+

WO2013047763A1 - Procédé de culture de cellule de mammifère dans système contenant de la laminine 511 - Google Patents

Procédé de culture de cellule de mammifère dans système contenant de la laminine 511 Download PDF

Info

Publication number
WO2013047763A1
WO2013047763A1 PCT/JP2012/075111 JP2012075111W WO2013047763A1 WO 2013047763 A1 WO2013047763 A1 WO 2013047763A1 JP 2012075111 W JP2012075111 W JP 2012075111W WO 2013047763 A1 WO2013047763 A1 WO 2013047763A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
laminin
activity
protein
stem cells
Prior art date
Application number
PCT/JP2012/075111
Other languages
English (en)
Japanese (ja)
Inventor
宗弘 山田
賀也 富盛
保田　尚孝
Original Assignee
オリエンタル酵母工業株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by オリエンタル酵母工業株式会社 filed Critical オリエンタル酵母工業株式会社
Publication of WO2013047763A1 publication Critical patent/WO2013047763A1/fr

Links

Images

Classifications

    • 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
    • C12N5/0068—General culture methods using substrates
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the present invention relates to a method for culturing mammalian cells in a system containing laminin 511.
  • Laminin is an extracellular matrix protein that is mainly localized in the basement membrane of various tissues and plays an important role in maintaining tissue structure and controlling cell function (Matrix Biol., 18: 19-28, 1999; Dev. Dyn., 218: 213-234, 2000).
  • laminin is a heterotrimeric molecule in which ⁇ chain, ⁇ chain, and ⁇ chain are linked by disulfide bonds, respectively, and has a characteristic cross structure. Each chain consists of multiple domains, and domains I and II form a triple helix.
  • laminin molecules Prior to this application, at least 19 laminin molecules were identified by different combinations of 5 types of ⁇ chains ( ⁇ 1 to ⁇ 5), 3 types of ⁇ chains ( ⁇ 1 to ⁇ 3), and 3 types of ⁇ chains ( ⁇ 1 to ⁇ 3). It has been suggested that there are actually several times as many types.
  • the nomenclature of Laminin was corrected in 1994 and 2005, and is different between before and after (Matrix Biol., 24: 326-332, 2005; Cell Tissue Res. 339 (1) 259-268, 2010).
  • Laminin ⁇ chain, ⁇ chain, and ⁇ chain are encoded by different genes, and each laminin isoform has a unique site and function, mainly through cell membrane receptor integrins. It regulates differentiation and the like (Dev. Dyn. 218, 213-234, 2000; Physiol. Rev. 85, 979-1000, 2005). If the ⁇ chain, ⁇ chain, and ⁇ chain constituting each laminin isoform are different, the functions and activities are completely different.
  • Laminin molecules associate with each other at the triple-stranded amino (N) terminal portion (short arm) or associate with other matrix molecules to construct a basement membrane.
  • N triple-stranded amino
  • LG1-LG5 domain homologous globular domains
  • the ⁇ chain is involved in cell adhesion and plays a major role in laminin.
  • Laminin 511 is a laminin molecule composed of ⁇ 5 chain, ⁇ 1 chain, and ⁇ 1 chain, and is a major isolator of laminin present in liver portal vein, hepatic artery, central vein, and bile duct basement membrane. It is a form. Although the in vivo function of laminin 511 has not yet been fully elucidated, the lack of laminin ⁇ 5 chain causes various tissue dysfunctions, so it plays an important role in fetal tissue formation. It is suggested that there is not. For laminin 511, see Dev. Dyn. 218, 213-234, 2000, and J. Org. Biol. Chem. 277 (15), 12741-12748, 2002 has a detailed description. The contents described in these documents are incorporated herein by reference.
  • An object of the present invention is to provide a method for culturing mammalian cells in a system containing laminin 511.
  • the present inventors cultured laminin 511 with a specific polypeptide and / or peptide under the condition that laminin 511 was immobilized.
  • the inventors have found that various activities on cells increase, and have conceived the present invention.
  • the present invention includes the following embodiments as preferred embodiments.
  • [Aspect 1] In a method of culturing mammalian cells in a system containing laminin 511, serum, serum albumin, prealbumin, immunoglobulin, ⁇ -globulin, ⁇ -globulin, ⁇ 1-antitrypsin ( ⁇ 1-AT), heptoglobin (Hp), ⁇ 2-macroglobulin ( ⁇ 2-M), ⁇ -fetoprotein (AFP), transferrin, retinol-binding protein (RBP) or blood protein other than extracellular matrix protein which is adiponectin, and gelatin, tumor necrosis factor (TNF) family
  • the method comprising culturing the cell under conditions in which laminin 511 is immobilized on a polypeptide and / or peptide selected from the group consisting of a protein belonging to the group, peptone.
  • [Aspect 2] The method according to aspect 1, wherein the polypeptide and / or peptide is serum albumin, a protein belonging to the tumor necrosis factor (TNF) family, or peptone.
  • TNF tumor necrosis factor
  • [Aspect 3] An aspect in which the activity of laminin 511 on cells selected from the group consisting of cell adhesion activity, cell dispersion activity, wound healing activity, growth promotion activity, undifferentiation maintenance activity, pluripotency maintenance activity and colony formation promotion activity is increased.
  • [Aspect 4] The method according to any one of aspects 1 to 3, wherein the cells are selected from the group consisting of pluripotent stem cells, tissue stem cells, somatic cells, germ cells, and sarcoma cells.
  • the pluripotent stem cells are selected from embryonic stem cells, induced pluripotent stem cells, embryonic germ cells, or germ stem cells;
  • the tissue stem cells are selected from mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, neural stem cells, skin stem cells, or hematopoietic stem cells; or Somatic cells are hepatocytes, pancreatic cells, muscle cells, bone cells, osteoblasts, osteoclasts, chondrocytes, adipocytes, skin cells, fibroblasts, pancreatic cells, kidney cells, lung cells, or lymphocytes Selected from erythrocytes, leukocytes, monocytes, macrophages or megakaryocyte blood cells, A method according to embodiment 4.
  • laminin 511 and a specific polypeptide and / or peptide are combined and used in a solid phase, thereby allowing cell adhesion activity, cell dispersion activity, wound healing activity, growth promoting activity, undifferentiation maintenance activity, and pluripotency maintenance.
  • the activity of the laminin 511 selected from the group consisting of activities is increased.
  • FIG. 1 shows the results of a cell adhesion assay of rLm511 using BRL cells.
