Microfluidic droplet culture device for culturing cell aggregates

Abstract

The present invention relates to a microfluidic hanging drop culture device for culturing cell aggregates, and compared to conventional organoid / spheroid culture techniques, the organoid / spheroid cultured in the device of the present invention has the effect of having further enhanced stem cell activity and high differentiation degree, can be reused repeatedly, and can be used as a platform for various purposes by changing the size and number of wells. By using an agitator in the device, the culture solution located in the reservoir and the wells in the culture chamber continues to flow through the microchannel, and the environment of the entire well is maintained to be the same, so that the cell aggregates can be cultured with high efficiency. In addition, the cell aggregate culture system can be used as a disease research and drug screening model through mass production of cell aggregates that maintain disease phenotypes, and can also be applied to transplantation therapy for disease treatment through mass production of therapeutic cell aggregates.

Description

Technical Field

[0001] The present invention relates to a microfluidic hanging drop culture device for culturing cell aggregates.

Background Art

[0002] The technology for culturing tissue-specific organoids is currently the most cutting-edge field in stem cell research, and its applicability can be infinitely expanded in the fields of regenerative medicine and new drug research, such as refractory disease models, patient-ordered drug screening platforms, and in vitro models for the development of new drugs.

[0003] The technology of culturing cells using a microfluidic device is different from macroscale culture. It provides a fine environment suitable for cells and precisely adjusts the culture conditions of cells that are sensitive to the surrounding environment, and has been in the spotlight in the recent field of cell tissue engineering.

[0004] However, dynamic culture is different from static culture in that fluid flow is required, so complex equipment such as syringe pumps and hydraulic pumps, and a high level of proficiency are required for users.

[0005] In existing organoid research, as methods used to impart flow to the culture medium, a culture dish was mounted on an orbital shaker or a bioreactor such as a spinner flask was used. However, since the fluid flow provided to each organoid is non-uniform, it can cause a significant batch-to-batch variation, which has been pointed out as the biggest problem in organoid research.

[0006] Existing droplet culture techniques are performed by raising a microliter (20-50 μl) volume of cell culture medium onto a glass or Petri dish. The glass or Petri dish is then inverted. Due to surface tension, the droplet is prevented from falling, and the cells suspended in the culture medium are fixed by gravity. These fixed cells interact with neighboring cells, forming bonds and creating a three-dimensional sploid or microtissue. The biggest problem in such techniques is the exchange of culture medium from the droplet.

[0007] Here, the inventors have developed a microfluidic suspension culture device that can generate fluid flow without additional equipment using a commonly used stirring device in the laboratory, and have confirmed that it can enhance the viability, differentiation ability, and functionality of organoids. [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention aims to provide a cell aggregate culture device comprising a culture chamber containing one or more wells, one or more reservoirs for storing culture medium, and a microchannel connecting the culture chamber and the reservoirs.

[0009] The present invention aims to provide a cell aggregate culture system comprising the aforementioned device, a rocker, and a culture medium shared via a microchannel.

[0010] The present invention aims to provide a method for culturing cell aggregates using the aforementioned culture system. [Means for solving the problem]

[0011] One aspect of the present invention provides a cell aggregate culture device comprising a culture chamber having one or more wells, one or more reservoirs for storing culture medium, and a microchannel connecting the culture chamber and the reservoirs.

[0012] As one specific example of the present invention, the culture chamber may further include microchannels connecting a plurality of wells.

[0013] As a specific example of the present invention, the diameter of the well may be 1.5 to 4 mm, and the spacing between the wells connected by the microchannel may be 1.5 to 5 mm.

[0014] As a specific example of the present invention, the reservoir may be located at both ends of the device.

[0015] As a specific example of the present invention, the cell aggregate may be a speroid or organoid derived from any one of the following: mesenchymal stem cells, neural stem cells, vascular endothelial cells, induced pluripotent stem cells, germinal stem cells, tissue stem cells, fetal stem cells, cancer stem cells, and cardiac cells.

[0016] As a specific example of the present invention, the cell aggregate may be derived from one of the group consisting of the brain, optic cup, kidney, liver, pancreas, neural tube, stomach, large intestine, prostate, breast, heart, salivary gland, endometrium, mammary gland, thyroid gland, tongue, small intestine, esophagus, spinal cord, skin, bile duct, lung, blood vessels, muscle, adrenal cortex, and thyroid organoids.

[0017] Another aspect of the present invention provides a cell aggregate culture system comprising the device, a rocker, and a culture medium shared via a microchannel.

[0018] As one specific example of the present invention, the device may undergo a swinging motion due to the agitator.

[0019] Another aspect of the present invention provides a method for culturing cell aggregates using the culture system described above. [Effects of the Invention]

[0020] The cell aggregate culture device of the present invention, the cell aggregate culture system including the same, and the method for culturing a cell aggregate using the same have enhanced stem cell activity and high differentiation efficiency compared to conventional cell aggregate culture techniques.

[0021] The cell aggregate culture device can be repeatedly reused and can be utilized as a platform adaptable to various applications by changing the well size and number.

[0022] In addition, the cell aggregate culture system utilizes a stirrer so that the culture solution located in the reservoir and the wells in the culture chamber continuously flows through the microchannel, maintaining the environment of the entire well to be the same, and enabling the cell aggregate to be cultured uniformly and efficiently.

[0023] The cell aggregate culture system can be utilized as a disease research and drug screening model through mass production of cell aggregates in which disease phenotypes are maintained. Furthermore, it can also be applied to transplantation therapy for treating diseases through mass production of therapeutic cell aggregates.

