WO2022071334A1 - 副甲状腺細胞 - Google Patents
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Definitions
- the present invention relates to a cell population containing parathyroid cells and a culture system thereof, which can be used to prepare organoids or three-dimensional organs of parathyroid glands.
- Such organoids which can also be called "mini-organs"
- mini-organs can be used as an evaluation system whose drug efficacy or toxicity is closer to that of a living body, as compared with conventional cells of an organ cultured alone, and can be used for transplant surgery and transplant surgery. It is extremely valuable because it can also be used for the production of plasma proteins.
- parathyroid organoids or similar cell aggregates there are the following prior art documents.
- Non-Patent Document 1 spheroids were obtained by culturing mesenchymal stem cells (MSCs) derived from tonsillar gland in a microwell in a medium containing a predetermined component (cytocytosis, etc.), and the spheroids were parathyroid glands. It has been described that high levels of the hormone (PTH) and N-cadherin were expressed, and that the spheroid was transplanted into a rat from which the parathyroid gland was removed. The spheroid is composed of almost only PTH-positive cells when viewed from the stained image.
- MSCs mesenchymal stem cells
- Non-Patent Document 2 parathyroid cells derived from patients with hyperparathyroidism were cultured in non-adherent plastic to obtain spheroids, and the spheroids secreted PTH for a long period of time (150 days or more). It is described that the spheroid was transplanted into mice.
- Non-Patent Document 3 describes that iPS cells were cultured in a medium containing a predetermined component (cytokine or the like) to prepare parathyroid cells.
- parathyroid organoids can be obtained by culturing a cell population containing parathyroid cells and mesenchymal cells in a predetermined ratio in a medium containing an appropriate component (cytokine, etc.). rice field.
- an appropriate component cytokine, etc.
- rice field a medium containing an appropriate component
- the parathyroid cells and mesenchymal cells used in such a culture method aggregates of progenitor cells (Anterior foregut) of aderior cells obtained by culturing iPS cells and their surroundings, respectively. It is possible to utilize existing cells (interstitial cells), and a cell population in which those cells are once dissected and then mixed at a predetermined ratio is suitable for use in the above-mentioned culture method. I found it.
- the present inventors have found that the above problems can be solved by such means, and have completed the present invention.
- the present invention provides the following [1] to [18].
- [1] A cell population containing 50% or more of parathyroid cells and 10% or more of mesenchymal cells.
- [1A] A cell population containing 50% or more of parathyroid cells and 5% or more of mesenchymal cells.
- Item 2. The cell population according to Item 1, further comprising vascular endothelial cells.
- a matrix composition comprising a matrix and the cell population of item 1 embedded in the matrix.
- a culture system comprising the cell population according to Item 1 or the matrix composition according to claim 3, a medium for culturing a cell population, and a culture vessel.
- Item 4 The culture system according to Item 4, wherein the culture container is a round bottom plate.
- Item 4 The culture system according to Item 4, wherein the culture vessel is for suspension culture, stirring suspension culture, or channel culture.
- Item 4. The culture system according to Item 4, wherein the medium comprises two or more components selected from the group consisting of SHH, FGF8, FGF10, Wnt3a, LDN193189, activin and CaCl2.
- a culturing method comprising culturing a cell population containing parathyroid cells at a rate of 50% or more and mesenchymal cells at a rate of 10% or more in a medium.
- Item 7 The culture method according to Item 7, further comprising vascular endothelial cells.
- Item 9 The culture method according to Item 9, wherein the matrix composition in which the cell population is embedded is cultured in a state of being suspended from an air-permeable membrane.
- Item 2. The culture method according to Item 7, wherein the cell population is cultured on a round bottom plate.
- Item 7 The culture method according to Item 7, wherein the medium contains two or more components selected from the group consisting of SHH, FGF8, FGF10, Wnt3a, LDN193189, activin and CaCl2.
- a method for producing a three-dimensional organ which comprises a step of carrying out the culture method according to any one of Items 7 to 12.
- Item 3. A method for evaluating a drug or a drug for treating or preventing a disease related to hyperparathyroidism or hyperparathyroidism using the organoid according to Item 13 or the three-dimensional organ according to Item 15 or 16.
- Item 3. The method for transplanting the organoid according to Item 13, or the three-dimensional organ according to Item 15 or Item 16, into a human or non-human animal.
- the cell population in the present invention contains parathyroid cells and mesenchymal cells in predetermined proportions, for example, an organoid having excellent engraftment when transplanted can be produced from such a cell population. It will be possible.
- FIG. 1 is a schematic view showing an example (procedure of the embodiment) of the embodiment of the present invention.
- FIG. 2 is a graph showing the observed images of the parathyroid organoids obtained in the examples and the amount of parathyroid hormone (PTH) produced when the organoids were cultured in different calcium concentration environments.
- FIG. 3 is an immunostaining image (aSMA, PTH and DAPI) obtained in the examples.
- parathyroid cells refers to the parenchymal cells that make up the parathyroid glands. More specifically, “parathyroid cells” is a term that includes (collectively) main cells and oxyphil cells. The main cells may be transformed into acidophilic cells (differentiation conversion).
- the "colloidal thyroid cells" (main cells of the accessory thyroid and acidophilic cells of the accessory thyroid) in the present specification include (i) a predetermined function as the accessory thyroid cells that have differentiated and matured or reached terminal differentiation.
- Sexual cells referred to herein as “differentiated parathyroid cells”
- undifferentiated parathyroid cells or at the stage of stem or progenitor cells, cells that do not yet have sufficient predetermined functionality as parathyroid cells (collectively referred to herein as “undifferentiated parathyroid cells”). Both are included.
- the undifferentiated parathyroid cells include, for example, parathyroid precursor cells contained in Anterior foregut obtained by inducing differentiation from iPS cells and the like, as well as foregut endoderm cells, anterior foregut endoderm cells and the like. ..
- a cell is a differentiated parathyroid cell is one or more of a mature parathyroid marker, such as parathyroid hormone (PTH) produced by mature parathyroid main cells, or a calcium ion receptor expressed on the cell surface. It can be discriminated by whether the expression of two or more kinds is positive or negative (if it is positive, it is regarded as a differentiated parathyroid cell). On the other hand, whether or not a cell is an undifferentiated parathyroid cell can be determined by whether the expression of one or more cell markers such as CXCR4, EYA1, Six1, and Pax1 is positive or negative.
- PTH parathyroid hormone
- stromal cells refer to connective tissue cells that exist mainly in connective tissue derived from the mesoderm and form a support structure for cells that function in the tissue.
- Mesenchymal cells include differentiated cells (differentiated mesenchymal cells) and cells whose fate for differentiation into mesenchymal cells has been determined but have not yet differentiated into mesenchymal cells. Both (undifferentiated mesenchymal cells), so-called mesenchymal stem cells, are included.
- vascular endothelial cells are a type of cells that differentiate from undifferentiated mesenchymal cells, they are excluded from the definition of “mesenchymal cells” in the present specification.
- the undifferentiated mesenchymal cells also include, for example, cells existing around the Anterior foregut obtained by inducing differentiation from iPS cells or the like.
