CN115885037A - Midbrain organoid, high-speed large-scale production method thereof, method for screening neurotoxic substance and method for screening dopaminergic neuron-related disease drug using the same - Google Patents
Midbrain organoid, high-speed large-scale production method thereof, method for screening neurotoxic substance and method for screening dopaminergic neuron-related disease drug using the same Download PDFInfo
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
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Abstract
The present invention relates to a midbrain organoid and a method for manufacturing the same, a method for screening neurotoxic substances using the same, and a method for screening drugs for dopaminergic neuron-related diseases, wherein the manufacturing method can rapidly manufacture the organoid at a high speed, thereby achieving rapid drug screening, blocking abnormal differentiation, minimizing the dead nucleus phenomenon, and reducing variation between organoids, and thus can be effectively used for organoid production.
Description
Technical Field
The present invention relates to a method for producing Midbrain (Midbrain) organoids at high speed and in large scale, a method for screening neurotoxic substances using the same, and a method for screening drugs for treating dopaminergic neuron-related diseases (drug screening), and more particularly, to a method for producing Midbrain organoids that requires a number of starting cells (starting cell number) and a culture method (culture format) and that can produce high-quality Midbrain organoids with less inter-individual variation at high speed and in large scale by adjusting the number of starting cells and the culture method.
Background
Organoids are a new stem cell differentiation technology, which utilizes the differentiation ability (differentiation), self-regeneration ability (self-regeneration), and self-organization ability (self-organization) of stem cells to reproduce the cell composition and structure similar to those of in vivo organs by three-dimensional culture. Furthermore, by producing patient-customized organoids, patient-customized therapeutic drugs for various diseases can be produced at low cost in a short period of time, which is evaluated as a promising technology.
Recently, the production of brain organoids reflecting various brain characteristics has been reported, and studies on the simulation of diseases and the development of new drugs using ZIKA virus (ZIKA virus) or patient-derived brain organoids are actively being conducted.
However, the production of patient-customized organoids requires a lot of expense and a long time. As shown in FIG. 1, the production of Induced Pluripotent Stem Cells (iPSCs) derived from real patients usually takes 6 to 8 weeks or more, and the efficiency thereof is usually low, reaching about 0.02%. In addition, the time required for organoid production varies from organ to organ, but most require more than 3 to 6 months. Therefore, it is realistically difficult to develop a new drug tailored to a patient and the like for a patient with an advanced age of degenerative brain disease and other patients who are in urgent need of drug treatment. In addition, most of the organoids including brain organoids have problems to be solved for practical use or industrialization of organoid research.
First, the production of organoids requires much expense and long time.
For differentiation and maturation, oxygen and nutrients are required to be supplied to the organoids, for which reason, most often, shaking bioreactors (shaking bioreactors) are used to provide the requisite CO 2 Continued shaking and differentiation in the incubator, for long-term specific organoid differentiation, necessitates long-term treatment with expensive differentiation-promoting factors. Therefore, the production of long-term specific organoids is costly (FIG. 1).
Second, inter-organoid inter-individual variation (batch variation) is very severe (fig. 2), and thus inaccurate results may be produced in disease simulation studies and new drug screening studies using organoids.
Third, organoid differentiation produces abnormal tissue called "abnormal differentiation (out-growth"), which is a serious problem that impedes organoid quality and functionality, etc. (fig. 2).
Fourth, there are no blood vessels inside the organoids at present, so oxygen and nutrients are not normally delivered to the core of the organoids, and the phenomenon of "dead core" (dead core) resulting in internal cell death is widespread, which hinders the maturation of organoids and therefore is a problem that must be solved (fig. 2).
Fifth, organoid-based high-speed batch screening (HTS); in order to perform drug screening (drug screening) or drug remanufacturing (drug remanufacturing) studies by HTS, it is necessary to develop an organ-like mass production system conforming to the HTS model (format). In addition, the integration of organoid production and drug screening in HTS mode, the one-step organoid production that can be performed, and the development of new drug development systems are an essential part of the establishment of a practical foundation by organoid automation in the future.
Disclosure of Invention
Technical challenge
Therefore, in order to produce midbrain organoids at high speed/in large scale, the present inventors produced midbrain organoids using a High Throughput Screening (HTS) platform (platform) using a microplate such as a 96-or 384-well plate without using a shocking bioreactor (shocking bioreactor). Thus, in addition to production of mesencephalic organoids on an HTS platform (as shown in fig. 3), high-speed, large-scale production of normal levels of mesencephalic organoids with low inter-individual variability, abnormal differentiation and suppressed dead nuclei can be achieved on an HTS platform, which exhibits significantly improved uniformity and functionality over the midbrain organoids produced by prior methods at high speed and large scale production. It has further been found that the time and expense required to produce midbrain organoids can be saved.
Accordingly, it is an object of the present invention to provide a method for high-speed large-scale culture of a three-dimensional cell mass from human stem cells, comprising the steps of:
a step of forming an Embryoid body (Embryoid body) from a cell isolated from a human;
inducing differentiation from the embryoid body into a specific tissue to form a three-dimensional cell aggregate; and
a step of inducing maturation of said three-dimensional cell aggregate,
characterized in that the steps from the step of forming the embryoid to the step of inducing the three-dimensional cell aggregate to mature are continuously carried out on a large scale in the same microplate.
It is another object of the present invention to provide a three-dimensional cell aggregate produced by a high-speed large-scale culture method of the three-dimensional cell aggregate.
It is another object of the present invention to provide a three-dimensional cell mass culture kit comprising a concave culture part of a three-dimensional cell mass; and a covering part covering the concave culture part.
It is another object of the present invention to provide a three-dimensional mesencephalon cell mass, which is a three-dimensional mesencephalon cell mass cultured from any one of human embryonic stem cells, human induced pluripotent stem cells and adult stem cells;
the three-dimensional midbrain cell aggregate is a three-dimensional midbrain cell aggregate forming neural melanin.
Another object of the present invention is to provide a high-speed culture method from human stem cells to a three-dimensional cell aggregate, comprising the steps of:
a step of forming embryoid bodies from cells isolated from a human;
inducing differentiation from the embryoid body into a specific tissue to form a three-dimensional cell aggregate; and
a step of inducing maturation of the three-dimensional cell aggregate;
continuously culturing in a same microplate in a non-shaking manner from the step of forming the embryoid to the step of inducing the three-dimensional cell aggregate to mature;
the high-speed culture method is characterized by being a high-speed culture method of a three-dimensional cell aggregate which is carried out in an automated manner including a pipette robot, an automatic transfer and a plate transportation means.
Another object of the present invention is to provide a screening method for a drug for a dopaminergic neuron related disease, comprising:
a step of generating a three-dimensional cell aggregate from induced pluripotent stem cells derived from a patient with a dopaminergic neuron related disease;
a step of treating the three-dimensional cell aggregate with a candidate substance;
a step of determining the survival rate of dopaminergic neurons from the three-dimensional cell aggregate.
It is another object of the present invention to provide a method for providing information for treating a dopaminergic neuron related disease, comprising the steps of:
a step of culturing a three-dimensional cell aggregate from cells derived from a patient with a dopaminergic neuron related disease;
a step of treating the three-dimensional cell aggregate with a candidate substance;
a step of determining the survival rate of dopaminergic neurons from the three-dimensional cell mass;
providing appropriate therapeutic drug information to the patient based on the dopaminergic neuron survival rate.
