[go: up one dir, main page]

WO2019189769A1 - Composition pour croissance cellulaire - Google Patents

Composition pour croissance cellulaire Download PDF

Info

Publication number
WO2019189769A1
WO2019189769A1 PCT/JP2019/014022 JP2019014022W WO2019189769A1 WO 2019189769 A1 WO2019189769 A1 WO 2019189769A1 JP 2019014022 W JP2019014022 W JP 2019014022W WO 2019189769 A1 WO2019189769 A1 WO 2019189769A1
Authority
WO
WIPO (PCT)
Prior art keywords
monomer
polymer
medium
mol
neutral monomer
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/JP2019/014022
Other languages
English (en)
Japanese (ja)
Inventor
陽子 栗山
学 北澤
尚 杉本
弘樹 小澤
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ajinomoto Co Inc
Original Assignee
Ajinomoto Co Inc
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 Ajinomoto Co Inc filed Critical Ajinomoto Co Inc
Publication of WO2019189769A1 publication Critical patent/WO2019189769A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor

Definitions

  • the present invention relates to a cell growth composition and the like.
  • regenerative medicine using stem cells such as iPS cells and ES cells, it is assumed that these cells are efficiently proliferated and then differentiated into target tissues and transplanted.
  • stem cells such as iPS cells and ES cells
  • a system capable of stably culturing and supplying cells is indispensable, and the development of a medium for producing the medium and a method for producing the medium is required.
  • a medium (culture solution) is essential for cell culture, but expensive components are often required, and it is difficult to secure a sufficient amount of cells. Accordingly, there is a need for efficient cell culture and low cost. Therefore, search for materials that can promote cell proliferation and that can be supplied in large quantities at low cost is underway.
  • Patent Document 1 Non-Patent Documents 1 to 3
  • the present invention provides a medium for proliferating stem cells, particularly iPS cells, which has good culturing results, using a material that is inexpensive and can be supplied in large quantities, and provides a composition for cell proliferation for producing the medium for proliferation. For the purpose.
  • the present inventors have found that the medium contains a polymer composed of a neutral monomer, an anionic monomer, and a cationic monomer, so that an excellent cell proliferation promoting effect, particularly stem cell proliferation, can be achieved. It has been found that a promoting effect can be obtained. Water-solubility that has the effect of promoting the proliferation of stem cells by adjusting the ratio of each monomer component as appropriate so that the balance with neutral monomers that have both cation and anion properties and neutralize the effects of ions is optimized The present invention was completed by successfully obtaining a polymer.
  • a composition for promoting cell growth comprising a random polymer comprising a neutral monomer, an anionic monomer and a cationic monomer as an active ingredient.
  • ClogP is 0.9 or less
  • the number of hydrogen accepting groups is 2 or less
  • the neutral monomer 1 is at least one selected from the group consisting of N-isopropylacrylamide, N-vinylpyrrolidone and vinyl acetate.
  • the cationic monomer is N, N-dimethylpropylaminoacrylamide and / or 2-N-morpholinoethyl methacrylate.
  • the ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) of the polymer is 1.0 to 3.5. .
  • a cell culture medium comprising a random polymer comprising a neutral monomer, an anionic monomer and a cationic monomer.
  • [II-3] Neutral monomer 1 is N-isopropylacrylamide, neutral monomer 2 is 2-hydroxypropyl acrylate, anionic monomer is acrylic acid, and cationic monomer is N, N-dimethylpropylamino
  • [II-4] The above [II-3], wherein the monomer ratio of neutral monomer 1, neutral monomer 2, anionic monomer and cationic monomer in the random polymer is 4: 2: 1.5: 2.5 The medium described.
  • [37] The medium according to any one of [20] to [36] and [II-1] to [II-4] above, wherein the cell is a pluripotent stem cell.
  • the medium described in [37] above, wherein the pluripotent stem cells are iPS cells.
