Neurobiohybrid manufactured using reduced graphene oxide nanoparticles, neuromuscular junction model using same, and drug screening method using same

Abstract

The present invention relates to a neurobiohybrid, a neuromuscular junction model using same, and a method for screening a drug related to motor neuron diseases using same. The present inventors have experimentally verified that the neurobiohybrid and the neuromuscular junction model using same of the present invention have an excellent effect on drug screening for treating amyotrophic lateral sclerosis (ALS), and thus the neurobiohybrid and the neuromuscular junction model using same are expected to be applicable to drug screening and toxicity evaluation for various neurological disorders.

Description

A neural biohybrid manufactured using reduced graphene oxide nanoparticles, a neuromuscular junction model using the same, and a drug screening method using 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 institution of the said 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 Assembled", the main institution is Sogang University Industry-Academic Cooperation Foundation, and the research period is 2023.01.01 ~ 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, and the research management specialized organization of the said task is the National Research Foundation of Korea, the research project name is "Nanomaterial Technology Development", the research project name is "Nano-biohybrid actuator chip for organoid-based drug screening", the main organization is Sogang University Industry-Academic Cooperation Foundation, and the research period is 2023.01.01~2023.12.31.

[0003] In addition, the present invention was made under the support of the Ministry of Science and ICT under the task identification number 1711198532 and the detailed task number RS-2023-00259341, and the research management specialized organization of the said task is the National Research Foundation of Korea, the research project name is "Establishment of an Overseas Excellent Research Institution Cooperation Hub", the research project name is "Sogang-UPenn Emerging Infectious Disease Theranostics Convergence Research Center", the main organization is Sogang University Industry-Academic Cooperation Foundation, and the research period is 2023.07.01~2023.12.31.

[0004] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0146841, 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 neural biohybrid, a neuromuscular junction model using the same, and a method for screening drugs related to motor neuron diseases using the same.

[0006] The neuromuscular junction (NMJ) is a site in the human body where chemical and electrical signals are transmitted between motor neurons and muscle fibers, playing a crucial role in smooth muscle contraction. Motor neurons transmit signals to muscle fibers through the NMJ, triggering muscle contraction. For smooth muscle contraction to function in a neuromuscular junction model, a 3D structure with a similar skeletal muscle structure and well-defined motor nerve innervation is essential. The association of multiple motor neurons in this skeletal muscle tissue facilitates connections between muscle fibers and motor neurons and increases synapse formation at the NMJ.

[0007] Recently, carbon nanotubes, graphene, and other nanomaterials have been used to create neuromuscular junction environments, such as those of muscles and nerves, due to their high electrical conductivity and biocompatibility. The development of three-dimensional in vitro models resembling human neuromuscular junctions could provide valuable tools for basic biological research, the development of regenerative medicine strategies, and drug screening for motor neuron diseases.

[0008] Unlike 2D cell culture, 3D spheroid-based systems promote the development of neural differentiation and maturation of human neural stem cells (hNSCs). Despite these advantages, current spheroid models often face necrosis during in vitro long-term culture due to a lack of microvasculature and limited oxygen diffusion. Consequently, insufficient cell-ECM interactions lead to inefficient neurogenesis and unwanted glial formation within the aggregated spheroids.

[0009] Currently, vascular cells within neurons directly support the efficient distribution of oxygen and nutrients and secrete factors such as glial-derived neurotrophic factor (GDNF), which promotes neuronal survival and axonal growth. Furthermore, graphene, due to its unique properties, is attracting significant attention as one of the most promising biomaterials for biomedical applications. Graphene-based materials can be used as scaffold materials to enhance neurogenesis, neural differentiation, and regeneration of neural stem cells (NSCs), enabling diverse applications.

[0010] Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a rare and devastating neurodegenerative disease affecting motor neurons. The cause is unknown. Motor neurons, which transmit signals from the brain to muscles, gradually deteriorate and are destroyed, leading to muscle weakness, loss of voluntary movement control, and premature death. Research is underway to create a three-dimensional neuromuscular junction (NJ) that functions similarly to the human form to understand the mechanisms of these motor cell diseases and develop drugs. This ALS-based NJ model could provide a valuable tool not only for ALS research but also for all fields of neurodegenerative and developmental neuroscience.

[0011] The present inventors manufactured a neuromuscular junction model containing a biohybrid derived from an ALS patient and performed a comparative experiment treating it with an ALS drug, and confirmed that the neuromuscular junction model of the present invention is excellent in ALS drug screening.

[0012] Accordingly, the purpose of the present invention is to provide a neural biohybrid comprising carbon materials, vascular cells, and neural stem cells.

