Method for producing neural cells derived from pluripotent stem cells formed by concave and shaking culture technique, and method for evaluating drug toxicity through 3D neural organoid produced thereby

Abstract

The present invention relates to a method for producing neural cells derived from pluripotent stem cells formed by a concave and shaking culture technique and a method for evaluating drug toxicity through a 3D neural organoid produced thereby. Neural cells produced by using the method for producing neural cells of the present invention, and 3D neural organoids produced by using a microfluidic chip for three-dimensional cell culture of the neural cells, are differentiated from embryoid bodies that form uniform cell aggregates through a concave and shaking culture technique, thereby enabling stable and high-purity purification of neural cells through the neural cells and the 3D neural organoids. In addition, the 3D neural organoid produced by the production method expresses the same marker factor as mature neurons, and thus can be effectively utilized in the evaluation of drug toxicity.

Description

Method for producing neural cells derived from pluripotent stem cells formed by concave and shaking culture techniques and method for evaluating drug toxicity using 3D neural organoids produced thereby

[0001] The present invention relates to a method for producing neural cells derived from pluripotent stem cells formed by concave and shaking culture techniques, and a method for evaluating drug toxicity using 3D neural organoids produced thereby.

[0002] Human pluripotent stem cells (hPSCs) are cells with infinite self-renewability and pluripotency that can differentiate into the three germ layers (endoderm, mesoderm, and ectoderm) that make up the human body under specific environments. As studies have been published that have induced their differentiation into neural cells, vascular cells, muscle cells, pancreatic cells, and hepatic cells, hPSCs have been recognized as important cells for toxicity assessment and research on developing disease treatments.

[0003] In general, in order to utilize pluripotent stem cells for purposes such as cell therapy and biological research to determine the characteristics of stem cells, a differentiation induction process is performed to induce differentiation into cells with specific functions. The most widely known and commonly used differentiation method is to culture pluripotent stem cells, then collect them to form embryoid bodies (EBs), culture the formed embryoid bodies in suspension, and then collect the embryoid bodies in the suspension culture and culture them in a differentiation medium for attachment, thereby inducing differentiation into the desired cells.

[0004] However, there are many differences between the differentiation of embryonic stem cells into cells of human organs and actual human cells, and thus there are clear limitations in applying the differentiation-induced cells to actual toxicity evaluation or new drug development.

[0005] In particular, in the case of nerve cells, research on nerve cells is being conducted to develop neuroprotective agents such as N-methyl-D-aspartate receptor antagonists for the treatment of Alzheimer's disease (AD), a neurodegenerative disorder. In addition, research on drug treatment and nerve cell treatment using stem cells is being actively conducted. In the process of such research, the development of new in vitro cell models is required to assess the toxicity risk of neurotoxic effects in response to drug treatment or cell therapy.

[0006] Currently, neurotoxicity assessments for various chemicals are performed using animal models. However, due to licensing and restriction regulations and problems in reproducing the functionality of nerve cells in animal models, there is a need to develop in vitro cell models to identify new cytotoxicities.

[0007] Accordingly, the inventors of the present invention developed a method for producing neural cells and neural organoids capable of reproducing the functions of mature neural cells using concave and shaking culture techniques, and confirmed the reproducibility of a human neural cell model using neural cells and neural organoids produced through the above production method, thereby completing the present invention.

[0008] The purpose of the present invention is to provide a method for producing 3D neural organoids by differentiating embryoid bodies formed by concave and shaking culture techniques to produce neural cells, and to reproduce mature neural cells using the produced neural cells using a 3D microfluidic chip.

[0009] Another object of the present invention is to provide a method for evaluating drug toxicity using neural organoids produced by the above neural organoid production method.

[0010] In order to achieve the above purpose, the present invention comprises the steps of: i) culturing human pluripotent stem cells in concaves having a diameter of 300 to 1,000 μm after enzyme treatment to form embryoid bodies;

[0011] ii) a step of culturing the embryonic body formed in step i) by shaking the embryonic body at 10 to 100 rpm;

[0012] iii) a step of forming a cell aggregate in the embryonic body cultured in step ii); and

[0013] iv) Provided is a method for producing neural cells, including a step of maturing the above cell aggregate and differentiating it into neural cells.

