Biohybrid robot including eye / brain organoid, motor nerve spheroid, and muscle bundle, and manufacturing method therefor

Abstract

The present invention relates to a biohybrid robot including an eye / brain organoid, a motor nerve spheroid, and a muscle bundle, and a manufacturing method therefor. The universal biohybrid robot according to the present invention can generate movement of muscle cells by an electrophysiological signal generated in the eye / brain organoid like a human motor system by using light stimulation and a neurotransmitter. It is expected that the present invention can be used in a disease model simulating a signal transmission system of the human body by combining various neurodegenerative diseases, such as Parkinson and Alzheimer's disease models, and eye tumor models in the future, and can be used to manufacture a drug screening platform that leverages the movement of biohybrid robots composed of living tissues.

Description

Biohybrid robots including eye / brain organoids, motor neuron spheroids, and muscle bundles, and methods for manufacturing the same

[0001] The present invention was made with the support of the Ministry of Science and ICT under the task identification number 1711180504 and the detailed task number 2019R1A2C3002300. The research management specialized organization of the task is the National Research Foundation of Korea, the research project name is "Individual Basic Research (Ministry of Science and ICT)", the research project name is "Biohybrid Robot with Life-Mimicking Sensing Function Based on Brain Assembler", the main organization is Sogang University, and the research period is 2023-03-01 ~ 2024-02-29.

[0002] In addition, the present invention was made under the support of the Ministry of Science and ICT under the task identification number 1711187608 and the detailed task number 2022M3H4A1A01005271. The research management specialized organization of the said task is the National Research Foundation of Korea, the research project name is "Nanomaterial Technology Development Project", the research project name is "Leading Diabetes and Metabolic Disease Research Center", the main organization is Sogang University, and the research period is from 2023-01-01 to 2023-12-31.

[0003] In addition, the present invention was made with the support of the Ministry of Science and ICT under the task identification number 1711180793 and the detailed task number 2022H1D3A2A02093530, and the research management specialized organization of the said task is the National Research Foundation of Korea, the research project name is "Support for Expansion of Talent Utilization", the research project name is "Ni-TiO2 / Photosystem II with Drug Screening Function and Muscle Bundle-Based Nano-Biohybrid Actuator", the main organization is Sogang University, and the research period is 2023-01-01 ~ 2023-12-31.

[0004] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0146883, filed with the Korean Intellectual Property Office on October 30, 2023, the disclosure of which is incorporated herein by reference.

[0005] The present invention relates to a biohybrid robot including an eye / brain organoid, a motor neuron spheroid, and a muscle bundle, and a method for manufacturing the same, and more particularly, to a method for manufacturing a pluripotent biohybrid robot by connecting an eye / brain organoid derived from human induced pluripotent stem cells (iPSCs), a motor neuron spheroid derived from human neural stem cells (hNSCs), and a muscle bundle derived from three-dimensional skeletal muscle cells (C2C12).

[0006] In the human motor system, muscle cells contract and relax through electrophysiological signals generated in the brain, transmitted to the muscle cells via motor neurons. However, most biorobots used in the laboratory are based on muscle cells, and muscle movement is achieved through electrical stimulation and motility drugs.

[0007] Alternatively, methods exist to induce muscle movement through genetic manipulation of muscle cells, stimulating them with external light. These methods of inducing muscle movement differ from actual human muscle movement, making it difficult to determine how brain-borne diseases, such as neurodegenerative diseases, affect muscle movement. Therefore, the introduction of methods that mimic the human motor system is necessary.

[0008] One representative method involves introducing brain organoids, which are structurally and functionally similar to the brain. Electrophysiological signals generated from brain organoids induce acetylcholine signaling in connected motor neurons, which then converts acetylcholine into electrical signals through the synaptic membrane, thereby inducing muscle contraction.

[0009] The conventional technologies for manufacturing bio-robots mentioned above are those related to electrical stimulation, introduction of nanomaterials, and treatment of chemicals and drugs.