It is another object of the present invention to provide a method for discriminating a three-dimensional cell aggregate for drug testing, comprising the steps of:
(i) A step of treating the three-dimensional cell mass with a candidate substance:
the step of treating with a candidate substance is carried out in one or more steps included in a high-speed large-scale culture method of a three-dimensional cell mass; and
(ii) A method for discriminating a three-dimensional cell mass used for a drug test, comprising the step of comparing the responses of the three-dimensional cell mass of each step in the presence and absence of a candidate substance.
It is another object of the present invention to provide a method for in vitro toxicity screening of drugs using a three-dimensional cell aggregate, comprising the steps of:
(i) A step of treating a three-dimensional cell mass with a candidate substance in one or more steps included in a high-speed large-scale culture method for a three-dimensional cell mass;
(ii) Comparing the reactions of the three-dimensional cell aggregates of the respective steps in the presence and absence of the candidate substance;
(iii) A step of determining whether or not cells present in the three-dimensional cell collection die;
to provide a method for screening toxicity of drugs in vitro by using a three-dimensional cell aggregate.
Means for solving the problems
The present invention relates to a method for producing Midbrain (Midbrain) organoids at high speed and on a large scale, a method for screening neurotoxic substances using the Midbrain (Midbrain) organoids, and a drug screening method for treating dopaminergic neuron-related diseases. According to the organoid manufacturing method of the present invention, organoids can be produced at high speed and in large scale, drug screening can be rapidly performed, abnormal differentiation (out-growth) can be blocked, dead core (dead core) phenomenon can be minimized, variation between organoids can be reduced, and excellent functionality can be induced by rapid maturation of midbrain organoids. The present inventors have achieved a result of shortening the time required for organoid production from the conventional 3 to 6 months or more to up to 30 days by applying a starting cell number of 50 to 3,000 in the organoid production process.
The present invention will be described in more detail below.
One aspect of the present invention is a method for high-speed culture of a three-dimensional cell aggregate from human stem cells, comprising:
a step of forming an Embryoid body (Embryoid body) from a cell isolated from a human;
inducing differentiation from the embryoid body into a specific tissue to form a three-dimensional cell aggregate; and
a step of inducing maturation of the three-dimensional cell aggregate;
characterized in that the steps from the step of forming the embryoid to the step of inducing the three-dimensional cell aggregate to mature are continuously cultured in the same microplate.
In the present invention, the cell isolated from a human is one of a human embryonic stem cell (hESC), an induced pluripotent stem cell (induced pluripotent stem cell) and an adult stem cell.
The term "stem cell" in the present specification refers to a cell having a self-replicating ability while having an ability to differentiate into two or more cells, and includes a pluripotent stem cell (totipotent stem cell), a pluripotency stem cell (pluripotent stem cell), and a multi-differentiated stem cell (multi-pluripotent stem cell), as well as an induced pluripotent stem cell.
In the present invention, the three-dimensional cell aggregate may be an organoid, such as a brain organoid.
The term "organoid" in the present specification refers to a cell aggregate formed by 3D culture to re-aggregate or recombine cells isolated from stem cells or organ derived cells, and may include organoids or cell clusters formed from suspension cell cultures. The organoids may also be named small similar organs, organ analogs, similar organs. The organoids include in particular one or more of the various cells that make up the organ or tissue, which must be able to reproduce the structure and function of the tissue or organ.
In the examples of the present invention, the brain organoids produced have a diameter mostly in the range of about 0.9 to 1.4mm, are uniform in shape and size, and thus have less variation among individuals and exhibit better quality.
In the present invention, the three-dimensional cell aggregate may be formed within 20 to 50 days or 20 to 40 days, for example, within 20 to 30 days, but is not limited thereto.
In the examples of the present invention, the activity of the midbrain gene was confirmed in the case of culturing for 7 days according to the method of the present invention. Specific midbrain dopaminergic neurons (mDA neurons) appear if cultured for 14 days, and the number of mDA neurons increases if cultured for 21 days.
In the present invention, after the three-dimensional cell aggregate is formed, shake culture may be further performed. By the further shake culture, a long-term culture can be performed.
In the examples of the present invention, the three-dimensional cell mass was cultured by performing shake culture for 300 days or more after the formation of the three-dimensional cell mass.
In the present invention, the microplate may be a multi-well plate having a concave portion. The multi-well plate having a well is a 96-well plate, a 384-well plate, or a1,536-well plate.
In the present invention, the initial starting cell number of the cells isolated from human may be 50 to 3,000.
If a 96-well plate is used, the initial starting cell number is preferably 300 to 3,000, preferably 300 to 1,000, preferably 500, but is not limited thereto.
If a 384 well plate is used, the initial starting cell number is preferably 50 to 500, preferably 50 to 200, preferably 100, but is not limited thereto.
In the present invention, the method of high-speed mass culture can be carried out without using a bioreactor, for example, without performing a shaking (shaking) process, in the entire process of forming a three-dimensional cell mass.
In the present invention, the three-dimensional cell aggregate may be a midbrain cell aggregate.
In the present invention, the three-dimensional cell aggregate may be prepared from a digested organ selected from the group consisting of liver, heart and lung.
In the present invention, the step of forming a three-dimensional cell aggregate may include an ectoderm-forming step or a brain tissue differentiation step.
The ectoderm-forming step may be performed in a medium including CHIR99021, dihydrodeoxymorphine (Dorsomorphin), and a83-01 (dual SMAD inhibitors).
The midbrain tissue differentiation step may be performed in a medium including CHIR99021, dihydrodeoxymorphine (Dorsomorphin), A83-01, IWP, SAG and FGF-8 b.
In the present invention, the step of inducing the maturation of the three-dimensional cell mass may be performed in a medium including SAG, FGF-8b, insulin (Insulin), laminin (Laminin), and further including Growth factor reduced matrigel (Growth factor reduced matrigel).
In the embodiment of the present invention, in performing the step of inducing the maturation of the three-dimensional cell mass, a method of culturing by directly adding matrigel to a culture medium is used. However, according to the known methods, a common method is to embed cells in matrigel on a plate, which is different from the culture method of the present invention.
In the present invention, in the step of forming a three-dimensional cell aggregate, no endoderm marker may be expressed and ectoderm-forming factors may be expressed within 7 days after the induction of tissue differentiation. The ectoderm-forming factor may be selected from one or more of, but is not limited to, N-CAD, PLZF, SOX1, SOX2, and NESTIN.
In the present invention, in the step of forming a three-dimensional cell aggregate, the forebrain or hindbrain marker may not be expressed and the mesencephalon marker may be expressed within 14 days after the induction of tissue differentiation. The midbrain markers may be selected from, but are not limited to, one or more of LMX1B, ASCL and TH.
In the present invention, it is possible to express markers for mesencephalic dopaminergic neurons after performing the step of inducing the maturation of the three-dimensional cell mass.
In embodiments of the invention, the midbrain marker is expressed at or before day 14, but the expression level of the marker is higher at day 21. It can be seen that the midbrain dopaminergic neuron marker TH, and the like are expressed at a high level after the step of inducing the three-dimensional cell aggregate to mature.
In the embodiment of the invention, the steps from the step of forming embryoid to the step of inducing the three-dimensional cell aggregate to mature are continuously carried out in the same microplate, the movement of the microplate is not required, and the microplate can be used for carrying out high-speed large-scale culture within 30 days.
The second aspect of the present invention is a three-dimensional cell aggregate produced by a high-speed large-scale culture method of a three-dimensional cell aggregate.
Another aspect of the invention is a three-dimensional cell aggregate culture kit comprising a concave culture section of a three-dimensional cell aggregate; and a covering part covering the concave culture part.
In the present invention, the three-dimensional cell mass culture kit may include a preservation solution.
