WO2022108117A1 - Cerebral organoid-motor neuron spheroid structure and fabrication method therefor - Google Patents

Cerebral organoid-motor neuron spheroid structure and fabrication method therefor Download PDF

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WO2022108117A1
WO2022108117A1 PCT/KR2021/014097 KR2021014097W WO2022108117A1 WO 2022108117 A1 WO2022108117 A1 WO 2022108117A1 KR 2021014097 W KR2021014097 W KR 2021014097W WO 2022108117 A1 WO2022108117 A1 WO 2022108117A1
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organoid
spheroid
brain
neuron
culture
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최정우
박수정
손주형
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서강대학교산학협력단
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • C—CHEMISTRY; METALLURGY
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
    • C12N2502/081—Coculture with; Conditioned medium produced by cells of the nervous system neurons
    • C—CHEMISTRY; METALLURGY
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening

Definitions

  • the present invention relates to a cerebral organoid-motoneuron spheroid structure and a method for manufacturing the same, and more particularly, by co-culturing cerebral organoids and motorneuron spheroids independently in a co-culture chip designed to be in contact with an appropriate culture medium. It relates to a method for fabricating a noid-motoneuron spheroid construct.
  • Tissue-specific organoid culture technology is a cutting-edge field that is currently receiving the most spotlight in stem cell research, and has unlimited potential in regenerative medicine and new drug research, such as intractable disease models, patient-specific drug screening platforms, and in vitro models for new drug development. can be expanded.
  • the present inventors fabricated a structure through cerebral organoid-motoneuron spheroid coupling and measured changes in bioelectrical signals. As a result, it was confirmed that the electrical signals were amplified by caffeine, known as a cranial nerve stimulant.
  • an object of the present invention is to provide a method for preparing a brain organoid-neuron spheroid structure comprising a co-culturing step of co-culturing brain organoids and neuronal spheroids.
  • Another object of the present invention is to provide a brain organoid-neuron spheroid structure comprising a brain organoid, and one or more neuronal spheroids bound to the bottom of the brain organoid.
  • Another object of the present invention is a brain organoid, and a brain organoid comprising one or more neuronal spheroids bound to the lower end of the brain organoid - a drug screening method comprising the step of treating a target drug to a neuron spheroid structure will provide
  • Another object of the present invention relates to the use of brain organoid-neuron spheroid constructs for drug screening.
  • the present invention relates to a cerebral organoid-motoneuron spheroid structure and a method for manufacturing the same, and the cerebral organoid-motoneuron spheroid structure according to the present invention is a cerebral organoid-motoneuron spheroid structure. indicates.
  • the present inventors prepared a co-culture chip in which each culture medium of cerebral organoids and motor neuron spheroids can be independently supplied to prepare such a cerebral organoid-motoneuron spheroid structure, and the structure was prepared using this .
  • One aspect of the present invention is a method for preparing a brain organoid-neuron spheroid construct comprising a co-culturing step of co-culturing brain organoids and neuronal spheroids.
  • organ is also called an organ-like organ, and by re-aggregating/recombining stem cells or cells isolated from an organ through three-dimensional culture, organization similar to an actual organ and reproduction of the function of a living organ are possible. It can be used as a model for various studies such as diseases.
  • spheroid is a three-dimensional aggregate of cells, which prevents spontaneous differentiation and facilitates differentiation of a desired lineage. In particular, since it is easy to induce differentiation into nerve cells in 3D, many studies are proceeding with nerve differentiation using spheroids.
  • the brain organoid may be at least one selected from the group consisting of cerebral organoids, thalamus organoids, cerebellar organoids and midbrain organoids, for example, it may be a cerebral organoid, but is limited thereto. it is not going to be In the case of an organoid that mimics the brain region involved in the transmission of movement-related signals, it can be applied as the brain organoid.
  • the neuron spheroid may be a motor neuron spheroid, but is not limited thereto.
  • spheroids differentiated into other neurons can also be applied.
  • the co-culturing step is performed in a co-culture chip
  • the co-culture chip is a co-culture chamber; a brain organoid culture reservoir connected by an independent channel to the co-culture chamber; and a neuron spheroid culture reservoir connected to an independent channel in the co-culture chamber.
  • the co-culturing step may be performed by placing one or more neuron spheroids in the co-culture chamber and then adding brain organoids.
  • the channel connected to the brain organoid culture reservoir may have a higher position connected to the co-culture chamber than the channel connected to the neuron spheroid culture reservoir.
  • the brain organoid culture medium is, for example, in a medium mixed with DMEM-F12 and Neurobasal medium, N2 supplement (supplement), insulin 1X GlutaMAX supplement, 0.5X MEM-NEAA, 1X penicillin-streptomycin (penicillin) -streptomycin) 2-mercaptoethanol (2-mercaptoethanol) and vitamin A (vitamin A) containing 1X B27 supplement may be included, but is not limited thereto.
  • Brain-derived neurotrophic factor (BDNF) and Glial cell-Derived Neurotrophic Factor (GDNF) may be added to the culture medium of the neuronal spheroids in StemPro hESC medium, but is limited thereto it is not
  • the co-culture chamber connection position of the channel through which the culture solution is introduced from the neuron spheroid culture medium reservoir is lower than the brain organoids is located in Accordingly, the neuronal spheroid culture is used for the culture of the neuronal spheroids underneath the brain organoids.
  • the co-culture chamber connection position of the channel through which the culture medium is introduced from the brain organoid culture medium reservoir is located above the brain organoid. Accordingly, brain organoid cultures are used for culturing brain organoids and not for culturing neuronal spheroids underneath brain organoids.
  • a combined structure can be prepared while independently using a culture medium suitable for each culture.
  • the manufacturing method may further include a brain organoid differentiation step of differentiating human induced pluripotent stem cells (hiPSCs) into brain organoids.
  • hiPSCs human induced pluripotent stem cells
  • human induced pluripotent stem cell refers to four specific genes (Oct4, Sox2, Klf4, c-Myc) that cause dedifferentiation into adult somatic cells that have been differentiated to thereby introduce pluripotency such as embryonic stem cells.
  • Oct4, Sox2, Klf4, c-Myc specific genes that cause dedifferentiation into adult somatic cells that have been differentiated to thereby introduce pluripotency such as embryonic stem cells.
  • the brain organoid differentiation step is a neurodifferentiation induction step of embedding the spheroid having the germ layer formed in the cells cultured in the nerve induction medium into MatriGel, the brain for neuronal differentiation from the embedded spheroid. It may consist of a patterning step of culturing in an organoid differentiation medium, and a final differentiation step of brain tissue producing brain organoids by inducing final differentiation of brain tissue when a neuroepithelial bud is formed.
  • the manufacturing method may further include a neuron spheroid differentiation step of differentiating human neural stem cells into neuronal spheroids.
  • Another aspect of the present invention is a brain organoid-neuron spheroid construct comprising a brain organoid and one or more neuronal spheroids bound to the underside of the brain organoid.
  • the brain organoid may be at least one selected from the group consisting of cerebral organoids, thalamus organoids, cerebellar organoids and midbrain organoids, for example, it may be a cerebral organoid, but is limited thereto. it is not going to be In the case of an organoid that mimics the brain region involved in the transmission of movement-related signals, it can be applied as the brain organoid.
  • the neuron spheroid may be a motor neuron spheroid, but is not limited thereto.
  • spheroids differentiated into other neurons can also be applied.
  • Another aspect of the present invention is a drug screening method comprising the step of treating a target drug to a brain organoid-neuron spheroid construct comprising a brain organoid, and one or more neuronal spheroids bound to the bottom of the brain organoid. .
  • the brain organoid may be at least one selected from the group consisting of cerebral organoids, thalamus organoids, cerebellar organoids and midbrain organoids, for example, it may be a cerebral organoid, but is limited thereto. it is not going to be In the case of an organoid that mimics the brain region involved in the transmission of movement-related signals, it can be applied as the brain organoid.
  • the neuron spheroid may be a motor neuron spheroid, but is not limited thereto.
  • spheroids differentiated into other neurons can also be applied.
  • the target drug may be a candidate substance for the prevention or treatment of brain diseases causing movement disorders including Parkinson's disease or Huntington's disease, and various brain nervous system diseases.
  • the drug screening method in the present invention may be performed by deriving an analysis result using a multi-channel electrode array (MEA) system for the brain organoid-neuron spheroid structure after the treatment of the target drug.
  • MEA multi-channel electrode array
  • multi-channel electrode array system is a device that simultaneously measures the action potential of a nerve cell with multiple electrodes. By measuring the change in the action potential of brain organoids, electrophysiological variation analysis, neuronal ion channel It can be used for analysis of changes, signal transduction rate, and signal transduction detection between nerve cells.
  • the drug screening method may be performed for the purpose of testing the toxicity of a target drug.
