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WO2018186420A1 - Population cellulaire contenant des cellules souches mésenchymateuses ainsi que procédé de fabrication de celle-ci, cellules souches mésenchymateuses, et composition pharmaceutique - Google Patents

Population cellulaire contenant des cellules souches mésenchymateuses ainsi que procédé de fabrication de celle-ci, cellules souches mésenchymateuses, et composition pharmaceutique Download PDF

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Publication number
WO2018186420A1
WO2018186420A1 PCT/JP2018/014325 JP2018014325W WO2018186420A1 WO 2018186420 A1 WO2018186420 A1 WO 2018186420A1 JP 2018014325 W JP2018014325 W JP 2018014325W WO 2018186420 A1 WO2018186420 A1 WO 2018186420A1
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mesenchymal stem
stem cells
cell population
cells
cell
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PCT/JP2018/014325
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Japanese (ja)
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渓太 稲生
悠太 喜田
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株式会社カネカ
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Priority to JP2019511269A priority Critical patent/JP7152389B2/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates to a method for producing a cell population containing mesenchymal stem cells.
  • the present invention further relates to a cell population containing mesenchymal stem cells, a mesenchymal stem cell, and a pharmaceutical composition.
  • Mesenchymal stem cells also called mesenchymal stromal cells, are somatic stem cells that have been reported to exist in bone marrow, adipose tissue, dental pulp, etc. Recently, placenta, umbilical cord, egg membrane It has been found that it also exists in fetal appendages. Mesenchymal stem cells have attracted attention as a promising cell source in regenerative medicine because they have the ability to differentiate into bone, cartilage, fat and the like.
  • mesenchymal stem cells have not only differentiation ability but also immunosuppression ability, it has been reported that immune related diseases and inflammatory diseases can be treated by intravenous administration.
  • Patent Document 1 describes a method for producing an amniotic mesenchymal cell composition, a cryopreservation method, and a therapeutic agent.
  • a cryopreservation method for producing an amniotic mesenchymal cell composition
  • a cryopreservation method for producing an amniotic mesenchymal cell composition
  • a cryopreservation method for producing an amniotic mesenchymal cell composition
  • a cryopreservation method a therapeutic agent.
  • Patent Document 2 (D) a step of collecting a cell population of mesenchymal cells from a mammal's amniotic membrane, and (E) seeding the collected cell population at a cell concentration of 400 to 35000 / cm 2 , A step of initial culture for 2 to 3 days, and (F) 3 to 4 subcultures inoculated at a cell concentration of 1/5000 or more and less than 1/10 of the initial culture and changing the medium twice a week And a step of (G) maintaining a culture in the same culture dish until the cells become confluent when a colony of cells having a spindle-like morphology is formed in the subculture.
  • a method for preparing a stem cell population has been described.
  • mesenchymal stem cells derived from fetal appendages are heterogeneous cell populations containing various cells having different differentiation ability, proliferation ability, and cytokine production ability.
  • Patent Document 1 discloses that a mixture containing amnion mesenchymal cells is cryopreserved in a specific cryopreservation solution, thereby suppressing a decrease in the survival rate of the amnion mesenchymal cells after thawing, and cryopreserved amniotic membrane. It is described that mesenchymal cells can be produced as cell preparations optimized for transplantation. However, by selectively preparing mesenchymal stem cells having specific excellent characteristics from mesenchymal stem cells, specifically, a cell population containing a lot of mesenchymal stem cells exhibiting a high immunosuppressive action, There is no description about selective preparation using the characteristics of mesenchymal stem cells as an index.
  • Patent Document 2 discloses that a mesenchymal stem cell population having high proliferation ability and differentiation ability is prepared by seeding cells at a low density, but mesenchymal stem cells included in the mesenchymal stem cell population. There is no description or suggestion of selecting a cell population containing a large amount of mesenchymal stem cells exhibiting a high immunosuppressive effect using the above characteristics as an index.
  • the present inventors have found that the cell population containing cells collected from fetal appendages contains mesenchymal stem cells that are positive for CD97 and CD358, Further, in the cell population including mesenchymal stem cells in which CD97 is positive and the proportion of mesenchymal stem cells is positive and CD358 is positive, a gene encoding an immunosuppression-related cytokine (TGFB2 gene, TNFAIP6) The gene, IL1A gene, IL1B gene, and CCL2 gene) were highly expressed, and it was found to exhibit high immunosuppressive action. The present invention has been completed based on these findings.
  • TGFB2 gene immunosuppression-related cytokine
  • a method for producing a cell population containing mesenchymal stem cells comprising obtaining a cell population having the following cell characteristics (a) and (b): (A) In the cell population, the proportion of mesenchymal stem cells that are positive for CD97 is 30% or more, and (b) the cell population includes mesenchymal stem cells that are positive for CD358. [2] A cell population of mesenchymal stem cells having the following cell characteristics (a) and (b): (A) In the cell population, the proportion of mesenchymal stem cells that are positive for CD97 is 30% or more, and (b) the cell population includes mesenchymal stem cells that are positive for CD358.
  • [4] Mesenchymal stem cells that are positive for CD97 and CD358.
  • [5] A pharmaceutical composition comprising the cell population according to [2] or [3] or the mesenchymal stem cell according to [4] and a pharmaceutically acceptable medium.
  • the relative expression level of the TGFB2 gene with respect to the expression level of the SDHA gene is 1.5 or more
  • the relative expression level of the TNFAIP6 gene with respect to the expression level of the SDHA gene is 0.2 or more
  • the relative expression level of the IL1A gene with respect to the expression level of the SDHA gene is 0.2 or more
  • the relative expression level of the IL1B gene with respect to the expression level of the SDHA gene is 1.3 or more
  • CCL2 with respect to the expression level of the SDHA gene The cell population according to [2] or [3], wherein the relative expression level of the gene satisfies any one or more of 1.0 or more.
  • [12] Use of the cell population according to [2] or [3] or the mesenchymal stem cell according to [4] for the production of a pharmaceutical composition.
  • the pharmaceutical composition is a pharmaceutical composition in which a single dose of mesenchymal stem cells to a human is 10 9 cells / kg body weight or less.
  • the pharmaceutical composition is an injectable preparation.
  • the pharmaceutical composition is a preparation for transplantation having a cell mass or a sheet-like structure.
  • [17] The cell population according to [2] or [3] or the mesenchymal stem cell according to [4] for use in the treatment of a disease.
  • [18] The cell population or mesenchymal stem cell according to [17], wherein a single dose of mesenchymal stem cells to human is 10 9 cells / kg body weight or less.
  • the cell population or mesenchymal stem cell according to [17] or [18] which is a preparation for transplantation having a cell mass or a sheet-like structure.
  • [21] The cell population or mesenchymal stem cell according to any one of [17] to [20], wherein the disease is an immune-related disease.
  • a method for treating a disease comprising administering the cell population according to [2] or [3] or the mesenchymal stem cell according to [4] to a patient in need of treatment.
  • [25] The method for treating a disease according to [22] or [23], which is a transplantation preparation having a cell mass or a sheet-like structure.
