MANUFACTURE OF STRUCTURE CAPABLE OF FORMING THREE-DIMENSIONAL NEURONAL SPHEROID AND GENERATING NEURITE THROUGH VARIOUS SURFACE PROCESSES

Abstract

The present invention relates to the manufacture of a platform for forming a neuronal spheroid and, more specifically, to the manufacture of a structure capable of simultaneously forming a test-tube three-dimensional neuronal spheroid and generating a neurite by forming a three-dimensional neuronal spheroid with a micro-platform and forming a neurite between the neuronal spheroid and the micro-platform so as to enable signal transduction, which is an essential function of a nerve cell.

Claims

1. A platform for three-dimensional (3D) nerve cells culture, comprising concave microwells surface-treated with 3-aminopropyltriethoxysilane (APTES).

2. The platform of claim 1, wherein the concave microwells are hemispherical with a diameter of 100 μm to 1,000 μm.

3. The platform of claim 1, wherein the platform for culturing 3D nerve cells promotes the formation of neuronal spheroids and neurites.

4. The platform of claim 1, wherein the substrate consists of a plurality of concave microwells, wherein open spaces are present between the adjacent concave microwells.

5. The platform of claim 1, wherein the substrate is further surface-treated with one or more of carbon nanotubes (CNTs), laminin, and poly-L-lysine (PLL).

6. The platform of claim 1, wherein the substrate consisting of the concave microwells is made of polydimethylsiloxane (PDMS).

7. A method of culturing 3D nerve cells in vitro, comprising: culturing nerve cells in the platform for culturing 3D nerve cells defined in claim 1.

8. A method of screening a drug for treatment of damaged nerves or neurological disorders, comprising: culturing nerve cells in the platform for culturing 3D nerve cells defined in claim 1; treating the cultured nerve cells with a candidate substance; treating the nerve cells treated with the candidate substance with a substance that causes neurological disorders; and comparing a degree of formation of neurites and neuronal spheroids in the nerve cells with that of the control (not treated with the candidate substance).

Description

DESCRIPTION OF DRAWINGS

[0031] FIG. 1 is a schematic diagram showing a process of surface-treating a concave microwell in which a neurite and a neuronal spheroid may be formed.

[0032] FIG. 2 shows different chemical properties of five surfaces obtained by performing a surface process using Fourier transform infrared spectroscopy.

[0033] FIG. 3 shows surface properties of concave microwells obtained by five surface processes using an atomic force microscope. Scale bar: 500 nm

[0034] FIG. 4 shows different physical properties of the concave microwells obtained by five surface processes by measuring a contact angle.

[0035] FIG. 5 shows different cell adhesive forces of the concave microwells obtained by five surface processes.

[0036] FIG. 6 shows that neuronal spheroids are formed in the concave microwells by five surface processes: A) Optical microscope image obtained by date; B) Days required to form neuronal spheroids; and C) Size of neuronal spheroids. Scale bar: 100 μm

[0037] FIG. 7 shows an image of transporting calcium in the neuronal spheroids cultured on the five surfaces using a calcium indicator (Fluo-4 AM). Scale bar: 50 μm

[0038] FIG. 8 shows whether the cultured neuronal spheroids are formed and neurites are generated in the manufactured concave microwells by the five surface processes: A) Electron microscope image of neuronal spheroids; B) Image of fluorescence-stained neuronal spheroids and neurites; C) Number of neurites formed in each concave microwell; D) Length of neurites formed in each concave microwell; E) Relationship between contact angle and cell adhesive force and formation of nerve cells; and F) Schematic diagram showing the relationship between each surface and adhesive force of nerve cells and formation of neurites. Scale bar: 200 μm

[0039] FIG. 9 is a microscope image showing the neurites connected to the neuronal spheroids cultured in a network structure in a uniform distance using a surface process. Scale bar: 500 μm

[0040] FIG. 10 shows a neurite reduction model for administering amyloid-beta to verify a drug screening model: A) Neuronal spheroids in concave microwells treated with APTES; B) Accumulation of amyloid-beta; C) Neuronal spheroids treated with amyloid-beta; D) Increased accumulation of amyloid-beta; E) Comparison of accumulation of amyloid-beta; F) Comparison of the number of neurites; and G) Comparison of lengths of neurites.

BEST MODE

[0041] Hereinafter, the present invention will be described in further detail with reference to Examples according to the present invention. However, it should be understood that the following Examples are not intended to limit the scope of the present invention.

[0042] An interaction force between cells and a neurite structure was optimized by a surface process so that a neurite structure could be generated and at the same time, neuronal spheroids could be formed in a regular shape and at regular intervals. Then, nerve cells extracted from a prenatal rat were cultured in concave microwells made of polydimethylsiloxane (PDMS) (FIG. 1).

