Basal ganglia-on-chip for screening therapeutic agents for brain and nervous system diseases

Abstract

The present disclosure provides a basal ganglia-on-a-chip for screening therapeutic agents for brain and nervous system diseases and a method for fabricating the same. The present invention provides a method for screening therapeutic agents for dopamine-dependent brain and nervous system diseases by using a basal ganglia-on-a-chip. When the basal ganglia-on-a-chip of the present invention is used in the effect evaluation of therapeutic agents for brain and nervous system diseases, the effect evaluation of therapeutic candidate substances can be economically and promptly carried out compared with an existing technique.

Claims

1. A basal ganglia-on-a-chip for screening therapeutic agents for dopamine-dependent brain and nervous system diseases, the basal ganglia-on-a-chip comprising: graphene-conjugated magnetic nanoparticles patterned on a substrate; a first layer comprising (i) a first hydrogel containing glutamatergic neurons and (ii) a second hydrogel containing GABAergic neurons, the first and second hydrogels being disposed in parallel on a pattern of the graphene-conjugated magnetic nanoparticles; a second layer comprising a third hydrogel in contact with the second hydrogel, the third hydrogel containing GABAergic neurons and neuronal membrane protein-specific antibody-conjugated magnetic nanoparticles; and a third layer comprising a fourth hydrogel in contact with the third hydrogel, the fourth hydrogel containing dopaminergic neurons and neuronal membrane protein-specific antibody-conjugated magnetic nanoparticles, wherein the third hydrogel is on the second hydrogel and the fourth hydrogel is on the third hydrogel, and wherein the GABAergic neuron of the third hydrogel and the dopaminergic neurons of the fourth hydrogel induced vertical growth towards the substrate, wherein the first hydrogel and the second hydrogel have a concentration of gelatin methacrylate (GeIMA) of more than 3.0 w/w % and less than 5.0 w/w %, and wherein the GeIMA is synthesized by adding 0.4 mL/g methacrylate to Dulbecco's phosphate-buffered saline (DPBS) in which 10 w/v % gelatin is dissolved.

2. The basal ganglia-on-a-chip of claim 1, wherein the third hydrogel to the fourth hydrogel contain at least one hydrogel monomer selected from the group consisting of gelatin methacrylate (GeIMA), acrylic acid, acrylamide, N-isopropylacrylamide (NIPAAM), and polyethylene glycol diacrylate (PEGDA).

3. The basal ganglia-on-a-chip of claim 1, wherein the first hydrogel to the fourth hydrogel further contain decellularized brain matrix (DECM).

4. The basal ganglia-on-a-chip of claim 1, wherein the graphene-conjugated magnetic nanoparticles are manufactured by combining a modified amine group on a surface of the magnetic nanoparticle and a carboxyl group of a graphene oxide.

5. The basal ganglia-on-a-chip of claim 1, wherein the antibody specific to cell membrane proteins which is bound to the antibody-conjugated magnetic nanoparticles specific to the neuronal membrane protein is an antibody specific to membrane receptors, transport proteins, membrane enzymes, or cell adhesion molecules in neurons.

6. A method for screening therapeutic agents for brain and nervous system diseases by using the basal ganglia-on-a-chip of claim 1, the method comprising: (a) treating dopaminergic neurons with a candidate of therapeutic agents for brain and nervous system diseases; and (b) investigating whether the dopaminergic neurons proliferate or are reduced.

7. The method of claim 6, wherein the dopaminergic neurons are induced to have damages.

8. The method of claim 7, wherein the damages are caused by oxidative stress.

9. The method of claim 6, wherein the dopaminergic neurons are induced to differentiate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A illustrates a neural circuit of the basal ganglia. The neural circuit is composed of an indirect neural circuit by glutamate and a direct neural circuit by GABA. In Parkinson's disease, the direct neural circuit is inhibited through specific apoptosis of dopamine-producing neurons.

(2) FIG. 1B illustrates a neural circuit simulating a Parkinson's disease-like model inducing specific apoptosis of dopamine-producing neurons.

(3) FIG. 2A schematically illustrates the vertical growth of neurons using magnetic nanoparticles with an anti-noradrenaline transporter antibody attached thereto.

