WO2015129764A1 - 目的細胞の純度が高い細胞集団の製造方法 - Google Patents
目的細胞の純度が高い細胞集団の製造方法 Download PDFInfo
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
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- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
Definitions
- the present invention relates to a method for producing a cell population with high purity of a target cell, a cell population obtained by the method, a pharmaceutical composition containing the cell population, and the like.
- Non-Patent Document 1 fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, etc. have been tried to repair myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction.
- Patent Document 1 cell structures formed using scaffolds and sheet-shaped cell cultures in which cells are formed into sheets have been developed.
- sheet cell culture For the application of sheet cell culture to the treatment, use of cultured epidermis sheet for skin damage caused by burns, use of corneal epithelial sheet cell culture for corneal injury, oral mucosa sheet for endoscopic resection of esophageal cancer Studies such as the use of cell cultures are ongoing.
- Cells used for cell transplantation are usually isolated from the tissue to be transplanted in order to avoid adverse events such as rejection, but the tissue often contains cells other than the target cell.
- the tissue often contains cells other than the target cell.
- cells other than the target cells proliferate, and the ratio of the target cells to the cells used for transplantation is low.
- the therapeutic effect is not as good as when the target cells are transplanted. Therefore, if the purity of the target cells is low, there is a possibility that sufficient therapeutic effects cannot be obtained even if cells obtained from tissues are transplanted.
- the skeletal muscle tissue is immersed in a proteolytic enzyme solution for a predetermined time as a measure for increasing the proportion of target cells contained in the cell population separated from the tissue.
- An object of the present invention is obtained by a method for producing a cell population having a high purity of a target cell from a cell population containing the target cell and a contaminated cell, in particular, a method for easily and efficiently producing such a cell population.
- the object is to provide a cell population with high purity of the target cell, a pharmaceutical composition containing the cell population, and the like.
- the present inventor is diligently researching to solve the above problems, in the cell population including the target cell and the contaminating cell, the trait of the contaminating cell is changed, and the contaminating cell is removed based on the changed trait, In addition, the inventors have found that by collecting target cells, a cell population having high target cell purity can be produced, and the present invention has been completed.
- the present invention relates to the following.
- ⁇ 1> (1) a step of changing the character of the contaminating cell in a cell population including the target cell and the contaminating cell; and (2) removing the contaminating cell based on the changed character and / or the target cell.
- a method for producing a cell population having a high purity of a target cell comprising a step of collecting.
- ⁇ 2> The method according to ⁇ 1> above, wherein the contaminating cells are fibroblasts.
- ⁇ 3> The method according to ⁇ 1> or ⁇ 2> above, wherein the character is selected from the group consisting of cell shape, size, specific gravity, surface charge, adhesion ability, and marker.
- ⁇ 4> The method according to any one of ⁇ 1> to ⁇ 3> above, wherein the target cell is a skeletal myoblast or a mesenchymal stem cell.
- ⁇ 5> A cell population produced by the method according to any one of ⁇ 1> to ⁇ 4> above, wherein the target cell has high purity.
- ⁇ 6> A method for producing a sheet-shaped cell culture, comprising the step of culturing the cell population according to ⁇ 5> above in a sheet shape.
- ⁇ 7> A sheet-shaped cell culture comprising the cell population according to ⁇ 5> above.
- ⁇ 8> A pharmaceutical composition comprising an active ingredient selected from the group consisting of the cell population according to ⁇ 5> and the sheet-shaped cell culture according to ⁇ 7>.
- the present invention makes it possible to increase the purity of target cells contained in a cell population containing a plurality of types of cells collected from a living body, increase the therapeutic effect by cell transplantation, etc., and produce a cell composition used for such treatment. Increase efficiency.
- cell size, specific gravity, surface charge, etc. as the character to be changed, it becomes possible to use a simple method such as filtration and centrifugation that can process a large amount of cells at once. It can be purified efficiently.
- FIG. 1 is a diagram showing the basic concept of the present invention.
- One aspect of the present invention is (1) a step of changing a character of a contaminating cell in a cell population containing a target cell and a contaminating cell, and (2) removing the contaminating cell based on the changed character, and / or And a method for producing a cell population having a high purity of the target cell (hereinafter, sometimes abbreviated as “cell population production method”), including a step of collecting the target cell.
- target cells and contaminating cells can be arbitrarily set according to the use of the obtained cell population. Since the cell population containing the target cell and the contaminating cell is typically prepared from a tissue collected from a living body, the target cell and the contaminating cell are typically selected from cells existing in the same tissue. For example, since a cell population prepared from skeletal muscle tissue is mainly composed of skeletal myoblasts and fibroblasts, one of these can be a target cell and the other can be a mixed cell.
- the target cell is a skeletal myoblast and the contaminating cell is a fibroblast
- the target cell is a fibroblast and the contaminating cell is a skeletal myoblast.
- the cell population prepared from bone marrow tissue is mainly composed of mesenchymal stem cells, hematopoietic stem cells, blood progenitor cells, fibroblasts, etc., any one or more of these cells are targeted cells, others Can be used as a mixed cell.
- the target cell is a mesenchymal stem cell
- the contaminating cells are hematopoietic stem cells, blood precursor cells, and fibroblasts
- the target cell is mesenchymal.
- Stem cells, hematopoietic stem cells, and contaminating cells may be blood precursor cells and fibroblasts.
- Target cells and contaminating cells can be selected from a part of cell types existing in living tissue. That is, it is not necessary to distribute all the cell types present in the living tissue into target cells and contaminating cells in the present invention, and cell types that can be easily separated from the target cells without changing the character are pre-treated by pretreatment.
- the target cells and contaminating cells can be selected from the cell types remaining after the pretreatment.
- Examples of the pretreatment include, but are not limited to, filtration (for example, filtration through a filter, a microchannel, etc.), centrifugation (for example, density gradient centrifugation, equal density centrifugation, countercurrent centrifugation (Countercurrent centrifugal elutriation): CCE), etc.), and separation of adherent cells and suspension cells by adhesion culture.
- filtration for example, filtration through a filter, a microchannel, etc.
- centrifugation for example, density gradient centrifugation, equal density centrifugation, countercurrent centrifugation (Countercurrent centrifugal elutriation): CCE), etc.
- CCE countercurrent centrifugation
- the cell population production method of the present invention may further include a step of removing cells other than the target cell and the contaminating cell from the tissue collected from the living body before the step of changing the character of the contaminating cell.
- the target cells are not limited, and are used for treatment of blood diseases, for example.
