Multi-organ model for diabetes

Abstract

A multi-organ model for type 2 diabetes according to the present invention comprises: a pancreatic model unit including pancreatic beta cells and a pancreatic tissue-derived decellularized extracellular matrix (PdECM); an adipose tissue model unit including adipocytes, macrophages, and a visceral adipose tissue-derived decellularized extracellular matrix (VadECM); and a liver model unit including hepatocytes and a liver tissue-derived decellularized extracellular matrix (LdECM).

Description

Multi-organ model for diabetes implementation

[0001] The present invention relates to a multi-organ model for implementing diabetes, and specifically, the present invention relates to a model capable of implementing diabetes, particularly Type 2 diabetes (T2D), which can implement characteristics of diabetes such as hyperglycemia and inflammation almost identically to reality, and also to a model capable of reproducing complex biological crosstalk between multiple organs, and a method for manufacturing the same.

[0002] Diabetes is a metabolic disease that affects multiple organs and causes various complications. Diabetic retinopathy (DR), one of the complications of diabetes, is a major cause of blindness and is a common disease among diabetic patients. Type 2 diabetes (T2D) is directly linked to obesity, and along with the rapid increase in the obese population, the number of T2D patients is also increasing significantly every year. However, the pathological mechanisms and causes of T2D are not precisely known, and there are currently no treatments available to completely cure T2D. Therefore, to study this, an in vitro model that accurately mimics the pathological mechanisms of T2D is required. To achieve this, it is necessary to simultaneously implement hyperglycemia and inflammation within the model and to create organ compartments that mimic the functions of organs directly associated with T2D, such as the pancreas, liver, fat, and muscle. The pancreas is directly linked to insulin secretion capable of regulating blood sugar, the liver is directly linked to blood sugar regulation through gluconeogenesis, and muscles are directly linked to regulating blood sugar by directly consuming glucose as an energy source. Therefore, to address this, Single Organ-on-a-Chips and Disease-on-a-Chips containing organ structures similar to actual organs have been developed. Most existing research on T2D-mimicking Disease-on-a-Chips has focused on single-organ mimicry, such as pancreas mimicry to simulate insulin secretion or fat mimicry to induce inflammatory responses. However, since these in vitro models of single organs mimic T2D pathological mechanisms to a limited extent, they are limited in fully replicating the micropathological environment of T2D caused by crosstalk among multiple organs. Furthermore, there are no models that simultaneously implement hyperglycemia and inflammation.

[0003] Accordingly, the inventors sought to solve these problems by developing a multi-organ model capable of demonstrating crosstalk between multiple biological organs in T2D pathophysiology, including hyperglycemia and inflammation.

[0004] Therefore, the main objective of the present invention is to provide a model capable of implementing diabetes mellitus, particularly T2D, which can implement the characteristics of diabetes mellitus, such as hyperglycemia and inflammation, almost identically to reality, and also reproduce complex biological crosstalk between various organs.

[0005] Another objective of the present invention is to provide a method for manufacturing a diabetes model as described above.

[0006] According to one aspect of the present invention, the present invention provides a multi-organ model for implementing type 2 diabetes (T2D), comprising: a pancreas model part comprising pancreatic beta cells and pancreas-derived decellularized extracellular matrix (PdECM); an adipose tissue model part comprising adipocytes, macrophages, and visceral adipose tissue-derived decellularized extracellular matrix (vadECM); and a liver model part comprising hepatocytes and liver-derived decellularized extracellular matrix (LdECM).

[0007] In the multi-organ model for T2D implementation of the present invention, it is preferable that each model part be compartmentalized so as not to come into direct contact.

[0008] In the multi-organ model for T2D implementation of the present invention, it is preferable that each model part is arranged in a row on a base plate in the order of a pancreas model part, an adipose tissue model part, and a liver model part.

[0009] In the multi-organ model for T2D implementation of the present invention, it is preferable that each model part additionally be provided with an endothelial cell layer.

[0010] In the multi-organ model for T2D implementation of the present invention, it is preferable to further include a retinal model part including retinal cells.

[0011] In the multi-organ model for T2D implementation of the present invention, it is preferable that each model part is arranged in a row on a base plate in the order of a pancreas model part, an adipose tissue model part, a liver model part, and a retina model part.

[0012] In the multi-organ model for T2D implementation of the present invention, it is preferable to further include muscle model parts, wherein each model part is connected to each other by a coronary blood vessel structure.

[0013] In the multi-organ model for T2D implementation of the present invention, it is preferable to further include one or more model parts selected from the retinal model part, the kidney model part, and the neural model part.

[0014] According to another aspect of the present invention, the present invention provides a method for manufacturing a multi-organ model for T2D implementation, comprising the steps of: preparing a first bioink comprising pancreatic beta cells and pancreatic-derived decellularized extracellular matrix (PdECM); preparing a second bioink comprising adipocytes, macrophages, and visceral adipose tissue-derived decellularized extracellular matrix (vadECM); preparing a third bioink comprising hepatocytes and liver-derived decellularized extracellular matrix (LdECM); printing a pancreas-mimicking structure on a base plate using the first bioink; printing an adipose tissue-mimicking structure on a base plate using the second bioink; and printing a liver-mimicking structure on a base plate using the third bioink.

[0015] In the method for manufacturing a multi-organ model for T2D implementation according to the present invention, it is preferable to compartmentalize each simulated structure so as not to come into direct contact and print it on a base plate.

[0016] In the method for manufacturing a multi-organ model for T2D implementation according to the present invention, it is preferable to print each simulated structure in a row on a base plate in the order of a pancreas simulated structure, an adipose tissue simulated structure, and a liver simulated structure.

[0017] In the method for manufacturing a multi-organ model for T2D implementation according to the present invention, it is preferable to further include the step of printing an endothelial cell layer on each simulated structure.

[0018] In the method for manufacturing a multi-organ model for T2D implementation according to the present invention, it is preferable to further include the step of arranging a retinal-mimicking structure comprising retinal cells.