  • the present invention relates to a cerebral organoid-motoneuron spheroid structure and a method for manufacturing the same, and the structure can be prepared by co-culturing cerebral organoids and motorneuron spheroids independently in a co-culture chip designed to contact an appropriate culture medium. And from this, it is possible to observe the change in the electrical signal of the motor neuron according to a specific substance that stimulates the cerebrum, so it can be effectively used for drug screening or toxicity test for the prevention or treatment of brain diseases that cause movement disorders.
  • 1a is a schematic diagram showing the differentiation process of cerebral organoids according to an embodiment of the present invention.
  • Figure 1b is a photograph showing the tissue change according to the differentiation progress of cerebral organoids according to an embodiment of the present invention.
  • 1C is a photograph confirming the differentiation result of cerebral organoids according to an embodiment of the present invention by fluorescence immunostaining method.
  • 1D is a photograph confirming the differentiation result of cerebral organoids according to an embodiment of the present invention by a three-dimensional fluorescence immunostaining method.
  • Figure 2a is a schematic diagram showing the differentiation process of motor neuron spheroids according to an embodiment of the present invention.
  • Figure 2b is a photograph confirming the differentiation result of the motor neuron spheroid according to the embodiment of the present invention by the fluorescence immunostaining method.
  • Figure 2c is a photograph confirming the gene expression level by performing a polymerase chain reaction (PCR) as a result of differentiation of motor neuron spheroids according to an embodiment of the present invention.
  • PCR polymerase chain reaction
  • Figure 3a is a schematic diagram of the manufacturing process and application examples of the co-culture chip manufactured to combine cerebral organoids and motor neuron spheroids.
  • Figure 3b is a photo of the implementation of the co-cultured chip fabricated to combine cerebral organoids and motor neuron spheroids.
  • Figure 4a is a graph showing the signal results appearing as a result of processing caffeine to the cerebral organoids attached to the multi-electrode array (Microelectrode array; MEA) substrate.
  • Figure 4b is a graph showing the signal results appearing as a result of the caffeine treatment on the motor neuron spheroids attached to the MEA substrate.
  • Figure 4c is a graph showing the signal results appearing as a result of caffeine treatment on the cerebral organoid-motoneuron spheroid structure attached to the MEA substrate.
  • a brain organoid comprising a co-culturing step of co-culturing brain organoids and neuronal spheroids - a method for preparing a neuron spheroid construct.
  • % used to indicate the concentration of a specific substance is (weight/weight)% solid/solid, (weight/volume)%, and (weight/volume)% for solid/solid, and Liquid/liquid is (vol/vol) %.
  • Example 1 Preparation of cerebral organoids from human induced pluripotent stem cells
  • hiPSC human induced pluripotent stem cells
  • NIP laminin-based iMatrix-511
  • NIP laminin-based iMatrix-511
  • hiPSC NCRM5AS1-iCAGcGFP.c9; National Stem Cell Regeneration Center, Korea Centers for Disease Control and Prevention, 1382D2; RIKEN BioResource Research Center purchase, 201B7; ATCC purchase
  • TrypLE Select (Gibco, Cat #. 12563-011) was treated to separate the cells into single cells. The separated cells were resuspended after centrifugation, and spheroids were prepared by dispensing at 100,000 cells/well in an ultra-low attachment 96 well plate (Corning®, Cat #. CLS7007).
  • the produced spheroids are about 500 to 600 ⁇ m in diameter, in DMEM-F12 with 1XN2 supplement, 1X GlutaMAX supplement, 1X MEM-NEAA and 1 ⁇ g/ml heparin in a neural induction medium containing 4-5 Incubated for one day.
  • the germ layer (Germ layer) was formed by embedding the spheroid in MatriGel (MatriGel) to induce nerve differentiation.
  • the embedded spheroids were mixed with DMEM-F12 and Neurobasal medium at a 1:1 ratio with 0.5X N2 supplement, 62.5 ⁇ l of insulin 1X GlutaMAX supplement, 0.5X MEM-NEAA, 1X penicillin-streptomycin (penicillin) -streptomycin) 10 ⁇ M 2-mercaptoethanol (2-mercaptoethanol) and vitamin A removed 1X B27 supplement added cerebral organoid differentiation medium (Cerebral organoid differentiation medium) was cultured for about 5 days and patterned.
  • 1X B27 supplement is replaced with a vitamin A-containing one in the cerebral organoid differentiation medium to induce differentiation for about 15 days to grow cerebral tissue and finally differentiate to complete cerebral organoids.
  • Fluorescent immunostaining was performed to confirm the expression of specific markers in the prepared cerebral organoids.
  • the cerebral organoids were fixed with 4% paraformaldehyde for more than 24 hours, and washed three times with PBS (phosphate-buffered saline) containing 0.2% Triton X 100. Thereafter, non-specific binding of the antibody was prevented by blocking with PBS containing bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • Antibodies such as FOXG1 and PROX1, which are markers of cerebral organoids, TUJ1, which are neuronal markers, and SOX2, which are stem cell markers, were each treated at a concentration of 1:100 and maintained at 4°C for more than 24 hours.
  • cerebral organoid markers FOXG1 and PROX1, neuronal cell marker TUJ1, stem cell marker SOX2, etc. were stained and fluorescence was observed with a confocal microscope, and cerebral organoids was confirmed to be well formed.
  • Example 3 Preparation of motor neuron spheroids from human neural stem cells
  • Motor neuron spheroids were obtained by dispensing human neural stem cells at 5.0 X 10 4 cells/well in an ultra-low attachment 96 well U-bottom plate (Corning®, Cat #. CLS7007) and cultured for 24 hours. Then, in StemPro hESC medium (Gibco, Cat #. A1000701), retinoic acid (RA) 50 ⁇ M, secretory signal factor (sonic hedgehog) 200 ng/ml, basic fibroblast growth factor (bFGF) A medium for motor neuron differentiation containing 8 ng/ml and 10 ng/ml of activin A was replaced by 100 ⁇ l per well.
  • StemPro hESC medium StemPro hESC medium (Gibco, Cat #. A1000701)
  • RA retinoic acid
  • secretory signal factor sonic hedgehog
  • bFGF basic fibroblast growth factor
  • BDNF brain-derived neurotrophic factor
  • GDNF glial cell-derived neurotrophic factor
  • Fluorescence immunostaining was performed to confirm the expression of specific markers of the prepared motor neuron spheroids. It was performed in the same manner as in Example 2, except that the antibody of TUJ1, a neuron marker and islet1, a motor neuron marker, was used in a ratio of 1:100, respectively.
  • cDNA was synthesized using the Superscript III kit, and PCR was performed using the FlexCycler2 system device. PCR was performed using PAX6 and SOX1 as undifferentiated markers, ChAT, HB9 and islet1 as motor neuron differentiation markers, and GAPDH primer to confirm the initial amount of cDNA.
  • Example 5 Induction of cerebral organoid-motoneuron spheroid binding
  • a structure was manufactured by combining the prepared cerebral organoids and motor neuron spheroids, and a co-culture chip (3D engineered nervous system, 3D-ENS) including two culture medium reservoirs and a co-culture chamber for culturing them was prepared.
  • the co-culture chip was manufactured by assembling an upper plate and a lower plate formed using 3D printing technology.
  • the co-culture chip was prepared for the purpose of minimizing the stimulus to be applied to the other tissue during the induction of connective tissue because the culture medium for each tissue is different.
  • the cerebral organoid culture medium storage The height connected to the co-culture chamber was designed to be higher than the height at which the motor neuron spheroid culture medium reservoir was connected to the co-culture chamber.
  • binding was induced by culturing each tissue to be exposed to a specific individual culture medium. Specifically, in performing the coupling of cerebral organoids and motor neuron spheroids using the co-culture chip, motor neuron spheroids were first placed on the lower plate of the co-culture chamber, and brain organoids were introduced.
  • Example 3 As the culture medium flowing into the co-culture chamber through the channel from the cerebral organoid culture medium, the same medium was used in which the 1X B27 supplement was replaced with one containing vitamin A in the cerebral organoid differentiation medium after neuroepithelial embryo formation in Example 1 As the culture medium flowing into the co-culture chamber from the motor neuron spheroid culture medium reservoir through the channel, the motor neuron culture medium used for the maturation of motor neurons in Example 3 was used.
  • Example 6 Confirmation of changes in bioelectrical signals of cerebral organoid-motoneuron spheroid structures
  • each electrical signal was confirmed through a microelectrode array (MEA).
  • MEA substrate was coated with polyimide (PI) and then re-coated with laminin to raise the structure and measure the signal.
  • PI polyimide
  • the motor neuron spheroid was placed in contact with the MEA substrate, and the signal change of the motor neuron spheroid according to the stimulation applied to the cerebral organoid was observed.
  • caffeine known as a cranial nerve stimulant, was treated in cerebral organoids, motor neurons, and cerebral organoid-motoneuron spheroid structures, respectively, and changes in electrical signals caused by caffeine were observed.
