WO2018190305A1 - Method for producing differentiated cell spheroids - Google Patents

Abstract

Provided is a method for producing differentiated cell spheroids having almost uniform sizes and high purity from stem cells. A method for producing differentiated cell spheroids by differentiating stem cells in the presence of a differentiation inducing factor, said method being characterized in that, after the formation of the spheroids, each of the spheroids is disaggregated into smaller spheroids or single cells and then the smaller spheroids or the single cells are reaggregated at an arbitrary time point.

Description

Method for producing differentiated cell spheroids

The present invention relates to a method for producing a large amount of differentiated cell spheroids (cell aggregates) of substantially uniform size and high purity from stem cells, a kit for producing the spheroids, and a test using the spheroids The present invention relates to a method for evaluating a drug efficacy or safety of a substance, and a method for screening a substance having a target activity using the spheroid.

In drug discovery research, the accuracy of predicting effects in clinical trials based on the results of nonclinical trials is very important. If a compound that was highly effective in non-clinical trials did not show sufficient efficacy in clinical trials, or if a compound that had no safety problems in non-clinical trials showed strong toxicity in clinical trials, Enormous development costs up to will be wasted. Therefore, there is a need for a test tool that can more accurately reflect the results of clinical trials.

In recent years, spheroid culture in which cells are cultured and aggregated three-dimensionally is attracting attention instead of monolayer culture in which cells are cultured two-dimensionally. Compared to monolayer culture, spheroid culture can construct a state closer to cells in a living body, and can extract specific functions that cells have in vivo. For this reason, spheroids are expected to be useful tools in drug discovery research, particularly in screening for compounds having the desired drug efficacy and in drug efficacy evaluation or safety assessment in non-clinical studies. For example, Patent Document 1 describes the use of spheroids to further increase drug sensitivity when evaluating drug efficacy or toxicity.

In order to obtain scientifically reliable test results, it is preferable to perform the test with a statistically reliable number of samples. For this reason, when using spheroids for non-clinical studies, it is important to be able to prepare a large amount of spheroids having a certain size and quality. For example, in Patent Document 2, there are a plurality of indentations forming a compartment in which a culture object is cultured and a bank portion interposed between adjacent indentations on the upper surface of the plate-shaped culture substrate. It is disclosed that spheroids having a uniform size can be produced in large quantities by using a culture substrate having a continuous curved surface of the matching bank portion and the hollow portion as a culture vessel.

Evaluation of drug efficacy and the like is preferably performed using cells of a tissue in which the target drug actually acts, but cells collected from animals are greatly affected by individual differences, and generally differentiated cells proliferate. Since the ability is lost, it is difficult to prepare a large number of cells having the same characteristics. On the other hand, using cultured cell lines, it is possible to prepare a large number of cells with the same characteristics, but because cultured cells are considerably altered during the process of establishment, prediction of clinical trial results from the results of cultured cells Sex is not so high. Therefore, in recent years, cells differentiated from stem cells have been used for drug discovery research. Stem cells can be prepared in large quantities while maintaining homogeneous cells for a long period of time because of their proliferative ability. In addition, differentiated cells having the same characteristics can be stably supplied by performing constant differentiation induction. For example, in Patent Document 3, a planar culture of mature hepatocyte-like cells differentiated from mesenchymal stem cells is used for screening for hepatitis C virus infection inhibitor or hepatitis C virus growth inhibitor. It is described. Patent Document 4 discloses that a retinal layer-specific nerve cell differentiated from a pluripotent stem cell is used as a toxicity evaluation reagent or a drug effect evaluation reagent for a therapeutic agent candidate compound for a disease caused by a disorder of a retinal layer-specific nerve cell. The use is described.

Special table 2016-136848 Japanese Patent No. 5921437 JP 2009-153383 A JP 2013-128477 A

Spheroids of mature cells with the desired function differentiated from stem cells are expected to be useful tools for drug efficacy evaluation or safety evaluation in drug discovery research. However, when stem cells are differentiated in a spheroid state, there is a problem that the types and maturity (differentiation degrees) of the cells constituting the spheroids vary greatly and the purity of the cells is low. In addition, there is a problem that the spheroids vary greatly and the reliability as an evaluation tool is low.

An object of the present invention is to provide a high-quality differentiated cell that is substantially uniform in size and satisfies the four items of viability, maturity, and purity in order to provide a high-function spheroid from a stem cell. A method for producing a spheroid, a method for evaluating the efficacy or safety of a substance using the spheroid produced by the method, a method for screening a substance having a desired activity using the spheroid, and the method To provide a kit for producing spheroids.

In the method for producing a differentiated cell spheroid by differentiating stem cell spheroids in the presence of a differentiation-inducing factor, the present inventors reaggregated after disaggregating the spheroid at any time after spheroid formation. By forming a spheroid, the present inventors have found that spheroids of differentiated cells having almost uniform size and high survival rate, maturity, and purity can be produced, and the present invention has been completed.

That is, the present invention provides the following [1] to [19].
[1] In a method for producing a differentiated cell spheroid by differentiating a stem cell in the presence of a differentiation-inducing factor,
A method for producing a differentiated cell spheroid, characterized in that the spheroid is disaggregated into smaller spheroids or single cells at any time after spheroid formation, and then reaggregated.
[2] The method for producing a differentiated cell spheroid according to [1], wherein stem cells are sequentially cultured in a medium containing a differentiation-inducing factor and differentiated into a target differentiated cell spheroid via a precursor cell spheroid.
[3] The method for producing a differentiated cell spheroid according to [1] or [2], wherein spheroid formation and reaggregation are performed in a culture container for spheroid formation.
[4] After preparing a cell suspension made into smaller spheroids or single cells generated by the disaggregation, the ratio of living cells to the whole cells in the cell suspension is measured,
When the proportion of the living cells is 90% or more, the cell suspension is reaggregated as it is in a spheroid-forming culture vessel,
When the ratio of the living cells is less than 90%, dead cells are removed from the cell suspension and the ratio of the living cells is adjusted to 90% or more, and then reconstituted in the spheroid-forming culture container. Agglomerate,
The method for producing a differentiated cell spheroid according to any one of [1] to [3].
[5] The method for producing a differentiated cell spheroid according to any one of [1] to [4], wherein smaller spheroids or single cells generated by the disaggregation are reaggregated in the presence of a differentiation-inducing factor.
[6] The disaggregation and reaggregation of the spheroid is performed at the time when the precursor cell spheroid is transferred to a medium containing a differentiation inducing factor for differentiating the precursor cell into a target differentiated cell, or the differentiated cell spheroid The method for producing a differentiated cell spheroid according to any one of the above [2] to [5], which is carried out during the culture of [2].
[7] The method for producing a differentiated cell spheroid according to any one of [1] to [6], wherein the stem cell is an embryonic stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a Muse cell, or a mesenchymal stem cell.
[8] The method for producing a differentiated cell spheroid according to any one of [1] to [7], wherein the differentiated cells are cardiomyocytes, neurons, or hepatocytes.
[9] The stem cell is an embryonic stem cell or an induced pluripotent stem cell,
The stem cells are cultured in a medium containing one or more differentiation-inducing factors that cause embryonic stem cells or induced pluripotent stem cells to differentiate into any germ layer of the three germ layers. After forming germ layer spheroids,
The formed early germ layer spheroid is cultured in a medium containing one or more differentiation-inducing factors for differentiating one of the three germ layers into a precursor cell of the target differentiated cell, and the precursor cell of the target differentiated cell Of spheroids,
Further, the formed precursor cell spheroids are cultured in a medium containing one or more differentiation inducers for differentiating the precursor cells into target differentiated cells to form spheroids of the target differentiated cells. [8] The method for producing a differentiated cell spheroid according to any one of [8].
[10] The progenitor cells are cardiac progenitor cells, and the differentiated cells are cardiomyocytes,
The differentiation-inducing factor that differentiates any germ layer of the three germ layers into a precursor cell of a target differentiated cell is one or more Wnt signal activators,
The differentiation-inducing factor for differentiating any germ layer of the three germ layers into a precursor cell of a target differentiated cell is one or more Wnt signal inhibitors,
[9] The method for producing a differentiated cell spheroid according to [9] above, wherein the differentiation inducing factors for differentiating the progenitor cells into target differentiated cells are vascular endothelial growth factor and basic fibroblast growth factor.
[11] Any one of the above [1] to [10], which produces a differentiated cell spheroid to be used for an evaluation test of a drug efficacy or safety of a test substance or to be used for screening a substance having a target activity Of producing differentiated cell spheroids.
[12] After producing differentiated cell spheroids by the method for producing differentiated cell spheroids according to any one of [1] to [11] above, using the produced differentiated cell spheroids, evaluation of the drug efficacy or safety of the test substance A method for evaluating a test substance.
[13] After producing differentiated cell spheroids by the method for producing differentiated cell spheroids according to any one of [1] to [11], screening of substances having the desired activity is performed using the produced differentiated cell spheroids. , Screening method of active substance.
[14] The inner bottom surface of the container has a plurality of indentations forming a compartment in which the culture object is cultured, and a bank portion interposed between the adjacent indentations, and the adjacent bank portion and the indentation portion are adjacent to each other. Is a continuous curved surface, and the spheroid-forming culture container in which the inner surface of the recess is coated with a cell adhesion inhibitor;
A differentiation-inducing factor for differentiating stem cells;
A kit for producing a differentiated cell spheroid, comprising:
[15] The differentiation-inducing factor that differentiates an embryonic stem cell or an induced pluripotent stem cell into any one of the three germ layers, and a differentiated cell intended for any one of the three germ layers [14] The kit for producing a differentiated cell spheroid according to [14], comprising a differentiation inducing factor for differentiating the progenitor cells into differentiation and a differentiation inducing factor for differentiating the precursor cells into the differentiated cells.