[0010] Electrical stimulation techniques require separate electrodes made of polymers and graphene to effectively deliver electrical stimulation to muscle cells, and the challenge lies in the need to induce muscle movement through direct stimulation. Genetic engineering, while promising in that it can induce muscle cell contraction and relaxation through external light stimulation, presents the challenge of requiring complex steps for actual genetic manipulation of muscle cells. Drug treatment, while relatively simple to use, has the disadvantage of requiring the control of various conditions, such as drug concentration and time, that influence muscle cell responses.

[0011] This method of inducing muscle movement is different from the actual human motor system in which muscle movement is controlled by signals generated from the brain.

[0012] Accordingly, the inventors of the present invention fabricated a biohybrid robot comprising an eye / brain organoid, a motor neuron spheroid, and a muscle bundle, and confirmed that a human motor system can be implemented therefrom.

[0013] Accordingly, the object of the present invention is to provide a conjugate in which an eye organoid and a brain organoid are conjugated;

[0014] Motor neuron spheroids connected to the above conjugate;

[0015] Muscle bundles connected to the above motor neuron spheroids; and

[0016] A robotic structure connected to the above muscle bundle;

[0017] It provides a biohybrid robot including:

[0018] Another object of the present invention is to provide a method for manufacturing a biohybrid robot comprising the following steps:

[0019] A joining step for preparing a junction that joins an eye organoid and a brain organoid;

[0020] A first connecting step of connecting a motor neuron spheroid to the above-mentioned junction;

[0021] A second connecting step of connecting a muscle bundle to a motor neuron spheroid connected to the above-mentioned junction; and

[0022] A third connecting step connecting the robot structure to the muscle bundle connected to the above joint.

[0023] Another object of the present invention is to provide a method for screening a candidate substance for treating a neurodegenerative disease or an ocular disease, comprising the following steps:

[0024] A motor system preparation step for preparing a biohybrid robot comprising a zygote in which an eye organoid and a brain organoid are fused; a motor neuron spheroid connected to the zygote; a muscle bundle connected to the motor neuron spheroid; and a robot structure connected to the muscle bundle;

[0025] A drug contact step of contacting a candidate substance with the biohybrid robot; and

[0026] A drug evaluation step in which the degree of contraction induced by light stimulation in muscle bundles after contact with a candidate substance is compared with that of muscle bundles not contacted with the candidate substance.

[0027] Another object of the present invention is to provide a conjugate comprising an eye organoid and a brain organoid;

[0028] Motor neuron spheroids connected to the above conjugate;

[0029] Muscle bundles connected to the above motor neuron spheroids; and

[0030] A robotic structure connected to the above muscle bundle;

[0031] It relates to the use of a biohybrid robot including a human movement system.

[0032] The present invention relates to a biohybrid robot comprising an eye / brain organoid, a motor neuron spheroid, and a muscle bundle, and a method for manufacturing the same. The biohybrid robot according to the present invention can implement a human motor system that can be utilized in screening candidate substances for treating nervous system diseases.

[0033] To improve the connection efficiency between eye / brain organoids and muscle bundles, the present inventors improved the growth and directionality of neurites in motor neuron spheroids by incorporating hyaluronic acid (HA)-modified gold-nickel-gold nanorods (GNRs) and human umbilical vein endothelial cells (HUVECs) into the motor neuron spheroids.

[0034] To immobilize hyaluronic acid onto the surface of gold-nickel-gold nanorods, hyaluronic acid was modified with thiol groups using cysteamine and immobilized onto the surface of gold-nickel-gold nanorods through SS bonds. The fabricated hyaluronic acid-modified gold-nickel-gold nanorods were mixed with human umbilical vein endothelial cells and extracellular matrix, and used to connect eye / brain organoids and muscle bundles with motor neuron spheroids. They were also immobilized onto polymeric structures for the fabrication of universal biohybrid robots.