Another aspect of the present invention is a three-dimensional mesencephalon cell mass cultured from one of human embryonic stem cells, human induced pluripotent stem cells and adult stem cells;
the three-dimensional midbrain cell aggregate is a three-dimensional midbrain cell aggregate forming neural melanin.
The three-dimensional mesencephalon cell aggregate may be, but is not limited to, forming neuromelanin 20 to 50 days, 20 to 40 days, 20 to 35 days, 25 to 50 days, or 25 to 40 days after culture, for example forming neuromelanin 25 to 35 days after culture.
In the present invention, the three-dimensional mesencephalon cell aggregate may not include other brain tissues than the mesencephalon.
In the present invention, the incidence of abnormal differentiation (out growth) of the three-dimensional mesencephalon cell mass may be 5% or less.
In the present invention, the occurrence rate of deep cell death phenomenon of the three-dimensional midbrain cell aggregate may be less than 40% of the total area of the cell aggregate.
In the present invention, the diameter of the three-dimensional midbrain cell aggregate may be 0.9 to 1.4mm, which has a uniform size and a uniform morphology.
In the present invention, the three-dimensional mesencephalic cell aggregate can include more than 10% mesencephalic dopaminergic neurons.
The three-dimensional mesencephalic cell aggregate can comprise more than 30% of mesencephalic dopaminergic neurons or more than 50% of mesencephalic dopaminergic neurons. At an early stage, approximately 80% of mesencephalic dopaminergic neurons can be involved, but in a mature three-dimensional mesencephalic cell aggregate, a variety of mesencephalic cells are formed, and the content of dopaminergic neurons in the whole cell population may be reduced.
In the present invention, the three-dimensional mesencephalon cell mass is a three-dimensional mesencephalon cell mass in which the variation rate of gene expression formed uniformly can be 10% or less.
In the examples of the present invention, the gene expression quality of the three-dimensional mesencephalon cell mass was very good, showing homogeneity of about 90% or more. The remaining about 10% of the cases observed had equivalent or better uniformity of gene expression than the cases made by the conventional method.
In the present invention, the three-dimensional mesencephalon cell aggregate may include inhibitory nerves and excitatory nerves.
In the present example, the three-dimensional mesencephalon cell aggregates include inhibitory nerves and excitatory nerves, which are identified as accounting for 10% to 30% or more of the entire cell aggregate. This can be confirmed from the experimental results of the tissue staining data of fig. 11 and 15a in the present specification.
In the present invention, electrophysiological activity was observed in the three-dimensional mesencephalon cell aggregates from 15 days after cell differentiation. The conventional method is such that the cell differentiation begins to show electrophysiological activity after several months, from which it can be demonstrated that the method of the invention, which has a very rapid maturation pattern, has significant advantages.
In the present invention, the three-dimensional mesencephalon cell aggregate may include any one selected from the group consisting of: substantia nigra, locus coerulea, akashiwo nucleus, central gray matter, internal test mother zone, oculomotor nucleus and trochlear nucleus.
In the present invention, in the three-dimensional midbrain cell aggregate, the glial cells may include oligodendrocytes, and the three-dimensional midbrain cell aggregate may include 1% to 10% of glial cells and oligodendrocytes.
In an embodiment of the present invention, the extent to which the three-dimensional midbrain cell aggregate includes glial cells and oligodendrocytes can be confirmed from the experimental results of the tissue staining data of fig. 11 and 15a on the present specification.
Another aspect of the present invention is a method for high speed culture of human stem cells into three-dimensional cell aggregates comprising the steps of:
a step of forming embryoid bodies from cells isolated from a human;
inducing differentiation from the embryoid body into a specific tissue to form a three-dimensional cell aggregate; and
a step of inducing maturation of the three-dimensional cell aggregate;
continuously culturing in a same microplate in a non-shaking manner from the step of forming the embryoid to the step of inducing the three-dimensional cell aggregate to mature;
the high-speed culture method is characterized by being a high-speed culture method of a three-dimensional cell aggregate which is carried out in an automated mode comprising a liquid-moving robot, automatic transportation and a plate transportation tool.
Another aspect of the present invention is a method for screening a drug for a dopaminergic neuron related disease, comprising:
a step of forming a three-dimensional cell aggregate from induced pluripotent stem cells derived from a patient with a dopaminergic neuron-related disease;
a step of treating the three-dimensional cell aggregate with a candidate substance;
a step of determining the survival rate of dopaminergic neurons from said three-dimensional cell mass.
In the present invention, the dopaminergic neuron related disease includes, but is not limited to, one of Parkinson's disease, alzheimer's disease, cerebral hemorrhage, stroke, huntington's disease, pick's disease, creutzfeldt-jakob disease, autism, and brain developmental disorder.
The information providing method can be used as a method for evaluating the toxicity and efficacy of a drug by applying the three-dimensional cell aggregate to the screening use of a drug for a dopaminergic neuron related disease.
The candidate substance may be determined to have a therapeutic effect as a therapeutic agent for a dopaminergic neuron-related disease, based on the fact that the candidate substance prevents or treats the onset of a dopaminergic neuron-related disease, or improves prognosis as compared with a control substance.
Another aspect of the invention is a method of providing information for treating a dopaminergic neuron related disease, comprising the steps of:
a step of culturing a three-dimensional cell aggregate from cells derived from a patient with a dopaminergic neuron related disease;
a step of treating the three-dimensional cell aggregate with a candidate substance; and
a step of determining the survival rate of dopaminergic neurons from the three-dimensional cell mass;
providing appropriate therapeutic drug information to the patient based on the dopaminergic neuron survival rate.
In the present invention, candidate substances include natural compounds, synthetic compounds, RNA, DNA, which may be selected from, but not limited to, one of polypeptides, enzymes, proteins, ligands, antibodies, antigens, metabolites of bacteria or fungi, and bioactive molecules.
Another aspect of the present invention is a method for discriminating a three-dimensional cell aggregate used for a drug test, comprising the steps of:
(i) A step of treating the three-dimensional cell mass with a candidate substance:
the step of treating with a candidate substance is carried out in one or more steps included in a high-speed large-scale culture method of a three-dimensional cell mass; and
(ii) A method for discriminating a three-dimensional cell mass used for a drug test, comprising the step of comparing the responses of the three-dimensional cell mass of each step in the presence and absence of a candidate substance.
Another aspect of the invention is a method for in vitro toxicity screening of drugs using a three-dimensional cell aggregate comprising the steps of:
(i) A step of treating the three-dimensional cell aggregate with a candidate substance in one or more steps included in the high-speed large-scale culture method for the three-dimensional cell aggregate;
(ii) Comparing the responses of the three-dimensional cell aggregates of the respective steps in the presence and absence of the candidate substance;
(iii) A step of determining whether or not cells present in the three-dimensional cell collection die;
the above is a method for in vitro toxicity screening of drugs using a three-dimensional cell aggregate.
The term "screening" in the present specification means that a substance having a specific property is screened by a specific procedure or evaluation method for the purpose of a candidate group consisting of a plurality of substances.
The in vitro toxicity screening method can be used as a drug toxicity evaluation platform for identifying toxic substances or identifying neurotoxicity of new drug candidate substances.
ADVANTAGEOUS EFFECTS OF INVENTION
The present invention relates to a midbrain organoid and a method for manufacturing the same, a method for screening neurotoxic substances using the same, and a drug screening (drug screening) method for treating diseases related to dopaminergic neurons, the manufacturing method enabling high-speed production of organoids, enabling rapid drug screening, blocking abnormal differentiation, minimizing the phenomenon of dead nuclei, reducing variation among organoids, inducing high functionality of midbrain organoids, and efficiently converting into organoids that can be used for production.
