WO2015129902A1 - Feuille de cellules souches dérivées de tissu adipeux, ayant un potentiel de différenciation osseuse et son procédé de production - Google Patents

Feuille de cellules souches dérivées de tissu adipeux, ayant un potentiel de différenciation osseuse et son procédé de production Download PDF

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WO2015129902A1
WO2015129902A1 PCT/JP2015/056005 JP2015056005W WO2015129902A1 WO 2015129902 A1 WO2015129902 A1 WO 2015129902A1 JP 2015056005 W JP2015056005 W JP 2015056005W WO 2015129902 A1 WO2015129902 A1 WO 2015129902A1
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bone
adscs
sheet
adipose
cell
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向 方
英樹 村上
弘行 土屋
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国立大学法人金沢大学
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • 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
    • 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
    • A61L27/3821—Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • 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
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins

Definitions

  • the present invention relates to the technical field of stem cell differentiation induction. Specifically, the present invention relates to a method for producing an adipose-derived stem cell sheet having bone differentiation ability from an adipose-derived stem cell, and an adipose-derived stem cell sheet obtained by the method.
  • This application claims priority from Japanese Patent Application No. 2014-038913, which is incorporated herein by reference.
  • Non-Patent Document 1 Bone marrow stromal cells (BMSCs) are attracting attention regarding bone regeneration (Non-patent Document 2), and embryonic stem cells (ESCs) are also materials for regenerative stem cells, and are attracting attention.
  • Non-patent Document 3 Artificial pluripotent stem cells (iPSCs) are also attracting attention in the field of regenerative medicine, and are considered as one of promising materials for regenerative stem cells (Non-patent Document 4).
  • Osteoblasts play an important role in bone formation. Osteoblasts are cells that form bone in bone tissue and produce and secrete proteins and the like to create a bone matrix. Specifically, osteoblasts secrete matrix proteins such as collagen in the living body and form matrix vesicles there. Calcium phosphate is deposited around the matrix vesicles to complete the bone matrix, and the osteoblasts eventually become bone cells in the matrix.
  • osteoblasts are differentiated from mesenchymal stem cells and can be induced to differentiate by allowing dexamethasone, ⁇ -glycerophosphate, and ascorbic acid to act on mesenchymal stem cells (Non-patent Document 5).
  • a mesenchymal stem cell differentiated from pluripotent stem cells in vitro is cultured containing bone morphogenetic protein (BoneBMorphogenetic Protein; BMP) -4, ascorbic acid-2-phosphate, dexamethasone and ⁇ -glycerophosphate. It has been disclosed that differentiation can be induced in osteoblasts by culturing in a culture plate coated with gelatin using a medium (Patent Document 1).
  • BMP bone morphogenetic Protein
  • Adipose-derived stem cells Adipose-derived stem cells (hereinafter abbreviated as ADSCs), which are recognized as one of mesenchymal stem cells, are also being investigated for use in bone regenerative medicine in addition to BMSCs, ESCs, and iPSCs. (Non-Patent Document 6). There are several options for using ADSCs to repair and reconstruct damaged tissues and organs.
  • Non-Patent Documents 7-10 A method of directly injecting ADSCs into the repair site (Non-Patent Documents 7-10).
  • a method is known in which ADSCs are mixed with a biological adhesive such as fibrin glue and injected into a repair site.
  • a biological adhesive such as fibrin glue
  • Non-Patent Document 11 a method for transplanting ADSCs together with various carriers.
  • ADSCs sheets in which ADSCs in individual dispersed states have achieved certain results in tissue regeneration in the cardiovascular field and plastic surgery field. It has been reported (Non-Patent Document 12-21).
  • Currently available ADSCs sheets can be roughly divided into two types, one of which is an ADSCs sheet (Non-patent Documents 19-23) that supports a special carrier such as a CellSeed product.
  • One is an ADSCs sheet transplanted to a transplant site together with a collagen protein carrier (Non-patent Documents 11 and 24).