  • the rHSA concentration was examined in a wide range of 0-100 ⁇ g / ml.
  • FIG. 1B reproducibility was confirmed at the rHSA concentration in which the cell adhesion activity increase was observed in FIG. 1A.
  • FIG. 1C the rHSA concentration was examined by finely dividing it at 0-12.5 ⁇ g / ml.
  • FIG. 1D shows the result of examination by further subdividing and setting the rHSA concentration range that showed a high cell adhesion effect in FIG. 1C.
  • FIG. 2 shows the results of rLm511 cell adhesion assay using HT1080 cells.
  • the rHSA concentration was examined in a wide range of 0-25 ⁇ g / ml.
  • FIG. 2B the effect of increasing cell adhesion activity when a recombinant human receptor activator NF ⁇ B ligand (sRANKL, ⁇ RA) was used was examined.
  • FIG. 2C the cell adhesion activity increasing action when cotton seed-derived peptone (peptide, Pep) was used was examined.
  • sRANKL recombinant human receptor activator NF ⁇ B ligand
  • FIG. 3 shows the results of cell adhesion assays of rLm511 and rLm211 using C2C12 cells.
  • the vertical axis represents OD 595
  • the horizontal axis represents laminin concentration ( ⁇ g / ml).
  • the rHSA concentration used is 5 ⁇ g / ml.
  • FIG. 4 is a photograph showing the results of rLm511 wound healing assay using HT1080 cells. The bar in the picture corresponds to the width of the wound.
  • FIG. 5 is a diagram showing the healing rate calculated from the width of the wound in the photograph of FIG. From the results of the wound healing assay in FIGS. 4 and 5, it was revealed that the wound healing activity of rLm511 can be increased by addition of rHSA in addition to the cell adhesion activity.
  • FIG. 6 shows the results of a culture experiment for examining colony formation using human iPS cells (201B7 strain). Specifically, the number of alkaline phosphatase (AP) positive colonies was shown. Mg indicates Matrigel. + And ⁇ indicate addition or non-addition of rHSA.
  • the present invention relates to a method for culturing mammalian cells in a system containing laminin 511.
  • laminin 511 is immobilized on a polypeptide and / or peptide selected from the group consisting of a protein belonging to the group, peptone.
  • Laminin 511 As shown in Table 1, “Laminin 511” (formerly “Laminin 10”) is a laminin molecule consisting of ⁇ 5 chain, ⁇ 1 chain, and ⁇ 1 chain, and is applied to the portal vein of the liver, hepatic artery, central vein, and bile duct basement membrane. It is the major isoform of laminin that exists. Although the in vivo function of laminin 511 has not yet been fully elucidated, laminin ⁇ 5 chain deficiency causes a variety of tissue dysgenesis, and thus plays an important role in fetal tissue formation. It is suggested that.
  • the laminin 511 protein may be a natural type or a modified type in which one or more amino acid residues are modified while retaining its biological activity, particularly cell adhesion promoting activity. Moreover, as long as the laminin 511 protein in this invention has the characteristics described in this specification, the origin, a manufacturing method, etc. are not limited. That is, the laminin 511 protein of the present invention may be any of naturally occurring proteins, proteins expressed from recombinant DNA by genetic engineering techniques, or chemically synthesized proteins.
  • laminin 511 protein is not particularly limited, but is preferably derived from human. When culturing human pluripotent stem cells for the purpose of obtaining materials for regenerative medicine, it is preferable to use human-derived laminin 511 in order to avoid the use of materials derived from other animals.
  • SEQ ID NOs: 1-6 in the sequence listing in this specification show the base sequence and amino acid sequence of ⁇ 5 chain, ⁇ 1 chain and ⁇ 1 chain of human laminin 511.
  • the laminin 511 protein used in the present invention is preferably an ⁇ 5 chain having an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which one or more amino acids are deleted, added, or substituted in this sequence (J. Biol.Chem.277 (15), 12741-12748, 2002; US 6,933,273 B2), the amino acid sequence of SEQ ID NO: 4, or one or more amino acids in this sequence have been deleted, added, or substituted ⁇ 1 chain having the amino acid sequence (J. Biol. Chem.
  • Each chain of laminin 511 may have an amino acid sequence in which one or more amino acid residues are deleted, added, or substituted in the amino acid sequence represented by the corresponding SEQ ID NO.
  • a protein having an amino acid sequence homologous to such a natural protein can also be used in the present invention.
  • the number of amino acids that can be changed is not limited in the amino acid sequences of ⁇ 5 chain, ⁇ 1 chain, and ⁇ 1 chain, but preferably 1 to 300 amino acid residues, 1 to 200 amino acid residues, and 1 to 150 amino acid residues.
  • amino acid residues 1 to 120 amino acid residues, 1 to 100 amino acid residues, 1 to 80 amino acid residues, 1 to 50 amino acid residues, 1 to 30 amino acid residues, 1 to 20 amino acid residues, 1 to 15 amino acid residues 1 to 10 amino acid residues and 1 to 5 amino acid residues.
  • the number of amino acid residues that can be modified by a known site-directed mutagenesis method for example, 1 to 10 amino acid residues, 1 to 8, 1 to 5, 1 to 3 amino acid residues is more preferable.
  • substitutions include replacing an amino acid with a residue having similar physicochemical properties, eg, replacing one fatty acid residue (Ile, Val, Leu or Ala) with another fatty acid residue, or Substitution between basic residues Lys and Arg, acidic residues Glu and Asp, amide residues Gln and Asn, hydroxyl residues Ser and Tyr, or aromatic residues Phe and Tyr are included.
  • laminin 511 used in the present invention has at least 80%, 85%, 90%, 95%, 98% or 99% identity with each amino acid sequence shown in SEQ ID NOs: 2, 4, and 6. And a protein that can promote cell adhesion activity.
  • The% identity between two amino acid sequences may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be determined by Needleman, S .; B. And Wunsch, C.I. D. (J. Mol. Biol., 48: 443-453, 1970) and determined by comparing sequence information using the GAP computer program available from the University of Wisconsin Genetics Computer Group (UWGCG). May be.
  • Preferred default parameters for the GAP program include: (1) Henikoff, S .; And Henikoff, J. et al. G. (Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992), scoring matrix, blossum 62; (2) 12 gap weights; (3) 4 gap length weights; And (4) no penalty for end gaps.
