by filling it with cells / mL).

[0053] The outer blood-retinal barrier portion was fabricated by sequentially stacking a porous membrane, a fourth bioink layer composed of RdECM bioink, and a retinal pigment epithelium (RPE monolayer).

[0054] Finally, an on-a-chip retinal model was completed by fitting the outer blood-retinal barrier portion onto the inner blood-retinal barrier portion.

[0055] Here, the fourth bioink was prepared by mixing type 1 collagen and RdECM in a 1:1 weight ratio (Hybrid RdECM), and the third bioink was prepared by mixing type 1 collagen and RdECM in a 1:1 weight ratio (Hybrid RdECM) and including retinal cells therein.

[0056] In addition, the RdECM bioink used here was prepared as follows:

[0057] 1. Within 2 hours of slaughter, the eyeball is separated from the pig, the surrounding tissue including the muscle attached to the separated retina is removed, and then the area is thoroughly washed.

[0058] 2. After immersing the washed eyeball in PBS solution, cut it in half to remove the cornea and vitreous humor, and carefully peel the retina off the eyeball from the remaining eye tissue using a disposable pipette.

[0059] 3. The collected retina was washed with 1% PBS (99% ultrapure DW, hereinafter omitted) for 24 hours to remove blood from the retina.

[0060] 4. Immerse the washed retina in 1% PBS and then treat with 0.1% SDS solution for 6 hours.

[0061] 5. After washing three times with 1% PBS, the retina was treated with a 1% PBS solution containing 2% Triton-X / 25mM EDTA for 72 hours.

[0062] 6. After washing the retina 6 times with 1% PBS, treat it with 100 U / ml DNase solution for 6 hours.

[0063] 7. After washing 6 times with 1% PBS, sterilize with 0.1% peracetic acid and 4% ethanol for 2 hours.

[0064] 8. Wash the sterile retina three times with 1% PBS, then wash it twice with ultrapure DW.

[0065] 9. Freeze-dry the produced RdECM and store it in powder form at -20℃.

[0066] 10. To prepare a hydrogel-type bioink, dissolve lyophilized RdECM in pepsin-acetic acid (2% RdECM; use 10N acetic acid; use pepsin at 1 / 10 the weight of the lyophilized RdECM powder).

[0067] 11. Finally, to prepare the Hybrid RdECM bioink, a collagen solution of the same volume and concentration (Dalim Tisen, Seoul, Korea) (final collagen concentration was 20 mg / mL based on total volume) was mixed and neutralized with 0.1N NaOH and 10× DMEM to finally prepare the Hybrid RdECM bioink.

[0068] In addition, the VdECM bioink used herein was prepared as follows (see Korean Registered Patent No. 10-1974716, the entire contents of which are incorporated herein by reference):

[0069] 1. Porcine aortic tissue was chopped into pieces approximately 2×2×2 mm in size, and then washed with 0.3% SDS (sodium dodecyl sulfate), 3% Triton, and 25 U / mL DNase to remove cells from the tissue.

[0070] 2. Subsequently, the solution was dissolved in a mixed acidic solution of 0.5 M acetic acid and 0.6 wt% pepsin and freeze-dried to obtain a 60 mg / ml VdECM pre-gel.

[0071] 3. Subsequently, the VdECM pre-gel was neutralized using 10M sodium hydroxide (NaOH) to produce a vascular tissue-specific VdECM bioink (final VdECM concentration was 20 mg / mL based on total volume).

[0072] Example 2. Preparation of a retinal model of retinal vascular occlusion

[0073] As shown in (C) of Fig. 1, the remaining part was manufactured in the same manner as in Example 1, but when creating the retinal blood vessel part, the printing speed was momentarily increased from 150 mm / min (the printing speed for creating a typical retinal blood vessel part) to 900 mm / min to produce a retinal blood vessel occlusion retinal model (RVO model) in which part of the blood vessel structure is narrow.

[0074] When operating the constructed RVO model, THP-1 (Human monocytic cell line) was differentiated into macrophages and then mixed with LDL and flowed into the culture medium to create an inflammatory environment.

[0075] Experimental Example 1. Analysis of RdECM-containing Bioink Characteristics

[0076] We reviewed RdECM bioinks for use in fabricating retinal models.