  • a method for culturing iPS cells comprising culturing in the culture medium according to [38].
  • the polymer of the present invention When the polymer of the present invention is added to the medium during cell culture, the proliferation of stem cells, particularly iPS cells, can be promoted, so that the culture time required to obtain a desired number of cells can be reduced. Therefore, necessary reagents and materials such as a culture solution can be reduced, and the efficiency and cost reduction of stem cell culture can be achieved. Furthermore, since the polymer of the present invention is water-soluble, it can be dissolved in a culture solution and is easy to handle.
  • R is an arbitrary functional group, and is appropriately determined depending on the monomer used.
  • R is a tert-butyl group
  • R is an isopropyl group
  • R is a hydroxyl group
  • R is N-dimethylpropylamino group.
  • NIPAm content polymer 12 (upper figure) and NIPAm content polymer 23 (lower figure). It is a graph which shows the result of having investigated the stem cell proliferation promotion effect of the polymer (HPA containing polymer 2, PAAm containing polymer 1, NIPAm containing polymer 12) of this invention at the time of using vitronectin as a scaffold. Three wells were measured and indicated by the average value and standard error. It is a graph which shows the result of having investigated the stem cell proliferation promotion effect of the polymer (PAAm containing polymer 1, NIPAm containing polymer 12) of this invention at the time of using Matrigel as a scaffold. Three wells were measured and indicated by the average value and standard error.
  • the type of cell is not limited, but the cell is preferably a stem cell, and more preferably a pluripotent stem cell.
  • the “stem cell” means an immature cell having self-renewal ability and differentiation / proliferation ability.
  • the stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, unipotent stem cells, etc., depending on the differentiation ability.
  • a pluripotent stem cell means a cell having an ability to differentiate into all tissues and cells constituting a living body.
  • a multipotent stem cell means a cell having the ability to differentiate into multiple types of tissues and cells, although not all types.
  • a unipotent stem cell means a cell having the ability to differentiate into a specific tissue or cell.
  • Pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), pluripotent stem cells induced and selected by stress or cell stimulation, etc. I can list them. Stem cells established by culturing early embryos produced by nuclear transfer of somatic cell nuclei are also preferred as pluripotent stem cells (Nature, 385, 810 (1997); Science, 280, 1256 (1998) Nature Biotechnology, 17, 456 (1999); Nature, 394, 369 (1998); Nature Genetics, 22, 127 (1999); Proc. Natl. Acad. Sci. USA, 96, 14984 (1999); Nature Genetics, 24, 109 (2000)).
  • multipotent stem cells examples include somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, nervous system stem cells, bone marrow stem cells, and reproductive stem cells.
  • the multipotent stem cell is preferably a mesenchymal stem cell, more preferably a bone marrow mesenchymal stem cell.
  • a mesenchymal stem cell broadly means a population of stem cells or precursor cells thereof that can differentiate into all or some mesenchymal cells such as osteoblasts, chondroblasts, and lipoblasts.
  • the basal medium used in the present invention may be any known per se and is not particularly limited as long as it does not inhibit the growth of cells, particularly stem cells.
  • a medium modified for stem cell culture a mixture of the above basal medium and another medium, or the like may be used.
  • StemFit registered trademark
  • AK medium Alkaolin
  • Essential 8 medium Life Technologies
  • mTeSR1 medium SERCA-1
  • TeSR2 medium basic medium for pluripotent stem cell culture ( STEMCELL Technologies)
  • RHB medium StemCells, Inc.
  • TeSRTM-E6 SERCAELL Technologies
  • hESF-GRO medium Nipro Corporation
  • HESF-DIF medium Nipro Corporation
  • CSTI-7 Cell Science Research Co., Ltd.
  • Essential-6 medium Life Technologies
  • the medium used in the present invention may contain additives known per se.
  • the additive is not particularly limited as long as it does not inhibit the growth of cells, particularly stem cells.