[0035] Through co-culture for a week, eye / brain organoids, motor neuron spheroids, and muscle bundles were connected on a polymeric structure. At this time, the implementation of the human motor system was confirmed through neurotransmitter or light stimulation in the connected eye / brain organoids. The generation of electrophysiological signals in the eye organoids was confirmed through light stimulation, and the generation of electrophysiological signals in the brain organoids was confirmed through neurotransmitter. The fabricated universal biohybrid robot showed movement for more than a week, and the magnitude of the movement was successfully controlled by neurotransmitters.

[0036] Hereinafter, the present invention will be described in more detail.

[0037] One aspect of the present invention is a conjugate comprising an eye organoid and a brain organoid;

[0038] Motor neuron spheroids connected to the above conjugate;

[0039] Muscle bundles connected to the above motor neuron spheroids; and

[0040] A robotic structure connected to the above muscle bundle;

[0041] It is a biohybrid robot that includes .

[0042] In the present invention, the muscle bundle may have two or more rings.

[0043] The above robot structure may have two or more connecting portions connected to two or more rings provided in the muscle bundle, and may have a node formed by extending from the connecting portion in a direction opposite to the eye organoid and the brain organoid.

[0044] The above-mentioned connecting portions may be, but are not limited to, column-shaped, independently arranged vertically spaced apart on a single plane for insertion into the ring of the muscle bundle.

[0045] Another aspect of the present invention is a method for manufacturing a biohybrid robot comprising the following steps:

[0046] A joining step for preparing a junction that joins an eye organoid and a brain organoid;

[0047] A first connecting step of connecting a motor neuron spheroid to the above-mentioned junction;

[0048] A second connecting step of connecting a muscle bundle to a motor neuron spheroid connected to the above-mentioned junction; and

[0049] A third connecting step connecting the robot structure to the muscle bundle connected to the above joint.

[0050] In the present invention, the bonding step may be performed by placing the eye organoid and brain organoid in contact with each other within the hydrogel, but is not limited thereto.

[0051] In the present invention, the hydrogel may be an alginate-based sacrificial hydrogel, and the alginate-based hydrogel may be sodium alginate, but is not limited thereto.

[0052] In the present invention, the method may additionally include a co-culturing step of co-culturing eye organoids and brain organoids after the bonding step, but is not limited thereto.

[0053] In one embodiment of the present invention, the bonding step is performed by placing eye organoids and brain organoids in contact with each other within an alginate-based sacrificial hydrogel, reacting them with a potassium chloride solution, and then performing a co-culture step in the bonded state when the sodium azide solution hardens, thereby completing the formation of the bond.

[0054] In the present invention, the first connection step may be performed by contacting a hydrogel prepared by mixing at least one selected from the group consisting of gold-nickel-gold nanorods (GNRs), human umbilical vein endothelial cells (HUVECs), and an extracellular matrix with a motor neuron spheroid, and the conjugate, and for example, a hydrogel prepared by mixing all of gold-nickel-gold nanorods (GNRs), human umbilical vein endothelial cells (HUVECs), and an extracellular matrix may be used, but is not limited thereto.

[0055] The above gold-nickel-gold nanorods may have their surface modified with hyaluronic acid (HA), but are not limited thereto.

[0056] In the present invention, the muscle bundle may have two or more rings, but is not limited thereto.

[0057] The above robot structure may have two or more connecting portions connected to two or more rings provided in the muscle bundle, and may have a node formed by extending from the connecting portion in a direction opposite to the eye organoid and the brain organoid.

[0058] The above-mentioned connecting portions may be, but are not limited to, column-shaped, independently arranged vertically spaced apart on a single plane for insertion into the ring of the muscle bundle.

[0059] Another aspect of the present invention is a method for screening a candidate substance for treating a neurodegenerative disease or an ocular disease, comprising the following steps:

[0060] A motor system preparation step for preparing a biohybrid robot comprising a zygote in which an eye organoid and a brain organoid are fused; a motor neuron spheroid connected to the zygote; a muscle bundle connected to the motor neuron spheroid; and a robot structure connected to the muscle bundle;

[0061] A drug contact step of contacting a candidate substance with the biohybrid robot; and

[0062] A drug evaluation step in which the degree of contraction induced by light stimulation in muscle bundles after contact with a candidate substance is compared with that of muscle bundles not contacted with the candidate substance.