(4) FIG. 2B illustrates the results that the treatment of cells with magnetic nanoparticles with an anti-noradrenaline transporter antibody attached thereto induced the vertical growth of the cells.

(5) FIG. 2C illustrates the results obtained by analyzing, using a confocal microscope, the induction of vertical growth of cells through the treatment of cells with magnetic nanoparticles with an anti-noradrenaline transporter antibody attached thereto.

(6) The top panel of FIG. 2D shows a schematic diagram illustrating the manufacturing of magnetic/graphene hybrid nanostructures. The magnetic/graphene hybrid nanostructures were manufactured by treating 10 nm-sized magnetic nanoparticles with 3-aminopropyl triethoxysilane (APTES) to modify a surface of the magnetic nanoparticles to exhibit an amine group and then allowing amine group-introduced magnetic nanoparticles to react with graphene oxide nanoflakes selected through ultrasonication and centrifugation. The bottom panel of FIG. 2d shows a schematic diagram illustrating a procedure in which the horizontal growth of neurons was induced by fine-contact printing magnetic/graphene hybrid nanostructures on a substrate, seeding neurons on the nanostructures, and then culturing the neurons under the application of electricity.

(7) FIG. 2E illustrates an SEM image (scale bar: 20 nm) of the magnetic/graphene hybrid nanostructures and a particle size thereof.

(8) FIG. 2F illustrates a micro-contact printing procedure using magnetic/graphene hybrid nanostructures depending on the presence or absence of magnetic force.

(9) FIG. 2G illustrates the results that the horizontal growth of neurons was induced in regions in which the magnetic/graphene hybrid nanostructures were micro-contact printed.

(10) FIG. 3A illustrates the results of confirming structural stability in culture media after neurons were printed on 3%, 4%, and 5% gelatin-methacrylate (Gel-MA).

(11) FIG. 3B illustrates the results that neurons printed on 4% Gel-MA formed neural networks.

(12) FIG. 3C shows H-NMR results of gelatin and Gel-MA. The red dotted circles represent a peak of methacrylate. The left graph shows an NMR peak of gelatin itself, and the right graph shows that the synthesis of gelatin and methacrylate was well done by confirming peaks of Gel-MA after methacrylate was synthesized in gelatin.

(13) FIG. 3D illustrates the cell morphology of SH-SY5Y according to the culture time in media containing decellularized porcine brain Matrix (DECM).

(14) FIG. 4A is a schematic diagram illustrating that a structure of the basal ganglia was mimicked and printed using a 3D cell printer.

(15) FIG. 4B shows printing images obtained utilizing a 3D printer. The left image illustrates printing using a 3D cell printer, and the right image shows a printed Gel-MA image.

(16) FIG. 4C is a schematic diagram of a Parkinson's disease-like basal ganglia structure.

(17) FIG. 4D shows a 3D printer-based basal ganglia-on-a-chip.

(18) FIG. 5A is a schematic diagram illustrating the treatment of a Parkinson's disease-like basal ganglia structure with L-dopa or 6-OHDA.

(19) FIG. 5B shows an image of a basal ganglia structure simulating neural structure observed by a confocal microscope. The right image confirms through a confocal microscope that dopaminergic neurons and GABAergic neurons are connected vertically relative to each other.

(20) FIG. 5C illustrates an increase in the proliferation of dopaminergic neurons by the treatment of Parkinson's disease-like basal ganglia structure with L-dopa.

DETAILED DESCRIPTION

(21) Hereinafter, the present invention will be described in more detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

(22) Throughout the present specification, the % used to express the concentration of a specific material, unless otherwise particularly stated, refers to (wt/wt) % for solid/solid, (wt/vol) % for solid/liquid, and (vol/vol) % for liquid/liquid.