  • [26] The method for treating a disease according to any one of [22] to [25], wherein the disease is an immune-related disease.
  • a composition comprising the cell population according to [2] or [3] or the mesenchymal stem cell according to [4] and a medium.
  • a cell population containing mesenchymal stem cells exhibiting a high immunosuppressive action can be prepared.
  • the positive rate of surface antigen and the relative expression level of various genes with respect to housekeeping genes can be used as an indicator of cell population formation including mesenchymal stem cells exhibiting a high immunosuppressive effect.
  • a cell preparation pharmaceutical composition
  • the result of having measured the ratio of the cell which becomes positive with respect to CD97 using the flow cytometer regarding the amniotic membrane MSC cultured in Example 1 is shown.
  • the result of having measured the ratio of the cell which becomes positive with respect to CD97 using the flow cytometer about the amniotic membrane MSC cultured by the comparative example 1 is shown.
  • the result of having measured the ratio of the cell which becomes positive with respect to CD358 using the flow cytometer regarding the amniotic membrane MSC cultured in Example 1 is shown.
  • the result of having measured the ratio of the cell which becomes positive with respect to CD358 about the amniotic membrane MSC cultured by the comparative example 1 using the flow cytometer is shown.
  • Fretal appendage in the present specification refers to egg membrane, placenta, umbilical cord and amniotic fluid.
  • the “egg membrane” is a fetal sac containing fetal amniotic fluid and consists of an amnion, chorion and decidua from the inside. Of these, the amnion and chorion originate from the fetus.
  • Amnion refers to a transparent thin film with poor blood vessels in the innermost layer of the egg membrane.
  • the inner layer of the amniotic membrane (also called the epithelial cell layer) is covered with a layer of secretory epithelial cells to secrete amniotic fluid, and the outer layer of the amniotic membrane (also called the extracellular matrix layer, which corresponds to the stroma) contains mesenchymal stem cells. Including.
  • MSC Mesenchymal stem cells
  • mesenchymal stem cells i) Adhesive to plastic under culture conditions in standard medium. ii) The surface antigens CD105, CD73, and CD90 are positive, and CD45, CD34, CD11b, CD79alpha, CD19, and HLA-DR are negative.
  • the “mesenchymal stem cell population” in the present specification means a cell population containing mesenchymal stem cells, and the form thereof is not particularly limited. For example, a cell pellet, a cell aggregate, a cell suspension or a cell suspension Etc.
  • amniotic mesenchymal stem cells refers to mesenchymal stem cells derived from amniotic membrane, and is used without distinction from “amniotic mesenchymal stromal cells”. In the present specification, “amniotic mesenchymal stem cells” may be described as “amniotic MSC”.
  • immunosuppressive effect refers to an effect of suppressing the response of the immune system in vivo.
  • the immunosuppressive effect can be evaluated by the expression level of one or more genes selected from the TGFB2 gene, the TNFAIP6 gene, the IL1A gene, the IL1B gene, or the CCL2 gene, and the evaluation method is described in Examples described later. Can be performed by microarray analysis.
  • the ratio of mesenchymal stem cells that are positive for CD97 refers to the ratio of cells that are positive for the surface antigen analyzed by flow cytometry, as described in the Examples below. In the present specification, the “ratio of mesenchymal stem cells that are positive for CD97” may be described as “positive rate”.
  • a cell population containing mesenchymal stem cells provided by the present invention includes: (A) In the cell population, the proportion of mesenchymal stem cells that are positive for CD97 is 30% or more, and (b) the cell population includes mesenchymal stem cells that are positive for CD358. It is characterized by that.
  • CD97 refers to the differentiation cluster 97 and is a protein also known as BL-Ac [F2] encoded by the ADGRE5 (Adhesion G protein-coupled receptor E5) gene.
  • CD358 means a differentiation cluster 358 and is a protein encoded by TNFRSF21 (tumor necrosis factor receptor family, member 21) gene.
  • the cell population containing mesenchymal stem cells comprises (a) a ratio of mesenchymal stem cells that are positive for CD97 in the cell population is 30% or more, and (b) the cell population is When the condition that the CD358 contains a mesenchymal stem cell that is positive for CD358 is satisfied, a cell population including a mesenchymal stem cell exhibiting a high immunosuppressive action is formed. Therefore, in the present invention, the above condition can be used as an index for forming a cell population exhibiting a high immunosuppressive action.
  • the proportion of mesenchymal stem cells that are positive for CD97 in the cell population is 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 51% or more, It may be 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, or 60% or more.
  • “Including mesenchymal stem cells that are positive for CD358” means that the mesenchymal stem cells that are positive for CD358 exist in the cell population, and the ratio is not particularly limited as long as the ratio is 5% or more. For example, it may be 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more.
  • the surface antigen markers can be detected by any detection method known in the art.
  • methods for detecting surface antigen markers include, but are not limited to, flow cytometry or cell staining.
  • flow cytometry using a fluorescence-labeled antibody when a cell that emits stronger fluorescence than a negative control (isotype control) is detected, the cell is determined to be “positive” for the marker.
  • the fluorescent labeled antibody any antibody known in the art can be used, and examples thereof include antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and the like. However, it is not limited to these.
  • the cell staining when cells that are colored or fluoresce are observed under a microscope, the cells are determined to be “positive” for the marker.
  • the cell staining may be immune cell staining using an antibody or non-immune cell staining without using an antibody.
  • the ratio (positive rate) of cells positive for the surface antigen markers is determined by the following procedures (1) to (8) using dot plot development analysis of flow cytometry. Can be measured.
  • PBS phosphate buffer
  • the timing for detecting the surface antigen marker is not particularly limited. For example, immediately after separating cells from a biological sample, during the culture process, after purification in the culture process, and immediately after N passages (N is 1 or more) An integer), during maintenance culture, before cryopreservation, after thawing, or before formulation as a pharmaceutical composition.
  • the immunosuppressive effect can be evaluated by the expression level of one or more genes selected from the TGFB2 gene, TNFAIP6 gene, IL1A gene, IL1B gene or CCL2 gene.
  • the immunosuppressive action may be evaluated as a relative expression level of each of the above genes with respect to the expression level of the SDHA gene by microarray analysis described in Examples described later.
  • the sequence of the SDHA (Succinate dehydrogenase complex, subunit A) gene is registered as ID: 6389 in the gene database of the National Center for Biotechnology Information.
  • the sequence of TGFB2 (Transforming growth factor, beta 2) gene is registered as ID: 7042 in the gene database of National Center for Biotechnology Information.
  • the sequence of the TNFAIP6 (Tumor necrosis factor, alpha-induced protein 6) gene is registered in the gene database of National Center for Biotechnology Information as ID: 7130.
  • IL1A Interleukin 1, alpha
  • IL1B Interleukin 1, beta
  • CCL2 Cyhemokine (C-C motif) ligand 2
  • the relative expression level of the TGFB2 gene with respect to the expression level of the SDHA gene is preferably 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 1.9 or more, 2.0 or more. It may be 1 or more, 2.2 or more, 2.3 or more, 2.4 or more, 2.5 or more, 2.6 or more, or 2.7 or more.