  • cerebral organoids were amplified electrical signals by caffeine, but motor neuron spheroids did not change.
  • the present invention relates to a cerebral organoid-motoneuron spheroid structure and a method for manufacturing the same, and more particularly, by co-culturing cerebral organoids and motorneuron spheroids independently in a co-culture chip designed to be in contact with an appropriate culture medium. It relates to a method for fabricating a noid-motoneuron spheroid construct.

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Abstract

The present invention relates to a cerebral organoid-motor neuron spheroid structure and a fabrication method therefor. The structure can be fabricated by co-culturing a cerebral organoid and motor neuron spheroids in a co-culture chip which is designed to bring the cerebral organoid and the motor neuron spheroids into independent contact with a proper culture medium. The structure can be effectively used for screening drugs for prevention or treatment of brain diseases causative of motor disturbance or for a toxicity assay because electrical signal change of motor neurons according to specific substances stimulating the cerebrum can be observed from the structure.

Description

대뇌 오가노이드-모터뉴런 스페로이드 구조체 및 이의 제조방법Cerebral organoid-motoneuron spheroid structure and manufacturing method thereof
본 특허출원은 2020년 11월 17일에 대한민국 특허청에 제출된 대한민국 특허출원 제10-2020-0154027호에 대하여 우선권을 주장하며, 상기 특허출원의 개시 사항은 본 명세서에 참조로서 삽입된다.This patent application claims priority to Korean Patent Application No. 10-2020-0154027 submitted to the Korean Intellectual Property Office on November 17, 2020, the disclosure of which is incorporated herein by reference.
본 발명은 대뇌 오가노이드-모터뉴런 스페로이드 구조체 및 이의 제조방법에 관한 것으로서, 더욱 상세하게는 대뇌 오가노이드와 모터뉴런 스페로이드를 독립적으로 적절한 배양액에 접촉되도록 설계된 공배양 칩에서 공배양함으로써 대뇌 오가노이드-모터뉴런 스페로이드 구조체를 제조하는 방법에 관한 것이다.The present invention relates to a cerebral organoid-motoneuron spheroid structure and a method for manufacturing the same, and more particularly, by co-culturing cerebral organoids and motorneuron spheroids independently in a co-culture chip designed to be in contact with an appropriate culture medium. It relates to a method for fabricating a noid-motoneuron spheroid construct.
실험동물 질환 모델은 인간과 여러 가지 면에서 차이가 존재하기 때문에, 이를 이용한 질환 기전 연구나 신약 개발에는 명확한 한계가 존재한다. 세계적으로 실험동물을 이용한 연구에 제한이 있어, 최근에는 인간 장기를 모사하는 오가노이드가 대안으로 제시되고 있으며, 현재 다양한 인체 장기를 모사하는 오가노이드가 개발되고 있다.Since experimental animal disease models differ from humans in many ways, there are clear limitations in disease mechanism research or drug development using them. Since there are limitations in research using experimental animals worldwide, organoids that mimic human organs have been proposed as an alternative, and organoids that mimic various human organs are currently being developed.
2013년 영국 연구진에 의해 대뇌와 기능 및 조직이 유사한 대뇌 오가노이드가 제작되었다. 이러한 대뇌 오가노이드는 제작 과정에서 특정 영역으로의 분화 과정 없이 일반적인 뇌와 유사한 특성을 지닌 조직 유사체로 조직화되었으며, 이후 많은 연구진들이 다양한 영역의 뇌 오가노이드를 제작하고 발표하였다. 그러나 이들은 뇌의 특정 영역과 유사한 개별 오가노이드로 생체의 뇌와 같은 복잡하고 유기적인 기능 구현이 불가능하여, 오가노이드 간의 결합을 통한 뇌 구조 및 기능의 구현이 필요하다.In 2013, a cerebral organoid similar in function and organization to the cerebrum was produced by British researchers. These cerebral organoids were organized into tissue analogs with characteristics similar to those of the general brain without differentiation into specific regions during the manufacturing process. Since then, many researchers have produced and published brain organoids of various regions. However, these are individual organoids similar to a specific area of the brain, and since it is impossible to implement complex and organic functions like the brain of a living body, it is necessary to implement the brain structure and function through the bonding between organoids.
조직 특이적 오가노이드 배양 기술은 현재 줄기세포 연구에서 가장 각광을 받고 있는 최첨단 분야로서 난치성 질환 모델, 환자 맞춤형 약물 스크리닝 플랫폼, 신약 개발을 위한 체외 모델 등 재생의학 및 신약 연구 분야에서 활용성이 무한하게 확장될 수 있다.Tissue-specific organoid culture technology is a cutting-edge field that is currently receiving the most spotlight in stem cell research, and has unlimited potential in regenerative medicine and new drug research, such as intractable disease models, patient-specific drug screening platforms, and in vitro models for new drug development. can be expanded.
이에 본 발명자들은 대뇌 오가노이드-모터뉴런 스페로이드 결합을 통한 구조체를 제작하여 생체 전기신호 변화를 측정한 결과, 뇌신경 자극 물질로 알려진 카페인에 의하여 전기신호가 증폭됨을 확인하였다.Accordingly, the present inventors fabricated a structure through cerebral organoid-motoneuron spheroid coupling and measured changes in bioelectrical signals. As a result, it was confirmed that the electrical signals were amplified by caffeine, known as a cranial nerve stimulant.
이에, 본 발명의 목적은 뇌 오가노이드 및 뉴런 스페로이드를 공배양하는 공배양 단계를 포함하는 뇌 오가노이드-뉴런 스페로이드 구조체의 제조방법을 제공하는 것이다.Accordingly, an object of the present invention is to provide a method for preparing a brain organoid-neuron spheroid structure comprising a co-culturing step of co-culturing brain organoids and neuronal spheroids.
본 발명의 다른 목적은 뇌 오가노이드, 및 뇌 오가노이드 하단에 결합된 1개 이상의 뉴런 스페로이드를 포함하는 뇌 오가노이드-뉴런 스페로이드 구조체를 제공하는 것이다.Another object of the present invention is to provide a brain organoid-neuron spheroid structure comprising a brain organoid, and one or more neuronal spheroids bound to the bottom of the brain organoid.
본 발명의 또 다른 목적은 뇌 오가노이드, 및 뇌 오가노이드 하단에 결합된 1개 이상의 뉴런 스페로이드를 포함하는 뇌 오가노이드-뉴런 스페로이드 구조체에 대상 약물을 처리하는 단계를 포함하는 약물 스크리닝 방법을 제공하는 것이다.Another object of the present invention is a brain organoid, and a brain organoid comprising one or more neuronal spheroids bound to the lower end of the brain organoid - a drug screening method comprising the step of treating a target drug to a neuron spheroid structure will provide
본 발명의 또 다른 목적은 뇌 오가노이드-뉴런 스페로이드 구조체의 약물 스크리닝 용도에 관한 것이다.Another object of the present invention relates to the use of brain organoid-neuron spheroid constructs for drug screening.
본 발명은 대뇌 오가노이드-모터뉴런 스페로이드(Spheroid) 구조체 및 이의 제조방법에 관한 것으로, 본 발명에 따른 대뇌 오가노이드-모터뉴런 스페로이드 구조체는 뇌신경 자극 물질로 알려진 카페인에 의하여 전기신호가 증폭됨을 나타낸다.The present invention relates to a cerebral organoid-motoneuron spheroid structure and a method for manufacturing the same, and the cerebral organoid-motoneuron spheroid structure according to the present invention is a cerebral organoid-motoneuron spheroid structure. indicates.
본 발명자들은 이러한 대뇌 오가노이드-모터뉴런 스페로이드 구조체를 제조하기 위하여 대뇌 오가노이드 및 모터뉴런 스페로이드의 각각의 배양액이 독립적으로 공급될 수 있는 공배양 칩을 제조하였고, 이를 이용하여 구조체를 제조하였다.The present inventors prepared a co-culture chip in which each culture medium of cerebral organoids and motor neuron spheroids can be independently supplied to prepare such a cerebral organoid-motoneuron spheroid structure, and the structure was prepared using this .
이하 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태는 뇌 오가노이드 및 뉴런 스페로이드를 공배양하는 공배양 단계를 포함하는 뇌 오가노이드-뉴런 스페로이드 구조체의 제조방법이다.One aspect of the present invention is a method for preparing a brain organoid-neuron spheroid construct comprising a co-culturing step of co-culturing brain organoids and neuronal spheroids.
본 명세서상의 용어 "오가노이드"는 유사장기체라고도 불리며, 줄기세포나 장기에서 분리한 세포를 3차원 배양을 통해 재응집/재조합하여 실제 장기와 유사한 조직화와 생체 장기의 기능 재현이 가능하여 발달 및 질병 등 다양한 연구에 모델로 활용 가능하다.As used herein, the term “organoid” is also called an organ-like organ, and by re-aggregating/recombining stem cells or cells isolated from an organ through three-dimensional culture, organization similar to an actual organ and reproduction of the function of a living organ are possible. It can be used as a model for various studies such as diseases.