[0019] In the method for manufacturing a multi-organ model for T2D implementation according to the present invention, it is preferable to arrange each mimic structure in a row on a base plate in the order of a pancreas mimic structure, an adipose tissue mimic structure, a liver mimic structure, and a retina mimic structure.

[0020] In the method for manufacturing a multi-organ model for T2D implementation according to the present invention, it is preferable to further include the step of arranging a muscle-mimicking structure including muscle cells, and further include the step of printing a coronary blood vessel structure connecting each of the mimicking structures.

[0021] In the method for manufacturing a multi-organ model for T2D implementation according to the present invention, it is preferable to further include the step of arranging one or more simulation structures selected from a retinal simulation structure, an elongation simulation structure, and a neuro-simulation structure.

[0022] According to the present invention, a model is provided that can reproduce characteristics of diabetes, such as hyperglycemia and inflammation, almost identically to reality, and in particular, can reproduce complex biological crosstalk between various organs to study correlations between organs, and can also accurately represent responses to related drugs, thereby serving as a platform for developing new drugs. The manufacturing method of the present invention has significant advantages not only in that it can produce such a model, but also in that it can easily produce such a model with uniform quality by applying 3D printing technology. The diabetes model of the present invention will be of great help in research to understand the pathophysiology of diabetes, particularly T2D, and in the development of related therapeutic agents.

[0023] Figures 1 and 2 show a schematic diagram of the fabrication of a multi-organ model for implementing diabetes mellitus (T2D) in the form of an on-a-chip according to one embodiment of the present invention.

[0024] FIGS. 3 to 6 show the results of comparative experiments on the effects of a T2D model according to an embodiment of the present invention. (Fig. 3) Effect of multiple organ interaction; (Fig. 4) Effect according to the arrangement order of each organ model part; (Fig. 5) Effect according to whether compartmentalization is performed; (Fig. 6) Effect according to the method of applying endothelial cells.

[0025] FIGS. 7 to 9 show the experimental results regarding the pathological characteristics of a T2D model according to an embodiment of the present invention. (Fig. 7 (A)) T2D model operation schedule; (Fig. 7 (B)) Insulin levels according to T2D model operation time; (Fig. 7 (C)) Glucose levels according to T2D model operation time; (Fig. 8 (A)) Analysis results of cell survival / apoptosis of the pancreas, visceral adipose tissue, and liver on days 1 and 2 of T2D model operation; (Fig. 8 (B)) VE-cadherin and CD31 immunofluorescence staining results of endothelial cell monolayers at 0, 12, and 24 hours; (Fig. 9) Quantitative measurement results of T2D-related cytokines.

[0026] FIGS. 10 to 13 illustrate experimental results related to T2D complications of a T2D model according to an embodiment of the present invention. (Fig. 10 (A)) Process for constructing a T2D complication model; (Fig. 10 (B)) Operating schedule of the T2D complication model; (Fig. 10 (C)) Schematic diagram of three models for comparison; (Fig. 11) Image of an actual T2D complication model; (Fig. 12) Immunofluorescence staining results to confirm hRPE tight junction breakdown and dysfunction; (Fig. 13 top) hRPE tight junction breakdown confirmed by TEER measurement; (Fig. 13 middle and bottom) Gene expression levels of ZO-1, occludin, E-cadherin, and RPE65 confirmed by qRT-PCR.

[0027] FIGS. 14 to 18 show the results of drug application experiments on a T2D model according to an embodiment of the present invention. (Fig. 14) Drug test schedule for three models for comparison; (left side of FIG. 15) Insulin levels according to the T2D model operation time; (right side of FIG. 15) Glucose levels according to the T2D model operation time; (Fig. 16 (A)) Immunofluorescence staining results of the pancreas model part on day 5 of T2D model operation; (Fig. 16 (B)) Immunofluorescence staining results of the liver model part on day 5 of T2D model operation; (Fig. 17) Confirmation results of TNF-alpha, IL-6, and albumin using ELISA on day 5 of T2D model operation; (Fig. 18) Expression levels of albumin, insulin, E-cadherin, and INSR confirmed by qRT-PCR.

[0028] The multi-organ model for implementing T2D according to the present invention is characterized by comprising: a pancreas model part including pancreatic beta cells and pancreas-derived decellularized extracellular matrix (PdECM); an adipose tissue model part including adipocytes, macrophages, and visceral adipose tissue-derived decellularized extracellular matrix (vadECM); and a liver model part including hepatocytes and liver-derived decellularized extracellular matrix (LdECM).

[0029] The T2D model of the present invention can not only realize pathological characteristics nearly identical to actual T2D through configurations such as applying model parts corresponding to the pancreas, adipose tissue, and liver, appropriately arranging them, and applying appropriate components to each model part, but can also reproduce complex biological crosstalk between various organs.

[0030] In the present invention, the PdECM may be obtained from pancreatic tissue through a conventional tissue decellularization method. For example, it may be obtained through a PdECM production process as described in Korean Registered Patent No. 10-2672136 (the entire contents of which are incorporated herein by reference). For example, the PdECM may be obtained by applying physical, chemical, and / or enzymatic treatments to the pancreatic tissue of an animal, e.g., a pig, so that the extracellular matrix of the pancreatic tissue is preserved and the genes are removed. The pancreatic tissue used for such PdECM production may be pancreatic tissue 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 pancreas, pancreatic tissue derived from pigs may be used. If the target being mimicked is a human pancreas, pancreatic tissue derived from humans, e.g., pancreatic tissue cultured through isolation and tissue culture from humans, may be used; however, if this is difficult, pancreatic tissue derived from other animals, e.g., pigs, may be used.