  • the percent identity can be determined by comparison with sequence information using, for example, the BLAST program described in Altschul et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997).
  • the program can be used on the Internet from the National Center for Biotechnology Information (NCBI) or the DNA Data Bank of Japan (DDBJ) website.
  • NCBI National Center for Biotechnology Information
  • DDBJ DNA Data Bank of Japan
  • Various conditions (parameters) for identity search by the BLAST program are described in detail on the same site, and some settings can be changed as appropriate, but the search is usually performed using default values.
  • the percent identity between two amino acid sequences can be determined by genetic information processing software GENETYX Ver. It may be determined using a program such as 7 (manufactured by Genetics) or the FASTA algorithm. At that time, the default value may be used for the search.
  • laminin 511 in the present invention has the characteristics described in the present specification, its origin, production method and the like are not limited. That is, laminin 511 may be a culture supernatant of human or animal cells that secrete laminin 511, or natural laminin 511 protein purified therefrom. However, laminin 511 can be effectively produced as a recombinant protein by expressing each subunit using recombinant DNA technology known in the art. However, human recombinant laminin 511 is particularly preferred because it avoids unnecessary animal factors.
  • a DNA sequence comprising nucleic acid residues 68-11155 of SEQ ID NO: 1 encoding the ⁇ 5 chain of laminin 511, nucleic acid residues 118-5478 of SEQ ID NO: 3 encoding ⁇ 1 chain and a nucleic acid of SEQ ID NO: 5 encoding ⁇ 1 chain
  • Primers can be designed based on the nucleotide sequence of residues 260-5089, and can be produced by amplifying the desired sequence by polymerase chain reaction (PCR) using an appropriate cDNA library as a template.
  • PCR polymerase chain reaction
  • a DNA encoding each chain gene of laminin 511 is incorporated into an appropriate vector and introduced into either a eukaryotic or prokaryotic cell using an expression vector that can be expressed in each host.
  • the desired protein can be obtained by expressing the chain.
  • Host cells that can be used to express laminin 511 are not particularly limited, and include prokaryotic host cells such as E. coli and Bacillus subtilis, yeast, fungi, insect cells, plants and plant cells, mammalian cells, and the like. Examples include eukaryotic hosts.
  • a vector constructed to express laminin 511 is transformed into the above host cell by transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, Agrobacterium method, direct microinjection, etc. Can be introduced inside.
  • Laminin 511 can be obtained by growing cells containing the vector in an appropriate medium to produce laminin 511 for use in the present invention and purifying the cells or medium. Purification can be performed using size exclusion chromatography, HPLC, ion exchange chromatography, immunoaffinity chromatography, and the like.
  • Laminin 511 or a commercially available one can be used.
  • a recombinant protein of laminin 511 can be purchased from BioLamina.
  • a typical laminin, laminin 111 (formerly called “laminin 1”) is a heterotrimeric molecule composed of ⁇ 1, ⁇ 1, and ⁇ 1, and laminin 511 used in the present invention is composed of ⁇ 5, ⁇ 1, and ⁇ 1.
  • laminin 1 a heterotrimeric molecule composed of ⁇ 1, ⁇ 1, and ⁇ 1
  • laminin 511 used in the present invention is composed of ⁇ 5, ⁇ 1, and ⁇ 1.
  • the identity of ⁇ 1 and ⁇ 5 is 35%. Even if the same laminin is named, the identity of ⁇ 1 and ⁇ 5 encoded by two different genes is about 35%, and laminin 111 and laminin 511 are considered to exhibit different properties.
  • Polypeptide and / or Peptide The present invention is intended to increase the activity of various laminin 511 by using a specific polypeptide and / or peptide in combination in a cell culture system containing laminin 511.
  • Polypeptides include serum, serum albumin, prealbumin, immunoglobulin, ⁇ -globulin, ⁇ -globulin, ⁇ 1-antitrypsin ( ⁇ 1-AT), heptoglobin (Hp), ⁇ 2-macroglobulin ( ⁇ 2-M), ⁇ -Selected from the group consisting of blood proteins other than extracellular matrix proteins which are fetoprotein (AFP), transferrin, retinol binding protein (RBP) or adiponectin, as well as gelatin, a protein belonging to the tumor necrosis factor (TNF) family, peptone Is done.
  • AFP fetoprotein
  • RBP retinol binding protein
  • TNF tumor necrosis factor
  • blood protein in the present invention, blood protein, more preferably blood protein other than extracellular matrix protein, is used together with laminin 511 protein.
  • the blood protein is preferably serum, serum albumin, prealbumin, immunoglobulin, ⁇ -globulin, ⁇ -globulin, ⁇ 1-antitrypsin ( ⁇ 1-AT), heptoglobin (Hp), ⁇ 2-macroglobulin ( ⁇ 2-M ), ⁇ -fetoprotein (AFP), transferrin, retinol binding protein (RBP) or adiponectin.
  • ⁇ 1-AT heptoglobin
  • Hp heptoglobin
  • ⁇ 2-M ⁇ 2-macroglobulin
  • AFP transferrin
  • RBP retinol binding protein
  • adiponectin adiponectin
  • Extracellular matrix is a substance that fills the extracellular space and at the same time has a skeletal role (eg animal cartilage and bone), a role of scaffold in cell adhesion (eg basement membrane and fibronectin), cell proliferation Responsible for holding and providing factors (eg, cell growth factor FGF that binds to heparan sulfate). It can be said that many of the individual cells constituting a multicellular organism live in a bed or nest of an extracellular matrix. Conspicuous components in the extracellular matrix of vertebrates including humans are glycoproteins (partially cell adhesion molecules) such as collagen, proteoglycan, fibronectin and laminin. “Extracellular matrix protein” means a protein constituting such an extracellular matrix.
  • blood protein other than extracellular matrix protein means a protein other than extracellular matrix protein involved in cell adhesion among blood proteins. These are all known proteins and can be appropriately obtained by those skilled in the art.
  • Bood proteins other than extracellular matrix proteins are not limited, but preferably human serum albumin (available from HSA / eg, Nacalai), recombinant human serum albumin (rHSA / eg, from SIGMA) Available), or bovine serum albumin (BSA / available from SIGMA, for example).
  • human serum albumin available from HSA / eg, Nacalai