  • growth factors for example, insulin
  • iron sources for example, transferrin
  • polyamines for example, putrescine
  • minerals for example, Sodium selenate etc.
  • saccharides eg glucose etc.
  • albumin eg human serum albumin
  • organic acids eg pyruvate, lactic acid etc.
  • amino acids eg L-glutamine etc.
  • reducing agents eg 2-mercaptoethanol etc.
  • Vitamins eg, ascorbic acid, d-biotin, etc.
  • steroids eg, ⁇ -estradiol, progesterone, etc.
  • antibiotics eg, streptomycin, penicillin, gentamicin, etc.
  • buffers eg, HEPES, etc.
  • the additive conventionally used for culture
  • the medium used in the present invention may contain serum.
  • the serum is not particularly limited as long as it is an animal-derived serum, as long as it does not inhibit the proliferation of stem cells, but is preferably a mammal-derived serum (eg, fetal bovine serum, human serum, etc.).
  • the serum concentration may be within a concentration range known per se. However, since serum components are known to contain human ES cell differentiation factors and the like, and the culture results may vary due to differences between serum lots, the serum content is A lower value is preferable, and most preferably no serum is contained. Further, when the cultured stem cells are used for medical purposes, it is preferable that the xenogeneic component does not contain serum since it may become an infection source of a blood-borne pathogen or a heterologous antigen. When serum is not included, serum substitute additives (eg, Knockout Serum Replacement (KSR) (Invitrogen), Chemically-defined Lipid concentrated (Gibco), Glutamax (Gibco), etc.) may be used.
  • the present invention provides a composition for promoting cell growth (hereinafter also referred to as the composition of the present invention) comprising a random polymer as an active ingredient.
  • the random polymer used in the present invention comprises a neutral monomer, an anionic monomer, and a cationic monomer, and specifically is a random polymer obtained by radical polymerization of these monomer components (hereinafter referred to as the present invention). Also referred to as a polymer).
  • the polymer of the present invention is water-soluble and can be dissolved in a culture solution.
  • the neutral monomer used in the present invention is not particularly limited as long as it can neutralize the influence of ions by the anionic monomer and the cationic monomer, which are other monomer components, and specifically, N-isopropyl.
  • Acrylamide abbreviation: NIPAm
  • NIPAm N-vinylpyrrolidone
  • VA vinyl acetate
  • TBAm t-butylacrylamide
  • PAAm phenylacrylamide
  • 2-hydroxypropyl acrylate abbreviation: HPA
  • HEA hydroxyethyl acrylate
  • two or more neutral monomer components may be used, but preferably contain at least one selected from the group consisting of NIPAm, VP and VA.
  • the neutral monomer has the following characteristics: (1) ClogP is 0.9 or less (2) A series of monomers having 2 or less hydrogen accepting groups (hereinafter also referred to as neutral monomer 1) and other monomers that do not contain ionic groups (hereinafter referred to as (Also referred to as neutral monomer 2).
  • neutral monomer 1 A series of monomers having 2 or less hydrogen accepting groups
  • Also referred to as neutral monomer 2 A series of monomers having 2 or less hydrogen accepting groups
  • Also referred to as neutral monomer 2 alpha referred to as neutral monomer 2
  • the hydrophobic nature of a compound can generally be represented by a hydrophobicity parameter, but can be represented, for example, by a partition coefficient, specifically logP.
  • logP For the calculation of logP, it is simply calculated using ClogP (predicted value obtained by software for estimating the hydrophobic parameter of a compound by a computer; for example, Corwin / Leo's program (ClogP, Daylight Chemical Information System Co., Ltd). Can be used). It means that hydrophobicity is so high that ClogP is large. If ClogP is large, that is, if the hydrophobicity is too high, the cell is toxic.