[0031] In the present invention, vadECM may be obtained from visceral adipose tissue through a conventional tissue decellularization method. For example, it may be obtained by separating and collecting white visceral fat attached to the pancreas, liver, and / or intestines of a pig, chopping it finely, and then freeze-drying it following isopropanol treatment, SDS treatment, and Triton X-100 treatment. The visceral adipose tissue used for the preparation of vadECM may be visceral adipose tissue derived from various animals and may be appropriately selected depending on the target being mimicked. For example, if the target being mimicked is pig visceral fat, pig-derived visceral adipose tissue may be used. If the target being mimicked is human visceral fat, human-derived visceral adipose tissue, for example, visceral adipose tissue isolated from a human and cultured through tissue culture, may be used; however, if this is difficult, visceral adipose tissue derived from other animals, for example, pig-derived visceral adipose tissue, may be used.

[0032] In the present invention, LdECM may be obtained from liver tissue through a conventional tissue decellularization method. For example, it may be obtained through an LdECM production process as described in Korean Patent Publication No. 10-2019-0096180 (the entire contents of which are incorporated herein by reference). For example, LdECM may be obtained by applying physical, chemical, and / or enzymatic treatments to liver tissue of an animal, e.g., a pig, so that the extracellular matrix of the liver tissue is preserved and genetic factors are removed. The liver tissue used for such LdECM production may be liver tissue 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 liver, pig-derived liver tissue may be used. If the target being mimicked is a human liver, human-derived liver tissue, e.g., liver tissue isolated from a human and cultured through tissue culture, may be used; however, if this is difficult, liver tissue derived from another animal, e.g., pig-derived, may be used.

[0033] In the present invention, the pancreas model part is characterized by comprising pancreatic beta cells and PdECM. Here, the pancreatic beta cells may be pancreatic beta 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 pancreas, pig-derived pancreatic beta cells may be used. If the target being mimicked is a human pancreas, human-derived pancreatic beta cells, for example, pancreatic beta cells cultured through isolation and tissue culture from humans, may be used; however, if this is difficult, pancreatic beta cells derived from other animals, for example, pig-derived pancreatic beta cells, may be used. Furthermore, for the expected effects of the present invention, the concentration of pancreatic beta cells is preferably 5 x 10⁶ based on the total volume of the pancreas model part. 5 Up to 5x10 6It is cells / mL. For the expected effects of the present invention, more preferably, the concentration of pancreatic beta cells is 7 x 10 based on the total volume of the pancreatic model part. 5 Up to 4x10 6 It is cells / mL, and more preferably 8x10 based on the total volume of the pancreatic model part. 5 Up to 2x10 6 Cells / mL (e.g., 10 6 It is cells / ml).

[0034] In the present invention, the adipose tissue model part is characterized by comprising adipocytes, macrophages, and vadECM. Here, the adipocytes and macrophages may be cells derived from various animals and may be appropriately selected depending on the target being simulated. For example, if the target being simulated is porcine adipose tissue, porcine-derived adipocytes and macrophages may be used. If the target being simulated is human adipose tissue, human-derived adipocytes and macrophages, for example, adipocytes and macrophages cultured from humans through isolation and tissue culture, may be used; however, if this is difficult, adipocytes and macrophages derived from other animals, for example, porcine-derived adipocytes and macrophages may be used. Furthermore, for the expected effects of the present invention, the concentrations of adipocytes and macrophages are preferably 5 x 10⁶, respectively, based on the total volume of the adipose tissue model part. 5 Up to 5x10 6 It is cells / mL. For the expected effects of the present invention, more preferably, the concentrations of adipocytes and macrophages are each separately 7x10 based on the total volume of the adipose tissue model part. 5 Up to 4x10 6 It is cells / mL, and more preferably 8x10 based on the total volume of the adipose tissue model part. 5 Up to 2x10 6 Cells / mL (e.g., 10 6 It is cells / ml).

[0035] In the present invention, the liver model part is characterized by comprising hepatocytes and LdECM. Here, the hepatocytes may be hepatocytes 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 liver, pig-derived hepatocytes may be used. If the target being mimicked is a human liver, human-derived hepatocytes, such as hepatocytes isolated from humans and cultured through tissue culture, such as HepG2, may be used; however, if this is difficult, hepatocytes derived from other animals, such as pig-derived hepatocytes, may be used. Furthermore, for the expected effects of the present invention, the concentration of hepatocytes is preferably 5 x 10⁶ based on the total volume of the liver model part. 5 Up to 5x10 6 It is cells / mL. For the expected effects of the present invention, more preferably, the concentration of hepatocytes is 7 x 10 based on the total volume of the liver model part. 5 Up to 4x10 6 It is cells / mL, and more preferably 8x10 based on the total volume of the liver model part. 5 Up to 2x10 6 Cells / mL (e.g., 10 6 It is cells / ml).

[0036] Preferably, for the expected effects of the present invention, each model part of the present invention is compartmentalized so as not to come into direct contact. Such compartmentalization can be achieved by forming a compartmentalized frame corresponding to each model part using, for example, a polymer, for example, a biocompatible polymer, and placing each model part within this frame.

[0037] In addition, for the expected effects of the present invention, each model part is preferably arranged in a line on a base plate in the order of pancreas model part, adipose tissue model part, and liver model part. This order is based on the direction in which the culture medium is flowed when operating the model. For example, when operating a T2D model, if a method is used where culture medium is introduced at point "A" and used culture medium is removed at point "B", to arrange them in the order above, the pancreas model part, adipose tissue model part, and liver model part are arranged in order from point "A" toward point "B".

[0038] In addition, for the expected effects of the present invention, each model part is preferably additionally provided with an endothelial cell layer. This can be achieved, for example, by forming each model part and then printing endothelial cells thereon. For the expected effects of the present invention, the endothelial cell layer is preferably a monolayer.

[0039] In addition, the multi-organ model for implementing T2D according to the present invention preferably further includes a retinal model part comprising retinal cells for the expected effects of the present invention. The retinal model part can be achieved, for example, by placing a container loaded with retinal cells, for example, retinal pigment epithelial cells.

[0040] In the case where a retinal model part is included, for the expected effects of the present invention, each model part is preferably arranged in a line on a base plate in the order of pancreas model part, adipose tissue model part, liver model part, and retinal model part. The order therein is based on the direction in which the culture medium is flowed when operating the model, as mentioned above.