EXAMPLE 1

Design of Simplified Neural Circuit in Basal Ganglia-on-Chip Model

(23) The basal ganglia are composed of various types of neurons, and have various complex neural circuits involved in motor and sensory regulation. The neural circuit has a direct neural circuit (direct pathway), in which the behavior evoking neural transmission is made through an excitatory neurotransmission pathway, and an indirect neural circuit (indirect pathway), in which the behavior evoking neural transmission is made through an inhibitory neurotransmission pathway, and it has been reported that such a neural circuit is involved in neurodegenerative brain diseases, such as Parkinson's disease and Huntington's chorea disease. Especially, Parkinson's disease, which is one of the neurodegenerative brain diseases, is caused by abnormal secretion of gamma-aminobutyric acid (GABA) from the globus pallidus interna (GPi) and substantia nigra reticulate (SNr) due to dysfunction of a direct neural circuit resulting from the apoptosis of dopamine-producing neurons in the substantia nigra pars compacta (SNc) located in the basal ganglia.

(24) As for a basal ganglia-on-a-chip model to be developed in the present study, the present inventors designed a neural circuit of a basal ganglia-on-a-chip model as shown in FIG. 1 in order to develop a basal ganglia-on-a-chip model in which a neural circuit associated with Parkinson's disease is simplified and simulated on a chip, and thus a normal neural circuit is simulated thereon, and a Parkinson's disease-like model in which the apoptosis of dopamine-producing neurons is simulated.

EXAMPLE 2

Control of Growth Direction of Magnetic Nanoparticle-Based Neurons

(25) In order to form vertical/horizontal neural networks for the fabrication of a basal ganglia simulating structure, a technique capable of controlling growth directivity of neurons is needed. A method was made that induces the vertical/horizontal growth of neurons by attaching magnetic nanoparticles to neurons, followed by culturing in hydrogel (4% gelatin methacrylate), and then applying a magnetic field in vertical/horizontal directions (FIG. 2).

(26) For the induction of the vertical/horizontal growth of neurons, zinc ferrite nanoparticles, corresponding to a strong magnetic substance having excellent reactivity to a magnetic field, were synthesized. To a 3-neck flask, 2 mmol Fe(acac).sub.3, 1 mmol ZnCl.sub.2, 6 mmol oleylamin, 6 mmol oleic acid, and 10 mmol 1,2-hexadecanediol were added, and then 20 ml of trioctylamine as a buffer was added. After being kept at 200? C. for 2 hours, the solution was heated at 300? C. and then kept for 1 hour. Thereafter, 100% ethanol and the synthesized material MNP (zinc ferrite nanoparticles) were mixed at a ratio of 3:1, followed by centrifugation. Then, the supernatant was discarded, and hexane as a buffer was added, and centrifugation was repeatedly performed three times to determine the size of particles. An amine group was formed on a surface of the synthesized zinc ferrite nanoparticles by the treatment with (3-aminopropyl)triethoxysilane (APTES). The amine group on the surface of the zinc ferrite nanoparticles and the N-terminus of the anti-noradrenaline transporter antibody (Sigma-Aldrich) were attached to each other using a 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-Hydroxysuccinimide (EDC/NHS) coupling method, thereby synthesizing magnetic nanoparticles capable of attaching to neurons.

(27) The magnetic nanoparticles-attached neurons were cultured on a hydrogel (4% gelatin methacrylate), and after 24 hours, a magnetic field was applied for 7 days to induce the vertical growth of neurons. According to an existing method, a magnetic field was formed using a neodymium magnet, but for the accuracy of an experiment, a magnetic field forming device was provided to apply a constant magnetic field at the level (400-450 mT) similar to that of a neodymium magnet, thereby inducing the vertical growth of neurons, and thus the accuracy of the experiment was improved. A magnetic field was applied using the magnetic field forming device for about 5-7 days to induce directivity, thereby forming neural networks between dopaminergic neurons and GABAergic neurons (FIG. 2b).

(28) After the vertical growth was induced, fluorescein isothiocyanate (FITC) attached to the anti-noradrenaline transporter antibody was imaged using a confocal microscope to conform that the neurons were induced to grow in a direction in which the magnetic field was applied, and it was confirmed that neurons continued without breakage upon the induction of vertical growth of neurons (FIG. 2).