본 명세서상의 용어 "스페로이드"는 세포의 3차원 집합체로, 자연 분화를 방지하고 원하는 계열의 분화를 용이하도록 한다. 특히 3차원상에서 신경세포로의 분화유도가 용이하여 많은 연구에서 스페로이드를 이용한 신경분화를 진행하고 있다.As used herein, the term “spheroid” is a three-dimensional aggregate of cells, which prevents spontaneous differentiation and facilitates differentiation of a desired lineage. In particular, since it is easy to induce differentiation into nerve cells in 3D, many studies are proceeding with nerve differentiation using spheroids.
본 발명에 있어서 뇌 오가노이드는 대뇌 오가노이드, 시상 오가노이드, 소뇌 오가노이드 및 중뇌 오가노이드로 이루어진 군으로부터 선택되는 1종 이상인 것일 수 있고, 예를 들어, 대뇌 오가노이드인 것일 수 있으나, 이에 한정되는 것은 아니다. 운동 관련 신호의 전달에 관여하는 뇌의 영역을 모사한 오가노이드의 경우, 상기 뇌 오가노이드로서 적용할 수 있다.In the present invention, the brain organoid may be at least one selected from the group consisting of cerebral organoids, thalamus organoids, cerebellar organoids and midbrain organoids, for example, it may be a cerebral organoid, but is limited thereto. it is not going to be In the case of an organoid that mimics the brain region involved in the transmission of movement-related signals, it can be applied as the brain organoid.
본 발명에 있어서 뉴런 스페로이드는 모터뉴런 스페로이드인 것일 수 있으나, 이에 한정되는 것은 아니다. 목적에 따라 모터뉴런 이외에 다른 신경세포로 분화한 스페로이드 또한 적용할 수 있다.In the present invention, the neuron spheroid may be a motor neuron spheroid, but is not limited thereto. Depending on the purpose, other than motor neurons, spheroids differentiated into other neurons can also be applied.
본 발명에 있어서 공배양 단계는 공배양 칩에서 수행되고, 상기 공배양 칩은공배양 챔버; 공배양 챔버에 독립적인 채널로 연결된 뇌 오가노이드 배양액 저장소; 및 공배양 챔버에 독립적인 채널로 연결된 뉴런 스페로이드 배양액 저장소;를 포함하는 것일 수 있다.In the present invention, the co-culturing step is performed in a co-culture chip, the co-culture chip is a co-culture chamber; a brain organoid culture reservoir connected by an independent channel to the co-culture chamber; and a neuron spheroid culture reservoir connected to an independent channel in the co-culture chamber.
상기 공배양 단계는 공배양 챔버에 뉴런 스페로이드를 1개 이상 배치한 후 뇌 오가노이드를 투입하여 수행되는 것일 수 있다.The co-culturing step may be performed by placing one or more neuron spheroids in the co-culture chamber and then adding brain organoids.
상기 뇌 오가노이드 배양액 저장소에 연결된 채널은 뉴런 스페로이드 배양액 저장소에 연결된 채널보다 공배양 챔버에 연결된 위치가 높은 것일 수 있다.The channel connected to the brain organoid culture reservoir may have a higher position connected to the co-culture chamber than the channel connected to the neuron spheroid culture reservoir.
상기 뇌 오가노이드의 배양액은, 예를 들어, DMEM-F12와 Neurobasal medium을 혼합한 배지에 N2 서플리먼트(supplement), 인슐린(insulin) 1X GlutaMAX 서플리먼트, 0.5X MEM-NEAA, 1X 페니실린-스트렙토마이신(penicillin-streptomycin) 2-머캅토에탄올(2-mercaptoethanol) 및 비타민 A(vitamin A) 포함 1X B27 서플리먼트가 포함된 것일 수 있으나, 이에 한정되는 것은 아니다.The brain organoid culture medium is, for example, in a medium mixed with DMEM-F12 and Neurobasal medium, N2 supplement (supplement), insulin 1X GlutaMAX supplement, 0.5X MEM-NEAA, 1X penicillin-streptomycin (penicillin) -streptomycin) 2-mercaptoethanol (2-mercaptoethanol) and vitamin A (vitamin A) containing 1X B27 supplement may be included, but is not limited thereto.
상기 뉴런 스페로이드의 배양액은 StemPro hESC 배지에 뇌유래신경성장인자(Brain-derived neurotrophic factor; BDNF) 및 교세포 신경성장인자(Glial cell-Derived Neurotrophic Factor; GDNF)가 첨가된 것일 수 있으나, 이에 한정되는 것은 아니다.Brain-derived neurotrophic factor (BDNF) and Glial cell-Derived Neurotrophic Factor (GDNF) may be added to the culture medium of the neuronal spheroids in StemPro hESC medium, but is limited thereto it is not
본 발명의 일 구현예에서, 뉴런 스페로이드를 공배양 챔버에 배치하고 그 위에 뇌 오가노이드를 투입하였을 때 뉴런 스페로이드 배양액 저장소로부터 배양액이 유입되는 채널의 공배양 챔버 연결 위치는 뇌 오가노이드보다 아래에 위치한다. 이에 따라, 뉴런 스페로이드 배양액은 뇌 오가노이드의 아래에 있는 뉴런 스페로이드의 배양에 사용된다. 이 때 뇌 오가노이드 배양액 저장소로부터 배양액이 유입되는 채널의 공배양 챔버 연결 위치는 뇌 오가노이드보다 위에 위치한다. 이에 따라, 뇌 오가노이드 배양액은 뇌 오가노이드의 배양에 사용되며 뇌 오가노이드의 아래에 있는 뉴런 스페로이드의 배양에 사용되지 않는다.In one embodiment of the present invention, when the neuron spheroids are placed in the co-culture chamber and brain organoids are put thereon, the co-culture chamber connection position of the channel through which the culture solution is introduced from the neuron spheroid culture medium reservoir is lower than the brain organoids is located in Accordingly, the neuronal spheroid culture is used for the culture of the neuronal spheroids underneath the brain organoids. At this time, the co-culture chamber connection position of the channel through which the culture medium is introduced from the brain organoid culture medium reservoir is located above the brain organoid. Accordingly, brain organoid cultures are used for culturing brain organoids and not for culturing neuronal spheroids underneath brain organoids.
상기와 같은 원리로 공배양 챔버를 이용하여 공배양 단계를 수행하면, 두 가지 종류의 오가노이드 및 스페로이드를 배양하면서 각각의 배양에 적합한 배양액을 독립적으로 사용하면서도 결합된 구조체를 제조할 수 있다.If the co-culture step is performed using the co-culture chamber in the same manner as described above, while culturing two types of organoids and spheroids, a combined structure can be prepared while independently using a culture medium suitable for each culture.
본 발명에 있어서 제조방법은 인간 유도만능 줄기세포(human induced Pluripotent Stem Cells, hiPSC)를 뇌 오가노이드로 분화시키는 뇌 오가노이드 분화 단계를 추가적으로 포함하는 것일 수 있다.In the present invention, the manufacturing method may further include a brain organoid differentiation step of differentiating human induced pluripotent stem cells (hiPSCs) into brain organoids.
본 명세서상의 용어 "인간 유도만능 줄기세포"는 분화가 끝난 성체의 체세포에 역분화를 일으키는 4가지 특정 유전자(Oct4, Sox2, Klf4, c-Myc)를 도입하여 배아줄기세포와 같은 만능분화능(Pluripotancy)을 갖도록 한 줄기세포이다. 유도만능줄기세포는 윤리적인 문제와 면역거부반응 등의 문제에서 자유로우며, 다양한 조직으로의 분화가 가능하여 발생학을 비롯한 다양한 질병 및 치료제 연구에 활용되는 세포이다.As used herein, the term "human induced pluripotent stem cell" refers to four specific genes (Oct4, Sox2, Klf4, c-Myc) that cause dedifferentiation into adult somatic cells that have been differentiated to thereby introduce pluripotency such as embryonic stem cells. ) is a stem cell that has Induced pluripotent stem cells are free from issues such as ethical and immune rejection, and because they can differentiate into various tissues, they are used for research on various diseases and therapeutics, including embryology.
상기 뇌 오가노이드 분화 단계는, 신경 유도 배지에서 배양된 세포 중 배엽층(Germ layer)이 형성된 스페로이드를 마트리겔(MatriGel)로 포매하는 신경분화 유도 단계, 포매된 스페로이드로부터 신경 분화를 위하여 뇌 오가노이드 분화 배지에서 배양하는 패턴화 단계, 및 신경상피배(Neuroepithelial bud)가 형성되면 뇌 조직의 최종 분화를 유도함으로써 뇌 오가노이드를 제조하는 뇌 조직 최종 분화 단계로 이루어진 것일 수 있다.The brain organoid differentiation step is a neurodifferentiation induction step of embedding the spheroid having the germ layer formed in the cells cultured in the nerve induction medium into MatriGel, the brain for neuronal differentiation from the embedded spheroid. It may consist of a patterning step of culturing in an organoid differentiation medium, and a final differentiation step of brain tissue producing brain organoids by inducing final differentiation of brain tissue when a neuroepithelial bud is formed.
