Patent Application
20240142438
The present disclosure relates to a multi-organ model.
Non-alcoholic fatty liver (NAFL) disease, whose basic lesion is fatty liver, is a disease state which exhibits tissue damage of the hepatic parenchyma such as inflammation, necrosis, and fibrosis that are similar to those in alcoholic liver injury, even without a long history of alcohol intake. NAFLD is basically asymptomatic. During the course of progression of the disease state, the fatty liver leads to steatohepatitis, liver cirrhosis, and then to liver cancer. The steatohepatitis in NAFLD is called non-alcoholic steatohepatitis (NASH). In particular, in recent years, metabolic syndromes due to obesity or diabetes have become a social problem, and NASH is also regarded as one of the metabolic syndromes. As complications of NAFLD and NASH, lifestyle-related diseases such as obesity, diabetes, hyperlipidemia, and hypertension are observed, and their major clinical manifestations characteristically include increases in the blood alanine aminotransferase (ALT) and hyaluronic acid levels, and on the other hand, decreases in the blood albumin level. However, the pathogenic mechanisms of NAFLD and NASH still remain largely unclear, and there is no established effective therapeutic method or therapeutic drug therefor at present. This is partly due to the fact that, since development of NAFLD and NASH is based on lifestyle-related diseases of humans, animal models suitable for research of NAFLD and NASH have not been established yet.
Elucidation of the disease pathology of NAFLD and NASH, which may progress to fatal diseases such as liver cirrhosis and liver cancer, is indispensable for development of effective therapeutic methods and therapeutic drugs therefor, and the elucidation requires appropriate models for NAFLD and NASH.
Animal models for NASH have so far been reported (for example, Patent Document 1). However, non-human animal models for NAFLD have been hardly reported. Further, due to recent issues related to animal ethics, the need for developing an effective in vitro model as an alternative to animal models has increased.
Furthermore, the occurrence and progression of various diseases in a human body and the reaction of the human body when a drug is administered are phenomena not occurring in one organ, but usually caused by complex interactions between various organs in the human body.
For example, metabolic diseases such as obesity, diabetes, and hypertension are caused by various factors such as eating habits, exercise, and stress, and various organ tissues such as the intestine, liver, immune system, and adipose tissue are known to be involved in the process.
Although the development of a test model capable of implementing the interactions between these organs can be of great help in researching disease mechanisms and developing therapeutic drugs, it cannot be achieved by conventional test methods since culture conditions are different for cells of each organ.
Thus, the inventors of the present disclosure completed the present disclosure as a result of researching a multi-organ model capable of reflecting the microenvironment of interactions between various organs.
An aspect of the present disclosure is conceived to provide a multi-organ model, including: a liver organoid well; and an intestinal organoid well, a pancreas organoid well, and a cardiac organoid well, each of which is directly or indirectly connected to the liver organoid well by microchannels.
Another aspect of the present disclosure is conceived to provide a non-alcoholic fatty liver multi-organ model in which the liver organoid well of the multi-organ model is treated with free fatty acid.
Yet another aspect of the present disclosure is conceived to provide a method of fabricating a non-alcoholic fatty liver multi-organ model, including: fabricating the multi-organ model; and injecting culture medium containing free fatty acid into the liver organoid well.
Still another aspect of the present disclosure is conceived to provide a method of screening a therapeutic drug for non-alcoholic fatty liver disease, including: treating the non-alcoholic fatty liver multi-organ model with a candidate substance; and comparing a group treated with the candidate substance to a control group, and a method of evaluating drug metabolism and drug toxicity on peripheral organs.
An aspect of the present disclosure provides a multi-organ model, including: a liver organoid well; and an intestinal organoid well, a pancreas organoid well, and a cardiac organoid well, each of which is directly or indirectly connected to the liver organoid well by microchannels.
In an embodiment of the present disclosure, the intestinal organoid well, the pancreas organoid well, and the cardiac organoid well may not be directly connected to each other.
In an embodiment of the present disclosure, the microchannels may have a cross-sectional width of 10 μm to 30 μm and a height of 5 μm to 20 μm.
In an embodiment of the present disclosure, the liver organoid well may include: a hydrogel containing decellularized liver tissue-derived extracellular matrix (Liver Extracellular Matrix; LEM); and liver organoids.
In an embodiment of the present disclosure, the intestinal organoid well may include a hydrogel containing decellularized intestinal tissue-derived extracellular matrix and intestinal organoids, the pancreas organoid well may include a hydrogel containing decellularized pancreas tissue-derived extracellular matrix and pancreas organoids, and the cardiac organoid well may include a hydrogel containing decellularized heart tissue-derived extracellular matrix and cardiac organoids.
In an embodiment of the present disclosure, the liver organoids may be derived from mouse tissue, human induced pluripotent stem cell (hiPSC) or human liver tissue.
Another aspect of the present disclosure provides a non-alcoholic fatty liver multi-organ model in which the liver organoid well of the multi-organ model is treated with free fatty acid.
In an embodiment of the present disclosure, the free fatty acid may have a concentration ranging from 100 μM to 900 μM.
Yet another aspect of the present disclosure provides a method of fabricating a non-alcoholic fatty liver multi-organ model, including: fabricating the multi-organ model; and injecting culture medium containing free fatty acid into the liver organoid well.
Still another aspect of the present disclosure provides a method of screening a therapeutic drug for non-alcoholic fatty liver disease, including: treating the non-alcoholic fatty liver multi-organ model with a candidate substance; and comparing a group treated with the candidate substance to a control group.
Still another aspect of the present disclosure provides a method of providing information about drug metabolism of a non-alcoholic fatty liver therapeutic drug on peripheral organs, including: treating the non-alcoholic fatty liver multi-organ model with a candidate substance; and comparing a group treated with the candidate substance to a control group.
Still another aspect of the present disclosure provides a method of evaluating drug toxicity of a non-alcoholic fatty liver therapeutic drug, including: treating the non-alcoholic fatty liver multi-organ model with a candidate substance; and comparing a group treated with the candidate substance to a control group.
A multi-organ model of the present disclosure is excellent in culturing each organoid due to linkage between organoids and properties of hydrogel and can more accurately reflect the interactions between organs and the microenvironment in vivo. Also, the multi-organ model is further subjected to free fatty acid treatment and thus can more accurately mimic the phenotypes of non-alcoholic fatty liver.
Further, since a non-alcoholic fatty liver multi-organ model of the present disclosure mimics non-alcoholic fatty liver, it can be used for screening a therapeutic drug for non-alcoholic fatty liver disease.
Hereafter, the present disclosure will be described in detail with reference to the accompanying drawings. However, it is to be noted that the present disclosure is not limited to examples described herein but can be embodied in various other ways. It is to be understood that the term “comprises or includes” and/or “comprising or including” used in the document means that one or more other components, steps, operation and/or existence or addition of elements are not excluded in addition to the described components, steps, operation and/or elements unless context dictates otherwise.
Unless otherwise indicated, the practice of the disclosure involves conventional techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, and recombinant DNA fields, which are within the skill of the art. Such techniques are known to a person with ordinary skill in the art and are described in numerous standard texts and reference works.
An aspect of the present disclosure provides a composition for hydrogel, including a phenol derivative-modified, tissue-derived extracellular matrix.
Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by a person with ordinary skill in the art to which this disclosure belongs.
Various scientific dictionaries that include the terms included herein are well known and available to those in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice or testing of the disclosure, some preferred methods and materials are described. It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context in which they are used by a person with ordinary skill in the art. Hereinafter, the present disclosure will be described in more detail.
An aspect of the present disclosure provides a multi-organ model, including: a liver organoid well; and an intestinal organoid well, a pancreas organoid well, and a cardiac organoid well, each of which is directly or indirectly connected to the liver organoid well by microchannels.
In an embodiment of the present disclosure, the intestinal organoid well, the pancreas organoid well, and the cardiac organoid well may not be directly connected to each other. In the past, attempts have been made to fabricate multi-organ models, but due to problems such as difficulty of fabrication/application, they were fabricated without considering the interactions between actual organs and the microenvironment. However, the multi-organ model of the present disclosure is composed of: the liver organoid well; the intestinal organoid well, the pancreas organoid well, and the cardiac organoid well, each of which is directly or indirectly connected to the liver organoid well by the microchannels, and is fabricated such that the intestinal organoid well, the pancreas organoid well, and the cardiac organoid well are not directly connected to each other. Thus, the multi-organ model of the present disclosure can more accurately reflect the interactions between organs in vivo, in particular, the in vivo microenvironment related to non-alcoholic fatty liver disease (see
In another embodiment of the present disclosure, the microchannels may have a cross-sectional width of 10 μm to 30 μm and a height of 5 μm to 20 μm, more specifically a width of 20 μm and a height of 10 μm. The dimensions of the microchannels are determined in consideration of the diffusion rate of paracrine factors in the microchannels, and the interactions between organs and the microenvironment may not be reflected in a microchannel having the sizes outside the above range. The number of microchannels between organoid wells may be one or more, and the microchannels may have a known shape and length.
In an embodiment of the present disclosure, the liver organoid well may include: a hydrogel containing decellularized liver tissue-derived extracellular matrix (Liver Extracellular Matrix; LEM); and liver organoids.
Also, in an embodiment of the present disclosure, the intestinal organoid well may include a hydrogel containing decellularized intestinal tissue-derived extracellular matrix and intestinal organoids, the pancreas organoid well may include a hydrogel containing decellularized pancreas tissue-derived extracellular matrix and pancreas organoids, and the cardiac organoid well may include a hydrogel containing decellularized heart tissue-derived extracellular matrix and cardiac organoids.
In an embodiment of the present disclosure, the LEM may be a matrix in which 95% to 99.9%, more specifically 96% to 98%, and most specifically 97.18% of liver tissue cells have been removed. Further, the decellularized intestinal tissue-derived extracellular matrix (Intestinal Extracellular Matrix; IEM) may be a matrix in which 95% to 99.9%, more specifically 96% to 98%, and most specifically 97.68% of intestinal tissue cells have been removed. Furthermore, the decellularized pancreas tissue-derived extracellular matrix (Pancreas Extracellular Matrix; PEM) may be a matrix in which 95% to 99.9%, more specifically 96% to 98%, and most specifically 96.03% of pancreas tissue cells have been removed. Also, the decellularized heart tissue-derived extracellular matrix (Heart Extracellular Matrix; HEM) may be a matrix in which 95% to 99.9%, more specifically 97% to 99%, and most specifically 98.72% of heart tissue cells have been removed. The organoids can be cultured in the respective tissue-derived extracellular matrices to better reflect the interactions between organs in vivo and the microenvironment specific to each organ.
The term “extracellular matrix” refers to a protein component found in mammals and multicellular organisms and a natural scaffold for cell growth that is prepared by decellularization of tissue. The extracellular matrix can be further processed through dialysis or crosslinking
The extracellular matrix may be a mixture of structural or non-structural biomolecules including, but not limited to, collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines and growth factors.
In mammals, the extracellular matrix may contain about 90% collagen in various forms. The extracellular matrices derived from various living tissues may differ in their overall structure and composition due to the unique role needed for each tissue.
The term “derive” or “derived” refers to a component obtained from any stated source by any useful method.
The term “organoid” refers to an ultraminiature body organ prepared in the form of an artificial organ by culturing cells derived from tissues or pluripotent stem cells in a 3D form.
The organoid is a three-dimensional tissue analog that contains organ-specific cells which originate from stem cells and self-organize (or self-pattern) in a similar manner to the in vivo condition. The organoid can be developed into a specific tissue by patterning restricted elements (for example, a growth factor).
The organoid can have the intrinsic physiological properties of the cells and can have an anatomical structure that mimics the original state of a cell mixture (multiple cell types including all remaining stem cells and the neighboring physiological niche). A three-dimensional culture method allows the organoid to be better arranged in terms of cell to cell functions and to have an organ-like form with functionality and tissue-specific functions.
In an embodiment of the present disclosure, the liver organoid may be derived from mouse tissue, human induced pluripotent stem cells (hiPSC) or human liver tissue, more specifically human induced pluripotent stem cells (hiPSC) or human liver tissue.
The multi-organ model may be prepared by fabricating a multi-organ model device, including: a liver organoid well; and an intestinal organoid well, a pancreas organoid well, and a cardiac organoid well, each of which is directly or indirectly connected to the liver organoid well by microchannels; and locating and culturing each organ-derived extracellular matrix and tissue cells in each organoid well.
Another aspect of the present disclosure provides a non-alcoholic fatty liver multi-organ model in which the liver organoid well of the multi-organ model is treated with free fatty acid.
The free fatty acid treatment may be performed directly onto a liver organoid well including the cultured liver organoids, or may be performed simultaneously with liver organoid culture or after a liver organoids are cultured and then mixed with culture medium.
In an embodiment of the present disclosure, the free fatty acid may have a concentration ranging from 100 μM to 900 μM, specifically 200 μM to 800 μM, and most specifically 500 μM. The liver organoid well is treated with the free fatty acid, which makes the liver organoids exhibit the characteristics of non-alcoholic fatty liver, and factors secreted by the non-alcoholic fatty liver organoids flow through the microchannels to the intestinal organoid well, the pancreas organoid well, and the cardiac organoid well and thus affect each organoid. If the free fatty acid is used at a concentration out of the above range, the characteristics of non-alcoholic fatty liver may not appear or the cells in the model may die.
Also, if the free fatty acid treatment is performed simultaneously with liver organoid culture or after liver organoids are cultured and then mixed with culture medium, the components of the culture medium may be a mixture of known substances used for liver organoid culture in addition to free fatty acids.
In an embodiment of the present disclosure, the free fatty acid may be any one selected from oleic acid, palmitic acid and linoleic acid, and may be specifically oleic acid.
Yet another aspect of the present disclosure provides a method of fabricating a non-alcoholic fatty liver multi-organ model, including: fabricating the multi-organ model; and injecting culture medium containing free fatty acid into the liver organoid well.
The fabricating the multi-organ model is to fabricate the multi-organ model. Specifically, it may be composed of fabricating a device (microfluidic chip) including each organoid chamber and microchannels by using PDMS polymer; and locating the tissue-derived extracellular matrices and tissue cells in the respective wells. Details of the extracellular matrix and organoid are the same as those of the non-alcoholic fatty liver artificial tissue model described above.