- a cell is an undifferentiated mesenchymal cell or a differentiated mesenchymal cell is, for example, Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, which are markers of undifferentiated mesenchymal cells. , CD271, Nestin, etc., can be discriminated by whether one or more of them are positive or negative (positive for undifferentiated mesenchymal cells, negative for differentiated mesenchymal cells). .. Whether or not a cell is a differentiated mesenchymal (stromal) cell depends on, for example, whether ⁇ -smooth muscle actin ( ⁇ SMA) is positive (if positive, it is a differentiated mesenchymal (stromal) cell). It can be identified (which is a cell).
- ⁇ SMA ⁇ -smooth muscle actin
- vascular endothelial cell is a concept of both hematopoietic vascular endothelial cell (HEC) and non-hemogenic endothelial cell (non-HEC). Is a term that includes.
- HEC is a vascular endothelial cell capable of producing hematopoietic stem cells (having hematopoietic ability), and is also called a blood cell-producing vascular endothelial cell.
- non-HEC is a vascular endothelial cell that does not have such hematopoietic capacity.
- Whether a cell is a vascular endothelial cell depends on whether one or more of the markers of the vascular endothelial cells such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3, CD41 are positive. It can be determined (if positive, it is vascular endothelial cell). Whether or not a cell is a differentiated vascular endothelial cell further depends on whether or not one or more of the markers such as CD31 and CD144 are positive (if positive, it is a differentiated vascular endothelial cell). And) can be determined.
- vascular endothelial cells in the present specification can also be read as “vascular cells” including vascular endothelial cells, vascular smooth muscle cells and pericytes.
- Hematopoietic vascular endothelial cells As used herein, the term “hematopoietic vascular endothelial cell (HEC)” means a hematopoietic vascular endothelial cell that is CD34-positive and CD73-negative as a cell marker for HEC. Further, the HEC used in the present invention may contain progenitor cells thereof. Such progenitor cells typically exist in the process of differentiation from progenitor cells (eg, lateral plate mesodermal cells) of vascular endothelial cells positive for the cell marker Flk-1 (CD309, KDR) to HEC cells. Examples include cells (see Cell Reports 2, 553-567, 2012).
- progenitor cells eg, lateral plate mesodermal cells
- Flk-1 CD309, KDR
- progenitor cells in the early stage of differentiation such as Flk-1 (CD309, KDR) positive are progenitor cells common to HEC cells and non-HEC cells, and unless otherwise specified, “HEC progenitor cells” are referred to as “HEC progenitor cells”. It also includes progenitor cells common to HEC cells and non-HEC cells.
- non-HEC -Non-hematopoietic vascular endothelial cells
- non-HEC non-hematopoietic vascular endothelial cell
- the term "non-hematopoietic vascular endothelial cell (non-HEC)” means a non-hematopoietic vascular endothelial cell that is positive for CD31, CD73 and CD144 as cell markers for non-HEC.
- the non-HEC used in the present invention may contain progenitor cells thereof.
- progenitor cells typically include progenitor cells of vascular endothelial cells positive for the cell marker Flk-1 (CD309, KDR) (eg, lateral plate mesodermal cells) in the process of differentiation from non-HEC cells.
- Flk-1 CD309, KDR
- progenitor cells in the early stage of differentiation such as Flk-1 (CD309, KDR) positive are progenitor cells common to HEC cells and non-HEC cells, and unless otherwise specified, "non-HEC precursor cells”. Also includes progenitor cells common to HEC cells and non-HEC cells.
- a "cell marker” is a gene that is specifically expressed (positive marker) or not expressed (negative marker) in a predetermined cell type, and is specifically by transcription of the gene in the genome.
- the cell marker is preferably labeled (stained) with a fluorescent substance, and is a protein expressed on the cell surface (cell surface marker) that can easily detect, concentrate, isolate, and the like the cells expressing the cell marker. Is.
- a marker gene is "positive” when the expression level of the mRNA or protein of the gene is detectable by a method commonly or known to those skilled in the art, or above a predetermined threshold (background level, etc.). It means high.
- a marker gene is "negative", the expression level of the mRNA or protein of the gene is undetectable or lower than a predetermined threshold (background level, etc.) by a method generally or known to those skilled in the art. Means that.
- Whether a cell marker is positive or negative can be determined with qualitative or quantitative results by a method generally or known to those skilled in the art.
- Cellular markers as proteins can be detected or their expression levels can be detected or measured using immunological assays using antibodies specific for the protein, such as ELISA, immunostaining, flow cytometry and the like.
- Cellular markers as mRNA are detected or their expression level is measured by utilizing an assay using nucleic acid specific to the mRNA, for example, RT-PCR (including quantitative PCR), microarray, biochip and the like. be able to.
- Cell composition refers to one having a predetermined cell composition (type and proportion) according to the present invention, which is prepared for embedding in a matrix (before cell aggregate formation).
- a “cell aggregate” is prepared from a “cell composition” prior to embedding in a matrix, or is obtained after embedding a “cell composition” in a matrix and culturing for some time (organoids). (Before formation), refers to pre-stage structures that do not have organoid-like structures or properties.
- Organoid refers to an artificially created organ or tissue-like tissue (three-dimensional structure).
- Organ organoids include not only those in the relatively mature stage of organoids that have functions and structures similar to various organs or tissues, but also “organ buds” in the early stages of such complications. Structures called “primordiums” are also included.
- Organ organoids include, for example, liver, pancreas, kidney, heart, lung, spleen, and esophagus, in addition to the "parathyroid organoid” (sometimes referred to simply as “organoid” herein), which is the subject of the present invention. , Stomach, thyroid gland, thoracic gland, gonad, brain, spinal cord, etc.
- the three-dimensional structure of organ organoids can be confirmed with the naked eye or by microscopic observation. In addition to confirming such a three-dimensional structure, depending on whether the markers corresponding to the cells contained in the organ, particularly the markers of the parenchymal cells of the organ, are positive, more preferably, the proteins of those markers are secreted into the culture supernatant. It can be determined by whether or not it is used.
- parathyroid hormone PTH
- PTH parathyroid hormone
- a "three-dimensional organ” is a structure containing a cell population or structure that is more mature than an organ organoid, which can be said to be a mature organ organoid.
- Whether or not a three-dimensional organ was obtained from an organ organoid is determined by, for example, the density of cells in the structure (whether it exceeds a predetermined standard, etc.), the three-dimensional shape of the structure (whether it is more three-dimensional than a certain level, etc.), and the like. Functions and traits (whether a predetermined function or trait such as metabolic function is acquired, etc.), cell marker (whether the expression of a gene or protein of the cell marker is positive, or whether the density of positive cells exceeds a predetermined standard, It can be determined from one or more viewpoints such as whether the amount of marker protein secreted in the culture supernatant exceeds a predetermined standard, etc.).
- the above-mentioned cell density, three-dimensional shape, function and trait, cell marker, etc. can be appropriately set according to the organ organoid and the three-dimensional organ, but for example, the same degree as the organ in the living body. Alternatively, whether or not a level close to that is achieved can be used as the basis for the above determination.