  • rHSA / eg, from SIGMA recombinant human serum albumin
  • BSA bovine serum albumin
  • the “blood protein other than extracellular matrix protein” may alternatively be an immunoglobulin.
  • Immunoglobulins are well known to those skilled in the art and include IgG, IgA, IgM, IgD, IgE and the like.
  • human immunoglobulin IgG / for example, available from Oriental Yeast Co., Ltd. can be used.
  • Gelatin Gelatin is extracted by applying heat to collagen, which is the main component of connective tissue such as animal skin, bones, and tendons, and contains protein as the main component.
  • TNF Tumor necrosis factor
  • TNF Tumor necrosis factor
  • LT lymphphotoxin
  • the “protein belonging to the TNF family” includes at least 19 kinds of molecules such as receptor activator NF ⁇ B ligand (RANKL), Fas ligand, CD40 ligand and the like.
  • RNKL receptor activator NF ⁇ B ligand
  • Fas ligand CD40 ligand
  • a receptor activator NF ⁇ B ligand (RANKL, sRANKL) can be preferably used.
  • Peptone “Peptone” is obtained by degrading a protein with a proteolytic enzyme. In vivo, proteins are digested by pepsin in the stomach to become peptone, and further digested to amino acids by pancreatic juice secreted by the pancreas and intestinal juice secreted by the jejunum.
  • Peptone as a nutrient source of this medium is a protein hydrolyzed to amino acids and low molecular weight peptides.
  • protein from milk milk casein
  • protes such as pancreatin extracted from porcine pancreas are used
  • peptone is preferably derived from a plant.
  • it is selected from the group consisting of cottonseed-derived peptone, soybean-derived peptone, wheat-derived peptone and pea-derived peptone.
  • the polypeptide and / or peptide is serum albumin, a protein belonging to the tumor necrosis factor (TNF) family, or peptone.
  • the polypeptide and / or peptide is an immunoglobulin or gelatin.
  • Mammalian cells The type and origin of mammalian cells cultured in the method of the present invention are not particularly limited.
  • the pluripotent stem cells are selected from embryonic stem cells, induced pluripotent stem cells, embryonic germ cells, or germ stem cells.
  • the tissue stem cells are selected from mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, neural stem cells, skin stem cells, or hematopoietic stem cells.
  • the somatic cells are hepatocytes, pancreatic cells, muscle cells, bone cells, osteoblasts, osteoclasts, chondrocytes, adipocytes, skin cells, fibroblasts, pancreatic cells, kidney cells, lung cells, or , Lymphocytes, erythrocytes, leukocytes, monocytes, macrophages or megakaryocyte blood cells.
  • the mammalian species from which the cells are derived is not particularly limited. Preferably, it is derived from mouse, rat, human, monkey, pig, dog, sheep, goat and the like. More preferably, it is derived from a species selected from the group consisting of mouse, rat and human.
  • pluripotent stem cell is intended to be a generic term for stem cells having the ability to differentiate into cells of any tissue (differentiation pluripotency). In the examples described later in this specification, examination is performed using ES cells (EB3 cells).
  • ES cells EB3 cells
  • pluripotent stem cells that can be used in the method of the present invention are not limited to embryonic stem cells, but also mammalian cells. Examples include all pluripotent stem cells having traits similar to embryonic stem cells derived from adult organ and tissue cells, bone marrow cells, blood cells, and embryonic and fetal cells.
  • embryonic stem cells traits similar to embryonic stem cells are specific to embryonic stem cells, such as the expression of genes specific to embryonic stem cells and the ability to differentiate into all germ layers of endoderm, mesoderm, and ectoderm. It can be defined with cell biological properties.
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • EG cells embryonic germ stem cells
  • GS cells germ stem cells
  • ES cells and iPS cells are preferred as the pluripotent stem cells in the present invention.
  • iPS cells are particularly preferred for reasons such as no ethical problems.
  • Any known pluripotent stem cell can be used.
  • the pluripotent stem cell described in International Publication WO2009 / 123349 (PCT / JP2009 / 057041) can be used.
  • tissue stem cell means a stem cell that has the ability to differentiate into various cell types (differentiated pluripotency) although the cell line that can be differentiated is limited to a specific tissue. For example, hematopoietic stem cells in the bone marrow become blood cells, and neural stem cells differentiate into nerve cells. In addition, there are various types such as liver stem cells that make the liver and skin stem cells that become skin tissue.
  • Somatic cells refers to cells other than germ cells among the cells constituting multicellular organisms. In sexual reproduction, it is not passed on to the next generation. In the present specification, it means various cells other than “pluripotent stem cells” and “tissue stem cells”.
  • system containing laminin 511 cells are cultured in a system containing laminin 511.
  • the “system containing laminin 511” means that laminin 511 is somehow included in the cell culture system, and its mode is not particularly limited.
  • cells are cultured under conditions in which a specific polypeptide and / or peptide and laminin 511 are immobilized.
  • Solid phase is not a state in which a polypeptide and / or peptide and laminin 511 are simply added and dissolved in a liquid medium, for example, in a cell culture system. It means the adsorbed state.
  • it means a state in which a cell culture container is treated with a specific polypeptide and / or peptide and laminin 511 and the culture container is coated. Therefore, in order to culture cells in a system containing laminin 511, it is preferable to use a culture vessel coated with a specific polypeptide and / or peptide and laminin 511.
  • the “cell culture container” is not particularly limited, and any material and container of any shape that is sterilized to prevent bacterial contamination and suitable for culturing cells is used. be able to.
  • Examples of such culture containers include culture dishes, culture flasks, culture petri dishes, 96-well, 48-well, 12-well, 6-well, 4-well, etc., commonly used in this technical field. Examples thereof include, but are not limited to, plates and culture bottles.
  • a specific polypeptide and / or peptide and laminin 511 are solid-phased (coated) on the surface of the cell culture container.