  • the hydrogen accepting group also referred to as a hydrogen bond accepting group
  • the hydrogen accepting group means a functional group having an electron accepting structure that accepts a hydrogen atom when a hydrogen bond is formed. Hydrogen accepting groups, like ClogP, are an important factor in drug development (CA Lipinski et al.
  • the number of hydrogen accepting groups can be easily calculated using chemical software such as Symyx Draw. Too many hydrogen accepting groups is not preferable because it causes the ion balance of the polymer to be lost.
  • Specific examples of the neutral monomer 2 include TBAm, PAAm, HPA, and HEA.
  • the neutral monomer component is preferably at least one neutral monomer 1 selected from the neutral monomer 1, more preferably at least one neutral monomer 1 selected from the group consisting of NIPAm, VP and VA. And optionally at least one neutral monomer 2 selected from neutral monomers 2, more preferably at least one neutral monomer 2 selected from the group consisting of TBAm, PAAm, HPA and HEA. More preferably, both neutral monomer 1 and neutral monomer 2 are included.
  • the neutral monomer 1 is preferably NIPAm.
  • the neutral monomer 2 is preferably TBAm or HPA, and more preferably HPA. When only the neutral monomer 1 is used, the neutral monomer 1 is preferably VP.
  • neutral monomer both the neutral monomer 1 and the neutral monomer 2 are included, and the amount thereof includes both the neutral monomer 1 and the neutral monomer 2 as neutral monomers. Means the total amount thereof.
  • the ratio of the neutral monomer in the polymer is not particularly limited as long as it can provide the polymer of the present invention with the desired property of neutralizing ionicity, and varies depending on the type of monomer used.
  • X mol (%) of neutral monomer is used for 100 mol (%) of polymer.
  • X satisfies the requirement of 20 ⁇ X ⁇ 65, preferably 30 ⁇ X ⁇ 60.
  • the ratio of the neutral monomer 1 and the neutral monomer 2 optionally contained in the polymer is not particularly limited as long as it can provide the desired properties to the polymer of the present invention, and varies depending on the type of monomer used.
  • neutral monomer 1 of X1 mol (%) and neutral monomer 2 of X2 mol (%) are used with respect to 100 mol (%) of the polymer.
  • X1 satisfies the requirement of X1 ⁇ 65, preferably 10 ⁇ X1 ⁇ 65.
  • X2 satisfies the requirement of 0 ⁇ X2 ⁇ 30, preferably 0 ⁇ X2 ⁇ 30. It is preferable that X1 ⁇ X2.
  • neutral monomer 1 contains at least VP and neutral monomer 2 is TBAm, it preferably satisfies the requirements of 20 ⁇ X1 ⁇ 65; 0 ⁇ X2 ⁇ 20; 20 ⁇ X1 + X2 ⁇ 65.
  • neutral monomer 1 contains at least VA and neutral monomer 2 is TBAm, it preferably satisfies the requirements of 20 ⁇ X1 ⁇ 65; 0 ⁇ X2 ⁇ 20; 20 ⁇ X1 + X2 ⁇ 65.
  • anionic monomer component examples of the anionic monomer used in the present invention include monomers having an anionic functional group.
  • the anionic functional group include a carboxylic acid group, a sulfonic acid group, a sulfuric acid group, a phosphoric acid group, and a boronic acid group.
  • Specific examples of the anionic monomer include acrylic acid (abbreviation: AAc), 2-carboxyethyl acrylate (abbreviation: CEAc), and N-methacryloylglycine. AAc is preferable.
  • the ratio of the anionic monomer in the polymer is not particularly limited as long as the desired characteristics can be provided to the polymer of the present invention, and varies depending on the type of monomer used, but is usually 100 mol (%) of the polymer. On the other hand, A mol (%) of an anionic monomer is used.
  • A satisfies the requirement of 10 ⁇ A ⁇ 40, preferably 15 ⁇ A ⁇ 40.
  • Examples of the cationic monomer used in the present invention include a monomer having a cationic functional group.