본 발명에 있어서 제조방법은 인간 신경줄기세포(human neural stem cells)를 뉴런 스페로이드로 분화시키는 뉴런 스페로이드 분화 단계를 추가적으로 포함하는 것일 수 있다.In the present invention, the manufacturing method may further include a neuron spheroid differentiation step of differentiating human neural stem cells into neuronal spheroids.
본 발명의 다른 양태는 뇌 오가노이드, 및 뇌 오가노이드 하단에 결합된 1개 이상의 뉴런 스페로이드를 포함하는 뇌 오가노이드-뉴런 스페로이드 구조체이다.Another aspect of the present invention is a brain organoid-neuron spheroid construct comprising a brain organoid and one or more neuronal spheroids bound to the underside of the brain organoid.
본 발명에 있어서 뇌 오가노이드는 대뇌 오가노이드, 시상 오가노이드, 소뇌 오가노이드 및 중뇌 오가노이드로 이루어진 군으로부터 선택되는 1종 이상인 것일 수 있고, 예를 들어, 대뇌 오가노이드인 것일 수 있으나, 이에 한정되는 것은 아니다. 운동 관련 신호의 전달에 관여하는 뇌의 영역을 모사한 오가노이드의 경우, 상기 뇌 오가노이드로서 적용할 수 있다.In the present invention, the brain organoid may be at least one selected from the group consisting of cerebral organoids, thalamus organoids, cerebellar organoids and midbrain organoids, for example, it may be a cerebral organoid, but is limited thereto. it is not going to be In the case of an organoid that mimics the brain region involved in the transmission of movement-related signals, it can be applied as the brain organoid.
본 발명에 있어서 뉴런 스페로이드는 모터뉴런 스페로이드인 것일 수 있으나, 이에 한정되는 것은 아니다. 목적에 따라 모터뉴런 이외에 다른 신경세포로 분화한 스페로이드 또한 적용할 수 있다.In the present invention, the neuron spheroid may be a motor neuron spheroid, but is not limited thereto. Depending on the purpose, other than motor neurons, spheroids differentiated into other neurons can also be applied.
본 발명의 또 다른 양태는 뇌 오가노이드, 및 뇌 오가노이드 하단에 결합된 1개 이상의 뉴런 스페로이드를 포함하는 뇌 오가노이드-뉴런 스페로이드 구조체에 대상 약물을 처리하는 단계를 포함하는 약물 스크리닝 방법이다.Another aspect of the present invention is a drug screening method comprising the step of treating a target drug to a brain organoid-neuron spheroid construct comprising a brain organoid, and one or more neuronal spheroids bound to the bottom of the brain organoid. .
본 발명에 있어서 뇌 오가노이드는 대뇌 오가노이드, 시상 오가노이드, 소뇌 오가노이드 및 중뇌 오가노이드로 이루어진 군으로부터 선택되는 1종 이상인 것일 수 있고, 예를 들어, 대뇌 오가노이드인 것일 수 있으나, 이에 한정되는 것은 아니다. 운동 관련 신호의 전달에 관여하는 뇌의 영역을 모사한 오가노이드의 경우, 상기 뇌 오가노이드로서 적용할 수 있다.In the present invention, the brain organoid may be at least one selected from the group consisting of cerebral organoids, thalamus organoids, cerebellar organoids and midbrain organoids, for example, it may be a cerebral organoid, but is limited thereto. it is not going to be In the case of an organoid that mimics the brain region involved in the transmission of movement-related signals, it can be applied as the brain organoid.
본 발명에 있어서 뉴런 스페로이드는 모터뉴런 스페로이드인 것일 수 있으나, 이에 한정되는 것은 아니다. 목적에 따라 모터뉴런 이외에 다른 신경세포로 분화한 스페로이드 또한 적용할 수 있다.In the present invention, the neuron spheroid may be a motor neuron spheroid, but is not limited thereto. Depending on the purpose, other than motor neurons, spheroids differentiated into other neurons can also be applied.
본 발명에 있어서 대상 약물은 파킨슨병 또는 헌팅턴병을 포함하는 운동장애를 유발하는 뇌질환 및 다양한 뇌 신경계 질환의 예방 또는 치료를 위한 후보물질인 것일 수 있다.In the present invention, the target drug may be a candidate substance for the prevention or treatment of brain diseases causing movement disorders including Parkinson's disease or Huntington's disease, and various brain nervous system diseases.
본 발명에 있어서 약물 스크리닝 방법은 대상 약물의 처리 후 뇌 오가노이드-뉴런 스페로이드 구조체에 대하여 다채널전극배열 시스템(Multielectrode Array (MEA) System)을 이용한 분석 결과를 도출하는 것으로 수행될 수 있다.The drug screening method in the present invention may be performed by deriving an analysis result using a multi-channel electrode array (MEA) system for the brain organoid-neuron spheroid structure after the treatment of the target drug.
본 명세서상의 용어 "다채널전극배열 시스템"은 여러 개의 전극이 신경세포의 활동전위를 동시에 측정하는 장비로 뇌 오가노이드의 활동전위의 변화를 측정함으로써 전기생리학적 변이 분석, 신경세포의 이온채널의 변화, 신호전달 속도, 신경세포간의 신호전달 검출 등의 분석에 활용 가능하다.As used herein, the term "multi-channel electrode array system" is a device that simultaneously measures the action potential of a nerve cell with multiple electrodes. By measuring the change in the action potential of brain organoids, electrophysiological variation analysis, neuronal ion channel It can be used for analysis of changes, signal transduction rate, and signal transduction detection between nerve cells.
본 발명에 있어서 약물 스크리닝 방법은 대상 약물의 독성을 테스트하는 용도로 실시될 수 있다.In the present invention, the drug screening method may be performed for the purpose of testing the toxicity of a target drug.
본 발명은 대뇌 오가노이드-모터뉴런 스페로이드 구조체 및 이의 제조방법에 관한 것으로서, 대뇌 오가노이드와 모터뉴런 스페로이드를 독립적으로 적절한 배양액에 접촉되도록 설계된 공배양 칩에서 공배양함으로써 상기 구조체를 제조할 수 있고, 이로부터 대뇌를 자극하는 특정 물질에 따른 모터뉴런의 전기신호 변화를 관찰할 수 있으므로, 이를 효과적으로 운동 장애를 유발하는 뇌질환의 예방 또는 치료를 위한 약물 스크리닝 또는 독성 테스트에 이용할 수 있다.The present invention relates to a cerebral organoid-motoneuron spheroid structure and a method for manufacturing the same, and the structure can be prepared by co-culturing cerebral organoids and motorneuron spheroids independently in a co-culture chip designed to contact an appropriate culture medium. And from this, it is possible to observe the change in the electrical signal of the motor neuron according to a specific substance that stimulates the cerebrum, so it can be effectively used for drug screening or toxicity test for the prevention or treatment of brain diseases that cause movement disorders.
도 1a는 본 발명의 실시예에 따른 대뇌 오가노이드의 분화 과정을 나타낸 모식도이다.1a is a schematic diagram showing the differentiation process of cerebral organoids according to an embodiment of the present invention.
도 1b는 본 발명의 실시예에 따른 대뇌 오가노이드의 분화 진행에 따른 조직 변화를 나타낸 사진이다.Figure 1b is a photograph showing the tissue change according to the differentiation progress of cerebral organoids according to an embodiment of the present invention.
도 1c는 본 발명의 실시예에 따른 대뇌 오가노이드의 분화 결과를 형광면역염색 방법으로 확인한 사진이다.1C is a photograph confirming the differentiation result of cerebral organoids according to an embodiment of the present invention by fluorescence immunostaining method.
도 1d는 본 발명의 실시예에 따른 대뇌 오가노이드의 분화 결과를 3차원 형광면역염색 방법으로 확인한 사진이다.1D is a photograph confirming the differentiation result of cerebral organoids according to an embodiment of the present invention by a three-dimensional fluorescence immunostaining method.
도 2a는 본 발명의 실시예에 따른 모터뉴런 스페로이드의 분화 과정을 나타낸 모식도이다.Figure 2a is a schematic diagram showing the differentiation process of motor neuron spheroids according to an embodiment of the present invention.
도 2b는 본 발명의 실시예에 따른 모터뉴런 스페로이드의 분화 결과를 형광면역염색 방법으로 확인한 사진이다.Figure 2b is a photograph confirming the differentiation result of the motor neuron spheroid according to the embodiment of the present invention by the fluorescence immunostaining method.