By using the method for producing a differentiated cell spheroid according to the present invention, it is possible to stably supply a sufficient amount of a differentiated cell spheroid having a substantially uniform size and a high purity with any size. For this reason, this manufacturing method is very useful as a tool used for screening drug candidate compounds in drug discovery research, and evaluating drug efficacy or safety in non-clinical studies.

In Reference Example 1, the particle size distribution of spheroids formed in EZSPHEREHER # 4000-900. In Reference Example 1, the particle size distribution of spheroids formed in EZSPHEREHER # 4000-905. In Example 1, it is the figure which showed the measurement result of the relative expression level of Nkx2-5 with respect to the expression level of GAPDH of each myocardial mature cell spheroid. In Example 1, it is the figure which showed the measurement result of the relative expression level of TNNT2 with respect to the expression level of GAPDH of each myocardial mature cell spheroid. In Example 1, it is the figure which showed the measurement result of the relative expression level of MYL7 with respect to the expression level of GAPDH of each myocardial mature cell spheroid. In Example 1, it is the figure which showed the measurement result of the relative expression level of MYL2 with respect to the expression level of GAPDH of each myocardial mature cell spheroid. In Example 1, it is the figure which showed the measurement result of the relative expression level of HCN4 with respect to the expression level of GAPDH of each myocardial mature cell spheroid. In Example 2, it is the figure which showed the measurement result of TD20, TD50, and TD90 after E-4031 process of 2D (planar culture | cultivation) iPS origin myocardial mature cell. In Example 2, it is the figure which showed the measurement result of TD20, TD50, and TD90 after E-4031 treatment of the myocardial mature cell spheroid. In Example 2, it is the figure which showed the measurement result of TD20, TD50, and TD90 after E-4031 process of myocardial mature cell / NHCF spheroid.

The method for producing a differentiated cell spheroid according to the present invention (hereinafter sometimes referred to as “the spheroid production method of the present invention”) produces a spheroid of a differentiated cell differentiated from a stem cell to a cell having a target function. Is the method. In the present invention and the specification of the present application, the “target differentiated cell” is a target differentiated cell produced by the spheroid production method of the present invention. The target differentiated cells may be any cells that have been differentiated to the extent that they have the desired function, and are not limited to cells that have been matured to the final stage of differentiation (final differentiated mature cells).

The target differentiated cell in the present invention may be a cell that differentiates and matures from the ectoderm, a cell that differentiates and matures from the mesoderm, or a cell that differentiates and matures from the endoderm. Specific examples of the differentiated cells in the present invention include, for example, nerve cells, lens cells, retinal pigment epithelial cells, corneal epithelial cells, corneal endothelial cells, conjunctival epithelial cells, lacrimal gland cells, neuroretinal cells, glial cells, Pigment cells, corneal endothelial cells, melanocytes, olfactory epithelial cells, osteoblasts, chondrocytes, cardiomyocytes, vascular endothelial cells, vascular wall cells, vascular smooth muscle cells, blood cells, hepatocytes, bile duct epithelial cells, kidney cells, Pancreatic β cells, enterocytes, goblet cells, enteroendocrine cells and the like can be mentioned. The target differentiated cells in the present invention are preferably cardiomyocytes, nerve cells, or hepatocytes.

In the present invention and the present specification, a stem cell means a cell having differentiation ability and self-replication ability. Stem cells used in the spheroid production method of the present invention may be embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), or somatic stem cells. As the somatic stem cells, mesenchymal stem cells, Muse cells or hematopoietic stem cells are preferable. Moreover, the biological species from which the stem cells used are derived is not particularly limited. The stem cells used in the present invention are preferably stem cells derived from animals, more preferably stem cells derived from mammals, further preferably stem cells derived from primates, and particularly preferably stem cells derived from humans.

In the spheroid production method of the present invention, spheroid formation and reaggregation are preferably performed in a spheroid-forming culture vessel. As a culture container for spheroid formation, any of various culture containers used for spheroid formation may be used.

In the present invention and the present specification, the spheroid-forming culture container means a culture container in which spheroids are formed by culturing with a suspension of cells constituting the spheroids.

The spheroid-forming culture container used in the spheroid production method of the present invention includes a plurality of indentations forming a compartment in which the culture object is cultured on the inner bottom surface of the container, and intervening between adjacent indentations. A culture vessel in which the adjacent bank portion and the dent portion are continuous curved surfaces, and the inner surface of the dent portion is coated with a cell adhesion inhibitor (hereinafter referred to as a “spheroid-forming culture vessel”). A ”) is preferred. When the cell suspension is poured into the spheroid-forming culture vessel A, the cells that have entered the recess are aggregated to form spheroids. Since the size of the spheroids formed depends on the number of cells that have entered the depressions, the shape and size of the depressions can be determined within one spheroid-forming culture container by aligning the size of the depressions. Many spheroids having a size corresponding to the height can be formed. In the spheroid-forming culture container A, the inner surface of the depression is coated with a cell adhesion inhibitor, and the adjacent bank and depression are continuous curved surfaces, and there is no flat part. It is possible to suppress the formation of spheroids having a random size that is not affected by the size of the cells and the size of the depressions, and the uniformity of the size is improved. That is, in the method for producing spheroids of the present invention, by using the spheroid-forming culture vessel A during spheroid formation, a sufficient amount of spheroids of differentiated cells having a substantially uniform size can be supplied.

Further, in the spheroid-forming culture vessel A, cells accumulate at the bottom of the dent, and the cells tend to aggregate, so that spheroids can be formed quickly. For many cells, the free state (single cell state) is stress and is likely to be damaged. However, by using the spheroid-forming culture vessel A, the time of the single cell state is short, and the damage to the cells is reduced. As a result, the cell viability can be increased. The “spheroid cell viability” means the ratio of living cells to all cells constituting the spheroids.

When the number of cells constituting the spheroid is too small, there is a possibility that a desired physiological function may not be generated, and there is a possibility that variation between spheroids may increase in response to a drug or the like. In addition, if the number of cells constituting the spheroid is too large, the central part of the spheroid will be necrotic, or the differentiation of the cells constituting the spheroid will not be induced by differentiation-inducing factors. Becomes larger. When forming spheroids in the method for producing spheroids of the present invention, it is preferable to adjust the number of cells constituting the spheroids within an appropriate range in consideration of these.

For example, the size of the recess of the spheroid-forming culture container A is 20 μm or more and 1500 μm or less in diameter (when the shape of the opening is approximately elliptical, it indicates the long diameter), and the depth is 10 μm or more and 1500 μm or less. More preferably, the diameter is 200 μm or more and 1400 μm or less, the depth is 100 μm or more and 400 μm or less, and the diameter is 400 μm or more and 1000 μm or less, and the depth is 100 μm or more and 400 μm or less. When the diameter of the dent is within the range, the number of cells constituting the spheroid can be adjusted within an appropriate range. Moreover, it can suppress effectively that the formed spheroid jumps out of a hollow part at the time of culture | cultivation or a culture medium exchange, and spheroid aggregates because the depth of a hollow part is in this range.

In the spheroid-forming culture vessel A, the number of depressions formed on the inner bottom surface of the vessel is preferably 10 / cm ² to 10,000 / cm ² , and 20 / cm ² to 8000 / cm ^2. More preferably, 20 pieces / cm ² to 3000 pieces / cm ² are further preferred. By providing a large number of depressions per one spheroid-forming culture container, a large number of spheroids can be formed in one spheroid-forming culture container. In addition, by forming small spheroids at high density, high quality spheroids with higher survival rate and maturity can be easily obtained.

The spheroid-forming culture container A can be formed, for example, by irradiating the inner bottom surface of the spheroid-forming culture container made of a synthetic resin such as polystyrene with laser light. When the laser beam is irradiated, the synthetic resin material that constitutes the inner bottom surface of the container is dissolved to form a recess. Further, the melted synthetic resin material rises around the opening of the recess to form a bank portion. By adjusting the irradiation conditions such as the laser beam irradiation position and output amount, the distance between adjacent recesses, the diameter and depth of the recesses, the width and height of the bank, etc. can be adjusted. A hollow part and a bank part can be formed so that a flat surface does not remain between the parts. In the culture container for spheroid formation produced by the production method, the shape of the laser light irradiation spot is circular, whereas the opening shape of the depression is flattened to be substantially elliptical.