[0077] First, as a result of performing a proteomic analysis on the RdECM prepared as in Example 1 above, as shown in Figure 2 (A), it was found that the RdECM contains a large amount of proteins related to nervous system development, vision, and angiogenesis.

[0078] Next, we examined the application of this RdECM to bioink, but since pure RdECM had a problem with thermal gelation, we prepared a bioink by mixing type 1 collagen and RdECM in a 1:1 weight ratio (hybrid RdECM) as in Example 1 above to solve this problem.

[0079] When retinal vascular endothelial cells were treated with the bioink of the present invention prepared as described above, immunofluorescence staining and qRT-PCR for VE (vascular endothelial)-cadherin and CD31 were performed and viability tests were conducted in comparison to cases treated with laminin or collagen. As a result, as shown in Figures 2 (B) to (D), it was found that the bioink of the present invention could provide more assistance in the growth and function improvement of retinal vascular endothelial cells compared to other hydrogels such as laminin or collagen.

[0080] [Correction pursuant to Rule 91 10.12.2025] Next, when the bioink of the present invention prepared as above was treated on retinoblastoma (Y79) and immunofluorescence staining and qRT-PCR were performed on markers related to differentiation into mature retinal cells, as shown in (A) to (B) of FIGS. 3 and 4, the bioink of the present invention was found to be able to greatly assist retinal stem cells in differentiating into mature retinal cells with specific functions, such as an increase in GFAP expression and a decrease in Nestin expression upon treatment with the bioink of the present invention, and in particular, it was found to have a superior effect compared to other hydrogels such as laminin or collagen.

[0081] The above results demonstrate that using the bioink utilizing RdECM of the present invention in the manufacture of retinal models can enhance endothelial cell function and help retinal cells perform their proper functions due to various proteins involved in the integrin signaling pathway for maintaining healthy blood vessels. Furthermore, these results suggest that precise model fabrication is possible by using the bioink of the present invention.

[0082] Experimental Example 2. Operation of the manufactured retinal model and confirmation of RVO pathology

[0083] [Correction pursuant to Rule 91 10.12.2025] A functional comparison was conducted between the normal retinal model prepared in Example 1 above and the RVO model prepared in Example 2 above. Each model was operated according to the schedule shown in Fig. 5 (A). On the 7th day of operation, through the two left photos in Fig. 5 (B) and the results in (D), it was confirmed that compared to the normal model, the expression of VE-cadherin, a mature endothelial cell-specific marker, was lower in the RVO model with a narrowed middle section of the blood vessel when normalized relative to the blood vessel area, and the junctions showed an overall broken shape. This suggests that the narrowed shape may affect the endothelial cells themselves. Furthermore, we confirmed that CD68, a macrophage-specific marker, is strongly expressed in the narrowed areas of the blood vessels in the RVO model. This indicates that a large accumulation of macrophages occurs due to deposition caused by vortices in the narrowed areas, a result consistent with the actual characteristics of RVO. In contrast, we confirmed that macrophage accumulation hardly occurs in the normal model. Additionally, we simulated an RVO-specific environment, such as vascular inflammation, by flowing LDL mixed into the culture medium into the RVO model for two days. It is known that when macrophages are exposed to LDL, inflammatory cytokines such as TNF-alpha and IL-6 are produced, and an increase in the concentration of inflammatory cytokines leads to an increase in the expression of ICAM-1 (intercellular adhesion molecules-1), which induces inflammation in blood vessels and macrophage accumulation.Looking at the ELISA results for TNF-alpha and IL-6 in Fig. 5 (E), it can be confirmed that the amounts of TNF-alpha and IL-6 significantly increased in the RVO model compared to the normal model. Consequently, in the two right-hand images of Fig. 5 (B), ICAM-1 was hardly expressed in the normal model where macrophages were hardly accumulated, whereas ICAM-1 was highly expressed in the RVO model where macrophages accumulated in large quantities in the narrowing area compared to the normal model. These results are also consistent with the qRT-PCR results for ICAM-1 in Fig. 5 (D). In other words, the results of Fig. 5 (B) and (D) confirm that the breakdown of vascular tight junctions due to inflammation occurred in the RVO model. Therefore, as shown in the Day 9 image of Fig. 5 (C), it was confirmed that leakage of intravascular substances occurred due to the breakdown of vascular tight junctions and that macrophages migrated out of the blood vessels. Macrophages leaking from such damaged blood vessels continuously generate inflammatory cytokines such as TNF-alpha and IL-6, similar to actual RVO lesions, sequentially affecting the entire internal BRB and the RPE region. In fact, when verifying whether such sequential reactions occur in the model of the present invention, as shown in Figures 6 (A) and (B), it was confirmed that in the RVO model, the expression levels of rhodopsin and GFAP in Y79 cells within the internal BRB region were reduced due to the influence of inflammatory cytokines leaking from blood vessels and macrophages, and it was inferred that light-sensitivity characteristics and the degree of differentiation into mature retinal cells were inhibited. Additionally, as shown in Figures 6 (C) and (D), it was confirmed that the expression levels of normal RPE markers, E-cadherin and RPE65, were reduced in the RVO model, and it was inferred that problems occurred in RPE cell function.Based on these results, it was shown that inflammatory cytokines and macrophages released from damaged blood vessels in the fabricated RVO model sequentially affected the entire internal BRB and the RPE, resulting in the destruction of the entire BRB; therefore, it can be inferred that the fabricated RVO model is a platform capable of accurately reflecting the characteristics of actual RVO lesions.