  • a processing technique for immobilizing laminin on the surface of a culture vessel is known in the art, and those skilled in the art employ any culture vessel according to the purpose of the present invention to treat the vessel, It can be used in the inventive method.
  • the amount of laminin 511 used for processing the cell culture container is not particularly limited. Preferably, good results are obtained when treated with a laminin 511 solution of 0.01 ⁇ g / ml or more, preferably 0.01 to 10 ⁇ g / ml, more preferably 0.01-2 ⁇ g / ml.
  • the laminin 511 of 0.01-10 ⁇ g / ml, 0.01 ⁇ g / ml-2 ⁇ g / ml has an amount of laminin 511 immobilized on the area of the culture vessel of 0.0015-1.5 ⁇ g / cm 2 , This corresponds to 0.0015-0.3 ⁇ g / cm 2 .
  • the amount of polypeptide or peptide used is not particularly limited. A person skilled in the art can appropriately select an appropriate amount according to factors such as the type of polypeptide or peptide used, the type of cells to be cultured, and the like. Preferably, but not exclusively, the polypeptide or peptide is used at a concentration between 0.4 ⁇ g / ml and 200 ⁇ g / ml. This corresponds to 0.06 to 30 ⁇ g / cm 2 as the amount of polypeptide or peptide to be immobilized per area of the culture vessel.
  • the culture container may be treated with laminin 511 by applying laminin 511 to the inner surface of the culture container and then drying.
  • a culture medium generally used for culturing cells such as GMEM (GIBCO) and DMEM is placed in a culture container treated with laminin 511, and pluripotent stem cells are added to the culture medium.
  • the cells are then cultured under known appropriate culture conditions, such as, but not limited to, 37 ° C., 5% carbon dioxide gas layer conditions.
  • the order in which the specific polypeptide and / or peptide and laminin 511 are immobilized is not particularly limited. In one embodiment, the polypeptide and / or peptide and laminin 511 are immobilized simultaneously. Alternatively, after immobilization with a polypeptide and / or peptide, immobilization with laminin 511 is performed.
  • Cell culture container, composition, agent, kit This invention relates to the cell culture container which solidified the polypeptide and / or peptide mentioned above and laminin 511 in one aspect.
  • the present invention also provides a composition for immobilizing a cell culture vessel for culturing cells in a system containing laminin 511, including the above polypeptide and / or peptide and laminin 511, or a cell culture vessel
  • the present invention relates to an agent for immobilizing a solid.
  • the composition or agent of the present invention is a coating composition or a coating agent.
  • the present invention further relates to a kit containing the above composition or agent.
  • the kit of the present invention may further contain a cell culture medium, a cell culture container and the like.
  • the cell culture container may be, for example, a precoat culture dish or a precoat culture plate.
  • the cell culture container of the kit may be in a state where the above polypeptide and / or peptide and laminin 511 are immobilized.
  • the cell culture container, composition, agent and kit of the present invention can be used in a method for culturing mammalian cells in a system containing laminin 511 of the present invention.
  • laminin 511 various activities exhibited by laminin 511 in cell culture are increased in combination with polypeptides and / or peptides.
  • the effects of laminin 511 include, but are not limited to, “cell adhesion activity” including cell adhesion activity, cell dispersion activity, wound healing activity, growth promoting activity, undifferentiation maintenance activity and pluripotency maintenance activity. Means the effect of adhering cells.
  • the use of the polypeptide or peptide preferably increases the cell adhesion activity by 4 times or more, more preferably 8 times or more, and most preferably 16 times or more compared to the case where the polypeptide or peptide is not used.
  • Cell dispersal activity means the effect of dispersing cells.
  • the use of the polypeptide preferably increases the cell dispersion activity more than twice as compared to the case where the polypeptide is not used.
  • “Wound healing activity” means the effect of healing a wound. That is, by applying laminin 511 or the like to a part where the cell has been physically damaged due to trauma, for example, the cell is allowed to migrate from around to the applied part. The effect of healing a wound can be confirmed, for example, by measuring the width of the wound after a certain period of time (for example, after 14 hours) after being damaged. For example, in Example 2, the wound healing rate increased from 32% to 55% by using rHSA in addition to human laminin 511 (0.125 ⁇ g / ml).
  • “Proliferation promoting activity” means an effect of promoting cell proliferation.
  • the effect of cell proliferation can be confirmed by measuring the number of cells after a certain period of time has passed since the cells were run.
  • Undifferentiated maintenance activity means that when the cells to be cultured are undifferentiated cells such as pluripotent stem cells and tissue stem cells, the undifferentiated state is maintained. When these cells are cultured with laminin 511, cell differentiation does not proceed and an undifferentiated state is maintained. For example, by measuring an undifferentiated marker such as Sox2, Nanog, Oct4, etc., it is possible to evaluate whether tissue stem cells are differentiated during culture.
  • undifferentiated maintenance activity means that when the cells to be cultured are undifferentiated cells such as pluripotent stem cells and tissue stem cells, the undifferentiated state is maintained. When these cells are cultured with laminin 511, cell differentiation does not proceed and an undifferentiated state is maintained. For example, by measuring an undifferentiated marker such as Sox2, Nanog, Oct4, etc., it is possible to evaluate whether tissue stem cells are differentiated during culture.
  • the “pluripotency maintenance activity” means to maintain the pluripotency when the cultured cell is a pluripotent cell, for example, a pluripotent stem cell.
  • pluripotency is maintained even when laminin 511 and a polypeptide and / or peptide are used in combination.