Brief Description of the Drawings

[0024] [Figure 1] It is a diagram showing the structure of the hanging drop chip (HD chip) of the present invention. [Figure 2] It is a diagram showing the structure and culture method of the hanging drop chip (HD chip). [Figure 3] It is a diagram showing the structure and culture method of the hanging drop chip (HD chip). [Figure 4] It is a photograph of the formation and culture of spheroids using the hanging drop chip (HD chip). [Figure 5] It is a diagram showing the reusability of the hanging drop chip (HD chip) fabricated with PDMS. [Figure 6]This is a photograph showing the formation methods of various cell aggregates using a hanging drop culture chip (HD chip). [Figure 7] This is a photograph showing the results of culturing adipose-derived stem cell spheroids (hADSC spheroids) for comparison with the existing Petri dish hanging drop culture method. [Figure 8] This is a photograph showing the results of culturing adipose-derived stem cell spheroids (hADSC spheroids) for comparison with the existing U-bottom well-plate culture method. [Figure 9] This is a photograph showing the results of culturing neural stem cell spheroids (hNSC spheroids) as an example of applying various spheroid / organoid cultures. [Figure 10] This is a photograph showing the results of culturing cardiac spheroids as an example of applying various spheroid / organoid cultures. [Figure 11] This is a photograph showing the results of culturing human iPSC-derived brain organoids as an example of applying various spheroid / organoid cultures. [Figure 12] This is a figure showing the analysis results of the difference in differentiation ability due to the cell composition of human iPSC-derived liver organoids (Human iPSC-derived liver organoid) cultured in an HD chip. [Figure 13] This is the result of comparing the differentiation ability of long-term culture of human iPSC-derived liver organoids (Human iPSC-derived liver organoid, HEM). [Figure 14] This is a figure showing the analysis results of the marker expression of human iPSC-derived liver organoids (Human iPSC-derived liver organoid, HEM). [Figure 15] This is a figure showing the analysis results of the marker expression of human iPSC-derived liver organoids (Human iPSC-derived liver organoid, HEM). [Figure 16]This figure shows the results of the analysis of marker expression and functionality in liver organoids derived from human iPSCs. [Figure 17] This figure shows the comparative results of marker expression during long-term culture of liver organoids derived from human iPSCs. [Figure 18] This figure shows the results of manufacturing a high-throughput HD chip. [Figure 19] This figure shows the results of manufacturing a high-throughput HD chip. [Figure 20] This figure shows the results of manufacturing a high-throughput HD chip. [Figure 21] This figure shows the results of an analysis of the homogeneity of human iPSC-derived liver organoids produced using a 100-well HD chip. [Figure 22] This figure shows the results of a comparison of the uniformity of gene expression in human iPSC-derived liver organoids cultured on high-throughput HD chips. [Figure 23] This figure shows the results of mass production and functional analysis of normal and non-alcoholic steatohepatitis organoids derived from human iPSCs. [Figure 24] This figure shows the mass production of non-alcoholic steatohepatitis organoids derived from human iPSCs, and the results of testing for effective drugs. [Figure 25] This figure shows the results of mass production of non-alcoholic steatohepatitis organoids derived from human iPSCs, and quantitative analysis of ROS. [Figure 26] This figure shows the results of large-scale culture of human iPSC-derived pancreas organoids compared to existing microwell and U-bottom well plate methods. [Figure 27]This figure shows the results of comparing the expression of markers and differentiation potential of human iPSC-derived pancreas organoids. [Figure 28] This figure shows the comparative results of insulin production from human iPSC-derived pancreas organoids. [Figure 29] This figure shows the results of spheroid (human adipose-derived stem cell (hADSC) spheroid) fusion via chip-to-chip transfer. [Figure 30] This figure shows the results of comparing the fusion methods between different organs: human iPSC-derived liver organoids and pancreatic and blunt organoids. [Figure 31] This figure shows the results of highly efficient fusion between adipose-derived stem cell spheroids (hADSC spheroids) using a high-throughput HD chip that was fabricated. [Figure 32] This figure shows the results of highly efficient fusion of liver-pancreatic-bone organoids on an HD chip substrate. [Figure 33] This figure shows the results of transplanting liver organoids cultured on an HD chip (Human iPSC-derived liver organoid) into a mouse model of steatohepatitis. [Figure 34] This figure shows the results of transplanting liver organoids cultured on an HD chip (Human iPSC-derived liver organoid) into a mouse model of steatohepatitis. [Figure 35] This figure shows the results of on-chip drug screening and fluorescence quantitative analysis (hADSC-human adipose-derived stem cell sperm) using a plate reader. [Figure 36] This figure shows the results of fabricating a prototype HD chip using 3D printing. [Figure 37]This figure shows the results of 3D speroid / organoid culture using accessories. [Figure 38] This figure shows the culture results of a multilayered, three-dimensional adipose-derived stem cell (ADSC) speroid using accessories. [Modes for carrying out the invention]

[0025] One aspect of the present invention provides a cell aggregate culture device comprising a culture chamber having one or more wells, one or more reservoirs for storing culture medium, and a microchannel connecting the culture chamber and the reservoirs.

[0026] The culture chamber includes one or more wells, each filled with a culture medium, allowing for the cultivation of cell aggregates. The wells in the culture chamber can be arranged in a single row, or they can be configured in multiple rows, depending on the scale and application of the culture.

[0027] The reservoir is a device that supplies and shares culture medium to the culture chamber via a microchannel, and can be manufactured in various forms and in various numbers, taking into consideration the purpose of the experiment.

[0028] The aforementioned microchannel connects the culture chamber and the reservoir, and its shape, size, length, etc., can be changed without restriction as long as the culture medium can flow through it.

[0029] As a concrete example of the present invention, the culture chamber may further include microchannels connecting a plurality of wells. Since the culture chamber includes one or more wells, it can share the culture medium between the plurality of wells and the reservoir and maintain a constant environment by including not only microchannels between the culture chamber and the reservoir, but also microchannels between the wells. On the other hand, microchannels between the plurality of wells may be formed only between some of the wells, taking into consideration the purpose of some experiments.

[0030] In one specific example of the present invention, the diameter of the well may be 1.5 to 4 mm, specifically 2 to 3.1 mm, and the spacing between the wells connected by the microchannel may be 1.5 to 5 mm, specifically 2.5 to 4.5 mm. More specifically, the diameter of the well may be 2.0 mm or 3.1 mm, and the spacing between the wells connected by the microchannel may be 2.5 mm or 4.5 mm, most specifically, if the diameter of the well is 2.0 mm, the spacing between the wells may be 2.5 mm, and if the diameter of the well is 3.1 mm, the spacing between the wells may be 4.5 mm.

[0031] As a specific example of the present invention, the reservoir may be located at both ends of the device. The reservoir is located at both ends of the device and is connected to one or more wells of the culture chamber by microchannels, and each well is also connected by microchannels, so that the reservoir-one or more wells-reservoirs are connected and the culture medium can be supplied and shared throughout.

[0032] As a specific example of the present invention, the cell aggregate may be a speroid or organoid derived from any one of the following: mesenchymal stem cells, neural stem cells, vascular endothelial cells, induced pluripotent stem cells, germinal stem cells, tissue stem cells, fetal stem cells, cancer stem cells, and cardiac cells.

[0033] The term "speroid" refers to an aggregate of spherical cells. In practice, "spherical" is not limited to perfectly spherical objects, but may also include forms that are slightly flattened.

[0034] The term "organoid" refers to a micro-organism fabricated in the form of an artificial organ by culturing cells derived from tissue or fully differentiated stem cells in a 3D morphology. The organoid can develop into a specific tissue through restricted element (e.g., growth factor) patterning, as a three-dimensional tissue analog containing organ-specific cells that originate from stem cells and self-organize (or self-pattern) in a manner similar to that in vivo. The organoid possesses the intrinsic physiological characteristics of the cells and can have an anatomical structure that mimics the original state of the cell mixture (including not only limited cell types but also residual stem cells and the surrounding physiological niche). Through the 3D culture method, the organoid can have a more well-arranged arrangement of cells and cell functions, resulting in a functional organ-like morphology and tissue-specific functions.

[0035] As a specific example of the present invention, the cell aggregate may be one or more selected from the group consisting of the brain, optic cup, kidney, liver, pancreas, neural tube, stomach, large intestine, prostate, breast, heart, salivary gland, endometrium, mammary gland, thyroid gland, tongue, small intestine, esophagus, spinal cord, skin, bile duct, lung, blood vessel, muscle, adrenal cortex, and thyroid organoid.

[0036] Another aspect of the present invention provides a cell aggregate culture system comprising the device, a rocker, and a culture medium shared via a microchannel.

[0037] The aforementioned "rocker" can impart a dynamic flow to the culture medium by moving the culture device at a constant period. It is sufficient for the rocker to be able to impart a dynamic flow to the culture medium by changing the position of the device, and the range and form of movement are not particularly limited.

[0038] The aforementioned "culture medium" is both a culture medium for cells and a transfer medium for nutrients, oxygen, and other substances. The culture medium can supply cells with the nutrients and oxygen they need and remove waste products.

[0039] As one specific example of the present invention, the device may undergo a swinging motion due to the agitator.

[0040] The aforementioned "swing motion" refers to the operating mode of a mechanical device, meaning that the drive part does not rotate around an axis, but rather moves back and forth over a certain distance.

[0041] Since the device oscillates at a constant frequency, the culture medium within the device can reciprocate at a constant frequency within the chamber, thereby creating an environment in which the cell aggregate can be cultured stably.

[0042] Another aspect of the present invention provides a method for culturing cell aggregates using the culture system described above.

[0043] The aforementioned culture refers to the process of maintaining and growing cells under suitable conditions, which may include, for example, the temperature at which the cells are maintained, the solubility of nutrients, the CO2 content of the atmosphere, and the cell density.