[0013] Another object of the present invention is to provide a neuromuscular junction model manufactured by co-culturing a neural biohybrid manufactured by culturing a mixture of carbon materials, vascular cells, and neural stem cells; and a muscle bundle manufactured by culturing a hydrogel containing muscle cells.

[0014] Another object of the present invention is to provide a method for producing a neural biohybrid comprising the following steps:

[0015] A culture step in which carbon materials, vascular cells, and neural stem cells are mixed and cultured.

[0016] Another object of the present invention is to provide a method for screening drugs related to motor neuron disease, comprising the following steps:

[0017] A neural biohybrid manufacturing step for manufacturing a neural biohybrid by culturing a mixture of carbon materials, vascular cells, and neural stem cells; a muscle bundle manufacturing step for manufacturing muscle bundles by culturing a hydrogel containing muscle cells; a neuromuscular junction formation step for co-culturing the hydrogel containing the neural biohybrid and the muscle bundle to form a neuromuscular junction; a drug contact step for contacting a candidate substance with the muscle bundle; and a drug evaluation step for comparing the degree of muscle contraction in a muscle bundle contacted with the candidate substance with a muscle bundle not contacted with the candidate substance.

[0018] The present invention relates to a neural biohybrid, a method for producing the same, a neuromuscular junction model using the same, and a method for screening drugs related to motor neuron diseases using the same.

[0019] The present inventors studied a method for producing neural biohybrids or brain organoids by integrating rGOp and HUVECs into hNSCs or IPSCs to mimic a neuromuscular junction similar to that of humans, and completed the present invention.

[0020] Hereinafter, the present invention will be described in more detail.

[0021] One aspect of the present invention relates to a neural biohybrid manufactured by culturing a mixture of carbon materials, vascular cells, and neural stem cells.

[0022] In the present invention, the carbon material may be at least one selected from the group consisting of reduced graphene oxide nanoparticles (rGOp), carbon nanotubes, carbon nanofibers, and graphite, and may be, for example, reduced graphene oxide nanoparticles, but is not limited thereto.

[0023] In the present invention, the vascular cell may be at least one selected from the group consisting of vascular endothelial cells (HUVEC), vascular endothelial progenitor cells, and vascular smooth muscle cells, and may be, for example, vascular endothelial cells, but is not limited thereto.

[0024] The rGO mixed in the above mixture can enhance neural development and differentiation of the neural biohybrid.

[0025] Vascular cells embedded within the above neural biohybrid can improve neural cell growth and neural network development through efficient distribution of oxygen and nutrients.

[0026] Another aspect of the present invention relates to a neuromuscular junction model manufactured by co-culturing a neural biohybrid manufactured by culturing a mixture of carbon materials, vascular cells, and neural stem cells; and a muscle bundle manufactured by culturing a hydrogel containing muscle cells.

[0027] In the present invention, the carbon material may be at least one selected from the group consisting of reduced graphene oxide nanoparticles (rGOp), carbon nanotubes, carbon nanofibers, and graphite, and may be, for example, reduced graphene oxide nanoparticles, but is not limited thereto.

[0028] In the present invention, the vascular cell may be at least one selected from the group consisting of vascular endothelial cells, vascular endothelial progenitor cells, and vascular smooth muscle cells, and may be, for example, vascular endothelial cells, but is not limited thereto.

[0029] In the present invention, the muscle cell may be at least one selected from the group consisting of myoblasts, myocytes, and myotubes, but is not limited thereto.

[0030] In the present invention, the co-culture of the neural biohybrid and the muscle bundle may be performed for 3 to 28 days, preferably 3 to 21 days, 3 to 14 days, 7 to 28 days, 7 to 21 days, and for example, 7 to 14 days, but is not limited thereto.

[0031] Another aspect of the present invention is a method for producing a neural biohybrid comprising the following steps:

[0032] A culture step in which carbon materials, vascular cells, and neural stem cells are mixed and cultured.

[0033] In the present invention, the carbon material may be at least one selected from the group consisting of reduced graphene oxide nanoparticles (rGOp), carbon nanotubes, carbon nanofibers, and graphite, and may be, for example, reduced graphene oxide nanoparticles, but is not limited thereto.

[0034] In the present invention, the vascular cell may be at least one selected from the group consisting of vascular endothelial cells, vascular endothelial progenitor cells, and vascular smooth muscle cells, and may be, for example, vascular endothelial cells, but is not limited thereto.