(29) Also, for the induction of horizontal growth, 10 nm-sized magnetic nanoparticles were treated with APTES such that a surface of the magnetic nanoparticles was modified to exhibit an amine group (FIG. 2d). The amine group-introduced magnetic nanoparticles were subjected to ultrasonication and centrifugation, so that the magnetic nanoparticles bunched together were separated from each other to utilize magnetic nanoparticles as small as possible (FIG. 2e). The selected magnetic nanoparticles were allowed to react with graphene oxide (combination of the amine group of APTES attached to the magnetic nanoparticles and the carboxyl group of GO) to manufacture magnetic/graphene hybrid nanostructures. Specifically, a 5% APTES solution was mixed at a ratio of 1:1 with a solution obtained by diluting 5 mg/ml magnetic nanoparticles to 1/10. The mixture was maintained at 25? C. for 30 min in a stirrer. In addition, a solution obtained by diluting a 1 g/L graphene oxide solution to 1/2 was allowed to react with a magnetic nanoparticle solution reacted with APTES at 25? C. for 30 min in a stirrer.

(30) Linear magnetic/graphene patterns were manufactured on a polydimethylsiloxane (PDMS) substrate using the magnetic/graphene hybrid nanostructures by micro-contact printing method using PDMS stamp. As a result of micro-contact printing, it was confirmed through the SEM images that the magnetic/graphene hybrid nanostructure linear patterns were more clearly formed in the presence of magnetic force, and thus the micro-contact printing method with the application of magnetic force is a more suitable method for inducing the horizontal growth of cells (FIG. 2f). It was confirmed through an optical microscope that neurons were well immobilized in regions in which the magnetic/graphene hybrid nanostructures were micro-contact printed, but the cells hardly grew in regions in which the magnetic/graphene hybrid nanostructures were not micro-contact printed.

(31) Linear magnetic/graphene patterns with different intervals and thicknesses were formed on the PDMS through a micro-contact printing technique utilizing PDMS stamps with different intervals and thicknesses. Thereafter, as a result of culturing neurons, it was confirmed that the neurons were cultured along the magnetic/graphene patterns (FIG. 2g).

EXAMPLE 3

Manufacturing of Bio-Ink to be Used in 3D Cell Printing and Fabrication of Basal Ganglia-on-a-Chip Model

(32) A hydrogel is needed to mimic the basal ganglia using a 3D cell printer on the basis of a vertical/horizontal network forming technique. The gelatin-methacrylate harmless to cells was synthesized. After 10% gelatin (Sigma-Aldrich) was completely dissolved in Dulbecco's phosphate-buffered saline (DPBS), 0.4 ml/g methacrylate (Sigma-Aldrich) was slowly added (0.1 mL/g), followed by reaction at 50? C. for about 4 hours. Thereafter, the total concentration was adjusted to 4.5% with DPBS, followed by dialysis. After the dialysis was completed, the resultant material was frozen at ?80? C. for about one day and then freeze-dried for 7 days. The synthesis was investigated through H-NMR (FIG. 3c). It is very important that the printed structure of the synthesized gelatin methacrylate is held without collapse until neurons can grow to form networks. Accordingly, it was investigated how long the structure was held after the structure was manufactured by preparing 3%, 4%, and 5% gelatin methacrylate and cell-printing the gelatin methacrylate using a 3D cell printer. It was confirmed that a structure of 4% gelatin methacrylate was held without collapse for 72 hours or longer (FIG. 3a). A factor that is as important as the structure being held is that neurons in the hydrogel well stretch and grow to form neural networks. Thus, the cell growth was observed by printing gelatin methacrylate in a grid pattern using a 3D cell printer. The cell printing was performed using 4% gelatin methacrylate, and then the neurons were grown for 4 days, stained, and observed through a confocal microscope. As a result, it was confirmed that the neurons well grew and stretch in the gelatin methacrylate (FIG. 3b). The 3% gelatin methacrylate was structurally unstable in culturing neurons due to a disadvantage that the structure collapses after about 2 days. The 5% gelatin methacrylate is unsuitable for the growth of neurons due to a disadvantage that the gelatin methacrylate floats in media. Therefore, a basal ganglia-on-a-chip was fabricated by using the 4% gelatin methacrylate having structural stability suitable to culture neurons and containing a hydrogel well attached to the bottom of a culture dish. The decellularized porcine brain matrix (DECM) extracted from the porcine brain was dissolved at a concentration of 0.1 mg/mL in a culture medium, and then the resultant medium, instead of an existing culture medium, was added to gelatin methacrylate every 12 hours to grow neurons.