[0044] Appropriate culture conditions for maintaining, proliferating, expanding, and differentiating different cell types are known and documented in the art. Conditions suitable for the formation of such cell aggregates may also facilitate or allow cell differentiation and the formation of multicellular structures. [Examples]

[0045] One or more specific examples will be described in more detail below through the examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[0046] Example 1: Manufacturing of an organoid culture device

[0047] A chip consisting of a culture chamber where organoids are cultured and a reservoir for the culture medium has been realized, allowing for the selection of well diameter and spacing depending on the experimental objective and the characteristics of the organoids. Specifically, if the goal is highly efficient organoid formation, the size and spacing of the wells can be reduced to increase culture efficiency. If the goal is not only organoid formation but also analysis, a standardized plate reader that matches the specifications of existing plates can be used.

[0048] On the other hand, the surface tension of the culture medium can be used to maintain the flow of the culture medium and the droplets of culture medium that enclose the cells. The HD chip was basically fabricated using PDMS polymer in the same way as a general microfluidic chip.

[0049] The specific method for fabricating HD chips involves designing the desired plate pattern (Figure 1), then creating a silicon wafer with the engraved pattern through a lithography process. Using this as a mold, a device pattern PDMS (Polydimethylsiloxane) is fabricated through a soft lithography process. The PDMS is then shaped with a blade, and holes for the well chamber and culture medium inlet are punched using a biopsy punch. The formed elements are activated by irradiating the surface with oxygen plasma at 60W for 1 minute, and then bonded together. For complete bonding, the elements are placed overnight in an oven at over 70 degrees Celsius, followed by sterilization through high-temperature, high-pressure sterilization and UV irradiation.

[0050] As a result, 96-well devices and 25-well devices were manufactured, as shown at the top of Figure 2.

[0051] Furthermore, it was confirmed that when human adipose-derived stem cells (hADSCs) were cultured in the fabricated device, the cells solidified and formed spleoids within one day (within 6 hours in the case of hADSCs).

[0052] Specifically, human adipose-derived stem cells (hADSCs) were placed in the 25-well device manufactured in Example 1 and cultured.

[0053] When the cell suspension was injected into the 25-well device, gravity caused the cells to sink, and droplets of culture medium accumulated at the ends. Over time, the cells clumped together, and a speleoid / organoid morphology at a level of 6000 cells / organoid was observed (bottom of Figure 2).

[0054] On the other hand, the device of the present invention can continuously mix the culture medium in both culture medium reservoirs with the culture medium in each culture chamber using a rocker, thereby exchanging the consumed culture medium with secreted waste products.

[0055] Through this, we confirmed that the droplet structure of the culture medium is stably maintained within the HD chip even on the agitator, and that the culture medium can be easily exchanged through both reservoirs (Figure 3).

[0056] Experimental Example 1: Formation of spermoid using a suspension culture chip (HD chip) and confirmation of culture results.

[0057] By increasing the number of culture chambers (96 wells), it was confirmed that it could be used as a highly efficient platform for speroid / organoid formation (left side of Figure 4). Through these results, it is possible to easily inject cells in a single step and form large quantities of speroids / organoids.

[0058] As shown on the right side of Figure 4, it was confirmed that by using the HD chip, it is possible to form speroids of uniform size, the size of the speroids can be adjusted according to the number of cells injected, and stable speroid culture is possible from the initial stage of speroid formation to long-term culture without adhering to or deforming the surface of the device.

[0059] Experimental Example 2: Confirmation of the reusability of suspension culture chips (HD chips) fabricated with PDMS.

[0060] The reusability of the suspension culture chips (HD chips) fabricated using PDMS was confirmed.

[0061] HD chip devices can be manufactured using a variety of materials, but those made from PDMS material, in particular, can be reused after sterilization.

[0062] When the suspension culture chips were fabricated using PDMS and reused more than 10 times for culturing human adipose-derived stem cells (hADSCs), hADSC speroids were consistently formed each time, and a high cell viability rate was confirmed without cell death (Figure 5).

[0063] Experimental Example 3: Confirmation of diverse cell aggregate formation methods using a suspension culture chip (HD chip) (Enabling the formation of three-dimensional speroids and organoids through various on-chip cell / gel loading methods)

[0064] While most existing speroid / organoid culture systems are simple three-dimensional cultures, the HD chip system developed in this invention enables a variety of on-chip three-dimensional culture methods that are difficult to achieve with existing culture systems.

[0065] Specifically, it was confirmed that it is possible to culture three-dimensional cell aggregates in various forms, including (1) cell spheroids, (2) culture on a hydrogel (gel bed), (3) culture within a hydrogel (gel encapsulation), (4) complex cultures combining these, and (5) conjugation of different hydrogels (gel+gel fusion) (Figure 6).

[0066] Furthermore, it is possible to inject additional cells during the culture process, which is expected to enable the production of advanced speleoids and organoids composed of diverse cell types.

[0067] Experimental Example 4: Comparison with Existing Culture Methods

[0068] Experimental Example 4-1. Comparison with existing Petri dish suspension culture method (culture of adipose-derived stem cell spheroids (hADSC spheroids))

[0069] The conventional hanging drop culture method using Petri dish lids has several drawbacks: it requires skill to form uniform culture droplets, changing the culture medium is very cumbersome, the total volume of culture medium is small, and the viability of cells sensitive to the culture environment, as well as cells that require time to form speleoids / organoids, is significantly reduced.

[0070] In contrast, when using an HD chip that stably supplies culture medium via microchannels, it is possible to form uniform sperm / organoids with improved cell viability and activity, as shown in Figure 7.

[0071] Experimental Example 4-2. Comparison with existing U-bottom well plate culture method (culture of adipose-derived stem cell spheroids (hADSC spheroids))

[0072] In the case of the currently widely used U-bottom well plate, unlike the suspension culture in the culture dish compared above, it showed high cell viability. However, after 6 days of culture, a live / dead assay revealed that the speroid morphology was irregular and had a relatively rough structure, which was judged to be due to contact with the bottom surface. In contrast, when adipose-derived stem cell speroids were cultured on an HD chip, which lacks a bottom surface to which cells can contact or adhere, a uniform spherical pattern and high cell density were observed, and excellent cell junctions were formed (Figure 8A).

[0073] mRNA was extracted from sperm cultured in each well of a U-bottom plate and from sperm cultured in each well of an HD chip. qPCR was performed on the stem cell gene (Oct4, stemness marker). The results showed that Oct4 expression was significantly increased in the HD chip group compared to the U-bottom group (Figure 8B), and the uniformity of expression was greatly improved (Figure 8C).

[0074] Therefore, these results demonstrate that using the HD chip culture system makes it possible to produce stem cell sperm spleroids of even higher quality compared to existing methods.

[0075] Experimental Example 5: Culturing of various speroids / organoids

[0076] Experimental Example 5-1. Culture of neural stem cell spheroids (hNSC spheroids)

[0077] Human neural stem cells (hNSCs) generally form spleroids by spontaneously clumping together during suspension culture in a Petri dish. However, because spleroids are formed randomly in a Petri dish, they exhibit diverse sizes and morphologies, resulting in low uniformity and significant differences in the proliferation and differentiation potential of the cells constituting each spleroid.

[0078] Comparative analysis of neural stem cell sperm cultured on HD chips and those cultured on the same Petri dish revealed that the sperm cultured on HD chips formed with greater uniformity in size (Figure 9A). When qPCR was performed on day 6 of culture to analyze stem cell gene (Oct4, stemness marker) expression, the expression of the Oct4 gene in individual sperm formed on HD chips was found to be more uniform than in sperm formed on Petri dishes (Figure 9B).