Drawings
FIG. 1 is a simulation of the generation of Induced Pluripotent Stem cells (iPSCs) for patient customized organoid production and organoid differentiation process.
FIG. 2 is a simulation showing various problems in organoid production.
FIG. 3 is a simulation showing the organoid production strategy of the present invention, as compared to the prior art.
Fig. 4 is a photograph of morphology (upper), differentiation rate and morphology classification (middle) and standard morphology (lower) of differentiated midbrain organoids produced in 96-well plates on day 7 according to an embodiment of the present invention.
FIG. 5a is a graph of total differentiation function (Pluripotency) marker gene expression for differentiated midbrain organoids at day 7 produced in 96-well plates, according to an embodiment of the present invention.
FIG. 5b is a graph of mesendoderm (Mesoendedrerm) marker gene expression for day 7 differentiated midbrain organoids produced in 96-well plates, according to an embodiment of the present invention.
FIG. 5c is a graph of Neuroectoderm (Neuroectoderm) marker gene expression of differentiated day 7 midbrain organoids produced in 96-well plates according to an example of the present invention.
Fig. 6 is a photograph of morphology (upper), differentiation rate and morphology classification (middle) and standard morphology (lower) of differentiated 14-day midbrain organoids produced in 96-well plates according to an embodiment of the present invention.
FIG. 7a is a graph of apoptosis marker gene expression for day 14 differentiated midbrain organoids produced in 96-well plates, according to an embodiment of the present invention.
FIG. 7b is a graph of the expression of Forebrain (Forebrain) marker genes of differentiated day 14 midbrain organoids produced in 96-well plates, according to an embodiment of the present invention.
FIG. 7c is a graph of Midbrain (Midbrain) marker gene expression for differentiated day 14 Midbrain organoids produced in 96-well plates, according to an embodiment of the present invention.
FIG. 7d is a graph of Hindbrain (Hindbrain) marker gene expression for differentiated day 14 midbrain organoids produced in 96-well plates according to an embodiment of the present invention.
Fig. 8 is a photograph of morphology (upper), differentiation rate and morphological classification (middle) and standard morphology (lower) of 14 day differentiated midbrain organoids produced in 96-well plates under an initial starting cell number of 500 or less, according to an embodiment of the present invention.
FIG. 9a is a graph of in vivo neuroectodermal marker gene expression of day 7 differentiated midbrain organoids produced in 96-well plates under an initial starting cell number of 500 or less, in accordance with an example of the present invention.
FIG. 9b is a graph of mesencephalon marker gene expression of the differentiated day 14 mesencephalon organoids produced in 96-well plates under conditions of an initial starting cell number of 500 or less, according to an embodiment of the present invention.
FIG. 9c is a graph of the expression of hindbrain marker genes of day 14 differentiated midbrain organoids produced in 96-well plates at an initial starting cell number of 500 or less, in accordance with an embodiment of the present invention.
FIG. 10a is a photograph comparing the sizes of midbrain organoids formed with different initial starting cell numbers in 384-well plates, according to an embodiment of the present invention.
FIG. 10b is a graph of midbrain marker gene expression for midbrain organoids produced in 384 well plates according to an embodiment of the present invention.
FIG. 11 is a photograph showing the results of immunostaining analysis of specific midbrain dopaminergic neurons (mDA neurons) with Tyrosine Hydroxalase (TH) in the midbrain organoids on day 14 of differentiation, according to an embodiment of the present invention.
Fig. 12 is a photograph of morphology (upper), differentiation rate and morphology classification (middle) and a photograph of standard morphology (lower) of the midbrain organoids differentiated on day 21 according to an embodiment of the present invention.
FIG. 13a is a graph of apoptosis marker gene expression at day 21 of differentiation, in accordance with an embodiment of the present invention.
FIG. 13b is a graph showing the expression of forebrain marker genes at day 21 of differentiation, in accordance with an embodiment of the present invention.
FIG. 13c is a graph of mesencephalon marker gene expression at day 21 of differentiation, according to an example of the present invention.
FIG. 13d is a graph of hindbrain marker gene expression at day 21 of differentiation, in accordance with an embodiment of the present invention.
FIG. 14a is a photograph of midbrain organoids of size compared by date with different initial starting cell numbers, according to an embodiment of the present invention.
FIG. 14b is a graph of midbrain organoid sizes formed with different initial starting cell numbers compared by date, in accordance with an embodiment of the present invention.
FIG. 15a is a photograph showing the result of immunostaining analysis of specific mesencephalic dopaminergic neurons in the mesencephalic organoids of day 21 of differentiation formed with different initial starting cell numbers, as TH, according to an embodiment of the present invention.
FIG. 15b is a graph of the results of an immunostaining assay showing the proportion of specific midbrain dopaminergic neurons in day 21 of differentiation organoids formed from different initial starting cell numbers, according to an embodiment of the present invention.
FIG. 16a is a photograph showing the results of immunostaining analysis of dead nucleus size in differentiated day 21 midbrain organoids formed with different initial starting cell numbers, according to an embodiment of the present invention.
FIG. 16b is a graph of immunostaining analysis results showing dead nucleus size in day 21 differentiated midbrain organoids formed with different initial starting cell numbers, according to an embodiment of the present invention.
FIG. 17 is a photograph showing the result of immunostaining analysis of neuronal types and other cellular components of midbrain organoids produced under the condition of 500 initial starting cell numbers according to an embodiment of the present invention.
FIG. 18 is a photograph of High Content Imaging (HCI) showing the results of analysis of midbrain organoids produced in a 96, 384 well plate high speed batch screening (HTS) platform, according to an embodiment of the present invention.
FIG. 19 is a photograph and a graph of the results of morphology (top) and size (bottom) of midbrain organoids produced by prior methods and high speed, large scale production of midbrain organoids according to embodiments of the present invention.
FIG. 20 is a graph comparing gene expression between individual midbrain organoids in order to confirm variation between individual midbrain organoids produced at high speed and on a large scale, according to an embodiment of the present invention.
Fig. 21 is a photograph analyzing the result of the production of neural melanin in midbrain organoids produced at high speed and on a large scale according to an embodiment of the present invention.
FIG. 22 is a photograph of melanin production in high-speed, large-scale production of midbrain organoids confirmed by melanin staining (Fontana-Masson) technique, according to an embodiment of the present invention.
FIG. 23 is a comparison of electrophysiological activities of midbrain organoids produced by prior methods and high-speed, large-scale production of midbrain organoids, in accordance with embodiments of the present invention.
FIG. 24 is a photograph (top) and a graph (bottom) showing morphological and size changes of high-speed, mass-produced mesencephalon organoids treated with the neurotoxic substance 6-OHDA and MPTP in accordance with an embodiment of the present invention.
FIG. 25 is a graph of apoptosis marker (upper) and midbrain marker (lower) gene expression for high speed, large scale production of midbrain organoids treated with neurotoxic substance 6-OHDA and MPTP in accordance with an embodiment of the present invention.
Fig. 26 is a schematic diagram of an organoid new drug development platform for the construction of HTS drug efficacy and toxicity assessment, according to an embodiment of the present invention.
Detailed Description
Embodiments in accordance with the present invention are further described. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.
In the present specification, "%" used to indicate the concentration of a specific substance, solid/solid is (weight/weight)%, solid/liquid is (weight/volume)%, and liquid/liquid is (volume/volume)%, unless otherwise specified.
Example 1 preparation of organoids
Organoids were prepared as follows.