In the spheroid-forming culture container A, the inner surface of the recess is coated with a cell adhesion inhibitor. The cell adhesion inhibitor plays a role of inhibiting cells from adhering to the inner bottom surface of the container, particularly the inner surface of the recess. As the cell adhesion inhibitor, for example, phospholipid polymer, polyhydroxyethyl methacrylate, polyethylene glycol or the like is used.

As the spheroid-forming culture vessel A, for example, the culture substrate described in Patent Document 2 can be used. Moreover, among commercially available culture containers, for example, “EZSPHERE” (manufactured by AGC Techno Glass Co., Ltd.) or the like is used.

The spheroid production method of the present invention is a method for producing a differentiated cell spheroid by differentiating stem cells in the presence of a differentiation-inducing factor, and the spheroid is converted into a smaller spheroid or a single spheroid at any time after spheroid formation. It is characterized by reaggregating after disaggregating into one cell.

When stem cells are treated with a differentiation-inducing factor, the finally obtained spheroids contain not only target differentiated cells but also undifferentiated cells and cells that have undergone differentiation other than the desired differentiation. End up. The low purity of this spheroid (the proportion of the differentiated cells of interest in the cells that make up the spheroids) results in low data reliability and low reproducibility when spheroids of differentiated cells are used in various tests. linked.

On the other hand, in the spheroid production method of the present invention, the purity can be increased by deaggregating and separating the formed spheroids and then reaggregating them. In general, cells of the same type are more likely to aggregate than cells of different types, and at the time of reaggregation, the target cells aggregate preferentially and spheroids are formed.

The deaggregation process is a process for converting spheroids into smaller spheroids or single cells. In the disaggregation treatment, it is not necessary to completely separate the cells, and small aggregates may be formed. In the present invention, since the effect of the reaggregation treatment can be obtained, it is preferable to disperse most cells into single cells, and it is more preferable to disperse almost all cells into single cells. preferable.

After disaggregating the spheroids to be treated, the obtained single cells or smaller spheroids are reaggregated. The reaggregation treatment is preferably performed by culturing in the spheroid-forming culture vessel, and more preferably by culturing in the spheroid-forming culture vessel A.

In the reaggregation treatment, reaggregation is preferably performed in a state where the proportion of living cells is high in order to form spheroids with a higher survival rate. For example, after preparing a cell suspension in which spheroids are smaller spheroids or single cells, the ratio of living cells to total cells in the cell suspension is measured. When the ratio of viable cells in the cell suspension is 90% or more, the cell suspension is dispensed and cultured as it is in a spheroid-forming culture vessel to form spheroids. On the other hand, when the proportion of viable cells in the cell suspension is less than 90%, dead cells are removed from the cell suspension, in other words, by selectively recovering live cells, It is preferable to adjust the ratio to 90% or more and then dispense and culture in a spheroid-forming culture vessel to form spheroids. In the determination of the viability of cells, for example, the ratio of viable cells in a cell suspension is examined using a reagent or the like that specifically stains dead cells. For cell suspensions with a low percentage of living cells, centrifugation can be used to precipitate live cells with dead cells suspended in the upper sperm. Enhanced.

In the re-aggregation treatment, high-quality spheroids with higher maturity are likely to be formed. Therefore, the number of cells seeded per depression of the spheroid-forming culture container is adjusted to be within an appropriate range. It is preferable to do. In the present invention, the cells of the cells prepared so that the number of cells seeded per one depression is about 100 to 3000, preferably about 200 to 2000, more preferably about 500 to 1000. The suspension is preferably seeded in a culture container for spheroid formation. The degree of maturity means the degree to which the differentiated cells obtained from stem cells are close to the corresponding differentiated cells in the adult body.

The spheroid disaggregation / reaggregation treatment is preferably performed in the presence of a differentiation-inducing factor. By reaggregating smaller spheroids or single cells generated by disaggregation in the presence of differentiation-inducing factors, sufficient differentiation-inducing factors can be applied not only to cells near the surface of spheroids but also to cells that were present inside. It becomes easy to arrange the maturity of the cells constituting the spheroid.

Stem cells are sequentially cultured in a medium containing a differentiation-inducing factor, and are differentiated into target differentiated cell spheroids via precursor cell spheroids. In the present invention, spheroid disaggregation / reaggregation treatment can be performed at any point in the process of differentiation from a stem cell to a target differentiated cell. However, the free state (single cell state) may be stressful for the cells. Therefore, in the spheroid production method of the present invention, the deaggregation / reaggregation treatment is preferably performed twice or less throughout the entire process, and more preferably performed only once.

In the spheroid production method of the present invention, spheroid disaggregation and reaggregation are performed at the time when the precursor cell spheroid is transferred to a medium containing a differentiation inducing factor for differentiating the precursor cell into a target differentiated cell, or It is particularly preferred to carry out during the culture of the differentiated cell spheroids. By performing spheroid disaggregation / reaggregation treatment in a medium containing a differentiation inducing factor for differentiating progenitor cells into target differentiated cells, the differentiation inducing factor is also present in cells that were present inside the spheroid. In addition to being able to contact sufficiently, it is easy to make the maturity of the cells constituting the spheroid easy, and in addition, the proportion of cells matured to the desired differentiated cells in the entire cells constituting the spheroid can be improved. In this way, the spheroid disaggregation / reaggregation process is performed only once in the differentiation process from the stem cell to the target differentiated cell, in the differentiation stage to the target differentiated cell, which is the final stage of differentiation. The spheroids of the target cells with high purity are more easily formed without applying excessive stress to the cells.

The method for producing a spheroid of the present invention comprises differentiating a stem cell in a spheroid state to a target differentiated cell, and disaggregation / reaggregation treatment at any differentiation stage for differentiation into a target differentiated cell, preferably at a final differentiation stage. Except for the step, it can be carried out in the same manner as a known method for differentiating a stem cell into a desired differentiated cell, or by appropriately modifying the known method.

For example, when the stem cells are ES cells or iPS cells, the stem cells are cultured to form spheroids of any of the three germ layers, and then the formed early germ layer spheroids (embryoid bodies) are cultured. Then, spheroids of the target differentiated cell precursor cells are formed, and the formed precursor cell spheroids are further cultured to form the target differentiated cell spheroids. At any point in the process of differentiating the embryoid body into a precursor cell of the target differentiated cell and further differentiating the precursor cell into the target differentiated cell, the spheroid is subjected to disaggregation / reaggregation treatment at least once. Do.

In order to form embryoid bodies from stem cells, the stem cells are cultured in a medium containing one or more differentiation-inducing factors that differentiate embryonic stem cells or induced pluripotent stem cells into any one of the three germ layers. To do. Similarly, in order to form a spheroid of a precursor cell of a target differentiated cell from an embryoid body, the embryoid body is differentiated from one of the three germ layers to a precursor cell of the target differentiated cell 1 Incubate in a medium containing a differentiation-inducing factor of more than one species. In order to form a spheroid of a target differentiated cell from a precursor cell spheroid, the precursor cell spheroid is cultured in a medium containing one or more differentiation inducers that differentiate the precursor cell into a target differentiated cell. Hereinafter, a differentiation-inducing factor that differentiates a stem cell into an embryonic stem cell or an induced pluripotent stem cell into any one of the three germ layers is sometimes referred to as a “first differentiation-inducing factor”. One or more differentiation-inducing factors that cause the three germ layers to differentiate into progenitor cells of the target differentiated cells may be referred to as “second differentiation-inducing factors”. One or more differentiation-inducing factors that cause the precursor cells to differentiate into target differentiated cells may be referred to as “third differentiation-inducing factors”.

In order to differentiate from a stem cell to a differentiated cell having a desired function, it is important to contact an appropriate differentiation-inducing factor with the cell at an appropriate timing. In addition, differentiation from stem cells into various mature cells by a differentiation-inducing factor is disclosed in a number of documents including Patent Document 3 and the like. The first differentiation-inducing factor, the second differentiation-inducing factor, and the third differentiation-inducing factor used in the present invention are described with reference to differentiation methods using differentiation-inducing factors described in various literatures and methods obtained by modifying them. In consideration of the type of stem cell to be used, the type of target differentiated cell, and the like, it can be appropriately determined from a wide variety of differentiation inducing factors.

For example, when the target differentiated cells are cardiomyocytes, one or more Wnt signal activators can be used as the first differentiation inducer, and one or more kinds of the second differentiation inducer can be used. A Wnt signal inhibitor can be used, and several growth factors are used as the third differentiation inducer. Examples of the Wnt signal activator include CHIR99021 (selective GSK-3 inhibitor, CAS No: 252917-06-9), BMP4 (bone morphogenetic factor 4), and activin A. Examples of Wnt signal inhibitors include IWR1 (CAS No: 1127442-82-3), IWP2 (CAS No: 687770-61-6), IWP4 (CAS No: 686772-17-8), and XAV939 (CAS: 284028). -89-3) and Dkk-1 (Dickkopf1). Furthermore, examples of the growth factor used as the third differentiation-inducing factor include VEGF (vascular endothelial cell growth factor), bFGF (basic fibroblast growth factor), and BMP4. By appropriately adjusting these differentiation-inducing factors, cardiomyocyte subtypes such as ventricular cells, atrial cells and pacemaker cells can be selectively matured.