[0035] Another aspect of the present invention is a method for screening drugs related to motor neuron disease, comprising the following steps:

[0036] A neural biohybrid manufacturing step for manufacturing a neural biohybrid by culturing a mixture of carbon materials, vascular cells, and neural stem cells; a muscle bundle manufacturing step for manufacturing muscle bundles by culturing a hydrogel containing muscle cells; a neuromuscular junction formation step for co-culturing the hydrogel containing the neural biohybrid and the muscle bundle to form a neuromuscular junction; a drug contact step for contacting a candidate substance with the muscle bundle; and a drug evaluation step for comparing the degree of muscle contraction in a muscle bundle contacted with the candidate substance with a muscle bundle not contacted with the candidate substance.

[0037] In the present invention, the carbon material may be at least one selected from the group consisting of reduced graphene oxide nanoparticles (rGOp), carbon nanotubes, carbon nanofibers, and graphite, and may be, for example, reduced graphene oxide nanoparticles, but is not limited thereto.

[0038] In the present invention, the vascular cell may be at least one selected from the group consisting of vascular endothelial cells, vascular endothelial progenitor cells, and vascular smooth muscle cells, and may be, for example, vascular endothelial cells, but is not limited thereto.

[0039] In the present invention, the muscle cell may be at least one selected from the group consisting of myoblasts, myocytes, and myotubes, but is not limited thereto.

[0040] In the present invention, the co-culture of the neural biohybrid and the muscle bundle may be performed for 3 to 28 days, preferably 3 to 21 days, 3 to 14 days, 7 to 28 days, 7 to 21 days, and for example, 7 to 14 days, but is not limited thereto.

[0041] In the present invention, the motor neuron disease may be at least one selected from the group consisting of amyotrophic lateral sclerosis (ALS), progressive bulbar palsy, pseudobulbar palsy, progressive muscular atrophy (PMA), progressive lateral sclerosis (PLS), and monomeric amyotrophy (MMA), but is not limited thereto.

[0042] The neural biohybrid manufactured in the present invention directly enhanced stem cell growth, neural network development, neurogenesis and differentiation, and when applied to a neuromuscular junction (NMJ) model, enhanced connectivity between NMJ motor nerve endings and muscle fibers was observed compared to simple neural spheroids.

[0043] Additionally, we compared NMJs containing ALS-biohybrids derived from ALS patients with normal biohybrids and confirmed that muscle contraction was reduced and that the degree of muscle contraction was restored through treatment with the ALS drug bosutinib.

[0044] Therefore, it is expected that the neurobio hybrid of the present invention can be applied to drug screening and toxicity evaluation for various neurological diseases.

[0045] Figure 1a is a schematic diagram showing a process for manufacturing a muscle bundle according to one embodiment of the present invention.

[0046] Figure 1b is a fluorescent image showing a muscle bundle manufactured according to one embodiment of the present invention stained using an immunostaining method.

[0047] Figure 1c is a graph showing the degree of voluntary contraction of a muscle bundle manufactured according to one embodiment of the present invention.

[0048] FIG. 2a is a schematic diagram illustrating a process for producing normal and ALS neural biohybrids according to one embodiment of the present invention.

[0049] Figure 2b is a fluorescent image showing a normal nerve biohybrid manufactured according to one embodiment of the present invention stained by immunostaining.

[0050] Figure 2c is a fluorescent image showing an ALS neural biohybrid manufactured according to one embodiment of the present invention stained by immunostaining.

[0051] FIG. 2d is a fluorescence image showing a neural biohybrid manufactured according to one embodiment of the present invention, which was immunostained using CD31, a HUVEC marker, to confirm the presence of vascular endothelial cells (HUVECs) in the neural biohybrid.

[0052] Figure 2e is a fluorescence image comparing the difference between an ALS neural biohybrid manufactured according to one embodiment of the present invention and a control group not treated with bosutinib after treating the ALS neural biohybrid with bosutinib for 7 days.

[0053] FIG. 3a is a schematic diagram illustrating a process for manufacturing a 3D neuromuscular junction model using a hydrogel embedded with normal and ALS neuronal biohybrids according to one embodiment of the present invention.

[0054] Figure 3b is a fluorescent image showing a 3D neuromuscular junction stained by immunostaining using a hydrogel in which a biohybrid and a control spheroid manufactured according to one embodiment of the present invention are embedded.

[0055] FIG. 3c is a graph comparing the degree of recovery of muscle movement over time after treating a 3D ALS-neuromuscular junction manufactured according to one embodiment of the present invention with conservative treatment.

[0056] Figure 3d is a graph comparing the movement of muscle bundles to confirm the recovery effect of muscle movement according to the concentration of conservativenib according to one embodiment of the present invention.