  • the cationic functional group include amino groups such as primary to quaternary amino groups, guanidine groups, and the like. Specific examples include N, N-dimethylpropylaminoacrylamide (abbreviation: DPAAAm) and 2-N-morpholinoethyl methacrylate (abbreviation: MEMA). DPAAm is preferable.
  • the proportion of the cationic monomer in the polymer is not particularly limited as long as it can provide the desired properties to the polymer of the present invention, and varies depending on the type of monomer used, but is usually 100 mol (%) of the polymer. In contrast, C mol (%) of cationic monomer is used.
  • C satisfies the requirement of 20 ⁇ C ⁇ 50, preferably 20 ⁇ C ⁇ 40.
  • the polymer of the present invention is synthesized by radical polymerization of each monomer component by adding a polymerization initiator to a solution in which each monomer component is dissolved in a predetermined solvent (see FIG. 1). A random polymer in which each monomer component is randomly introduced is generated by radical polymerization.
  • radical polymerization a known polymerization initiator such as a radical thermal polymerization initiator or a radical photopolymerization initiator can be used as the polymerization initiator.
  • a radical thermal polymerization initiator is a compound that generates radicals when heated to a temperature equal to or higher than the decomposition temperature.
  • radical thermal polymerization initiator examples include diacyl peroxide (acetyl peroxide, benzoyl peroxide, etc.), ketone peroxide (methyl ethyl ketone peroxide, cyclohexanone peroxide, etc.), hydroperoxide (hydrogen peroxide, tert-butyl hydro gen).
  • Such radical thermal polymerization initiators may be used alone or in combination of two or more.
  • the radical polymerization method is not particularly limited, and an emulsion polymerization method, a suspension polymerization method, a bulk polymerization method, a solution polymerization method, and the like can be applied.
  • a polymerization initiator is used, the amount of the polymerization initiator used is preferably 0.1 to 10% by mass relative to the mass of the monomer to be polymerized (the total when two or more monomers are used). 2 to 5% by mass is more preferable.
  • Examples of preferred solvents for radical polymerization include alcohols such as ethanol, isopropanol and butanol, ethers such as dibutyl ether, ethylene glycol dimethyl ether, tetrahydrofuran (THF) and dioxane, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone and cyclohexanone. , Esters such as ethyl acetate, butyl acetate, amyl acetate and ⁇ -butyrolactone, aromatic hydrocarbons such as benzene, toluene and xylene, dimethylacetamide, dimethylformamide, N-methylpyrrolidone and the like.
  • alcohols such as ethanol, isopropanol and butanol
  • ethers such as dibutyl ether, ethylene glycol dimethyl ether, tetrahydrofuran (THF) and dioxane
  • these organic solvents can be used individually by 1 type or in combination of 2 or more types. Furthermore, from the viewpoint of solubility of the monomer and the polymer to be produced, a water-mixed organic solvent in which water is used in combination with the above organic solvent is also applicable. In the case of emulsion polymerization, an emulsifier such as sodium dodecyl sulfate is mixed.
  • the radical polymerization temperature is preferably in the range of 30 ° C to 170 ° C, more preferably in the range of 40 ° C to 150 ° C.
  • the polymerization reaction is preferably carried out in an inert atmosphere.
  • materials necessary for the reaction such as monomers, a solvent, a polymerization initiator, and the like are added to a sealed container, and an inert gas can be bubbled to create an inert atmosphere in the container.
  • the ratio of each monomer in the polymer of the present invention should be adjusted so that the balance between the polymer and the neutral monomer that has both cation and anion properties and neutralizes the influence of ions is optimized. Specifically, for a polymer containing 100 mol (%) of a polymer, a X mol (%) neutral monomer, an A mol (%) anionic monomer, and a C mol (%) cationic monomer, Preferably, the requirements of 20 ⁇ X ⁇ 65 and 35 ⁇ A + C ⁇ 80 are satisfied.