  • Cell sheet There are various reports on methods for producing cell sheets. For example, a method for producing chondrocytes, cartilage precursor cells, synovial cells, synovial stem cells, osteoblasts, mesenchymal stem cells, fat-derived cells, or fat-derived stem cells by culturing them on a support or carrier. Have been reported (Patent Documents 2 and 3). Also reported is a method of culturing a cell selected from mesenchymal stem cells, synovial cells, and embryonic stem cells on a cell culture support and producing the cultured cells as a sheet-like three-dimensional structure. (Patent Document 4).
  • a method for producing a transplant material for treating heart disease comprising a cell sheet containing a fat cell, wherein a) a group of cells containing a fat cell on a cell culture support coated with a temperature-responsive polymer B) a step of bringing the temperature of the culture solution above the upper critical solution temperature or lower than the lower critical solution temperature, c) a step of peeling the cell group from the cell culture support as a cell sheet, Furthermore, a method including a step of adding ascorbic acid or a derivative thereof to the culture broth before step c) is disclosed (Patent Document 5).
  • Non-patent Documents 25-27 The application of ADSCs in the orthopedic field is based on a method in which a mixture of dispersed ADSCs and other substances such as standard saline, fibrin gel, and collagen gel is injected into cancellous bone (Non-patent Documents 8 and 28). It is only done.
  • Non-patent Documents 29 and 30 it has been reported that the combined use of ADSCs and ⁇ -tricalcium phosphate ( ⁇ -TCP) -based bone cement scaffolds is promising for bone defects.
  • ADSCs are used as a mixture containing a fibrin glue-like substance as a component in the orthopedic field in order to fix at an implantation site, the concentration of ADSCs contained in such a mixture is limited. .
  • ADSCs adipose-derived stem cells
  • An object of the present invention is to provide a simple and inexpensive method for producing a cell construct that can be a bone tissue regeneration material using ADSCs, and a cell construct having bone forming ability obtained by the method. is there.
  • ADSCs sheets are produced by culturing ADSCs in a production medium prepared by adding only ascorbic acid-2-phosphate to the cell culture medium. It was found that the produced ADSCs sheet was effectively induced in a short period of time when bone differentiation was induced, compared to ADSCs cultured in a conventional cell culture medium.
  • the ADSCs sheet prepared by such a method has high mechanical strength, and when transplanted to a bone damage site, the ADSCs sheet has good engraftment on the bone surface and is considered to induce bone differentiation effectively even in vivo. be able to.
  • the present invention has been achieved by these findings.
  • the present invention relates to the following.
  • a solution prepared by adding ascorbic acid or a salt thereof to a cell culture medium not containing a cell differentiation inducer is added to a culture vessel to which no scaffold is added, and adipose-derived stem cells are cultured in the solution.
  • a method for producing an adipose-derived stem cell sheet having bone differentiation ability. 2.
  • the production method according to item 1, wherein the ascorbic acid or a salt thereof is added to the cell culture medium so as to have a concentration of 50 ⁇ M to 500 ⁇ M.
  • 3. 3 The production method according to item 1 or 2, wherein culturing the adipose-derived stem cells is culturing the adipose-derived stem cells for 3 to 15 days.
  • the present invention has bone differentiation ability including culturing ADSCs in a solution prepared by adding only ascorbic acid or a salt thereof to a normal cell culture medium without using a cell culture support.
  • An ADSCs sheet production method and an ADSCs sheet obtained by the production method and having bone differentiation ability can be provided.
  • seat which can be used as a bone tissue reproduction
  • the ADSCs sheet of the present invention has higher bone forming ability and can rapidly form bone as compared with dispersed ADSCs.
  • the dispersed ADSCs need to be used together with a fixing material such as fibrin glue or a scaffold for fixation and proliferation at the bone tissue regeneration site, but the ADSCs sheet of the present invention is used together with the scaffold.