[0079] Experimental Example 5-2. Culture of cardiac spheroids

[0080] The HD chip culture system was applied to produce cardiac spermoids using cardiomyocytes induced by direct reprogramming from mouse fibroblasts. As shown in Figure 10, compared to existing methods such as suspension culture in petri dishes or culture in U-bottom well plates, the cardiac spermoids formed through HD chip culture were structurally more developed, and the expression of the cardiomyocyte marker (α-actinin) was significantly improved, resulting in a clearer α-actin pattern.

[0081] Furthermore, the cardiac sperm cultured on the HD chip exhibited a more clearly defined α-actin structure and a more patterned arrangement with adjacent cells. This indicates that using the HD chip allows for the production of cardiac sperm with higher maturity and functionality in electrophysiological aspects, such as cardiac beating, compared to existing methods.

[0082] Experimental Example 5-3. Culture of brain organoids (human iPSC-derived brain organoids)

[0083] Human-induced pluripotent stem cell (human iPSC)-derived brain organoids were formed under various conditions, and their morphology was observed (Figures 11A and 11B) while they were cultured. The results of qPCR to analyze the expression of neural differentiation markers (Figure 11C) were also confirmed (Pax6 - 25 days of culture, and MAP2 - 27 days of culture).

[0084] (Figures 11A and 11B) Compared to brain organoids cultured in a control group (U-bottom well plate), the size of brain organoids cultured in the HD chip was confirmed to be even larger. (Figure 11C) When the gene expression of vascular cells (ECs) and brain organoids cultured in empty cells on the HD chip was examined, it was confirmed that the expression of the neural progenitor cell marker (Pax6) and the neuronal cell marker (MAP2) was significantly increased.

[0085] Experimental Example 6: Confirmation of liver organoid culture (Human iPSC-derived liver organoid)

[0086] Experimental Example 6-1. Analysis of differences in differentiation potential based on the cell composition of liver organoids cultured on an HD chip.

[0087] To analyze the differences in differentiation potential between two types of human iPSC-derived liver organoids cultured on HD chips (HM-iPSC-derived hepatocytes (H): mesenchymal stem cells (M) = 10:2, HEM-iPSC-derived hepatocytes (H): vascular endothelial cells (E): mesenchymal stem cells (M) = 10:7:2), marker expression was compared and analyzed using immunohistochemistry and quantitative PCR (qPCR) on days 5 and 7 of culture.

[0088] As a result, as shown in Figure 11A, both HM and HEM liver organoids cultured on HD chips expressed HNF4A and ALB, which are liver-specific differentiation markers, and in the case of HEM, it was confirmed that CD31, a vascular marker, was also well expressed.

[0089] Furthermore, as shown in Figure 11B, when comparing gene expression between the HM and HEM groups on day 7 of culture, we observed a tendency for increased expression of liver differentiation markers (AFP, FOXA2, HNF4A, ALB) in the HEM group containing vascular endothelial cells, confirming that liver organoids containing vascular endothelial cells develop into more mature liver organoids.

[0090] These results demonstrate that the HD chip enables efficient culture of liver organoids composed of diverse cell types.

[0091] Experimental Example 6-2. Comparison of Differentiation Potential of Human iPSC-Derived Liver Organoids (HEMs) in Long-Term Culture

[0092] Human iPSC-derived liver organoids (HEMs) containing vascular endothelial cells were cultured for 20 days in various culture systems, and then gene expression against diverse markers was compared.

[0093] As a result, as shown in Figure 13, a significant increase in differentiation markers was observed in liver organoids cultured on HD chips compared to the control group (U-bottom plate and microwell). Both liver differentiation-related markers (AFP, ALB) and vascular markers (PECAM1, CD34, CDH5) showed increased expression in the HD chip group, while the cell death marker CASP3 showed a slight decrease in the HD chip group.

[0094] Therefore, compared to existing organoid culture systems such as U-bottom well-plates and microwells, it was confirmed that using HD chips can enhance the differentiation of hepatocytes and the maturation of blood vessels in liver organoids (HEMs), reduce cell death, and enable the production of liver organoids of superior quality.

[0095] Experimental Example 6-3. Analysis of Marker Expression in Human iPSC-Derived Liver Organoids (HEMs)

[0096] Human iPSC-derived liver organoids (HEMs) containing vascular endothelial cells were cultured for 15 days in each culture system, and then the expression of liver differentiation markers and vascular markers among the groups was compared by immunohistochemistry.

[0097] As a result, as shown in Figure 14, compared to the control group (U-bottom plate and microwell), the liver organoids cultured in the HD chip showed even higher expression levels of HNF4A, a liver differentiation marker, and also showed increased expression of CD31, a vascular marker. The patterns and morphology of the formed liver organoids were also found to be more uniform compared to the control group.

[0098] Therefore, compared to existing organoid culture systems such as U-bottom well-plates and microwells, the use of HD chips can enhance the differentiation of hepatocytes and the maturation of blood vessels in liver organoids (HEMs), thus confirming that it is possible to produce liver organoids of superior quality.

[0099] After culturing human iPSC-derived liver organoids (HEMs) containing vascular endothelial cells in each culture system for 15 days, the expression of liver differentiation markers and vascular markers among the groups was compared by immunohistochemistry.

[0100] As a result, as shown in Figure 15, compared to the control group (U-bottom plate and microwell), liver organoids cultured in HD chips showed even higher expression levels of ALB, a liver differentiation marker, and increased expression of CD31, a vascular marker. The patterns and morphologies of the formed liver organoids were also found to be more uniform compared to the control group.

[0101] Therefore, compared to existing organoid culture systems such as U-bottom well-plates and microwells, the use of HD chips can enhance the differentiation of hepatocytes and the maturation of blood vessels in liver organoids (HEMs), thus confirming that it is possible to produce liver organoids of superior quality.

[0102] Experimental Example 6-4. Analysis of marker expression and functionality in liver organoids derived from human iPSCs.

[0103] After culturing human iPSC-derived liver organoids in each culture system for 15 days, the expression of markers between groups was compared via immunohistochemistry, and the ability to synthesize elements, one of the important indicators of liver function, was compared.

[0104] As a result, as shown in Figure 16A, compared to the control group (U-bottom well-plate and microwell), liver organoids cultured on HD chips showed even higher expression of the liver differentiation markers AFP and HNF4A. Furthermore, when the actin filament structure was examined through F-actin staining, it was confirmed that organoids with a more uniform pattern were formed in the HD chip group.

[0105] When comparing the ability to synthesize elements, an indicator of liver function, Figure 16B shows that the ability to synthesize elements in liver organoids cultured in HD chips was significantly increased compared to liver organoids cultured in the control system.

[0106] Therefore, it was confirmed that using HD chips makes it possible to produce liver organoids with improved functionality compared to existing organoid culture systems such as U-bottom well-plates and microwells.

[0107] Experimental Example 6-5. Comparison of marker expression during long-term culture of liver organoids derived from human iPSCs.

[0108] Human iPSC-derived liver organoids were cultured in various culture systems for 10, 15, and 30 days, and then the expression of markers was compared by immunohistochemistry.

[0109] As a result, as shown in Figure 17, it was confirmed that the pattern of liver organoids cultured in HD chips remained uniform over a long period compared to the control group (U-bottom plate and microwells), and that HNF4A and ALB, which are mature liver differentiation markers (mid- and late-stage markers), were expressed at even higher levels. Furthermore, when cultured for more than 30 days, liver organoids cultured in U-bottom plates and microwells died, but liver organoids cultured in HD chips formed liver tissue-specific structures (high distribution image of HD chip group), and it was confirmed that more mature organoids were formed.