  • the relative expression level of the TNFAIP6 gene with respect to the expression level of the SDHA gene is preferably 0.2 or more, and may be 0.3 or more.
  • the relative expression level of the IL1A gene with respect to the expression level of the SDHA gene is preferably 0.2 or more, and may be 0.3 or more.
  • the relative expression level of the IL1B gene with respect to the expression level of the SDHA gene is preferably 1.3 or more, and may be 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, or 1.8 or more.
  • the relative expression level of the CCL gene with respect to the expression level of the SDHA gene is preferably 1.0 or more, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more. It may be 6 or more, or 1.7 or more.
  • the timing for measuring the above gene expression level is not particularly limited. For example, immediately after separating cells from a biological sample, during the culture process, after purification in the culture process, and immediately after N passages (N is 1 or more). An integer), during maintenance culture, before cryopreservation, after thawing, or before formulation as a pharmaceutical composition.
  • the origin of the mesenchymal stem cells is not particularly limited, but for example, mesenchymal stem cells derived from fetal appendages, bone marrow, fat, or dental pulp can be used.
  • the mesenchymal stem cell is preferably a mesenchymal stem cell derived from a fetal appendage, and more preferably a mesenchymal stem cell derived from an amniotic membrane.
  • the mesenchymal stem cell population of the present invention can be stored in a frozen state until just before use.
  • the mesenchymal stem cell population may contain any component other than mesenchymal stem cells. Examples of such components include salts, polysaccharides (eg, HES, dextran, etc.), proteins (eg, albumin, etc.), DMSO, amino acids, medium components (eg, components contained in RPMI 1640 medium, etc.), and the like. Although it can, it is not limited to these.
  • the cell population of the present invention may be provided as a composition in combination with a medium.
  • a medium for example, a medium or a pharmaceutically acceptable medium described later
  • a liquid medium for example, a medium or a pharmaceutically acceptable medium described later
  • the cell population of the present invention can comprise any number of mesenchymal stem cells.
  • the cell population of the present invention is, for example, 1 ⁇ 10 1 , 2 ⁇ 10 1 , 5 ⁇ 10 1 , 1 ⁇ 10 2 , 2 ⁇ 10 2 , 5 ⁇ 10 2 , 1 ⁇ 10 3 2 ⁇ 10 3 pieces, 5 ⁇ 10 3 pieces, 1 ⁇ 10 4 pieces, 2 ⁇ 10 4 pieces, 5 ⁇ 10 4 pieces, 1 ⁇ 10 5 pieces, 2 ⁇ 10 5 pieces, 5 ⁇ 10 5 pieces, 1 ⁇ 10 6 , 2 ⁇ 10 6 , 5 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 5 ⁇ 10 7 , 1 ⁇ 10 8 , 2 ⁇ 10 8 , 5 ⁇ 10 8 units, 1 ⁇ 10 9 units, 2 ⁇ 10 9 units, 5 ⁇ 10 9 units, 1 ⁇ 10 10 units, 2 ⁇ 10 10 units, 5 ⁇ 10 9 units, 1 ⁇ 10 10 units, 2 ⁇ 10 10
  • the method for producing a cell population containing mesenchymal stem cells according to the present invention obtains a cell population having the following cell characteristics (a) and (b). It is a method including. (A) In the cell population, the proportion of mesenchymal stem cells that are positive for CD97 is 30% or more, and (b) the cell population includes mesenchymal stem cells that are positive for CD358.
  • the cell population containing mesenchymal stem cells has a ratio of mesenchymal stem cells that are positive for CD97 is 30% or more and CD358 is positive. It is a method including the step of preparing under conditions that maintain the presence of presenting mesenchymal stem cells.
  • the above-mentioned condition that “the ratio of mesenchymal stem cells that are positive for CD97 is 30% or more and that mesenchymal stem cells that are positive for CD358 are included” is a cell containing mesenchymal stem cells that exhibits a high immunosuppressive effect. It is an index of group formation, and the production method of the present invention is not particularly limited as long as the index is satisfied.
  • the production method of the present invention includes a cell population obtaining step of obtaining a cell population containing mesenchymal stem cells by enzymatic treatment of a sample containing mesenchymal stem cells (for example, fetal appendages such as amniotic membrane). But you can.
  • Amniotic membrane is composed of an epithelial cell layer and an extracellular matrix layer, and the latter includes amniotic membrane MSC.
  • Amniotic epithelial cells like other epithelial cells, express epithelial cadherin (E-cadherin: CD324) and epithelial adhesion factor (EpCAM: CD326), whereas amniotic MSCs express these epithelial specific surface antigen markers. Is not expressed and can be easily distinguished by flow cytometry.
  • the cell population acquisition step may include a step of obtaining an amniotic membrane by cesarean section.
  • the cell population containing mesenchymal stem cells in the present invention is preferably a cell population obtained by treating a sample containing an epithelial cell layer and an extracellular matrix layer collected from fetal appendages with at least collagenase.
  • Enzymatic treatment of a sample collected from a fetal appendage preferably a sample including an epithelial cell layer and an extracellular matrix layer
  • a sample collected from a fetal appendage preferably a sample including an epithelial cell layer and an extracellular matrix layer
  • an enzyme that does not degrade the epithelial cell layer.
  • Such an enzyme is not particularly limited, and examples thereof include collagenase and / or metal proteinase.
  • metal proteinases include, but are not particularly limited to, thermolysin and / or dispase, which are metal proteinases that cleave the N-terminal side of nonpolar amino acids.
  • the active concentration of collagenase is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, further preferably 200 PU / ml or more, further preferably 300 PU / ml or more, and further preferably 400 PU / ml or more.
  • the active concentration of collagenase is not particularly limited, and is, for example, 1000 PU / ml or less, 900 PU / ml or less, 800 PU / ml or less, 700 PU / ml or less, 600 PU / ml or less, 500 PU / ml or less.
  • PU Protease Unit
  • PU Protease Unit
  • the active concentration of the metalloproteinase is preferably 50 PU / ml or more, more preferably 100 PU / ml or more, further preferably 200 PU / ml or more, more preferably 300 PU / ml or more, further preferably 400 PU / ml or more.
  • the activity concentration of the metal proteinase is preferably 1000 PU / ml or less, more preferably 900 PU / ml or less, further preferably 800 PU / ml or less, more preferably 700 PU / ml or less, further preferably 600 PU / ml or less, and further preferably Is 500 PU / ml or less.
  • PU Protease Unit
  • PU is defined as the amount of enzyme that liberates amino acid corresponding to 1 ug of tyrosine from lactate casein per minute at pH 7.5 and 30 ° C.
  • the mesenchymal stem cells contained in the extracellular matrix layer can be efficiently released while preventing contamination of the epithelial cells contained in the fetal appendage epithelial cell layer.
  • a combination of preferred concentrations of collagenase and / or metalloproteinase can be determined by microscopic observation of fetal appendages after enzyme treatment or flow cytometry of the obtained cells.
  • thermolysin and / or dispase can be used, but are not limited thereto.