- Hematopoietic stem cells immune cells such as lymphocytes used for immunotherapy, dendritic cells, cells capable of forming a sheet-like cell culture, such as myoblasts (for example, skeletal myoblasts), mesenchymal stem cells ( Bone marrow, adipose tissue, peripheral blood, skin, hair root, muscle tissue, endometrium, placenta, umbilical cord blood, etc.), cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, synoviocytes, cartilage Cells, epithelial cells (eg, oral mucosal epithelial cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells,
- the contaminating cells are selected from cells other than the target cells present in the tissue in which the target cells are present, in particular, cells that are difficult to separate from the target cells and have a high proliferation ability and / or a high ratio in the tissues. be able to.
- the contaminating cells are fibroblasts.
- the target cell is a myoblast, preferably a skeletal myoblast.
- the target cell is a skeletal myoblast and the contaminating cell is a fibroblast.
- the target cell is a mesenchymal stem cell.
- the target cell is a mesenchymal stem cell
- the contaminating cell is a fibroblast.
- the trait of the contaminated cell to be changed one or more known arbitrary traits can be selected, but the trait after the change is preferably useful for separation from the target cell.
- traits include, but are not limited to, cell size, specific gravity, surface charge, presence / absence of marker expression, adhesion ability, proliferation ability, chemotaxis and the like.
- the alteration of the character can be performed by any known technique or a combination thereof.
- one cell type can be converted to another cell type by introducing a gene such as a transcription factor or administering a drug (for example, Nizzardo et al., Cell Transplant. 2013; 22 (6): 921 -44, Zhu et al., Cell Reprogram. 2012 Apr; 14 (2): 99-105). Therefore, the step of changing the trait of the contaminating cell in the present invention includes using this technique to convert the contaminating cell into a cell type that can be easily separated from the target cell.
- a technique for converting fibroblasts into adipocytes, megakaryocytes, platelets, hepatocyte-like cells, macrophage-like cells, nerve cells, cardiomyocytes and the like is known (for example, the above-mentioned Nizzardo et al., 2013). , Zhu et al., 2012, Ono et al., Blood.
- fibroblasts as contaminating cells
- desired target cells such as skeletal myoblasts or Transformed into any of these cells that have different traits from mesenchymal stem cells
- the contaminated cells after the transformation are separated and removed from skeletal myoblasts or mesenchymal stem cells based on the difference in traits
- skeletal myoblasts or mesenchymal stem cells can be separated and removed from the contaminating cells after conversion.
- the cell diameter of skeletal myoblasts is about 11-18 ⁇ m
- the cell diameter of megakaryocytes is about 35-160 ⁇ m
- the cell diameter of platelets is about 2-4 ⁇ m
- the cell diameter of hepatocytes is about 20-30 ⁇ m
- the cell diameter of macrophages is about 25-50 ⁇ m
- fibroblasts are converted into these cells, they can be separated from skeletal myoblasts using the difference in cell size.
- adipocytes have a specific gravity different from that of skeletal myoblasts or mesenchymal stem cells
- the difference in specific gravity of the cells is used to convert skeletal myoblasts or mesenchymal cells. Can be separated from stem cells.
- fibroblasts into adipocytes is not limited, for example, PPAR ⁇ (Tontonoz et al., Cell. 1994 Dec 30; 79 (7): 1147-56), C / EBP ⁇ (Freytag et al., Genes Dev. 1994 Jul 15; 8 (14): 1654-63), ADD1 / SREBP-1 (Kim and Spiegelman, Genes Dev.
- fibroblasts into hepatocyte-like cells is not limited, for example, a combination of introduction of Gata4, Hnf1 ⁇ and Foxa3 and inactivation of p19Arf (Huang et al., Nature.
- a TGF ⁇ signaling inhibitor eg, a TGF- ⁇ RI kinase inhibitor such as SB431542
- a ROCK signaling inhibitor eg, thiazobibin
- the conversion of fibroblasts into hepatocyte-like cells is not limited, for example, a combination of introduction of Gata4, Hnf1 ⁇ and Foxa3 and inactivation of p19Arf (Huang et al., Nature.
- fibroblasts into macrophage-like cells is not limited, for example, PU. 1 and introduction of C / EBP ⁇ or C / EBP ⁇ (Feng et al., Proc Natl Acad Sci U S A. 2008 Apr 22; 105 (16): 6057-62) and the like. Conversion of fibroblasts into megakaryocytes and platelets can be performed without limitation, for example, by introduction of p45NF-E2, Maf G and Maf K (Ono et al., 2012).
- the conversion of fibroblasts into neurons is not limited, for example, introduction of Asc11, Brn2 and Mytll, introduction of Asc11, Brn2, Mytll and NeuroD1, miR-9 / 9, miR-124, NeuroD2, Asc11 and Introduction of Mytll, introduction of Mash1, Nurr1 and Lmx1a, introduction of Asc11, Pitx3, Lmx1a, Nurr1, Foxa2 and EN1, Asc11, Brn2, Nyt11, Lhx3, Hb9, Is11, Ngn2 and NEUROd1 2013).
- Conversion of fibroblasts into cardiomyocytes can be performed by introduction of Gata4, Tbx5 and Mef2c, introduction of Oct4, Sox2, Klf4 and c-Myc (Nizzardo et al., 2013).
- Other techniques for changing the character of the contaminated cell include, but are not limited to, expression or suppression of a desired marker, conversion to iPS cells, and subsequent differentiation induction into desired cells.
- the desired marker include, but are not limited to, cell surface markers, fluorescent proteins such as GFP, and the like. Marker expression or expression suppression can be performed by any known technique.
- the marker expression method include introduction of a nucleic acid encoding the marker into a contaminated cell. The introduction of the nucleic acid is not limited, and for example, calcium phosphate method, lipofection method, ultrasonic introduction method, electroporation method, particle gun method, vector (for example, adenovirus vector, adeno-associated virus vector, retrovirus vector, etc.
- RNAi methods may include introduction of RNAi molecules into contaminated cells
- antisense methods may include introduction of antisense nucleic acids into contaminated cells.
- Conversion to iPS cells can be performed by introducing genes such as OCT3 / 4, SOX2, KLF4, C-MYC, LIN28, NANOG, GLIS1, etc. into cells (for example, OCT3 / 4, SOX2, KLF4 and C-MYC combinations, OCT3 / 4, SOX2, LIN28 and NANOG combinations, OCT3 / 4, SOX2, KLF4 and GLISI combinations, OCT3 / 4, SOX2 and KLF4 combinations, OCT3 / 4 and SOX2 combinations, etc.).