도 2c는 본 발명의 실시예에 따른 모터뉴런 스페로이드의 분화 결과 중합효소연쇄반응(PCR)을 수행하여 유전자 발현량을 확인한 사진이다.Figure 2c is a photograph confirming the gene expression level by performing a polymerase chain reaction (PCR) as a result of differentiation of motor neuron spheroids according to an embodiment of the present invention.
도 3a는 대뇌 오가노이드와 모터뉴런 스페로이드를 결합하기 위해 제작한 공배양 칩의 제조 과정 및 활용예의 모식도이다.Figure 3a is a schematic diagram of the manufacturing process and application examples of the co-culture chip manufactured to combine cerebral organoids and motor neuron spheroids.
도 3b는 대뇌 오가노이드와 모터뉴런 스페로이드를 결합하기 위해 제작한 공배양 칩의 실시 사진이다.Figure 3b is a photo of the implementation of the co-cultured chip fabricated to combine cerebral organoids and motor neuron spheroids.
도 4a는 다중 전극 어레이(Microelectrode array; MEA) 기판에 부착한 대뇌 오가노이드에 카페인을 처리한 결과 나타나는 신호 결과를 나타낸 그래프이다.Figure 4a is a graph showing the signal results appearing as a result of processing caffeine to the cerebral organoids attached to the multi-electrode array (Microelectrode array; MEA) substrate.
도 4b는 MEA 기판에 부착한 모터뉴런 스페로이드에 카페인을 처리한 결과 나타나는 신호 결과를 나타낸 그래프이다.Figure 4b is a graph showing the signal results appearing as a result of the caffeine treatment on the motor neuron spheroids attached to the MEA substrate.
도 4c는 MEA 기판에 부착한 대뇌 오가노이드-모터뉴런 스페로이드 구조체에 카페인을 처리한 결과 나타나는 신호 결과를 나타낸 그래프이다.Figure 4c is a graph showing the signal results appearing as a result of caffeine treatment on the cerebral organoid-motoneuron spheroid structure attached to the MEA substrate.
뇌 오가노이드 및 뉴런 스페로이드를 공배양하는 공배양 단계를 포함하는 뇌 오가노이드-뉴런 스페로이드 구조체의 제조방법.A brain organoid comprising a co-culturing step of co-culturing brain organoids and neuronal spheroids - a method for preparing a neuron spheroid construct.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 범위가 이들 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 "%"는 별도의 언급이 없는 경우, 고체/고체는 (중량/중량)%, 고체/액체는 (중량/부피)%, 그리고 액체/액체는 (부피/부피)%이다.Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight)% solid/solid, (weight/volume)%, and (weight/volume)% for solid/solid, and Liquid/liquid is (vol/vol) %.
실시예 1: 인간 유도만능줄기세포로부터 대뇌 오가노이드 제작Example 1: Preparation of cerebral organoids from human induced pluripotent stem cells
도 1a 및 1b와 같이, 인간 유도만능 줄기세포(human induced Pluripotent Stem Cells, hiPSC)를 라미닌(Laminin)계열의 iMatrix-511(NIP, Cat #. T311)로 코팅된 배양 용기에 StemFit Basic002(Ajinomoto) 배지를 이용하여 배양하였다. (hiPSC: NCRM5AS1-iCAGcGFP.c9; 질병관리본부 국립줄기세포재생센터 분양, 1382D2; RIKEN BioResource Research Center 구입, 201B7; ATCC 구입)1a and 1b, human induced pluripotent stem cells (hiPSC) in a culture vessel coated with laminin-based iMatrix-511 (NIP, Cat #. T311) StemFit Basic002 (Ajinomoto) The culture medium was used. (hiPSC: NCRM5AS1-iCAGcGFP.c9; National Stem Cell Regeneration Center, Korea Centers for Disease Control and Prevention, 1382D2; RIKEN BioResource Research Center purchase, 201B7; ATCC purchase)
세포가 배양 용기의 70~80%로 자라면 TrypLE Select(Gibco쪠, Cat #. 12563-011)를 처리하여 단일 세포로 분리하였다. 분리한 세포는 원심분리 후 재부유시키고, Ultra-low attachment 96 웰 플레이트(well plate)(Corning®, Cat #. CLS7007)에 100,000 cells/well 로 분주하여 스페로이드를 제작하였다.When the cells grew to 70-80% of the culture vessel, TrypLE Select (Gibco, Cat #. 12563-011) was treated to separate the cells into single cells. The separated cells were resuspended after centrifugation, and spheroids were prepared by dispensing at 100,000 cells/well in an ultra-low attachment 96 well plate (Corning®, Cat #. CLS7007).
제작한 스페로이드는 지름이 500 내지 600 μm 정도일 때, DMEM-F12에 1XN2 서플리먼트, 1X GlutaMAX 서플리먼트, 1X MEM-NEAA와 1 μg/ml heparin 이 포함된 신경 유도 배지(neural induction medium)에서 4~5일 간 배양하였다. 배엽층(Germ layer)이 형성된 스페로이드를 마트리겔(MatriGel)로 포매하여 신경분화를 유도하였다.The produced spheroids are about 500 to 600 μm in diameter, in DMEM-F12 with 1XN2 supplement, 1X GlutaMAX supplement, 1X MEM-NEAA and 1 μg/ml heparin in a neural induction medium containing 4-5 Incubated for one day. The germ layer (Germ layer) was formed by embedding the spheroid in MatriGel (MatriGel) to induce nerve differentiation.
이후 포매된 스페로이드를 DMEM-F12와 Neurobasal medium을 1:1로 혼합한 배지에 0.5X N2 서플리먼트, 62.5 μl의 인슐린(insulin) 1X GlutaMAX 서플리먼트, 0.5X MEM-NEAA, 1X 페니실린-스트렙토마이신(penicillin-streptomycin) 10 μM 2-머캅토에탄올(2-mercaptoethanol) 및 비타민 A가 제거된 1X B27 서플리먼트가 첨가된 대뇌 오가노이드 분화 배지(Cerebral organoid differentiation medium)에서 약 5일간 배양하여 패턴화하였다.Afterwards, the embedded spheroids were mixed with DMEM-F12 and Neurobasal medium at a 1:1 ratio with 0.5X N2 supplement, 62.5 μl of insulin 1X GlutaMAX supplement, 0.5X MEM-NEAA, 1X penicillin-streptomycin (penicillin) -streptomycin) 10 μM 2-mercaptoethanol (2-mercaptoethanol) and vitamin A removed 1X B27 supplement added cerebral organoid differentiation medium (Cerebral organoid differentiation medium) was cultured for about 5 days and patterned.
신경상피배(Neuroepithelial bud)가 형성되면 대뇌 오가노이드 분화 배지에서 1X B27 서플리먼트를 비타민 A가 포함된 것으로 교체하여 약 15일간 분화를 유도하여 대뇌 조직을 성장시키고 최종 분화시켜 대뇌 오가노이드를 완성하였다.When a neuroepithelial bud is formed, 1X B27 supplement is replaced with a vitamin A-containing one in the cerebral organoid differentiation medium to induce differentiation for about 15 days to grow cerebral tissue and finally differentiate to complete cerebral organoids.
실시예 2: 대뇌 오가노이드의 분화 확인Example 2: Confirmation of differentiation of cerebral organoids
제작한 대뇌 오가노이드의 특이적 마커 발현을 확인하기 위해 형광면역염색을 진행하였다.Fluorescent immunostaining was performed to confirm the expression of specific markers in the prepared cerebral organoids.
구체적으로, 분화가 끝난 대뇌 오가노이드는 4% 파라포름알데하이드로 24시간 이상 고정하고, 0.2% Triton X 100이 포함된 PBS(phosphate-buffered saline)로 3차례 세척하였다. 이후 소혈청알부민(Bovine serum albumin; BSA)이 포함된 PBS로 블로킹(blocking)하여 항체의 비특이적 결합을 방지하였다. 대뇌 오가노이드의 마커인 FOXG1과 PROX1, 신경세포 마커인 TUJ1, 줄기세포 마커인 SOX2 등의 항체를 각각 1:100의 농도로 처리하여 4℃에서 24시간 이상 유지하였다.Specifically, After differentiation, the cerebral organoids were fixed with 4% paraformaldehyde for more than 24 hours, and washed three times with PBS (phosphate-buffered saline) containing 0.2% Triton X 100. Thereafter, non-specific binding of the antibody was prevented by blocking with PBS containing bovine serum albumin (BSA). Antibodies such as FOXG1 and PROX1, which are markers of cerebral organoids, TUJ1, which are neuronal markers, and SOX2, which are stem cell markers, were each treated at a concentration of 1:100 and maintained at 4°C for more than 24 hours.