  • the enzyme treatment of the fetal appendage is preferably carried out by immersing the amniotic membrane washed with a washing solution such as physiological saline or Hank's balanced salt solution in the enzyme solution and stirring with stirring means.
  • stirring means for example, a stirrer or a shaker can be used from the viewpoint of efficiently releasing mesenchymal stem cells contained in the extracellular matrix layer of the fetal appendage, but is not limited thereto.
  • the stirring speed is not particularly limited, but when a stirrer or shaker is used, it is, for example, 5 rpm or more, 10 rpm or more, 20 rpm or more, 30 rpm or more, 40 rpm or more, or 50 rpm or more.
  • stirring speed is not specifically limited, When a stirrer or a shaker is used, it is 100 rpm or less, 90 rpm or less, 80 rpm or less, 70 rpm or less, or 60 rpm or less, for example.
  • the enzyme treatment time is not particularly limited, and is, for example, 10 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, 60 minutes or more, 70 minutes or more, 80 minutes or more, or 90 minutes or more.
  • the enzyme treatment time is not particularly limited, and is, for example, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 110 minutes or less, or 100 minutes or less.
  • the enzyme treatment temperature is not particularly limited.
  • the enzyme treatment temperature is 15 ° C or higher, 16 ° C or higher, 17 ° C or higher, 18 ° C or higher, 19 ° C or higher, 20 ° C or higher, 21 ° C or higher, 22 ° C or higher, 23 ° C or higher, 24 ° C. 25 ° C or higher, 26 ° C or higher, 27 ° C or higher, 28 ° C or higher, 29 ° C or higher, 30 ° C or higher, 31 ° C or higher, 32 ° C or higher, 33 ° C or higher, 34 ° C or higher, 35 ° C or higher, or 36 ° C or higher. is there.
  • the enzyme treatment temperature is not particularly limited, and is, for example, 40 ° C. or lower, 39 ° C. or lower, 38 ° C. or lower, or 37 ° C. or lower.
  • the mesenchymal stem cells released from the enzyme solution containing the released mesenchymal stem cells are separated and / or collected by a known method such as a filter, centrifugation, hollow fiber separation membrane, cell sorter or the like. can do.
  • the enzyme solution containing mesenchymal stem cells released by the filter is filtered.
  • the enzyme solution is filtered with a filter, only the released cells pass through the filter, and the undegraded epithelial cell layer cannot pass through the filter and remains on the filter, so that the released mesenchymal stem cells can be easily removed.
  • the risk of contamination with bacteria and viruses can also be reduced.
  • the pore size (mesh size) of the mesh filter is not particularly limited, and is, for example, 40 ⁇ m or more, 50 ⁇ m or more, 60 ⁇ m or more, 70 ⁇ m or more, 80 ⁇ m or more, or 90 ⁇ m or more.
  • the pore size of the mesh filter is not particularly limited, and is, for example, 200 ⁇ m or less, 190 ⁇ m or less, 180 ⁇ m or less, 170 ⁇ m or less, 160 ⁇ m or less, 150 ⁇ m or less, 140 ⁇ m or less, 130 ⁇ m or less, 120 ⁇ m or less, 110 ⁇ m or less, or 100 ⁇ m or less.
  • the filtration rate is not particularly limited, but by setting the pore size of the mesh filter within the above range, the enzyme solution containing mesenchymal stem cells can be filtered by natural fall, thereby preventing a decrease in cell viability. be able to.
  • Nylon is preferably used as the mesh filter material.
  • a tube containing a 40 ⁇ m, 70 ⁇ m, 95 ⁇ m or 100 ⁇ m nylon mesh filter such as a Falcon cell strainer widely used for research can be used.
  • the medical mesh cloth nylon and polyester
  • an arterial filter polyyester mesh filter, pore size: 40 ⁇ m or more and 120 ⁇ m or less
  • Other materials such as a stainless mesh filter can also be used.
  • mesenchymal stem cells When mesenchymal stem cells are passed through a filter, natural fall (free fall) is preferred. Although forced filter passage such as suction using a pump or the like is possible, it is desirable to make the pressure as weak as possible in order to avoid damaging the cells.
  • the mesenchymal stem cells that have passed through the filter can be recovered by centrifugation after diluting the filtrate with a double or more medium or a balanced salt buffer.
  • a balanced salt buffer Dulbecco's phosphate buffer (DPBS), Earl balanced salt solution (EBSS), Hanks balanced salt solution (HBSS), phosphate buffer (PBS) and the like can be used, but not limited thereto.
  • the cell population obtained in the above cell population acquisition step is prepared under the condition that the ratio of mesenchymal stem cells positive for CD97 is 30% or more and contains mesenchymal stem cells positive for CD358. .
  • the above conditions are useful as an index for obtaining a cell population containing mesenchymal stem cells exhibiting a high immunosuppressive action.
  • the preparation method is not particularly limited as long as it satisfies the above index. Such a method includes, for example, sorting a cell population that satisfies the above conditions using a cell sorter.
  • the cell population includes a mesenchymal stem cell in which the ratio of mesenchymal stem cells that are positive for CD97 is 30% or more and that contains mesenchymal stem cells that are positive for CD358. And culturing.
  • Examples of the culture method satisfying the index include a step of repeatedly inoculating and culturing a cell population in an uncoated plastic culture vessel at a density of 400 to 5,000 cells / cm 2. Can do.
  • the density at the time of seeding the cell population is more preferably 500 cells / cm 2 or more, more preferably 600 cells / cm 2 or more, further preferably 700 cells / cm 2 or more, more preferably 800 cells.
  • the density at the time of seeding the cell population is more preferably 4800 cells / cm 2 or less, further preferably 4600 cells / cm 2 or less, further preferably 4400 cells / cm 2 or less, more preferably 4200 cells.
  • / Cm 2 or less more preferably 4000 cells / cm 2 or less, further preferably 3800 cells / cm 2 or less, more preferably 3600 cells / cm 2 or less, and further preferably 3400 cells / cm 2.
  • 2 or less more preferably 3200 cells / cm 2 or less, further preferably 3000 cells / cm 2 or less, more preferably 2800 cells / cm 2 or less, and further preferably 2600 cells / cm 2 or less.
  • More preferably 2400 cells / cm 2 or less Preferably it is 2200 cells / cm 2 or less.
  • a coating agent at a density of 400 to 5,000 cells / cm 2.
  • the preferable conditions for the density when seeding the cell population are the same as those described above.
  • coating agent examples include extracellular matrix, fibronectin, vitronectin, osteopontin, laminin, entactin, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, gelatin, poly-L-ornithine, poly-D. -Lysine, Matrigel® matrix may be mentioned, but not limited to.
  • Still another culture method that satisfies the above-mentioned index includes, for example, culturing by adding basic fibroblast growth factor (bFGF) to a basal medium used for culture.
  • the concentration of the basic fibroblast growth factor is preferably 2 ng / mL or more, more preferably 4 ng / mL or more, further preferably 6 ng / mL or more, more preferably 8 ng / mL or more, More preferably, it is 10 ng / mL or more.