[0084] Experimental Example 3. Confirmation of the Applicability of the RVO Model as a Drug Testing Platform

[0085] [Correction pursuant to Rule 91 10.12.2025] To confirm the applicability of the RVO model as a drug testing platform, dexamethasone and avastin, which are used in the treatment of RVO, were administered to the RVO model prepared in Example 2 above to confirm drug responsiveness. Dexamethasone is known to play a role in preventing retinal inflammation that causes dysfunction of retinal neurons and RPE cells, and avastin is known to bind to VEGF-A to block the activity of VEGF receptors, and subsequently stop downstream signaling pathways to inhibit abnormal neovascularization and inhibit retinal inflammation. As shown in Fig. 7, the "Normal model" was created by connecting the normal model of Example 1 to a syringe pump and conducting dynamic culture for 7 days; the "Stenotic model" was created using the RVO model of Example 2, which had the morphology of the RVO model but was not treated with macrophages and LDL; the "model" was created by exposing the RVO model of Example 2 to macrophages and LDL for 2 days and then not treating it with any drugs for 5 days; and the "+ drugs model" was created by exposing the RVO model of Example 2 to macrophages and LDL for 2 days and then treating it with 50 ng / ml of dexamethasone and 250 ng / ml of Avastin for 5 days. As a result, as shown in Fig. 8 (A), it was confirmed that all markers were significantly higher in the "model" compared to the "Normal model," thereby confirming that the typical characteristics of RVO were well expressed. In the "+ drugs model," it was confirmed that the concentrations of inflammatory cytokines such as TNF-alpha and IL-6 secreted from macrophages and the concentration of VEGF-A were significantly reduced compared to the "model."In other words, this result implies that an anti-inflammatory response and blockade of the VEGF signaling pathway occurred upon drug treatment, indicating that the drugs are functioning normally within the constructed RVO model. The results in Figure 8 (B) show the results of qRT-PCR performed on each model on day 7; the trend of VEGF-A is the same as that in (A), and regarding the qRT-PCR results for CD31 and VE-cadherin, it was confirmed that they were significantly reduced in the "stenotic model" compared to the "normal model," suggesting that the narrowed shape may affect the endothelial cells themselves. By confirming that the two markers were expressed at lower levels in the "model" compared to the "normal model," it was confirmed that the typical characteristics of RVO are well expressed. In particular, in the "+ drugs model," compared to the "model," it was observed that the expression levels of CD31 and VE-cadherin markers did not increase, while the expression level of VEGF-A was significantly reduced. This result accurately reflects the situation in which, when dexamethasone and Avastin are administered to actual RVO patients, already damaged blood vessels are not restored, and only the development of abnormal neovascularization is suppressed. In other words, it can be inferred that the developed RVO model is suitable as a drug testing platform.

