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WO2018190305A1 - Procédé de production de sphéroïdes de cellules différenciées - Google Patents

Procédé de production de sphéroïdes de cellules différenciées Download PDF

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Publication number
WO2018190305A1
WO2018190305A1 PCT/JP2018/014951 JP2018014951W WO2018190305A1 WO 2018190305 A1 WO2018190305 A1 WO 2018190305A1 JP 2018014951 W JP2018014951 W JP 2018014951W WO 2018190305 A1 WO2018190305 A1 WO 2018190305A1
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cells
cell
spheroid
differentiation
spheroids
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Japanese (ja)
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達明 三輪
アリムジャン イディリス
博道 熊谷
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AGC Inc
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Asahi Glass Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • 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.
  • Patent Document 1 describes the use of spheroids to further increase drug sensitivity when evaluating drug efficacy or toxicity.
  • 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.
  • 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.
  • differentiated cells having the same characteristics can be stably supplied by performing constant differentiation induction.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present inventors 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.
  • 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.
  • [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.
  • 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.
  • 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
  • [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.
  • the differentiated cells are cardiomyocytes, neurons, or hepatocytes.
  • 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.
  • 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.
  • the method for producing a differentiated cell spheroid according to any one of [8].
  • 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 [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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Example 2 it is the figure which showed the measurement result of TD20, TD50, and TD90 after E-4031 process of 2D (planar culture
  • 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.
  • 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.
  • 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 ⁇ cells, enterocytes, goblet cells, enteroendocrine cells and the like can be
  • 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.
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • somatic stem cells mesenchymal stem cells, Muse cells or hematopoietic stem cells are preferable.
  • 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.
  • spheroid formation and reaggregation are preferably performed in a spheroid-forming culture vessel.
  • a culture container for spheroid formation any of various culture containers used for spheroid formation may be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • the free state (single cell state) is stress and is likely to be damaged.
  • 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.
  • the number of cells constituting the spheroid 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.
  • the size of the recess of the spheroid-forming culture container A is 20 ⁇ m or more and 1500 ⁇ m or less in diameter (when the shape of the opening is approximately elliptical, it indicates the long diameter), and the depth is 10 ⁇ m or more and 1500 ⁇ m or less. More preferably, the diameter is 200 ⁇ m or more and 1400 ⁇ m or less, the depth is 100 ⁇ m or more and 400 ⁇ m or less, and the diameter is 400 ⁇ m or more and 1000 ⁇ m or less, and the depth is 100 ⁇ m or more and 400 ⁇ m or less.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the shape of the laser light irradiation spot is circular, whereas the opening shape of the depression is flattened to be substantially elliptical.
  • 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.
  • the cell adhesion inhibitor for example, phospholipid polymer, polyhydroxyethyl methacrylate, polyethylene glycol or the like is used.
  • the culture substrate described in Patent Document 2 can be used.
  • EZSPHERE manufactured by AGC Techno Glass Co., Ltd.
  • 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.
  • 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 results in low data reliability and low reproducibility when spheroids of differentiated cells are used in various tests. linked.
  • the purity can be increased by deaggregating and separating the formed spheroids and then reaggregating them.
  • 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.
  • the disaggregation treatment it is not necessary to completely separate the cells, and small aggregates may be formed.
  • 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.
  • 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.
  • 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.
  • the ratio of living cells to total cells in the cell suspension is measured.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • spheroid disaggregation / reaggregation treatment can be performed at any point in the process of differentiation from a stem cell to a target differentiated cell.
  • the free 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.
  • 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.
  • the differentiation inducing factor is also present in cells that were present inside the spheroid.
  • 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.
  • 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.
  • 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.
  • the spheroid is subjected to disaggregation / reaggregation treatment at least once.
  • 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.
  • 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.
  • 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.
  • 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”.
  • 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.
  • 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.
  • 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).
  • VEGF vascular endothelial cell growth factor
  • bFGF basic fibroblast growth factor
  • BMP4 basic fibroblast growth factor
  • a basal medium that is a nutrient medium not containing a differentiation-inducing factor and a target differentiation-inducing factor added thereto are used.
  • a basal medium generally, a medium used for maintaining or growing stem cells or a medium used for culturing animal cells is used.
  • the medium examples include Eagle's minimum essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), Eagle's minimum essential medium ⁇ -modified (MEM- ⁇ ), 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the formed ectodermal embryoid body, mesoderm embryoid body, or endoderm embryoid body is differentiated into a target differentiated cell precursor cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • the second differentiation-inducing factor 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.
  • 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.
  • 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.
  • the disaggregation / reaggregation treatment may be performed on spheroids at an arbitrary time after the initial germ layer 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.
  • the medium containing the second differentiation-inducing factor in the spheroid-forming culture vessel may be reaggregated to form spheroids.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • spheroids mixed spheroids
  • 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.
  • 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 are substantially uniform in size and are composed of highly differentiated cells. It is very useful as a drug discovery research tool.
  • 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.
  • 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.
  • the kit may include various reagents or devices used in the implementation of the spheroid production method of the present invention.
  • 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.
  • 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.
  • Laminin-521 manufactured by BioLamina, product number: LN521-03
  • mTeSR1 modified Tenneille Serum Replacer 1
  • CORNING product number: 353046
  • DPBS Dynamic Host Cell
  • Wako Wako, product number: 045-29795
  • DPBS Dynamic Host Cell
  • a cell dissociation enzyme “TrypLE select” manufactured by Thermo Fisher Scientific, product number: 12563-011
  • a cell scraper manufactured by AGC Techno Glass, product number: 9000.
  • BMP4 bone morphogenetic factor 4
  • BSA bovine serum albumin
  • 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 ⁇ g / mL was used.
  • 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 ⁇ g / mL is used. It was.
  • 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 ⁇ g / mL.
  • IWP4 manufactured by Reprocell, product number: 04-0036
  • 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.
  • 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%.
  • MTG monothioglycerol
  • Ascorbic acid was added after thawing just before use.
  • 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.
  • BMP4 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 ⁇ M.
  • the added medium was used.
  • VEGF 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.
  • 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 ⁇ m and a depth of 100 ⁇ m.
  • EZSPHERE 35mmDish manufactured by AGC Techno Glass Co., Ltd., product number: 4000-905
  • EZSPHERE # 4000-905 having a diameter of 1400 ⁇ m and a depth of 600 ⁇ m 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 ⁇ M Y-27632 was added to the iPS cells, incubated at 37 ° C. for 5 minutes, and dispersed in a single cell.
  • 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 ⁇ M Y -27632, 10 ⁇ M SB-431542, and 2 ⁇ M 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.
  • 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 ⁇ 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.
  • FIGS. 1 and 2 The results of examining the particle size distribution of spheroids in each dish are shown in FIGS. 1 and 2, respectively.
  • the particle size distribution of the spheroid formed in EZSPHERE # 4000-900 had only one peak, the average diameter was 226.9 ⁇ 50.8 ⁇ m, 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 ⁇ m.
  • 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.
  • 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 ⁇ m). 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.
  • EZ-bindshut II manufactured by AGC Techno Glass, product number: 4020-800LP
  • EZSPHERE # 4020 product number: 4020-800LP
  • 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.
  • second 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 ⁇ m). 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 ⁇ m, depth: 400 ⁇ m) (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.
  • 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.
  • 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.
  • myocardial mature cell formation medium 10 mL 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.
  • FIGS. The measurement results of the relative expression level of the myocardial marker are shown in FIGS.
  • “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 relative expression levels of Nkx2-5 and HCN4 were not different between those without reaggregation and those with reaggregation, but as shown in FIGS.
  • 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.
  • 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.
  • 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.
  • Nkx2-5 starts from about day 6 (cardiac progenitor cells).
  • 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.
  • 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.
  • 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 ⁇ m).
  • 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 ⁇ L.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ L) from each well and then adding half of the same type of medium (100 ⁇ L).
  • ⁇ 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.
  • a cell suspension of 8 ⁇ 10 5 cells / mL 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”).
  • EZSPHERE # 4860-900 a spheroid-forming culture container
  • 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.
  • 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 ⁇ L) from each well and then adding half of the same type of medium (100 ⁇ L).
  • ⁇ 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.
  • 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.
  • 200 ⁇ L 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.
  • 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 ⁇ L) from each well and then adding half of the same type of medium (100 ⁇ L).
  • DMEM manufactured by Nacalai, product number: 08459-59
  • FBS fetal bovine serum
  • Nonionic surfactant “Pluonic F127” manufactured by Sigma, product number: P2443-250G
  • Fluo-4AM manufactured by Dojindo, product number: F311
  • 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.
  • 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.