  • the concentration of basic fibroblast growth factor is preferably 20 ng / mL or less, more preferably 18 ng / mL or less, 16 ng / mL or less, more preferably 14 ng / mL or less, and more preferably 12 ng / mL or less.
  • the timing for adding the basic fibroblast growth factor is not particularly limited. For example, at the beginning of the culture process, during the culture process, immediately after purification in the culture process, and immediately after N passages (N is an integer of 1 or more) In the middle of maintenance culture, before cryopreservation, or after thawing.
  • Examples of the culture period of the above-mentioned single culture include 4 to 10 days, and more specifically, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. Can be mentioned.
  • the medium used for the above culture can be prepared by using any animal cell culture liquid medium as a basal medium and adding other components (serum, serum replacement reagent, growth factor, etc.) as necessary. .
  • BME medium BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199Mem, AlF Minimum Essential Medium Eagle medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham F10 medium, Ham F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media (for example, DMEM / F12 medium) Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)) but the medium and the like can be used is not particularly limited.
  • IMDM medium Iscove's Modified Dulbecco's Medium
  • Medium 199Mem AlF Minimum Essential Medium Eagle medium
  • DMEM medium Dulbecco's Modified Eagle's Medium
  • Ham F10 medium Ham F12 medium
  • RPMI 1640 medium RPMI 1640 medium
  • Fischer's medium and mixed media
  • a commercially available serum-free medium may be used.
  • examples include STK1 and STK2 (DS Pharma Biomedical), EXPREP MSC Medium (Biomimetics Sympathies), Corning stemgro human mesenchymal stem cell medium (Corning), but are not particularly limited.
  • albumin a reagent for stabilizing the growth factor in the medium
  • serum a reagent for stabilizing the growth factor in the medium
  • growth factor a reagent for stabilizing the growth factor in the medium
  • the growth factor may be further added in addition to the growth factor, or the growth factor may be preliminarily added with a gel or a polysaccharide. It may be stabilized and then a stabilized growth factor may be added to the basal medium.
  • Culture of mesenchymal stem cells can be performed, for example, by the following steps. First, the cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in a medium. Next, the cells are seeded in a plastic culture vessel and cultured in a CO 2 concentration of 3% or more and 5% or less at 37 ° C. using a culture medium so that the confluence rate is 95% or less. Examples of the medium include, but are not limited to, ⁇ MEM, M199, or a medium based on these. The cells obtained by the culture as described above are cells cultured once.
  • the cells cultured once can be further subcultured and cultured as follows, for example. First, the cells cultured once are treated with ethylenediaminetetraacetic acid (EDTA) and then treated with trypsin and detached from the plastic culture vessel. Next, the obtained cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in a medium. Finally, the cells are seeded in a plastic culture vessel and cultured in a CO 2 concentration of 3% or more and 5% or less at 37 ° C. using a culture medium so that the confluence rate is 95% or less. Examples of the medium include, but are not limited to, ⁇ MEM, M199, or a medium based on these.
  • Cells obtained by passage and culture as described above are cells that have been passaged once. By performing similar passage and culture, cells that have been passaged N times can be obtained (N represents an integer of 1 or more).
  • the lower limit of the number N of passages is, for example, 1 or more times, preferably 2 times or more, more preferably 3 times or more, further preferably 4 times or more, further preferably 5 times or more, from the viewpoint of producing a large amount of cells.
  • the upper limit of the number N of passages is preferably, for example, 50 times or less, 45 times or less, 40 times or less, 35 times or less, or 30 times or less from the viewpoint of suppressing cell aging.
  • a cell population containing mesenchymal stem cells exhibiting a high immunosuppressive effect can be obtained.
  • the lower limit of the number of cells acquired per batch of culture varies depending on the number of seeded cells, the seeding density, etc., for example, 1.0 ⁇ 10 5 (cells / cm 2 / day) or more, 2.0 ⁇ 10 5 (pieces / cm 2 / day) or more, 3.0 ⁇ 10 5 (pieces / cm 2 / day) or more, 4.0 ⁇ 10 5 (pieces / cm 2 / day) or more, 5.0 ⁇ 10 5 (pieces / cm 2 / day) or more, 6.0 ⁇ 10 5 (pieces / cm 2 / day) or more, 7.0 ⁇ 10 5 (pieces / cm 2 / day) Or more, 8.0 ⁇ 10 5 (pieces / cm 2 / day) or more, 9.0 ⁇ 10 5 (
  • the upper limit of the number of obtained cells per batch of culture is not particularly limited. For example, 10.0 ⁇ 10 8 (cells / cm 2 / day) or less, 9.0 ⁇ 10 8 (cells / cm 2 / day).
  • 8.0 ⁇ 10 8 (pieces / cm 2 / day) or less 7.0 ⁇ 10 8 (pieces / cm 2 / day) or less, 6.0 ⁇ 10 8 (pieces / cm 2 / day) or less , 5.0 ⁇ 10 8 (pieces / cm 2 / day) or less, 4.0 ⁇ 10 8 (pieces / cm 2 / day) or less, 3.0 ⁇ 10 8 (pieces / cm 2 / day) or less, 2 0.0 ⁇ 10 8 (pieces / cm 2 / day) or less or 1.0 ⁇ 10 8 (pieces / cm 2 / day) or less.
  • the mesenchymal stem cells obtained by the production method of the present invention are preferably cultured after in vitro, preferably after 40 days, more preferably after 45 days, until 50 days, until 55 days, Until 60 days, 65 days and later, 70 days and later, 75 days and later, 80 days and later, 85 days and later, 90 days and later, 95 days and later, 100 days and later, 105 days and later, Alternatively, it can be cultured until 110 days or later.
  • the mesenchymal stem cells obtained by the production method of the present invention preferably have a doubling number of 10 times or more, more preferably 15 times or more, 20 times or more, 25 times or more, 30 times after the start of in vitro culture. As described above, the culture can be performed until 35 times, 40 times, 45 times, or 50 times or more.
  • the production method of the present invention comprises: (A) In the cell population, the ratio of mesenchymal stem cells that are positive for CD97 is 30% or more, and (b) the cell population includes mesenchymal stem cells that are positive for CD358. As an index, an identification step for identifying a cell population containing mesenchymal stem cells exhibiting a high immunosuppressive action may be included.
  • the means for identifying the cell population containing the mesenchymal stem cells is preferably flow cytometry.
  • the ratio (positive rate) of mesenchymal stem cells that are positive for CD97 and CD358 in the cell population can be measured according to the procedure described in paragraph 0029 using dot plot development analysis of flow cytometry.
  • timing for performing the above identification is not particularly limited, for example, immediately after separating cells from a biological sample, in the middle of a culture process, after purification in the culture process, and immediately after N passages (N represents an integer of 1 or more) ), During maintenance culture, before cryopreservation, after thawing, or before formulation as a pharmaceutical composition.
  • the production method of the present invention can include a step of selectively separating the identified cell population after identifying the cell population containing the mesenchymal stem cells using the above (a) and (b) as an index.
  • the means for selectively separating the identified cell population is not particularly limited, and examples thereof include sorting of the cell population by a cell sorter, purification of the cell population by culture, and the like.