- Methods for conversion to iPS cells are well known in the art (eg, Miyazaki et al., Jpn J Clin Oncol. 2012 Sep; 42 (9): 773-9, Bayart and Cohen-Haguenauer, Curr Gene Ther. 2013 Apr; 13 (2): 73-92, etc.), any known conversion method or variations thereof can be used in the present invention.
- Methods for converting iPS cells to other cell types are also well known in the art, and any known conversion method or modification thereof can be used in the present invention for converting iPS cells to megakaryocytes or platelets.
- a medium containing STEMspan-ACF, BMP4, VEGF and bFGF then a medium containing STEM-diff APEL Medium, TPO, SCF, Flt-3 ligand, IL-3, IL-6 and heparin, and then STEMspan -A method of sequentially culturing cells in a medium containing ACF, TPO, SCF, IL-6, IL-9 and heparin (Feng et al., Stem Cell Reports.
- an RNAi molecule refers to any molecule that causes RNA interference, including, but not limited to, siRNA (small interfering RNA), miRNA (micro RNA), shRNA (short hairpin RNA), ddRNA (DNA- directed RNA), piRNA (Piwi-interacting RNA), rasiRNA (repeat associated siRNA) and the like, and variants thereof. These RNAi molecules are commercially available or can be designed and produced based on known sequence information.
- Antisense nucleic acid as used herein includes RNA, DNA, PNA, or a composite thereof.
- a delivery method specific to the contaminated cell can be used.
- Such delivery techniques include, for example, active targeting (Marcucci and Lefoulon, Drug Discov Today. 2004 Mar 1; 9 (5): 219-) using a targeting agent (such as a targeting ligand) specific to contaminating cells. 28, Torchilin, Eur J Pharm Sci. 2000 Oct; 11 Suppl 2: S81-91 etc.).
- Many targeting agents specific to specific cells are known. Active targeting allows nucleic acids and drugs to act only on contaminating cells without acting on the target cells.
- the targeting agent specific for fibroblasts is not limited, but includes, for example, TE-7, which is a fibroblast-specific cell surface marker (eg Rosendal et al., J Cell Sci. 1994 Jan; 107 (Pt 1): 29-37, Goodpaster et al., J Histochem Cytochem.
- antibody or antigen recognition against TE-7 examples thereof include fragments of the antibody containing the site (for example, F (ab ′) 2 , Fab ′, Fab, Fv, scFv fragments, etc.).
- the nucleic acid When the nucleic acid is expressed in a contaminated cell and used, the nucleic acid can be placed under the control of a regulatory sequence specific to the contaminated cell for expression specific to the contaminated cell.
- the nucleic acid can be expressed only in the contaminating cell without being expressed in the target cell. For example, the same action (for example, conversion to the same cell type) on both the target cell and the contaminating cell. It is possible to use a nucleic acid that brings about Many regulatory sequences that are specific to a particular cell are known. Regulatory sequences that can be used for specific expression of nucleic acids in fibroblasts are not limited, and include, for example, the ST2 gene proximal promoter (Iwahana et al., Eur J Biochem.
- the trait of the mixed cells to be changed may be one or a combination of two or more. For example, when converting contaminated cells to another cell type, multiple traits may be altered simultaneously. In addition, a plurality of markers can be expressed at the same time, or the expression of a plurality of markers can be suppressed.
- the removal of the contaminated cells or the collection of the target cells can be performed by any known method according to the target trait.
- the trait to be changed is cell size
- remove contaminated cells or collect target cells using appropriate size filters for example, nylon mesh filters
- filtration through microchannels, centrifugation, etc. can do.
- the character to be changed is cell specific gravity
- centrifugation eg, density gradient centrifugation, iso-density centrifugation, counter-flow centrifugation, etc.
- the altered trait is a cell surface marker
- remove contaminating cells or remove target cells using flow cytometry, affinity separation eg, affinity column method, magnetic cell separation, immunopanning, etc.
- affinity separation eg, affinity column method, magnetic cell separation, immunopanning, etc.
- the character after the change is the expression of fluorescent protein
- the contaminated cells can be removed or the target cells can be collected using a flow cytometry method or the like.
- the character to be changed is a surface charge
- contaminating cells can be removed or target cells can be collected using ion exchange chromatography, electrophoresis, dielectrophoresis, or the like.
- the above method may be applied once or multiple times for the same sample. Moreover, when two or more techniques exist for the same character, only one of them may be applied, or two or more may be applied in combination. Furthermore, when there are a plurality of traits to be changed, even if removal of the contaminating cells or collection of the target cells is focused on any one of them, the removal of the contaminated cells or the purpose is focused on two or more. Cells may be collected.
- the purity of the target cell is high means higher than the purity of the target cell in a natural tissue.
- the purity of the target cell after the method of the present invention is 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85 % Or more, 90% or more, 95% or more, 98% or more, or 99% or more, and most preferably 100%.
- the purity of the target cell can be determined using any known technique. As such a technique, for example, the target cell is labeled with an antibody specific to the same cell, and the number of positive cells bound by the antibody is divided by the total number of cells counted.
- the cells can be counted by microscopic observation of a specimen stained with a specific antibody, image analysis of a microscopic image, flow cytometry analysis of a cell population stained with a specific antibody, and the like.
- the marker specific to the cell is not limited, and for example, CD56, ⁇ 7 integrin, myosin heavy chain IIa, myosin heavy chain IIb, myosin heavy chain IId (IIx) , MyoD, Myf5, myogenin, etc.
- the marker specific to the cell is not limited, and examples thereof include CD29, CD73, CD90, CD105, CD166, etc. .
- the cell population production method of the present invention may further include a step of culturing the collected target cells and a step of subcultured the cultured cells.
- Cell culture and passage can be performed using any known method.
- the cell population production method of the present invention may further include a step of introducing a gene into the collected cells.
- the gene to be introduced is not particularly limited as long as it is useful for the treatment of the disease to be treated, and may be a cytokine such as HGF, for example.
- the gene can be introduced by any known method such as calcium phosphate method, lipofection method, ultrasonic introduction method, electroporation method, particle gun method, adenovirus vector, retrovirus vector or other viral vector method, or microinjection method. Can be used.
- the present invention relates to a cell population produced by the cell population production method of the present invention and having a high target cell purity (hereinafter sometimes abbreviated as “high purity cell population”).
- the high-purity cell population of the present invention is obtained by the cell population production method of the present invention, and has a high target cell purity.