As the medium used in the present invention, a basal medium that is a nutrient medium not containing a differentiation-inducing factor and a target differentiation-inducing factor added thereto are used. As the basal medium, generally, a medium used for maintaining or growing stem cells or a medium used for culturing animal cells is used. Examples of the medium include Eagle's minimum essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), Eagle's minimum essential medium α-modified (MEM-α), mesenchymal cell basal medium (MSCBM), Ham's F -12 medium, Ham's F-10 medium, DMEM / F12 medium, Williams medium E, RPMI-1640 medium, MCDB medium, 199 medium, Fisher medium, Iscove modified Dulbecco medium (IMDM), McCoy modified medium, etc. . You may add an amino acid, inorganic salts, vitamins, antibiotics, etc. to these culture media as needed. In addition, commercially available culture media for various stem cells can also be used. The basal medium used is a step of forming embryoid bodies from stem cells (early germ layer spheroid formation step), a step of differentiating embryoid bodies into precursor cells of target differentiated cells (progenitor cell spheroid formation step), and It may be common in each process of the process (differentiated cell spheroid formation process) which differentiates this progenitor cell to the target differentiated cell, and may be changed.

Culture conditions other than the composition of the culture medium in each step can be generally culture conditions for culturing animal cells, and may be appropriately modified as necessary. For example, the culture can be performed at a culture temperature of 30 to 40 ° C., a CO ₂ concentration of 1 to 10% by volume, and an O ₂ concentration of 0.1 to 25% by volume. The conditions of temperature, CO ₂ concentration, and O ₂ concentration may be common in each step or may be changed.

First, in the early germ layer spheroid formation step, the cell suspension of stem cells is dispensed into the aforementioned specific spheroid formation culture vessel and cultured in a medium containing the first differentiation-inducing factor. When the cell suspension is dispensed into a culture vessel and cultured for several hours, an embryoid body is formed, then further differentiated, and according to induction by the first differentiation-inducing factor used, the ectoderm, mesoderm, or inner A germ layer is formed.

In the early germ layer spheroid formation step, a cell suspension of stem cells is prepared in a basal medium that does not contain the first differentiation-inducing factor or a medium in which only a part of the first differentiation-inducing factor is added to the basal medium. After the body is formed, the remaining first differentiation inducing factor can be added. A cell suspension of pluripotent stem cells may be prepared in a medium in which all of the first differentiation-inducing factors are added to a basal medium and dispensed into the culture container for spheroid formation.

When the spheroid-forming culture vessel A is used as a spheroid-forming culture vessel, in order to form spheroids of an appropriate size that can obtain a desired physiological function when differentiated into target differentiated cells, It is preferable to adjust the cell suspension so that the number of cells seeded per one depression of the spheroid-forming culture vessel A is within an appropriate range. In the present invention, stem cell cells prepared so that the number of stem cells seeded per one depression is about 100 to 3000, preferably about 150 to 2000, more preferably about 200 to 1000. The suspension is preferably seeded in the spheroid-forming culture vessel A.

In order to form spheroids with higher survival rate and homogeneous characteristics, the cell suspension of stem cells dispensed into the spheroid-forming culture vessel has a ratio of viable cells to the whole cells in the cell suspension of 90. % Or more is preferable. The ratio of the living cells in the cell suspension is determined by staining with a reagent or the like that specifically stains dead cells and measuring the ratio of dead cells to the whole cells. When the ratio of living cells in the prepared cell suspension of stem cells is less than 90%, the ratio of living cells is adjusted to 90% or more by removing dead cells or the like, and then a spheroid-forming culture container It is preferable to sow.

Then, in the precursor cell spheroid formation step, the formed ectodermal embryoid body, mesoderm embryoid body, or endoderm embryoid body is differentiated into a target differentiated cell precursor cell. At this time, the embryoid body may be continuously cultured and differentiated in the same spheroid-forming culture vessel as in the early germ layer spheroid formation step, or transferred to a low cell adhesion plate culture vessel to differentiate into progenitor cells. You may let them.

The low cell adhesion plate culture container is a culture container having a flat bottom surface used generally for cell culture, and the bottom surface of the container is coated with the cell adhesion inhibitor. Specifically, the embryoid body formed from the spheroid-forming culture vessel is collected in a tube or the like, washed with phosphate physiological saline or the like as necessary, and then the second differentiation inducing factor is added to the basal medium. Suspension is added so that the spheroid structure is not impaired. The obtained spheroid suspension is dispensed into a low cell adhesion plate culture container and cultured to differentiate the cells constituting the spheroid into progenitor cells.

By performing the progenitor cell spheroid formation process in the culture container for spheroid formation in the same manner as in the early germ layer spheroid formation process, adhesion between adjacent spheroids is effectively suppressed, and the spheroid size uniformity is maintained. It's easy to do. However, the specific spheroid-forming culture container has a large number of depressions and banks formed on the bottom of the container, so that it is difficult to change the culture medium. In particular, when changing from a medium containing the first differentiation-inducing factor to a medium containing the second differentiation-inducing factor, if the medium containing the first differentiation-inducing factor remains, the second differentiation-inducing factor is There is a possibility that differentiation induction is not performed properly due to dilution. Also, even during differentiation induction, it is preferable to change the medium every few days, but if the medium change is not successful, the nutrient state in the medium can be biased, and the size of the spheroids formed in the container The homogeneity of thickness and characteristics may be impaired. By performing the treatment with the second differentiation-inducing factor in a plate culture vessel, sufficient differentiation induction and nutrition can be given to all spheroids.

Differentiation from an embryoid body into a precursor cell of a target differentiated cell may be performed through two or more differentiation phases. For example, the embryoid body is transferred into a plate culture vessel with low cell adhesion, cultured for several days in a medium containing a specific combination of differentiation-inducing factors, and then replaced with a medium containing another combination of differentiation-inducing factors, By culturing for several days, it can be differentiated into progenitor cells. A series of differentiation induction processes from embryoid bodies to progenitor cells are included in the progenitor cell spheroid formation step.

Then, as a differentiated cell spheroid formation step, spheroids of progenitor cells are cultured in a medium in which a third differentiation inducer is added to a basal medium to form spheroids of the desired differentiated cells. Whether or not the target differentiated cell has been differentiated and matured can be confirmed by examining the expression of the marker of the differentiated cell.

In the present invention, the disaggregation / reaggregation treatment may be performed on spheroids at an arbitrary time after the initial germ layer spheroid formation step. For example, after the progenitor spheroid formation step, the embryoid bodies formed in the early germ layer spheroid formation step are disaggregated and then cultured in a medium containing a second differentiation-inducing factor in a spheroid formation culture vessel. May be re-aggregated to form spheroids, and after the spheroids are disaggregated during the precursor cell spheroid formation step, the medium containing the second differentiation-inducing factor in the spheroid-forming culture vessel And may be reaggregated to form spheroids. In addition, before the differentiated cell spheroid formation step, the spheroids formed in the precursor cell spheroid formation step are disaggregated, and then cultured in a medium containing a third differentiation inducer in a culture container for spheroid formation. The spheroids may be aggregated to form a spheroid, and after the spheroids are disaggregated in the middle of the differentiated cell spheroid formation step, the medium containing the third differentiation-inducing factor described later in the culture container for spheroid formation And may be reaggregated to form spheroids.

When the spheroid disaggregation / reaggregation treatment is performed in the middle of the differentiated cell spheroid formation step, after the precursor cell spheroid formation step, the medium of the plate culture container in which the precursor cell spheroid is formed is used as the third differentiation. The medium is exchanged with a medium containing an inducer and cultured to induce differentiation into the desired differentiated cells. During the differentiation induction or after differentiation into the desired differentiated cells, the spheroids are collected from the plate culture vessel, washed with phosphate physiological saline or the like as necessary, and then separated by enzyme treatment or the like. A cell suspension is prepared by resuspending the obtained cells or small cell mass in a medium containing a third differentiation-inducing factor. Next, the cell suspension is dispensed into the above-described specific spheroid-forming culture vessel and cultured to form spheroids of target differentiated cells.

When the disaggregation / reaggregation treatment is performed before the differentiated cell spheroid formation step, after the precursor cell spheroid formation step, the spheroid is collected from the plate culture vessel in which the precursor cell spheroid is formed, and if necessary Cell suspension by washing with phosphoric saline, etc., and then resuspending the cells or small cell mass obtained by separating the spheroids by enzyme treatment or the like in a medium containing a third differentiation-inducing factor. To prepare. Next, the cell suspension is dispensed into the above-described specific spheroid-forming culture vessel and cultured to form spheroids of target differentiated cells.