[0063] In the present invention, the neurodegenerative disease may be selected from the group consisting of Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and epilepsy, but is not limited thereto.

[0064] In the present invention, the eye disease may be selected from the group consisting of macular degeneration, retinitis pigmentosa, Stargardt disease, choroideremia, gyrate-atrophy, dry eye syndrome, eye tumor, and eye strain, but is not limited thereto.

[0065] The present invention relates to a biohybrid robot comprising an eye / brain organoid, a motor neuron spheroid, and a muscle bundle, and a method for manufacturing the same. The universal biohybrid robot according to the present invention can induce muscle cell movement by electrophysiological signals generated from the eye / brain organoid, similar to the human motor system, using light stimulation and neurotransmitters. In the future, this can be utilized for disease models that mimic the signal transmission system of the human body by combining various neurodegenerative diseases such as Parkinson's disease models and Alzheimer's disease models and eye tumor models, and it is expected that it can be utilized for manufacturing a drug screening platform using the operation of the universal biohybrid robot composed of biological tissues.

[0066] Figure 1a is a schematic diagram of the production of brain organoids and eye organoids according to one embodiment of the present invention.

[0067] Figure 1b is a photograph showing differentiation markers of eye organoids produced according to one embodiment of the present invention confirmed by immunostaining.

[0068] Figure 1c is a graph showing the results of confirming the degree of differentiation of eye organoids produced according to one embodiment of the present invention using qPCR.

[0069] Figure 1d is a fluorescent image showing differentiation markers (S100β, TuJ1, and SOX2) of brain organoids produced according to one embodiment of the present invention confirmed by immunostaining.

[0070] Figure 1e is a graph showing the results of confirming the degree of differentiation of brain organoids produced according to one embodiment of the present invention using qPCR.

[0071] FIG. 2A is an optical image of an eye organoid (RO) and brain organoid (BO) conjugate fabricated using an alginate-based sacrificial hydrogel according to one embodiment of the present invention.

[0072] FIG. 2b is an image confirming the bonding of a brain organoid with an eye organoid containing GFP according to one embodiment of the present invention.

[0073] FIG. 3a is a schematic diagram of the growth direction control of motor neuron spheroids produced using gold-nickel-gold nanorods modified with hyaluronic acid and human umbilical vein endothelial cells according to one embodiment of the present invention.

[0074] FIG. 3b is a photograph showing the surface of a gold-nickel-gold nanorod (left) and a gold-nickel-gold nanorod (right) modified with hyaluronic acid, obtained by observing the surface using a transmission electron microscope (TEM) according to one embodiment of the present invention.

[0075] Figure 3c is a fluorescent image showing the degree of directionality control of a motor neuron spheroid (TuJ1) produced according to one embodiment of the present invention, confirmed by immunostaining.

[0076] FIG. 3D is a graph confirming the degree of growth directionality of nerve cells in the 0-180 degree direction in a motor neuron spheroid produced according to one embodiment of the present invention.

[0077] FIG. 4A is a graph analyzing electrophysiological signals before and after light stimulation of an eye / brain organoid conjugate manufactured according to one embodiment of the present invention.

[0078] FIG. 4b is a graph confirming changes in electrophysiological signals using glutamate, a neurotransmitter, in an eye / brain organoid conjugate produced according to one embodiment of the present invention.

[0079] FIG. 5A is a schematic diagram of eye / brain organoids, motor nerve spheroids, and muscle bundle connections for fabricating a universal biohybrid robot according to one embodiment of the present invention.

[0080] FIG. 5b is a fluorescent image showing the connection of eye / brain organoids (GFP), motor neuron spheroids (TuJ1), and muscle bundles (F-actin) produced according to one embodiment of the present invention using immunostaining.

[0081] Figure 5c is an image of a universal biohybrid robot manufactured according to one embodiment of the present invention.

