WO2003087350A1 - Regulation de la differentiation cellulaire par formation d'agregat tridimensionnel - Google Patents

Regulation de la differentiation cellulaire par formation d'agregat tridimensionnel Download PDF

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Publication number
WO2003087350A1
WO2003087350A1 PCT/JP2003/004900 JP0304900W WO03087350A1 WO 2003087350 A1 WO2003087350 A1 WO 2003087350A1 JP 0304900 W JP0304900 W JP 0304900W WO 03087350 A1 WO03087350 A1 WO 03087350A1
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Prior art keywords
cells
culture
chondrocytes
dimensional
present
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PCT/JP2003/004900
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English (en)
Japanese (ja)
Inventor
Katsuko Sakai
Takashi Ushida
Yasuyuki Sakai
Tetsuya Tateishi
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National Institute Of Advanced Industrial Science And Technology
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Priority to AU2003235200A priority Critical patent/AU2003235200A1/en
Publication of WO2003087350A1 publication Critical patent/WO2003087350A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—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
    • 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
    • C12N5/0655—Chondrocytes; Cartilage

Definitions

  • the present invention forms a three-dimensional aggregate of cells maintaining a normal phenotype in a short time in a large amount by floating culture of adherent animal cells such as bone and chondrocytes in a non-adherent container. About the method. Furthermore, the present invention relates to a method for producing a tissue reconstruction type implant using the three-dimensional aggregate formed by the method. Background technology
  • Damage to bone and cartilage tissue due to accident, aging, or disease can significantly impair human motor function.
  • joint disorders occur in many elderly people and have a significant impact on their lives, so today's aging society is a major social issue today.
  • adhesive cells are generally cultured with an adhesive carrier or adhered and cultured in a container having an improved surface adhesiveness.
  • a polystyrene container whose surface has been hydrophilized
  • an adhesive container whose surface is coated with a basic polymer such as polyethyleneimine / polylysine, or a cell adhesive protein such as fibronectin, laminin, various collagens, and proteoglycan.
  • Culture in BHK Some intact cultured cell lines, such as CHO, CHL, etc., which are easy to culture in suspension, are also known to be cultured in suspension by physical agitation. Suspension culture of sexual animal cells is generally said to be difficult and has not been performed. Disclosure of the invention
  • the present invention promotes the differentiation of isolated bone / chondrocytes, and SUMMARY OF THE INVENTION
  • the present inventors have solved the above-mentioned problems by constructing a three-dimensional aggregate in a large amount and in a short time while maintaining a normal type, and providing a tissue reconstruction type implant using this.
  • suspension culture of normal bone, chondrocytes, and undifferentiated stem cells in an environment that enhances the cohesive strength of cells can form large amounts of three-dimensional aggregates while maintaining the normal phenotype of the cells That is, the present invention has been completed.
  • the present invention provides the following (1) to (7).
  • a method of producing a three-dimensional aggregate of cells by suspending and culturing primary cells of normal bone and chondrocytes or undifferentiated stem cells in a non-adherent container.
  • suspension culture is a suspension culture in a system in which there is a flow that enhances cell aggregation.
  • the present invention provides a method for tertiary culturing of cells in which phenoquine cells are normally maintained in a short time and in large amounts by suspending and culturing primary cells of normal bone / chondrocytes or undifferentiated stem cells in a non-adhesive container.
  • the present invention relates to a method for producing a primary aggregate.
  • the cells used in the present invention are normal bone / chondrocytes or primary cultured cells of undifferentiated stem cells.
  • the origin of the cells is not particularly limited as long as they are animals, but mammals are preferred, and primates are most preferred.
  • normal bone / chondrocytes refers to histologically and genetically normal bone cells and chondrocytes, and excludes osteocytes / chondrocytes that have been affected by disease or the like.
  • the bone and chondrocytes also include osteoblasts, periosteal cells, osteoclasts, and chondroblasts, as well as osteocytes and chondrocytes.
  • undifferentiated stem cells are undifferentiated cells having differentiation and proliferation ability, and include, for example, embryonic stem cells (ES cells), mesenchymal stem cells, hematopoietic stem cells, skeletal muscle stem cells, neural stem cells and Although liver stem cells and the like can be mentioned, embryonic stem cells and mesenchymal stem cells are particularly preferred. These undigested stem cells need to be primary cultured cells.
  • ES cells embryonic stem cells
  • mesenchymal stem cells hematopoietic stem cells
  • skeletal muscle stem cells skeletal muscle stem cells
  • neural stem cells and Although liver stem cells and the like can be mentioned, embryonic stem cells and mesenchymal stem cells are particularly preferred. These undigested stem cells need to be primary cultured cells.