- the three-dimensional organ of the "parathyroid gland", which is the subject of the present invention, can be determined by, for example, whether or not it has a function of regulating the amount of PTH produced according to the calcium concentration in the culture environment.
- Each cell contained in the cell composition, cell aggregate and organ organoid of the present invention may be derived from human or mammals such as non-human animals such as mice, rats, dogs, pigs and monkeys. It may be of animal origin. For example, if the use of organ organoids is transplantation into humans or development of human drugs (detection of drugs that cause drug poisoning that are difficult to detect in conventional animal experiments or human cell tests, etc.), each cell. Is preferably derived from humans.
- the cell population of the present invention may contain parathyroid cells and mesenchymal cells in predetermined proportions, respectively, and may further contain vascular endothelial cells in arbitrary proportions, if necessary.
- the term “cell population” includes both the “cell composition” and the “cell aggregate” described above. That is, the “cell population” may refer to the “cell composition” or “cell aggregate” in the state before being embedded in the matrix, or the “cell composition” or the “cell composition” immediately after being embedded in the matrix. It may also refer to the "cell composition” obtained after culturing for a while.
- the "cell population” in the present specification may broadly refer to a cell population included in (constituting) the above-mentioned "organ organoid” and "three-dimensional organ".
- the proportion of parathyroid cells contained in the cell population of the present invention is 50% or more, preferably 70% or more.
- the proportion of mesenchymal cells contained in the cell population of the present invention is 5% or more, preferably 10% or more.
- the proportion of mesenchymal cells contained in the cell population of the present invention is, for example, 1% or more, preferably 2% or more.
- Parathyroid cells can include main cells, oxyphil cells, or both, and can also include differentiated parathyroid cells, undifferentiated parathyroid cells, or both.
- the ratio of each cell constituting the parathyroid cells is arbitrary and can be appropriately adjusted according to the embodiment.
- the parathyroid cells may be collected from a living body, or differentiate into pluripotent stem cells such as ES cells and iPS cells, and other parathyroid cells according to a known method or the method of the present invention. It may be made from capable cells.
- the parathyroid cells are contained in a cell aggregate (Anterior foregut in FIG. 1) obtained by inducing differentiation from iPS cells (or other cells having a predetermined differentiation potential).
- the parathyroid cells in such an embodiment mainly consist of undifferentiated parathyroid cells (parathyroid progenitor cells).
- Mesenchymal cells can include differentiated mesenchymal cells, undifferentiated mesenchymal cells, or both.
- the ratio of each cell constituting the mesenchymal cells is arbitrary and can be appropriately adjusted according to the embodiment.
- the mesenchymal cells may be collected from a living body, or according to a known method or the method of the present invention, to pluripotent stem cells such as ES cells and iPS cells, and other mesenchymal cells. It may be made from cells capable of differentiating.
- the mesenchymal cells are cell aggregates containing parathyroid cells obtained by inducing differentiation from iPS cells (or other cells having a predetermined differentiation potential) (Anterior in FIG. 1). It exists around foregut).
- the mesenchymal cells (stromal cells) in such an embodiment mainly consist of undifferentiated mesenchymal cells.
- the cell population of the present invention can be prepared by preparing parathyroid cells and mesenchymal cells (and, if necessary, vascular endothelial cells), and then mixing the cells at a predetermined ratio.
- the cell population is a pipetting operation or the like of parathyroid cells contained in cell aggregates obtained from iPS cells as described above and mesenchymal cells existing around the parathyroid cells. After dissociating once, the cells are mixed and prepared so that the number of each cell satisfies the above-mentioned predetermined ratio.
- Vascular endothelial cells can include hematopoietic vascular endothelial cells (HEC), non-hematopoietic vascular endothelial cells (non-HEC), or both.
- HEC hematopoietic vascular endothelial cells
- non-HEC non-hematopoietic vascular endothelial cells
- the ratio of each cell constituting the vascular endothelial cell is arbitrary and can be appropriately adjusted according to the embodiment.
- the vascular endothelial cells may be those collected from a living body (for example, microvessel endothelial cells (MVEC), umbilical-vein endothelial cells (UVEC), etc.) or ES cells. It may be obtained by differentiating pluripotent stem cells such as iPS cells and other cells having the ability to differentiate into vascular endothelial cells.
- MVEC microvessel endothelial cells
- UVEC umbilical-vein endothelial cells
- Hematopoietic vascular endothelial cells may be collected from a living body or have the ability to differentiate into pluripotent stem cells such as ES cells and iPS cells, and other vascular endothelial cells according to known methods. It may be obtained by differentiating the cells having (for example, lateral plate mesophyll lineage cells).
- pluripotent stem cells such as ES cells and iPS cells
- iPS cells vascular endothelial cells
- Non-hemogenic vascular endothelial cells may be collected from a living body, or differentiate into pluripotent stem cells such as ES cells and iPS cells, and other vascular endothelial cells according to known methods. It may be obtained by differentiating a cell having the ability to (for example, a side plate mesophyll lineage cell). For example, for a method of producing non-HEC from iPS cells, Nat Cell Biol. 2015; 17 (8): 994-1003, Cell Rep. 2017; 21 (10): 2661-2670 and the like can be referred to.
- the vascular endothelial cell is HEC or non-HEC obtained by inducing differentiation from iPS cells (or other cells having a predetermined differentiation potential).
- the matrix composition of the present invention comprises (1) a matrix and (2) the cell composition of the present invention described above.
- the extracellular matrix in the present invention, a general extracellular matrix that is solid at room temperature or higher and is used for cell culture, particularly three-dimensional cell culture, can be used.
- the extracellular matrix is basically composed mainly of fibrous protein and proteoglycan, and such components include elastin, entactin, osteonectin, collagen (type IV collagen, etc.), tenascin, thrombospondin, perlecan, and vitronectin. , Fibrillin, fibronectin, heparin (sulfate), laminin and the like.
- laminin for example, it contains laminin, collagen (type IV collagen) and entactin, known as the trade name "Matrigel” (Corning), and also contains growth factors such as EGF, IGF-1, PDGF and TGF- ⁇ .
- the basement membrane matrix is an example of a preferred matrix in the present invention.
- self-assembling peptides known as hydrogels such as hydrogels containing arginine, glycine and asparagine, can also be used as the matrix in the present invention.
- any one type may be used, or two or more types may be mixed or not mixed (so as to form different layers when solidified).
- the composition and concentration of the matrix can be appropriately adjusted by those skilled in the art so that the cells have an appropriate hardness after embedding the cell population.
- the matrix eg, Matrigel
- the matrix composition is immersed and embedded therein in the "culture system” and "culture method" of the present invention, which are described separately in the present invention.
- the same liquid medium for culturing the cell population can be used.
- the matrix composition generally comprises adding an appropriate amount of the matrix containing the appropriate components to a liquid medium containing a cell population and then solidifying the matrix (for solidifying the hydrogel, if necessary). It can be prepared by adding an ionic solution or an ionic molecule).
- a matrix composition can be obtained by stirring and mixing a matrix (eg, Matrigel) in a liquid state at 4 ° C. or lower and a cell population, and then allowing the cells to stand at 37 ° C. or higher to solidify the matrix.