  • the production method of the present invention can include a step of cryopreserving a cell population containing the mesenchymal stem cells.
  • the cell population may be identified, separated, collected and / or cultured as necessary after thawing the cell population.
  • the cell population may be used as it is after thawing.
  • the means for cryopreserving the cell population containing the mesenchymal stem cells is not particularly limited, and examples thereof include a program freezer, a deep freezer, and immersion in liquid nitrogen.
  • the freezing temperature is preferably -30 ° C or lower, -40 ° C or lower, -50 ° C or lower, -60 ° C or lower, -70 ° C or lower, -80 ° C or lower, -90 ° C or lower.
  • preferable freezing rates when freezing are, for example, -1 ° C / min, -2 ° C / min, -3 ° C / min, -4 ° C / min, -5 ° C / min, -6 ° C / min, -7 ° C / min, -8 ° C / min, -9 ° C / min, -10 ° C / min, -11 ° C / min, -12 ° C / min, -13 ° C / min, -14 ° C / min Min or ⁇ 15 ° C./min.
  • the temperature is between ⁇ 50 ° C. and ⁇ 30 ° C.
  • the temperature is further lowered to a temperature of ⁇ 100 ° C. to ⁇ 80 ° C. (eg, ⁇ 90 ° C.) at a freezing rate of ⁇ 11 ° C./min to ⁇ 9 ° C./min (eg, ⁇ 10 ° C./min).
  • the temperature can be rapidly lowered to ⁇ 196 ° C. and frozen, and then frozen and stored in liquid nitrogen (gas phase).
  • the above cell population may be frozen in an arbitrary storage container.
  • storage containers include, but are not limited to, cryotubes, cryovials, freezing bags, infusion bags, and the like.
  • cryopreservation solution When freezing by the above freezing means, the above cell population may be frozen in any cryopreservation solution.
  • a cryopreservation solution a commercially available cryopreservation solution may be used, and examples thereof include CryoNovo (Acron Biotechnology), MSC Freezing Solution (Biological Industries), CryoStor (HemaCare), and the like. It is not limited.
  • the above cryopreservation solution can contain a predetermined concentration of polysaccharide.
  • the preferable concentration of the polysaccharide is, for example, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, 8% by mass or more, 9% It is 10 mass% or more, 11 mass% or more, or 12 mass% or more.
  • concentration of polysaccharide is 40 mass% or less, 35 mass% or less, 30 mass% or less, 25 mass% or less, 20 mass% or less, 19 mass% or less, 18 mass% or less, 17 mass% or less, for example.
  • polysaccharides include, but are not limited to, hydroxylethyl starch (HES) or dextran (such as Dextran 40).
  • HES hydroxylethyl starch
  • dextran such as Dextran 40
  • the above-mentioned cryopreservation solution can contain a predetermined concentration of dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • the preferred concentration of DMSO is, for example, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, 8% by mass or more, or 9% by mass. % Or more.
  • concentration of DMSO is 20 mass% or less, 19 mass% or less, 18 mass% or less, 17 mass% or less, 16 mass% or less, 15 mass% or less, 14 mass% or less, 13 mass% or less, It is 12 mass% or less, 11 mass% or less, or 10 mass% or less.
  • the above cryopreservation solution may contain a predetermined concentration of albumin greater than 0% by mass.
  • the preferred concentration of albumin is, for example, 0.5 mass% or more, 1 mass% or more, 2 mass% or more, 3 mass% or more, 4 mass% or more, 5 mass% or more, 6 mass% or more, 7 mass% or more, or It is 8 mass% or more.
  • concentration of albumin is 40 mass% or less, 35 mass% or less, 30 mass% or less, 25 mass% or less, 20 mass% or less, 15 mass% or less, 10 mass% or less, or 9 mass% or less, for example. is there.
  • albumin include, but are not limited to, bovine serum albumin, mouse albumin, human albumin and the like.
  • compositions The cell population containing mesenchymal stem cells according to the present invention can be used as a pharmaceutical composition. That is, according to the present invention, there is provided a pharmaceutical composition comprising the cell population according to the present invention and a pharmaceutically acceptable medium.
  • the pharmaceutical composition of the present invention is preferably a liquid, and more preferably an injectable liquid.
  • the pharmaceutical composition of the present invention can be used as a cell therapeutic agent, for example, an intractable disease therapeutic agent.
  • the pharmaceutical composition of the present invention can be used as a therapeutic agent for immune-related diseases.
  • the above-mentioned diseases can be treated by administering the pharmaceutical composition of the present invention to the treatment site in such an amount that the effect can be measured.
  • a cell population comprising mesenchymal stem cells according to the present invention used for a pharmaceutical composition.
  • a cell population comprising mesenchymal stem cells according to the present invention used for a cell therapeutic agent.
  • a cell population containing mesenchymal stem cells according to the present invention which is used for treatment of immune-related diseases.
  • a cell population containing mesenchymal stem cells according to the present invention which is administered to a patient or a subject and used for suppressing an immune response.
  • a method of transplanting cells into a patient or subject comprising the step of administering to the patient or subject a therapeutically effective amount of a cell population comprising mesenchymal stem cells according to the present invention, and a disease of the patient or subject.
  • a method of treatment is provided.
  • a cell population comprising mesenchymal stem cells according to the present invention for the manufacture of a pharmaceutical composition.
  • a cell population comprising mesenchymal stem cells according to the present invention for the manufacture of a cell therapeutic agent.
  • a cell population containing mesenchymal stem cells for the manufacture of a therapeutic agent for immune related diseases.
  • a cell population containing mesenchymal stem cells for the manufacture of a therapeutic agent required for suppression of an immune response by administration to a patient or subject.
  • the dosage of the pharmaceutical composition of the present invention is such an amount of cells that, when administered to a patient or subject, can obtain a therapeutic effect on the disease compared to a non-administered patient or subject.
  • the specific dose can be appropriately determined depending on the administration form, administration method, purpose of use, age of patient or subject, body weight, symptoms and the like.
  • the single dose of mesenchymal stem cells to humans is not particularly limited, and is, for example, 10 4 cells / kg body weight or more, 10 5 cells / kg body weight or more, or 10 6 cells / kg body weight or more.
  • the dose of mesenchymal stem cells to human being is not particularly limited, and is, for example, 10 9 cells / kg body weight or less, 10 8 cells / kg body weight or less, or 10 7 cells / kg body weight or less.
  • the administration method of the pharmaceutical composition of the present invention is not particularly limited, and for example, subcutaneous injection, intralymphatic injection, intravenous injection, intraarterial injection, intraperitoneal injection, intrathoracic injection or direct local injection, or local injection And transplanting directly.
  • administration method of the pharmaceutical composition see, for example, JP-A-2015-61520, Onken JE, tal. American College of Gastroenterology Conference 2006 Las Vegas, NV, Abstract 121. Garcia-Olmo D, et al. Dis Colon Rectum 2005; 48: 1416-23.
  • Intravenous injection, intravenous drip injection, local direct injection, direct local transplantation and the like are known.
  • the pharmaceutical composition of the present invention can also be administered by various methods described in the above literature.