- the degree of target cell purity is as described above. Since the high-purity cell population of the present invention contains target cells at a high ratio, it is useful as a source of the cells used for medical use and the like.
- the high purity cell population of the present invention is preferably sterile.
- the high-purity cell population of the present invention may be attached to a culture vessel, suspended in a physiologically acceptable liquid, or may be cryopreserved.
- the “sheet-shaped cell culture” refers to a sheet in which cells are connected to each other and is typically composed of one cell layer, but is composed of two or more cell layers. Including those that are made.
- the cells may be linked to each other directly (including those via cell elements such as adhesion molecules) and / or via intervening substances.
- the intervening substance is not particularly limited as long as it is a substance that can connect cells at least physically (mechanically), and examples thereof include an extracellular matrix.
- the intervening substance is preferably derived from cells, particularly derived from cells constituting the sheet-shaped cell culture.
- the cells are at least physically (mechanically) connected, but may be further functionally, for example, chemically or electrically connected.
- the sheet-shaped cell culture of the present invention preferably does not contain a scaffold (support) from the viewpoints of biocompatibility and high therapeutic effect.
- Scaffolds may be used in the art to attach cells on and / or within its surface and maintain the physical integrity of sheet-like cell cultures, for example, polyvinylidene difluoride ( PVDF) membranes and the like are known, but preferred sheet-like cell cultures of the present invention can maintain their physical integrity even without such a scaffold.
- the sheet-shaped cell culture of the present invention is preferably composed only of cells derived from the cells constituting the sheet-shaped cell culture, and does not contain other substances.
- the step of culturing the high-purity cell population of the present invention in a sheet form can be performed by any known technique. Such a method is not limited and is, for example, Patent Document 1, JP 2010-081829, JP 2010-226962, JP 2010-226991, JP 2011-110368, JP 2011-115058, JP 2011-150 172925 etc. are mentioned.
- the step of culturing the high-purity cell population in a sheet form may include a step of seeding the cell population on a culture substrate and a step of forming the seeded cell population into a sheet.
- the culture substrate is not particularly limited as long as the cell population can form a sheet-shaped cell culture thereon, and includes, for example, containers of various materials, solid or semi-solid surfaces in containers, and the like.
- the container preferably has a structure / material that does not allow permeation of a liquid such as a culture solution. Examples of such materials include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl. Examples include acrylamide and metals (for example, iron, stainless steel, aluminum, copper, brass).
- the container preferably has at least one flat surface.
- Examples of such containers include, but are not limited to, cell culture dishes and cell culture bottles. Further, the container may have a solid or semi-solid surface therein. Examples of solid surfaces include plates and containers of various materials as described above, and examples of semi-solid surfaces include gels and soft polymer matrices.
- the culture substrate may be prepared using the above materials, or commercially available materials may be used. Preferable culture substrates include, but are not limited to, substrates having an adhesive surface suitable for the formation of sheet cell cultures.
- a substrate having a hydrophilic surface for example, a substrate coated with a hydrophilic compound such as polystyrene subjected to corona discharge treatment, collagen gel or hydrophilic polymer, and further, collagen, fibronectin, laminin , Substrates coated with an extracellular matrix such as vitronectin, proteoglycan and glycosaminoglycan, and cell adhesion factors such as cadherin family, selectin family and integrin family.
- a hydrophilic compound such as polystyrene subjected to corona discharge treatment, collagen gel or hydrophilic polymer, and further, collagen, fibronectin, laminin , Substrates coated with an extracellular matrix such as vitronectin, proteoglycan and glycosaminoglycan, and cell adhesion factors such as cadherin family, selectin family and integrin family.
- base materials are commercially available (for example, Corning (R) TC-Treated Culture Dish, Corning,
- the surface of the culture substrate may be coated with a material whose physical properties change in response to stimulation, for example, temperature or light.
- materials include, but are not limited to, (meth) acrylamide compounds, N-alkyl-substituted (meth) acrylamide derivatives (eg, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylate Amide), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethyl
- the seeding of the cell population on the culture substrate can be performed by any known method and condition.
- the seeding of the cell population on the culture substrate may be performed, for example, by injecting a cell suspension obtained by suspending the cell population in the culture solution into the culture substrate (culture vessel).
- an apparatus suitable for the operation of injecting the cell suspension such as a dropper or a pipette, can be used.
- the step of forming the seeded cell population into a sheet can also be performed by any known technique and condition.
- Non-limiting examples of such methods include, for example, Patent Document 1, JP 2010-081829, JP 2010-226962, JP 2010-226991, JP 2011-110368, JP 2011-115058, JP 2011-172925, and the like. It is described in. It is considered that the sheet formation of the cell population is achieved by the cells adhering to each other via an adhesion molecule or an intercellular adhesion mechanism such as an extracellular matrix. Therefore, the step of forming the seeded cells into a sheet can be achieved, for example, by culturing a cell population under conditions that form cell-cell adhesion.
- Such conditions may be any as long as cell-cell adhesion can be formed, but cell-cell adhesion can usually be formed under the same conditions as general cell culture conditions.
- a person skilled in the art can select optimal conditions according to the type of cell population to be seeded.
- the culture for forming a seeded cell population into a sheet may be referred to as “sheet culture”.
- the cell culture medium used for the culture (sometimes simply referred to as “culture medium” or “medium”) is not particularly limited as long as it can maintain cell survival, but typically, amino acids, vitamins, electrolytes are used. Can be used.
- a culture solution based on a basal medium for cell culture may be used.
- Such a basal medium is not limited, for example, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), F12 / DMEM, L15, SkBM, RITC80 -7 etc. are included. Many of these basal media are commercially available, and their compositions are also known.
- the basal medium may be used in a standard composition (for example, as it is commercially available), or the composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to those having a known composition, and includes one in which one or more components are added, removed, increased or decreased.
- the cell population can be cultured under conditions usually used in the art.
- typical culture conditions include culture at 37 ° C. and 5% CO 2 .
- Culturing can be performed in containers of any size and shape.
- the size and shape of the sheet-shaped cell culture can be adjusted by adjusting the size and shape of the cell adhesion surface of the culture vessel, or by placing a mold of the desired size and shape on the cell adhesion surface of the culture vessel, It can be arbitrarily adjusted by, for example, culturing cells therein.
- the sheet-shaped cell culture production method of the present invention may further include a step of collecting the sheet-shaped cell culture.
- the recovery of the sheet-shaped cell culture is not particularly limited as long as the sheet-shaped cell culture can be released (peeled) from the culture substrate serving as a scaffold while at least partially maintaining the sheet structure. Enzymatic treatment with an enzyme (for example, trypsin) and / or mechanical treatment such as pipetting can be performed.