- a matrix eg, Matrigel
- the matrix should be sufficiently firm by adding a sufficient amount of the matrix, and the cell population should not settle to the bottom. It is appropriate to keep it in the matrix.
- the hardness of the matrix can be adjusted, for example, in the range of 0.05 to 50 kPa.
- the use of the matrix composition of the present invention is not particularly limited, but it can be used, for example, for producing parathyroid organoids (further, three-dimensional organs of the parathyroid gland) by the culture method of the present invention.
- the matrix composition of the present invention is transplanted into a non-human animal (eg, mouse, rabbit, pig, dog, monkey) and differentiated and matured into a parathyroid organoid (further, a three-dimensional organ of the parathyroid gland) in the body. It can also be used to produce non-human chimeric animals.
- the matrix composition may be transplanted by peeling off what is formed on the breathable membrane, or may be transplanted as it is formed on the breathable membrane of the biodegradable material. You can also.
- the matrix composition can have a multi-layer structure.
- multilayer structures include those that include a first matrix and a second matrix, the first matrix enclosing the second matrix, and the first matrix having at least one opening.
- the compositions of the first matrix and the second matrix may be the same or different from each other.
- Each cell constituting the cell population of the present invention may be contained in the same matrix or may be contained in a different matrix.
- vascular endothelial cells vascular cells
- the "opening" of the first matrix refers to a portion that does not enclose the second matrix. That is, the second matrix can receive oxygen, nutrients, and other substances required by cells contained in the second matrix from the outside at the "opening" without going through the first matrix.
- the matrix composition having a multilayer structure as described above can be prepared according to a known method. For example, when it is formed on a breathable membrane (details will be described later), the second matrix is dropped onto the breathable membrane. Then, if desired, a method of dropping a first matrix containing cells onto the solidified second matrix and solidifying the cells can be mentioned. In the above manufacturing method, the contact portion between the second matrix and the breathable membrane is not covered by the first matrix and corresponds to the above "opening".
- the culture system of the present invention comprises the above-mentioned cell population or matrix composition of the present invention, a medium for culturing the cell population, and a culture vessel.
- the system of the present invention is typically for carrying out the culture method of the present invention described later, and the technical matters concerning the "medium” and the "culture container” defined in the system of the present invention are , Can be applied as a technical matter relating to the "medium” and “culture container” defined (used) in the culture method of the present invention.
- the "matrix composition" in the culture system of the present invention can be "in a state of being suspended from a breathable membrane", which will be described later in relation to the culture method of the present invention.
- the "medium” is usually a liquid medium, and an appropriate medium may be used depending on the cell population or the matrix composition (the cell population in the state of being embedded therein).
- an appropriate medium may be used depending on the cell population or the matrix composition (the cell population in the state of being embedded therein).
- a mixture of media suitable for culturing each cell contained in a cell population can be used as the medium in the culture system of the present invention.
- the media suitable for culturing the parathyroid cells and mesenchymal cells contained in the cell population of the present invention and the vascular endothelial cells contained as needed are known, and an appropriate medium is selected from the media. By mixing at an appropriate ratio, the medium in the culture system of the present invention can be prepared.
- basal medium for parathyroid cells examples include RPMI (Fujifilm), DMEM (Gibco), EGM (Lonza) and the like.
- Additives for parathyroid cells include, for example, activin A, BMP4, FGF4, GSK3 inhibitor (CHIR99021), TGFb / Smad signaling pathway inhibitor inhibitor (SB431542), BMP signaling pathway inhibitor (LDN193189), One or more selected from the group consisting of SHH, FGF8, FGF10, Wnt3 and CaCl 2 can be mentioned.
- a basal medium supplemented with a predetermined component in advance for example, Advanced DMEM (Gibco) can also be used.
- basal medium for vascular endothelial cells examples include DMEM / F-12 (Gibco), Stempro-34 SFM (Gibco), Essential 6 medium (Gibco), Essential 8 medium (Gibco), EGM (Lonza), and BulletKit ( Lonza), EGM-2 (Lonza), BulletKit (Lonza), EGM-2 MV (Lonza), VascuLife EnGS Comp Kit (LCT), Human Endothelial-SFM Basal Growth Medium (Invitrogen), Human Microvascular Endothelial Cell Growth Medium (Invitrogen) TOYOBO) and so on.
- Additives for vascular endothelial cells include, for example, B27 Supplements (GIBCO), BMP4 (bone formation factor 4), GSK ⁇ inhibitor (eg, CHIR99021), VEGF (vascular endothelial cell growth factor), FGF2 (Fibroblast Growth Factor).
- GBCO B27 Supplements
- BMP4 bone formation factor 4
- GSK ⁇ inhibitor eg, CHIR99021
- VEGF vascular endothelial cell growth factor
- FGF2 Fibroblast Growth Factor
- bFGF basic fibroblast growth factor
- Folskolin SCF (Stem Cell Factor), TGF ⁇ receptor inhibitor (eg SB431542), Flt-3L (Fms-related tyrosine kinase 3 ligand), IL-3 (Interleukin) 3), IL-6 (Interleukin 6), TPO (thrombopoietin), hEGF (recombinant human epithelial cell growth factor), hydrocortisone, ascorbic acid, IGF1, FBS (bovine fetal serum), antibiotics (eg, gentamycin, One or more selected from the group consisting of amphotericin B), heparin, L-glutamine, phenol red and BBE can be mentioned.
- SCF Stems-related tyrosine kinase 3 ligand
- IL-3 Interleukin 3
- IL-6 Interleukin 6
- TPO thrombopoietin
- hEGF recombin
- the medium comprises two or more components selected from the group consisting of SHH, FGF8, FGF10, Wnt3a, BMPi, activin and CaCl2.
- the combination of these components can be appropriately adjusted according to, for example, the culture stage of the cell population of the present invention (days from the start of culture, properties of the cell population, etc.).
- the above components can be added to the medium in the following combination. [I] SHH, FGF8, FGF10 and Wnt3a [Ii] SHH, FGF8 and BMPi [Iii] SHH and activin [iv] SHH, activin and CaCl 2
- the "culture container” (device for cell culture) is not particularly limited as long as it can culture a cell population or a matrix composition (a cell population embedded therein). not.
- Various culture vessels (or culture systems) for producing organoids, artificial organs, etc. from various cell populations are known, and the present invention also uses them, or the present invention is based on them. It can be used if it is adapted to.
- the culture vessel may be made of or surface treated with a material to which the cell population or matrix composition does not adhere.
- a plate with one or more wells can be used.
- cell aggregates can be formed by using, for example, a round bottom plate (Corning elplasia or the like). Further, various culture containers such as those for suspension culture, stirring suspension culture (spinner flask, etc.), and channel culture (organ-on-chip, etc.) can also be used.
- the system of the present invention is an instrument for holding a desired component according to an embodiment, for example, a breathable membrane (details will be described later) in a state in which a matrix composition is suspended.
- a culture device for performing suspension culture, stirring suspension culture, etc., a culture device for culturing at an appropriate atmosphere and temperature, and the like can be further provided.