[0014] The present invention also provides a method for producing a 3D neural organoid, which includes a step of culturing neural cells produced by the above-described production method using a microfluidic chip for 3D cell culture.

[0015] The present invention also provides a 3D neural organoid manufactured according to the above manufacturing method,

[0016] The above neural organoid provides a 3D neural organoid that expresses at least one selected from the group consisting of SOX2, Nestin, TUBB3, MAP2, FOXO4, GFAP, LMX1A, CXCR4, CD113, CD15, and TuJ1.

[0017] The present invention also provides a method for evaluating drug toxicity using a 3D neural organoid manufactured according to the above manufacturing method, the method comprising the following steps:

[0018] i) a step of treating the neural organoid with a drug; and

[0019] ii) A step of measuring neural cell expression markers, cell viability or cytotoxicity in the above neural organoids.

[0020] The neural cells produced using the neural cell production method of the present invention and the 3D neural organoids produced using a 3D cell culture microfluidic chip are differentiated from embryonic bodies that form uniform cell aggregates through concave and shaking culture techniques, enabling stable and high-purity neural cell purification through the neural cells and 3D neural organoids. In addition, the 3D neural organoids produced by the above production method express the same markers as mature neural cells, and thus can be usefully utilized in drug toxicity assessment.

[0021] Figure 1 is a schematic diagram showing the process of producing neural cells from human pluripotent stem cells.

[0022] Figure 2 is a drawing showing the observation of embryoid bodies cultured using the pipetting technique and the hang-in-drop technique for the production of uniform embryoid bodies.

[0023] Figure 3 is a drawing comparing and observing each embryoid body cultured by differentiating human pluripotent stem cells in 300 μm, 500 μm, and 1,000 μm concave.

[0024] Figure 4 is a drawing showing the cell number measured through DAPI staining of each embryoid body cultured in 300 μm, 500 μm, and 1,000 μm concave.

[0025] Figure 5 is a drawing showing the morphological changes observed in each embryoid body cultured with 300 μm, 500 μm, and 1,000 μm concave cultured in a culture medium containing N2 and B27.

[0026] Figure 6 is a graph comparing the expression of Oct4, Nestin, TUBB3, and MAP2 in each embryoid body cultured in 300 μm, 500 μm, and 1,000 μm concave cultured in a medium containing N2 and B27.

[0027] Figure 7 is a drawing showing the results of morphological observation (yellow box: lens-shaped embryoid body and white box: black stone-shaped embryoid body) and immunostaining observation of embryoid bodies cultured in 500 μm concave for 10 days.

[0028] Figure 8 is a drawing showing the results of maintaining the culture of embryonic cells cultured in 500 μm concave through shaking culture.

[0029] Figure 9 shows the process of manufacturing 3D neural organoids using a microfluidic chip for 3D cell formation and the expression patterns of TuJ1 and MAP2 genes in the manufactured 3D neural organoids.

[0030] Figure 10 is a diagram showing the neurotoxicity evaluation experiment process in which the manufactured 3D neural organoids were treated with the neurotoxic drug vincristine, and the toxicity observed at each concentration of vincristine through immunostaining.

[0031] Hereinafter, the present invention will be described in detail.

[0032] Method for manufacturing nerve cells

[0033] In one aspect, the present invention comprises the steps of: i) culturing human pluripotent stem cells in concaves having a diameter of 300 to 1,000 μm after enzyme treatment to form embryoid bodies;

[0034] ii) a step of culturing the embryonic body formed in step i) by shaking the embryonic body at 10 to 100 rpm;

[0035] iii) a step of forming a cell aggregate in the embryonic body cultured in step ii); and

[0036] iv) It relates to a method for producing neural cells, including a step of maturing the above cell aggregate and differentiating it into neural cells.

[0037] In this specification, the term “human Pluripotent Stem Cell (hPSC)” may refer to a cell that has the ability to differentiate into all cells that constitute the human body.

[0038] In one embodiment of the present invention, the human pluripotent stem cell may be a human embryonic stem cell (ESC) or a human induced pluripotent stem cell (iPSC), preferably a human induced pluripotent stem cell (iPSC).