[0041] In addition, the multi-organ model for T2D implementation of the present invention preferably further includes a muscle model part for the expected effects of the present invention, and each model part is connected to each other by a coronary blood vessel structure.

[0042] A coronary blood vessel structure can be formed by a method of printing a coronary blood vessel structure using an inner layer bioink containing a vascular-derived decellularized extracellular matrix (VdECM) and endothelial cells, and an outer layer bioink containing a vascular-derived decellularized extracellular matrix (VdECM) and perivascular cells, such that the inner layer bioink forms the inner layer and the outer layer bioink forms the outer layer.

[0043] A coronary blood vessel structure can be formed, more specifically, by printing using 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, and by a printing method in which a support ink is ejected through the first nozzle, an inner bioink is ejected through the second nozzle, and an outer bioink is ejected through the third nozzle. At this time, as the support ink, a support ink commonly used for printing in a tubular form using bioink can be used, for example, CPF-127 can be used as the support ink.

[0044] In addition, the multi-organ model for implementing T2D of the present invention preferably further includes one or more model parts selected from the retinal model part, the kidney model part, and the neural model part for the expected effects of the present invention. These model parts may be connected to each other by a coronary vascular structure together with the pancreas model part, the adipose tissue model part, the liver model part, and the muscle model part.

[0045] In addition, the multi-organ model for T2D implementation of the present invention may be a model for diabetic retinopathy and / or diabetic nephropathy.

[0046] In addition, the multi-organ model for implementing T2D of the present invention may include the implementation of a hyperglycemic environment and / or a hypoxic environment. In this case, the hyperglycemic environment can be implemented by directly flowing a medium containing glucose at a high concentration into each model part or by flowing it through a coronary blood vessel structure, and the hypoxic environment can be implemented by using cobalt chloride.

[0047] The method for manufacturing a multi-organ model for T2D implementation according to the present invention comprises the steps of: preparing a first bioink comprising pancreatic beta cells and pancreatic-derived decellularized extracellular matrix (PdECM); preparing a second bioink comprising adipocytes, macrophages, and visceral adipose tissue-derived decellularized extracellular matrix (vadECM); preparing a third bioink comprising hepatocytes and liver-derived decellularized extracellular matrix (LdECM); printing a pancreas-mimicking structure on a base plate using the first bioink; printing an adipose tissue-mimicking structure on the base plate using the second bioink; and printing a liver-mimicking structure on the base plate using the third bioink. This may be a method for manufacturing a T2D model according to the present invention.

[0048] In the method for manufacturing a T2D model of the present invention, matters concerning each component such as PdECM, vadECM, and LdECM may be the same as those described above in relation to the T2D model of the present invention.

[0049] In the present invention, the first bioink is characterized by comprising pancreatic beta cells and PdECM. Here, the pancreatic beta cells may be pancreatic beta 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 pancreas, pig-derived pancreatic beta cells may be used. If the target being mimicked is a human pancreas, human-derived pancreatic beta cells, for example, pancreatic beta cells cultured from humans through isolation and tissue culture, may be used; however, if this is difficult, pancreatic beta cells derived from other animals, for example, pig-derived pancreatic beta cells, may be used. Furthermore, for the expected effects of the present invention, the concentration of pancreatic beta cells is preferably 5 x 10⁻¹⁰ based on the total volume of the first bioink. 5 Up to 5x10 6 It is cells / mL. For the expected effects of the present invention, more preferably, the concentration of pancreatic beta cells is 7 x 10 based on the total volume of the first bioink. 5 Up to 4x10 6 It is cells / mL, and more preferably 8x10 based on the total volume of the first bioink. 5 Up to 2x10 6 Cells / mL (e.g., 10 6 It is cells / ml).

[0050] In the present invention, the second bioink is characterized by comprising adipocytes, macrophages, and vadECM. Here, the adipocytes and macrophages may be cells of various animal origin and may be appropriately selected depending on the target being mimicked. For example, if the target being mimicked is porcine adipose tissue, porcine-derived adipocytes and macrophages may be used. If the target being mimicked is human adipose tissue, human-derived adipocytes and macrophages, for example, adipocytes and macrophages cultured from humans through isolation and tissue culture, may be used; however, if this is difficult, adipocytes and macrophages of other animal origin, for example, porcine-derived adipocytes and macrophages may be used. Furthermore, for the expected effects of the present invention, the concentrations of the adipocytes and macrophages are preferably 5 x 10⁻¹⁰, respectively, based on the total volume of the second bioink. 5 Up to 5x10 6 It is cells / mL. For the expected effects of the present invention, more preferably, the concentrations of adipocytes and macrophages are each separately 7x10 based on the total volume of the second bioink. 5 Up to 4x10 6 It is cells / mL, and more preferably 8x10 based on the total volume of the second bioink. 5 Up to 2x10 6 Cells / mL (e.g., 10 6 It is cells / ml).

[0051] In the present invention, the third bioink is characterized by comprising hepatocytes and LdECM. Here, the hepatocytes may be hepatocytes of various animal origin and may be appropriately selected depending on the target being mimicked. For example, if the target being mimicked is a pig's liver, pig-derived hepatocytes may be used. If the target being mimicked is a human liver, human-derived hepatocytes, for example, hepatocytes isolated from humans and cultured through tissue culture, such as HepG2, may be used; however, if this is difficult, hepatocytes of other animal origin, for example, pig-derived hepatocytes, may be used. Furthermore, for the expected effects of the present invention, the concentration of hepatocytes is preferably 5 x 10⁻⁶ based on the total volume of the third bioink. 5 Up to 5x10 6 It is cells / mL. For the expected effects of the present invention, more preferably, the concentration of hepatocytes is 7 x 10 based on the total volume of the third bioink. 5 Up to 4x10 6 It is cells / mL, and more preferably 8x10 based on the total volume of the third bioink. 5 Up to 2x10 6 Cells / mL (e.g., 10 6 It is cells / ml).

[0052] In the present invention, the base plate may be made of a polymer, for example, a biocompatible polymer.