At the time of reaggregation treatment, the suspension of other cells is mixed with the cell suspension in which the cells constituting the spheroids are separated, and this mixed suspension is dispensed into the above-mentioned specific spheroid-forming culture vessel. By culturing, spheroids (mixed spheroids) containing both the target differentiated cells and the other cells are formed. For example, mixed spheroids of target differentiated cells and other cells that are present in close proximity to the target differentiated cells in vivo are more in vivo than spheroids formed only from target differentiated cells. Therefore, it is useful as a drug discovery research tool. When the target differentiated cells are cardiomyocytes, mixed spheroids with cardiac fibroblasts are false positives when used as evaluation cells in evaluation tests for the efficacy or safety of test substances that affect the function of the heart. It can be expected that a more reliable evaluation result can be obtained.

The differentiated cell spheroids produced by the spheroid production method of the present invention (hereinafter sometimes referred to as “the spheroids of the present invention”) are substantially uniform in size and are composed of highly differentiated cells. It is very useful as a drug discovery research tool. For example, the spheroid of the present invention is very useful as an evaluation cell in a test for evaluating the efficacy or safety of a test substance. An evaluation result with high statistical reliability can be obtained by evaluating the efficacy or safety of the test substance using the spheroid of the present invention. The spheroid of the present invention is also useful as a material for screening for a substance having a target activity. By screening a substance having the target activity using the spheroid of the present invention, the activity of the candidate compound can be examined more appropriately, and a screening result with high statistical reliability can be obtained.

For example, spheroids of matured myocardial cells produced by the method for producing spheroids of the present invention have functions of myocardial cells in vivo rather than planarly cultured cardiomyocytes and spheroids of matured myocardial cells formed by conventional methods. The characteristics are more fully reflected. For this reason, the candidate compound of a pharmaceutical product is brought into contact with a spheroid of a matured myocardial cell produced by the spheroid production method of the present invention, and the contraction movement, intracellular calcium ion concentration change, membrane potential, etc. are measured, thereby the candidate compound The cardiotoxicity and medicinal properties of can be accurately examined.

The above-described spheroid-forming culture vessel A used in the spheroid production method of the present invention and a differentiation-inducing factor for differentiating stem cells can be kitted to make the spheroid production method of the present invention easier. The differentiation-inducing factor provided in the kit may be only one type or two or more types among a plurality of differentiation-inducing factors used in the step of differentiating from a stem cell to a target differentiated cell, All types may be used. The kit includes a differentiation-inducing factor for differentiating stem cells into three germ layers, a differentiation-inducing factor for differentiating three germ layers into precursor cells of target differentiated cells, and a differentiation-inducing factor for differentiating the precursor cells into the differentiated cells, It is preferable that all of these are included.

In addition, the kit may include various reagents or devices used in the implementation of the spheroid production method of the present invention. For example, the kit further includes a stem cell, a culture medium, a flat cell culture vessel with low cell adhesion, a reagent for determining whether a cell is alive (a reagent that specifically stains dead cells), and the like.

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited by the following description.

<IPS cells>
In subsequent experiments, the human iPS cell 253G1 strain purchased from iPS Academia and licensed was used as the iPS cell.
IPS cells used for differentiation induction were prepared according to the product protocol of Laminin-521 (manufactured by BioLamina, product number: LN521-03). Specifically, mTeSR1 (modified Tenneille Serum Replacer 1) medium (STEMCELL TECHNOLOGIES, product number: 05850) is used in a 6-well plate coated with Laminin-521 (CORNING, product number: 353046). IPS cells in a state of 60% to 100% confluence after 4 to 7 days of culture were used. For washing of iPS cells, DPBS (Dulbeccoline physiological saline) (manufactured by Wako, product number: 045-29795) was used. For detachment of iPS cells from the plate, the cells were treated with a cell dissociation enzyme “TrypLE select” (manufactured by Thermo Fisher Scientific, product number: 12563-011), and then a cell scraper (manufactured by AGC Techno Glass, product number: 9000). -220) It was done by scratching.

<Cytokine and low molecular weight compounds>
In the subsequent experiments, the following cytokines and the like were used.
BMP4 (bone morphogenetic factor 4) is human BMP4 (manufactured by R & D systems, product number: 314-BP-010) and 0.1% BSA (bovine serum albumin) so that the final concentration is 10 μg / mL. A BMP4 stock solution diluted with 4 mM hydrochloric acid was used.
As bFGF, a bFGF stock solution obtained by diluting human bFGF (manufactured by Wako, product number: 064-04541) with DPBS containing 0.1% BSA so as to have a final concentration of 10 μg / mL was used.
As for VEGF, bFGF stock solution obtained by diluting human VEGF (manufactured by R & D systems, product number: 293-VE-010) with DPBS containing 0.1% BSA so as to have a final concentration of 5 μg / mL is used. It was.
For activin A (R & D systems, product number: 338-AC-010), an activin A stock solution diluted with DPBS containing 0.1% BSA was used so that the final concentration was 10 μg / mL.
For IWP4 (manufactured by Reprocell, product number: 04-0036), an IWP4 stock solution diluted with dimethyl sulfoxide so as to have a final concentration of 1.2 mM was used.

<Preparation of myocardial differentiation medium>
All media were prepared on the day and warmed in a 37 ° C. water bath for at least 10 minutes before use.
As a basal medium used for differentiation induction, “StemPro-34” (manufactured by Thermo Fisher Scientific, product number: 10639-011), penicillin / streptomycin mixture “Pen / Strep (10 U / L)” (Thermo Fisher Scientific) Product number: 15140-122) to a final concentration of 1%, and transferrin “holo-” L-glutamine (manufactured by Thermo Fisher Scientific, product number: 25030-081) to a final concentration of 1%. Transferrin human "(manufactured by Sigma, product number: T0665-100MG) has a final concentration of 0.5%, and ascorbic acid (manufactured by Sigma, product number: A92902-100MG) has a final concentration of 1%. , MTG (monothioglycerol) (manufactured by Sigma, product number: M6145-25ML) was added to each so as to obtain a final concentration of 0.0039% (mStemPro-34 medium). Ascorbic acid was added after thawing just before use.

As an EB (embryoid body) formation medium, Y-27632 ROCK inhibitor was added to mStemPro-34 medium to a final concentration of 0.5 to 1.0 ng / mL to a final concentration of 0.5 to 1.0 ng / mL. Medium was used.

As a differentiation induction medium at the mesoderm stage (medium for forming mesoderm at a double concentration), in mStemPro-34 medium, BMP4 has a final concentration of 20 ng / mL, and bFGF has a final concentration of 10 ng / mL. Each medium added with activin A to a final concentration of 12 ng / mL was used.

The differentiation precursor medium for cardiac progenitor cells (cardiac progenitor cell formation medium) is mStemPro-34 medium, so that VEGF has a final concentration of 10 ng / mL and IWP4 has a final concentration of 2.5 μM. The added medium was used.

As a differentiation induction medium for myocardial maturation stage (medium for forming myocardial mature cells), VEGF was added to mStemPro-34 medium to a final concentration of 10 ng / mL and bFGF was added to a final concentration of 5 ng / mL. Medium was used.

[Reference Example 1]
IPS cells were seeded in two types of spheroid-forming culture containers “EZSPHERE” having different dent portions, and the size of the formed spheroids was examined. Specifically, “EZSPHERE 35mmDish” (product number: 4000-900, manufactured by AGC Techno Glass Co., Ltd.) (hereinafter referred to as “EZSPHERE # 4000-900”) having a hollow portion diameter of 500 μm and a depth of 100 μm. “EZSPHERE 35mmDish” (manufactured by AGC Techno Glass Co., Ltd., product number: 4000-905) (hereinafter referred to as “EZSPHERE # 4000-905”) having a diameter of 1400 μm and a depth of 600 μm was used.

CTK solution was added to iPS cells cultured on SNL feeder cells and incubated at 37 ° C. for 1 minute. Accutase containing 50 μM Y-27632 was added to the iPS cells, incubated at 37 ° C. for 5 minutes, and dispersed in a single cell. Subsequently, the obtained dispersion of iPS cells was centrifuged at 190 × g for 3 minutes to remove the supernatant, and then the EB formation medium (5% KSR (KnockOut Serum Replacement) (ThermoFisher Scientific), 50 μM Y -27632, 10 μM SB-431542, and 2 μM dorsomorphin (manufactured by Wako Pure Chemicals Industries) were added to a primate ES cell medium) to prepare a cell suspension. The cell suspension was cultured in EZSPHERE # 4000-900 for 4.6 days with 4.6 × 10 ⁶ cells and EZSPHERE # 4000-905 for 1.8 days with 1.8 × 10 ⁶ cells. The amount of medium added per container was 2 to 3 mL. For each container, half of the medium was changed on the first and fourth days of culture.