- the culturing method of the present invention comprises a step of culturing the above-mentioned cell population of the present invention or the matrix composition of the present invention obtained by embedding the cell population in a matrix in a medium.
- the matrix composition is cultured in a suspended state on the breathable membrane.
- the "breathable membrane” is a membrane having at least oxygen permeability and, if necessary, carbon dioxide and other desired gas permeability.
- Various breathable films are known, for example, polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), fluorocarbon, polytetrafluoroethylene (PTFE), fibers such as polyurethane, or in vivo decomposition of collagen and the like.
- PET polyethylene terephthalate
- PDMS polydimethylsiloxane
- fluorocarbon fluorocarbon
- PTFE polytetrafluoroethylene
- fibers such as polyurethane, or in vivo decomposition of collagen and the like.
- -Examples are films made of absorbent materials.
- the breathable membrane may be surface-treated to increase or decrease cell adhesion, if necessary, and may be coated with ECM such as collagen.
- the breathable membrane may be a laminated membrane (hybrid membrane) with
- the matrix composition "suspended on the breathable membrane” is a solidified matrix composition in which the matrix composition before solidification is dropped onto the breathable membrane, solidified, and then adhered to the breathable membrane. It can be formed by turning the object upside down so that it faces downward (convex downward).
- the matrix composition is immersed in the medium while the breathable membrane is immersed.
- the composition of the medium and various culture conditions can be appropriately adjusted according to the purpose.
- the culturing method of the present invention is carried out in a medium containing the appropriate components (as necessary, the composition of the medium) until organoids are formed from the cell population (cell composition or cell aggregate). It can be carried out for a sufficient period of time (eg, 1-10 days), at a suitable temperature (eg, 30-40 ° C, preferably about 37 ° C), and at a suitable CO 2 concentration (eg, 5%). ..
- the culture method of the present invention is typically for producing organoids of the parathyroid gland.
- the organoid of the present invention is obtained by the culture method of the present invention.
- the culture method of the present invention can be further carried out (as part of the process of the production method) for producing a three-dimensional organ of the parathyroid gland.
- the organoid of the present invention is obtained by a production method including a step of carrying out the culture method of the present invention.
- composition of the medium and various culture conditions (atmosphere, temperature, period, etc.) in the method for producing a three-dimensional organ of the present invention can be basically the same as those in the above-mentioned culture method of the present invention. , If necessary, can be appropriately adjusted, for example, a sufficient culture period for the formation of a three-dimensional organ.
- the cell population of the present invention should be cultured for 20 to 40 days from the day when the culture of the cell population of the present invention is started in order to produce the parathyroid organoid, or the three-dimensional structure as a parathyroid organoid. From the day when the formation is confirmed, culturing for another 10 to 20 days for maturity may be mentioned.
- a three-dimensional organ can be obtained by transplanting the cell population or matrix composition of the present invention or the organoid of the parathyroid gland obtained by the culture method of the present invention into a non-human animal and maturing it.
- the parathyroid organoids and three-dimensional organs of the present invention can be transplanted into human or non-human animals.
- an artificial organ can be created by connecting with the extracorporeal circulation.
- Such artificial organs can be used as a model of organ failure or used to evaluate the function of an organ.
- WO2013 / 047720 can be referred to.
- the organoids and three-dimensional organs of the present invention obtained as described above may be used, for example, for transplantation into human or non-human animals to treat or prevent diseases associated with increased or decreased parathyroid function. Can be done.
- diseases related to hyperparathyroidism include primary hyperparathyroidism, secondary hyperparathyroidism, and hypoparathyroidism.
- the organoids, three-dimensional organs, and non-human chimeric animals of the present invention obtained as described above are, for example, agents (formulated forms) for treating or preventing diseases related to the enhancement or decrease of parathyroid function. Or it can be used to carry out various evaluation methods such as prediction of human drug metabolism profile, drug efficacy evaluation, toxicity evaluation, drug interaction evaluation, etc. for drugs (form before formulation, active ingredient). can.
- the "drug” is not particularly limited as long as it acts on organoids, three-dimensional organs, non-human chimeric animals, or is used for analyzing whether or not it acts, and can be selected according to the purpose.
- the “drug” is not particularly limited as long as it can be used as an active ingredient of a drug such as a small molecule drug, an antibody drug, a peptide drug, and a nucleic acid drug. Further, the “drug” can be obtained by using the “drug” as an active ingredient, combining it with an additive or the like according to a desired dosage form, and formulating it by a general method.
- the method for evaluating a drug using an organoid or a three-dimensional organ of the present invention in vitro is, for example, a step of culturing an organoid or a three-dimensional organ in a culture medium supplemented with the drug to be evaluated, and an organoid or a three-dimensional organ. It includes a step of evaluating desired items such as efficacy and safety of a drug for an organ.
- a method for evaluating a drug using a non-human chimeric animal equipped with a three-dimensional organ of the present invention includes, for example, a step of administering the drug to be evaluated to the non-human chimeric animal, and a non-human chimeric animal (a three-dimensional organ provided with the three-dimensional organ). Includes a step of evaluating desired items such as efficacy and safety of the drug.
- the medium was Actibin A (100 ng / mL) and Actibin A (100 ng / mL) in a basal medium (hereinafter referred to as basal medium A) in which 1% Penicillin-Streptomycin, HEPES (10 mM) (all Gibco) was added to RPMI (Fujifilm) (2 ml).
- basal medium A 1% Penicillin-Streptomycin, HEPES (10 mM) (all Gibco) was added to RPMI (Fujifilm) (2 ml).
- the cells were replaced with BMP4 (50 ng / mL) added and cultured at 5% CO 2 , 37 ° C. for 1 day (D1-D2).
- basal medium A (2 ml) supplemented with activin A (100 ng / mL) and 0.2% FBS gold (MP Biomedicals), culture at 5% CO 2 , 37 ° C for 1 day, and then add FBS.
- the medium was replaced with a similar medium in which gold was changed to 2%, and the cells were cultured for another day (D2-D4).
- the medium was a basal medium (hereinafter referred to as basal medium B) in which 1% Penicillin-Streptomycin, HEPES (10 mM) 2% B27, 1% N2, and 1% GlutaMAX (all Gibco) were added to Advanced DMEM (Gibco) (2 ml).
- FGF4 100 ng / mL
- GSK3 inhibitor CH1 + TGFb / Smad signaling pathway inhibitor
- SB431542 TGFb / Smad signaling pathway inhibitor
- BMP signaling pathway inhibitor LDN193189
- [2] Cell population preparation step and matrix composition preparation step
- Anterior foregut cell population containing the mesenchymal cells obtained in the above [1] is separated by a pipetting operation, collected in a 1.5 mL tube, and then further.
- Anterior foregut cells and mesenchymal cells were isolated by separating into 20-100 ⁇ m cell clamps or spheroids by repeating the pipetting operation about 10 to 20 times.
- matrix preparation was prepared by mixing the above-mentioned Anterior foregut medium and Matrigel (BD Harmingen) in a volume ratio of 1: 1.