[0039] The term "embryonic stem cell" as used herein refers to a cell derived from the inner cell mass of a blastocyst prior to implantation, and the derived cell is a cell capable of unlimited culture and pluripotent differentiation.

[0040] The term "induced pluripotent stem cell" as used herein refers to a pluripotent differentiated cell created by dedifferentiation from a somatic cell of the body, and is a cell that is formed by making the somatic cell into a state similar to an embryonic stem cell through a reprogramming process such as cell fusion, nuclear transfer, and overexpression of a pluripotent regulatory factor.

[0041] As used herein, the term "concave" refers to a shape with a concave surface, and more specifically, refers to a hemispherical shape with a concave surface. The concave may have a plurality of "concave patterns" arranged in a certain direction, and the concave patterns may be arranged in multiseriate rows in the horizontal and vertical directions.

[0042] In another embodiment of the present invention, the concave pattern may be a multi-row arrangement of a plurality of concaves, with each row of the pattern being arranged in parallel.

[0043] In another embodiment of the present invention, the concave may preferably have a diameter of 500 μm. When human pluripotent stem cells are cultured in a concave having the above diameter, the rate of embryoid body formation can be maximized.

[0044] In another embodiment of the present invention, the enzyme of step i) may be at least one selected from the group consisting of accutase, trypsin, collagenase, hyaluronidase, heparinase, elastase, pronase, DNase, dispase, papain, chymotrypsin, and EDTA, and preferably may be trypsin and EDTA.

[0045] In another embodiment of the present invention, step i) may be performed in a medium containing 2% FBS.

[0046] In another embodiment of the present invention, the medium in step i) may be DMEM / F12.

[0047] In another embodiment of the present invention, step i) may be performed through 3D floating culture.

[0048] In another embodiment of the present invention, step i) may be performed in a culture dish coated with any one selected from the group consisting of Pluronic F-68, Pluronic F-127, Pluronic F-188, Pluronic F-237, Pluronic F-338 and Pluronic F-407.

[0049] In another embodiment of the present invention, step i) may preferably be performed in a culture dish coated with Pluronic F-127.

[0050] The term "shaking culture" in this specification may mean a method of performing culture by seeding cultured cells in a medium and continuously shaking them on a shaker incubator.

[0051] In another embodiment of the present invention, step ii) may be performed by placing a culture vessel containing a culture medium and a culture body formed in a concave containing a culture body on a shaking culture device and operating the device at 10 to 100 rpm, preferably 30 rpm.

[0052] The above shaking culture device includes a culture plate on which culture vessels are positioned. The culture plate may be a square plate or shelf on which several to several dozen culture vessels can be placed, and may be provided with pillars or side plates to prevent the culture vessels from tilting.

[0053] In another embodiment of the present invention, step ii) may be performed in a medium containing N2 and B27.

[0054] In another embodiment of the present invention, the medium in step ii) may be RPMI1640.

[0055] In another embodiment of the present invention, step ii) may be performed in a culture dish coated with Pluronic F-127.

[0056] In another embodiment of the present invention, step ii) may be performed for 1 to 3 days, preferably 1 day.

[0057] In another embodiment of the present invention, step iii) may be performed in a medium containing N2 and bFGF.

[0058] In another embodiment of the present invention, the concentration of bFGF may be 1 to 30 ng / ml, preferably 20 ng / ml.

[0059] In another embodiment of the present invention, the medium in step iii) may be RPMI1640.

[0060] In another embodiment of the present invention, step iii) may be performed through 2D attachment culture.

[0061] In another embodiment of the present invention, step iii) may be performed in a culture dish coated with poly-D-lysine.

[0062]

[0063] In another embodiment of the present invention, step iii) may be performed for 10 to 18 days, preferably 14 days.

[0064] In another embodiment of the present invention, the cell aggregate of step iii) may be enzyme-treated.

[0065] In another embodiment of the present invention, the enzyme of step iii) may be at least one selected from the group consisting of accutase, trypsin, collagenase, hyaluronidase, heparinase, elastase, pronase, DNase, dispase, papain, chymotrypsin, and EDTA, and preferably, accutase.

[0066] In the present invention, the culture medium of step iv) may use a known neural cell culture solution, and preferably, Sigma's neural cell differentiation medium (Neuronal Differentiation Medium) (Cat. No. SCM111) may be used.