[0053] In the present invention, printing of each organ-mimicking structure, namely the pancreas-mimicking structure, the adipose tissue-mimicking structure, and the liver-mimicking structure, can be achieved by using the bioink of the present invention in a conventional printing method that prints structures using bioink. For example, a 3D printing technique may be used.

[0054] Preferably, for the expected effects of the present invention, each model structure is compartmentalized so as not to come into direct contact and printed on a base plate. This can be achieved by, for example, using a polymer, for example, a biocompatible polymer, to form a compartmentalized frame corresponding to each model part, and then printing the corresponding structure within this frame.

[0055] In addition, for the expected effects of the present invention, each mimic structure is preferably printed in a row on a base plate in the order of a pancreas mimic structure, an adipose tissue mimic structure, and a liver mimic structure. The order at this time is based on the direction in which the culture medium is flowed when operating the model, as mentioned above.

[0056] In addition, for the expected effects of the present invention, the method preferably further includes the step of printing an endothelial cell layer on each mimic structure. This can be achieved, for example, by printing each structure and then printing endothelial cells thereon. For the expected effects of the present invention, the endothelial cell layer is preferably a single layer.

[0057] In addition, the method for manufacturing a multi-organ model for T2D implementation according to the present invention preferably further includes the step of placing a retinal-mimicking structure containing retinal cells to achieve the expected effects of the present invention. This can be achieved, for example, by additionally placing a container loaded with retinal cells, for example, retinal pigment epithelial cells.

[0058] When additionally arranging retinal mimic structures, for the expected effects of the present invention, each mimic structure is preferably arranged in a line on a base plate in the order of pancreas mimic structure, adipose tissue mimic structure, liver mimic structure, and retina mimic structure. The order at this time is based on the direction in which the culture medium is flowed when operating the model, as mentioned above.

[0059] In addition, the method for manufacturing a multi-organ model for T2D implementation of the present invention preferably further includes the step of arranging a muscle-mimicking structure containing muscle cells for the expected effect of the present invention, and further includes the step of printing a coronary blood vessel structure connecting each mimic structure.

[0060] The step of printing a coronary blood vessel structure may be a step of printing a coronary blood vessel structure using an inner layer bioink containing a vascular-derived decellularized extracellular matrix (VdECM) and endothelial cells and an outer layer bioink containing a vascular-derived decellularized extracellular matrix (VdECM) and perivascular cells, such that the inner layer bioink forms an inner layer and the outer layer bioink forms an outer layer.

[0061] The step of printing a coronary blood vessel structure may, more specifically, be a step of printing 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 a support ink is ejected through the first nozzle, the inner layer bioink is ejected through the second nozzle, and the outer layer bioink is ejected through the third nozzle. At this time, as the support ink, a support ink commonly used for printing in the form of a tube using bioink may be used, for example, CPF-127 may be used as the support ink.

[0062] In addition, the method for manufacturing a multi-organ model for T2D implementation according to the present invention preferably further comprises the step of arranging one or more mimic structures selected from a retinal mimic structure, a kidney cap structure, and a neuromimicking structure for the expected effects of the present invention.

[0063] 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.

[0064]

[0065] [Example]

[0066] Example 1. Manufacture of a multi-organ model for implementing type 2 diabetes

[0067] To simulate type 2 diabetes (T2D) in an in vitro model, the following on-a-chip multi-organ model was fabricated using 3D bioprinting.

[0068] It consists of a pancreas model section, an adipose tissue model section, and a liver model section. The pancreas model section is composed of pancreatic-derived decellularized extracellular matrix (PdECM) encapsulated with pancreatic beta cells; the adipose tissue model section is composed of visceral adipose tissue-derived decellularized extracellular matrix (vadECM) encapsulated with adipocytes and macrophages; and the liver model section is composed of liver-derived decellularized extracellular matrix (LdECM) encapsulated with the human liver cancer cell line HepG2. The size of each model section compartment was manufactured to match the size ratio of actual human organs. Additionally, it was manufactured so that Human Umbilical Vein Endothelial Cells (HUVECs) were disposed as a monolayer on all model section compartments.

[0069] Specifically, first, a first bioink containing pancreatic beta cells in PCL (Polycaprolactone) bioink and PdECM, a second bioink containing adipocytes and macrophages in PdECM, and a third bioink containing HepG2 in LdECM were prepared, and then, as shown in FIG. 2, frames for a pancreas model part, an adipose tissue model part, and a liver model part were printed using PCL bioink on a base plate (a 150 pi culture dish made of polystyrene) so that each was compartmentalized, and then a pancreas mimic structure was printed using the first bioink on the pancreas model part frame, then an adipose tissue mimic structure was printed using the second bioink on the adipose tissue model part frame, and then a liver mimic structure was printed using the third bioink on the liver model part frame.

[0070] Next, an endothelial cell layer was created by printing HUVEC as a monolayer on top of it, and then covered and stabilized with a PDMS (polydimethylsiloxane) cover to produce a T2D model.

[0071] The PdECM used herein was manufactured as follows (see Korean Registered Patent No. 10-2672136, the entire contents of which are incorporated herein by reference):

[0072] Porcine pancreases were decellularized, freeze-dried, and lysed. The pancreases were sliced, treated with reagents (1% Triton X-100, isopropyl alcohol, and 0.1% peracetic acid in 4% ethanol), and washed with 1X phosphate-buffered saline (PBS). The decellularized tissue was freeze-dried at -50°C, then lysed with 10 w / w% pepsin (P7215; Sigma-Aldrich, St. Louis, MO, USA) in 0.5 M acetic acid for 96 hours, and the pH was adjusted to 7. Solubilized PdECM bioink (20 mg / ml) was mixed with the cell suspension at a final concentration of 1 w / v%.

[0073] In addition, the vadECM used here was prepared as follows:

[0074] 1. After receiving pig pancreases, livers, intestines, etc. from a slaughterhouse, the white visceral fat attached to each organ is separated, gathered into one mass, and then cut into small pieces.