Spheroids of each dish were stained with Calcein-AM, and the green fluorescence emitted from the stained living cells was observed with a fluorescence microscope. As a result, each dish had a spheroid of approximately the same size in each depression. It was confirmed that was formed. That is, it was confirmed that a large number of spheroids can be formed with a high survival rate by using a culture container having a large number of dents per dish and the dents being separated by a bank.

The spheroid particle size distribution of each dish was measured as follows. First, the spheroids were transferred together with the medium to a flat culture vessel, and a micrograph was taken. Using the Particle の analyzer function of the image analysis software Image J (NIH; http://rsbweb.nih.gov/ij/), the area of the spheroid in the micrograph is measured, and the diameter of the spheroid is calculated based on the area value. The particle size distribution was obtained by calculation.

The results of examining the particle size distribution of spheroids in each dish are shown in FIGS. 1 and 2, respectively. As a result, when using either dish, there was no excessively large spheroid, and the size of most spheroids was relatively uniform. In particular, the particle size distribution of the spheroid formed in EZSPHERE # 4000-900 had only one peak, the average diameter was 226.9 ± 50.8 μm, and the half-width of the peak was relatively small. The average diameter of the spheroids formed in EZSPHERE # 4000-905 was 381.3 ± 115.7 μm. From these results, by using a culture container for forming spheroids having a diameter of the recess of 500 μm and a depth of 100 μm, rather than using a culture container for forming spheroids having a diameter of the recess of 1400 μm and a depth of 600 μm. It was found that spheroids having a more uniform size can be formed.

[Example 1]
Spheroids differentiated from iPS cells into cardiomyocytes were formed. Myocardial differentiation from iPS cells was performed by improving the method of Lei Yang et al. (Nature, 2008, vol. 453, p. 524-528). “Day of differentiation X” means the number of days that have elapsed since the cell suspension of iPS cells made into single cells was seeded in a culture container for spheroid formation.

<Embryoid body formation>
IPS cells that reached 80% to 100% confluence were washed with 1 mL of DPBS per well of a 6-well plate, 1 mL of TrypLE select was added, and the mixture was incubated at 37 ° C. for 4 minutes, and then TrypLE select was removed by aspiration. Next, 1 mL of EB formation medium was added per well of a 6-well plate, iPS cells were peeled off with a cell scraper, and pipetted 2-5 times to make iPS cells into single cells. The obtained suspension of iPS cells was centrifuged (100 × g, 4 minutes), the supernatant was removed, EB formation medium was added, and a TC20 (registered trademark) fully automatic cell counter (Bio-Rad) was added. Cell number was measured (particle size: 8-30 μm). The cell viability was confirmed to be 90% or more, and used for the subsequent experiments. Aggregates that could not be single-celled were removed using a cell strainer.

The prepared cell suspension of iPS cells is placed in “EZSPHERE 100 mmDish” (manufactured by AGC Techno Glass, product number: 4020-900) (hereinafter referred to as “EZSPHERE # 4020-900”) in a culture container for spheroid formation. The dish was dispensed at 3 × 10 ⁶ cells and 5 to 10 mL of medium. The dish on which the iPS cells were seeded was shaken 5 times at a time to disperse the cells evenly in the dish, and then allowed to stand in a 37 ° C. 5% CO ₂ incubator for 24 hours.

<Mesodermal differentiation phase>
Between 24 hours ± 2 hours after seeding with iPS cells, each dish is added with an EB formation medium equivalent to the EB formation medium that has already been added, in a 5% CO ₂ incubator at 37 ° C. And incubated for 3 days.

<Cardiac progenitor cell differentiation phase>
After the mesoderm differentiation phase (4th day of differentiation), the medium was gently transferred from each dish to a 15 mL or 50 mL tube using a 5 mL pipette so as not to disrupt the spheroids. Then, in order to precipitate spheroids, the tube was allowed to stand at 37 ° C. for 2 to 10 minutes, and then the medium supernatant was carefully removed. To the spheroids in the tube, 3 mL of a cardiac progenitor cell-forming medium was added and centrifuged (50 × g, 3 minutes, room temperature), and then the supernatant was carefully removed. Thereafter, 10 mL or 20 mL of cardioprogenitor cell-forming medium was added to the tube, and a 100 mm low adhesion dish “EZ-bindshut II” (manufactured by AGC Techno Glass, product number: 4020-800LP) (hereinafter referred to as “EZSPHERE # 4020”). -800LP "), and incubated in a 5% CO ₂ incubator at 37 ° C for 3 days.

<Reaggregation of spheroids>
After the cardiac progenitor cell differentiation phase (7th day of differentiation), spheroids from each dish are collected in a 50 mL tube together with the medium, and in order to precipitate the spheroids, the tube is allowed to stand at 37 ° C. for 2 to 10 minutes, and then carefully cultured on the medium. The supernatant was removed. After adding 10 mL of DPBS to the spheroids in the tube and centrifuging (50 × g, 3 minutes, room temperature), the supernatant was carefully removed. Thereafter, 2 mL of cell dispersion (“Accumax”: 0.05% Trypsin / EDTA = 1: 3) was added to the tube and incubated at 37 ° C. for 8 minutes. The tube was rocked with a vortex mixer every 4 minutes during this incubation period. After the incubation, the tube was allowed to stand for 1 to 2 minutes to precipitate a large mass and collect only small dispersed cells (first cell suspension). After adding 2 mL of the cell dispersion to the precipitated large mass and repeating the same operation to separate the cells, only the small dispersed cells are collected (second cell suspension), 1 After mixing with the second cell suspension, an equivalent amount of a medium for forming myocardial mature cells was added and centrifuged (140 × g, 4 minutes), and the supernatant was removed. Next, an appropriate amount of a medium for forming mature myocardial cells was added to the precipitated cells, and the number of cells was measured with a TC20 (registered trademark) fully automatic cell counter (manufactured by Bio-Rad) (particle size: 8-30 μm). The cell suspension having a cell viability of 90% or more was used as it was in the subsequent experiments. A cell suspension with a cell viability of less than 90% was prepared so that the proportion of viable cells was 90% or more by removing dead cells and aggregates that could not be made into single cells, and then the subsequent experiments. Used for.

<Myocardial maturation phase>
The resulting cell suspension, using a medium for myocardial mature cell formation, prepared in 3.3 × 10 ⁵ cells /ML,6.7×10 ⁵ cells /ML,1.0×10 ⁶ cells / mL did. Each cell suspension was spheroid-forming culture container “EZSPHERE 35mmDish” (manufactured by AGC Techno Glass, product number: 4000-903, hollow diameter: 800 μm, depth: 400 μm) (hereinafter referred to as “EZSPHERE # 4000 -903 ") was dispensed at a rate of 3 mL per dish. The dish was shaken 5 times at a time to disperse the cells evenly in the dish and then incubated in a 5% CO ₂ incubator at 37 ° C. for 11 days to obtain spheroids of myocardial mature cells. During the incubation, the medium was changed every 2-3 days with the medium having the same composition. The medium was changed by inclining the dish by 15 to 30 °, slowly removing the whole medium, and then slowly adding 3 mL of a new medium. Beating was observed 7-8 days after replacement with the medium for forming myocardial mature cells. By seeding EZSPHERE # 3.3 × 10 ⁵ cells per dish 4000-903 /mL,6.7×10 ⁵ cells /ML,1.0×10 ⁶ cells / mL of the cell suspension 3 mL, container In theory, the number of cells spread per dent, that is, the number of cells that form one spheroid is approximately 1000, 2000, or 3000, respectively.

<Transition to the myocardial maturation phase without reaggregation>
As a control, after the mesoderm differentiation phase, the spheroids were not separated and moved directly to the myocardial maturation phase.
Specifically, after the cardiac progenitor cell differentiation phase (7th day of differentiation), spheroids are collected from each dish in a 15 mL or 50 mL tube together with the medium, and the tube is precipitated at 37 ° C. for 2-10. After allowing to stand for 5 minutes, the medium supernatant was carefully removed. Next, 10 mL of myocardial mature cell formation medium was added to the tube, transferred to a new 100 mm low adhesion dish EZ-BindshutII # 4020-800LP, and incubated in a 5% CO ₂ incubator at 37 ° C. for 11 days. Got spheroids. During the incubation, the medium was changed in the same manner with a medium having the same composition every 2-3 days. The medium change did not replace the low adhesion dish.

<Measurement of relative expression level of myocardial marker>
Regarding the spheroids of the obtained myocardial mature cells, myocardial markers Nkx2-5 (NK-2 transcription factor related, locus 5), TNNT2 (Cardiac troponin-T), MYL7 (myosin light chain7), MYL2 (myosin light chain2) The expression levels of HCN4 and GAPDH, which is a housekeeping enzyme, were measured, and the relative expression levels of each myocardial marker with respect to the expression levels of GAPDH were determined by the qRT-PCR method. Specifically, using a “Pure Link RNA Mini Kit” (Ambion, product model number: 12183018A), total RNA was extracted from cryopreserved cells, and “High Capacity cDNA Reverse Transcription Kit” (Thermo Fisher) , Product model: 4368814), reverse transcription to cDNA, measurement reagent “THUNDERBIRD (registered trademark) SYBR qPCR Mix” (manufactured by TOYOBO, product model: QPS-201) and measuring instrument “Mx3000P” (Agilent Technologies) The relative expression level was examined.