  • a method for increasing the activity of laminin 511 on cells also comprises culturing the cell under the condition that laminin 511 is immobilized with a specific polypeptide and / or peptide, and the activity on the cell of laminin 511 Provide a way to raise.
  • the activity on cells is selected from the group consisting of cell adhesion activity, cell dispersion activity, wound healing activity, growth promotion activity, undifferentiation maintenance activity, pluripotency maintenance activity and colony formation promotion activity. .
  • the definition of each activity is also as described above.
  • Example 1 Cell Adhesion Assay This example shows the results of an adhesion assay when various additives are added to rLm511 and rLm511.
  • Recombinant human laminin 511 (rLm511) used was purchased from Biolamina.
  • rat liver cell line (BRL), human sarcoma cell line (HT1080), and mouse myoblast cell line (C2C12).
  • BRL was obtained from the Human Science Promotion Foundation (JCRB0025).
  • HT1080 was obtained from RIKEN BioResource Center (RCB1956).
  • C2C12 was obtained from DS Pharma Biomedical (EC-91031101).
  • BMEM was DMEM / F12 (GIBCO) supplemented with 10% fetal calf serum
  • HT1080 was MEM (SIGMA) supplemented with 10% fetal calf serum
  • C2C12 was DMEM (SIGMA) supplemented with 15% fetal calf serum.
  • a serum-free medium obtained by removing serum from these media was used.
  • a 96-well plate (Corning) was treated with rLm511 at a adjusted concentration for 2 hours at 37 ° C. or overnight at 4 ° C., and the treated surface was washed with PBS ( ⁇ ), and then washed with 1.2% BSA (SIGMA) solution. Blocking treatment was performed at 1 ° C. for 1 hour.
  • Treatment with rLm511 as necessary may involve recombinant human serum albumin (rHSA / SIGMA), recombinant human receptor activator NF ⁇ B ligand (sRANKL / OYC), cottonseed-derived peptone (Pep / DMV), glycine (Gly / Nacalai) ) Was mixed and processed.
  • FIGS. 2A to 2D show the results of the adhesion assay using HT1080
  • FIG. 3 shows the results of the adhesion assay using C2C12.
  • rLm211 did not reach the activity of 8 ⁇ g / of laminin 211 alone even when 2 ⁇ g / ml of rHSA was added. Similarly, even when 8 ⁇ g / ml rHSA was added, the activity did not reach 32 ⁇ g / of laminin 211 alone. From this, it was judged that the increase in the adhesion activity of rLm211 by the combined use of rHSA was 4 times or less.
  • Example 2 Wound healing assay using HT1080 cells
  • the results of wound healing assay when rHSA was added to rLm511 and rLm511 for various cells are shown.
  • HT1080 cells were used as cells.
  • HT1080 was obtained from RIKEN BioResource Center (RCB1956).
  • HT1080 cells were cultured and grown using MEM (SIGMA) supplemented with 10% fetal bovine serum.
  • MEM SIGMA
  • a medium supplemented with a serum-free medium obtained by removing serum from the medium was used in addition to the above medium.
  • Cells were seeded in a 24-well plate (Nunc) at 400000 cells / well in 10% fetal calf serum medium, and cultured for 3 hours at 37 ° C., 5% CO 2 , 95% air.
  • the reason why the cells were cultured using the serum-added medium is to first adhere the cells uniformly to the culture surface. After culturing, the adherent cell population in each well was washed twice with a serum-free medium in which a fixed width was scratched using a blue chip and serum was removed.
  • rLm511 was treated with rLm511 adjusted in serum-free medium at 37 ° C. for 30 minutes to immobilize rLm511. If necessary, rLm511 was mixed with rHSA (5 ⁇ g / ml), and rHSA was also immobilized. After treatment with rLm511, the treated surface was washed twice with serum-free medium, and serum-free medium was added.
  • FIG. 5 shows the results of the cell dispersion assay.
  • rHSA was added to 0.125 ⁇ g / ml-0.5 ⁇ g / ml Lm511, an increase in wound healing activity was observed.
  • Example 3 Colony Formation Test Using Human iPS Cells In this example, the results of a colony formation test using human iPS cells are shown.
  • the cell used was 201B7 strain.
  • the 201B7 strain was obtained from iPS Academia Japan.
  • MEF-CM mouse embryonic fibroblast conditioned medium
  • WAKO mouse embryonic fibroblast conditioned medium
  • KSR Knockout TM Serum Replacement Additive
  • GBCO Knockout TM Serum Replacement Additive
  • GBCO 2 mM glutamine
  • GIBCO 1% non-essential amino acid
  • WAKO 10 ⁇ 4 M 2-mercaptoethanol
  • a 12-well plate (NUNC) treated with rLm511 at a adjusted concentration for 2 hours at 37 ° C. or overnight at 4 ° C. was used. If necessary, rLm511 was treated by mixing rHSA at 5 ⁇ g / ml.
  • an experimental group of 300 ⁇ g / ml Matrigel (BD, Becton Dickinson) was also set as a positive control.
  • 201B7 is recovered in the form of cell mass, seeded in an equal amount in a 12-well plate, cultured for 6 days while changing the medium at 37 ° C., 5% CO 2 , 95% air. Colonies were formed in the absence. After colony formation, the cells were fixed with 4% paraformaldehyde and stained with alkaline phosphatase (Vector Laboratories) to detect only normal colonies retaining undifferentiated properties. As a result, when rLm511 was used alone, colony formation was hardly observed at a concentration of 0.5 ⁇ g / ml or less, but it was found that colonies were sufficiently formed even at 0.5 ⁇ g / ml when rHSA was used in combination. (FIGS. 6 and 7).
  • polypeptides including rHSA not only increases the cell adhesion activity and wound healing activity of rLm511, but also enhances the function of rLm511 in pluripotent stem cell culture / proliferation / colony formation. I understood.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un procédé de culture d'une cellule de mammifère dans un système contenant de la laminine 511. Le procédé selon la présente invention est caractérisé par la culture de la cellule dans des conditions telles qu'une protéine sanguine, autre qu'une protéine de la matrice extracellulaire, qui est le sérum, la sérumalbumine, la pré-albumine, une immunoglobuline, l'α-globuline, la β-globuline, l'α1-antitrypsine (α1-AT), l'haptoglobine (Hp), l'α2-macroglobuline (α2-M), l'α-fétoprotéine (AFP), la transferrine, une protéine de liaison au rétinol (RBP) ou une adiponectine, un polypeptide et/ou un peptide choisi dans le groupe constitué par la gélatine, une protéine appartenant à la famille du facteur de nécrose tumorale (TNF) et une peptone, et la laminine 511 sont en phase solide.
PCT/JP2012/075111 2011-09-29 2012-09-28 Procédé de culture de cellule de mammifère dans système contenant de la laminine 511 WO2013047763A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2011-214589 2011-09-29
JP2011214589 2011-09-29