[0110] Experiment Example 7: Fabrication of a high-throughput HD chip

[0111] Experimental Example 7-1. Fabrication of a high-throughput HD chip

[0112] To improve the culture efficiency of the 25-well HD chip, we fabricated a high-efficiency HD chip that, like the existing 25-well HD chip, has the specifications of a 384-well plate and an increased number of culture chambers (Figure 18).

[0113] The highly efficient HD chip allows for simultaneous suspension culture of speroids / organoids in 100 wells and is designed to be stacked perpendicularly to each other, similar to existing 25-well HD chips, and can be cultured on a tray plate.

[0114] To investigate the fluid flow characteristics in a high-efficiency HD chip, we conducted simulations, specifically a time-dependent study under a 10 rpm condition.

[0115] We confirmed that fluid fluidity was relatively high near the channel, while the shear stress was low on the organoid surface (Figure 19).

[0116] This demonstrates that the HD chip is suitable for culturing delicate organoids because it uses a continuous flow to enable the exchange of substances through smooth circulation of the culture medium, while also allowing organoids to move to other chambers or avoid direct damage from the flow.

[0117] Simulations were performed to compare the oxygen transfer rate into the organoid using a U bottom plate and an HD chip. The results of the simulation under a steady state condition through average flow represent the overall average values ​​simulated during culture.

[0118] Figure 20A shows the oxygen concentration in the HD chip and the oxygen concentration in the U bottom plate well (Figure 20B) in three dimensions (left) and cross-section (right), respectively. Figure 20C shows the numerical oxygen concentration graph corresponding to the red arrows in each cross-sectional image.

[0119] Simulation results confirmed that the oxygen concentration in the center of the organoid was higher when cultured on an HD chip than when cultured on a U-bottom plate. Therefore, it is predicted that the HD chip culture system will be even more effective in organoid culture, where substance transfer to the center of the organoid, where cells are densely concentrated, is important.

[0120] Experimental Example 7-2. Analysis of the homogeneity of human iPSC-derived liver organoids produced using a high-efficiency chip (100-well HD chip).

[0121] To confirm whether uniform mass production of human iPSC-derived liver organoids is possible in a highly efficient 100-well HD chip, iPSC hepatocytes (H):vascular endothelial cells (E):intermediate lobe stem cells (M) were seeded in a ratio of 10:7:2, with 6,000 cells per well, for a total of 600,000 cells per chip. To confirm whether the formed liver organoids possessed proliferative capacity, this was confirmed by Ki67 immunostaining.

[0122] As a result, as shown in Figure 21A, when 600,000 cells were sown on the HD chip, it was confirmed that the cells uniformly gathered and solidified in the culture medium droplets inside all the wells, and within 24 hours, 100 organoids of uniform size were formed.

[0123] When 100 organoids, one formed per well, were stained with the cell proliferation marker Ki67, it was confirmed that active and uniform cell proliferation occurred within the organoids (Figure 21B).

[0124] Therefore, the HD chip is expected to be more suitable than existing organoid culture systems such as U-bottom well plates and microwells for the simultaneous mass production of uniform liver organoids.

[0125] Experimental Example 7-3. Comparison of the uniformity of expression of human iPSC-derived liver organoid genes cultured on high-throughput HD chips.

[0126] We compared the uniformity of gene expression for liver tissue markers between 100 human iPSC-derived liver organoids (HEMs) cultured simultaneously in a high-efficiency 100-well HD chip and liver organoids cultured in an existing U-bottom well plate.

[0127] As a result, as shown in Figure 22, quantitative PCR analysis was performed on representative liver differentiation markers AFP, HNF4A, and ALB. Compared to the gene expression distribution of liver organoids cultured in existing U-bottom well plates, the gene expression deviations in liver organoids produced using HD chips were smaller, and it was confirmed that they were meaningfully uniform. This validates that it is possible to produce liver organoids with uniform expression of major differentiation markers through highly efficient suspension culture using HD chips.

[0128] Experimental Example 7-4. Mass production and functional analysis of normal and non-alcoholic steatohepatitis organoids derived from human iPSCs.

[0129] After producing human iPSC-derived liver organoids (HEMKS) in a highly efficient 100-well HD chip, the normal group was cultured in normal culture medium for 7 days, while the non-alcoholic steatohepatitis (NASH) group was cultured for an additional 2 days after 5 days of normal culture by mixing oleic acid, a free fatty acid, into the culture medium and culturing for an additional 2 days to induce steatohepatitis. After a total of 7 days of culture, the degree of fat accumulation was compared between the normal and steatohepatitis groups, and cytochrome activity (CYP3A4 activity), one of the important indicators of liver function, was compared. For more accurate steatohepatitis modeling and drug screening, liver organoids were produced including epidemic cells (Kupffer cells) and stellate cells (hepatic stellate cells), which constitute the microenvironment of liver tissue. Therefore, liver organoids derived from human iPSCs were created using the following ratio (HEMKS): iPSC-derived hepatocytes (H): vascular endothelial cells (E): mesenchymal stem cells (M): iPSC-derived Kupffer cells (K): iPSC-derived hepatic stellate cells (S) = 10:7:2:2:1.

[0130] As a result, as shown in Figure 23A, in the case of mass-produced normal liver organoids, actin filaments (F-actin) were well distributed within the organoids, and the BODIPY fluorescence signal that labels lipids was hardly observed. On the other hand, in the case of NASH organoids, the F-actin structure was observed abnormally, confirming that lipid accumulation had occurred inside the organoids.

[0131] When comparing cytochrome activity, an indicator of liver function, as shown in Figure 23B, we confirmed that the normal organoid group showed approximately twice the activity compared to the NASH-induced organoid group. This confirmed that we successfully created NASH organoids in which liver function was inhibited by the induction of fatty liver disease.

[0132] Therefore, it was confirmed that by utilizing a highly efficient HD chip, it is possible to mass-produce not only normal liver organoids but also non-alcoholic steatohepatitis organoids exhibiting disease phenotypes.

[0133] Experimental Example 7-5. Mass Production and Effective Drug Testing of Non-Alcoholic Steatohepatitis Organoids Derived from Human iPSCs

[0134] After producing human iPSC-derived liver organoids (HEMKS) in a high-efficiency 100-well HD chip, the normal group was cultured in a normal culture medium for 7 days, while the non-alcoholic steatohepatitis (NASH) group was cultured for an additional 2 days after 5 days of normal culture by mixing oleic acid (a free fatty acid) at a concentration of 500 μM into the culture medium and culturing for an additional 2 days to induce steatohepatitis. Ezetimibe (Eze), an effective drug for treating fatty liver, is a candidate drug that has been used as a cholesterol absorption inhibitor to treat hyperglycemia, high cholesterol, and dyslipidemia. After 5 days of normal culture, oleic acid (500 μM) and Ezetimibe (50 μM) were mixed into the culture medium and cultured for an additional 2 days to perform a drug test for steatohepatitis. After a total of 7 days of culture, the degree of fat accumulation was compared, and the expression of major markers and genes between the groups was compared through immunohistochemistry and quantitative PCR analysis. For more accurate steatohepatitis modeling and drug screening, liver organoids were created including epidemic cells (Kupffer cells) and stellate cells (hepatic stellate cells) that constitute the liver tissue microenvironment. Therefore, human iPSC-derived liver organoids were created in the following ratio (HEMKS): iPSC-derived hepatocytes (H): vascular endothelial cells (E): mesenchymal stem cells (M): iPSC-derived Kupffer cells (K): iPSC-derived hepatic stellate cells (S) = 10:7:2:2:1.

[0135] As a result, as shown in Figure 24A, when gene expression was confirmed by quantitative PCR analysis of each organoid group (Normal, NASH, and NASH+Eze) cultured on HD chips, it was confirmed that the expression levels of PLIN2 (a fatty acid accumulation marker), TNF-α (an inflammatory marker), and SMA and VIM (liver fibrosis markers) increased in the NASH group and decreased in the group treated with Eze drug.