이를 0.2% Triton X 100이 포함된 PBS로 세척 후, BSA가 포함된 PBS로 형광염료를 포함한 2차 항체의 비특이적 결합을 방지하였고, 2차 항체를 1:100의 농도로 처리하여 4℃에서 2시간 이상 유지한 후, 세척하여 핵 염색을 위한 Hochest를 처리하고 공초점 현미경을 이용하여 형광을 관찰하였다.After washing this with PBS containing 0.2% Triton X 100, non-specific binding of the secondary antibody containing the fluorescent dye was prevented with PBS containing BSA, and the secondary antibody was treated at a concentration of 1:100 at 4°C for 2 After maintaining for more than an hour, it was washed and treated with Hochest for nuclear staining, and fluorescence was observed using a confocal microscope.
도 1c 및 1d에서 확인할 수 있듯이, 대뇌 오가노이드의 마커인 FOXG1과 PROX1, 신경세포 마커인 TUJ1, 줄기세포 마커인 SOX2 등으로 염색하고 공초점 현미경(Confocal microscope)으로 형광을 관찰하였고, 대뇌 오가노이드가 잘 형성되었음을 확인하였다.As can be seen in FIGS. 1c and 1d, cerebral organoid markers FOXG1 and PROX1, neuronal cell marker TUJ1, stem cell marker SOX2, etc. were stained and fluorescence was observed with a confocal microscope, and cerebral organoids was confirmed to be well formed.
실시예 3: 인간 신경줄기세포로부터 모터뉴런 스페로이드 제작Example 3: Preparation of motor neuron spheroids from human neural stem cells
모터뉴런 스페로이드의 제조 과정은 도 2a와 같다. 모터뉴런 스페로이드는 인간 신경줄기세포(human neural stem cells)를 ultra-low attachment 96 well U-bottom plate(Corning®, Cat #. CLS7007)에 5.0 X 104 cells/well로 분주하여 24시간 동안 배양한 후, StemPro hESC 배지(Gibco, Cat #. A1000701)에 레티노산(Retinoic Acid; RA) 50 μM, 분비성 신호인자(sonic hedgehog) 200 ng/ml, 섬유아세포 성장인자(basic fibroblast growth factor; bFGF) 8 ng/ml 및 액티빈(activin) A 10 ng/ml가 포함된 모터뉴런 분화용 배지를 웰 당 100 μl씩 교체하였다.The manufacturing process of the motor neuron spheroid is shown in Figure 2a. Motor neuron spheroids were obtained by dispensing human neural stem cells at 5.0 X 10 4 cells/well in an ultra-low attachment 96 well U-bottom plate (Corning®, Cat #. CLS7007) and cultured for 24 hours. Then, in StemPro hESC medium (Gibco, Cat #. A1000701), retinoic acid (RA) 50 μM, secretory signal factor (sonic hedgehog) 200 ng/ml, basic fibroblast growth factor (bFGF) A medium for motor neuron differentiation containing 8 ng/ml and 10 ng/ml of activin A was replaced by 100 μl per well.
20일 이후에는, StemPro hESC medium에 뇌유래신경성장인자(Brain-derived neurotrophic factor; BDNF) 10 ng/ml 및 교세포 신경성장인자(Glial cell-Derived Neurotrophic Factor; GDNF) 10 ng/ml가 포함된 모터뉴런 배양용 배지에서 모터뉴런의 숙성화를 위해 7~8일간 배양하였다.After 20 days, a motor containing 10 ng/ml of brain-derived neurotrophic factor (BDNF) and 10 ng/ml of glial cell-derived neurotrophic factor (GDNF) in StemPro hESC medium For maturation of motor neurons in neuron culture medium, cultured for 7 to 8 days.
실시예 4: 모터뉴런 스페로이드의 분화 확인Example 4: Confirmation of differentiation of motor neuron spheroids
제작한 모터뉴런 스페로이드의 특이적 마커 발현을 확인하기 위하여 형광면역염색을 진행하였다. 실시예 2의 방법과 동일하게 수행하되, 신경세포 마커인 TUJ1과 모터뉴런 마커인 islet1의 항체를 각각 1:100의 비율로 사용하였다.Fluorescence immunostaining was performed to confirm the expression of specific markers of the prepared motor neuron spheroids. It was performed in the same manner as in Example 2, except that the antibody of TUJ1, a neuron marker and islet1, a motor neuron marker, was used in a ratio of 1:100, respectively.
이어서 모터뉴런 스페로이드의 분화를 확인하기 위하여 역전사 중합효소 연쇄반응(reverse transcription PCR)을 진행하였다. 신경줄기세포와 분화유도한 모터뉴런 스페로이드를 TRIzol 용액으로 처리하여 RNA를 분리하였다. Superscript III kit을 이용하여 cDNA를 합성하고 FlexCycler2 system 기기를 이용하여 PCR을 진행하였다. 미분화상태의 마커로 PAX6와 SOX1, 모터뉴런 분화 마커로 ChAT, HB9과 islet1을, 초기 cDNA양을 확인하기 위해 GAPDH의 프라이머(primer)를 이용하여 PCR을 수행하였다.Subsequently, reverse transcription PCR was performed to confirm the differentiation of motor neuron spheroids. RNA was isolated by treating neural stem cells and differentiation-induced motor neuron spheroids with TRIzol solution. cDNA was synthesized using the Superscript III kit, and PCR was performed using the FlexCycler2 system device. PCR was performed using PAX6 and SOX1 as undifferentiated markers, ChAT, HB9 and islet1 as motor neuron differentiation markers, and GAPDH primer to confirm the initial amount of cDNA.
도 2b 및 2c에서 확인할 수 있듯이, 모터뉴런 스페로이드의 특이적 마커의 발현을 확인함으로써 모터뉴런 스페로이드가 잘 형성되었음을 확인하였다.As can be seen in Figures 2b and 2c, it was confirmed that the motor neuron spheroid was well formed by confirming the expression of a specific marker of the motor neuron spheroid.
실시예 5: 대뇌 오가노이드-모터뉴런 스페로이드 결합 유도Example 5: Induction of cerebral organoid-motoneuron spheroid binding
제작한 대뇌 오가노이드와 모터뉴런 스페로이드를 결합하여 구조체를 제작하였고, 이를 배양하기 위한 2개의 배양액 저장소와 공배양 챔버를 포함하는 공배양 칩(3D engineered nervous system, 3D-ENS)을 제조하였다. 상기 공배양 칩은 3D 프린팅 기술을 이용하여 형성된 상판과 하판을 조립하여 제조하였다.A structure was manufactured by combining the prepared cerebral organoids and motor neuron spheroids, and a co-culture chip (3D engineered nervous system, 3D-ENS) including two culture medium reservoirs and a co-culture chamber for culturing them was prepared. The co-culture chip was manufactured by assembling an upper plate and a lower plate formed using 3D printing technology.
도 3a에서 확인할 수 있듯이, 공배양 칩은 각각의 조직을 배양하는 배지가 상이하여 결합 유도시 상대 조직에 가해질 자극을 최소화하기 위한 목적으로 제조하였으며, 이러한 목적을 달성하기 위하여 대뇌 오가노이드 배양액 저장소가 공배양 챔버에 연결되는 높이는 모터뉴런 스페로이드 배양액 저장소가 공배양 챔버에 연결되는 높이보다 높게 설계되었다.As can be seen in Figure 3a, the co-culture chip was prepared for the purpose of minimizing the stimulus to be applied to the other tissue during the induction of connective tissue because the culture medium for each tissue is different. To achieve this purpose, the cerebral organoid culture medium storage The height connected to the co-culture chamber was designed to be higher than the height at which the motor neuron spheroid culture medium reservoir was connected to the co-culture chamber.
이러한 설계에 따라 제조된 공배양 칩을 이용함으로써, 각각의 조직이 특이적인 개별 배양 배지에 노출되도록 배양하여 결합을 유도하였다. 구체적으로, 상기 공배양 칩을 이용한 대뇌 오가노이드 및 모터뉴런 스페로이드의 결합을 수행함에 있어서, 먼저 공배양 챔버의 하판에 모터뉴런 스페로이드를 배치하고 뇌 오가노이드를 투입하였다.By using a co-culture chip manufactured according to this design, binding was induced by culturing each tissue to be exposed to a specific individual culture medium. Specifically, in performing the coupling of cerebral organoids and motor neuron spheroids using the co-culture chip, motor neuron spheroids were first placed on the lower plate of the co-culture chamber, and brain organoids were introduced.
대뇌 오가노이드 배양액 저장소로부터 채널을 통하여 공배양 챔버로 유입되는 배양액으로는 실시예 1에서 신경상피배 형성 후 대뇌 오가노이드 분화 배지에서 1X B27 서플리먼트를 비타민 A가 포함된 것으로 교체한 것과 같은 배지를 사용하였고, 모터뉴런 스페로이드 배양액 저장소로부터 채널을 통하여 공배양 챔버로 유입되는 배양액으로는 실시예 3에서 모터뉴런의 숙성화에 사용된 모터뉴런 배양용 배지를 사용하였다.As the culture medium flowing into the co-culture chamber through the channel from the cerebral organoid culture medium, the same medium was used in which the 1X B27 supplement was replaced with one containing vitamin A in the cerebral organoid differentiation medium after neuroepithelial embryo formation in Example 1 As the culture medium flowing into the co-culture chamber from the motor neuron spheroid culture medium reservoir through the channel, the motor neuron culture medium used for the maturation of motor neurons in Example 3 was used.