The injecting is to inject the culture medium containing the free fatty acid into the multi-organ model. As described above, the culture medium containing the free fatty acid flows into the well in which the liver organoid is located, and the liver organoids are exposed to the free fatty acid and thus exhibit the phenotypes of non-alcoholic fatty liver.
Still another aspect of the present disclosure provides a method of screening a therapeutic drug for non-alcoholic fatty liver disease, including: treating the non-alcoholic fatty liver multi-organ model with a candidate substance; and comparing a group treated with the candidate substance to a control group.
The treating with the candidate substance is to treat the non-alcoholic fatty liver multi-organ model with the candidate substance, and the treatment with the candidate substance may vary depending on the intended route of administration and dosage of the candidate substance.
Further, the comparing the group treated with the candidate substance to the control group may be to compare the non-alcoholic fatty liver multi-organ model treated with the candidate substance to the control group. The control group may be a non-alcoholic fatty liver multi-organ model treated with or not treated with a conventionally known non-alcoholic fatty liver therapeutic drug or a known substance within the range that does not inhibit or increase the physiological activity of the liver organoid in the non-alcoholic fatty liver multi-organ model.
The group treated with the candidate substance may be compared to the control group by analyzing the levels of fat accumulation in the liver organoids, the differentiation and functionality of the liver organoids, the levels of inflammation and fibrosis, and the viability of the liver organoids and/or by checking various indicators in the liver organoids or culture media.
The method of screening may further include selecting a non-alcoholic fatty liver therapeutic drug. The selecting may be to select a non-alcoholic fatty liver therapeutic drug when a reduction of fat accumulation in the liver organoids, the recovery of differentiation and functionality of the liver organoids, a decrease in levels of inflammation and fibrosis, an increase in viability of the liver organoids, and/or an increase in improved indicators in the liver organoids or culture media are confirmed in the above-described process of comparing. If a conventionally known non-alcoholic fatty liver therapeutic drug is used as a control group, any non-alcoholic fatty liver therapeutic drug can be determined and selected as having an improved effect compared to the conventionally known non-alcoholic fatty liver therapeutic drug when it shows an improved effect compared to the control group. Also, the multi-organ model of the present disclosure includes organoids of major human organs, i.e. the liver, intestine, pancreas, and heart and reflects their microenvironment. Thus, it is possible to not only select a non-alcoholic fatty liver therapeutic drug but also check the effect of the selected therapeutic drug on other organs. Further, it is possible to select a drug capable of reducing, or improving stress applied to other organs in the environment of non-alcoholic fatty liver.
Still another aspect of the present disclosure provides a method of providing information about drug metabolism of a non-alcoholic fatty liver therapeutic drug on peripheral organs, including: treating the non-alcoholic fatty liver multi-organ model with a candidate substance; and comparing a group treated with the candidate substance to a control group, and a method of evaluating drug toxicity of the non-alcoholic fatty liver therapeutic drug.
The treating and the comparing are the same as described above.
Meanwhile, the method may further include evaluating drug metabolism of a non-alcoholic fatty liver therapeutic drug on peripheral organs; or evaluating drug toxicity of the non-alcoholic fatty liver therapeutic drug. The selecting may be to evaluate the method of providing information about drug metabolism of a therapeutic drug on peripheral organs and drug toxicity when a reduction of fat accumulation in the liver organoids, the recovery of differentiation and functionality of the liver organoids, a decrease in levels of inflammation and fibrosis, an increase in viability of the liver organoids, and/or an increase in improved indicators in the liver organoids or culture media are confirmed in the above-described process of comparing. If a conventionally known non-alcoholic fatty liver therapeutic drug is used as a control group, any non-alcoholic fatty liver therapeutic drug can be determined and selected as having an improved effect compared to the conventionally known non-alcoholic fatty liver therapeutic drug when it shows an improved effect compared to the control group.
Hereinafter, one or more specific embodiments will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present disclosure is not limited to these examples.
To implement a multi-organ non-alcoholic steatohepatitis model, a microdevice in which liver, intestinal, pancreas and cardiac organoids can be organically connected and co-cultured was fabricated.
In actual non-alcoholic steatohepatitis (NASH) patients, various organs are affected by fatty liver with excessive fat accumulation. Particularly, it is known that the intestine, heart, and pancreas are affected. Actually, NASH patients may often develop an inflammatory bowel disease in the small intestine or have leaky gut due to damage to the wall of the small intestine. Also, NASH patients may develop cardiovascular disease and arrhythmia in the heart. Further, NASH patients may develop acute pancreatitis in the pancreas or have a decrease in function of insulin-secreting beta cells. Therefore, it is very important to consider the effects on other organs together in the development of non-alcoholic steatohepatitis drugs and the treatment of actual patients.
To this end, a multi-organ NASH co-culture model, which cannot be implemented in a conventional well-plate, was implemented by using a microfluidic device. The organoids were respectively cultured in divided compartments in one device, and each organoid was cultured in the culture medium suitable for the corresponding organoid. Also, the organoids were connected to mimic the locations, sequence, and metabolic process of the respective organs in the body, and could interact with each other through microfluidic channels (
Specifically, the microfluidic device had a horizontal length of 48 mm and a vertical length of 32 mm, and was fabricated to have a total of three layers. The lowermost layer is a non-patterned bottom layer, and has a thickness of 1 mm to 2 mm. The intermediate layer is a patterned layer for seeding organoids, and has a thickness of 1 mm to 2 mm and a pattern diameter of 5 mm or 8 mm. The uppermost layer is a layer including microfluidic channels and organoid culture chambers, and has a thickness of 7 to 8 mm and an organoid culture chamber diameter of 10 mm Basically, the microfluidic device can be fabricated using a polydimethylsiloxane (PDMS) polymer by soft lithography (
There are two types of microfluidic channels: (i) straight channel and (ii) curved channel. The both channels have a width of 20 μm, an average length of 8 mm, and a height of 10 μm. The organoid chambers were connected through a total of 9 microfluidic channels (
When non-alcoholic steatohepatitis occurs, various harmful substances such as inflammatory cytokines are produced and these harmful substances affect the peripheral organs. Therefore, it is essentially required to check the effects of these harmful substances on other organs through a multi-organ non-alcoholic steatohepatitis model. Simulation analysis was conducted to check the transfer of these harmful substances in the fabricated microfluidic device.
In the microfluidic device, the substances are transferred between the organoid chambers mainly by means of diffusion (
The organoid culture medium contains various growth factors, and in the non-alcoholic steatohepatitis model, cells at a disease site secrete various inflammatory cytokines. Simulation analysis was conducted to predict the transfer of these growth factors and paracrine factors in the microfluidic device.