[0082] FIG. 5d is a drawing confirming changes in movement due to glutamate, a neurotransmitter, in a universal biohybrid robot manufactured according to one embodiment of the present invention.

[0083] The present invention relates to a biohybrid robot comprising: a zygote in which an eye organoid and a brain organoid are zygote; a motor neuron spheroid connected to the zygote; a muscle bundle connected to the motor neuron spheroid; and a robot structure connected to the muscle bundle.

[0084] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0085] Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, and (vol / vol)% for liquid / liquid, unless otherwise stated.

[0086]

[0087] Example 1: Production of eye / brain organoids, motor neuron spheroids, and muscle bundles.

[0088] Eye / brain organoids that receive light and generate electrophysiological signals similar to human eyes were produced using human induced pluripotent stem cells (iPSCs) as shown in Figure 1a.

[0089]

[0090] 1-1. Production of eye organoids

[0091] Human induced pluripotent stem cells were formed into eye organoids through three differentiation stages. First, 1.0 × 10 4 Embryonic bodies (CBDs) consisting of 10 cells / mL were cultured in DMEM / F12 (1:1) medium containing 1% N2 supplement, 1% minimum essential amino acids (NEAA), and 2 mg / mL heparin. After attachment to Matrigel-coated plates on day 7 of culture, the culture was continued, and on day 16, the medium was switched to DMEM / F12 (3:1) containing 2% B27 (vitamin A-free), 1% NEAA, and 1% antibiotic-antimycotic for differentiation into oocytes.

[0092] At 4 weeks of differentiation, the horseshoe-shaped bud region was isolated using a sharp tungsten needle and cultured to gradually form 3D eye organoids. For long-term culture, the medium was supplemented with 10% fetal bovine serum, 100 mM taurine, and 2 mM Glutamax from day 42. The production of eye organoids was confirmed through immunohistochemistry and electrophysiological signal measurement.

[0093] As can be seen in Figure 1b, MITF (microphthalmia-associated transcription factor), a visual cell differentiation marker observed in the intermediate stage of eye organoid formation, was observed.

[0094] As can be seen in Figure 1c, quantitative polymerase chain reaction (quantitative PCR; qPCR) measurement results showed that the expression levels of PAX6, LHX2, and SIX6, genes related to visual cells, increased with eye organoid differentiation.

[0095]

[0096] 1-2. Production of brain organoids

[0097] Human induced pluripotent stem cells were formed into brain organoids through four differentiation stages. 1.0 × 10 4Embryoid bodies composed of cells at 10 cells / mL were cultured for 4 days in StemFit Basic04 containing 50 μM Rho-associated protein kinase (ROCK) inhibitor and 4 ng / mL basic fibroblast growth factor (bFGF). On the 5th day of culture, they were cultured for 5 days in DMEM / F-12 containing 1% N2 supplement, 1% GlutaMAX supplement, 1% NEAA solution, and 1 μg / mL heparin. On the 9th day of culture, the differentiating brain organoids were embedded in 5 μL of Matrigel and cultured for 4 days in DMEM / F-12: Neurobasal media (1:1) containing 1% N2 supplement, 1% B-27 supplement without vitamin A, 1% GlutaMAX supplement, 1% NEAA solution, 50 μM 2-mercaptoethanol, and 2.5 μg / mL insulin, 100 μg / mL streptomycin, and 100 U / mL penicillin.

[0098] Finally, on day 13, the 1% B-27 supplement in the existing media was replaced with a 1% B-27 supplement containing vitamin A, and long-term culture was performed. The structure and function of the produced brain organoids were confirmed through immunohistochemistry and electrophysiological signal measurements.

[0099] As can be seen in Fig. 1d, the neural cell differentiation markers S100β (S100 calcium-binding protein β), TuJ1 (Neuron-specific class III beta-tubulin), and SOX2 (SRY (sex-determining region Y)-box 2) observed during brain organoid formation showed that neural network formation and structure were formed inside the brain organoid.