Claims

1. A step of printing a coronary blood vessel structure using a first bioink comprising a blood vessel-derived decellularized extracellular matrix (VdECM) and endothelial cells and a second bioink comprising a blood vessel-derived decellularized extracellular matrix (VdECM) and perivascular cells, wherein the first bioink forms an inner layer and the second bioink forms an outer layer; A step of printing an inner blood-retinal barrier (BRB) structure around the above-mentioned coronary blood vessel structure so that a third bioink containing retinal-derived decellularized extracellular matrix (RdECM) and retinal cells is filled; A step of printing an outer blood-retinal barrier (BRB) structure such that a layer of a fourth bioink containing retinal-derived decellularized extracellular matrix (RdECM) and not containing retinal cells is formed on a porous membrane, and a monolayer of retinal pigment epithelial (RPE) cells is formed thereon; and A step of arranging the outer blood retinal barrier structure so that the porous membrane contacts the inner blood retinal barrier structure; A method for manufacturing a retinal model including 2. In Paragraph 1, A method for manufacturing a retinal model, wherein the step of printing the above-described coronary blood vessel structure is performed through a triple-coaxial nozzle comprising a first nozzle positioned at the center of the axis, a second nozzle surrounding the first nozzle, and a third nozzle surrounding the second nozzle, wherein a support ink is ejected through the first nozzle, the first bioink is ejected through the second nozzle, and the second bioink is ejected through the third nozzle.

3. In Paragraph 2, A method for manufacturing a retinal model, wherein the step of printing the above-mentioned coronary blood vessel structure is a step of printing by temporarily increasing the ejection speed of the above-mentioned support ink, the above-mentioned first bioink, and the above-mentioned second bioink, and the retinal model is a retinal vascular occlusion (RVO) disease model.

4. In Paragraph 1, A method for manufacturing a retinal model, wherein the third bioink is a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen to which retinal cells are added.

5. In Paragraph 4, A method for preparing a retinal model, wherein the mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen is a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen in a weight ratio of 1:0.5 to 2.

6. In Paragraph 1, A method for preparing a retinal model, wherein the above-mentioned fourth bioink is a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen.

7. In Paragraph 6, A method for preparing a retinal model, wherein the mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen is a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen in a weight ratio of 1:0.5 to 2.

8. A structure in which an outer blood-retinal barrier structure is disposed on an inner blood-retinal barrier structure, The inner blood-retinal barrier structure is composed of a coronary blood vessel structure and a matrix structure formed around the coronary blood vessel structure, and The above-described coronary blood vessel structure is printed such that a first bioink comprising a vascular-derived decellularized extracellular matrix (VdECM) and endothelial cells and a second bioink comprising a vascular-derived decellularized extracellular matrix (VdECM) and perivascular cells are printed such that the first bioink forms an inner layer and the second bioink forms an outer layer. The above matrix structure is printed such that a third bioink comprising retinal-derived decellularized extracellular matrix (RdECM) and retinal cells is filled around the above coronary blood vessel structure, and The above-mentioned outer blood-retinal barrier structure comprises a porous membrane, an extracellular matrix layer formed on the porous membrane, and a monolayer of retinal pigment epithelial (RPE) cells formed on the extracellular matrix layer, and The extracellular matrix layer formed on the above porous membrane comprises retinal-derived decellularized extracellular matrix (RdECM) and is printed with a fourth bioink that does not contain retinal cells, and A structure in which the porous membrane of the outer blood-retinal barrier structure is positioned to be in contact with the inner blood-retinal barrier structure, Retina model.

9. In Paragraph 8, The above-mentioned coronary vascular structure forms a tubular shape in which a part is thinner than another part, and the above-mentioned retinal model is a retinal vascular occlusion (RVO) disease model.

10. In Paragraph 8, The above-mentioned third bioink is a retinal model in which retinal cells are added to a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen.

11. In Paragraph 10, A retinal model, wherein the mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen is a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen in a weight ratio of 1:0.5 to 2.

12. In Paragraph 8, The above-mentioned fourth bioink is a retinal model that is a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen.

13. In Paragraph 12, A retinal model, wherein the mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen is a mixture of retinal-derived decellularized extracellular matrix (RdECM) and type 1 collagen in a weight ratio of 1:0.5 to 2.

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