To simulate concentration changes of growth factors and paracrine factors that diffuse over time, simulation analysis was conducted for 24 hours on concentration changes caused by diffusion, through microfluidic channels, of FITC-dextran of 4 kDa, 40 kDa and 70 kDa corresponding to a molecular weight range of growth factors and paracrine factors present in the body. FITC-dextran of each molecular weight was set to be present in one side culture chamber (left chamber) of the device at a concentration of 0.01 mol/m3 at Time=0 h. FITC-dextran of 4 kDa has a diffusion coefficient of 1.35×10−10 m2/s in the culture medium, FITC-dextran of 40 kDa has a diffusion coefficient of 4.5×10−11 m2/s in the culture medium, and FITC-dextran of 70 kDa has a diffusion coefficient of 2.3×10−11 m2/s in the culture medium. At Time=6 h, the concentration of FITC-dextran of 4 kDa was first checked in the opposite side (right) culture chamber, and at Time=12 h, the concentration of FITC-dextran of 40 kDa was checked in the opposite side (right) culture chamber, and at Time=18 h, the concentration of FITC-dextran of 70 kDa was finally checked in the opposite side (right) culture chamber (
Quantitative analysis was conducted on diffusion patterns of FITC-dextran of 4 kDa, 40 kDa and 70 kDa in the microfluidic channel for 24 hours. In
Further, to predict material transfer in the microfluidic device applied to the multi-organ non-alcoholic steatohepatitis model, simulation analysis was conducted on a diffusion pattern of 40 kDa FITC-dextran from in an intermediate culture chamber (chamber where liver organoids were cultured) to peripheral culture chambers over time.
As a result, as shown in
Furthermore, to predict material transfer in the microfluidic device applied to the multi-organ non-alcoholic steatohepatitis model, diffusion of 40 kDa FITC-dextran from the intermediate culture chamber (chamber where liver organoids were cultured) to peripheral culture chambers for 24 hours was simulated, followed by quantitative analysis.
Specifically, the concentration of 40 kDa FITC-dextran diffused through the microfluidic channel for 24 hours was checked at the point indicated by the red dot in the device diagram on the left of
Further, the concentration of 40 kDa FITC-dextran diffused through the microfluidic channel at Time=24 h was analyzed. It was confirmed that at Time=24 h, the concentration at Distance=0 mm, the left end point of the microfluidic channel in the red line in the device diagram of
Furthermore, to identify diffusion patterns of the paracrine factors secreted from the peripheral culture chambers of the microfluidic device applied to the multi-organ non-alcoholic steatohepatitis model to the intermediate culture chamber (chamber where liver organoids were cultured), diffusion of 40 kDa FITC-dextran having a concentration of 0.01 mol/m3 at Time=0 for 24 hours was simulated and analyzed.
As a result, it was confirmed that 40 kDa FITC-dextran secreted from the culture chamber located at the top left was diffused into the intermediate culture chamber at Time=24 h (
As a result of analyzing this series of simulations, it was confirmed that various growth factors and paracrine factors, such as inflammatory cytokines, secreted by cells in the non-alcoholic steatohepatitis model can be diffused through the microfluidic channel. Therefore, it was verified that the fabricated microfluidic device can be used to evaluate interactions between organs and drug metabolism/toxicity in multi-organ non-alcoholic steatohepatitis modeling.
In order to construct a multi-organ non-alcoholic steatohepatitis model, growth factors and paracrine factors need to be diffused at an optimal rate through a channel connecting organoid chambers. In the microfluidic device, the diffusion rates of growth factors and paracrine factors are determined by channel dimensions (height, width, length, and number of channels). When a multi-organoid-based non-alcoholic steatohepatitis (NASH) model is constructed, four different types of organoids are affected by paracrine factors, such as inflammatory cytokines, secreted from non-alcoholic steatohepatitis-induced organoids. However, growth factors required for culture and included in each organoid culture chamber need to be maintained at certain levels in order to normally culture the four different types of organoids. To this end, organoids were cultured in a device equipped with diffusion channels of various dimensions and an optimal device for a multi-organ model was selected. Liver and pancreas organoids were obtained by extracting adult stem cells from a mouse tissue, encapsulated in a decellularized liver tissue-derived matrix (6 mg/ml) and a decellularized pancreas tissue-derived matrix (4 mg/ml), respectively, at a cell density of 70,000 cells/30 μL gel, and cultured. An intestinal organoid was obtained by extracting intestinal crypts from a mouse intestinal tissue and encapsulated in a decellularized intestinal tissue-derived matrix (2 mg/ml) at a cell density of 800 crypts/30 μL gel, and cultured. A cardiac organoid composed of cardiomyocytes was prepared by three-dimensionally culturing mouse embryonic fibroblasts in microwells, followed by direct reprogramming chemically induced by the culture medium components. The cardiac organoid was cultured on a hydrogel (20 organoids/70 μL gel bed) prepared by crosslinking of a decellularized cardiac tissue-derived matrix (5 mg/ml) in the form of a 70 μL gel bed at the bottom of the device. The decellularized matrix for culturing each organoid was applied at the optimal concentration for differentiation of each organoid as determined through previous research.
Three devices with different channel dimensions shown in
The device with the standard diffusion channel dimensions (i) had a channel width of 0.020 mm, a channel length of 8.000 mm, and a channel height of 0.010 mm, and in the device, the organoid culture chambers were connected by nine channels. Further, the total volume of the channels was 0.014 mm3. The device with the wider diffusion channel dimensions (ii) had a channel width of 0.400 mm, a channel length of 105.803 mm, and a channel height of 0.175 mm, and in the device, the organoid culture chambers were connected by one channel. Further, the total volume of the channel was 7.406 mm3. The device with the widest diffusion channel dimensions (iii) had a channel width of 1.000 mm, a channel length of 2.222 mm, and a channel height of 0.300 mm, and in the device, the organoid culture chambers were connected by 15 channels. Further, the total volume of the channels was 9.999 mm3 (
In order to construct a multi-organ non-alcoholic steatohepatitis model, growth factors and paracrine factors need to be diffused at an optimal rate through a channel connecting organoid chambers. In the microfluidic device, the diffusion rates of growth factors and paracrine factors are determined by channel dimensions (height, width, length, and number of channels). When a multi-organoid-based non-alcoholic steatohepatitis (NASH) model is constructed, four different types of organoids are affected by paracrine factors, such as inflammatory cytokines, secreted from non-alcoholic steatohepatitis-induced organoids. However, growth factors required for culture and included in each organoid culture chamber need to be maintained at certain levels in order to normally culture the four different types of organoids. To this end, organoids were cultured in a device equipped with diffusion channels of various dimensions and an optimal device for a multi-organ model was selected. Liver and pancreas organoids were obtained by extracting adult stem cells from a mouse tissue, encapsulated in a decellularized liver tissue-derived matrix (6 mg/ml) and a decellularized pancreas tissue-derived matrix (4 mg/ml), respectively, at a cell density of 70,000 cells/30 μL gel, and cultured. An intestinal organoid was obtained by extracting intestinal crypts from a mouse intestinal tissue and encapsulated in a decellularized intestinal tissue-derived matrix (2 mg/ml) at a cell density of 800 crypts/30 μL gel, and cultured. A cardiac organoid composed of cardiomyocytes was prepared by three-dimensionally culturing mouse embryonic fibroblasts in microwells, followed by direct reprogramming chemically induced by the culture medium components. The cardiac organoid was cultured on a hydrogel (20 organoids/70 μL gel bed) prepared by crosslinking of a decellularized cardiac tissue-derived matrix (5 mg/ml) in the form of a 70 μL gel bed at the bottom of the device. The decellularized matrix for culturing each organoid was applied at the optimal concentration for differentiation of each organoid as determined through previous research.