  • the pharmaceutical composition of the present invention can also be used as an injectable preparation, a cell mass or a sheet-form transplant preparation, or a gel preparation mixed with any gel for the purpose of treating other diseases.
  • the patient or subject of the present invention is typically a human but may be other animals.
  • Other animals include, for example, dogs, cats, cows, horses, pigs, goats, sheep, monkeys (cynomolgus monkeys, rhesus monkeys, common marmosets, Japanese monkeys), ferrets, rabbits, rodents (mouses, rats, gerbils, guinea pigs, Mammals such as hamster), and birds such as chicken and quail, but are not limited thereto.
  • the pharmaceutical composition of the present invention can be stored in a frozen state until just before use.
  • the pharmaceutical composition of the present invention can be used by rapidly thawing at 37 ° C.
  • the pharmaceutical composition of the present invention may contain any component used for human treatment.
  • components include salts, polysaccharides (eg, HES, dextran, etc.), proteins (eg, albumin, etc.), DMSO, amino acids, medium components (eg, components contained in RPMI 1640 medium, etc.), and the like. Although it can, it is not limited to these.
  • the pharmaceutical composition of the present invention may be obtained by diluting a cell population containing mesenchymal stem cells with an infusion preparation used as a pharmaceutically acceptable medium.
  • the “infusion preparation (pharmaceutically acceptable medium)” in the present specification is not particularly limited as long as it is a solution used in human treatment.
  • physiological saline, 5% glucose solution, Ringer's solution, lactic acid Ringer's solution, Ringer's acetate solution, starting solution (No. 1 solution), dehydration replenisher (No. 2 solution), maintenance infusion (No. 3 solution), postoperative recovery solution (No. 4 solution) and the like can be mentioned.
  • the pharmaceutical composition of the present invention comprises various additives for increasing storage stability, sterility, isotonicity, absorbability and / or viscosity, such as emulsifiers, dispersants, buffers, preservatives, wetting agents. , Antibacterial agents, antioxidants, chelating agents, thickeners, gelling agents, pH adjusting agents, and the like.
  • the thickener include, but are not limited to, HES, dextran, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, and the like.
  • the concentration of the thickener depends on the selected thickener, but can be arbitrarily set within a range of concentrations that are safe when administered to a patient or subject and achieve a desired viscosity.
  • the pH of the pharmaceutical composition of the present invention can be a neutral pH, for example, pH 6.5 or more or pH 7.0 or more, and can be pH 8.5 or less or pH 8.0 or less. It is not limited to these.
  • Step 1 Collection of amniotic membrane
  • the egg membrane and placenta were aseptically collected from a pregnant woman (donor) with a waiting cesarean section who obtained informed consent.
  • the obtained egg membrane and placenta were housed in a sterile vat containing physiological saline, and the amniotic membrane was manually peeled from the stump of the egg membrane.
  • the amniotic membrane was washed with Hanks balanced salt solution (not containing Ca ⁇ Mg) to remove the adhering blood and clot.
  • Step 2 Enzymatic treatment of amniotic membrane and recovery of amniotic membrane MSC
  • the amniotic membrane containing the epithelial cell layer and the extracellular matrix layer is immersed in a Hank's balanced salt solution (containing Ca ⁇ Mg) containing 300 PU / mL collagenase and 200 PU / mL dispase I, and incubated at 37 ° C. for 90 minutes at 50 rpm.
  • the amniotic membrane was enzymatically treated by stirring under conditions.
  • the solution after the enzyme treatment was filtered through a nylon mesh having an opening of 95 ⁇ m to remove undigested amniotic membrane, and a cell suspension containing amniotic membrane MSC was recovered.
  • the ratio of cells positive for the expression of CD90 which is one of the surface antigens known as typical positive markers of MSC, was analyzed using a flow cytometer. It was confirmed that the amniotic membrane MSC was separated from the amniotic membrane by purity.
  • BD Accu TM C6 Flow Cytometer manufactured by Becton Dickinson (BD) was used, and the measurement conditions were the number of analyzed cells: 10,000 cells, and the flow rate setting: Slow (14 ⁇ L / min).
  • FITC Mouse Anti-Human CD90 (BD / Model: 561969) was used as an antibody corresponding to the CD90 antigen
  • FITC Mouse IgG1, ⁇ Isotype Control (BD / Model: 349041) was used as an antibody for isotype control.
  • Step 3 Cryopreservation of amniotic membrane MSC
  • the cell population obtained in the above “Step 2: Enzymatic treatment of amniotic membrane and recovery of amniotic MSC” is suspended in a bunker (Lymphtec Corporation) so as to be 1.0 ⁇ 10 7 cells / mL, Dispense into tubes.
  • the cryotube was placed in a bicell (freezing container) (Nippon Freezer), stored at ⁇ 80 ° C. for 12 hours, and then stored frozen at liquid nitrogen temperature.
  • Step 4 Culture of amniotic membrane MSC
  • the cell population obtained in the above “Step 3: Cryopreservation of amniotic MSC” is seeded in an uncoated plastic culture container, and 10% fetal bovine serum (FBS) (inactivated) and 1 ⁇ Antibiotic- Adhesion culture was performed until ⁇ -MEM (Alpha Modification of Minimum Essential Medium) containing Anticotic (manufactured by Thermo Fisher Scientific) until subconfluent. Thereafter, the cells are detached using TrypLE Select (1 ⁇ ) (manufactured by Thermo Fisher Scientific), and 1 ⁇ 4 amount of cells is seeded in an uncoated plastic culture vessel of the same scale as the previous culture. Subculture was performed. Medium exchange was performed twice a week. In this way, subculture was continued.
  • FBS fetal bovine serum
  • Step 1 Collection of amniotic membrane
  • the amniotic membrane was obtained in the same manner as in Step 1 of Comparative Example 1 except that the egg membrane and placenta as fetal appendages were aseptically collected from a donor different from the donor in Comparative Example 1.
  • Step 2 Enzymatic treatment of amniotic membrane and recovery of amniotic membrane MSC
  • a cell suspension containing amniotic membrane MSC was collected by the same method as in Step 2 of Comparative Example 1.
  • cells with positive expression of CD90 which is one of surface antigens known as typical positive markers of MSC, using a flow cytometer in the same manner as in Comparative Example 1 This ratio was analyzed, and it was confirmed that the amniotic membrane MSC was separated from the amniotic membrane with high purity.
  • Step 3 Cryopreservation of amniotic membrane MSC
  • Step 2 Enzymatic treatment of amniotic membrane and recovery of amniotic membrane MSC
  • Step 4 Culture of amniotic membrane MSC
  • the cell population obtained in “Step 3: Cryopreservation of amniotic membrane MSC” was adherently cultured until it became subconfluent by the same method as in Step 4 of Comparative Example 1. Subculture was performed in the same manner as in Step 4 of Comparative Example 1.
  • Anti-CD97-Mouse Mono IgG1 (Miltenyi Biotec / Model: 130-097-102) as an antibody corresponding to the CD97 antigen
  • Mouse IgG1 (Miltenyi Biotec / Model: 130-098-) as an antibody for isotype control. 845) was used.