도 3b에서 확인할 수 있듯이, 공배양 챔버에 모터뉴런 스페로이드를 적절히 배치 후 뇌 오가노이드를 투입하여 7일 동안 배양한 결과 대뇌 오가노이드-모터뉴런 스페로이드 구조체를 형성하였다.As can be seen in Figure 3b, after properly disposing the motor neuron spheroids in the co-culture chamber, the brain organoids were added and cultured for 7 days to form a cerebral organoid-motoneuron spheroid structure.
실시예 6: 대뇌 오가노이드-모터뉴런 스페로이드 구조체의 생체 전기신호 변화의 확인Example 6: Confirmation of changes in bioelectrical signals of cerebral organoid-motoneuron spheroid structures
대뇌 오가노이드-모터뉴런 스페로이드 결합을 통한 구조체 제작 후, 다중 전극 어레이(Microelectrode array; MEA)를 통해 각각의 전기신호를 확인하였다. MEA 기판은 폴리이미드(Polyimide; PI)로 코팅 후, 라미닌으로 재코팅하여 구조체를 올리고 신호를 측정하였다. 특히 대뇌 오가노이드-모터뉴런 스페로이드 구조체는 모터뉴런 스페로이드를 MEA 기판과 접하게 배치하고, 대뇌 오가노이드에 가한 자극에 따른 모터뉴런 스페로이드의 신호 변화를 관찰하였다.After fabrication of the structure through cerebral organoid-motoneuron spheroid bonding, each electrical signal was confirmed through a microelectrode array (MEA). The MEA substrate was coated with polyimide (PI) and then re-coated with laminin to raise the structure and measure the signal. In particular, in the cerebral organoid-motoneuron spheroid structure, the motor neuron spheroid was placed in contact with the MEA substrate, and the signal change of the motor neuron spheroid according to the stimulation applied to the cerebral organoid was observed.
또한 뇌신경 자극 물질로 알려진 카페인을 대뇌 오가노이드, 모터뉴런, 대뇌 오가노이드-모터뉴런 스페로이드 구조체에 각각 처리하여 카페인에 의한 전기신호 변화를 관찰하였다.In addition, caffeine, known as a cranial nerve stimulant, was treated in cerebral organoids, motor neurons, and cerebral organoid-motoneuron spheroid structures, respectively, and changes in electrical signals caused by caffeine were observed.
도 4a 및 4b에서 확인할 수 있듯이, 대뇌 오가노이드는 카페인에 의해 전기 신호가 증폭되었으나 모터뉴런 스페로이드는 변화가 없었다.As can be seen in Figures 4a and 4b, cerebral organoids were amplified electrical signals by caffeine, but motor neuron spheroids did not change.
도 4c에서 확인할 수 있듯이, 대뇌 오가노이드-모터뉴런 스페로이드 구조체에 카페인을 처리했을 때, 기판에 접한 모터뉴런 스페로이드에서 측정되는 전기 신호 증폭은 두 조직간 신경망이 연결되어 대뇌 오가노이드의 흥분에 의한 신호가 전달되므로 모터뉴런 스페로이드의 전기신호를 증폭시킨 것으로 나타났다.As can be seen in Figure 4c, when the cerebral organoid-motoneuron spheroid structure was treated with caffeine, the electrical signal amplification measured in the motorneuron spheroid in contact with the substrate was stimulated by the neural network between the two tissues connected to the excitation of the cerebral organoids. It was shown that the electric signal of the motor neuron spheroid was amplified because the signal by
본 발명은 대뇌 오가노이드-모터뉴런 스페로이드 구조체 및 이의 제조방법에 관한 것으로서, 더욱 상세하게는 대뇌 오가노이드와 모터뉴런 스페로이드를 독립적으로 적절한 배양액에 접촉되도록 설계된 공배양 칩에서 공배양함으로써 대뇌 오가노이드-모터뉴런 스페로이드 구조체를 제조하는 방법에 관한 것이다.The present invention relates to a cerebral organoid-motoneuron spheroid structure and a method for manufacturing the same, and more particularly, by co-culturing cerebral organoids and motorneuron spheroids independently in a co-culture chip designed to be in contact with an appropriate culture medium. It relates to a method for fabricating a noid-motoneuron spheroid construct.

Claims (13)

  1. 뇌 오가노이드 및 뉴런 스페로이드를 공배양하는 공배양 단계를 포함하는 뇌 오가노이드-뉴런 스페로이드 구조체의 제조방법.A brain organoid comprising a co-culturing step of co-culturing brain organoids and neuronal spheroids - a method for preparing a neuron spheroid construct.
  2. 제1항에 있어서, 뇌 오가노이드는 대뇌 오가노이드인 것인, 제조방법.The method according to claim 1, wherein the brain organoid is a cerebral organoid.
  3. 제1항에 있어서, 뉴런 스페로이드는 모터뉴런 스페로이드인 것인, 제조방법.The method according to claim 1, wherein the neuron spheroid is a motor neuron spheroid.
  4. 제1항에 있어서, 공배양 단계는 공배양 칩에서 수행되고, The method of claim 1, wherein the co-culturing step is performed in a co-culture chip,
    상기 공배양 칩은,The co-culture chip,
    공배양 챔버;co-culture chamber;
    공배양 챔버에 독립적인 채널로 연결된 뇌 오가노이드 배양액 저장소; 및a brain organoid culture reservoir connected by an independent channel to the co-culture chamber; and
    공배양 챔버에 독립적인 채널로 연결된 뉴런 스페로이드 배양액 저장소;a neuron spheroid culture reservoir connected by an independent channel to the co-culture chamber;
    를 포함하는 것인, 제조방법.A manufacturing method comprising a.
  5. 제4항에 있어서, 공배양 단계는 공배양 챔버에 뉴런 스페로이드를 1개 이상 배치한 후 뇌 오가노이드를 투입하여 수행되는 것인, 제조방법.The method according to claim 4, wherein the co-culturing step is performed by placing one or more neuronal spheroids in the co-culture chamber and then adding brain organoids.
  6. 제4항에 있어서, 뇌 오가노이드 배양액 저장소의 채널은 뉴런 스페로이드 배양액 저장소의 채널보다 공배양 챔버에 연결된 위치가 높은 것인, 제조방법.The method according to claim 4, wherein the channel of the brain organoid culture reservoir has a higher position connected to the co-culture chamber than the channel of the neuron spheroid culture reservoir.
  7. 뇌 오가노이드, 및 뇌 오가노이드 하단에 결합된 1개 이상의 뉴런 스페로이드를 포함하는 뇌 오가노이드-뉴런 스페로이드 구조체.A brain organoid-neuron spheroid construct comprising a brain organoid, and one or more neuronal spheroids bound to the underside of the brain organoid.
  8. 제7항에 있어서, 뇌 오가노이드는 대뇌 오가노이드인 것인, 구조체.8. The construct of claim 7, wherein the brain organoid is a cerebral organoid.
  9. 제7항에 있어서, 뉴런 스페로이드는 모터뉴런 스페로이드인 것인, 구조체.The construct of claim 7 , wherein the neuron spheroid is a motor neuron spheroid.
  10. 뇌 오가노이드, 및 뇌 오가노이드 하단에 결합된 1개 이상의 뉴런 스페로이드를 포함하는 뇌 오가노이드-뉴런 스페로이드 구조체에 대상 약물을 처리하는 단계를 포함하는 약물 스크리닝 방법.A drug screening method comprising the step of treating a target drug to a brain organoid-neuron spheroid construct comprising a brain organoid, and one or more neuronal spheroids bound to the bottom of the brain organoid.
  11. 제10항에 있어서, 뇌 오가노이드는 대뇌 오가노이드인 것인, 스크리닝 방법.11. The method of claim 10, wherein the brain organoid is a cerebral organoid.
  12. 제10항에 있어서, 뉴런 스페로이드는 모터뉴런 스페로이드인 것인, 스크리닝 방법.The screening method according to claim 10, wherein the neuronal spheroid is a motor neuron spheroid.
  13. 제10항에 있어서, 대상 약물은 파킨슨병 또는 헌팅턴병을 포함하는 운동장애를 유발하는 뇌질환의 예방 또는 치료를 위한 후보물질인 것인, 스크리닝 방법.The screening method according to claim 10, wherein the target drug is a candidate substance for the prevention or treatment of brain diseases causing movement disorders including Parkinson's disease or Huntington's disease.
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