Multi-organoids were cultured for 3 days in three types of devices with different diffusion channel dimensions and chamber arrangement, followed by quantitative PCR analysis to compare the expression of each organ-specific differentiation marker gene. It was confirmed that the liver organoid cultured in the standard diffusion channel (i) showed the highest expression levels of hepatic differentiation markers, AFP and ALB, and the liver organoids cultured in the wider diffusion channel (ii) and the widest diffusion channel (iii) showed remarkably low expression levels of the differentiation markers. Also, it was confirmed that CASP3, an apoptosis marker, had the lowest expression level in a multi-organ chip with the standard channel design (i) selected as the optimal device in the present development, and the expression level of the apoptosis marker increased in the order of the wider channel device (ii) and the widest channel device (iii) (
As for intestinal organoids, it was confirmed that the expression levels of intestinal differentiation markers, MUC2 and CHGA, and a gut barrier marker, OCLN, were the highest in the multi-organ chip with the standard channel (i), and gradually decreased in the order of the wider channel device (ii) and the widest channel device (iii) (
As for pancreas organoids, it was confirmed that there was no significant difference in the expression of PDX1, a pancreatic endoderm marker, among the three types of devices, but the expression levels of KRT19 and HNF1B were the highest in the standard channel multi-organ device (i) selected as the optimal design in the present disclosure, and decreased in the order of the wider channel device (ii) and the widest channel device (iii) (
As a result of comparing the gene expression of cardiac organoids cultured in the three types of devices, it was confirmed that the expression levels of cardiac differentiation markers, ACTC1 and MYH7, were the highest in the standard channel device (i), and decreased in the order of the wider channel device (ii) and the widest channel device (iii). On the contrary, the expression level of CASP3, an apoptosis marker, was the lowest in the standard channel device (i), and increased as the size of the channel increased (
Accordingly, it was confirmed that the device with the standard diffusion channel dimensions and design (i) is capable of performing co-culture while maintaining high differentiation potency of each of the four different types of multi-organoids (liver, intestine, pancreas, and heart) and minimizing apoptosis. Therefore, it was used later as a device for multi-organ culture.
Liver, intestinal and pancreas organoids were cultured by using the standard culture medium components, which have been most widely used in culture media for mouse adult stem cell-derived organoids, and the composition thereof is shown in
A cardiac organoid was cultured by using the composition of a representative culture medium used in a chemically induced protocol for direct reprogramming from mouse embryonic fibroblasts to cardiomyocytes. Since each organoid has different culture medium composition and growth factor concentration optimized for growth and differentiation, optimized channel dimensions for appropriate degree of diffusion during multi-organoid co-culture are required. It seems that in the wider diffusion channel device (ii) and the widest diffusion channel device (iii) used as control groups for the microfluidic device [standard diffusion channel (i)] developed in the present disclosure, excessive diffusion of the culture media among the culture chambers of the four different types of organoids occurred and affected proliferation and differentiation of the peripheral organoids, which made it difficult to achieve appropriate co-culture.
The expression of each organ-specific differentiation marker was compared by comparing liver, intestinal, pancreas and cardiac organoids each cultured in a plate. In addition to the comparison between well-plate and multi-organ microfluidic chip conditions, there was a comparison between culture in Matrigel (MAT), a commercially available culture scaffold mainly used for conventional organoid culture, and culture in an organ-specific decellularized matrix. As for MAT (plate) and MAT (chip) multi-organoids, each organoid was initially cultured in Matrigel and seeded during subculture, and as for LEM (plate) and LEM (chip) multi-organoids, organoids were initially cultured in each organ-specific decellularized matrix and seeded during subculture. The gene expression was compared by quantitative PCR analysis on day 4 of culture after the organoids were seeded into each culture platform (
As a result of comparing differentiation markers of liver organoids by quantitative PCR analysis, it was confirmed that no significant difference in the expression of Krt18 and Hnf4a was observed in the organoids of the four groups, but Krt19 and Afp had the highest expression levels in the group in which liver organoids were cultured in the decellularized liver tissue-derived matrix (6 mg/ml LEM) in the multi-organ microfluidic device (
As a result of comparing differentiation markers of intestinal organoids by quantitative PCR analysis, it was confirmed that the expression levels of a tight junction marker, Ocln, and intestinal differentiation markers, Muc2 and Lyz, were similar in the organoids cultured under the MAT (plate) and MAT (chip) conditions or slightly higher in the organoid cultured under the MAT (chip) conditions. It was confirmed that the expression levels of differentiation markers were significantly higher overall in the intestinal organoids cultured under the decellularized intestinal tissue-derived matrix (2 mg/ml IEM)-based IEM (plate) and IEM (chip) conditions than in the intestinal organoid cultured in MAT, a commercially available culture scaffold (
As a result of comparing differentiation markers of pancreas organoids by quantitative PCR analysis, it was confirmed that no significant difference in the expression of Krt19 and Hnf1b was observed in the organoids of the four groups, but Pdx1 and Foxa2 showed higher expression levels in the pancreas organoids cultured in the well-plate and the decellularized pancreas tissue-derived matrix (4 mg/ml PEM) in the multi-organ microfluidic device than in the pancreas organoid cultured in Matrigel under each condition (
As a result of comparing differentiation markers of cardiac organoids by quantitative PCR analysis, it was confirmed that the expression levels of cardiac differentiation markers, Actc1, Mef2c and Scn5a, were significantly higher in the cardiac organoid cultured in the decellularized heart tissue-derived matrix (5 mg/ml HEM) than in the cardiac organoid cultured under the MAT conditions, and the expression levels of the differentiation markers was similar in the organoids cultured under the HEM (plate) and HEM (chip) conditions or higher in the organoid cultured under the HEM (chip) conditions (
Accordingly, it was confirmed that the conventional culture scaffold can be replaced by a decellularized matrix specific to each organ, and organoids can be cultured well without a decrease in differentiation and functionality of each organoid when the organoids are co-cultured in a multi-organ microfluidic chip in the same way as when each organoid is independently cultured in a well-plate.
A multi-organ NASH model was constructed by applying not only conventional Matrigel, a commercially available culture scaffold, but also a decellularized tissue-derived hydrogel scaffold to culture of each organoid in a multi-organ device. Liver and pancreas organoids were obtained by extracting adult stem cells from a mouse tissue, encapsulated in a decellularized liver tissue-derived matrix (6 mg/ml) and a decellularized pancreas tissue-derived matrix (4 mg/ml), respectively, at a cell density of 70,000 cells/30 μL gel, and cultured. An intestinal organoid was obtained by extracting intestinal crypts from a mouse intestinal tissue and encapsulated in a decellularized intestinal tissue-derived matrix (2 mg/ml) at a cell density of 800 crypts/30 μL gel, and cultured. A cardiac organoid composed of cardiomyocytes was prepared by three-dimensionally culturing mouse embryonic fibroblasts in microwells, followed by direct reprogramming chemically induced by the culture medium components. The cardiac organoid was cultured on a hydrogel (20 organoids/70 μL gel bed) prepared by crosslinking of a decellularized cardiac tissue-derived matrix (5 mg/ml) in the form of a 70 μL gel bed at the bottom of the device. The decellularized matrix for culturing each organoid was applied at the optimal concentration for differentiation of each organoid as determined through previous research.