  • Mouse Anti HumanCD358 (Bio-Rad / Model: MCA2333) is used as the primary antibody corresponding to CD358 antigen
  • Mouse Mono IgG1 (Bio-Rad / Model: MCA928) is used as the isotype control antibody
  • CD358 antigen is supported.
  • Mouse F (ab) 2 IgG (H + L) APC-conjugated Antibody (R & D Systems / Model No .: F0101B) was used as a secondary antibody corresponding to the primary antibody and its isotype control antibody.
  • the positive rate of CD97 and CD358 improved in the cell population of Example 1 compared to the cell population of Comparative Example 1.
  • the proportion of mesenchymal stem cells that are positive for CD97 is less than 30% (specifically 20.6%), whereas in the cell population of Example 1, CD97 is positive.
  • the ratio of mesenchymal stem cells exhibiting the above was 30% or more (specifically 60.6%).
  • the cell population of Comparative Example 1 does not contain mesenchymal stem cells that are positive for CD358 (specifically, 0.0%), whereas the cell population of Example 1 is positive for CD358. It was revealed to contain the presenting mesenchymal stem cells (specifically 7.1%).
  • Microarray analysis was performed according to the following procedures (1) to (5).
  • the following procedures (3) to (5) were performed by entrusting RIKEN GENESIS.
  • (1) The sixth passage cell population cultured in Comparative Example 1 and Example 1 was detached from the plastic culture vessel using a cell scraper (manufactured by Corning) and collected by centrifugation.
  • (2) RNAlater (manufactured by Thermo Fisher Scientific) was added to the obtained cell pellet to stably preserve the RNA, and then total RNA was extracted and purified using an RNeasy Plus Mini kit (manufactured by QIAGEN).
  • cDNA was synthesized from 100 ng of total RNA by reverse transcription, transferred from cDNA to cRNA by in vitro transcription, and labeled with biotin (using 3'IVT PLUS Reagent Kit).
  • the expression level of each gene was determined as a relative expression level with respect to the expression level of the SDHA gene. The results are shown in the table below.
  • Example 2 shows that the cell population of Example 1 has a higher relative expression level of the TGFB2 gene, the TNFAIP6 gene, the IL1A gene, the IL1B gene, and the CCL2 gene compared to the cell population of Comparative Example 1.
  • TGFB2 plays a key role in regulating the initiation and resolution of inflammatory responses as well as inducing and maintaining immune tolerance by affecting leukocyte proliferation, differentiation, activation and survival. It functions as a mediator (Kyrkchiev D, et al. World J Stem Cells. 2014; 6 (5): 552-70.).
  • TGFB2 There are various regulatory activities of TGFB2 on immune cell function, such as inhibition of proliferation and function of effector T cells, generation of regulatory T cells from naive T lymphocytes, attenuation of cytokine production and cytolytic activity by NK cells , Suppression of B cells, dendritic cells and macrophages (Kyrkchiev D, et al. World J Stem Cells. 2014; 6 (5): 552-70.).
  • TNFAIP6 produced by mesenchymal stem cells is involved in a decrease in macrophage activation in an in vitro co-culture system and an alleviation of inflammation in a zymosan-induced peritonitis model (Bartosh TJ, et al.
  • mesenchymal stem cells must be stimulated with inflammatory cytokines to acquire anti-inflammatory properties, but IL1A, IL1B and CCL2 produced by mesenchymal stem cells are anti-inflammatory properties of mesenchymal stem cells themselves. Functions as a self-inflammatory cytokine to enhance (Cesarz Z, Tamama K. Stem Cells Int. 2016; 2016: 9176357.).
  • the mesenchymal stem cells of the present invention can produce TGFB2, TNFAIP6, IL1A, IL1B and CCL2 at high levels, and can exert high anti-inflammatory and / or immunosuppressive ability by these cytokines. Therefore, the mesenchymal stem cells of the present invention can improve the immune status of patients by being administered to patients with immune-related diseases (eg, graft-versus-host disease, Crohn's disease). From the above, it was revealed that the cell population of Example 1 exhibits a higher immunosuppressive effect than the cell population of Comparative Example 1.
  • immune-related diseases eg, graft-versus-host disease, Crohn's disease
  • the cell population having the following cell characteristics (a) and (b) exhibits a high immunosuppressive action.
  • the proportion of mesenchymal stem cells that are positive for CD97 is 30% or more
  • the cell population includes mesenchymal stem cells that are positive for CD358.
  • Example 2 From a pregnant woman of a waiting cesarean section who obtained informed consent (a donor different from Comparative Example 1 and Example 1), in the same manner as in Example 1, “Step 1: Collection of amniotic membrane”, “Step 2: Amniotic membrane Enzyme treatment and collection of amniotic membrane MSC ”,“ step 3: cryopreservation of amniotic membrane MSC ”and“ step 4: culture of amniotic membrane MSC ”were performed. In the culture of amniotic membrane MSC, a part of the cell population was collected at each passage, and the following conditions (a) and (b) were evaluated for each collected cell population.
  • a cell population that satisfies the conditions (a) and (b) has a TGFB2 gene, TNFAIP6 gene, IL1A gene, IL1B gene, and It was found that the relative expression level of the CCL2 gene was large.
  • the cell population that satisfies the conditions (a) and (b) exhibits a high immunosuppressive action. If the condition of b) is used as an index, a cell population exhibiting a high immunosuppressive action can be selectively obtained.
  • Example 3 Production of pharmaceutical composition> Part of the cell population obtained in Example 1 above is used for the preparation of a pharmaceutical composition.
  • a pharmaceutical composition (cell preparation) comprising 20 mL of RPMI1640 medium containing 8 amniotic MSC 4.0 ⁇ 10 8 cells, HES 800 mg, DMSO 0.7 mL and human serum albumin 800 mg is prepared.
  • the pharmaceutical composition is enclosed in a freezing bag and stored in a frozen state. In use, the pharmaceutical composition can be thawed and provided to the patient.

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Abstract

L'invention a pour objet de fournir une population cellulaire contenant des cellules souches mésenchymateuses présentant une immunosuppression élevée, un procédé de fabrication de cette population cellulaire, des cellules souches mésenchymateuses présentant une immunosuppression élevée, et une composition pharmaceutique contenant ladite population cellulaire. Plus précisément, l'invention concerne un procédé de fabrication de population cellulaire contenant des cellules souches mésenchymateuses, qui inclut l'acquisition d'une population cellulaire présentant les caractéristiques cellulaires (a) et (b) suivantes. Ainsi, le procédé de l'invention est tel que (a) la proportion de cellules souches mésenchymateuses présentant une valeur CD97 positive dans ladite population cellulaire, est supérieure ou égale à 30%, et (b) ladite population cellulaire contient des cellules souches mésenchymateuses présentant une valeur CD358 positive.
PCT/JP2018/014325 2017-04-03 2018-04-03 Population cellulaire contenant des cellules souches mésenchymateuses ainsi que procédé de fabrication de celle-ci, cellules souches mésenchymateuses, et composition pharmaceutique WO2018186420A1 (fr)

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