[0067] In another embodiment of the present invention, step iv) may be performed for 2 to 6 days, preferably 4 days.

[0068] In another embodiment of the present invention, the nerve cells may be dissociated with trypsin-EDTA enzyme, preferably with TrypLE enzyme from Gibco.

[0069] In another embodiment of the present invention, the neural cells may be passaged.

[0070] In another embodiment of the present invention, the neural cells may exhibit a high survival rate while maintaining their shape even after freezing and thawing.

[0071] In another embodiment of the present invention, the neural cells may express at least one selected from the group consisting of SOX2, Nestin, TUBB3, MAP2, FOXO4, GFAP, LMX1A, CXCR4, CD113, CD15, and TuJ1.

[0072] In another embodiment of the present invention, the neuron may express Nestin, TUBB3 and MAP2.

[0073] In one embodiment of the present invention, the expression of Oct4, Nestin, TUBB3, and MAP2, which are markers of the ectoderm-early neural cell-neuron-maturation stages, was confirmed during the differentiation process of embryonic bodies (EBs) formed from iPSCs using concave and shaking culture techniques (Fig. 6). The expression of TuJ1 and MAP2, which are markers of mature neural cells, was confirmed through accutase enzyme treatment and subculture (Fig. 7).

[0074] That is, the neural cells produced by the neural cell production method according to the present invention exhibit the characteristics of mature neural cells, and high-purity neural cells can be produced through the concave and shaking culture techniques.

[0075] Method for producing neural organoids

[0076] Another aspect of the present invention relates to a method for producing a 3D neural organoid, comprising a step of culturing the neural cells produced by the above production method using a microfluidic chip for 3D cell culture.

[0077]

[0078] As used herein, the term "organoid," also called an organoid, refers to a three-dimensional cell aggregate formed through self-renewal and self-organization from adult stem cells (ASCs), embryonic stem cells, and induced pluripotent stem cells (iPSCs). Organoids are small, simplified in vitro three-dimensional (3D) organs that mimic the anatomical structure of actual tissues. By constructing organoids from patient tissues, disease modeling and drug toxicity assessment based on the patient's genetic information are possible.

[0079] In one embodiment of the present invention, the method for producing a 3D neural organoid by differentiating the neural cells into organoids may specifically use the method disclosed in Korean Patent No. 10-2155868 (Patent Document 0001) for forming organoids from neural cells, and may start culturing by seeding neural cells onto the microfluidic chip. (Figure 9)

[0080] In one embodiment of the present invention, the method for producing a 3D neural organoid may additionally include a step of aggregating neural cells after performing the culturing step.

[0081] 3D neural organoids manufactured by the manufacturing method

[0082] In another aspect, the present invention provides a 3D neural organoid manufactured according to the neural organoid manufacturing method,

[0083] The above neural organoid relates to a neural organoid expressing at least one selected from the group consisting of SOX2, Nestin, TUBB3, MAP2, FOXO4, GFAP, LMX1A, CXCR4, CD113, CD15, and TuJ1.

[0084]

[0085] In one embodiment of the present invention, the neural organoid may preferably express MAP2 and TuJ1.

[0086] In one embodiment of the present invention, the neural organoids are differentiated from embryoid bodies optimized for concave and shaking culture techniques, and are produced by 3D culture using a 3D cell culture microfluidic chip. This allows for stable production of organoids without separate cell transfer, maintaining a normal karyotype, and maintaining the characteristics and functions of mature neural cells. In particular, the 3D neural organoids were confirmed to exhibit the characteristics of mature neural cells by performing specific morphological analysis and confirming the expression of neural cell-specific factors MAP2 and TuJ1 (Fig. 9).

[0087] Drug toxicity evaluation method using neural organoids

[0088] In another aspect, the present invention provides a method for evaluating drug toxicity using a 3D neural organoid manufactured according to the above 3D neural organoid manufacturing method,

[0089] A method comprising the following steps:

[0090] i) a step of treating the neural organoid with a drug; and

[0091] ii) A step of measuring the expression of neural cell markers, cell viability, or cytotoxicity in the above neural organoids.