In the multi-organ microfluidic device, steatohepatitis was induced by treating the liver organoid with oleic acid (500 μM), which is a fatty acid, for 3 days, and the changes of and effects on the peripheral organoids were compared on day 3. When the organoids were cultured by using the decellularized tissue-derived hydrogel in the multi-organ NASH model, inflammation and fat accumulation occurred in the liver organoid in the same way as when the organoids were cultured by using Matrigel (MAT). The intestinal organoid as a peripheral organ had damage to the gut barrier of the intestinal tissue, and the pancreas organoid was changed in shape and induced with internal inflammation. The cardiac organoid was changed in shape as the conditions got worse or structurally deformed as cardiomyocytes inside the organoid extended into the matrix (
While each organoid was cultured under the Matrigel conditions in a multi-organ chip, steatohepatitis was induced by treating the liver organoid with oleic acid (500 μM), which is a type of free fatty acid, for 3 days, and the effects on the peripheral organoids were compared on day 3 after treatment with the fatty acid by comparing the expression of each marker by immunostaining. A multi-organ device in which a normal liver organoid (Normal) not treated with the fatty acid was used as a control group.
As shown in
These results demonstrate that when a fatty liver organoid model is induced in a multi-organ chip, not only a liver organoid but also the peripheral organs are affected, and, thus, the fatty liver organoid model serves as a disease model platform that reflects fluidal interactions between tissues. Therefore, it is expected that in vivo drug response and efficacy/toxicity can be evaluated more accurately than when a single fatty liver organoid model is applied.
In a decellularized tissue-derived scaffold-based multi-organ chip, steatohepatitis was induced by treating the liver organoid with oleic acid (500 μM), which is a fatty acid, for 3 days, and the effects on the peripheral organoids were compared on day 3 after treatment with the fatty acid by comparing the expression of each marker by immunostaining. A multi-organ device in which a normal liver organoid (Normal) not treated with the fatty acid was used as a control group.
As shown in
These results demonstrate that when a fatty liver organoid model is induced in a decellularized tissue-derived scaffold-based multi-organ chip, not only a liver organoid but also the peripheral organs are affected, and, thus, the fatty liver organoid model serves as a disease model platform that reflects fluidal interactions between tissues. Therefore, it is expected that in vivo drug response and efficacy/toxicity can be evaluated more accurately than when a single fatty liver organoid model is applied.
In a decellularized tissue-derived scaffold-based multi-organ chip, steatohepatitis was induced by treating the liver organoid with oleic acid (500 μM), which is a fatty acid, for 3 days, and the effects on the peripheral organoids were compared on day 3 after inducement of steatohepatitis by comparing the expression of each marker by quantitative PCR analysis. A multi-organ device in which a normal liver organoid (Normal) not treated with the fatty acid was used as a control group.
As shown in
These results demonstrate that when a fatty liver organoid model is induced in a decellularized tissue-derived scaffold-based multi-organ chip, not only a liver organoid but also the peripheral organs are affected, and, thus, the fatty liver organoid model serves as a disease model platform that reflects fluidal interactions between tissues. Therefore, it is expected that in vivo drug response and efficacy/toxicity can be evaluated more accurately than when a single fatty liver organoid model is applied.
In a decellularized tissue-derived scaffold-based multi-organ chip, steatohepatitis was induced by treating the liver organoid with a fatty acid, and the effects of four representative drugs, which have been recently eliminated after entering clinical trials as candidate drugs for non-alcoholic steatohepatitis, on the liver organoid were evaluated. Obeticholic acid (OCA), a semi-synthetic bile acid analog, is an agonist for FXR (farnesoid X receptor) to regulate bile acid metabolism, inflammation, fibrosis, and sugar/lipid metabolism. Ezetimibe (Eze), a therapeutic drug for hyperlipidemia, is a drug that selectively inhibits cholesterol absorption in the small intestine. Elafibranor (Ela), a dual agonist for PPARα/δ, is a drug that inhibits fatty acid biosynthesis and glucose biosynthesis in the liver and has been used as a drug for cardiometabolic diseases. Liraglutide (Lira), an agonist for glucagon-like peptide-1 (GLP-1), is a drug that has been used to treat diabetes. Each organoid cultured in a decellularized tissue-derived matrix was seeded into a multi-organ microfluidic device and then cultured in a normal culture medium suitable for each organ for 2 days. Then, the NASH group was treated with oleic acid (500 μM) and cultured for 3 days only in the chamber in which the liver organoid was cultured, and the groups treated with the respective drugs were treated with oleic acid (500 μM) and the respective drugs (50 μM) for 3 days in the chamber in which the liver organoid was cultured. A multi-organ device in which a normal liver organoid (Normal) not treated with the fatty acid was used as a control group.
As a result of optical microscopy on each group, it was confirmed that the liver organoids in the Normal group were cultured normally without internal inflammation or fatty acid accumulation. Also, it was confirmed that in the NASH-induced group, the inside of the organoids turned dark due to inflammation and fatty acid accumulation, and in the groups treated with the four candidate drugs whose efficacy in improving non-alcoholic steatohepatitis was demonstrated to some extent, inflammation and fatty acid accumulation were partially improved (
Further, the liver organoids of each group were disrupted, followed by cholesterol analysis to check whether the four candidate drugs are effective in improving low-density cholesterol (LDL) levels in the liver organoids. As a result, it was confirmed that the LDL levels in an OCA-treated group were similar to or higher than those in a No Treatment (NT) group, which confirmed that an increase of in vivo LDL level, which is known as a side effect of OCA, appeared in the multi-organ NASH model. Meanwhile, Eze is known as being effective in reducing LDL by acting as a cholesterol absorption inhibitor in the liver and intestine, and the LDL levels in an Eze-treated group were significantly lower than those in the No Treatment (NT) group. Ela and Lira also showed an improvement in LDL levels compared to the No Treatment (NT) group, and it was confirmed that Ela showed a greater improvement than Lira (
Furthermore, as a result of comparing the gene expression of each group by quantitative PCR analysis, it was confirmed that α-SMA and COL1A1, fibrosis and drug toxicity-related markers, were increased in the NASH group compared to the normal group, and improved by treatment with OCA, Eze, Ela, and Lira (
These results demonstrate that the multi-organ non-alcoholic steatohepatitis organoid model prepared in the present disclosure can mimic the effect of drug treatment in an actual NASH patient in vitro, and can serve as a disease model platform that reflects fluidal interactions between organs.
Further, in a decellularized tissue-derived scaffold-based multi-organ chip, steatohepatitis was induced by treating the liver organoid with a fatty acid, and the effects of four representative drugs, which have been recently eliminated after entering clinical trials as candidate drugs for non-alcoholic steatohepatitis, on the liver organoid were evaluated by immunostaining for key markers. Each organoid cultured in a decellularized tissue-derived matrix was seeded into a multi-organ microfluidic device and then cultured in a normal culture medium suitable for each organ for 2 days. Then, the NASH group was treated with oleic acid (500 μM) and cultured for 3 days only in the chamber in which the liver organoid was cultured, and the groups treated with the respective drugs were treated with oleic acid (500 μM) and the respective drugs (50 μM) for 3 days in the chamber in which the liver organoid was cultured. A multi-organ device in which a normal liver organoid (Normal) not treated with the fatty acid was used as a control group.