- the isolated parathyroid cells and mesenchymal cells were mixed in a matrix preparation at 4 ° C. 9 ⁇ L of the obtained mixed solution was dropped onto the second matrix layer formed as described above, and the mixture was solidified by raising the temperature to 37 ° C. to form a “first matrix layer”.
- the medium was a mixture of basal medium B and EGM (Lonza) in a volume ratio of 1: 1 (hereinafter referred to as "basal medium for parathyroid organoids"), SHH (50 ng / mL), FGF8 (50 ng /). mL), FGF10 (50 ng / mL) and Wnt3a (50 ng / mL) were added, and the cells were cultured at 5% CO 2 , 37 ° C. for 2 days (D9-D11) to obtain pharyngeal pouches.
- the medium was replaced with a basal medium for organoids supplemented with SHH (100 ng / mL), FGF8 (50 ng / mL) and LDN 193189 (5 ⁇ M), and cultured at 5% CO 2 , 37 ° C. for 10 days (D11-D21). ).
- the medium was replaced with a basal medium for organoids supplemented with SHH (100 ng / mL) and actibin (100 ng / mL) (hereinafter referred to as "media for maturation of parathyroid organoids"), 5% CO 2 , 37 ° C. was cultured for 4 days (D21-D25) and matured into Parathyroid (parathyroid) organoids.
- SHH 100 ng / mL
- actibin 100 ng / mL
- Parathyroid organoid calcium-free medium 5% CO 2 , 37. It was cultured at ° C for 2 days (D25-D27) to promote the production of Parathyroid organoid parathyroid hormone (PTH).
- PTH Parathyroid organoid parathyroid hormone
- the hanging drop insert to which the matrix composition containing the organoid cultured at 5% CO 2 , 37 ° C. was taken out was taken out, and the organoid was peeled off by a cell scraper.
- Organoids were placed in a 2 mL tube containing a 4% paraformaldehyde / PBS fixation solution and fixed by allowing to stand at 4 ° C. for 2 hours. After fixation, the fixation solution was removed and then replaced with PBS.
- the fixed organoids were cleared and immunofluorescently stained using SCALEVIEW-S (Fuji Film Wako).
- the implementation method used was the AbSca / e method (https://labchem-wako.fujifilm.com/jp/category/00593.html).
- Primary antibody for immunofluorescent staining anti-alpha smooth muscle Actin (aSMA) antibody (Abcam, ab7817), anti-PTH antibody (Abcam, ab166631) and fluorescently labeled secondary antibody (Novex Donkey anti-Mouse) that binds to these antibodies.
- aSMA anti-alpha smooth muscle Actin
- Abcam Abcam, ab166631
- fluorescently labeled secondary antibody Novex Donkey anti-Mouse
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Abstract
Description
各種の臓器の疾患に対する医薬品開発や再生医療の実現を目指すために、培養細胞を自己組織化させ、それぞれの臓器の三次元的な構造や、血管、胆管、その他の脈管の複雑な構造を再現した細胞構造体、すなわち臓器オルガノイドを作製するための研究開発が進められている。そのような臓器オルガノイドの発展的な細胞構造体として、未分化の細胞を含む特定の種類の細胞を組み合わせて培養することにより、発生の初期に形成される臓器の原基(器官芽)を作製することも可能となっている。このような「ミニ臓器」とも呼べるオルガノイド等は、従来の単独で培養された臓器の細胞等に比べて、薬効または毒性の評価が生体により近い評価系として利用することができる上、移植手術や、血漿タンパク質の産生にも利用することができるなど、極めて利用価値が高い。
[1]
副甲状腺細胞を50%以上の割合で、かつ間葉系細胞を10%以上の割合で含む、細胞集団。
[1A]
副甲状腺細胞を50%以上の割合で、かつ間葉系細胞を5%以上の割合で含む、細胞集団。
[2]
さらに血管内皮細胞を含む、項1に記載の細胞集団。
[3]
マトリックスと、当該マトリックス中に包埋された状態の項1に記載の細胞集団とを含む、マトリックス組成物。
[4]
項1に記載の細胞集団、あるいは請求項3に記載のマトリックス組成物と、細胞集団培養用の培地と、培養容器とを備えた、培養システム。
[5]
前記培養容器が、ラウンドボトムプレートである、項4に記載の培養システム。
[5A]
前記培養容器が、浮遊培養用、撹拌浮遊培養用または流路培養用のものである、項4に記載の培養システム。
[6]
前記培地が、SHH、FGF8、FGF10、Wnt3a、LDN193189、アクチビンおよびCaCl2からなる群より選ばれる2以上の成分を含む、項4に記載の培養システム。
[7]
副甲状腺細胞を50%以上の割合で、かつ間葉系細胞を10%以上の割合で含む細胞集団を培地中で培養する工程を含む、培養方法。
[8]
さらに血管内皮細胞を含む、項7に記載の培養方法。
[9]
前記細胞集団をマトリックス中に包埋し、得られたマトリックス組成物を培養する、項7に記載の培養方法。
[10]
前記細胞集団が包埋されているマトリックス組成物を、通気性膜に懸下させた状態で培養する、項9に記載の培養方法。
[11]
前記細胞集団をラウンドボトムプレートで培養する、項7に記載の培養方法。
[12]