  • the ADSCs sheet contains cells having the ability to differentiate into bone, and can be fixed to a repair site by itself. Therefore, the ADSCs sheet is more useful as a bone tissue regeneration material compared to dispersed ADSCs.
  • the method according to the present invention can be carried out simply and inexpensively because an ADSCs sheet as a bone tissue regeneration material can be produced without using other substances in addition to ascorbic acid or a salt thereof.
  • Osteoblasts were detected by ALP staining (Alkaline Phosphatase Staining; ALP staining) and alizarin red staining (Alizarin red staining).
  • ADSCs showed positive ALP staining from 2 weeks after bone induction and positive alizarin red staining from 3 weeks.
  • ic indicates the ADSCs group in the osteoinduction medium
  • nc indicates the ADSCs group in the normal medium. It is a figure explaining that the ADSCs sheet
  • the ADSCs sheet was positive for ALP staining from day 5 after bone induction and positive for alizarin red staining from day 7.
  • “is” indicates the ADSCs sheet group in the osteoinduction medium
  • “ns” indicates the ADSCs sheet group in the normal medium.
  • ALP activity was expressed as activity per 1 ⁇ 10 5 cells.
  • Panel A shows a daily ALP activity curve.
  • Panel B shows the results of comparing the ALP activity on day 3, day 5, day 7 and day 10 of the culture with a control group cultured in a normal medium.
  • ⁇ , ⁇ , ⁇ , and x indicate is, ns, ic, and nc, respectively.
  • the data are is, ns, ic and nc in order from the left.
  • the ALP activity of the ADSCs sheet was measured by absorbance at a wavelength of 405 nm.
  • the graph in the figure compares the absorbance of ADSCs sheets prepared in media with different ascorbic acid concentrations with those prepared at an ascorbic acid concentration of 50 ⁇ M.
  • the absorbance of the ADSCs sheet prepared at an ascorbic acid concentration of 50 ⁇ M on each day was 1. 0 day means that the ADSCs reached overconfluence, and the ADSCs sheet medium was used for the measurement from that point.
  • the left of the graph of each day shows an ascorbic acid concentration of 50 ⁇ M
  • the middle shows an ascorbic acid concentration of 150 ⁇ M
  • the right shows an ascorbic acid concentration of 450 ⁇ M.
  • the present invention relates to a method for producing an ADSCs sheet having bone differentiation ability (more specifically, a single-layer ADSCs sheet), and an ADSCs sheet produced by the production method.
  • a solution prepared by adding ascorbic acid or a salt thereof to a cell culture medium not containing a cell differentiation inducer is added to a culture vessel to which a cell culture support is not added, Culturing the derived stem cells.
  • the ADSCs sheet is preferably used for bone formation.
  • the ADSCs sheet production method according to the present invention has an advantage that an ADSCs sheet that can be used as a bone tissue regeneration material for bone regeneration and bone reconstruction can be produced simply, rapidly, and inexpensively.
  • the ADSCs sheet prepared by the preparation method according to the present invention includes cells having bone differentiation ability, and since the mechanical strength thereof is high, the site when the cell itself is transplanted to a repair site alone. It has the advantage that it can be fixed to.
  • the present invention further provides a method for producing a cell sheet containing osteoblasts, comprising culturing an ADSCs sheet prepared by the production method of the present invention in a cell culture medium containing an osteoinduction agent.
  • stem cell refers to a cell that has both the ability to differentiate into cells of multiple lineages (multipotency) and the ability to maintain pluripotency even after cell division (self-renewal ability).
  • Stem cells play a role in supplying cells in the process of development and maintenance of tissues and organs.
  • Stem cells can be differentiated into embryonic stem cells that can differentiate into cells of all lineages, usually adult stem cells with limited differentiation lineages (tissue stem cells, somatic stem cells), and all lineages except extraembryonic tissues Artificially induced pluripotent stem cells having pluripotency and the like are known.