- an enzyme for example, trypsin
- a mechanical treatment such as pipetting
- a sheet-like cell culture is formed. Can also be liberated non-enzymatically.
- a particularly preferred embodiment of the method for producing a sheet-shaped cell culture of the present invention includes the following steps: (1) changing the character of the contaminating cell in a cell population containing the target cell and the contaminating cell collected from the subject; (2) removing contaminating cells based on the altered trait and / or collecting target cells to obtain a cell population with high purity of the target cells; (3) freezing the cell population; (4) thawing the frozen cell population; (5) culturing the cell population in a sheet form to form a sheet-like cell culture; (6) A step of collecting the formed sheet-shaped cell culture. Steps (1) to (2) and (5) to (6) have already been described above.
- the target cell is preferably a myoblast (particularly skeletal myoblast) or a mesenchymal stem cell
- the contaminating cell is preferably a fibroblast.
- Step (3) can be performed by any known method.
- a technique include, but are not limited to, subjecting the cell population in the container to a freezing means such as a freezer, a deep freezer, and a low-temperature medium (for example, liquid nitrogen).
- the temperature of the freezing means is not particularly limited as long as it is a temperature at which a part of the cell population in the container, preferably the whole can be frozen, but is typically 0 ° C. or lower, preferably ⁇ 20 ° C. or lower, more preferably ⁇ 40 ° C. or lower, more preferably ⁇ 80 ° C. or lower.
- the cooling rate in the freezing operation is not particularly limited as long as it does not significantly impair the viability and function of the cells after freezing and thawing.
- the cooling rate is from 1 to 5 until cooling begins at 4 ° C. and reaches ⁇ 80 ° C.
- the cooling rate is about a time, preferably 2 to 4 hours, particularly about 3 hours.
- cooling can be performed at a rate of 0.46 ° C./min.
- Such a cooling rate can be achieved by providing a container containing a cell population directly or by storing it in a freezing container in a freezing means set to a desired temperature.
- the freezing treatment container may have a function of controlling the temperature lowering speed in the container to a predetermined speed.
- any known container such as BICELL (R) (Japan Freezer) can be used.
- the freezing operation may be performed while the cell population is immersed in a culture solution or physiological buffer solution.
- a cryoprotectant for protecting cells from freezing and thawing operations may be added to the culture solution, or the culture solution may be frozen.
- You may perform after performing the process of replacing with the cryopreservation liquid containing a protective agent. Therefore, the sheet-shaped culture production method of the present invention may further include a step of adding a cryoprotectant to the culture solution or a step of replacing the culture solution with a cryopreservation solution.
- the solution in which cells are immersed during freezing contains an effective concentration of cryoprotectant, remove the culture solution before adding the cryopreservation solution.
- the cryopreservation solution may be added while leaving a part of the culture solution.
- the “effective concentration” means that the cryoprotectant exhibits a cryoprotective effect without exhibiting toxicity, for example, the viability, vitality, and function of the cell after freeze-thawing compared to the case where the cryoprotectant is not used. This means a concentration that exhibits a decrease-suppressing effect. Such a concentration is known to those skilled in the art or can be appropriately determined by routine experimentation.
- the cryoprotectant is not particularly limited as long as it exhibits a cryoprotective action on cells, for example, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, sericin, propanediol, dextran, polyvinylpyrrolidone, Polyvinyl alcohol, hydroxyethyl starch, chondroitin sulfate, polyethylene glycol, formamide, acetamide, adonitol, perseitol, raffinose, lactose, trehalose, sucrose, mannitol and the like.
- Cryoprotectants may be used alone or in combination of two or more.
- the concentration of the cryoprotectant added to the culture solution or the concentration of the cryoprotectant in the cryopreservation solution is not particularly limited as long as it is an effective concentration as defined above. 2 to 20% (v / v) with respect to the whole stock solution. However, although outside this concentration range, alternative use concentrations known or experimentally determined for each cryoprotectant may be employed, and such concentrations are within the scope of the present invention.
- Step (4) can be performed by any known cell thawing technique, typically involving, for example, thawing a frozen cell population into a thawing means, eg, solid, liquid or gaseous, at a temperature above the freezing temperature.
- a medium for example, water
- a water bath for example, water
- an incubator for example, an incubator
- a medium for example, a culture solution
- the temperature of the thawing means or the immersion medium is not particularly limited as long as the cell population can be thawed within a desired time, but typically 4 to 50 ° C., preferably 30 ° C.
- the thawing time is not particularly limited as long as it does not significantly impair the viability and function of the cells after thawing, but it is typically within 2 minutes, and in particular within 20 seconds can reduce the viability. It can be greatly suppressed.
- the thawing time can be adjusted, for example, by changing the temperature of the thawing means or the immersion medium, the volume or composition of the culture solution or cryopreservation solution at the time of freezing.
- the above-mentioned sheet-shaped cell culture production method may include a step of washing the cell population between step (4) and step (5).
- Washing of the cell population can be performed by any known technique, and typically includes, for example, a culture or physiological solution that contains or does not contain the cell population in liquid (eg, serum or serum components (such as serum albumin)). This is achieved by, but not limited to, suspending in a buffer or the like, centrifuging, discarding the supernatant, and collecting the precipitated cell population.
- the suspension, centrifugation, and recovery cycle may be performed once or a plurality of times (for example, 2, 3, 4, 5, etc.).
- the step of washing the cell population is performed immediately after step (4) of thawing the frozen cell population.
- all the steps of the production method of the present invention are performed in vitro.
- the production method of the present invention includes a step performed in vivo, without limitation, for example, collecting a cell population or a tissue serving as a source of the cell population from a subject.
- the production method of the present invention is performed under aseptic conditions in all steps.
- the production method of the present invention is performed so that the finally obtained cell population or sheet-shaped cell culture is substantially sterile.
- the production method of the present invention is performed so that the finally obtained cell population or sheet-shaped cell culture is sterile.
- the sheet-shaped cell culture of the present invention has a high purity of target cells.
- the high purity is as described above for the high purity cell population of the present invention.
- the target cell is preferably a myoblast, particularly a skeletal myoblast, or a mesenchymal stem cell.
- the sheet-shaped cell culture of the present invention may be produced by the sheet-shaped cell culture production method of the present invention.
- the sheet-shaped cell culture of the present invention is preferably sterile. Since the sheet-shaped cell culture of the present invention contains target cells with high purity, the therapeutic effect and the like are higher than those of the sheet-shaped cell culture not including the high-purity cell population of the present invention.