前記培地が、SHH、FGF8、FGF10、Wnt3a、LDN193189、アクチビンおよびCaCl2からなる群より選ばれる2以上の成分を含む、項7に記載の培養方法。
[13]
項7~12のいずれか一項に記載の培養方法により得られたオルガノイド。
[14]
項7~12のいずれか一項に記載の培養方法を実施する工程を含む、立体臓器の製造方法。
[15]
項14に記載の製造方法により得られた立体臓器。
[16]
項3に記載のマトリックス組成物、あるいは項7~12のいずれか一項に記載の培養方法により得られたオルガノイドを、非ヒト動物に移植して成熟化させることにより得られた立体臓器。
[17]
項13に記載のオルガノイド、あるいは項15または項16に記載の立体臓器を使った、副甲状腺機能の亢進または低下に関係する疾患を治療または予防するための薬物または薬剤の評価方法。
[18]
項13に記載のオルガノイド、あるいは項15または項16に記載の立体臓器を、ヒトまたは非ヒト動物に移植する方法。
・副甲状腺細胞
本明細書において「副甲状腺細胞」は、副甲状腺を構成している実質細胞を指す。より具体的には、「副甲状腺細胞」は、主細胞および好酸性細胞を包含する(総称する)用語である。なお、主細胞は好酸性細胞に変化(分化転換)することもある。
本明細書において「間葉系細胞」(間質細胞)は、主として中胚葉に由来する結合組織に存在し、組織で機能する細胞の支持構造を形成する結合組織細胞を指す。本明細書における「間葉系細胞」には、分化した細胞(分化間葉系細胞)と、間葉系細胞への分化運命が決定しているがまだ間葉系細胞へ分化していない細胞(未分化間葉系細胞)、いわゆる間葉系幹細胞の両方が包含される。ただし、「血管内皮細胞」は未分化間葉系細胞から分化する細胞の一種であるが、本明細書における「間葉系細胞」の定義から除外されるものとする。この未分化間葉系細胞(間質細胞)には、例えば、iPS細胞等からの分化誘導により得られるAnterior foregutの周囲に存在する細胞も包含される。
本明細書において「血管内皮細胞」とは、造血性血管内皮細胞(hemogenic endothelial cell;HEC)および非造血性血管内皮細胞(non-hemogenic endothelial cell;non-HEC)の両方の概念を包含する用語である。HECは、造血幹細胞を産生することのできる(造血能を有する)血管内皮細胞であり、血球産生型血管内皮細胞とも呼ばれる。一方で、non-HECは、そのような造血能を有さない血管内皮細胞である。
本明細書において「造血性血管内皮細胞(HEC)」とは、HECの細胞マーカーとしてCD34陽性かつCD73陰性である、造血能を有する血管内皮細胞を意味する。また、本発明に用いられるHECは、その前駆細胞を含んでいてもよい。このような前駆細胞としては、代表的には、細胞マーカーFlk-1(CD309、KDR)陽性の血管内皮細胞の前駆細胞(例、側板中胚葉系細胞)からHEC細胞までの分化過程に存在する細胞が挙げられる(Cell Reports 2, 553-567, 2012参照)。なお、Flk-1(CD309、KDR)陽性のような分化早期の段階の前駆細胞は、HEC細胞とnon-HEC細胞で共通する前駆細胞であり、特に述べない限り、「HEC前駆細胞」は、HEC細胞とnon-HEC細胞で共通する前駆細胞も含む。
本明細書において「非造血性血管内皮細胞(non-HEC)」とは、non-HECの細胞マーカーとしてCD31、CD73およびCD144が陽性である、造血能を有しない血管内皮細胞を意味する。また、本発明に用いられるnon-HECは、その前駆細胞を含んでいてもよい。このような前駆細胞としては、代表的には、細胞マーカーFlk-1(CD309、KDR)陽性の血管内皮細胞の前駆細胞(例、側板中胚葉系細胞)からnon-HEC細胞までの分化過程に存在する細胞が挙げられる(Cell Reports 2, 553-567, 2012参照)。なお、Flk-1(CD309、KDR)陽性のような分化早期の段階の前駆細胞は、HEC細胞とnon-HEC細胞で共通する前駆細胞であり、特に述べない限り、「non-HEC前駆細胞」は、HEC細胞とnon-HEC細胞で共通する前駆細胞も含む。
本明細書において「細胞マーカー」は、所定の細胞型において特異的に発現する(陽性マーカー)または発現しない(陰性マーカー)遺伝子であり、具体的にはゲノム中の当該遺伝子の転写によるmRNAとして、またはそのmRNAの翻訳によるタンパク質として、生成する(陽性マーカー)または生成しない(陰性マーカー)物質を指す。細胞マーカーは、好ましくは蛍光物質により標識(染色)可能であり、当該細胞マーカーを発現している細胞の検出、濃縮、単離等を容易に行える、細胞表面に発現するタンパク質(細胞表面マーカー)である。
本明細書において「細胞組成物」は、マトリックスに包埋するために調製される(細胞凝集体形成前の)、本発明に従う所定の細胞組成(種類および割合)を有するものを指す。
本明細書において「細胞凝集体」は、マトリックスに包埋される前に「細胞組成物」から調製される、またはマトリックスに「細胞組成物」を包埋してしばらく培養した後に得られる(オルガノイド形成前の)、オルガノイドのような構造や特性を有さない前段階の構造体を指す。
「臓器オルガノイド」は、人為的に創出された臓器または組織に類似した組織体(三次元構造体)を指す。「臓器オルガノイド」には、各種の臓器または組織に類似した機能や構造を有する、オルガノイドとして比較的成熟した段階にあるもののみならず、そのような複雑化の初期段階にある「器官芽」、「原基」などと呼ばれている構造体も包含される。臓器オルガノイドには、本発明の対象である「副甲状腺オルガノイド」(本明細書では単に「オルガノイド」と表記することがある。)の他、例えば肝臓、膵臓、腎臓、心臓、肺臓、脾臓、食道、胃、甲状腺、胸腺、生殖腺、脳、脊髄など、様々な種類の臓器オルガノイドが公知になっている(例、https://www.nejm.org/doi/pdf/10.1056/NEJMra1806175、https://www.nature.com/articles/s41568-018-0007-6、http://www.amsbio.com/brochures/organoid-culture-handbook.pdf参照)。
「立体臓器」は、成熟臓器オルガノイドともいえる、臓器オルガノイドよりも一層成熟した細胞集団または構造を含む構造体である。
本発明の細胞集団は、副甲状腺細胞および間葉系細胞をそれぞれ所定の割合で含み、必要に応じてさらに血管内皮細胞を任意の割合で含んでもよい。本明細書における「細胞集団」は、前述した「細胞組成物」および「細胞凝集体」の両方を包含する。すなわち、「細胞集団」は、マトリックスに包埋する前の状態の「細胞組成物」または「細胞凝集体」を指す場合もあるし、マトリックスに包埋された直後の「細胞組成物」またはそれをしばらく培養した後に得られる「細胞組成物」を指す場合もある。また、本明細書における「細胞集団」は、広義には、前述した「臓器オルガノイド」および「立体臓器」に含まれる(それらを構成している)細胞集団を指す場合もある。
「副甲状腺細胞」は、主細胞、好酸性細胞、またはその両方を含むことができ、また分化副甲状腺細胞、未分化副甲状腺細胞、またはその両方を含むことができる。副甲状腺細胞を構成する各細胞の比率は任意であり、実施形態に応じて適宜調節することができる。
「間葉系細胞」は、分化間葉系細胞、未分化間葉系細胞、またはその両方を含むことができる。間葉系細胞を構成する各細胞の比率は任意であり、実施形態に応じて適宜調節することができる。
「血管内皮細胞」は、造血性血管内皮細胞(HEC)、非造血性血管内皮細胞(non-HEC)、またはその両方を含むことができる。血管内皮細胞を構成する各細胞の比率は任意であり、実施形態に応じて適宜調節することができる。
本発明のマトリックス組成物は、(1)マトリックスと、(2)上述した本発明の細胞組成物とを含む。