  • Non-adhesive container The culture vessel used in the present invention is a non-adhesive vessel.
  • the “non-adhesive container” may be a container to which cells are unlikely to adhere, for example, a plastic container having low adhesion such as polyethylene terephthalate, polypropylene, polyethylene, polycarbonate, and polystyrene.
  • a plastic or glass container having a hydrophobic surface treatment such as a fluorine treatment or a silicon treatment may be used.
  • the size and shape of the container are not particularly limited, and an arbitrary shape and size such as a dish shape, a flask shape, a jewel shape and the like can be selected according to cells to be cultured.
  • cells are suspended and cultured in the non-adhesive container.
  • the suspension culture may be static culture or not static culture, but is preferably culture under conditions that increase the cohesive strength of cells.
  • the culture under conditions that enhance the cohesion of cells refers to, for example, culture under flow conditions that enhance cell aggregation.
  • condition under flow that enhances cell aggregation examples include a method of culturing cells at a single point by stress (centrifugal force, centripetal force) due to a flow such as a swirling flow. Can be.
  • spiral culture is mentioned as a preferred example.
  • the swirling speed when performing the swirling culture is not particularly limited, and is appropriately set depending on the size of the container and the like. For example, if the diameter is about 35 mm, a sufficient three-dimensional aggregate can be formed at about 100 to 100 Ori.
  • the medium used in the suspension culture is not particularly limited, and can be appropriately selected according to the cells to be cultured.
  • MEM, a MEM, DME M, RPM1, c RDF, ERDF, F12, MCDB131, F12 / Known media such as DMEM and WE can be mentioned.
  • the medium includes antibiotics such as FBS (manufactured by Sigma) and Antibiotic-Antimycotic (manufactured by GIBCO BRL), growth factors such as TGFj3, aFGF, bFGF, BMP, insulin, dexamethasone, proline, Sodium pyruvate, dalcose, transferrin, serine, retinoic acid, or the like may be appropriately added.
  • antibiotics such as FBS (manufactured by Sigma) and Antibiotic-Antimycotic (manufactured by GIBCO BRL)
  • growth factors such as TGFj3, aFGF, bFGF, BMP, insulin, dexamethasone, proline, Sodium pyruvate, dalcose, transferrin, serine, retinoic acid, or the like may be appropriately added.
  • the cells are seeded and cultured in the medium at an appropriate seeding density.
  • the number of cells to be seeded is desirably adjusted appropriately according to the cells and the container to be used in order to maintain the cell morphology and perform the culture efficiently.
  • the seeding density is preferably about 10 2 to 10 6 cells / ml.
  • Culture conditions may suitably set in accordance with the cells used, typically. 3 to 10% C0 2 under, 30 to 40 ° C, especially 4% to 6% C_ ⁇ 2, intends row under the conditions of. 34 to 38 ° C It is desirable.
  • the culture period is not particularly limited as long as the target three-dimensional aggregate is formed, but is preferably at least 0.5 hour or more.
  • a three-dimensional aggregate of cells (aggregates, spheroids) starts to be formed in a few hours, and a large three-dimensional aggregate of several hundreds / is formed in about 24 to 36 hours.
  • the term “three-dimensional aggregate” means a cluster formed by three-dimensionally gathering cells.
  • the cells forming the three-dimensional aggregate formed in this way are more differentiated than cells attached and cultured, and maintain a normal phenotype. That is, according to the method of the present invention, cells having a normal phenotype are promoted by promoting differentiation. 3D aggregates can be produced in large quantities in a short time.
  • the present invention also provides a method for promoting cell differentiation by suspending and culturing primary cells of normal bone / chondrocytes or undifferentiated stem cells in a non-adhesive container.
  • the three-dimensional aggregate formed by the method of the present invention is suitable for producing a tissue reconstruction type implant.
  • the present invention also provides a method for producing an implant using the three-dimensional aggregate formed by the culture method of the present invention.
  • the two-dimensional aggregate formed by the method of the present invention cell differentiation is promoted, and a normal phenotype is maintained.
  • a large amount of three-dimensional aggregates can be obtained in a short period of time, unlike in the past.
  • the implant may be composed only of the three-dimensional aggregate formed by the method of the present invention, or may be composed in combination with a suitable biodegradable material.
  • the biodegradable material used is, for example, hydroxy.
  • examples include porous ceramics such as apatite, 6-TCP (tricalcium phosphate), ⁇ -TCP, collagen, polylactic acid, and polyglycolic acid, and complexes thereof.
  • the biodegradable material may be appropriately selected depending on the purpose and application site of the implant. For example, for a transplant site where strength is required, a hydroxyapatite is preferable, and For a transplant site that is not required, bioabsorbable; 6-TCP or the like is preferable.