[0136] Furthermore, as shown in Figure 24B, in the normal organoid group, F-actin, an actin filament marker, was well distributed within the organoids, and it was confirmed that there was almost no BODIPY fluorescence signal that labels lipids. In the case of NASH organoids, damaged F-actin structures were observed, and it was confirmed that a large accumulation of lipids occurred inside the organoids. In the group treated with the effective drug (Eze), it was confirmed that fatty acid accumulation decreased and functionality was restored. In the case of ALB expression, a liver differentiation marker, the expression level was decreased in NASH organoids compared to the normal group, while it was observed that a certain level was restored in the group treated with Eze. In the case of Vimentin, a liver fibrosis marker, it was confirmed that its expression was significantly increased in the NASH organoid group, and in particular, immunohistochemistry confirmed that myofibroblast cells, which induce fibrosis while activating hepatic stellate cells included during the production of liver organoids, were distributed inside the organoids.

[0137] Therefore, it was confirmed that non-alcoholic steatohepatitis modeling with maintained disease phenotype is possible using human liver organoids fabricated on a highly efficient HD chip substrate, and it was further verified that drug efficacy screening is also possible.

[0138] Experimental Example 7-6. Mass Production of Non-Alcoholic Steatohepatitis Organoids Derived from Human iPSCs and Quantitative Analysis of ROS

[0139] Using the same culture method as described above, human iPSC-derived liver organoids (HEMKS) were fabricated in a highly efficient 100-well HD chip. Normal, NASH, and NASH+Eze groups were analyzed for ROS (reactive oxygen species) accumulation due to oxidative stress by performing ROS activity and quantitative analysis between the groups using CM-H2DCFDA staining, an oxidative stress detection analysis method.

[0140] As a result, as shown in Figure 25A, in the mass-produced normal organoid group, oxidative stress due to ROS was hardly observed in 100 organoids, whereas in the NASH organoids, ROS-related oxidative stress was significantly increased. In the NASH organoid group treated with the effective drug (Eze), this oxidative stress was reduced to below a certain level, confirming the potential of HD chips for highly efficient screening of liver organoid substrates for evaluating the efficacy of fatty liver disease treatments.

[0141] Furthermore, as shown in Figure 25B, on-chip analysis was performed by applying HD chips on which liver organoids were cultured to a commonly used plate reader device in order to quantitatively evaluate ROS oxidative stress for each organoid. In the case of high-efficiency HD chips, they were designed and manufactured to the same specifications as existing commercially available 384-well plates, so they can be used interchangeably with existing analytical instruments such as plate readers. When quantitative analysis was performed, it was confirmed that the fluorescence intensity increased significantly in NASH organoids and decreased in the organoid group treated with Eze drug, confirming that oxidative stress in fatty liver organoids can be reduced through drug treatment.

[0142] Therefore, it was confirmed that by using highly efficient HD chips manufactured to the same specifications as existing 384-well plates, large-scale drug screening and quantitative efficacy evaluation of mass-produced non-alcoholic steatohepatitis organoid substrates are possible.

[0143] Experimental Example 8: Results of a study on the culture of human iPSC-derived pancreas organoids (pancreatic and ulcerative colloids).

[0144] Experimental Example 8-1. Large-scale culture of pancreatic and intestinal organoids derived from human iPSCs (comparison with existing microwell and U-bottom well plate methods)

[0145] Using the fabricated HD chip, we cultured pancreatic and intestinal organoids derived from human iPSCs. The control group used U-bottom well plates and microwells, which are the most widely used methods for fabricating 3D organoids.

[0146] In the case of pancreatic organoids, iPSC-derived pancreatic progenitor cells (P): vascular endothelial cells (E): mesophyll stem cells (M) were mixed in a ratio of 10:7:2, and 6000 cells were seeded per well. After the organoids were formed, further differentiation was induced in the pancreatic organoids using beta cell differentiation medium.

[0147] As a result, as shown in Figure 26A, the pancreatic organoids cultured in HD chips were even more uniform than those in the control group, confirming that the organoids were formed with a consistent morphology.

[0148] Furthermore, as shown in Figure 26B, when the size of organoids formed in each culture environment was measured on the second day of organoid formation, it was confirmed that the pancreatic and gallbladder organoids produced on the HD chip showed the least individual variation compared to the control group. This indicates that it is possible to produce pancreatic and gallbladder organoids with even more uniform size through HD chip culture.

[0149] Experimental Example 8-2. Comparison of marker expression and differentiation potential of pancreatic and ventricular organoids derived from human iPSCs.

[0150] After culturing human iPSC-derived pancreatic organoids (PEMs) containing vascular endothelial cells for 5 days in each culture system, the expression of pancreatic and vascular differentiation markers among the groups was compared through gene expression analysis via immunohistochemistry and qPCR.

[0151] As a result, as shown in Figure 27A, compared to the control groups (U-bottom plate and microwell group), the pancreatic and gallbladder organoids cultured in HD chips showed even higher expression of the endodermal differentiation marker SOX17, the pancreatic and gallbladder progenitor cell markers PDX1 and NKX6.1, and the beta cell markers CHGA and insulin, as well as further increased expression of the vascular marker CD31.

[0152] As shown in Figure 27B, when comparing the gene expression levels of pancreatic and gallbladder organoids cultured on HD chips with those of the control group, it was confirmed that the expression of pancreatic and gallbladder differentiation markers (PDX1, KRT19), vascular markers (PECAM1), and proliferative capacity-related markers (KI67) tended to be most increased in the HD chip group.

[0153] Through this process, we were able to confirm that the fabricated HD chip is a device that can enhance not only the uniformity of pancreatic and gallbladder organoids but also the expression level of pancreatic and gallbladder-specific differentiation markers compared to existing commonly used platforms.

[0154] Experimental Example 8-3. Comparison of insulin production from human iPSC-derived pancreatic and intestinal organoids.

[0155] Pancreatic organoids (PEMs) derived from human iPSCs were fabricated on a highly efficient 100-well HD chip. After additional 5 days of culture in beta cell induction medium, the uniformity of the organoids and beta cell-specific insulin production were confirmed by immunohistochemistry.

[0156] As a result, as shown in Figure 28, human iPSC-derived pancreatic and bladder organoids, like liver organoids, can be mass-produced uniformly on a 100-well HD chip. It was confirmed that 100 pancreatic and bladder organoids differentiated in beta cell culture medium were uniform and produced insulin at a high level.

[0157] This demonstrates that it is possible to mass-produce uniform human pancreatic organoids with the crucial insulin production / secretion capacity for diabetes treatment through the cultivation of highly efficient HD chips.

[0158] Experimental Example 9: Fabrication of multiple organoids through high-efficiency HD chip bonding

[0159] Experimental Example 9-1. Spheroid fusion via chip-to-chip transfer

[0160] The upward-facing HD chips (25-well version) are designed to be superimposed perpendicularly to each other, and it has been confirmed that they can be joined at their perpendicular coordinates.

[0161] After culturing different sperroids on separate HD chips, the two chips can be joined together in a 1:1 ratio at once to immediately induce the migration of sperroids from one chip to the other (left side of Figure 29).

[0162] Because the movement was from one HD chip to another, the two speroids that had gathered on one chip were positioned at the apex of the depression in the culture medium droplets that accumulated in the well, and it was confirmed that efficient fusion between the speroids was possible (right side of Figure 29).

[0163] Recently, there has been active research aimed at creating more advanced organ analogs through the fusion of different organoids. Therefore, HD chips can be utilized for such applications and can be applied to culture systems for the production of organoids with multi-tissue structures.