本発明の培養システムは、前述した本発明の細胞集団またはマトリックス組成物と、細胞集団培養用の培地と、培養容器とを備える。なお、本発明のシステムは、典型的には、後述する本発明の培養方法を実施するためのものであり、本発明のシステムにおいて規定される「培地」および「培養容器」に関する技術的事項は、本発明の培養方法において規定される(使用される)「培地」および「培養容器」に関する技術的事項として適用することができる。逆に、本発明の培養システムにおける「マトリックス組成物」は、本発明の培養方法との関係で後述する、「通気性膜に懸下させた状態」のものとすることができる。
「培地」は、通常は液体培地であり、細胞集団またはマトリックス組成物(そこに包埋された状態の細胞集団)に応じた適切なものを用いればよい。一般的には、細胞集団に含まれる細胞それぞれの培養に適した培地を混合したものを、本発明の培養システムにおける培地として用いることができる。本発明の細胞集団に含まれる副甲状腺細胞および間葉系細胞、ならびに必要に応じて含まれる血管内皮細胞、それぞれの培養に適した培地は公知であり、その中から適切な培地を選択し、適切な割合で混合することにより、本発明の培養システムにおける培地を調製することができる。
[i]SHH、FGF8、FGF10およびWnt3a
[ii]SHH、FGF8およびBMPi
[iii]SHHおよびアクチビン
[iv]SHH、アクチビンおよびCaCl2
「培養容器」(細胞培養用デバイス)は、細胞集団またはマトリックス組成物(そこに包埋された状態の細胞集団)を培養することができるものであれば、特に限定されるものではない。各種の細胞集団からオルガノイド、人工臓器等を作製するための培養容器(または培養システム)としては様々なものが公知になっており、本発明でもそれらを利用すること、またはそれらに準じて本発明に適合させたもの利用することができる。例えば、培養容器は、細胞集団またはマトリックス組成物が付着しないような材質で作製されているまたは表面処理がされているものであってもよい。マトリックス組成物に包埋された状態の細胞集団を培養する場合は、そのマトリックス組成物(後述するように、通気性膜に懸下された状態であってもよい。)の形状やサイズに対応したウェルを1つまたは複数備えたプレートを用いることができる。マトリックス組成物に包埋されていない状態の細胞集団を培養する場合は、例えば、ラウンドボトムプレート(Corning elplasia等)を用いることにより、細胞凝集体を形成させることができる。さらに、浮遊培養用、撹拌浮遊培養用(スピナーフラスコ等)、流路培養用(organ-on-chip等)のような、様々な培養容器を利用することもできる。
本発明の培養方法は、前述した本発明の細胞集団、またはそれをマトリックス中に包埋して得られた本発明のマトリックス組成物を、培地中で培養する工程を含む。
本発明の好ましい一実施形態において、マトリックス組成物は、通気性膜に懸下させた状態で培養される。
ヒトiPS細胞(1383D2もしくは511-3E;京都大学iPS研究所)を、0.5μg/mL iMatrix 511でコーティングした24ウェルプレートに1.5-1.8×10^5cells/wellで播種し、ROCK阻害剤(Y-27632, Wako)(10μM)を加えたAK02N(味の素)(8 ml)の下、5%CO2、37℃で24時間前培養を行い、ROCK阻害剤を含まないAK02Nに交換し、1日間培養した(D0-D1)。培地を、RPMI(富士フイルム)(2ml)に1% Penicillin-Streptomycin、HEPES(10mM)(全てGibco)を添加した基礎培地(以下、基礎培地Aと呼ぶ。)にアクチビンA(100ng/mL)およびBMP4(50ng/mL)を添加したものに交換し、5%CO2、37℃で1日間培養した(D1-D2)。培地を、基礎培地A(2ml)にアクチビンA(100ng/mL)、0.2% FBS gold(MP Biomedicals)を添加したものに交換し、5%CO2、37℃で1日間培養後、添加するFBS goldを2%に変更した同様の培地に交換し、さらに1日間培養した(D2-D4)。培地を、Advanced DMEM(Gibco)(2 ml)に1% Penicillin-Streptomycin、HEPES(10mM)2% B27、1% N2、1% GlutaMAX(全てGibco)を加えた基礎培地(以下、基礎培地Bと呼ぶ。)にFGF4(100ng/mL)、GSK3阻害剤(CHIR99021)(1μM)、TGFb/Smadシグナル伝達経路阻害剤(SB431542)(5μM)およびBMPシグナル伝達経路阻害剤(LDN193189)(5μM)を添加したもの(以下「Anterior foregut用培地」と呼ぶ。)に交換し、5%CO2、37℃で3日間培養し(D4-D7)、間葉系細胞を含むAnterior foregut細胞集団を得た。
上記[1]で得られた間葉系細胞を含むAnterior foregut細胞集団をピペッティング操作により乖離して、1.5mLチューブに回収後、さらに10-20回程度ピペッティング操作をくり返すことにより、20-100μmの細胞クランプもしくはスフェロイドに分離することで、Anterior foregut細胞および間葉系細胞を単離した。
低吸着24-well plateに、上記Anterior foregut用培地を600μL加えた。上記のようにしてhanging drop insert上に形成した、第一~第二マトリックス層からなるマトリックス組成物を、ウェル内の培地方向に向けて挿入し、5%CO2、37℃で2日間培養した(D7-D9)。
Claims (18)
- 副甲状腺細胞を50%以上の割合で、かつ間葉系細胞を10%以上の割合で含む、細胞集団。
- さらに血管内皮細胞を含む、請求項1に記載の細胞集団。
- マトリックスと、当該マトリックス中に包埋された状態の請求項1に記載の細胞集団とを含む、マトリックス組成物。
- 請求項1に記載の細胞集団、あるいは請求項3に記載のマトリックス組成物と、細胞集団培養用の培地と、培養容器とを備えた、培養システム。
- 前記培養容器が、ラウンドボトムプレートである、請求項4に記載の培養システム。
- 前記培地が、SHH、FGF8、FGF10、Wnt3a、LDN193189、アクチビンおよびCaCl2からなる群より選ばれる2以上の成分を含む、請求項4に記載の培養システム。
- 副甲状腺細胞を50%以上の割合で、かつ間葉系細胞を10%以上の割合で含む細胞集団を培地中で培養する工程を含む、培養方法。
- さらに血管内皮細胞を含む、請求項7に記載の培養方法。
- 前記細胞集団をマトリックス中に包埋し、得られたマトリックス組成物を培養する、請求項7に記載の培養方法。
- 前記細胞集団が包埋されているマトリックス組成物を、通気性膜に懸下させた状態で培養する、請求項9に記載の培養方法。
- 前記細胞集団をラウンドボトムプレートで培養する、請求項7に記載の培養方法。
- 前記培地が、SHH、FGF8、FGF10、Wnt3a、LDN193189、アクチビンおよびCaCl2からなる群より選ばれる2以上の成分を含む、請求項7に記載の培養方法。
- 請求項7~12のいずれか一項に記載の培養方法により得られたオルガノイド。
- 請求項7~12のいずれか一項に記載の培養方法を実施する工程を含む、立体臓器の製造方法。
- 請求項14に記載の製造方法により得られた立体臓器。
- 請求項3に記載のマトリックス組成物、あるいは請求項7~12のいずれか一項に記載の培養方法により得られたオルガノイドを、非ヒト動物に移植して成熟化させることにより得られた立体臓器。
- 請求項13に記載のオルガノイド、あるいは請求項15または16に記載の立体臓器を使った、副甲状腺機能の亢進または低下に関係する疾患を治療または予防するための薬物または薬剤の評価方法。
- 請求項13に記載のオルガノイド、あるいは請求項15または16に記載の立体臓器を、ヒトまたは非ヒト動物に移植する方法。
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JP2013524836A (ja) * | 2010-04-25 | 2013-06-20 | マウント・シナイ・スクール・オブ・メディスン | 多能性細胞からの前部前腸内胚葉の生成 |
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