  • the requirements of 20 ⁇ X1 + X2 ⁇ 65 and 35 ⁇ A + C ⁇ 80 are preferably satisfied.
  • Preferred requirements of the polymer of the present invention are shown below.
  • Requirement 1 10 ⁇ X1 ⁇ 65, 0 ⁇ X2 ⁇ 30, 10 ⁇ A ⁇ 65, 20 ⁇ C ⁇ 50
  • Requirement 2 When the anionic monomer is Aac and the cationic monomer is DPAAAm, 10 ⁇ X1 ⁇ 65, 0 ⁇ X2 ⁇ 30, 10 ⁇ A ⁇ 40, 20 ⁇ C ⁇ 50
  • Requirement 3 When the anionic monomer is Aac and the cationic monomer is DPAAAm, 10 ⁇ X1 ⁇ 60, 0 ⁇ X2 ⁇ 30, 15 ⁇ A ⁇ 40, 20 ⁇ C ⁇ 40
  • Requirement 4 When the neutral monomer 1 is NIPAm and the optionally included neutral monomer 2 is TBAm (corresponding to the NIPAm-containing polymer in Examples described later), 20 ⁇ X1 + X2 ⁇ 60, 10 ⁇ X1 ⁇ 60, 0 ⁇ X2 ⁇ 20, 15 ⁇ A
  • the monomer ratio of neutral monomer 1 (NIPAm), neutral monomer 2 (HPA), anionic monomer (Aac) and cationic monomer (DPAAm) is 4: 2: 1.5: 2.5.
  • a random polymer (HPA-containing polymer 2 described in Examples) Preferably, the polymer satisfying the requirement 6 includes a random polymer in which the neutral monomer 1 is VP, the neutral monomer 2 is not included, the anionic monomer is Aac, and the cationic monomer is DPAAAm.
  • a random polymer in which the monomer ratio of the neutral monomer (VP), the anionic monomer (Aac), and the cationic monomer (DPAAm) is 5: 2: 3 can be used.
  • the molecular weight of the polymer of the present invention is not particularly limited, but varies depending on the constituent monomer components, and the acrylic polymer tends to have a large molecular weight, whereas the vinyl (VA, VP) polymer has a small molecular weight.
  • the number average molecular weight (Mn) is preferably in the range of 1000 to 50000, more preferably in the range of 3000 to 40000, and in the range of 4000 to 30000 in consideration of solubility in the culture medium and influence on the cells. Is more preferable.
  • the weight average molecular weight (Mw) is preferably in the range of 3000 to 100,000, more preferably in the range of 5000 to 80000, and still more preferably in the range of 10,000 to 60000.
  • the ratio of Mw to Mn (Mw / Mn, polydispersity) is preferably in the range of 1.0 to 3.5, more preferably in the range of 1.0 to 3.0.
  • Mw / Mn 1, it means that the obtained polymer has a uniform molecular length. That is, the larger the Mw / Mn value, the more uneven the molecular length, which may have an unintended adverse effect on the culture environment.
  • the molecular weight can be measured by gel permeation chromatography (GPC), and specifically can be measured and calculated by the method described in the examples.
  • the structure of the polymer of the present invention is not particularly limited, and may be linear or non-linear (eg, branched) selected depending on the monomer component and polymerization method used.
  • a polymer having a linear structure is preferable.
  • the polymer of the present invention has a chelate-forming ability as shown in Examples described later.
  • the chelate-forming ability is the ability to form a complex by coordination with a metal ion. In the present invention, for example, this corresponds to the ability to form a chelate with an inorganic salt in the medium.
  • the chelate rate varies depending on the configuration of the polymer and the type of metal ion to be coordinated, and is not particularly limited. In the case of forming a chelate with zinc ions, a chelate rate of about 20% or more is exemplified.
  • composition of the present invention can be in any shape when provided or stored.