(day 0) the number of cells was 5X 10 1 、1×10 2 、2.5×10 2 、5×10 2 、1×10 3 、3×10 3 、5×10 3 、7×10 3 、1×10 4 Human embryonic stem cells (human embryonic stem cells; hESCs) in COB1 medium: seeding was performed in U-shaped 96-well plates with 50. Mu. M Y-27632 and 4ng/ml primary fibroblast growth factor (bfgf). (GOB 1: mixture of DMEM/F12 (Corning) and Neurobasal Medium (Gibco) 1:1 including supplements 100 XN 2 supplement (Gibco), 50 XB 27w/o vitamin A (Gibco), 1% penicillin/streptomycin (penicilin/streptomycin; PS) (Gibco), 1% GlutaMAX TM (Gibco), 1% NEAA (non-essential amino acids; gibco), 55. Mu.M. Beta. -mercaptoethanol (mercaptoethanol; gibco), 1. Mu.g/ml heparin (Sigma).
(days 1-4) formation of Embryoid Bodies (EB), COB1 was supplemented with 3. Mu.M CHIR99021, 2. Mu.M dihydrodeoxymorphine (Dorsomorphin), 2. Mu. M A83-01, and 1. Mu.M WNT signaling system inhibitor IWP2. Treatment times for this period were limited to days 2,3 to 4.
(days 4-7) COB1 was supplemented with 3. Mu.M CHIR99021, 2. Mu.M Dorsomorphin, 2. Mu. M A83-01, 1. Mu.MWNT signaling system inhibitor IWP2, 2. Mu.g/ml SAG (static growth factor) and 100ng/ml FGF8 (fibroblast growth factor 8. The medium was changed to day 7, and the treatment time in this period was day 5, day 6 to day 7.
(day 7) Matrigel (Matrigel) was dissolved in ice for about 4 hours. 100ng/ml FGF8, 2. Mu.g/ml SAG, 2.5. Mu.g/ml insulin (insulin) and 200ng/ml Laminin (Lamin) were added to COB1 medium. The solubilized Matrigel (Matrigel) was added to the medium. The organoid medium is then replaced.
(day 9) the Medium was replaced with BMM (basic Mineral Medium) Medium including 10ng/ml Brain-derived neurotrophic factor (BDNF), 10ng/ml Glial cell line-derived neurotrophic factor (GDNF), 200. Mu.M ascorbic acid, and 125. Mu.M cAMP together with COB2 Medium. The medium was changed every two days. (COB 2: mixture of DMEM/F12 (Corning) and Neurobasal Medium (Gibco) 1:1 including supplements 100 XN 2 supplement (Gibco), 50 XB 27w/o vitamin A (Gibco), 1% PS (Gibco), 1% GlutaMAX TM (Gibco), 1% NEAA (Gibco), 55. Mu.M. Beta. -mercaptoethanol (Gibco), 1. Mu.g/ml heparin (Sigma).
Example 2 confirmation of organoid Properties on day 7 of differentiation
2-1. Determination of the survival and quality of organoids
Different initial cell numbers were tested using a High Throughput Screening (HTS) platform (platform), i.e., EB was formed from a 96-well plate, induced midbrain organoid differentiation, which is the first stage for achieving unification of application studies, such as maturation and new drug development. For this purpose, isolated single cell hescs were seeded into 96-well plates at 500, 1,000, 3,000, 5,000, 7,000, and 10,000 per well. Differentiation was observed and the results are shown in FIG. 4. The quality of organoids is divided into: class 1 (developing early Neural rosette), class 2 (light circular EBs) and class 3 (disrupted EBs).
As can be seen from fig. 4, comparing the survival rate and EB quality at day 7, the results showed that the overall quality was better, although the organoid size was slightly smaller, when the numbers of seeded isolated cells were 500, 1,000 and 3,000, respectively; however, when the number of the isolated cells to be seeded is 5,000 or more, the organoids are often classified into class 2. The decision criteria, most excellent in category 1.
2-2. Determination of organoid Gene expression characteristics
To understand organoid gene expression, the fully differentiated functional (Pluripotency) markers include OCT4 and NANOG, the mesendoderm (mesoenderm) markers include EOMES, MXL1 and T, and the neuroectoderm (neuroectoderm) markers include N-CAD, PLZF, SOX1, SOX2 and NESTIN. The expression level of the marker in the organoid was confirmed, and the results are shown in FIGS. 5a to 5 c. The primer sequences used for qPCR are shown in table 1.
TABLE 1
| Serial number | Name (R) | Sequence (5 '→ 3') | |
| 1 | Oct4_ | GACAGGGGGAGGGAGGGAGGCTAGG | |
| 2 | Oct4_ | CTTCCCTCCAACCAGTTGCCCCAAAC | |
| 3 | Nanog _ | TGCAACCTGAAGACGTGTGA | |
| 4 | Nanog _ | CTATGAGGGATGGGAGGA | |
| 5 | Eomes _ | CTCAAAAGGCATGGGAGGGTA | |
| 6 | Eomes _ | CACCACCAAGTCCATCTGCAA | |
| 7 | | ACAACGCTCTTGAACGACGAA | |
| 8 | MXL1R primer | CGGCAACATCAACTGACAAC | |
| 9 | T _ F primer | CGCCTCATAGCCTCATGGAC | |
| 10 | T _ | CACTGGCTGCCACGACAAA | |
| 11 | NCAD- | TGATGAAGAAGGTGGAGGAGAAGA | |
| 12 | NCAD- | ATTCGTCGGATTCCCACAGG | |
| 13 | PLZF-F primers | TCCCGCCCGACTGGAGGATA | |
| 14 | PLZF- | TTCTTTCCTGTGCTCCCCGCTC | |
| 15 | SOX1-F primer | GCCGAGTGGAAGGTCATGTC | |
| 16 | SOX1- | TTCTTGAGCAGCGTCTTGGTC | |
| 17 | SOX2-F primer | AGACTGCACATGAGCCAGACA | |
| 18 | SOX2- | CGTCTCCAGCCAGCTTCAAC | |
| 19 | NESTIN- | AGGAAAAAGACCATCTGCCCG | |
| 20 | NESTIN-R primer | GCCTCTTCAGCCAGAAACCAT |
As can be seen from FIGS. 5a to 5c, the expression pattern of the genes also exhibited a substantially similar pattern. The full differentiation function and the mesendoderm marker are not expressed, only the neuroectoderm marker is expressed, and the normal differentiation of early nerves in the midbrain organoid produced by the HTS platform is confirmed.
Example 3 confirmation of organoid Properties at day 14 of differentiation
3-1. Organoid survival and quality characterization
The day 14 differentiated organoids prepared according to the method of example 1 varied in survival rate and morphology depending on the initial starting cell number. Organoid qualities include class 1 (Bright edge with neural rosette), class 2 (abnormal differentiation), class 3 (covered by abnormal differentiation), and class 4 (deep and dark EB). The results are shown in table 2 and fig. 6.
TABLE 2
| Number of |
500 | 1,000 | 3,000 | 5,000 | 7,000 | 10,000 |
| Class 1 (main) | 30 | 25 | 26 | 16 | 15 | 15 |
| Class 2 (ones) | 0 | 5 | 1 | 1 | 3 | 1 |
| Class 3 (main) | 0 | 0 | 3 | 11 | 12 | 11 |
| Class 4 (main) | 0 | 0 | 0 | 2 | 0 | 3 |
| Is totaled | 30 | 30 | 30 | 30 | 30 | 30 |
As can be seen from table 1 and fig. 6, in the case where the number of cells is more than 5,000, a large number of abnormal differentiation (out-growth) occurs on the surface of the midbrain organoid, but in the case where 500 to 3,000 isolated cells are used, a high-quality midbrain organoid is produced without abnormal differentiation.