[0075] 2. Future processing steps will be carried out at a cool temperature of 18℃ to prevent spoilage.

[0076] 3. Finely chopped visceral fat was washed with 1X PBS for 2 hours, then washed three times for 30 minutes each in ultrapure DW.

[0077] 4. Treated in 100% isopropanol for 3 days.

[0078] 5. Wash twice for 30 minutes each in ultrapure DW.

[0079] 6. Treated for 3 days using 0.5% SDS detergent.

[0080] 7. Wash twice for 30 minutes each in ultrapure DW.

[0081] 8. Treated with 1% Triton X-100 for 2 days until the visceral fat tissue turned completely white.

[0082] 9. Wash 3 times for 30 minutes each in ultrapure DW.

[0083] 10. The prepared vadECM is freeze-dried and stored in powder form at -20℃.

[0084] In addition, the LdECM used here was prepared as follows:

[0085] 1. Receive pig liver from a slaughterhouse and cut the received liver into pieces approximately 0.5 mm thick.

[0086] 2. Wash with ultrapure DW for 2 hours.

[0087] 3. 2% Triton X-100 was placed in 1M NaCl and treated for 36 hours.

[0088] 4. Wash with ultrapure DW for 24 hours.

[0089] 5. Sterilize with 0.1% peracetic acid in PBS for 1 hour.

[0090] 6. Wash with ultrapure DW for 2 hours.

[0091] 7. The produced decellularized liver dECM (LdECM) is freeze-dried and stored in powder form at -20℃.

[0092]

[0093] Comparative Examples 1 to 6. Manufacture of single or dual-organ models

[0094] Using the same method as in Example 1 above, a T2D model in the form of an on-a-chip was manufactured, consisting of one or two selected model parts among a pancreas model part, an adipose tissue model part, and a liver model part, as shown in FIG. 3: Comparative Example 1, a T2D single-organ model consisting only of a pancreas model part; Comparative Example 2, a T2D single-organ model consisting only of a liver model part; Comparative Example 3, a T2D single-organ model consisting only of an adipose tissue model part; Comparative Example 4, a T2D dual-organ model consisting of a pancreas model part and a liver model part; Comparative Example 5, a T2D dual-organ model consisting of a liver model part and an adipose tissue model part; Comparative Example 6, a T2D dual-organ model consisting of a pancreas model part and an adipose tissue model part.

[0095]

[0096] Examples 2 to 6. Manufacture of multi-organ models according to various batches

[0097] A T2D multi-organ model was manufactured using the same method as in Example 1, but with a different arrangement of each model part as shown in FIG. 4: Example 2, a T2D multi-organ model configured in the order of adipose tissue model part → pancreas model part → liver model part; Example 3, a T2D multi-organ model configured in the order of pancreas model part → liver model part → adipose tissue model part; Example 4, a T2D multi-organ model configured in the order of liver model part → pancreas model part → adipose tissue model part; Example 5, a T2D multi-organ model configured in the order of adipose tissue model part → liver model part → pancreas model part; Example 6, a T2D multi-organ model configured in the order of liver model part → adipose tissue model part → pancreas model part (Example 1 is in the order of pancreas model part → adipose tissue model part → liver model part).

[0098]

[0099] Example 7. Manufacture of a Non-Compartmentalized Multi-Organization Model

[0100] A T2D multi-organ model was manufactured using the same method as in Example 1 above, but without compartmentalizing each model part as shown in Fig. 5.

[0101]

[0102] Example 8. Preparation of a model with a different endothelial cell application method

[0103] A T2D multi-organ model was manufactured using the same method as in Example 1, but as shown in Fig. 6, instead of forming a single layer of endothelial cells (HUVEC), the endothelial cells were contained in each organ model part.

[0104]

[0105] Example 9. Preparation of a model without an endothelial cell layer

[0106] A T2D multi-organ model was manufactured using the same method as in Example 1 above, but without applying endothelial cells (HUVEC) as shown in Fig. 6.

[0107]

[0108] Experimental Example 1. Comparison of the effects of each model

[0109] It has been revealed that the process of T2D pathology involves the metabolic activity of one organ directly influencing the responses of other organs, and that there is a strong correlation between the organs. Therefore, to accurately simulate the T2D pathology process on an on-a-chip T2D model, it is necessary to consider multiple organs and the order of crosstalk between them. To verify this, the release of metabolites was analyzed in the multi-organ model of Example 1 and the single or dual-organ models of Comparative Examples 1 to 6. As shown in Figure 3, it was confirmed that the release of all metabolites increased in the multi-organ model compared to the single or dual-organ model, which implies that interconnection between the organs was implemented in the multi-organ model. Next, the models of Examples 1 to 6 were compared to determine the most accurate order of organs. As shown in Figure 4, the arrangement in which the pancreas model and the adipose tissue model were close together and the pancreas model was positioned at the very front best simulated T2D. This accurately reflects the pathological phenomenon occurring in actual T2D patients, where the T2D cascade begins in the pancreas and the close interaction between the pancreas and adipose tissue induces inflammatory responses and insulin resistance, thereby exacerbating T2D. It can be inferred that the order of pancreas model → adipose tissue model → liver model is most suitable for creating the T2D model. Next, by comparing the models of Example 1 and Example 7 to verify the compartmentalization effect of each organ, and as shown in Figure 5, the increase in each marker when spatial compartmentalization occurs, it was found that compartmentalization of each organ is necessary to more accurately simulate T2D.Since physiological phenomena occurring in the endocrine system, such as paracrine effects and hormone secretion, take place when each organ occupies an independent space, it was inferred that organ compartmentalization could more effectively simulate T2D. Finally, the effect of an additional endothelial cell layer (HUVEC monolayer) on each organ compartment was confirmed by comparing the models of Examples 1, 8, and 9. A monolayer arrangement of endothelial cells is essential for the formation of mature vascular structures, which is crucial for the normal functioning of each organ; therefore, an endothelial cell layer is necessary to simulate mature organs. Therefore, when comparing the three types of models—Example 1 ("W / Layered HUVEC"), in which HUVECs were arranged in a single layer in each organ compartment; Example 8 ("W / Mixed HUVEC"), in which HUVECs were randomly mixed in the organ compartment; and Example 9 ("W / O HUVEC"), in which HUVECs were not mixed—as shown in Figure 6, it was confirmed that all metabolites increased in Example 1 ("W / Layered HUVEC"), which means that for a mature organ to be simulated, endothelial cells must be arranged in a layered form, specifically a single layer, on each organ compartment.