  • the composition can be formulated solids such as tablets, pills, capsules, granules, or liquid dissolved in a suitable solvent, or bound to a substrate or carrier.
  • a necessary amount of the composition of the present invention containing the polymer of the present invention is added to a basal medium or the like.
  • the amount of the composition of the present invention added to the basal medium is appropriately selected depending on the desired effect and the type of polymer to be included, but is usually added to the basal medium for growth culture of stem cells (particularly iPS cells).
  • the amount of polymer as an active ingredient is usually added so that the final concentration is 0.001 to 10 mg / ml, preferably 0.002 to 10 mg / ml, more preferably 0.002 to 5 mg / ml. Is done.
  • composition of the present invention may contain various factors that are preferably added to the medium in addition to the polymer that is an active ingredient.
  • the additives known per se included in the medium of the present invention exemplified above can be included.
  • the present invention is a growth promoting medium for cells, stem cells, pluripotent stem cells, particularly iPS cells, and contains a random polymer comprising a neutral monomer, an anionic monomer and a cationic monomer ( Hereinafter, the medium of the present invention is also provided).
  • the growth promoting medium is a medium that enables replication (ie, proliferation) of the cells while maintaining the replication ability, pluripotency, and unipotency of the stem cells.
  • the medium of the present invention can be suitably used for the growth of any cell, but is preferably a stem cell, particularly a pluripotent stem cell, preferably an ES cell or iPS cell, more preferably an iPS cell.
  • a stem cell particularly a pluripotent stem cell, preferably an ES cell or iPS cell, more preferably an iPS cell.
  • the culture medium of the present invention can be suitably used for the growth of cells derived from any animal.
  • Cells that can be cultured using the medium of the present invention include, for example, rodents such as mice, rats, hamsters and guinea pigs, rabbit eyes such as rabbits, ungulates such as pigs, cows, goats, horses and sheep, Cells derived from cats such as dogs and cats, humans, monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, and the like, preferably human-derived cells.
  • the random polymer used in the culture medium of the present invention has the same meaning as that used in the above “1. Composition of the present invention”.
  • the content of the polymer in the medium is not particularly limited as long as it is an amount that can be usually added to the cell culture medium, and varies depending on the type of polymer used. It is added to the basal medium for cell culture so as to be 001 to 10 mg / ml, preferably 0.002 to 10 mg / ml, more preferably 0.002 to 5 mg / ml.
  • the present invention provides a method of culturing stem cells (hereinafter also referred to as the culture method of the present invention).
  • the culture method of the present invention includes a step of culturing cells, particularly stem cells (preferably iPS cells), in the medium of the present invention (see the section of “2. Medium of the present invention” above).
  • the incubator used for culturing cells is not particularly limited as long as cells can be cultured. Flask, tissue culture flask, dish, petri dish, tissue culture dish, multi-dish, microplate, microwell Plates, multiplates, multiwell plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, and roller bottles may be mentioned.
  • the incubator may be cell-adhesive or non-cell-adhesive and is appropriately selected according to the purpose.
  • the cell-adhesive incubator can be coated with any cell-supporting substrate such as an extracellular matrix (ECM) for the purpose of improving adhesion with cells on the surface of the incubator.
  • ECM extracellular matrix
  • the cell support matrix can be any substance intended to adhere cells or feeder cells (if used).
  • the culture temperature is not particularly limited, but may be about 30-40 ° C., preferably about 37 ° C.
  • the CO 2 concentration can be about 1-10%, preferably about 2-5%.
  • the oxygen partial pressure can be 1-10%.
  • the timing of cell culture using the medium of the present invention or the addition of the composition of the present invention to the cell culture is not particularly limited as long as the desired effect of promoting stem cell proliferation can be obtained.
  • stem cells may be seeded in the medium of the present invention, or stem cells are seeded in a growth medium not containing a polymer and cultured for 1 to several days, preferably 1 day, and then cultured on the medium.