The measurement results of the relative expression level of the myocardial marker are shown in FIGS. In the figure, “without reaggregation” indicates the result of spheroids formed by transitioning to the myocardial maturation phase without reaggregation, “1000 cells”, “2000 cells”, and “3000 cells” of “with reaggregation”. "it is added reaggregation after 3.3 × 10 ⁵ cells /ML,6.7×10 ⁵ cells / mL, respectively spheroid, and 1.0 × 10 ⁶ cells / mL of cell suspension 3mL dish The results of spheroids formed by transitioning to the myocardial maturation phase are shown respectively. As a result, as shown in FIGS. 3 and 7, the relative expression levels of Nkx2-5 and HCN4 were not different between those without reaggregation and those with reaggregation, but as shown in FIGS. In particular, the relative expression levels of TNNT2, MYL7, and MYL2 are clearly higher with reaggregation than without aggregation, and in particular, less depending on the number of cells that form spheroids. The spheroids of matured myocardial cells that had formed TNNT2, MYL7, and MYL2 had the highest relative expression levels.

Here, the increase in the expression level of TNNT2 suggests that the cardiomyocytes are purified, that is, the ratio of cells differentiated from iPS cells other than the myocardium and undifferentiated cells is sufficiently low. In addition, an increase in the expression level of MYL2 suggests that the rate of maturation of cardiomyocytes, that is, the proportion of cardiac progenitor cells before maturation is sufficiently low. In particular, in the case of spheroids in which the spheroids formed by seeding iPS cells are continuously cultured in a differentiation-inducing medium without re-aggregation treatment as before, Nkx2-5 starts from about day 6 (cardiac progenitor cells). On the other hand, MYL2 begins to express slightly from about the 14th day, and the expression level increases until about the 21st day (not shown). Considering this point, the expression level of the Nkx2-5 gene expressed before the reaggregation treatment does not change even after the reaggregation treatment (FIG. 3). As for MYL2 that rises, the expression level clearly increased in cells after reaggregation treatment compared to cells that did not undergo reaggregation treatment (FIG. 6), indicating that maturation was promoted by reaggregation treatment. That is, it was found that maturation and purification of cardiomyocytes are promoted by separating and reaggregating spheroids once before differentiation into myocardial mature cells.

Moreover, when the reaggregated spheroids were compared, the expression level of MYL2 was highest in spheroids formed from about 1000 cells, and the spheroids formed from about 3000 cells had the highest rate of increase. Was small (FIG. 6). From this result, the effect of promoting the spheroid maturation and purification by the reaggregation treatment is affected by the number of cells per depression of the spheroid-forming culture container, that is, the number of cells that form spheroids during reaggregation. It has been found that spheroids seeded and formed so that the number of cells per depression in the spheroid-forming culture container is about 1000 can more effectively achieve the purification and maturation promoting effects by reaggregation.

[Example 2]
Myocardial mature cells differentiated from planarly cultured iPS cells, myocardial mature cell spheroids differentiated from iPS cells, and myocardial mature cells and cardiac fibroblasts (NHCF) differentiated from iPS cells Were examined for pharmacological response to E-4031 (Wako, product number: 059-08451), a hERG (potassium channel) blocker.

<2D iPS-derived myocardial mature cells>
2D iPS-derived matured myocardial cells (myocardial mature cells differentiated from planarly cultured iPS cells) were obtained as follows.
IPS cells that reached 80% to 100% confluence were washed with 1 mL of DPBS per well of a 6-well plate, 1 mL of TrypLE select was added, and the mixture was incubated at 37 ° C. for 4 minutes, and then TrypLE select was removed by aspiration. Next, 1 mL of EB formation medium was added per well of a 6-well plate, iPS cells were peeled off with a cell scraper, and pipetted 2-5 times to make iPS cells into single cells. The obtained suspension of iPS cells was centrifuged (100 × g, 4 minutes), the supernatant was removed, EB formation medium was added, and a TC20 (registered trademark) fully automatic cell counter (Bio-Rad) was added. Cell number was measured (particle size: 8-30 μm). The cell suspension having a cell viability of 90% or more was used as it was in the subsequent experiments. A cell suspension with a cell viability of less than 90% was prepared so that the proportion of viable cells was 90% or more by removing dead cells and aggregates that could not be made into single cells, and then the subsequent experiments. Used for.
The prepared cell suspension of iPS cells was dispensed into a plate culture container (6-well plate) coated with fibronectin so that the cell volume was 1.5 × 10 ⁵ cells and the medium amount was 200 μL. The 6-well plate seeded with iPS cells was shaken 5 times at a time to disperse the cells evenly in the wells, and then allowed to stand in a 5% CO ₂ incubator at 37 ° C. for 24 hours. Then, the same mesoderm-forming medium as the EB-forming medium already added was added to the 6-well plate and incubated in a 5% CO ₂ incubator at 37 ° C. for 3 days. Thereafter, the cells in the 6-well plate were washed with DPBS, a medium for forming myocardial mature cells was added, and incubated in a 5% CO ₂ incubator at 37 ° C. for 3 days. After that, the cells in the 6-well plate were washed with DPBS, a medium for forming myocardial mature cells was added, and incubated in a 5% CO ₂ incubator at 37 ° C. for 11 days. Obtained. During the culture for 11 days with the addition of the medium for forming myocardial mature cells, the medium was replaced with a medium having the same composition every 2-3 days. The medium was exchanged by removing half of the medium (100 μL) from each well and then adding half of the same type of medium (100 μL).

<Myocardial mature cell spheroid>
Formation of myocardial mature cell spheroids was performed as follows. First, <embryoid body formation>, <mesoderm differentiation phase>, and <cardiac progenitor cell differentiation phase> are performed from iPS cells in the same manner as in Example 1, and cardiac progenitor cells are contained in EZ-bindshutII # 4020-800LP. Spheroids were formed.
Subsequently, after the cardiac progenitor cell differentiation phase (7th day of differentiation), spheroids from each dish are collected in a 15 mL or 50 mL tube together with the medium, and the tube is allowed to stand at 37 ° C. for 2 to 10 minutes in order to precipitate the spheroids. After that, the medium supernatant was carefully removed. Next, 10 mL of myocardial mature cell formation medium was added to the tube, transferred to a new 100 mm low-adhesion dish EZ-bindshutII # 4020-800LP, and incubated for 7 days in a 37 ° C. 5% CO ₂ incubator. Got spheroids.
On the 14th day of differentiation, in the same manner as in <Spheroid reaggregation> in Example 1, spheroids were separated and 4 × 10 ⁵ cells / mL, 6 × 10 ⁵ cells / mL using a medium for forming myocardial mature cells. Or a cell suspension of 8 × 10 ⁵ cells / mL.
The obtained cell suspension (200 μL) is placed in a spheroid-forming culture container “EZSPHERE microplate 96 well” (manufactured by AGC Techno Glass, product number: 4860-900) (hereinafter referred to as “EZSPHERE # 4860-900”). After dispensing, the 96-well plate was shaken 5 times at a time to disperse the cells evenly in the wells, then incubated in a 5% CO ₂ incubator at 37 ° C. for 7 days to obtain spheroids of myocardial mature cells. Got. During the incubation, the medium was changed every 2-3 days with the medium having the same composition. The medium was exchanged by removing half of the medium (100 μL) from each well and then adding half of the same type of medium (100 μL).

<Myocardial mature cell / NHCF spheroid>
The formation of myocardial mature cells / NHCF spheroids was performed as follows. First, <embryoid body formation>, <mesoderm differentiation phase>, and <cardiac progenitor cell differentiation phase> are performed from iPS cells in the same manner as in Example 1, and cardiac progenitor cells are contained in EZ-bindshutII # 4020-800LP. Spheroids were formed.
Next, after the cardiac progenitor cell differentiation phase (7th day of differentiation), spheroids from each dish are collected in a 15 mL or 50 mL tube together with the medium, and the tube is allowed to stand at 37 ° C. for 2 to 10 minutes in order to precipitate the spheroids. After placing, the medium supernatant was carefully removed. Next, 10 mL of myocardial mature cell formation medium was added to the tube, transferred to a new 100 mm low-adhesion dish EZ-bindshutII # 4020-800LP, and incubated for 7 days in a 37 ° C. 5% CO ₂ incubator. Got spheroids.
On the 14th day of differentiation, in the same manner as in <Spheroid reaggregation> in Example 1, spheroids were separated and a cell suspension was prepared using a medium for forming myocardial mature cells.
To the obtained cell suspension, NHCF washed with a medium for forming myocardial mature cells was added so that the number of iPS-derived myocardial mature cells and NHCF was 75:25, and then the iPS-derived myocardial mature cells and A cell suspension having a combined concentration of NHCF of 4 × 10 ⁵ cells / mL was prepared. After 200 μL of the cell suspension was dispensed into EZSPHERE # 4860-900, the 96-well plate was shaken 5 times at a time to disperse the cells evenly in the well, and then 5% CO _{2 at} 37 ° C. After incubation for 7 days in an incubator, spheroids of matured myocardium were obtained. During the incubation, the medium was changed every 2-3 days with the medium having the same composition. The medium was exchanged by removing half of the medium (100 μL) from each well and then adding half of the same type of medium (100 μL).