(33) As for the DECM, the fresh porcine brain was directly purchased, and added to penicillin-containing PBS and sodium dodecyl sulfate (SDS, 0.1 wt/vol), and decellularized for 3-5 days while the supernatant was exchanged. After centrifugation using a centrifuge at 10,000 rpm/5 min, the supernatant was removed, followed by filling with tertiary distilled water. After this procedure was repeated about 12 times, the DECM was freeze-dried, and used if needed while stored at ?80? C. The prepared DECM at 0.1 mg/ml was added to DMEM containing 10% FBS and 1% penicillin, which was replaced for an existing culture medium every 24 hours while neurons were cultured. Compared with the neurons grown in an existing culture medium without the supplementation of DECM, the neurons (SH-S5SY) cultured in the DECM-supplemented culture medium showed a different growing form, a drop in the neuron proliferation rate as if the neurons differentiated, and long stretched axons (FIG. 3d).

(34) In order to fabricate a basal ganglia-on-a-chip, SH-SY5Y (ATCC CRL-2266) was used as dopaminergic neurons, and F3-NG1 (provided from professor Hong-Joon Lee at Chung-Ang University) were allowed to differentiate and then used as GABAergic neurons and glutamatergic neurons.

(35) The SH-SY5Y was cultured using DMEM/F12 containing 3% FBS, 1% penicillin/streptomycin, and 1 ?M retinoic acid (RA) while the medium was exchanged at intervals of 2 days.

(36) For the differentiation of F3-NG1 into GABAergic neurons, the cells were cultured in a medium for differentiation (DMEM/F12 supplemented with 10% FBS and 1 penicillin) for 1 day, a medium for differentiation containing B27 (1?), N2 (1?), 20 ng/ml basic fibroblast growth factor (bFGF), and 5 ?M valproic acid (VPA) for 2 days, and a medium for differentiation containing B27 (1?), 20 ng/ml brain-derived neurotrophic factor (BDNF), 20 ng/ml glial cell line-derived neurotrophic factor (GDNF), 20 ng/ml IGF, and 1 mM AA for 4-10 days.

(37) For the differentiation of F3-NG1 into glutamatergic neurons, the cells were cultured in a medium for differentiation containing B27 (1?), N2 (1?), and 20 ng/ml basic fibroblast growth factor (bFGF) for 1 day, and a medium for differentiation containing B27 (1?), N2 (1?), 20 ng/ml BDNF, and 100 ng/ml GDNF for 4-10 days.

(38) As for the bio-ink used in the experiments in the present invention, the synthesized Gel-MA was made to 4% by addition of DPBS, and the 4% Gel-MA was used by mixing with cells (0.033 mg/mL). The use of the bio-ink containing DECM was expected to significantly accelerate the stretching of cells or the growth rate thereof.

(39) Based on such a technique, a basal ganglia-on-a-chip can be fabricated by using neurons and gelatin methacrylate through a 3D cell printer.

EXAMPLE 4

Fabrication of Basal Ganglia-on-a-Chip-Based Parkinson's Disease-Like Model and Drug Screening

(40) Parkinson's disease is caused by the gradual loss of dopaminergic neurons distributed in the substantia nigra. 6-Hydroxydopamine (6-OHDA) causes oxidative stress to induce mitochondrial migration of c-Jun N-terminal kinase (JNK), and the activated JNK after mitochondrial migration causes mitochondrial dysfunction, contributing to apoptosis of dopaminergic neurons, occurring in Parkinson's disease.

(41) Based on this, a Parkinson's disease model was developed using 100 ?M 6-OHDA. After the Parkinson's disease-like model was established, 50 ?M Levodopa (L-dopa), and the proliferation of dopaminergic neurons was confirmed by artificially increasing the amount of L-dopa. These results indicate that the efficacy of therapeutic agents for treating dopamine-related diseases could be evaluated by using the Parkinson's disease-like model-based basal ganglia-on-a-chip of the present invention.

(42) Considering the above results, the basal ganglia are well simulated, and drug screening for a short time can be attained using a basal ganglia-on-a-chip model without animal experiments or clinical tests. Furthermore, on the basis of this technique, organs-on-chips could be fabricated, and several disease models can be implemented, such as a cerebral cortex-on-a-chip.