[0110]

[0111] Comparative Examples 7 and 8. Manufacturing of models in which vadECM was replaced with another

[0112] A T2D multi-organ model was prepared using the same method as in Example 1 above, but using subcutaneous adipose tissue-derived decellularized extracellular matrix (sadECM) (Comparative Example 7) or collagen (Comparative Example 8) instead of vadECM.

[0113]

[0114] Experimental Example 2. Analysis of Pathological Characteristics of the T2D Model

[0115] It was verified whether typical T2D characteristics were realized in the on-a-chip T2D model fabricated in Example 1 above. As shown in Fig. 7 (A), after fabricating the T2D model, a stabilization period of 5 days was observed, and then the T2D model was operated at a specific glucose concentration for 2 days. As a result of investigating the changes in insulin and glucose concentrations according to the operating time, as shown in Fig. 7 (B) and (C), it was confirmed that glucose intolerance and insulin resistance, which are typical characteristics of T2D, occurred in the model treated with high concentrations of glucose. In addition, as a result of confirming the cell death of each organ, it was confirmed that the cells of each organ died due to glucotoxicity when exposed to hyperglycemia for a long period, as shown in Fig. 8 (A). Furthermore, as a result of performing VE-cadherin and CD31 immunofluorescence staining on a monolayer of endothelial cells, it was confirmed that the vascular endothelium was destroyed due to exposure to a state of hyperglycemia, as shown in Fig. 8 (B). In addition, to determine whether the model could replicate different functions of subcutaneous adipose tissue (sAT) and visceral adipose tissue (vAT), the models of Example 1, Comparative Examples 7 and 8 were operated, and ELISA was performed on the release amounts of inflammatory molecules directly associated with T2D.The inflammatory molecules analyzed at this time were the pro-inflammatory cytokines IL-6 (Interleukin 6), TNF-alpha (Tumor necrosis factor-alpha), and IL-1 beta; the adipose tissue-specific secretory factor Resistin; Adiponectin / acrp30, which is involved in glucose regulation; and FactorD / Adipsin, an adipokine associated with insulin secretion and fat accumulation. As a result, as shown in Figure 9, unlike models loaded with sadECM or collagen, the secretion pattern of inflammatory molecules generated in the model loaded with vadECM matched the pattern observed in T2D patients and appeared strongly. As such, through the results of Figures 7 to 9, the difference between the results of vadECM and sadECM implies that the tissue specificity of dECM can induce T2D, and by confirming that various pathological characteristics of T2D are clearly evident in the T2D model loaded with vadECM, it can be inferred that the fabricated T2D model accurately reflects the pathological environment of T2D.

[0116]

[0117] Example 10. Manufacturing of a model with an additionally applied retinal model part

[0118] A T2D multi-organ model was manufactured by using the same method as in Example 1 above, but by combining a transwell loaded with retinal cells (Retinal Pigment Epithelium (RPE) cells) on a chip so that a retinal model part is additionally applied after the liver model part as shown in FIGS. 10 and 11.

[0119]

[0120] Comparative Examples 9 and 10. Preparation of a model in which vadECM was replaced with something else in a model with an additional retinal model part.

[0121] A T2D multi-organ model was prepared using the same method as in Example 10, but using sadECM (Comparative Example 9) or collagen (Comparative Example 10) instead of vadECM as in (C) of FIG. 10.

[0122]

[0123] Experimental Example 3. Confirmation of the applicability of the T2D model to the study of T2D complications

[0124] There are numerous complications affecting organs related to T2D, and in particular, eye-related complications are common in T2D patients. When RPE cells present in the retina are exposed to a T2D environment, various types of RPE connections are destroyed and the production of RPE65 is inhibited. Therefore, to verify whether this phenomenon occurs in the T2D model, the model of Example 10 was operated, and at the same time, the models of Comparative Examples 9 and 10 were operated together to determine which bioink is suitable for simulating T2D complications. The operation schedule is as shown in (B) of Fig. 10. Immunofluorescence staining and qRT-PCR were performed on tight junction markers zonula occludens-1 (ZO-1) and occludin, and normal RPE cell markers E-cadherin and RPE 65 while operating each model. Additionally, TEER values, which can quantitatively measure the degree of tight junction disruption, were measured. As shown in Figures 12 and 13, the most significant decline in RPE cell function was confirmed in the model loaded with vadECM and treated with high concentrations of glucose, indicating that the strongest T2D characteristics were expressed in that model. In other words, by confirming the occurrence of distinct retinal dysfunction caused by T2D conditions implemented in the fabricated T2D model, it can be inferred that the fabricated T2D model is suitable for studying T2D complications.