[0092] The term "drug" in this specification may refer to an individual nucleic acid, protein, other extract or natural product that is presumed or randomly selected to have the potential to lower the expression level of a neuronal marker or to impair the function of a neuronal cell according to a conventional selection method; and a drug that is presumed or confirmed to have toxic side effects that is currently on the market.

[0093] The term "cytotoxicity" in this invention may refer to the property of damaging or abnormally altering the function and / or structure of a cell. The cytotoxicity may specifically be apoptosis.

[0094] In one embodiment of the present invention, the toxicity assessment of a drug may be performed by treating the neural organoid with the drug, measuring the expression of neural cell markers, cell viability, or cytotoxicity, and comparing it with a control group that was not treated with the drug.

[0095] In another embodiment of the present invention, when treating the neuroorganoid with a drug, if the expression of a neuronal marker or cell viability decreases or if cytotoxicity is present, the drug may be determined to be a neurotoxic substance.

[0096] In another embodiment of the present invention, the neuronal cell marker may be at least one selected from the group consisting of SOX2, Nestin, TUBB3, MAP2, FOXO4, GFAP, LMX1A, CXCR4, CD113, CD15, and TuJ1, and preferably MAP2 and TuJ1.

[0097] In one embodiment of the present invention, when the neural organoid was treated with the neurotoxic drug vincristine, the expression of neural cell markers TuJ1 and MAP2 was confirmed to be reduced in the neural organoid, and apoptosis was observed (Fig. 10). Through this, it was confirmed that the neural organoid exhibited sensitivity and accuracy to drug toxicity unique to neural cells, and thus could be used as a neural model for evaluating drug toxicity.

[0098] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0099] Example 1. Formation of homogenized embryoid bodies and differentiation of neural cells

[0100] 1.1 Exploring methods for producing homogenized embryonic stem cells from pluripotent stem cells

[0101] To explore an optimized method for producing embryoid bodies from undifferentiated human pluripotent stem cells (iPSCs), we first compared the pipetting technique and the hang-in-drop technique.

[0102] For the experiment, iPSCs (produced in-house at Chung-Ang University) were maintained in undifferentiated culture medium StemMACS TM iPSCs were cultured in iPS-Brew XF (Miltenyi Biotec). After treating iPSCs with dispase (Gibco), only undifferentiated colonies were isolated. Subsequently, embryoid bodies were produced in the form of disrupted cells using the pipetting technique. Next, single cells were dissociated by treating with trypsin-EDTA (Gibco), and embryoid bodies were produced using the hang-in-drop technique.

[0103] The pipetting technique allowed the formation of embryoid bodies after 24 hours, but the embryoid bodies were not formed in a uniform shape. The hanging drop culture technique allowed the formation of embryoid bodies after 72 hours, allowing the production of embryoid bodies in a uniform shape. However, the formation of embryoid bodies took time and the size of the embryoid bodies was difficult to control. (Figure 2)

[0104] 1.2 Manufacturing of a uniformed concave body using a concave system

[0105] To produce more homogenous embryoid bodies, a concave system (Moon, S.-H., Ju, J., Park, S.-J., Bae, D., Chung, H.-M., & Lee, S.-H. (2014). Optimizing human embryonic stem cells differentiation efficiency by screening size-tunable homogenous embryoid bodies. Biomaterials, 35(23), 5987-5997.) was utilized.

[0106] After dissociating iPSC (Chung-Ang University, self-produced) with trypsin-EDTA (Gicbo), iPSC was dissociated with trypsin-EDTA (Gicbo) into concave sizes of 300 μm, 500 μm, and 1,000 μm produced by the above system, and each concave size was plated together with DMEM / F-12 (Gibco) culture medium containing 2% FBS (Hyclone).

[0107] As a result of the culture, it was observed that uniformly shaped embryos were formed in each size of concave after 24 hours, confirming that the size of the embryos could be uniformly controlled according to the size of the concave. (Fig. 3)

[0108] Furthermore, to determine the cell density of the homogenized embryoid bodies formed in the concave, the embryoid bodies were treated with trypsin-EDTA, dissociated into single cells, and stained with DAPI. Observation of the stained cells revealed that the embryoid bodies formed in the 300 μm concave contained approximately 320 cells, those formed in the 500 μm concave contained approximately 660 cells, and those formed in the 1,000 μm concave contained approximately 1,700 cells (Fig. 4).