Publications (1)

Publication Number Publication Date
WO2013047763A1 true WO2013047763A1 (fr) 2013-04-04

Family

ID=47995786

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2012/075111 WO2013047763A1 (fr) 2011-09-29 2012-09-28 Procédé de culture de cellule de mammifère dans système contenant de la laminine 511

Country Status (2)

Country Link
JP (1) JPWO2013047763A1 (fr)
WO (1) WO2013047763A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014168157A1 (fr) * 2013-04-08 2014-10-16 独立行政法人医薬基盤研究所 Procédé pour la culture de cellules de type hépatoblaste et produit de culture associé
WO2015080297A1 (fr) 2013-11-27 2015-06-04 Kyoto Prefectural Public University Corporation Application de laminine à une culture de cellules endothéliales de la cornée
CN105378054A (zh) * 2013-06-12 2016-03-02 国立大学法人大阪大学 以干燥状态包被有层粘连蛋白片段的细胞培养器具
CN106559994A (zh) * 2014-07-16 2017-04-05 国立大学法人大阪大学 层粘连蛋白片段的细胞培养基质活性增强方法
WO2017082220A1 (fr) * 2015-11-10 2017-05-18 国立大学法人京都大学 Procédé de culture cellulaire utilisant un milieu contenant un fragment de laminine
JPWO2018131491A1 (ja) * 2017-01-13 2019-11-21 国立大学法人大阪大学 角膜上皮細胞集団の製造方法
JP2020503878A (ja) * 2017-01-11 2020-02-06 ザ フランシス クリック インスティチュート リミティッド 多能性幹細胞の培養のための組成物
WO2021157421A1 (fr) * 2020-02-05 2021-08-12 味の素株式会社 Procédé de production d'une protéine
EP3875578A4 (fr) * 2018-10-31 2022-08-10 Kyoto University Procédé de production d'une cellule souche pluripotente ayant une résistance à la différenciation mésendodermique libérée
CN115516096A (zh) * 2020-05-08 2022-12-23 国立大学法人大阪大学 含有纤维蛋白原片段和层粘连蛋白片段的嵌合蛋白及其利用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0751049A (ja) * 1993-08-12 1995-02-28 Fujitsu Ltd 細胞接着試験器具
WO2002050111A2 (fr) * 2000-12-21 2002-06-27 Biostratum, Inc. Laminine 10 isolee
WO2008084401A2 (fr) * 2007-01-04 2008-07-17 Karl Tryggvason Composition et procédé pour permettre la prolifération de cellules souches pluripotentes
US20110039336A1 (en) * 2008-04-28 2011-02-17 Ge Healtgcare Bio-Sciences Ab Product for cell culture