  • implants of these implants include, for example, use in orthopedic fields such as artificial cartilage, artificial joints, bone and cartilage replacement implants such as human bones, and dental fields such as dental implants such as artificial roots. It can be used in orthopedic fields such as artificial cartilage, artificial joints, bone and cartilage replacement implants such as human bones, and dental fields such as dental implants such as artificial roots. It can be used in orthopedic fields such as artificial cartilage, artificial joints, bone and cartilage replacement implants such as human bones, and dental fields such as dental implants such as artificial roots. It can be used in orthopedic fields such as artificial cartilage, artificial joints, bone and cartilage replacement implants such as human bones, and dental fields such as dental implants such as artificial roots. It can be used in dental fields such as artificial cartilage, artificial joints, bone and cartilage replacement implants such as human bones, and dental fields such as dental implants such as artificial roots. It can be used in dental fields such as artificial cartilage, artificial joints, bone and cartilage replacement implants such as human bones, and dental fields such as dental implants such as artificial roots. It can be used in dental fields such as artificial cartilage, artificial
  • the shape and shape of the implant are not particularly limited, and any shape and shape such as a sponge, a mesh, a nonwoven fabric, a disk, a film, a rod, a particle, and a paste can be used. it can. Such a form and shape are appropriately selected according to the purpose of the implant. For example, an implant in which a three-dimensional aggregate of chondrocytes is embedded in a gel base material such as collagen gel and applied to an affected area is also used. It is possible.
  • an autologous cell taken out of a living body is cultured in vitro to produce an implant, it can be a useful material for regenerative medicine as an implant as close as possible to the living body.
  • FIG. 1 is a photograph showing a three-dimensional aggregate formed from chondrocytes by swirling culture in a non-adhesive container.
  • Fig. 2 shows chondrocytes adherently cultured (Monolayer) and chondrocytes cultured in rotation.
  • 1 is a photograph showing the expression of a type I collagen gene in (Aggregates).
  • FIG. 3 is a photograph showing a three-dimensional aggregate formed from mesenchymal stem cells by swirling culture in a non-adherent container. This description includes part or all of the contents as disclosed in the description of Japanese Patent Application No. 2002-115528, which is a priority document of the present application. BEST MODE FOR CARRYING OUT THE INVENTION
  • chondrocytes were extracted overnight with 0.2% collagenase / F12 medium (containing 10% fetal serum), they were seeded on a tissue culture dish (Falcon, 625 cm 2 — square dish). at 5% C0 2, 37 ° C and adherent culture. After the chondrocytes adhered and spread on the tissue culture dish, further passage was performed to increase the number of cells. After growing the cells to a sufficient number, the cells were detached and recovered using trypsin.
  • non-adhesive 6 The collected chondrocytes at a concentration of 1.5xl0 7 cells / dish - wells- plates (Costar Corporation, Ultra Low Attachment Polystylene: diameter 35 ⁇ , made of polystyrene) were seeded in. Then, the plate is cultured in E. coli. Use municipal force (TAITEC Co., NR- 2) placed on top of, under conditions of 5% C 0 2, 37 ° C, it was gyratory at a speed of 80 rpm.
  • TITEC Co., NR- 2 Use municipal force
  • Three-dimensional aggregates of chondrocytes were observed in the plate 12 hours after the start of the spin culture (Fig. 1), and a large number of three-dimensional aggregates with a diameter of several hundred m were formed 24 to 36 hours later. .
  • RNA from chondrocytes adhered (monolayer) cultured in a tissue culture dish and chondrocytes that formed three-dimensional aggregates by swirling culture were extracted using Isogen (Nippon Gene), and RT-PCR was performed.
  • Isogen Nippon Gene
  • RT-PCR was performed.
  • type I collagen gene which is known to be secreted from dedifferentiated chondrocytes, was observed.
  • GP3DH housekeeping gene
  • chondrocytes that formed three-dimensional aggregates by swirling culture produced significantly less type I collagen genes than chondrocytes (Monolayer) adherently cultured in a tissue culture dish. ( Figure 2 ). From this, it was confirmed that the cells in the three-dimensional aggregates subjected to the swirling culture were promoted to differentiate more than the cells subjected to the adherent culture.
  • Mesenchymal stem cells were prepared from the bone marrow of the limb of an adult cow according to a conventional method. Then, the mesenchymal cells were adhered and cultured in a tissue culture dish (Falcon, 625 cm 2- square dish) at 5% CO 2 and 37, and then treated with trypsin. And peeled and collected. The recovered cells, non-adhesive 6- Wells- plates at a concentration of 1.5xl0 7 cells / dish: seeded into (Costar Corp., Ultra Low Attachment 10 polystyrene diameter 35 ⁇ , polystyrene), 5% C0 2, 37 Rotating culture was performed at a speed of 80 rpm under the conditions of ° C.
  • three-dimensional aggregates derived from primary cultured normal bone / chondrocytes or undifferentiated stem cells, in which cell differentiation is promoted and phenotypes are normally maintained, are formed in a large amount in a short time. can do. Therefore, by using the culture method of the present invention, a tissue culture-type inplant that is a useful tool for regenerative medicine can be efficiently produced.