3-2. Determining organoid Gene expression profiles
In addition, to understand the expression of genes, apoptosis (Apoptosis) markers include BAX, BAD and PMA1P1, forebrain (Forebrain) markers include FOXGL, LHX2 and SIX3, midbrain (Midbrain) markers include LMX1B, ASCL, TH, and Hindbrain (Hindbrain) markers include HA1, HB4 and HC9. The results are shown in fig. 7a to 7d, respectively, and the primer sequences used in qPCR are shown in table 3.
TABLE 3
As can be seen from fig. 7, only the midbrain marker was specifically expressed in all cases, confirming that the expression of the apoptosis marker was increased as the initial cell number was increased.
3-3. Determination of the Effect of initial cell number on organoid formation
If less than 500 cells are used, the above experiment was performed again using 100, 250 and 500 cells in order to investigate the effect on midbrain organoid formation, and the results are shown in FIGS. 8, 9a to 9 c. The primer sequences used in qPCR are shown in tables 1, 3 and 4.
TABLE 4
| Serial number | Name(s) | Sequence (5 '→ 3') |
| 45 | KROX20-F primer | ACCGCCTCTCTCTCTCTTATT |
| 46 | KROX20-R primer | GGGTAGGCCAGAGAGGAAGA |
As can be seen in fig. 8, the organoids differentiated on day 14 did not differ much in morphology from the organoids differentiated on day 7. However, as can be seen from fig. 9a to 9c, in organoids with cell numbers of 100 and 250, expression of neuroectodermal markers N-CAD, SOX2 and PLZF was found at an early stage (day 7), expression of brain markers LMX1B, ASCL1 and TH was found to be low at day 14, and in contrast, expression of hindbrain markers HC9, HAL, HB4 and KROX20 was increased. Therefore, the above results indicate that the production efficiency of the midbrain organoids is the highest when 500 cells are used.
Effect of initial starting cell number in 3-4.384 well plates on organoid formation
For the mass production of midbrain organoids, production conditions for midbrain organoids based on 384-well plates were confirmed. In order to produce midbrain organoids in 384 well plates (-90. Mu.l) at a smaller volume than 96 well plates (-330. Mu.l), the initial cell numbers were confirmed at 50, 100, 200, 300 and 500, and the results are shown in FIGS. 10a and 10 b.
As can be seen from fig. 10a, the midbrain organoids at day 30 of differentiation showed similar morphology and size regardless of the number of starting cells.
However, as shown in fig. 10B, when the number of initial cells was 50, 100 and 200, the expression of midbrain markers TH, ACSL1 and LMX1B was higher than that of 500. Thus, the above results indicate that if a 384 well plate is used to produce midbrain organoids, the efficiency of using 50, 100 and 200 starting cells is the highest.
3-5, determining the number of organoid-specific mesencephalic dopaminergic neurons
Specific midbrain dopaminergic neurons (mdaneuronon) were observed in midbrain organoids on day 14 of differentiation and the results are shown in fig. 11. As shown in FIG. 11, if 500 initial starting cells were used, the mDA neurons were determined to exist most uniformly by the expression of Tyrosine Hydroxylase (TH).
Example 4 determination of organoid Properties at day 21 of differentiation
4-l. determination of organoid survival and quality
The survival rate and quality of the produced differentiated midbrain organoids on day 21 were investigated according to the method of example 1. Organoids are classified by mass into class 1 (early Neural stem cell development), class 2 (smooth organoids), class 3 (covered with abnormal differentiation), and class 4 (disrupted organoids). The results are shown in table 5 and fig. 12.
TABLE 5
| Number of |
500 | 1,000 | 3,000 | 5,000 | 7,000 | 10,000 |
| Class 1 (main) | 24 | 8 | 0 | 0 | 0 | 0 |
| Class 2 (main) | 6 | 17 | 10 | 10 | 11 | 16 |
| Class 3 (main) | 0 | 5 | 20 | 15 | 14 | 8 |
| Class 4 (main) | 0 | 0 | 0 | 5 | 5 | 6 |
| Total up to | 30 | 30 | 30 | 30 | 30 | 30 |
As shown in table 5 and fig. 12, the possibility of abnormal differentiation was completely reduced when 500 or 1,000 were used, and the formation of a mesencephalon organoid formed of a neuroepithelial cell layer was observed, whereas when 3,000 or more were used, the abnormal differentiation phenomenon was very severe, and only the mesencephalon organoid of low quality was formed.
4-2. Determination of organoid Gene expression characteristics
To understand the expression of the genes, the apoptotic markers BAX, BAD and PMA1P1, the forebrain markers FOXG1, LHX2 and SIX3, the midbrain markers LMX1B, ASCL1 and TH, and the hindbrain markers HA1, HB4 and HC9 were selected. After confirming the expression amount, the results are shown in fig. 13a to 13d, respectively, and the primer sequences used in qPCR are shown in table 3.
FIGS. 13a to 13d show that the expression of apoptosis markers was increased when 3,000 cells were used, and that the expression level in part of midbrain genes was decreased when 3,000 to 10,000 cells were used.
Thus, it was confirmed on the HTS platform that the initial starting cell number plays a very important role in the production of midbrain organoids.
4-3 comparison of organoid sizes
The sizes of the midbrain organoids formed using 500 to 10,000 cells on days 7, 14 and 21 of differentiation were compared and the results are shown in fig. 14a and 14 b.
As shown in fig. 14a and 14b, under conditions of 500 to 10,000, the sizes were all about, which means that 500 to 1,000 initial starting cell numbers were sufficient for midbrain organoid production.
4-4. Determination of the number of organoid-specific mesencephalic dopaminergic neurons
The number of specific mesencephalic dopaminergic neurons was determined in the mesencephalic organoids on day 21 of differentiation and the results are shown in table 6, fig. 15a and 15 b.
TABLE 6
| Number of |
500 | 1,000 | 3,000 | 5,000 | 7,000 | 10,000 |
| Intracellular ratio (%) | 12.6 | 7.0 | 4.9 | 3.0 | 3.9 | 1.4 |
As shown in table 6, fig. 15a and 15b, at 500 and 1,000 cell counts, there were more specific mesencephalic dopaminergic neurons and the neurons were present homogeneously. This indicates that the most suitable initial starting cell number is 500, 1,000 on the HTS platform.
In particular, as shown in FIGS. 16a and 16b, in the case where the initial starting cell number is 1,000 or less, it was confirmed that the phenomenon of "dead nuclei" which is a common problem occurring in organoids was very effectively suppressed.
In addition, as shown in FIG. 17, 500 initial starting cells produced midbrain organoids all containing TH positive mDA neurons, as well as mature A9 type (type) mDA neurons expressing GIRK2 (G protein-activated influx potassium channel protein 2. It was further found that astrocytes (astrocytes) confirmed the presence of oligodendrocytes (oligodendrocytes) and multi-subtype neurons.
4-5 determining organoid morphology
Finally, organoids were produced using 500 cells on a 96, 384-well HTS platform and their morphology was observed using High Content Imaging (HCI) equipment, as shown in FIG. 18.
As can be seen from fig. 19, most of the organoids in the 96-well plate on day 30 of culture had diameters in the range of 0.9 to 1.4mm, and the same midbrain organoids were produced very uniformly. In contrast, the midbrain organoids produced by the conventional method were found to exhibit a very nonuniform morphology and size without using a microplate. Similar results were obtained in other types of human embryonic stem cells or human induced pluripotent stem cells.