  • Adipose-derived stem cells are stem cells present in adipose tissue. It is mainly composed of mesenchymal stem cells and differentiates into many types of cells such as osteoblasts, chondrocytes, cardiomyocytes, adipocytes, hepatocytes, vascular endothelial cells, and insulin-secreting cells, similar to bone marrow-derived stem cells. Have pluripotency. Since adipose tissue is easy to collect and contains a large amount of stem cells, ADSCs can be prepared easily and in large quantities.
  • Adipose-derived stem cell sheet “Adipose-derived stem cell sheet (ADSCs sheet)” refers to a cell construct in which ADSCs in separate dispersion form a sheet. “Single-layered adipose-derived stem cell sheet” refers to a cell construct in which ADSCs are formed in a single sheet state and do not form a multi-layer structure or a three-dimensional structure.
  • ADSCs that are in individual dispersed states are simply referred to as ADSCs or distributed ADSCs, and ADSCs that form a sheet are referred to as ADSCs sheets.
  • the ADSCs and ADSCs sheets may be any one derived from mammalian fat, and examples of mammals include humans, monkeys, pigs, pigs, horses, cows, rabbits, sheep, goats, cats, dogs, guinea pigs, etc. it can. Preferred are human fat-derived ADSCs and ADSCs sheets. Further, when the ADSCs and ADSCs sheets are used as a bone tissue regeneration material, the same kind of ADSCs and the same kind of ADSCs sheet are preferable, and it is more preferable that they are self ADSCs and self ADSCs sheets.
  • Homogeneous ADSCs and homologous ADSCs sheets mean ADSCs and ADSCs sheets derived from the same animal species.
  • In-house ADSCs and in-house ADSCs sheets mean ADSCs and ADSCs sheets derived from the subject who is subjected to bone tissue regeneration.
  • Bone differentiation ability refers to the ability to differentiate into osteoblasts, bone cells or their precursor cells. That is, it refers to the ability to differentiate into osteoblasts, bone cells or their progenitor cells under conditions that induce bone differentiation.
  • the condition for inducing bone differentiation may be any of in vivo and in vitro conditions.
  • culture in a medium containing dexamethasone, ⁇ -glycerophosphate, and ascorbic acid (Non-patent Document 5), BMP-4, ascorbate-2-phosphate, dexamethasone and ⁇ -glycerophosphate Examples thereof include culture in a culture medium containing a salt.
  • fat-derived stem cells are cultured in the solution.
  • the container used for cell culture is not particularly limited as long as it is generally used for cell culture, and examples include cell culture containers such as petri dishes and flasks. If it is an adhesive cell, it grows by adhering to the wall of such a cell culture container, and if it is a floating cell, it grows in a free state in the culture solution.
  • a cell culture container may be referred to as a substrate.
  • Cell culture support refers to a structure formed of a coating material that is coated or laminated on a substrate for the purpose of cell attachment and proliferation, or construction of a three-dimensional structure of cells.
  • Preferred examples of the cell culture support include hydrogel and collagen matrix (Non-patent Document 31), which are three-dimensional network structures in which polymers are crosslinked.
  • the term “cell culture support” does not include a substrate.
  • Examples of the culture container to which the cell culture support is not added include, for example, commercially available petri dishes and cell culture flasks.
  • the ascorbic acid used in the method according to the present invention is L-ascorbic acid or a derivative thereof such as L-ascorbic acid-2-glucoside and L-ascorbic acid-2-phosphate, preferably L-ascorbic acid- 2-phosphate.
  • the salt of ascorbic acid may be any salt as long as it is a pharmacologically acceptable salt such as sodium salt or calcium salt.
  • Ascorbic acid-2-phosphate has been reported to increase collagen protein secretion in mesenchymal cells, but has no other effect on the cells (Non-patent Document 32).
  • Ascorbic acid or a salt thereof added to the cell culture medium is added to the cell culture medium so that the final concentration is 10 ⁇ M to 800 ⁇ M, preferably 50 ⁇ M to 500 ⁇ M, more preferably 100 ⁇ M to 200 ⁇ M, and most preferably about 150 ⁇ M.