- the high-purity cell population and sheet-shaped cell culture of the present invention are useful for the treatment of various diseases, particularly diseases related to tissue abnormalities. Accordingly, in one aspect, the high purity cell population and sheet cell culture of the present invention are for use in the treatment of diseases associated with tissue abnormalities.
- tissue to be treated include, but are not limited to, myocardium, cornea, retina, esophagus, skin, joint, cartilage, liver, pancreas, gingiva, kidney, thyroid, skeletal muscle, middle ear, bone marrow, and the like. It is done.
- the disease to be treated is not limited, and for example, heart disease (eg, myocardial injury (myocardial infarction, cardiac injury), cardiomyopathy, etc.), corneal disease (eg, corneal epithelial stem cell exhaustion, cornea) Injury (heat / chemical corrosion), corneal ulcer, corneal opacity, corneal perforation, corneal scar, Stevens-Johnson syndrome, pemphigoid, etc., retinal diseases (eg retinitis pigmentosa, age-related macular degeneration) , Esophageal diseases (for example, prevention of esophageal inflammation / stenosis after esophageal surgery (esophageal cancer removal)), skin diseases (for example, skin damage (trauma, burn), etc.), joint diseases (for example, osteoarthritis, etc.) Cartilage disease (eg, cartilage damage), liver disease (eg, chronic liver disease), pancreatic disease (eg, diabetes), dental disease (eg, periodon,
- Patent Document 1 Non-Patent Document 1, Arauchi ⁇ et al., ⁇ Tissue Eng Part A. 2009 Dec; 15 (12 ): 3943-9, Ito et al., Tissue Eng. 2005 Mar-Apr; 11 (3-4): 489-96, Yaji et al., Biomaterials. 2009 Feb; 30 (5): 797-803, Yaguchi et al., Acta Otolaryngol. 2007 Oct; 127 (10): 1038-44, Watanabe et al., Transplantation. 2011 Apr 15; 91 (7): 700-6, Shimizu et al., Biomaterials.
- the high-purity cell population and sheet-shaped cell culture of the present invention can be applied to a tissue to be treated and used to repair and regenerate the tissue, but as a source of a physiologically active substance such as a hormone, It can also be transplanted to a site other than the tissue to be treated (for example, subcutaneous tissue) (for example, Arauchi et al., Tissue Eng Part A. 2009 Dec; 15 (12): 3943-9, Shimizu et al ., Biomaterials. 2009 Oct; 30 (30): 5943-9). It is also possible to fragment the sheet-like cell culture of the present invention into an injectable size and inject it into a site requiring treatment (Wang et al., Cardiovasc Res. 2008 Feb 1; 77 (3) : 515-24).
- the high-purity cell population and sheet-shaped cell culture of the present invention can be prepared by adding various additional components such as a pharmaceutically acceptable carrier, and the viability and engraftment of the high-purity cell population and / or the sheet-shaped cell culture.
- it may further contain a component that enhances the function and the like, another active component useful for the treatment of the target disease, and the like. Any known additional components can be used, and those skilled in the art are familiar with these additional components.
- the high-purity cell population and sheet-shaped cell culture of the present invention include components that enhance the viability, engraftment and / or function of the high-purity cell population and / or sheet-shaped cell culture, and treatment of target diseases. It can be used in combination with other active ingredients useful for.
- compositions comprising an active ingredient selected from the group consisting of the high-purity cell population and sheet-shaped cell culture of the present invention.
- the pharmaceutical composition of the present invention can be prepared by adding various additional components such as a pharmaceutically acceptable carrier, high-purity cell population and / or It may contain components that enhance the viability, engraftment and / or function of the sheet-shaped cell culture, other active ingredients useful for the treatment of the target disease, and the like. Any known additional components can be used, and those skilled in the art are familiar with these additional components.
- the pharmaceutical composition of the present invention is a component that enhances the viability, engraftment and / or function of a high-purity cell population and / or sheet-shaped cell culture, and other active ingredients useful for the treatment of the target disease. Etc. can be used together.
- the pharmaceutical composition of the present invention is for use in the treatment of diseases associated with tissue abnormalities. The tissues and diseases to be treated are as described above for the high-purity cell population and sheet-shaped cell culture of the present invention.
- kits for producing cell populations, sheet-like cell cultures or compositions (eg, pharmaceutical compositions), etc., or for treating diseases (eg, diseases related to tissue abnormalities, etc.) (Hereinafter sometimes referred to as “production kit of the present invention”).
- production kit of the present invention the terms “set” and “pack” are used interchangeably with “kit”, and the description relating to “kit” in this specification also applies to “set” and “pack”.
- the kit of the present invention is not limited, for example, an agent for changing the character of a contaminated cell (for example, an agent for converting a cell type, an agent for expressing or suppressing a marker, agents for conversion to iPS cells, agents for inducing differentiation of iPS cells into other cell types, etc.), cell populations used to produce high purity cell populations (eg, cell populations isolated from living organisms), Cells used for culturing a sheet-shaped cell culture (for example, the high-purity cell population of the present invention), a culture solution, a culture dish, a washing solution, an agent used for purification of target cells and / or elimination of contaminating cells (for example, antibodies, Cleaning solutions, etc.), instruments (eg, pipettes, droppers, tweezers, beads, affinity columns,
- the kit for producing a high-purity cell population of the present invention or a composition containing the same comprises an agent for altering the trait of contaminating cells, and purification of target cells and / or elimination of contaminating cells. Including the agent used for.
- a kit for producing a high-purity cell population of the present invention or a composition comprising the same is provided, a cell population isolated from a living body, an agent for altering the trait of contaminating cells, and an object Contains agents used for cell purification and / or elimination of contaminating cells.
- kits for producing a sheet-shaped cell culture of the present invention or a composition containing the same includes the high-purity cell population of the present invention, a culture medium, and a culture dish.
- the kit for producing the sheet-shaped cell culture of the present invention or the composition containing the same comprises a cell population isolated from a living body, an agent for changing the character of contaminating cells, a target cell And / or agents used for the purification and / or elimination of contaminating cells.
- Another aspect of the invention is a method of treating a disease in a subject comprising administering to a subject in need thereof an effective amount of a high purity cell population, sheet-like cell culture or pharmaceutical composition of the invention.
- the method includes (hereinafter may be abbreviated as “treatment method”).
- the tissues and diseases to be treated by the treatment method of the present invention are as described above for the high-purity cell population and sheet-shaped cell culture of the present invention.