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Abstract

L'invention concerne un procédé de production de sphéroïdes de cellules différenciées ayant des tailles presque uniformes et une pureté élevée, à partir de cellules souches. L'invention concerne également un procédé de production de sphéroïdes de cellules différenciées par différenciation de cellules souches en présence d'un facteur d'induction de différenciation, ledit procédé étant caractérisé en ce que, après la formation des sphéroïdes, chacun des sphéroïdes est désagrégé en sphéroïdes plus petits ou en cellules individuelles, puis les sphéroïdes plus petits ou les cellules individuelles sont ré-agrégés à un moment arbitraire.
PCT/JP2018/014951 2017-04-12 2018-04-09 Procédé de production de sphéroïdes de cellules différenciées Ceased WO2018190305A1 (fr)

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JPWO2020230856A1 (fr) * 2019-05-14 2020-11-19
JPWO2022138101A1 (fr) * 2020-12-23 2022-06-30
EP4183867A4 (fr) * 2020-07-14 2024-11-06 Kaneka Corporation Procédé de production d'une masse de cellules cardiomyocytaires

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WO2023282423A1 (fr) * 2021-07-06 2023-01-12 건국대학교 산학협력단 Cellule souche mésenchymateuse, vésicule extracellulaire isolée à partir de celle-ci et utilisation de celle-ci

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JPWO2022138101A1 (fr) * 2020-12-23 2022-06-30

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