  • Cell culture medium a culture medium generally used for culturing ADSCs can be used.
  • DMEM Dulbecco's modified Eagle's medium
  • DMEM Dulbecco's modified Eagle's medium
  • culture conditions for ADSCs generally known culture conditions can be employed.
  • the number of days of ADSCs culture for preparing the ADSCs sheet is 3 to 15 days, preferably 4 to 13 days, more preferably 5 to 10 days, still more preferably 7 to 10 days, and most preferably 7 days. is there.
  • a cell sheet containing osteoblasts can be produced and provided by culturing the adipose stem cell sheet produced by the production method according to the present invention in a cell culture medium containing an osteodifferentiation inducer.
  • the bone cell differentiation-inducing agent refers to a drug that can differentiate stem cells and bone lineage progenitor cells into osteoblasts or bone cells. Any known osteoclast differentiation inducer can be used as the osteoclast differentiation inducer.
  • a mixture containing dexamethasone, ⁇ -glycerophosphate, and ascorbic acid, and BMP-4, ascorbyl-2-phosphate Preferred examples include a mixture containing a salt, dexamethasone and ⁇ -glycerophosphate.
  • Bone cell differentiation induction in vitro can be performed by culturing stem cells and bone lineage progenitor cells in a culture medium to which an osteoblast differentiation inducer is added.
  • Any culture medium to which a bone cell differentiation inducer is added can be used as long as it is a commonly used medium, and an ⁇ -Minimum Essential Media ( ⁇ -MEM) can be preferably exemplified.
  • the culture conditions are appropriately determined by culturing the adipose stem cell sheet produced by the production method according to the present invention in a cell culture medium containing an osteoinduction agent and detecting differentiation induction of osteoblasts in the sheet. Can be changed and set. Detection of osteoblast differentiation induction can be performed by a known method such as ALP staining (ALP staining), alizarin red staining, or measurement of ALP activity.
  • the bone tissue regeneration material refers to a material used for bone regeneration and bone reconstruction.
  • the bone tissue regeneration material of the present invention is an adipose stem cell sheet having the ability to differentiate into bone of the present invention, or a cell sheet containing the osteoblast of the present invention (or osteoblasts produced by the production method according to the present invention). Cell sheet).
  • the bone tissue regeneration material of the present invention is characterized in that a cell sheet containing osteoblasts has mechanical strength and is fixed to a bone repair site by itself, so that no scaffold is required. .
  • hADSCs human adipose-derived stem cells
  • DMEM Wired Eagle's medium
  • FBS fetal bovine serum
  • P / S penicillin-streptomycin solution
  • hADSCs were cultured in DMEM supplemented with 10% FBS and 1% P / S (Non-patent Document 33) and subcultured until they grew to cover 90% of the petri dish area. The third passage of hADSCs was used in subsequent experiments.
  • hADSCs were cultured in a known osteoinduction medium for differentiation induction.
  • the osteoinduction medium consists of ⁇ -MEM (Wako Pure Chemical Industries) containing 10% FBS, 0.1 ⁇ M dexamethasone, 50 ⁇ M ascorbic acid-2-phosphate, 10 mM ⁇ -glycerophosphate, 1% P / S (non-Wako Pure Chemical Industries).
  • Patent Document 6, 34-37 An ⁇ -MEM solution containing only 10% FBS and 1% P / S was used as a control medium for each condition.
  • a group of cells in which differentiation of hADSCs is induced in an osteoinduction medium may be referred to as a hADSCs induction group
  • a group of cells cultured in a hADSCs control medium may be referred to as a hADSCs control group.
  • Non-Patent Documents 38-40 were performed on the 1st day, 3rd day, 5th day, 7th day, 10th day, 2nd week and 3rd week during the differentiation induction culture period. .