[0125]

[0126] Experimental Example 4. Confirmation of the Applicability of the T2D Model as a Drug Testing Platform

[0127] To confirm the applicability of the T2D model of the present invention as a drug test platform, drug responsiveness was verified by treating the model of Example 1 with tolbutamide (Tol) and metformin (Met). Tol promotes insulin secretion from the pancreas, while Met interferes with the gluconeogenesis pathway in the liver and activates the AMPK pathway to promote the influx of glucose into cells. Therefore, using Tol and Met can ultimately result in a reduction of the amount of glucose in the body. As shown in Fig. 14, a "Normal model" operated at normal glucose concentrations for 5 days, a "T2D model" exposed to a hyperglycemic environment for 5 days, and a "T2D+Tol+Met model" treated with Tol+Met for 3 days after exposure to a hyperglycemic environment for 2 days were each constructed and operated. As a result of investigating changes in insulin and glucose concentrations according to operating time, as shown in Figure 15, it was confirmed that glucose intolerance and insulin resistance occurred in the "T2D model" but not in the "Normal model." Furthermore, in the "T2D+Tol+Met model," glucose concentration decreased and insulin concentration increased rapidly starting from the time of Tol+Met treatment, whereas this phenomenon did not occur in the "T2D model" without drug treatment. In other words, this implies that cell function was restored in the "T2D+Tol+Met model" by treating with the T2D drug, which means that the drug can function properly in the fabricated T2D model. Next, the functions of the organs included in each model were verified.To confirm pancreatic function, immunofluorescence staining was performed on Insulin and E-cadherin. To confirm liver function, immunofluorescence staining was performed on albumin, INSR (Insulin receptor), and Phospho-IR (phospho-Insulin receptor), the expression level of which increases during glucose uptake. Additionally, qRT-PCR was performed on the same markers. As a result, as shown in Figures 16 to 18, in the "T2D model," cell function was lost, and each marker was hardly detected; however, in the "T2D+Tol+Met model," cell function was restored, and it was confirmed that each marker was expressed at a level similar to that of the "Normal model." Furthermore, it was confirmed that in the "T2D model," the prolonged hyperglycemic environment caused cell membrane breakdown, and consequently, the expression level of INSR present on the cell membrane decreased compared to the "Normal model." In addition, although INSR phosphorylation was inhibited due to an increase in inflammatory cytokines (see Fig. 18), it was confirmed that the expression level of INSR in the "T2D+Tol+Met model" increased compared to the "T2D model" due to the recovery of cellular functions, including cell membrane reconstruction. Furthermore, it was confirmed that glucose metabolism proceeded normally, leading to INSR phosphorylation and a reduction in glucose levels. Fig. 18 shows the ELISA results for inflammatory cytokines and albumin. Due to the T2D drug, the inflammatory response was suppressed in the "T2D+Tol+Met model," resulting in a significant decrease in the levels of TNF-alpha and IL-6 compared to the "T2D model," and the release of albumin also significantly increased as liver function recovered. Thus, through the results shown in Figs. 15 to 18, it was confirmed that the T2D drugs function properly in the fabricated T2D model and that it can sufficiently serve as a drug testing platform.

Claims

1. A pancreatic model part comprising pancreatic beta cells and pancreatic-derived decellularized extracellular matrix (PdECM); Adipose tissue model part comprising adipocytes, macrophages, and visceral adipose tissue-derived decellularized extracellular matrix (vadECM); and A liver model comprising hepatocytes and liver-derived decellularized extracellular matrix (LdECM); including Multi-organ model for Type 2 diabetes implementation.

2. In Paragraph 1, A multi-organ model for implementing type 2 diabetes, wherein each of the above model parts is compartmentalized so as not to come into direct contact.

3. In Paragraph 1, A multi-organ model for implementing type 2 diabetes, wherein each of the above model parts is arranged in a line on a base plate in the order of pancreas model part, adipose tissue model part, and liver model part.

4. In Paragraph 1, A multi-organ model for implementing type 2 diabetes, wherein each of the above model parts is additionally equipped with an endothelial cell layer.

5. In Paragraph 1, A multi-organ model for implementing type 2 diabetes, further comprising a retinal model part including retinal cells.

6. In Paragraph 5, A multi-organ model for implementing type 2 diabetes, wherein each of the above model parts is arranged in a line on a base plate in the order of pancreas model part, adipose tissue model part, liver model part, and retina model part.

7. In Paragraph 1, It further includes a muscle model section, A multi-organ model for implementing type 2 diabetes, wherein each of the above model parts is connected to one another by a coronary blood vessel structure.

8. In Paragraph 7, A multi-organ model for implementing type 2 diabetes, further comprising one or more model parts selected from a retinal model part, a kidney model part, and a neural model part.

9. A step of preparing a first bioink comprising pancreatic beta cells and pancreatic-derived decellularized extracellular matrix (PdECM); A step of preparing a second bioink comprising adipocytes, macrophages, and visceral adipose tissue-derived decellularized extracellular matrix (vadECM); A step of preparing a third bioink comprising hepatocytes and liver-derived decellularized extracellular matrix (LdECM); A step of printing a pancreas-mimicking structure on a base plate using the first bioink; A step of printing an adipose tissue mimicking structure on the base plate using the second bioink; and A step of printing a liver-mimicking structure on the base plate using the third bioink; including Method for manufacturing a multi-organ model for implementing type 2 diabetes.

10. In Paragraph 9, A method for manufacturing a multi-organ model for implementing type 2 diabetes, wherein each of the above-mentioned simulated structures is compartmentalized so as not to come into direct contact and printed on the base plate.

11. In Paragraph 9, A method for manufacturing a multi-organ model for implementing type 2 diabetes, wherein each of the above-mentioned mimic structures is printed in a row on the base plate in the order of a pancreas mimic structure, an adipose tissue mimic structure, and a liver mimic structure.

12. In Paragraph 9, A multi-organ model for implementing type 2 diabetes, further comprising the step of printing an endothelial cell layer in addition to each of the above-mentioned simulated structures.

13. In Paragraph 9, A multi-organ model for implementing type 2 diabetes, further comprising the step of placing a retinal-mimicking structure containing retinal cells.

14. In Paragraph 13, A multi-organ model for implementing type 2 diabetes, wherein each of the above-mentioned mimic structures is arranged in a line on the base plate in the order of pancreas mimic structure, adipose tissue mimic structure, liver mimic structure, and retina mimic structure.

15. In Paragraph 9, The method further includes the step of placing a muscle-mimicking structure containing muscle cells, and A multi-organ model for implementing type 2 diabetes, further comprising the step of printing a coronary blood vessel structure connecting each of the above-mentioned simulated structures.

16. In Paragraph 15, A multi-organ model for implementing type 2 diabetes, further comprising the step of placing one or more mimic structures selected from a retinal mimic structure, a kidney mimic structure, and a neuromimicking structure.

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