- the high-purity cell population, sheet-shaped cell culture or pharmaceutical composition of the present invention can be used in combination.
- the treatment method of the present invention may further include a step of producing a high-purity cell population, a sheet-shaped cell culture or a pharmaceutical composition according to the production method of the present invention.
- the treatment method of the present invention provides a source of whole blood, cells or cells to produce a high purity cell population from a subject prior to the step of producing a high purity cell population, sheet-like cell culture or pharmaceutical composition.
- the method may further include collecting the tissue.
- the subject from whom the whole blood and / or cells or tissue that is the source of cells is collected is the same individual as the subject receiving the high-purity cell population, sheet cell culture, or pharmaceutical composition.
- the subject from whom the whole blood and / or cells or tissue that is the source of cells is collected is a separate species of the same type as the subject receiving the high-purity cell population, sheet-like cell culture, or pharmaceutical composition. is there.
- the subject from whom whole blood and / or cells or tissue that is the source of cells is collected is an individual different from the subject receiving the high-purity cell population, sheet-like cell culture, or pharmaceutical composition. .
- the subject may be healthy or afflicted with some disease, but when treatment of the disease is intended, the subject is typically afflicted with or affected by the disease. Means a subject at risk.
- treatment is also intended to encompass all types of medically acceptable prophylactic and / or therapeutic interventions intended to cure, temporarily ameliorate or prevent disease.
- treatment encompasses medically acceptable interventions for various purposes, including delaying or stopping the progression of the disease, regression or disappearance of the lesion, prevention of the onset of the disease or prevention of recurrence, etc. .
- the effective amount is, for example, an amount that can suppress the onset or recurrence of a disease, reduce symptoms, or delay or stop progression (for example, a high-purity cell population, a sheet-shaped cell culture, or a pharmaceutical composition).
- an amount that does not cause adverse effects exceeding the benefits of administration is preferred.
- Such an amount can be appropriately determined by, for example, testing in laboratory animals such as mice, rats, dogs or pigs, and disease model animals, and such test methods are well known to those skilled in the art.
- the size of the tissue lesion to be treated can be an important index for determining the effective amount.
- the high-purity cell population, sheet-shaped cell culture or pharmaceutical composition of the present invention is administered from various routes such as intravenous, intramuscular, subcutaneous, topical, intraarterial, intraportal, intraventricular, intraperitoneal, etc. Can do.
- the administration method typically includes direct application to tissues, but when a fragment of the sheet-shaped cell culture is used, an injection is used. It may be administered from various routes that can be administered by, for example, intravenous, intramuscular, subcutaneous, topical, intraarterial, intraportal, intraventricular, intraperitoneal, and the like.
- the frequency of administration is typically once per treatment, but multiple administrations are possible if the desired effect is not obtained.
- Example 1 Purification of skeletal myoblasts by conversion of fibroblasts into adipocytes
- a cell suspension of a cell population isolated from skeletal muscle is 0.8 x 10 ⁇ 5 > / well. Inoculate to a cell culture plate and culture overnight in a CO 2 incubator. 30 ⁇ L of serum-free medium was placed in a microtube, and 1.0 ⁇ g of a PPAR ⁇ -expressing adenovirus vector (AdRGD-PPAR ⁇ , Gastroenterology. 2003 May; 124 (5): 1315-24) was added thereto, followed by mixing by pipetting.
- AdRGD-PPAR ⁇ a PPAR ⁇ -expressing adenovirus vector
- AdRGD-PPAR ⁇ -HilyMax complex is added to the cultured cell population, and the plate is gently shaken. The cells are cultured for 24 hours in a CO 2 incubator. The cell population was washed with PBS, centrifuged, seeded in an 80 cm 2 flask at a density of 2 to 3 ⁇ 10 4 cells / cm 2 , and 5 to 7 in 15 mL of medium (medium for adipocyte differentiation induction: Cosmo Bio). Incubate for days.