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Abstract

L'invention porte sur un procédé de formation d'un agrégat tridimensionnel de cellules conservant un phénotype normal, en grande quantité et sur une courte durée, par mise en culture flottante de cellules animales adhésives telles que des chondro-ostéocytes, dans un récipient sans adhérence. L'invention porte également sur un procédé de préparation d'un implant de reconstruction de tissu au moyen d'un agrégat tridimensionnel formé par le procédé précité.
PCT/JP2003/004900 2002-04-17 2003-04-17 Regulation de la differentiation cellulaire par formation d'agregat tridimensionnel WO2003087350A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003235200A AU2003235200A1 (en) 2002-04-17 2003-04-17 Control of cell differentiation through formation of three-dimensional aggregate

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002115282A JP2003304866A (ja) 2002-04-17 2002-04-17 三次元凝集塊形成による細胞の分化制御
JP2002-115282 2002-04-17

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Cited By (2)

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WO2008136733A1 (fr) * 2007-05-04 2008-11-13 Ascendia Ab Procédé et moyens pour cultiver des ostéoblastes
US8246570B2 (en) 2007-12-21 2012-08-21 Japan Medical Materials Corporation Device for cell transplantation

Families Citing this family (13)

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KR101183066B1 (ko) * 2003-07-31 2012-09-20 고이치 나카야마 인공 관절의 제작 방법
WO2006003802A1 (fr) * 2004-06-30 2006-01-12 Nihon University Plaque d’agarose pour culture de cellule es
WO2007004469A1 (fr) * 2005-07-04 2007-01-11 Osaka University Procede de culture pour la differenciation d'un preadipocyte en adipocyte mature et criblage d'une substance susceptible d'influer sur le processus de differenciation du preadipocyte en adipocyte mature
JP4044579B2 (ja) 2005-08-02 2008-02-06 株式会社Ｐｇリサーチ 人工軟骨組織
JP4739286B2 (ja) * 2007-07-17 2011-08-03 株式会社Ｐｇリサーチ 人工軟骨組織の製造方法
JP5103573B2 (ja) * 2007-09-07 2012-12-19 独立行政法人産業技術総合研究所 浮遊培養システム及び浮遊培養方法
EP3021880A1 (fr) * 2013-07-17 2016-05-25 Institut National de la Santé et de la Recherche Médicale Treillis tridimensionnel fonctionnalisé avec des micro-tissus pour la régénération tissulaire
WO2019131941A1 (fr) 2017-12-28 2019-07-04 株式会社カネカ Inhibiteur d'agrégation cellulaire
US20220267736A1 (en) 2017-12-28 2022-08-25 Kaneka Corporation Pluripotent stem cell aggregation suppressor
WO2019131942A1 (fr) 2017-12-28 2019-07-04 株式会社カネカ Agent favorisant l'agrégation de cellules
JP2019118279A (ja) * 2017-12-28 2019-07-22 株式会社カネカ 細胞凝集促進剤
JP7398114B2 (ja) * 2018-07-11 2023-12-14 学校法人松本歯科大学 親水性の違いを利用したスフェロイドの製造方法
WO2020184350A1 (fr) 2019-03-08 2020-09-17 株式会社カネカ Culture de cellules souches pluripotentes en masse

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008136733A1 (fr) * 2007-05-04 2008-11-13 Ascendia Ab Procédé et moyens pour cultiver des ostéoblastes
US10010649B2 (en) 2007-05-04 2018-07-03 Ascendia Ab Method and means for culturing osteoblastic cells
US8246570B2 (en) 2007-12-21 2012-08-21 Japan Medical Materials Corporation Device for cell transplantation

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AU2003235200A1 (en) 2003-10-27
JP2003304866A (ja) 2003-10-28

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