[0109] 1.3 Analysis of morphological changes and gene expression of homogenized embryos

[0110] The uniform embryoid bodies formed in the above-mentioned concaves of each size were cultured for 10 days in RPMI1640 medium (Gibco) supplemented with N2 (Gibco) and B27 (Gibco), and the morphology and gene expression of the embryoid bodies were observed.

[0111] Morphological observations revealed that the uniformity of the embryoid bodies in each size of concave decreased on Day 4. Subsequently, on Day 7, the embryoid bodies in the 300 μm concave rapidly increased in size due to cell proliferation, while the embryoid bodies in the 500 μm and 1,000 μm concaves all became similar in size. This suggests that the embryoid bodies in the 500 μm concave proliferated, while no further cell proliferation occurred in the embryoid bodies in the 1,000 μm concave. In addition, lens morphologies were observed in the 500 μm and 1,000 μm concaves on Day 7, suggesting that differentiation was promoted. On Day 10, the sizes of the embryoid bodies in each concave were similar, indicating that no further proliferation of the embryoid bodies occurred (Fig. 5).

[0112] To compare the degree of differentiation of each stage of the embryonic body, gene expression in the embryonic body was analyzed using Oct4 (undifferentiated cells), Nestin (ectoderm and early neural cells), TUBB3 (neurons), and MAP2 (mature neural cells), which are genes expressed at each stage of neural cell differentiation.

[0113] For genetic analysis, embryoid bodies were first collected at each differentiation stage, and RNA was isolated from each sample using TRIzol (Invitrogen). cDNA was synthesized using a cDNA reverse transcription kit (Applied Biosystems) using the isolated RNA. qRT-PCR was performed using FastStart Essential DNA Green Master (Roche) with a Light Cycler 96 system (Roche). Primers for Oct4 (undifferentiated cells), Nestin (ectoderm and early neural cells), TUBB3 (neurons), and MAP2 (mature neural cells) along with GAPDH were designed using the Primer 3 program, and relative gene expression was analyzed based on the average Ct value for the technique.

[0114] Gene expression analysis results showed that Oct4 showed the fastest differentiation of embryoid bodies in 500 μm concaves, and in the case of Nestin, expression increased rapidly after Day 7 in embryoid bodies in 300 μm concaves, expression in embryoid bodies in 500 μm concaves was rapid until Day 7, and then the expression level plateaued on Day 10. As of Day 4 of embryoid body formation, embryoid bodies in 1,000 μm concaves showed a similar expression pattern to embryoid bodies in 500 μm concaves, but the expression rate was approximately 200 times lower compared to embryoid bodies in 500 μm concaves.

[0115] For TUBB3, the expression levels of the 300 μm and 1,000 μm concave embryos showed a pattern of rapid increase after Day 7, but the 500 μm concave embryos showed a consistently high gene expression pattern, showing the highest expression rate compared to the 300 μm and 1,000 μm concave embryos. For MAP2, the expression levels of the 300 μm concave embryos gradually increased until Day 10, but the expression rate was relatively low compared to the other concave embryos, and the expression levels of both the 500 μm and 1,000 μm concave embryos increased rapidly after Day 7, but the expression rate of the 500 μm concave embryos was about 8 times higher than that of the 1,000 μm concave embryos. (Fig. 6)

[0116] As a result, it was confirmed that the embryoid bodies of 500 μm concave showed the fastest differentiation level compared to the embryoid bodies of other sizes of concave, and were effective in differentiating neural cells. In the case of the expression pattern of each gene, the expression of Nestin, a marker of ectoderm and early neural cell development, increased rapidly on Day 7, and the expression of TUBB3, a representative neural cell marker, continuously increased until Day 10, and at the same time, the expression of MAP2, a mature neural cell marker, showed a pattern of rapid increase on Day 10.

[0117] That is, through analysis of the expression of markers for each differentiation stage of neural cells, i.e., ectoderm-early neural cell-neuron-maturation stage, it was found that the 500 μm concave embryoid body was most effective in the differentiation of neural cells.