[0100] As can be seen in Figure 1e, quantitative polymerase chain reaction (qPCR) measurements showed that the expression level of OCT4, a stem cell marker, decreased and the expression levels of PAX6 and TuJ1, genes related to neural cells, increased with brain organoid differentiation.

[0101]

[0102] 1-3. Production of eye organoids and brain organoids

[0103] Eye organoids and brain organoids were connected using an alginate-based sacrificial hydrogel. Brain and eye organoids were placed in a 1 g / 100 ml sodium alginate solution, and tweezers were used to physically fuse the retinal organoids with the brain organoids. Then, 4 g / 100 ml potassium chloride solution was sprayed onto the retina. This allowed the sodium alginate solution to harden, solidifying the brain and eye organoids. After culturing the brain and eye organoid fusions in co-culture media for 3 days, the solidified sodium alginate naturally decomposed, forming well-attached brain and eye organoid fusions, as shown in Figures 2a and 2b.

[0104]

[0105] 1-4. Production of motor neuron spheroids

[0106] To produce motor neuron spheroids, human neural stem cells (hNSCs) were cultured in KnockOut™ DMEM / F-12 containing 20 ng / mL of bFGF (Basic Fibroblast Growth Factor) and 20 ng / mL of EGF. Spheroids were grown at a density of 7.0 × 10 4 It was produced using human neural stem cells at 1 cell / well, and after 48 hours, the culture medium was replaced with motor neuron differentiation medium containing 8 ng / mL bFGF, 200 ng / mL sonic hedgehog, 10 ng / mL activin A, and 50 μM retinoic acid. On the 20th day of differentiation, the existing motor neuron differentiation medium was replaced with motor neuron differentiation medium containing 10 ng / mL BDNF (Brain-derived neurotrophic factor) and 10 ng / mL GDNF (Glial cell-Derived Neurotrophic Factor) for maturation of motor neuron spheroids, and cultured for 8 days.

[0107]

[0108] 1-5. Creation of muscle bundles

[0109] To create muscle bundles, a structure was created using a stereolithography 3D printer, and then PDMS was poured into the structure to create a mold for creating muscle bundles. The muscle bundles were 5 X 10 6Skeletal muscle cells (C2C12) (350 μL) at 100 μL / mL, Matrigel (300 μL), fibrinogen (4 mg / mL), and thrombin (2 U / mL) were mixed and placed in a mold for producing muscle bundles. 200 μL was added to the mold and allowed to gel for 30 minutes. The produced muscle bundles were able to demonstrate movement after approximately 2 weeks of differentiation. DMEM containing 2% Horse Serum, 1 mg / mL Aminocaproic acid, 1 ng / mL Insulin Growth Factor-1, and 1% Penicillin / Streptomycin was used for differentiation. Differentiation of the produced muscle bundles was confirmed through immunohistochemistry.

[0110]

[0111] Example 2: Directional control of motor neuron spheroid growth using gold-nickel-gold nanorods modified with hyaluronic acid.

[0112] In order to control the growth direction of the motor neuron spheroids produced in the above examples 1-4, as shown in Fig. 3a, gold-nickel-gold nanorods (GNRs) and hyaluronic acid (HA) were used to induce the growth of axons of nerve cells.

[0113] Specifically, to immobilize hyaluronic acid on the surface of gold-nickel-gold nanorods, thiol-modified hyaluronic acid was fabricated using cysteamine, as shown in Fig. 3b. To fabricate gold-nickel-gold nanorods modified with hyaluronic acid, gold-nickel-gold nanorods were first synthesized using a porous membrane with a size of 200 nm. For the synthesis, silver was physically deposited on the surface of the porous membrane, and then gold, nickel, and gold were sequentially deposited through electrochemical deposition. The porous membrane and silver used for fabrication were dissolved using 3 M sodium hydroxide and nitric acid, and then the gold-nickel-gold nanorods were washed and recovered using a magnet.