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0751049A (ja) * 1993-08-12 1995-02-28 Fujitsu Ltd 細胞接着試験器具
WO2002050111A2 (fr) * 2000-12-21 2002-06-27 Biostratum, Inc. Laminine 10 isolee
WO2008084401A2 (fr) * 2007-01-04 2008-07-17 Karl Tryggvason Composition et procédé pour permettre la prolifération de cellules souches pluripotentes
US20110039336A1 (en) * 2008-04-28 2011-02-17 Ge Healtgcare Bio-Sciences Ab Product for cell culture

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DOMOGATSKAYA,A. ET AL.: "Laminin-511 but not -332, -111, or -411 enables mouse embryonic stem cell self-renewal in vitro", STEM CELLS, vol. 26, no. 11, 1 November 2008 (2008-11-01), pages 2800 - 9, XP009109423 *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014168157A1 (fr) * 2013-04-08 2014-10-16 独立行政法人医薬基盤研究所 Procédé pour la culture de cellules de type hépatoblaste et produit de culture associé
JPWO2014168157A1 (ja) * 2013-04-08 2017-02-16 国立研究開発法人医薬基盤・健康・栄養研究所 肝幹前駆様細胞の培養方法及びその培養物
CN105378054A (zh) * 2013-06-12 2016-03-02 国立大学法人大阪大学 以干燥状态包被有层粘连蛋白片段的细胞培养器具
EP3009502A4 (fr) * 2013-06-12 2017-01-25 Osaka University Matériel de culture cellulaire enduit à l'état sec de fragments de laminines
US10287541B2 (en) 2013-06-12 2019-05-14 Osaka University Cell culture vessel coated with laminin fragment in dry state
CN105378054B (zh) * 2013-06-12 2018-09-21 国立大学法人大阪大学 以干燥状态包被有层粘连蛋白片段的细胞培养器具
WO2015080297A1 (fr) 2013-11-27 2015-06-04 Kyoto Prefectural Public University Corporation Application de laminine à une culture de cellules endothéliales de la cornée
JPWO2016010082A1 (ja) * 2014-07-16 2017-06-22 国立大学法人大阪大学 ラミニンフラグメントの細胞培養基質活性増強方法
US20170159020A1 (en) * 2014-07-16 2017-06-08 Osaka University Method for enhancing activity of laminin fragments as cell culture matrix
CN106559994A (zh) * 2014-07-16 2017-04-05 国立大学法人大阪大学 层粘连蛋白片段的细胞培养基质活性增强方法
US10428311B2 (en) 2014-07-16 2019-10-01 Osaka University Method for enhancing activity of laminin fragments as cell culture matrix
WO2017082220A1 (fr) * 2015-11-10 2017-05-18 国立大学法人京都大学 Procédé de culture cellulaire utilisant un milieu contenant un fragment de laminine
US11505785B2 (en) 2015-11-10 2022-11-22 Kyoto University Cell culture method using laminin fragment-containing medium
JP2020503878A (ja) * 2017-01-11 2020-02-06 ザ フランシス クリック インスティチュート リミティッド 多能性幹細胞の培養のための組成物
JPWO2018131491A1 (ja) * 2017-01-13 2019-11-21 国立大学法人大阪大学 角膜上皮細胞集団の製造方法
EP3875578A4 (fr) * 2018-10-31 2022-08-10 Kyoto University Procédé de production d'une cellule souche pluripotente ayant une résistance à la différenciation mésendodermique libérée
WO2021157421A1 (fr) * 2020-02-05 2021-08-12 味の素株式会社 Procédé de production d'une protéine
CN115516096A (zh) * 2020-05-08 2022-12-23 国立大学法人大阪大学 含有纤维蛋白原片段和层粘连蛋白片段的嵌合蛋白及其利用

Also Published As

Publication number Publication date
JPWO2013047763A1 (ja) 2015-03-30

Similar Documents

Publication Publication Date Title
WO2013047763A1 (fr) Procédé de culture de cellule de mammifère dans système contenant de la laminine 511
KR101921350B1 (ko) 인간 다능성 줄기세포를 유지하기 위한 시스템
JP6518878B2 (ja) 網膜色素上皮細胞の製造方法
CN104884468B (zh) 附加有胶原结合性分子的改造层粘连蛋白及其利用
JP6278324B2 (ja) ラミニンフラグメントの細胞培養基質活性増強方法
KR20180132786A (ko) 피브로인형 단백질 개변체 및 세포 배양 방법
CN105378054B (zh) 以干燥状态包被有层粘连蛋白片段的细胞培养器具
EP3492592B1 (fr) Fragment de fibronectine à utiliser pour la production de cellules souches
CN104364263A (zh) 含有层粘连蛋白和钙粘蛋白的细胞培养基底
CN107208064A (zh) 细胞的培养方法、细胞的凝集体、细胞凝集控制剂以及培养基
JP5001155B2 (ja) ラミニン５を利用した間葉系幹細胞の培養技術
JP7285520B2 (ja) 皮膚由来多能性前駆細胞の作製方法
JP5882198B2 (ja) ラミニン５を含んだ系で細胞を培養する方法
JP6916523B2 (ja) 筋衛星細胞培養用材料および筋衛星細胞の培養方法
JP6822668B2 (ja) 多能性幹細胞の胚様体形成方法および多能性幹細胞の胚様体形成用組成物
WO2019030524A1 (fr) Protéines de fusion s'assemblant en échafaudages et favorisant le renouvellement des cellules souches
CN115516096B (zh) 含有纤维蛋白原片段和层粘连蛋白片段的嵌合蛋白及其利用
JP2024533113A (ja) ヒトビトロネクチン断片及びその使用
Hill Regulating stem cell behaviour using bioengineered culture substrates
WO2021025122A1 (fr) Procédé de culture de cellules de papille dermique
JP2020015708A (ja) 幹細胞接着性ペプチド及びその利用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12835793

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2013536434

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12835793

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载