- the culture surface is washed with PBS, and the cell population obtained by trypsin treatment is filtered with a mesh having a pore size of 70 ⁇ m (cell strainer: manufactured by Corning), and the cell population passing through the mesh is obtained as the target cell population. .
- the obtained cell population is labeled with an anti-CD56 antibody, and the proportion of CD56 positive cells (skeletal myoblast purity) is measured using a flow cytometer.
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Abstract
Description
シート状細胞培養物の治療への応用については、火傷などによる皮膚損傷に対する培養表皮シートの利用、角膜損傷に対する角膜上皮シート状細胞培養物の利用、食道ガン内視鏡的切除に対する口腔粘膜シート状細胞培養物の利用などの検討が進められている。
<1> (1)目的細胞と混入細胞とを含む細胞集団において、混入細胞の形質を変更するステップ、および
(2)変更された形質に基づいて混入細胞を除去、および/または、目的細胞を採取するステップ
を含む、目的細胞の純度の高い細胞集団を製造する方法。
<2> 混入細胞が線維芽細胞である、上記<1>に記載の方法。
<3> 形質が、細胞の形状、サイズ、比重、表面電荷、付着能、マーカーからなる群から選択される、上記<1>または<2>に記載の方法。
<4> 目的細胞が骨格筋芽細胞または間葉系幹細胞である、上記<1>~<3>のいずれかに記載の方法。
<6> 上記<5>に記載の細胞集団を、シート状に培養するステップを含む、シート状細胞培養物の製造方法。
<7> 上記<5>に記載の細胞集団を含む、シート状細胞培養物。
<8> 上記<5>に記載の細胞集団および上記<7>に記載のシート状細胞培養物からなる群から選択される有効成分を含む、医薬組成物。
<9> 組織の異常に関連する疾患を処置するための、上記<8>に記載の医薬組成物。
<10> 上記<5>に記載の細胞集団、上記<7>に記載シート状細胞培養物または上記<8>もしくは<9>に記載の医薬組成物の有効量を、それを必要とする対象に投与するステップを含む、前記対象における疾患の処置方法。
本発明の一態様において、混入細胞は線維芽細胞である。本発明の一態様において、目的細胞は筋芽細胞、好ましくは骨格筋芽細胞である。本発明の好ましい態様において、目的細胞は骨格筋芽細胞であり、混入細胞は線維芽細胞である。本発明の別の態様において、目的細胞は間葉系幹細胞である。本発明の好ましい態様において、目的細胞は間葉系幹細胞であり、混入細胞は線維芽細胞である。
本発明において、「シート状細胞培養物」は、細胞が互いに連結してシート状になったものをいい、典型的には1の細胞層からなるものであるが、2以上の細胞層から構成されるものも含む。細胞同士は、直接(接着分子などの細胞要素を介するものを含む)および/または介在物質を介して、互いに連結していてもよい。介在物質としては、細胞同士を少なくとも物理的(機械的)に連結し得る物質であれば特に限定されないが、例えば、細胞外マトリックスなどが挙げられる。介在物質は、好ましくは細胞由来のもの、特に、シート状細胞培養物を構成する細胞に由来するものである。細胞は少なくとも物理的(機械的)に連結されるが、さらに機能的、例えば、化学的、電気的に連結されてもよい。
基礎培地は、標準的な組成のまま(例えば、市販されたままの状態で)用いてもよいし、細胞種や細胞条件に応じてその組成を適宜変更してもよい。したがって、本発明に用いる基礎培地は、公知の組成のものに限定されず、1または2以上の成分が追加、除去、増量もしくは減量されたものを含む。
(1)対象から採取した目的細胞と混入細胞とを含む細胞集団において、混入細胞の形質を変更するステップ、
(2)変更された形質に基づいて混入細胞を除去、および/または、目的細胞を採取し、目的細胞の純度の高い細胞集団を得るステップ、
(3)前記細胞集団を凍結するステップ、
(4)凍結した細胞集団を解凍するステップ、
(5)細胞集団をシート状に培養し、シート状細胞培養物を形成するステップ、
(6)形成されたシート状細胞培養物を回収するステップ。
(1)~(2)、(5)~(6)のステップについては、すでに上記したとおりである。上記態様において、目的細胞は好ましくは筋芽細胞(特に骨格筋芽細胞)もしくは間葉系幹細胞であり、混入細胞は好ましくは線維芽細胞である。
本発明のシート状細胞培養物は、目的細胞の純度が高い。純度の高さは、本発明の高純度細胞集団に関して上記したとおりである。目的細胞は、好ましくは筋芽細胞、特に骨格筋芽細胞、もしくは間葉系幹細胞である。本発明のシート状細胞培養物は、本発明のシート状細胞培養物製造方法によって製造されたものであってもよい。本発明のシート状細胞培養物は無菌であることが好ましい。本発明のシート状細胞培養物は、目的細胞を高い純度で含むため、本発明の高純度細胞集団を含まないシート状細胞培養物に比べ、治療効果などが高い。
本発明の医薬組成物は、本発明の高純度細胞集団および/またはシート状細胞培養物に加えて、種々の追加成分、例えば、薬学的に許容し得る担体や、高純度細胞集団および/またはシート状細胞培養物の生存性、生着性および/または機能などを高める成分、対象疾患の処置に有用な他の有効成分などを含んでいてもよい。かかる追加成分としては、既知の任意のものを使用することができ、当業者はこれらの追加成分について精通している。また、本発明の医薬組成物は、高純度細胞集団および/またはシート状細胞培養物の生存性、生着性および/または機能などを高める成分や、対象疾患の処置に有用な他の有効成分などと併用することができる。一態様において、本発明の医薬組成物は、組織の異常に関連する疾患の処置に用いるためのものである。処置の対象となる組織や疾患は、本発明の高純度細胞集団およびシート状細胞培養物について上記したとおりである。
また、用語「処置」は、疾患の治癒、一時的寛解または予防などを目的とする医学的に許容される全ての種類の予防的および/または治療的介入を包含するものとする。例えば、「処置」の用語は、疾患の進行の遅延または停止、病変の退縮または消失、当該疾患発症の予防または再発の防止などを含む、種々の目的の医学的に許容される介入を包含する。
投与頻度は、典型的には1回の処置につき1回であるが、所望の効果が得られない場合には、複数回投与することも可能である。
例1:線維芽細胞の脂肪細胞への転換による骨格筋芽細胞の精製
骨格筋から単離した細胞集団の細胞懸濁液を0.8×105個/ウェルとなるように細胞培養プレートへ播種し、CO2インキュベーターにて一晩培養する。マイクロチューブに無血清培地を30μL入れ、これに、PPARγ発現アデノウィルスベクター(AdRGD-PPARγ、Gastroenterology. 2003 May;124(5):1315-24)を1.0μg添加し、ピペッティングにより混合した後、HilyMax溶液(同仁化学研究所製)を5μL添加し、室温にて15分間静置して、AdRGD-PPARγ-HilyMax複合体を調製する。培養後の細胞集団に、AdRGD-PPARγ-HilyMax複合体を添加し、プレートを穏やかに振とうする。CO2インキュベーターにて細胞を24時間培養する。細胞集団をPBSで洗浄、遠心後、2~3×104個/cm2の密度で80cm2フラスコに播種し、15mLの培地(脂肪細胞分化誘導用メディウム:コスモ・バイオ製)で5~7日間培養する。培養後、培養表面をPBSで洗浄し、トリプシン処理にて得られた細胞集団を孔径70μmのメッシュ(セルストレーナー:Corning製)でろ過し、メッシュを通過した細胞集団を目的とする細胞集団として得る。得られた細胞集団を抗CD56抗体でラベルし、フローサイトメーターを用い、CD56陽性細胞の割合(骨格筋芽細胞純度)を測定する。
Claims (10)
- (1)目的細胞と混入細胞とを含む細胞集団において、混入細胞の形質を変更するステップ、および
(2)変更された形質に基づいて混入細胞を除去、および/または、目的細胞を採取するステップ
を含む、目的細胞の純度の高い細胞集団を製造する方法。 - 混入細胞が線維芽細胞である、請求項1に記載の方法。
- 形質が、細胞の形状、サイズ、比重、表面電荷、付着能、マーカーからなる群から選択される、請求項1または2に記載の方法。
- 目的細胞が骨格筋芽細胞または間葉系幹細胞である、請求項1~3のいずれか一項に記載の方法。
- 請求項1~4のいずれか一項に記載の方法で製造された、目的細胞の純度が高い細胞集団。
- 請求項5に記載の細胞集団を、シート状に培養するステップを含む、シート状細胞培養物の製造方法。
- 請求項5に記載の細胞集団を含む、シート状細胞培養物。
- 請求項5に記載の細胞集団および請求項7に記載のシート状細胞培養物からなる群から選択される有効成分を含む、医薬組成物。
- 組織の異常に関連する疾患を処置するための、請求項8に記載の医薬組成物。
- 請求項5に記載の細胞集団、請求項7に記載シート状細胞培養物または請求項8もしくは9に記載の医薬組成物の有効量を、それを必要とする対象に投与するステップを含む、前記対象における疾患の処置方法。
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