<Pharmacological response>
When potassium efflux is suppressed in cardiomyocytes by E-4031, the repolarization process is delayed, so the QT interval (time from ventricular excitement to repolarization) is extended. Therefore, the response to the hERG blocker was compared for the obtained three iPS-derived myocardial mature cells.

Specifically, first, DMEM (manufactured by Nacalai, product number: 08459-59) and FBS (fetal bovine serum) (manufactured by MP Biomedicals, product number: 2916154) are adjusted to a final concentration of 10%. Nonionic surfactant “Pluonic F127” (manufactured by Sigma, product number: P2443-250G) so that the final concentration of Fluo-4AM (manufactured by Dojindo, product number: F311) as a Ca ²⁺ probe was 20 μmol / L. ) Were added so that the final concentration would be 0.04% to prepare a double concentration medium.
Next, after removing a half amount (100 μL) of the medium from each well, a half amount (100 μL) of a 2-fold concentration medium was added and incubated at 37 ° C. for 20 minutes to allow the Ca ²⁺ probe to permeate inside each iPS-derived myocardial mature cell. It was.

Next, after removing half of the medium (100 μL) from each well, half of the medium containing E-4031 (100 μL) was added and incubated at 37 ° C. for 30 minutes. A medium containing E-4031 was prepared by adding E-4031 to DMEM containing 10% FBS so that the final concentration was 0, 60, or 120 nmol / L.

Next, changes in intracellular calcium ion concentration of each iPS-derived myocardial mature cell were observed using a cell imaging system “EVOS (registered trademark) FL Auto” (manufactured by Thermo Fisher Scientific). A 96-well plate to which E-4031 was added was taken out of the incubator, placed as it was, observed with EVOS, and a video was taken with an AG desktop recorder. The system settings were as follows.

EVOS FL Auto setting (lens: 10x, light cube: GFP, Lihgt: 74, EXP: 40 ms, GAIN: 10 db).
Setting of EVOS on Stage Incubator (temperature: 37 ° C., CO ₂ concentration: 5%, saturated steam atmosphere).
AG desktop recorder settings (rate: 25 FPS, main codec: RGB24).

The luminance analysis of the captured video was performed using the image analysis software “Image J”. Specifically, the captured video was opened with Image J, one spheroid was surrounded with Image J selection tool, and then image J's plot Z axis profile was executed to obtain the brightness (numerical value) of each spheroid.

From the obtained intracellular calcium ion concentration change, the extension of the intracellular calcium concentration transient decay time (CaT Decay Time: TD) was examined. Specifically, TD20 of each iPS-derived myocardial mature cell (time taken to decay to 20% with respect to the increase in intracellular calcium concentration), TD50 (time taken to decay to 50%), and TD90 (90% The results are shown in Figures 8 to 10. As a result, no extension of TD by E-4031 was observed in 2D iPS-derived myocardial mature cells (Figure 8). In contrast, in the matured myocardial cell spheroid (FIG. 9) and matured myocardial cell / NHCF spheroid (FIG. 10), TD50 and TD90 were clearly longer in the E-4031-treated cells, and the animals were actually administered E-4031. As in the case, an extension of TD50 and TD90 by E-4031 (an index indicating QT extension) was observed, and from the results, myocardial growth cultured in a plane was observed. It is not possible to reflect the physiology of the actual heart in cells but, by forming spheroids was found to be to better reflect the actual physiology of the heart.
It should be noted that the entire contents of the specification, claims, abstract and drawings of Japanese Patent Application No. 2017-079093 filed on Apr. 12, 2017 are cited here as disclosure of the specification of the present invention. Incorporated.

Claims (15)

1. In a method for producing a differentiated cell spheroid by differentiating a stem cell in the presence of a differentiation-inducing factor,
   A method for producing a differentiated cell spheroid, characterized in that the spheroid is disaggregated into smaller spheroids or single cells at any time after spheroid formation, and then reaggregated.
2. The method for producing a differentiated cell spheroid according to claim 1, wherein stem cells are sequentially cultured in a medium containing a differentiation-inducing factor, and differentiated into a target differentiated cell spheroid via a precursor cell spheroid.
3. The method for producing a differentiated cell spheroid according to claim 1 or 2, wherein spheroid formation and reaggregation are performed in a culture container for spheroid formation.
4. After preparing a cell suspension into smaller spheroids or single cells generated by the disaggregation, measure the ratio of viable cells to total cells in the cell suspension;
   When the proportion of the living cells is 90% or more, the cell suspension is reaggregated as it is in a spheroid-forming culture vessel,
   When the ratio of the living cells is less than 90%, dead cells are removed from the cell suspension and the ratio of the living cells is adjusted to 90% or more, and then reconstituted in the spheroid-forming culture container. Agglomerate,
   The method for producing a differentiated cell spheroid according to any one of claims 1 to 3.
5. The method for producing a differentiated cell spheroid according to any one of claims 1 to 4, wherein smaller spheroids or single cells generated by the disaggregation are reaggregated in the presence of a differentiation-inducing factor.
6. The spheroids are disaggregated and reaggregated at the time when the precursor cell spheroids are transferred to a medium containing a differentiation inducer for differentiating the precursor cells into target differentiated cells, or during the culture of the differentiated cell spheroids The method for producing a differentiated cell spheroid according to any one of claims 2 to 5, wherein
7. The method for producing a differentiated cell spheroid according to any one of claims 1 to 6, wherein the stem cells are embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, Muse cells, or mesenchymal stem cells.
8. The method for producing differentiated cell spheroids according to any one of claims 1 to 7, wherein the differentiated cells are cardiomyocytes, nerve cells, or hepatocytes.
9. The stem cell is an embryonic stem cell or an induced pluripotent stem cell;
   The stem cells are cultured in a medium containing one or more differentiation-inducing factors that cause embryonic stem cells or induced pluripotent stem cells to differentiate into any germ layer of the three germ layers. After forming germ layer spheroids,
   The formed early germ layer spheroid is cultured in a medium containing one or more differentiation-inducing factors for differentiating one of the three germ layers into a precursor cell of the target differentiated cell, and the precursor cell of the target differentiated cell Of spheroids,
   The formed precursor cell spheroid is cultured in a medium containing one or more differentiation-inducing factors that cause the precursor cell to differentiate into a target differentiated cell to form a spheroid of the target differentiated cell. The method for producing a differentiated cell spheroid according to any one of the above.
10. The progenitor cells are cardiac progenitor cells, and the differentiated cells are cardiomyocytes,
    The differentiation-inducing factor that differentiates any germ layer of the three germ layers into a precursor cell of a target differentiated cell is one or more Wnt signal activators,
    The differentiation-inducing factor for differentiating any germ layer of the three germ layers into a precursor cell of a target differentiated cell is one or more Wnt signal inhibitors,
    The method for producing a differentiated cell spheroid according to claim 9, wherein the differentiation-inducing factors for differentiating the progenitor cells into target differentiated cells are vascular endothelial growth factor and basic fibroblast growth factor.
11. The differentiation according to any one of claims 1 to 10, wherein a differentiated cell spheroid used for an evaluation test of a drug efficacy or safety of a test substance or used for screening a substance having a target activity is produced. A method for producing cell spheroids.
12. A differentiated cell spheroid is produced by the method for producing a differentiated cell spheroid according to any one of claims 1 to 11, and then the medicinal efficacy or safety of the test substance is evaluated using the produced differentiated cell spheroid. , Evaluation method of test substance.
13. An activity for producing a differentiated cell spheroid by the method for producing a differentiated cell spheroid according to any one of claims 1 to 11 and then screening a substance having a target activity using the produced differentiated cell spheroid. Substance screening method.
14. The inner bottom surface of the container has a plurality of indentations forming a compartment in which the culture object is cultured, and a bank portion interposed between the adjacent indentations, and the adjacent bank portion and the indentation portion are continuous. A spheroid-forming culture container in which the inner surface of the depression is coated with a cell adhesion inhibitor,
    A differentiation-inducing factor for differentiating stem cells;
    A kit for producing a differentiated cell spheroid, comprising:
15. The differentiation-inducing factor is a differentiation-inducing factor that causes embryonic stem cells or induced pluripotent stem cells to differentiate into any germ layer of the three germ layers, and a precursor cell of a target differentiated cell of any of the three germ layers The kit for producing a differentiated cell spheroid according to claim 14, comprising a differentiation inducer that differentiates into a differentiated cell and a differentiation inducer that differentiates the progenitor cell into the differentiated cell.

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