[0164] Experimental Example 9-2. Comparison with the U-bottom well-plate bonding method.

[0165] Human iPSC-derived liver organoids (HEMKS) and pancreatic and intestinal organoids (PEM) were cultured on their respective HD chips. The two chips were then joined vertically in a 1:1 ratio. Organoids were transferred from one chip to the other, and liver-pancreatic and intestinal fused organoids were fabricated through organoid fusion.

[0166] In the control group, when organoid conjugation is performed using a U-bottom plate, each cultured organoid must be transferred one by one in a 1:1 ratio before conjugation (left side of Figure 30A). On the other hand, when conjugation is performed using an HD chip, it is possible to efficiently move and conjugate all organoids in all wells at once (right side of Figure 30B).

[0167] In the case of liver-pancreatic-gill organoids cultured on HD chips, it was confirmed that conjugated organoids formed within 24 hours after migration (Figure 30B).

[0168] It was confirmed that liver-pancreatic-gill organoids were formed in both the control group (U-bottom plate and HD chip) (Figure 30C).

[0169] When the expression levels of liver and pancreatic-bone differentiation markers were compared via qPCR analysis, it was confirmed that the expression levels of liver differentiation markers (ALB, HNF4A), pancreatic-bone differentiation markers (NKX6.1, PDX1), and liver-pancreatic-bone bile duct marker (KRT19) were significantly increased in the HD chip group compared to the control group (U-bottom plate) (Figure 30D).

[0170] Experimental Example 9-3. Verification of speroid bonding results for high-throughput HD chip substrates.

[0171] Using a highly efficient HD chip, we were able to form speroids with ADSC and confirm that 100 speroids of uniform size could be formed and cultured simultaneously (left side of Figure 31).

[0172] Similar to existing 25-well HD chips, by superimposing two 100-well HD chips perpendicularly to each other and shifting the speroid to one side, we were able to achieve a success rate of over 80% for speroid shifting. Subsequently, it is expected that efficiency will be further increased by changing the material used to manufacture the chips (right side of Figure 31).

[0173] Experimental Example 9-4. High-efficiency joining of liver-pancreatic-bone organoids on an HD chip substrate.

[0174] Using a highly efficient 100-well HD chip, human iPSC-derived liver organoids (HEMKS) were uniformly mass-produced, and human iPSC-derived pancreatic organoids (PEM) were uniformly mass-produced using another HD chip. Then, the liver organoids and pancreatic organoids were fused in a 1:1 ratio through chip-to-chip fusion, in which the two HD chips were superimposed perpendicularly onto each other.

[0175] As a result, as shown in Figure 32, by superimposing two 100-well HD chips perpendicularly to each other and moving the liver organoids toward the pancreatic and gallbladder organoids, we were able to move the liver organoids with a success rate of over 95%. Thus, it was confirmed that not only is it possible to mass-produce single-organ organoids, but also to mass-produce liver-pancreatic and gallbladder multi-organoids with high efficiency through efficient organoid fusion.

[0176] Immunostaining was performed to analyze organ-specific differentiation markers in the formed hepatobiliary-gum junctioned multiple organoids. It was confirmed that HNF4A, a liver-specific differentiation marker, was expressed only in the liver organoid portion and not in the pancreatic-gum portion. The Kupffer cell marker (CD68), a hepatic rabbit epidemic cell marker, was also expressed only in the liver organoids, and the pancreatic-gum differentiation marker NKX6.1 was specifically expressed only in the pancreatic-gum organoid portion. SOX17, an endodermal marker expressed in both the liver and pancreas, and CD31, a vascular marker, were also confirmed to be well expressed in the multiple organoids junctioned with HD chips.

[0177] Experimental Example 10: Transplantation of liver organoids cultured on an HD chip into a mouse steatohepatitis model.

[0178] To confirm the in vivo transplantability and therapeutic effect of human iPSC-derived liver organoids (HEM-hepatocytes:vascular endothelial cells:intermediate lobe stem cells = 10:7:2) mass-produced on HD chips, liver organoid transplantation was performed into a mouse NASH model in which steatohepatitis was induced using an MCD (methionine-choline-deficient) diet. The MCD diet, which is deficient in methionine and choline, is a diet that readily induces the lesions of non-alcoholic steatohepatitis (NASH) by inducing steatohepatitis, oxidative stress, inflammation, and fibrosis, and is widely used in inducing mouse steatohepatitis models.

[0179] The normal group was fed a normal diet (Normal Chow) for 4 weeks, while the non-steatohepatitis-induced group (NASH) was fed MCD diet for 4 weeks. After the first two weeks, only liver organoid culture medium (100 μL) was injected via the hepatic portal vein, and NASH disease was induced while the mice were continuously fed MCD diet for the remaining two weeks. The group of mice with induced steatohepatitis that received liver organoid transplantation (NASH + Organoid) was fed MCD diet for 4 weeks. After the first two weeks, liver organoids produced from HD chips were injected via the hepatic portal vein (transplanted in 100 μL of culture medium), and NASH disease was continuously induced while the mice were continuously fed MCD diet for the remaining two weeks. A total of 400 liver organoids produced from 4 HD chips per mouse were collected in an insulin syringe and transplanted via the hepatic portal vein (Figure 33A).

[0180] As a result, as shown in Figure 33B, when blood analysis was performed separately for each group after model induction, the hepatotoxicity indicators ALT and LDH levels were measured at significantly higher levels in the mouse group in which NASH disease was induced with MCD diet therapy compared to the normal group on the day before organoid transplantation (Post-operative day (POD) - 1 day) two weeks after model induction. After injecting the culture medium and organoids through the hepatic portal vein, the NASH group also received organoid culture medium until day 3. Since factors contained in the culture medium helped restore liver function, ALT and LDH levels showed increasing recovery immediately after injection, similar to the NASH+Organoid group in which liver organoids were transplanted. However, from 7 days after injection, the effect of the culture medium on restoring liver function was not significant. In the NASH group that received only the culture medium, liver function steadily declined (increased ALT and LDH levels), while in the NASH group that received liver organoid transplants, ALT and LDH levels decreased to almost normal levels, confirming a clear improvement in liver function. In the case of ALB levels, an indicator of liver function, in the NASH group, liver function declined steadily from 3 days after the culture medium was injected, while in the NASH group that received liver organoid transplants, ALB levels recovered to near-normal levels.

[0181] These results confirm that homogeneous liver organoids mass-produced via the HD chip can reduce liver damage and contribute to the recovery of liver function when applied to a non-alcoholic steatohepatitis mouse model. In other words, the highly efficient High-throughput HD chip can be utilized as an efficient culture system for the mass production of organoid-based cell therapy agents for the treatment of liver damage.

[0182] To determine whether hepatic efficacy in treating non-steatohepatitis (NASH) could be induced after transplanting human iPSC-derived liver organoids (HEMs), mass-produced using HD chips, through the hepatic portal vein in a mouse model of NASH, histological analysis and immunohistochemical staining were performed two weeks after transplantation.

[0183] As a result, as shown in Figure 34A, the group that was fed MCD feed for 4 weeks to induce non-alcoholic steatohepatitis (NASH) and then injected with culture medium every 2 weeks showed more severe fatty liver lesions compared to normal livers. On the other hand, in the group that received liver organoids produced from HD chips, partial recovery of the lesions was visually confirmed.

[0184] Furthermore, as shown in Figure 34B, histological analysis via H&E staining confirmed that a large amount of fat was accumulated in the NASH-induced model compared to normal liver. In the group injected with liver organoids, it was confirmed that the transplanted liver organoids engrafted in the areas where fat was accumulated, resulting in a decrease in fat accumulation and a reduction in damaged areas. When collagen accumulated due to fibrosis was stained using MT (Masson's Trichrome) staining, it was confirmed that fibrosis progressed in some liver tissue in the NASH group that was only injected with culture medium, but in the group that received liver organoid transplants, collagen accumulation decreased and the progression of fibrosis was inhibited.