[0114] To attach hyaluronic acid to the fabricated gold-nickel-gold nanorods, 4 mg of thiol-group-attached hyaluronic acid was mixed with 10 mL of gold-nickel-gold nanorods at a concentration of 0.1 mg / mL and reacted at room temperature for 24 h. After the reaction, the nanorods were recovered and washed using a magnet. The fabricated gold-nickel-gold nanorods modified with hyaluronic acid were mixed with motor neuron spheroids, human umbilical vein endothelial cells (HUVECs), and extracellular matrix to fabricate a hybrid hydrogel, which was used to connect eye / brain organoids and motor neuron spheroids. The hybrid hydrogel was prepared by mixing 60 μL of Matrigel, 20 μL of gold-nickel-gold nanorods modified with hyaluronic acid (0.5 mg / mL), and 68 μL of 1 × 10 6 Human umbilical vein endothelial cells and 50 μL of fibrinogen (16 mg / mL) were mixed and used to connect eye / brain organoids and motor neuron spheroids.

[0115] To confirm the control of the growth direction of motor neuron spheroids by the hyaluronic acid-modified gold-nickel-gold nanorods used, three additional groups of motor neuron spheroids were fabricated [(i) motor neuron spheroids (w / o hyaluronic acid-modified gold-nickel-gold nanorods, human umbilical vein endothelial cells), (ii) motor neuron spheroids (w / o hyaluronic acid-modified gold-nickel-gold nanorods, w / human umbilical vein endothelial cells), (iii) motor neuron spheroids (w / hyaluronic acid-modified gold-nickel-gold nanorods, w / human umbilical vein endothelial cells)]. The neuronal growth and growth direction of the motor neuron spheroids in the three fabricated groups were confirmed through TuJ1 staining after approximately 7 days.

[0116] As can be seen in Figures 3c and 3d, group (iii) showed a 1.34-fold higher directionality of neurons in the 0-10° direction compared to group (i).

[0117]

[0118] Example 3: Control of electrophysiological signals in eye / brain organoids through light stimulation and neurotransmitters.

[0119] The formation of brain organoid and eye organoid fusions in Examples 1-3 was confirmed through signal measurement using a multielectrode array. Eye / brain organoids were placed on signal-measuring electrodes constituting the multielectrode array, and the generation of electrophysiological signals was measured by illuminating the eye organoid portion with light. Furthermore, glutamate, a neurotransmitter, was added to the co-culture medium used on the multielectrode array, and electrophysiological signals generated from the brain organoids were measured.

[0120] As can be seen in Figure 4a, light stimulation in the visible light range (98 mW / mm 2 , 1 s) was given to generate an electrophysiological signal by light stimulation, and the electrophysiological signal was transmitted to the adjacent brain organoid.

[0121] Additionally, as can be seen in Fig. 4b, the electrophysiological signal generated by stimulating brain organoids by mixing 100 μM glutamate into the co-culture medium was also transmitted to the brain organoids.

[0122]

[0123] Example 4: Fabrication and Operation of a Universal Biohybrid Robot

[0124] To fabricate a universal biohybrid robot as shown in Fig. 5a, a structure was fabricated using a stereolithography 3D printer, and Ecoflex:PDMS (5:1) was poured onto the structure to fabricate a universal biohybrid robot structure. After attaching a muscle bundle on the 7th day of differentiation to the fabricated universal biohybrid robot structure, it was connected to an eye / brain organoid using a hybrid hydrogel containing hyaluronic acid-modified gold-nickel-gold nanorods and motor neuron spheroids.

[0125] As shown in Figure 5b, the connection of eye / brain organoids (GFP), motor neuron spheroids (TuJ1), and muscle bundles (F-actin) from the universal hybrid robot was confirmed using immunohistochemistry.

[0126] The omnipotent biohybrid robot, such as 5c, was co-cultured for 7 days in a co-culture medium consisting of eye / brain organoid co-culture media and motor neuron spheroid / muscle bundle co-culture media. After 7 days, when the eye / brain organoids, motor neuron spheroids, and muscle bundles were connected, the fabricated omnipotent biohybrid robot moved forward on the co-culture medium.