[0057] Figure 3e is a graph comparing the movement of muscle bundles to confirm the recovery effect of muscle movement according to the concentration of conservativenib according to one embodiment of the present invention.

[0058] The present invention relates to a neural biohybrid manufactured by culturing a mixture of carbon materials, vascular cells, and neural stem cells.

[0059] Hereinafter, the present invention is described in detail through 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.

[0060]

[0061] Example 1. Preparation of muscle bundles and confirmation of differentiated muscle bundles.

[0062] We fabricated 3D muscle bundles mimicking the similar skeletal muscle structure of human cells using C2C12, a type of skeletal muscle cell.

[0063] First, the hydrogel used for the 3D muscle bundle was prepared using a mixture of C2C12 340 μl, 30% Matrigel, 250 μl 16 mg / ml fibronectin, 10 μl thrombin (0.5 U / 1 mg fibrinogen), and 100 μl DMEM.

[0064] Then, as shown in Fig. 1a, the hydrogel was spread into a pre-made PDMS mold, and DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin-G, 100 μg / mL streptomycin, and 1 mg / mL aminocaproic acid (ACA) was added as a muscle cell growth culture medium.

[0065] After culturing for 4 days, the medium was replaced with DMEM medium containing 2% horse serum, 100 U / mL penicillin-G, 100 μg / mL streptomycin, 1 mg / mL aminocaproic acid, and 1 ng / mL insulin growth factor-1 (IGF-1) as a muscle cell differentiation medium. The medium was replaced every two days, and differentiation was performed for more than 14 days.

[0066] To confirm muscle cell differentiation within 3D muscle bundles, immunostaining was performed using the differentiation marker α-actinin. As shown in Figure 1b, the development of myofibers within the muscle bundles was confirmed. Furthermore, after differentiation for more than 14 days, spontaneous muscle contraction was confirmed, as shown in Figure 1c.

[0067]

[0068] Example 2. Preparation of normal and ALS neural biohybrids and confirmation of differentiated neural biohybrids.

[0069] To generate normal and ALS neural biohybrids, human neural stem cells (hNSCs) and neural stem cells derived from pluripotent stem cells from an ALS patient (ALS-hNSCs, a 55-year-old Caucasian female diagnosed with ALS) were grown on laminin-coated tissue culture plates with KnockOut™ DMEM / F-12 medium containing 20 ng / ml bFGF and 20 ng / ml EGF.

[0070] Neural spheroids were cultured with hNSCs and ALS-hNSCs (7.0 x 10 per well) 4 ) and vascular endothelial cells (HUVEC) (7.0 x 10 per well) 3 (Dog cells) and reduced graphene oxide nanoparticles (rGOp) 0.1 mg / ml were mixed and seeded in a 96-well plate as shown in Fig. 2a. Forty-eight hours after cell seeding, the culture medium was replaced with motor neuron differentiation medium containing 8 ng / ml bFGF, 200 ng / ml shh, 10 ng / ml activin A, and 50 μM retinoic acid. After 20 days of motor neuron differentiation, the medium was replaced with motor neuron differentiation medium containing 10 ng / ml BDNF, 10 ng / ml and GDNF for maturation of neural biohybrids, and cultured for 8 days.

[0071] To confirm differentiation of normal and ALS neural biohybrid cells, immunostaining was performed using differentiation markers islet1 and Tuj1. As shown in Figures 2b and 2c, motor neuron gene expression was significantly higher than before. Furthermore, as shown in Figure 2d, expression of the vascular marker CD31 was confirmed in neural biohybrid cells containing HUVECs.

[0072] In addition, to confirm the ALS neural biohybrid, immunostaining was performed to confirm abnormal TDP-43 protein aggregation that was not found in normal neural biohybrid, and after treatment with the ALS drug bosutinib for 7 days, the amount of TDP-43 protein aggregation was confirmed to be reduced as shown in Fig. 2e as confirmed through immunostaining.

[0073]

[0074] Example 3. Manufacturing and Confirmation of 3D Neuromuscular Junctions Using Normal and ALS Neuronal Biohybrids

[0075] After muscle bundles were generated through the formation and differentiation of muscle bundles in a PDMS mold as shown in Fig. 3a, pre-differentiated normal and ALS neural biohybrids and CNT-COOH (0.1 mg / ml) were added to a hydrogel prepared using ECM proteins (30% Matrigel, 4 mg / ml fibronectin, 0.5 U / 1 mg thrombin) and further seeded on top of the muscle bundles for co-culture. The co-culture differentiation medium was supplemented with brain-derived and glial cell line-derived neurotrophic factors (10 ng / ml BDNF and 10 ng / ml GDNF) to support the viability of multiple neural biohybrids along with the muscle differentiation medium. The differentiation medium was changed every 2 days for 2 weeks.