  • the medium was removed, the cell layer was rinsed 3 times with PBS, and fixed with 4% paraformaldehyde-phosphate buffer (Wako Pure Chemical Industries, Ltd) for 5 minutes at room temperature. Thereafter, the cell layer was washed with deionized water. The fixed cells were then incubated with 1-Step NBT / BCIP plus Suppressor Solution (Thermo Fisher Scientific). After incubation at 37 ° C. for 30 minutes, the cell layer was washed with deionized water and observed with both the naked eye and light microscope.
  • 1-Step NBT / BCIP plus Suppressor Solution Thermo Fisher Scientific
  • hADSCs sheet of the present invention The 3rd generation hADSCs were seeded in a 10 cm dish at 1 ⁇ 10 6 cells / dishlet and cultured in DMEM supplemented with 10% FBS and 1% P / S to prepare an hADSCs sheet. .
  • the production medium contains 10% FBS, 50 ⁇ M ascorbic acid-2-phosphate, 1% P / S. The medium was changed every 3 days for 1 week. This is an important process developed to produce ADSCs sheets in the present application. Samples were randomly taken and examined to see if hADSCs sheets were created.
  • the osteoinduction medium consists of ⁇ -MEM containing 10%, 0.1 ⁇ M dexamethasone, 50 ⁇ M ascorbic acid-2-phosphate, 10 mM ⁇ -glycerophosphate, 1% P / S.
  • An ⁇ -MEM solution containing only 10% FBS and 1% P / S was used as a control medium for each condition.
  • a cell group in which the hADSCs sheet is differentiated with the osteoinduction medium may be referred to as a hADSCs sheet induction group
  • a cell group in which the hADSCs sheet is cultured in a control medium may be referred to as a hADSCs sheet control group.
  • ALP activity was measured for each of the hADSCs control group, the hADSCs induction group, the hADSCs sheet control group, and the hADSCs sheet induction group.
  • the ALP activity was measured using a TRACP & ALP assay kit (MK301, TaKaRa Bio) according to the manufacturer's instructions. Checkpoints were set on the 1st, 3rd, 5th, 7th, 10th, 2nd and 3rd weeks of the culture period. The significance of the difference in ALP activity between the four groups was evaluated by statistical analysis using the Student t-test. The p value was determined to be significant when it was 0.05 or less.
  • the average value of ALP activity in the hADSCs induction group indicates that the ALP activity of hADSCs increased rapidly from 5 days after bone induction (FIG. 4). Consistent with Life Technologies' user instructions, this increase reached a maximum on day 14 and is consistent with reports from other studies (42). The ALP activity in the hADSCs control group remained in the low range throughout the experimental period.
  • Both the hADSCs sheet induction group and the hADSCs sheet control group showed higher ALP activity on the first day than the hADSCs induction group and the hADSCs control group, respectively (P ⁇ 0.05).
  • the hADSCs sheet induction group showed a significantly higher value than the hADSCs induction group (P ⁇ 0.05).
  • the linear increase in ALP activity in the hADSCs sheet-induced group began on day 1 and reached a maximum on day 10. However, the ALP activity in the hADSCs sheet-induced group was still increased on the 14th and 21st days.
  • the ALP activity of the hADSCs sheet control group remained in the low range over the entire experimental period compared to the hADSCs sheet induction group, but the value was much greater than that of the hADSCs control group (P ⁇ 0.05). (FIG. 4).
  • the ADSCs sheet induction group since the ALP activity of the ADSCs sheet induction group was higher than that of the dispersed ADSCs group at each time point, it was confirmed that the ADSCs sheet acts more effectively and rapidly on bone formation than the dispersed ADSCs. . That is, the ADSCs sheet graft of the present invention is useful for clinical treatment because it has the advantage that it can play its role in bone formation sooner after transplantation than individual discrete dispersed cells.