[0127] In addition, as can be confirmed in Fig. 5d, when the brain organoid was stimulated using glutamate, the movement was confirmed to improve from 0.27 cm / min before the action to 0.52 cm / min after the action, and the movement was confirmed to appear for more than a week.

[0128] The present invention relates to a biohybrid robot including an eye / brain organoid, a motor neuron spheroid, and a muscle bundle, and a method for manufacturing the same, and more particularly, to a method for manufacturing a pluripotent biohybrid robot by connecting an eye / brain organoid derived from human induced pluripotent stem cells (iPSCs), a motor neuron spheroid derived from human neural stem cells (hNSCs), and a muscle bundle derived from three-dimensional skeletal muscle cells (C2C12).

Claims

1. A zygote in which an eye organoid and a brain organoid are joined; Motor neuron spheroids connected to the above conjugate; Muscle bundles connected to the above motor neuron spheroids; and A robotic structure connected to the above muscle bundle; A biohybrid robot including:

2. A biohybrid robot according to claim 1, wherein the muscle bundle has two or more rings.

3. A biohybrid robot in the second paragraph, wherein the robot structure has two or more connecting portions connected to two or more rings provided in the muscle bundle, and has a node formed by extending from the connecting portion in a direction opposite to the eye organoid and the brain organoid.

4. A method for manufacturing a biohybrid robot comprising the following steps: A joining step for preparing a junction that joins an eye organoid and a brain organoid; A first connecting step of connecting a motor neuron spheroid to the above-mentioned junction; A second connecting step of connecting a muscle bundle to a motor neuron spheroid connected to the above-mentioned junction; and A third connecting step connecting the robot structure to the muscle bundle connected to the above joint.

5. A method for manufacturing a biohybrid robot, wherein in the fourth paragraph, the bonding step is performed by placing the eye organoid and the brain organoid in contact with each other within the hydrogel.

6. A method for manufacturing a biohybrid robot, wherein the hydrogel in paragraph 5 is an alginate-based sacrificial hydrogel.

7. A method for manufacturing a biohybrid robot, wherein the method further comprises a co-culturing step of co-culturing eye organoids and brain organoids after the bonding step.

8. A method for manufacturing a biohybrid robot in claim 4, wherein the first connection step is performed by contacting a hydrogel manufactured by mixing at least one selected from the group consisting of gold-nickel-gold nanorods (GNRs), human umbilical vein endothelial cells (HUVECs), and an extracellular matrix with the motor neuron spheroid, to the conjugate.

9. A method for manufacturing a biohybrid robot, wherein the gold-nickel-gold nanorods in paragraph 8 are surface-modified with hyaluronic acid (HA).

10. A method for manufacturing a biohybrid robot, wherein the muscle bundle in paragraph 4 has two or more rings.

11. A method for manufacturing a biohybrid robot, wherein in the 10th paragraph, the robot structure has two or more connecting portions connected to two or more rings provided in the muscle bundle, and has a node formed by extending from the connecting portion in a direction opposite to the eye organoid and the brain organoid.

12. A method for screening a candidate substance for the treatment of a neurodegenerative disease or ocular disease, comprising the following steps: A motor system preparation step for preparing a biohybrid robot comprising a zygote in which an eye organoid and a brain organoid are fused; a motor neuron spheroid connected to the zygote; a muscle bundle connected to the motor neuron spheroid; and a robot structure connected to the muscle bundle; A drug contact step of contacting a candidate substance with the biohybrid robot; and A drug evaluation step in which the degree of contraction induced by light stimulation in muscle bundles after contact with a candidate substance is compared with that of muscle bundles not contacted with the candidate substance.

13. A method for screening a candidate substance for treating a neurodegenerative disease, wherein the neurodegenerative disease is selected from the group consisting of Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and epilepsy.

14. A method for screening a candidate substance for treating a neurodegenerative disease, wherein the eye disease in claim 12 is selected from the group consisting of macular degeneration, retinitis pigmentosa, Stargardt disease, choroideremia, gyrate-atrophy, dry eye syndrome, eye tumor, and eye strain.

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