[0076] As shown in Fig. 3b, after 10 days of co-culture, NMJ formation was confirmed through immunostaining using α-BTX, an NMJ marker, and it was confirmed that the highest amount of α-BTX was expressed in NMJs manufactured using neural biohybrid than in existing neural spheroids.

[0077] NMJs manufactured using ALS neural biohybrids showed significantly reduced muscle bundle movement compared to normal ones, and as shown in Figure 3c, after 5 days of treatment with the drug bosutinib, muscle bundle contraction was confirmed to have recovered to approximately 85% of the muscle movement of normal NMJs. In addition, muscle contraction according to the concentration of bosutinib was measured, and as shown in Figures 3d and 3e, it was confirmed that muscle movement recovery was greatest at 100 μM.

[0078] The present invention relates to a neural biohybrid, a neuromuscular junction model using the same, and a method for screening drugs related to motor neuron diseases using the same.

Claims

1. A neural biohybrid manufactured by culturing a mixture of carbon materials, vascular cells, and neural stem cells.

2. A biohybrid according to claim 1, wherein the carbon material is at least one selected from the group consisting of reduced graphene oxide nanoparticles (rGOp), carbon nanotubes, carbon nanofibers, and graphite.

3. A biohybrid according to claim 1, wherein the vascular cells are at least one selected from the group consisting of vascular endothelial cells (HUVEC), vascular endothelial progenitor cells, and vascular smooth muscle cells.

4. A neural biohybrid manufactured by culturing a mixture of carbon materials, vascular cells, and neural stem cells; and Muscle bundles manufactured by culturing hydrogels containing muscle cells; A neuromuscular junction model manufactured by co-culturing.

5. A neuromuscular junction model in the fourth paragraph, wherein the carbon material is at least one selected from the group consisting of reduced graphene oxide nanoparticles (rGOp), carbon nanotubes, carbon nanofibers, and graphite.

6. A neuromuscular junction model in the fourth paragraph, wherein the vascular cells are at least one selected from the group consisting of vascular endothelial cells (HUVEC), vascular endothelial progenitor cells, and vascular smooth muscle cells.

7. A neuromuscular junction model in paragraph 4, wherein the muscle cell is at least one type selected from the group consisting of myoblasts, myocytes, and myotubes.

8. A method for producing a neural biohybrid comprising the following steps: A culture step in which carbon materials, vascular cells, and neural stem cells are mixed and cultured.

9. A method for producing a biohybrid in claim 8, wherein the carbon material is at least one selected from the group consisting of reduced graphene oxide nanoparticles (rGOp), carbon nanotubes, carbon nanofibers, and graphite.

10. A method for producing a biohybrid in claim 8, wherein the vascular cells are at least one selected from the group consisting of vascular endothelial cells (HUVEC), vascular endothelial progenitor cells, and vascular smooth muscle cells.

11. A method for screening drugs related to motor neuron disease, comprising the following steps: A neural biohybrid manufacturing step for manufacturing a neural biohybrid by culturing a mixture of carbon materials, vascular cells, and neural stem cells; A muscle bundle manufacturing step for manufacturing muscle bundles by culturing a hydrogel containing muscle cells; A neuromuscular junction formation step in which a hydrogel containing the above-mentioned neural biohybrid and the above-mentioned muscle bundle are co-cultured to form a neuromuscular junction; a drug contact step of contacting a candidate substance with the muscle bundle; and A drug evaluation step that compares the degree of muscle contraction in muscle bundles that have been contacted with the candidate substance with muscle bundles that have not been contacted with the candidate substance.

12. A drug screening method according to claim 11, wherein the carbon material is at least one selected from the group consisting of reduced graphene oxide nanoparticles (rGOp), carbon nanotubes, carbon nanofibers, and graphite.

13. A drug screening method according to claim 11, wherein the vascular cells are at least one selected from the group consisting of vascular endothelial cells (HUVEC), vascular endothelial progenitor cells, and vascular smooth muscle cells.

14. A drug screening method according to claim 11, wherein the muscle cell is at least one selected from the group consisting of myoblasts, myocytes, and myotubes.

15. A drug screening method according to claim 11, wherein the motor neuron disease is at least one selected from the group consisting of amyotrophic lateral sclerosis (ALS), progressive bulbar palsy, pseudobulbar palsy, progressive muscular atrophy (PMA), progressive lateral sclerosis (PLS), and monomeric amyotrophy (MMA).

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