[Example 1] Surface Process

[0043] Surfaces of concave microwells were processed using an aminosilane functional group of APTES, carbon nanotubes, poly-L-lysine, and laminin. First, the concave microwells were surface-treated with oxygen plasma (100 W, 30 s), and then further surface-treated with a 10% (volume ratio relative to water) APTES solution or 0.01% (volume ratio relative to water) carbon nanotubes, or 20 μg/ml laminin or 20 μg/ml poly-L-lysine for approximately 1 hour.

[Example 2] Confirmation of Chemical, Surface and Physical Properties of Surface

[0044] To determine a chemical difference in surfaces of concave microwells coated with several substances, it was confirmed that the five concave microwells showed different chemical properties using Fourier transform infrared (FTIR) spectroscopy (FIG. 2). Also, it was confirmed that the different substances were attached to the surfaces of the concave microwells using an atomic force microscope (AFM) (FIG. 3). In addition, it was confirmed that the highest contact angle was formed on a surface of polydimethylsiloxane itself and the smallest contact angle was formed on the surface treated with poly-L-lysine when the contact angle was measured (FIG. 4).

[Example 3] Experiment for Attachment of Nerve Cells

[0045] To measure an interaction force between the cells and the structure, an experiment for attachment of nerve cells to five surfaces having different properties, which were manufactured by a surface process, was performed. 2*10.sup.5 nerve cells were seeded under each surface condition, and cultured for 3 hours, and a culture medium was then replaced three times to remove non-adherent cells. To measure an amount of the remaining cells, the cells were quantified using Cell Counting Kit-8 (Dojindo, USA). As a result, it was confirmed that the smallest amount of cells were attached to the surface of polydimethylsiloxane itself, and the highest amount of cells were attached to the surface treated with poly-L-lysine (FIG. 5).

[Example 4] Formation of Neuronal Spheroids

[0046] A formation period of the neuronal spheroids was determined under different surface conditions. The neuronal spheroids were formed within one day after cell seeding in the concave microwells made of polydimethylsiloxane and the concave microwells surface-treated with carbon nanotubes. It was shown that the neuronal spheroids were formed on day 5 after cell seeding in the case of the concave microwells treated with APTES, and the neuronal spheroids were not formed even after 10 days in the case of the concave microwells treated with laminin or poly-L-lysine. Meanwhile, it was confirmed that there was no significant difference in size between the generated neuronal spheroids (FIG. 6).

[Example 5] Confirmation of Signal Transduction via Neurons

[0047] To verify the neurotransmission activated by potassium, signal transduction was measured in a fluorescence image using an intracellular calcium indicator Fluo-4 AM. The neuronal spheroids into which Fluo-4 was injected in advance were stimulated with a high concentration of potassium to confirm calcium transport. In this way, it was verified that abnormalities in the nerve cells grown on the surface under the five different conditions were not induced by the surface process when the nerve cells were subjected to the surface process (FIG. 7).

[Example 6] Confirmation of Generation of Neurites and Formation of Neuronal Spheroids

[0048] The nerve cells were incubated for 10 days in the concave microwells under the five different surface conditions, fluorescence-stained with β-III tubulin, and then observed with an electron microscope to determine whether the neuronal spheroids were formed and the neurites were generated. As a result, the neuronal spheroids were formed in the concave microwells made of polydimethylsiloxane and the concave microwells treated with carbon nanotubes and APTES. It was confirmed that the highest amount of neurites were formed in the concave microwells treated with poly-L-lysine, and the neurites were formed longest in the concave microwells treated with APTES. Therefore, it was concluded that it was most suitable to generate the neurites and form the neuronal spheroids in the concave microwells treated with APTES. Based on the results, it was confirmed that an repulsive force between the cells and the structure decreased, a contact angle decreased, hydrophobicity decreased, the affinity between cells and the structure increased, surface wettability increased, the time taken to form a cell structure increased, and the number of formed neurites increased in the order of the concave microwells made of polydimethylsiloxane, and the concave microwells treated with carbon nanotubes, APTES, laminin, and poly-L-lysine (FIG. 8). Also, it was confirmed that neurites between the neuronal spheroids were formed and connected through the network between the concave microwells treated with APTES (FIG. 9).

[Example 7] Experiment for Administration of Amyloid-Beta for Drug Screening

[0049] After confirming that it was optimal to form the neuronal spheroids and generate the neurites in the concave microwells treated with APTES, amyloid-beta that is known to be a representative substance that causes Alzheimer's disease was administered to determine whether the concave microwells treated with APTES can be used to screen drugs. To determine the accumulation of amyloid-beta into the neuronal spheroids, thioflavin-S was used to obtain a fluorescence image. As a result, it was confirmed that the number and length of the neurites in the neuronal spheroids treated with amyloid-beta decreased dramatically. Based on the results, it was verified that the method provided in the present invention is able to be used to screen drugs (FIG. 10).