As a result of checking the expression of key markers in the liver organoids by immunofluorescence staining analysis, it was confirmed as shown in
These results demonstrate that the multi-organ non-alcoholic steatohepatitis organoid model prepared in the present disclosure can mimic the effect of drug treatment on an actual NASH patient in vitro, and can serve as a disease model platform that reflects fluidal interactions between organs. Therefore, it is expected that in vivo drug response and efficacy/toxicity can be evaluated more accurately than when a single fatty liver organoid model is applied.
In a decellularized tissue-derived scaffold-based multi-organ chip, steatohepatitis was induced by treating the liver organoid with a fatty acid, and the effects of four representative drugs, which have been recently eliminated after entering clinical trials as candidate drugs for non-alcoholic steatohepatitis, on the intestinal organoid were evaluated. Each organoid cultured in a decellularized tissue-derived matrix was seeded into a multi-organ microfluidic device and then cultured in a normal culture medium suitable for each organ for 2 days. Then, the NASH group was treated with oleic acid (500 μM) and cultured for 3 days only in the chamber in which the liver organoid was cultured, and the groups treated with the respective drugs were treated with oleic acid (500 μM) and the respective drugs (50 μM) for 3 days in the chamber in which the liver organoid was cultured. A multi-organ device in which a normal liver organoid (Normal) not treated with the fatty acid was used as a control group.
As a result of optical microscopy on each group, it was confirmed that the gut barrier of the intestinal organoid was well maintained in the Normal group and gut organoid-specific budding was observed (
As a result of comparing the gene expression of the intestinal organoids of the groups treated with the respective drugs by quantitative PCR analysis, it was confirmed as shown in
MUC2, intestinal differentiation markers, were decreased remarkably in the intestinal organoids of the NASH-induced No Treatment (NT) group, and recovered to some extent in the OCA-treated group. Meanwhile, it was confirmed that the expression levels of the differentiation markers were not much recovered in the Eze-, Ela- and Lira-treated groups, and in particular, the expression levels of the differentiation markers were lower in the Ela-treated group than in No Treatment (NT) group. When NASH is induced, the gut barrier of the intestine collapses and the permeability is greatly affected. Therefore, the gene expression for OCLN, a tight junction marker of the gut barrier, was compared. As a result, it was confirmed that the expression level of OCLN was decreased greatly in the No Treatment (NT) group, and recovered to some extent in the OCA-, Eze- and Lira-treated groups, but not improved in the Ela-treated group. FGF15 is highly expressed in intestinal tissue in liver-intestine interactions and is known as an endocrine factor that regulates bile acid synthesis in the liver. The expression of FGF15 is regulated by FXR (Farnesoid X receptor), and OCA acts as an FXR agonist and is known as recovering the decreased expression of FGF15 in the intestine and liver due to NASH. Actually, when NASH was induced, the gene expression of FGF15 in the intestinal organoid decreased in the multi-organ-based NASH organoid model, but was recovered to some extent in the Eze-treated group and recovered to a significant extent in the OCA-treated group.
These results demonstrate that the multi-organ non-alcoholic steatohepatitis organoid model prepared in the present disclosure can mimic the effect of drug treatment on other organs in an actual NASH patient in vitro, and can serve as a disease model platform that reflects fluidal interactions between organs.
Further, in a decellularized tissue-derived scaffold-based multi-organ chip, steatohepatitis was induced by treating the liver organoid with a fatty acid, and the effects of four representative drugs, which have been recently eliminated after entering clinical trials as candidate drugs for non-alcoholic steatohepatitis, on the liver organoid were evaluated by immunostaining for key markers. Specifically, each organoid cultured in a decellularized tissue-derived matrix was seeded into a multi-organ microfluidic device and then cultured in a normal culture medium suitable for each organ for 2 days. Then, the NASH group was treated with oleic acid (500 μM) and cultured for 3 days only in the chamber in which the liver organoid was cultured, and the groups treated with the respective drugs were treated with oleic acid (500 μM) and the respective drugs (50 μM) for 3 days in the chamber in which the liver organoid was cultured. A multi-organ device in which a normal liver organoid (Normal) not treated with the fatty acid was used as a control group.
As a result of checking the expression of key markers in the intestinal organoids by immunofluorescence staining analysis, it was confirmed as shown in
These results demonstrate that the multi-organ non-alcoholic steatohepatitis organoid model prepared in the present disclosure can mimic the effect of drug treatment on other organs in an actual NASH patient in vitro, and can serve as a disease model platform that reflects fluidal interactions between organs.
In a decellularized tissue-derived scaffold-based multi-organ chip, steatohepatitis was induced by treating the liver organoid with a fatty acid, and the effects of four representative drugs, which have been recently eliminated after entering clinical trials as candidate drugs for non-alcoholic steatohepatitis, on the pancreas organoid were evaluated. Each organoid cultured in a decellularized tissue-derived matrix was seeded into a multi-organ microfluidic device and then cultured in a normal culture medium suitable for each organ for 2 days. Then, the NASH group was treated with oleic acid (500 μM) and cultured for 3 days only in the chamber in which the liver organoid was cultured, and the groups treated with the respective drugs were treated with oleic acid (500 μM) and the respective drugs (50 μM) for 3 days in the chamber in which the liver organoid was cultured. A multi-organ device in which a normal liver organoid (Normal) not treated with the fatty acid was used as a control group.
As a result of optical microscopy on each group, it was confirmed as shown in
As a result of immunostaining of each group, it was confirmed that the expression levels of KRT19 and PDX1, pancreatic differentiation markers, were higher in the Normal group than in the other groups, and α-SMA and COL1, fibrosis and drug toxicity-related markers, were hardly expressed. However, it was confirmed that in the NASH-induced No Treatment (NT) group, the expression levels of KRT19 and PDX1 decreased and the expression levels of α-SMA and COL1 greatly increased. The expression levels of α-SMA and COL1 were significantly decreased in the Eze-treated group among the groups treated with the candidate drugs, and the groups treated with the other groups did not show remarkable improvement in fibrosis and recovery of expression of differentiation/functional markers (
Furthermore, as a result of comparing the gene expression of the pancreas organoids of the groups treated with the respective drugs by quantitative PCR analysis, it was confirmed as shown in
These results demonstrate that the multi-organ non-alcoholic steatohepatitis organoid model prepared in the present disclosure can mimic the effect of drug treatment on other organs in an actual NASH patient in vitro, and can serve as a disease model platform that reflects fluidal interactions between organs.
In a decellularized tissue-derived scaffold-based multi-organ chip, steatohepatitis was induced by treating the liver organoid with a fatty acid, and the effects of four representative drugs, which have been recently eliminated after entering clinical trials as candidate drugs for non-alcoholic steatohepatitis, on the cardiac organoid were evaluated. Each organoid cultured in a decellularized tissue-derived matrix was seeded into a multi-organ microfluidic device and then cultured in a normal culture medium suitable for each organ for 2 days. Then, the NASH group was treated with oleic acid (500 μM) and cultured for 3 days only in the chamber in which the liver organoid was cultured, and the groups treated with the respective drugs were treated with oleic acid (500 μM) and the respective drugs (50 μM) for 3 days in the chamber were cultured by treating with oleic acid (500 μM) and each drug (50 μM) for 3 days in the chamber in which the liver organoid was cultured. A multi-organ device in which a normal liver organoid (Normal) not treated with the fatty acid was used as a control group.