[0118] 1.4 Analysis of the morphology formed during the attachment culture of 500 μm concave embryos

[0119] After the initiation of differentiation of the embryoid bodies with 500 μm concave, the different differentiation forms, lens-shaped embryoid bodies and black stone-shaped embryoid bodies, observed on Day 7 and Day 10, were respectively distinguished and adherently cultured for 4 days in RPMI1640 medium (Gibco) supplemented with N2 (Gibco) and 20 ng / ml bFGF (RnD systems) and their morphology was observed. The differentiated embryoid bodies were fixed with 4% paraformaldehyde (Sigma), immunostained with TuJ1 and MAP2 antibodies, which are neuronal cell markers, and then the three-dimensional structure was photographed and analyzed using a confocal microscope (Ziess).

[0120] As a result, after the attachment culture of the lens-shaped embryoid bodies, no cells expressing the stained antibody were observed in the proliferated area on Day 7, and some cells expressing TuJ1 were confirmed on Day 10, but no cells expressing MAP2 were observed. After the attachment culture of the black stone-shaped embryoid bodies, cells expressing TuJ1 were observed on Day 7, and on Day 10, most of the stained cells were found to express TuJ1 or MAP2 (Fig. 7).

[0121] Therefore, it was found that when the embryonic body appeared in the form of a lens through the differentiation process, it was not suitable for neural cell differentiation, but when the embryonic body appeared in the form of a black stone, it was effective for the differentiation and production of neural cells.

[0122] 1.5 Maintaining uniform embryonic body through shaking culture

[0123] Based on the results of the above 1.4, a shaking incubator (Biofree) was used to culture the black stone-shaped embryoid bodies that are effective for the differentiation of neural cells, and the morphology of the embryoid bodies after 7 days of culture was observed using an inverted microscope (Nikon).

[0124] As a result of culturing the embryonic body for 7 days under each condition (30, 60, and 90 rpm) of the shaking incubator, the uniformity of the embryonic body was most stable at 30 rpm, so the embryonic body was continuously cultured at 30 rpm for 10 days and the morphology was compared with that of the embryonic body that was not cultured by shaking.

[0125] First, in the case of embryoid bodies differentiated using only 500 μm concave, lens-shaped embryoid bodies were observed from Day 7 and were continuously observed until Day 10. In the case of 500 μm concave and 30 rpm shaking culture conditions, it was confirmed that a uniform shape of embryoid bodies was maintained, and at the same time, the shape of the lens was not observed in the embryoid bodies on Day 10, and only black stone-shaped embryoid bodies were observed (Fig. 8).

[0126] Therefore, it was found that the 30 rpm shaking culture technique was effective in maintaining the culture of black stone embryoid bodies and inhibiting the formation of lens embryoid bodies.

[0127] 1.6 Optimized neural cell differentiation protocol from homogenized embryonic stem cells

[0128] Based on the above experimental results, neural cells were produced using 500 μm concave and shaking culture conditions, which are culture conditions for homogenized embryonic stem cells. The optimized neural cell differentiation protocol was performed according to Figure 1 of the present invention.

[0129] First, using 500 μm concave, embryoid bodies were formed in DMEM / F12 (Gibo) medium supplemented with 2% FBS (hclone), and then cultured for 24 hours at 30 rpm with shaking in RPMI1640 (Gibo) medium mixed with N2 (Gibco) and B27 (Gibco) to form uniform embryoid bodies. Then, for 2D adherent culture, uniformly formed embryoid bodies were attached to culture dishes coated with poly-D-lysine (Gibco) and cultured for 14 days using RPMI1640 medium mixed with N2 and 20 ng / ml bFGF (RnD systems). For cell purification, attached cultured neurons were dissociated into single cells using Accutase (SC Tech) enzyme, and the dissociated cells were cultured in neuronal cell culture medium (Sigma) for 4 days, then dissociated into single cells again using TrypLE (Gibco) enzyme, and the dissociated cells were subcultured or frozen.

[0130] Example 2. Preparation of neural organoids

[0131] To produce 3D neural organoids using neural cells differentiated through the above-mentioned optimized neural cell differentiation protocol, neural cells were seeded and aggregated using a 3D cell culture microfluidic chip disclosed in Korean Patent No. 10-2155868 to produce 3D neural organoids (Fig. 9).