As can be seen from fig. 20, the gene expression profile of the midbrain organoids in the 96-well plate produced at high speed and in large scale was confirmed in a single midbrain organoid, and it was observed that more than 90% of the midbrain organoids were better than those produced by the prior art, and the remaining about 10% had gene expression profiles at similar levels to those produced by the prior art. This means that high-speed, large-scale production of midbrain organoids has very uniform properties, with no inter-individual variation. Therefore, the high-speed and large-scale production technology of the midbrain organoids can be used as a standardized technology for industrialization of the midbrain organoids in the future.
Example 5 functional validation of high-speed, mass production of midbrain organoids
To verify the functionality of brain organoids in high-speed, large-scale production, neuromelanogenesis and electrophysiological analysis were performed, and the results are shown in fig. 21 and 22.
As can be seen from fig. 21, in the case of high-speed, large-scale production of midbrain organoids, neural melanin is produced by day 30 of differentiation. Considering that the formation of neural melanin in midbrain organoids produced by a general method is observed in the case where differentiation is induced for more than 100 days, it was confirmed that the midbrain organoids induced to differentiate within 30 days, which are produced at a high speed and in a large scale, have similar functionality to the midbrain organoids produced by a general method for more than several months.
The formation of neural melanin in the midbrain organoids was again confirmed by Fontana-Masson staining at high speed and on a large scale as shown in fig. 22.
Electrophysiological analysis was performed by measuring the firing rate of neural signals as part of functional validation of midbrain organoids in high-speed, large-scale production. As shown in fig. 23, the midbrain organoids produced by the general method were shown to have no electrophysiological activity until day 70 of differentiation, but the high-speed, large-scale production of midbrain organoids showed a marked increase in the neuro-discharge rate from day 20 of differentiation and a sharp increase in the neuro-discharge rate at day 30 of differentiation.
This means that the functional maturation pattern of high-speed, large-scale production of midbrain organoids produced by the present technology is very fast compared to conventional methods, and therefore, functionally mature midbrain organoids can be produced by the present technology on day 30 of differentiation.
Example 6 in vitro drug toxicity evaluation based on high speed, large scale production of midbrain organoids.
In order to verify the possibility of application of midbrain organoids in high-speed and large-scale production as a platform for evaluating drug efficacy and toxicity, evaluation of drug toxicity in vitro was performed on the basis of midbrain organoids in high-speed and large-scale production, and the results are shown in fig. 24 and 25.
Specifically, in midbrain organoids produced at high speed on a large scale, the areas of the midbrain organoids were measured after treatment with 6-OHDA (6-hydroxydopamine; 6-hydroxydopamine) and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine), which are neurotoxic substances that induce apoptosis of dopamine neurons, for 10 days at concentrations of 100, 250 and 500. Mu.M, respectively.
As can be seen from fig. 24, the size of the midbrain organoids decreased according to the concentration of each neurotoxic substance.
As shown in fig. 25, when the mesencephalon organoids were treated with a neurotoxic substance, the expression of apoptosis-related markers increased in proportion to the concentration of the toxic substance, and a decrease in the expression of mesencephalon markers was confirmed. The results show that the midbrain organoids have the utilization potential as a new drug development platform for evaluating the curative effect and toxicity of the in vitro drugs.
In summary, as shown in fig. 26, it is possible to develop various industrial linkage technologies based on the midbrain organoid technology, such as a new drug development platform for evaluating drug efficacy and toxicity, based on the ultra-high speed, mass production, and standardized technology of the midbrain organoids.
Sequence listing
<110> organoid factory Co
<120> midbrain organoid, method for high-speed large-scale production thereof, method for screening neurotoxic substance and method for screening drug for dopaminergic neuron-related disease using the same
<130> P22JM1NN04819CN
<150> KR 10-2020-0079093
<151> 2020-06-29
<150> KR 10-2021-0010149
<151> 2021-01-25
<160> 46
<170> KoPatentIn 3.0
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<213> Artificial sequence
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<223> HC9-R primer
<400> 44
<210> 45
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> KROX20-F primer
<400> 45
<210> 46
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> KROX20-R primer
<400> 46
Claims (36)
1. A high-speed large-scale culture method of a three-dimensional cell mass, which is a method for culturing a three-dimensional cell mass from human stem cells at a high speed, comprising the steps of:
a step of forming an Embryoid body (Embryoid body) from a cell isolated from a human;
inducing differentiation from the embryoid body into a specific tissue to form a three-dimensional cell aggregate; and
a step of inducing maturation of the three-dimensional cell aggregate;
characterized in that the steps from the step of forming the embryoid to the step of inducing the three-dimensional cell aggregate to mature are continuously cultured in the same microplate.
2. The method for high-speed mass culture of a three-dimensional cell aggregate according to claim 1, wherein the cells isolated from human being are one of human embryonic stem cells (hESC), induced pluripotent stem cells (induced pluripotent stem cells) and adult stem cells.
3. The high-speed large-scale culture method of a three-dimensional cell mass according to claim 1, the three-dimensional cell mass being formed within 20 to 50 days.
4. The method for culturing a three-dimensional cell mass at a high speed in a large scale according to claim 1, wherein the three-dimensional cell mass can be cultured for a long period of time after formation thereof by further performing shake culture.
5. The method for high-speed mass culture of a three-dimensional cell aggregate according to claim 1, wherein the microplate is a multi-well plate having a concave portion.
6. The method for high-speed large-scale culture of a three-dimensional cell aggregate according to claim 1, wherein the initial starting cell number of the cells isolated from human is 50 to 3000.
7. The high-speed large-scale culture method of a three-dimensional cell aggregate according to claim 1, which does not use a bioreactor throughout the formation of the three-dimensional cell aggregate.
8. The method for high-speed large-scale culture of the three-dimensional cell mass of claim 1, wherein the three-dimensional cell mass is a midbrain cell mass.
9. The method for high-speed large-scale culture of the three-dimensional cell mass of claim 1, wherein the three-dimensional cell mass is prepared from a digested organ selected from the group consisting of liver, heart and lung.
10. The method for high-speed large-scale culture of a three-dimensional cell aggregate according to claim 1, wherein the step of forming a three-dimensional cell aggregate comprises an ectoderm-forming step or a brain tissue differentiation step.
11. The method for high-speed large-scale culture of a three-dimensional cell aggregate according to claim 10, wherein the ectoderm-forming step is performed in a medium comprising CHIR99021, dihydrodeoxymorphine and a83-01 (dual SMAD inhibitor).
12. The method for high-speed large-scale culture of a three-dimensional cell mass according to claim 10, wherein the midbrain tissue differentiation step is performed in a medium comprising CHIR99021, dihydrodeoxymorphine, A83-01, IWP, SAG and FGF-8 b.
13. The method for high-speed large-scale culture of a three-dimensional cell mass according to claim 1, wherein the step of inducing maturation of the three-dimensional cell mass is performed in a culture medium comprising SAG, FGF-8b, insulin (Insulin), laminin (lamin), and further comprising matrigel that inhibits growth factors.
14. The method for high-speed large-scale culture of a three-dimensional cell mass according to claim 1, wherein in the step of forming the three-dimensional cell mass, no endodermal marker is expressed and an ectodermal formation factor is expressed within 7 days after the induction of tissue differentiation.
15. The method for high-speed large-scale culture of a three-dimensional cell mass according to claim 1, wherein the step of forming the three-dimensional cell mass expresses a mesencephalon marker without expressing a forebrain or hindbrain marker within 14 days after the induction of tissue differentiation.
16. The method for culturing a three-dimensional cell mass according to claim 1, wherein the step of inducing maturation of the three-dimensional cell mass comprises expressing a marker for mesencephalic dopaminergic neurons.
17. The three-dimensional cell mass produced by the high-speed large-scale culture method for the three-dimensional cell mass according to claim 1.