As a result of optical microscopy on each group, it was confirmed as shown in
As a result of immunostaining of each group, it was confirmed as shown in
As a result of comparing the gene expression of the cardiac organoids of the groups treated with the respective drugs by quantitative PCR analysis, it was confirmed as shown in
organoid model prepared in the present disclosure can mimic the effect of drug treatment on other organs in an actual NASH patient in vitro, and can serve as a disease model platform that reflects fluidal interactions between organs.
Human iPSC-derived liver organoids were differentiated into hepatic endoderm cells through endoderm, and then a total of 500,000 cells including the hepatic endoderm cells, vascular endothelial cells (HUVEC) and mesenchymal stem cells (hMSC) at a ratio of 10:7:2 were mixed in 10 μL of culture medium. The mixture was coated onto 10 μL of a decellularized liver tissue-derived matrix (LEM) to induce the formation of three-dimensional organoids. Human iPSC-derived intestinal organoids were differentiated into hindgut spheroids through endoderm, encapsulated in Matrigel, and matured for about 20 to 30 days. The mature intestinal organoids were extracted from Matrigel, re-encapsulated in a decellularized intestinal tissue-derived matrix (IEM), and further cultured. Human iPSC-derived pancreas organoids were differentiated into beta cells through endoderm, gut endoderm, pancreatic endoderm, and pancreatic progenitor cells, and then a total of 500,000 cells including the beta cells, vascular endothelial cells (HUVEC) and mesenchymal stem cells (hMSC) at a ratio of 10:7:2 were mixed in 10 μL of culture medium. The mixture was coated onto 10 μL of a decellularized pancreas tissue-derived matrix (PEM) to induce the formation of three-dimensional organoids. Human iPSC-derived cardiac organoids were induced to mature into cardiomyocytes through mesenchyma and cardiac progenitor cells, and then 400,000 cells were mixed in 20 μL of a decellularized heart tissue-derived matrix (HEM) to prepare three-dimensional organoids. In a decellularized tissue-derived scaffold-based multi-organ chip, steatohepatitis was induced by treatment of the liver organoids with a fatty acid, and the effects of representative drugs, which have been recently eliminated after entering clinical trials as candidate drugs for non-alcoholic steatohepatitis, on the liver and pancreas organoids were evaluated. Each organoids cultured in a decellularized tissue-derived matrix was seeded into a multi-organ microfluidic device and then cultured in a normal culture medium suitable for each organ for 2 days. Then, the NASH group was treated with oleic acid (500 μM) and cultured for 3 days only in the chamber in which the liver organoid was cultured, and the groups treated with the respective drugs were treated with oleic acid (500 μM) and the respective drugs (50 μM) for 3 days in the chamber in which the liver organoid was cultured. A multi-organ device in which a normal liver organoid (Normal) not treated with the fatty acid was used as a control group.
As a result of optical microscopy and immunofluorescence staining analysis of the human iPSC-derived liver organoids in each group, it was confirmed as shown in
As a result of optical microscopy and immunofluorescence staining analysis of the human iPSC-derived pancreas organoids in each group, it was confirmed as shown in
These results demonstrate that even when the multi-organ non-alcoholic steatohepatitis organoid model developed in the present disclosure is fabricated using human iPSC-derived organoids, it can mimic the effect of drug treatment on other organs in an actual NASH patient in vitro, and can serve as a disease model platform that reflects fluidal interactions between organs.
Human iPSC-derived liver organoids were differentiated into hepatic endoderm cells through endoderm, and then a total of 500,000 cells including the hepatic endoderm cells, vascular endothelial cells (HUVEC) and mesenchymal stem cells (hMSC) at a ratio of 10:7:2 were mixed in 10 μL of culture medium. The mixture was coated onto 10 μL of a decellularized liver tissue-derived matrix (LEM) to induce the formation of three-dimensional organoids. Human iPSC-derived intestinal organoids were differentiated into hindgut spheroids through endoderm, encapsulated in Matrigel, and matured for about 20 to 30 days. The mature intestinal organoids were extracted from Matrigel, re-encapsulated in a decellularized intestinal tissue-derived matrix
(IEM), and further cultured. Human iPSC-derived pancreas organoids were differentiated into beta cells through endoderm, gut endoderm, pancreatic endoderm, and pancreatic progenitor cells, and then a total of 500,000 cells including the beta cells, vascular endothelial cells (HUVEC) and mesenchymal stem cells (hMSC) at a ratio of 10:7:2 were mixed in 10 μL of culture medium. The mixture was coated onto 10 μL of a decellularized pancreas tissue-derived matrix (PEM) to induce the formation of three-dimensional organoids. Human iPSC-derived cardiac organoids were induced to mature into cardiomyocytes through mesenchyma and cardiac progenitor cells, and then 400,000 cells were mixed in 20 μL of a decellularized heart tissue-derived matrix (HEM) to prepare three-dimensional organoids. In a decellularized tissue-derived scaffold-based multi-organ chip, steatohepatitis was induced by treatment of the liver organoids with a fatty acid, and the effects of representative drugs, which have been recently eliminated after entering clinical trials as candidate drugs for non-alcoholic steatohepatitis, on the intestinal and cardiac organoids were evaluated. Each organoids cultured in a decellularized tissue-derived matrix was seeded into a multi-organ microfluidic device and then cultured in a normal culture medium suitable for each organ for 2 days. Then, the NASH group was treated with oleic acid (500 μM) and cultured for 3 days only in the chamber in which the liver organoid was cultured, and the groups treated with the respective drugs were treated with oleic acid (500 μM) and the respective drugs (50 μM) for 3 days in the chamber in which the liver organoid was cultured. A multi-organ device in which a normal liver organoid (Normal) not treated with the fatty acid was used as a control group.
As a result of optical microscopy and immunofluorescence staining analysis of the human iPSC-derived intestinal organoids in each group, it was confirmed as shown in
As a result of optical microscopy and immunofluorescence staining analysis of the human iPSC-derived cardiac organoids in each group, it was confirmed as shown in
These results demonstrate that even when the multi-organ non-alcoholic steatohepatitis organoid model developed in the present disclosure is fabricated using human iPSC-derived organoids, it can mimic the effect of drug treatment on other organs in an actual NASH patient in vitro, and can serve as a disease model platform that reflects fluidal interactions between organs.
The present disclosure has been described with reference to the preferred exemplary embodiments thereof. It can be understood by a person with ordinary skill in the art that the present disclosure can be implemented as being modified and changed within the scope departing from the spirit and the scope of the present disclosure. Accordingly, the above-described exemplary embodiments should be considered in descriptive sense only and not for purposes of limitation. Also, the technical scope of the present disclosure is defined not by the detailed description of the invention but by the appended claims, and all differences within the scope will be construed as being comprised in the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2021-0077660 | Jun 2021 | KR | national |
| 10-2022-0071910 | Jun 2022 | KR | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/KR2022/008401 | Jun 2022 | US |
| Child | 18539379 | US |