  • Example 1 (Optimization of added ascorbic acid concentration) In Example 1 above, it was confirmed that the adipose stem cell sheet having the ability to differentiate into bone and the cell sheet containing osteoblasts have an excellent ability to induce bone differentiation. Further, in this example, the optimum concentration of ascorbic acid to be added was examined. Details are as follows.
  • ADSCs sheets prepared under medium conditions of 50 ⁇ M, 150 ⁇ M or 450 ⁇ M ascorbic acid concentration were measured by absorbance at 405 nm wavelength.
  • ADSCs sheets prepared with 150 ⁇ M ascorbic acid-containing medium had higher ALP activity than ADSCs sheets prepared with 50 ⁇ M and 450 ⁇ M ascorbic acid-containing media (see FIG. 5).
  • the optimum range of the ascorbic acid concentration in the ADSCs sheet medium was in the range of 50 ⁇ M to 500 ⁇ M, more preferably in the range of 100 ⁇ M to 200 ⁇ M.
  • ADSCs sheet production usually takes 7 days. However, at an ascorbic acid concentration of 150 ⁇ M, ADSCs sheets could be produced from about 5 days.
  • the bone differentiation of the ADSCs sheet of the present invention is fast because collagen secretion is high.
  • Ascorbic acid-2-phosphate has been reported to increase the secretion of collagen protein in mesenchymal cells (Non-patent Document 32), and collagen protein is a natural carrier that has a good cell sheet in the osteogenic lineage. It is considered (Non-Patent Document 31).
  • the ADSCs sheets of the present invention exhibit significant mechanical strength due to the secretion of collagen proteins, and they can be directly held by conventional laboratory tweezers and conventional surgical instruments (Non-patent Documents 43 and 44). Have Also from this point, the ADSCs sheet of the present invention has high effectiveness for bone regeneration and bone reconstruction in vitro and in vivo.
  • the present invention provides a material for bone regeneration and reconstruction and a rapid and effective production method thereof in the field of bone tissue regeneration, for example, orthopedics, and is extremely useful in such field.

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Abstract

[Problème] La présente invention concerne : un procédé aisé et économique de production d'une construction cellulaire qui peut servir de matériau de restauration de tissu osseux ; et une construction cellulaire ayant un potentiel ostéogénique obtenue par ce procédé. [Solution] Procédé de production d'une feuille de cellules souches provenant de tissu adipeux présentant un potentiel de différenciation osseuse, le procédé comprenant l'ajout d'une solution, préparée par ajout d'acide ascorbique ou un sel de celui-ci à un milieu de culture de cellules exempt d'inducteur de différenciation cellulaire, à un récipient de culture ne comportant pas d'échafaudage ajouté à celui-ci et la culture de cellules souches dérivées de tissu adipeux dans cette solution ; et une feuille de cellules souches dérivées de tissu adipeux produite par ce procédé.
PCT/JP2015/056005 2014-02-28 2015-02-28 Feuille de cellules souches dérivées de tissu adipeux, ayant un potentiel de différenciation osseuse et son procédé de production WO2015129902A1 (fr)

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Cited By (2)

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WO2019075101A1 (fr) * 2017-10-10 2019-04-18 Los Angeles Biomedical Research Institute At Harbor-Ucla Medical Tissus dérivés ex vivo comprenant des multicouches de cellules souches dérivées de tissu adipeux et utilisations de ceux-ci
JP2020162565A (ja) * 2019-03-29 2020-10-08 国立大学法人金沢大学 脂肪由来幹細胞シート由来の骨三次元構造体及び該構造体の作製方法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019075101A1 (fr) * 2017-10-10 2019-04-18 Los Angeles Biomedical Research Institute At Harbor-Ucla Medical Tissus dérivés ex vivo comprenant des multicouches de cellules souches dérivées de tissu adipeux et utilisations de ceux-ci
JP2020162565A (ja) * 2019-03-29 2020-10-08 国立大学法人金沢大学 脂肪由来幹細胞シート由来の骨三次元構造体及び該構造体の作製方法

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