[0185] To confirm whether the transplanted liver organoids engrafted well in fatty liver tissue and functioned effectively, immunohistochemical staining was performed using antibodies that specifically react only to human proteins for liver differentiation markers (ALB, SOX17) and a tight ligation marker (ZO1). It was confirmed that human protein differentiation markers were expressed only in the liver tissue of mice transplanted with human iPSC-derived organoids (Figure 34C). In this experiment, the liver organoids were injected via the hepatic portal vein, allowing for uniform distribution within the liver tissue. Therefore, this is expected to be effective in restoring liver function impaired by fatty liver disease. In NASH models where only culture medium was injected, fibrotic tissue positive for SMA was observed within the liver tissue. However, in NASH models treated with liver organoid transplantation, the fibrotic sites expressing SMA were significantly reduced.

[0186] Two weeks after transplantation, when the body weight of mice in each group was measured, the NASH mouse group that received only culture medium experienced a significant decrease in body weight, as previously known. In contrast, the NASH mouse group that received liver organoid transplants recovered to above a certain level. Furthermore, in the NASH group that received only culture medium, the liver-to-body weight ratio was measured to be lower than that of the normal group, while in the NASH group that received liver organoid transplants, the liver-to-body weight ratio recovered to a certain level (Figure 34D). Therefore, it was confirmed that the transplanted liver organoids restored to some extent the liver function inhibited by the induction of NASH and reduced liver damage.

[0187] These results confirm that when homogeneous liver organoids mass-produced on the HD chip are transplanted into a non-alcoholic steatohepatitis (NASH) mouse model, they induce recovery of liver function and inhibit fibrosis. In other words, the highly efficient High-throughput HD chip can be utilized as an efficient culture system for the mass production of organoid-based cell therapy agents for the treatment of liver injury.

[0188] Experimental Example 11: Additional Applications of HD Chips

[0189] Experimental Example 11-1. Analysis of fluorescence quantification using on-chip drug screening and a plate reader.

[0190] The living cells were stained with calcein for 10 minutes, and the hADSC speroid was then fluorescently labeled.

[0191] Different concentrations of calcein were injected into each line within a single chip, and hADSC speroids cultured on a single HD chip were labeled to exhibit different fluorescence intensities. The fluorescence intensity was then immediately imaged and analyzed on the chip.

[0192] The HD chip (25-well version) fabricated in this invention is designed to conform to the specifications of a 384-well plate, allowing for immediate analysis using commercially available, general-purpose plate reader devices. Specifically, spermoids or organoids can be cultured in the chip, then fluorescently stained, and immediately quantified by measuring the fluorescence intensity through a plate reader (Figure 35).

[0193] When comparing the results of actual fluorescence image analysis with the results of fluorescence intensity measurement using a plate reader, a high correlation can be observed.

[0194] Experimental Example 11-2. Confirmation of the feasibility of device fabrication through 3D printing

[0195] The device of the present invention, as described above, was made from PDMS polymer material, but it has limitations in terms of mass production. Therefore, in order to overcome these problems, we introduced a 3D printing process to confirm whether it is possible to 3D print and manufacture the device of the present invention in a design that will actually be commercialized.

[0196] As a result, as shown in Figure 36, it was confirmed that the device of the present invention can be easily manufactured through 3D printing. It was also found that using 3D printing makes it easy to change the design, and that it has the advantage of being able to be produced using plastic-based materials that are easy to standardize and mass-produce.

[0197] Furthermore, even without an external coupling device, it is possible to culture organisms in a suspension culture device while reducing differences between individual organisms through continuous mixing of the culture medium on a rocker. Depending on the purpose of the experiment and the type of analysis, an additional tube can be connected and a syringe pump can be used as needed.

[0198] Experimental Example 11-3. Utilization of accessories applicable to the device of the present invention.

[0199] We manufactured an accessory that can enhance the potential applications of the device of the present invention, and we were able to confirm its potential applications.

[0200] Specifically, the present invention's device culture method (Figure 6), which simply involves adding a gel solution to a culture medium to induce gelation, can be further modified by using a ring-shaped accessory to allow the gel solution to take on a specific form using its viscosity and surface tension even before it comes into contact with the culture medium. This enables the use of hydrogels with diverse properties, allowing for the application of a wide variety of gel types used in sperm / organoid culture (for example, low-viscosity gels, gels that should not come into contact with the culture medium before solidifying, etc.).

[0201] The accessories are designed to fit the shape of the chip body and are detachable. After some time, once the speroid / organoid has formed well, the accessories can be removed and the culture can continue (Figure 37).

[0202] When fabricating multilayer 3D speraids using existing methods, additional microfluidic devices are generally required to form the core and outer layers separately, and most of these methods are only suitable for fabricating small-sized multilayer speraids.

[0203] These existing methods have relatively high loss rates and are difficult to apply to culturing large speroids or cell aggregates at a similar level to organoids.

[0204] In the HD chip developed in this invention, it was confirmed that a speroid in the form of a capsule with a multilayer structure can be formed very easily and efficiently by injecting the central portion and the outer layer portion separately through the attachment of additional accessories (Figure 38).

[0205] The present invention has been described above, focusing on its preferred embodiments. Those with ordinary skill in the art to which the present invention pertains will understand that the present invention can be embodied in modified forms, without deviating from its essential characteristics. Therefore, the disclosed embodiments should be considered in an explanatory rather than restrictive manner. The scope of the present invention is disclosed in the claims, not in the foregoing description, and all differences within an equivalent scope should be interpreted as being included within the present invention.

Claims

1. A culture chamber comprising a plurality of wells arranged in a matrix in a first direction and a second direction perpendicular to the first direction, A reservoir for storing the culture medium, The culture chamber and the reservoir are connected by a microchannel, Each of the aforementioned wells is open at the top and bottom, Two adjacent wells in the first direction are connected to each other via microchannels. Two adjacent wells in the second direction are connected to each other via microchannels. The microchannels connected to the two adjacent wells are connected to the top of the culture chamber. The aforementioned reservoir is A pair of first reservoirs are arranged at both ends of the culture chamber in the first direction, The present invention further includes a pair of second reservoirs positioned at both ends of the culture chamber in the second direction, The microchannels connected to the first and second reservoirs are connected to the upper part of the culture chamber, forming a cell aggregate culture device.

2. The diameter of the well is 1.5 to 4 mm. The cell aggregate culture device according to claim 1, wherein the distance between two adjacent wells connected by the aforementioned microchannel is 1.5 to 5 mm.

3. The cell aggregate culture device according to claim 1, wherein the cell aggregate is a speroid or organoid derived from any one of the following: mesenchymal stem cells, neural stem cells, vascular endothelial cells, induced pluripotent stem cells, germinal stem cells, tissue stem cells, fetal stem cells, cancer stem cells, and cardiac cells.

4. The cell aggregate is derived from one of the group consisting of the brain, optic cup, kidney, liver, pancreas, intestine, neural tube, stomach, large intestine, prostate, breast, heart, salivary gland, endometrium, mammary gland, thyroid gland, tongue, small intestine, esophagus, spinal cord, skin, bile duct, lung, blood vessels, muscle, adrenal cortex, and thyroid organoids, as described in claim 1.

5. A culture device according to any one of claims 1 to 4, A rocker and The culture medium shared via the aforementioned microchannel, A cell aggregate culture system, including the following:

6. The cell aggregate culture system according to claim 5, wherein the culture device is subjected to a swinging motion by the agitator.

7. A method for culturing a cell aggregate using the culture system described in claim 5.

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