18. A three-dimensional cell aggregate culture kit comprising a concave culture part containing the three-dimensional cell aggregate according to claim 17; and a covering part covering the concave culture part.
19. The three-dimensional cell aggregate culture kit of claim 18, comprising a preservation solution.
20. A three-dimensional mesencephalon cell aggregate which is a three-dimensional mesencephalon cell aggregate cultured from any one of human embryonic stem cells, human induced pluripotent stem cells and adult stem cells, the three-dimensional mesencephalon cell aggregate being a three-dimensional mesencephalon cell aggregate forming neural melanin.
21. The three-dimensional mesencephalon cell aggregate according to claim 20, which is a three-dimensional mesencephalon cell aggregate excluding other brain tissues than the mesencephalon.
22. The three-dimensional mesencephalon cell aggregate according to claim 20, which has an incidence of abnormal differentiation (out growth) of 5% or less.
23. The three-dimensional mesencephalon cell aggregate of claim 20, which has an incidence of deep apoptosis less than 40% of the total area of the cell aggregate.
24. The three-dimensional mesencephalon cell aggregate of claim 20, which is a three-dimensional mesencephalon cell aggregate having a diameter of 0.9 to 1.4mm, which has a uniform size and a uniform morphology.
25. The three-dimensional mesencephalon cell aggregate of claim 20, which comprises greater than 10% mesencephalon dopaminergic neurons.
26. The three-dimensional mesencephalon cell aggregate according to claim 20, which is a three-dimensional mesencephalon cell aggregate in which the variation rate of gene expression is 10% or less, and which is uniformly formed.
27. The three-dimensional mesencephalon cell aggregate of claim 20, which comprises inhibitory nerves and excitatory nerves.
28. The three-dimensional mesencephalon cell aggregate according to claim 20, which is observed in electrophysiological activity from 15 days after cell differentiation.
29. The three-dimensional mesencephalon cell aggregate according to claim 20, which comprises any one selected from the group consisting of: substantia nigra, locus coeruleus, akabane nucleus, central gray matter, internal test mother zone, oculomotor nucleus and pulley nucleus.
30. The three-dimensional mesencephalon cell aggregate of claim 20, which comprises glial cells and oligodendrocytes.
31. A high-speed culture method from human stem cells to three-dimensional cell aggregates comprises the following steps:
a step of forming embryoid bodies from cells isolated from a human;
inducing differentiation from the embryoid body into a specific tissue to form a three-dimensional cell aggregate;
a step of inducing maturation of the three-dimensional cell aggregate;
continuously culturing the embryoid to induce the three-dimensional cell aggregate to mature in the same micropore plate in a non-shaking way from the step of forming the embryoid to the step of inducing the three-dimensional cell aggregate to mature;
the high-speed culture method is characterized by being a high-speed culture method of a three-dimensional cell aggregate which is carried out in an automated manner including a pipette robot, an automatic transfer and a plate transportation means.
32. A method for screening a drug for a dopaminergic neuron related disease, comprising:
a step of forming a three-dimensional cell aggregate from induced pluripotent stem cells derived from a patient with a dopaminergic neuron-related disease;
a step of treating the three-dimensional cell aggregate with a candidate substance;
a step of determining the survival rate of dopaminergic neurons from said three-dimensional cell mass.
33. The method for screening a dopaminergic neuron related disease drug according to claim 32, wherein the dopaminergic neuron related disease is one of parkinson's disease, alzheimer's disease, cerebral hemorrhage, stroke, huntington's disease, pick's disease, creutzfeldt-jakob disease, autism, and brain development disorder syndrome.
34. A method of providing information for treating a dopaminergic neuron related disorder, comprising the steps of:
the method according to claim 1, comprising the step of culturing a three-dimensional cell mass from cells derived from a patient with a dopaminergic neuron-related disease;
a step of treating the three-dimensional cell aggregate with a candidate substance;
a step of determining the survival rate of dopaminergic neurons from the three-dimensional cell mass;
providing appropriate therapeutic drug information to the patient based on the dopaminergic neuron survival rate.
35. The method for discriminating the three-dimensional cell aggregate for drug testing comprises the following steps:
(i) A step of treating the three-dimensional cell mass with a candidate substance:
the step of treating with a candidate substance is carried out in one or more steps included in the method for high-speed large-scale culture of a three-dimensional cell mass according to claim 1; and
(ii) A method for discriminating a three-dimensional cell mass used for a drug test, comprising the step of comparing the responses of the three-dimensional cell mass of each step in the presence and absence of a candidate substance.
36. The method for screening the toxicity of the drugs in vitro by using the three-dimensional cell aggregate comprises the following steps:
(i) The method for high-speed large-scale culture of a three-dimensional cell mass according to claim 1, comprising a step of treating the three-dimensional cell mass with a candidate substance in one or more steps;
(ii) Comparing the reactions of the three-dimensional cell aggregates of the respective steps in the presence and absence of the candidate substance;
(iii) A step of determining whether cells present in the three-dimensional collection of cells die.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2020-0079093 | 2020-06-29 | ||
| KR20200079093 | 2020-06-29 | ||
| KR1020210010149A KR102346243B1 (en) | 2020-06-29 | 2021-01-25 | Midbrain organoids, high-speed and large-scale manufacturing method thereof, neurotoxic screening methods, and drug screening methods for dopaminergic neuron-related diseases |
| KR10-2021-0010149 | 2021-01-25 | ||
| PCT/KR2021/006590 WO2022005023A1 (en) | 2020-06-29 | 2021-05-27 | Midbrain organoid, method for rapidly producing same in large quantities, and method for screening neurotoxins and drug screening method for dopaminergic neuron-associated disease using same |
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| Publication Number | Publication Date |
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| CN115885037A true CN115885037A (en) | 2023-03-31 |
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| CN202180046126.0A Pending CN115885037A (en) | 2020-06-29 | 2021-05-27 | Midbrain organoid, high-speed large-scale production method thereof, method for screening neurotoxic substance and method for screening dopaminergic neuron-related disease drug using the same |
Country Status (3)
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| KR (1) | KR20220001503A (en) |
| CN (1) | CN115885037A (en) |
| WO (1) | WO2022005023A1 (en) |
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| WO2024020587A2 (en) | 2022-07-22 | 2024-01-25 | Tome Biosciences, Inc. | Pleiopluripotent stem cell programmable gene insertion |
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| EP2354217B1 (en) * | 2010-02-03 | 2015-09-02 | Universität Leipzig | Integrated cultivation and measurement device for label-free detection and classification of cellular alterations, in particular for generation and characterisation of cell-spheroids, components and uses thereof |
| IL251855B2 (en) * | 2014-10-24 | 2023-09-01 | Sumitomo Pharma Co Ltd | Production method for nerve tissue |
| LU92845B1 (en) * | 2015-10-08 | 2017-05-02 | Univ Du Luxembourg Campus Belval | Means and methods for generating midbrain organoids |
| SG11201807487WA (en) * | 2016-03-14 | 2018-09-27 | Agency Science Tech & Res | Generation of midbrain-specific organoids from human pluripotent stem cells |
| WO2019066059A1 (en) * | 2017-09-29 | 2019-04-04 | 国立大学法人東京医科歯科大学 | Organ organoid and method for producing same |
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- 2021-05-27 CN CN202180046126.0A patent/CN115885037A/en active Pending
- 2021-05-27 WO PCT/KR2021/006590 patent/WO2022005023A1/en active Application Filing
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| WO2022005023A1 (en) | 2022-01-06 |
| KR20220001503A (en) | 2022-01-05 |
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