[0132] The manufactured neural organoids were fixed with 4% paraformaldehyde (Sigma), immunostained with TuJ1 and MAP2 antibodies, which are neuronal markers, and observed using a confocal microscope (Ziess) to confirm the expression of TuJ1 and MAP2 (Fig. 9).

[0133] Example 3. Drug toxicity analysis using neural organoids

[0134] The neurotoxicity of drugs was analyzed using 3D neural organoids fabricated using a microfluidic chip. To this end, neural organoids cultured on the Concave microfluidic chip were treated with different concentrations (0.01 μM, 0.05 μM, 0.1 μM, and 0.5 μM) of vincristine (Sigma), an anticancer drug known to exhibit neurotoxicity, for 24 hours, and immunostaining was performed (Fig. 10). Antibodies targeting the neuronal markers TuJ1 and MAP2 were used, and 0.2% DMSO (Sigma) served as a control.

[0135] As a result of the toxicity analysis, it was confirmed that the expression of neuronal cell-specific antibodies TuJ1 and MAP2 was reduced at a concentration of 0.05 μM, and at concentrations of 0.1 and 0.5 μM, the expression of TuJ1 and MAP2 was not confirmed, and apoptosis phenomena such as enlargement of the cell nucleus were confirmed.

[0136] Therefore, from the above results, it was confirmed that the 3D neural organoid of the present invention, in which the expression of neural cell markers decreases and apoptosis occurs when treated with a neurotoxic drug, can be used to evaluate the neurotoxicity of drugs.

Claims

1. i) A step of forming embryoid bodies by culturing human pluripotent stem cells in concaves with a diameter of 300 to 1,000 μm after enzyme treatment; ii) a step of culturing the embryonic body formed in step i) by shaking the embryonic body at 10 to 100 rpm; iii) a step of forming a cell aggregate in the embryonic body cultured in step ii); and iv) A method for producing neural cells, comprising a step of maturing the above cell aggregate and differentiating it into neural cells.

2. In paragraph 1, A method for producing neural cells, wherein the human pluripotent stem cells are human embryonic stem cells (hESCs) or human induced pluripotent stem cells (iPSCs).

3. In paragraph 1, A method for producing neural cells, wherein the enzyme is at least one selected from the group consisting of accutase, trypsin, collagenase, hyaluronidase, heparinase, elastase, pronase, DNase, dispase, papain, chymotrypsin, and EDTA.

4. In paragraph 1, A method for producing neural cells, wherein step i) above is performed in a medium containing 2% FBS.

5. In paragraph 1, A method for producing neural cells, wherein the step i) above is performed through 3D floating culture.

6. In paragraph 1, A method for producing neural cells, wherein step ii) above is performed in a medium containing N2 and B27.

7. In paragraph 1, A method for producing neural cells, wherein step iii) above is performed in a medium containing N2 and bFGF.

8. In paragraph 1, A method for producing neural cells, wherein step iii) above is performed through 2D attachment culture.

9. In paragraph 1, A method for producing neural cells, wherein the cell aggregate of step iii) above is treated with an enzyme.

10. In paragraph 9, A method for producing neural cells, wherein the enzyme is at least one selected from the group consisting of accutase, trypsin, collagenase, hyaluronidase, heparinase, elastase, pronase, DNase, dispase, papain, chymotrypsin, and EDTA.

11. A method for manufacturing a 3D neural organoid, comprising a step of culturing neural cells manufactured according to the manufacturing method of Article 1 using a microfluidic chip for 3D cell culture.

12. A 3D neural organoid manufactured according to the manufacturing method of Article 11, A 3D neural organoid, wherein the neural organoid expresses at least one selected from the group consisting of SOX2, Nestin, TUBB3, MAP2, FOXO4, GFAP, LMX1A, CXCR4, CD113, CD15, and TuJ1.

13. A method for evaluating drug toxicity using a 3D neural organoid manufactured according to the manufacturing method of Article 11, A method comprising the following steps: i) a step of treating the neural organoid with a drug; and ii) a step of measuring the expression of neural cell markers, cell viability or cytotoxicity in the neural organoids.

14. In paragraph 13, A method wherein the above neuronal cell marker is at least one selected from the group consisting of SOX2, Nestin, TUBB3, MAP2, FOXO4, GFAP, LMX1A, CXCR4, CD113, CD15, and TuJ1.

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