  • Additives may be added.
  • the medium may be replaced with the medium of the present invention.
  • IPS cells grown by the culture method of the present invention maintain undifferentiated properties.
  • a method for confirming whether iPS cells have undifferentiated properties there is a method for confirming using an undifferentiated marker as an index.
  • undifferentiated markers include alkaline phosphatase, Oct3 / 4, Sox2, Nanog, ERas, Esgl and the like.
  • Examples of a method for detecting these undifferentiated markers include a method for detecting mRNA, an immunological detection method, and the like.
  • Example 1 Polymer synthesis After adding NIPAm (1.36g) and TBAm (0.38g) to an eggplant flask, THF (15mL), acrylic acid (0.41mL), DPAAAm (1.42mL), AIBN (12mg) were added. It was. The upper mouth was closed with a septum, and a three-way cock coated with grease on the top of the connection with the eggplant flask was attached to the eggplant flask. A syringe was inserted into the portion closed with a septum, and the tip of the syringe was immersed in the solution, and then bubbled with argon gas for 30 minutes.
  • the reaction solution was returned to room temperature, and MilliQ water in the same amount as THF was added to dissolve the precipitate as much as possible. Reduce the amount of solvent to about half by using an evaporator, put the concentrated solution obtained by removing THF into a dialysis membrane tube (Spectra Por, MWCO: 8000), and dialyze overnight with an aqueous NaOH solution adjusted to pH 11 or more. After that, the external solution was exchanged with water, and dialysis was performed for two days while changing the water every day.
  • a dialysis membrane tube Spectra Por, MWCO: 8000
  • GPC analysis A PBS solution of each polymer was prepared at 2 mg / mL and filtered through a 0.45 ⁇ m filter to prepare a sample for analysis. The analysis was performed using Shodex GF-7B, GF-310, and GF-510 columns at a column temperature of 30 ° C., a flow rate of 0.6 mL / min, and a cycle time of 70 minutes. Samples were injected at 50 ⁇ L. The molecular weight was calculated by a calibration curve method using polyethylene glycol / polyethylene oxide standard (Sigma-Aldrich, 02393-1EA) as a standard product. A GPC analysis is performed on the NIPAm-containing polymer 12 and the NIPAm-containing polymer 23, and the resulting chart is shown in FIG.
  • Example 3 Screening evaluation (cell proliferation activity) As the iPS cell, 201B7 strain purchased from iPS Academia Japan was used. Cell culture was performed under the conditions of 5% CO 2 and 37 ° C. using a culture vessel (24-well cell culture plate manufactured by Corning) coated with vitronectin (Life Technologies). Each polymer (prepared as an aqueous solution: test compound) synthesized in Example 1 was added to Essential8 medium (manufactured by Invitrogen) at a concentration of 0.006 mg / mL to 0.06 mg / mL to prepare a test medium. The effect was examined by using this for culture immediately or after storing it at 4 ° C. for 2 to 4 weeks.
  • Essential8 medium manufactured by Invitrogen
  • the iPS cell suspension was seeded at 600 cells / well.
  • Y-27632 final concentration 10 ⁇ M, manufactured by Nacalai Tesque: 08945-84 was added to the medium used at the time of seeding.
  • the medium was replaced with a test medium to which Y-27632 was not added.
  • the phase of the adhered cells was photographed using a fluorescence microscope BZ-X700 (Keyence Co., Ltd.) in accordance with the instruction manual for 9 visual fields / well (area 0.57 cm 2 ).
  • the number of colonies in each well was measured using the analysis software attached to the apparatus, and compared with the number of colonies when the same culture was performed without adding the polymer.
  • the activity intensity is expressed as A in which colonies of 2 times or more are formed relative to the test compound non-added group, B in which colonies of 1.2 times or more are formed, and C less than 1.2 times expressed as C, The addition concentration of the test compound is also shown. The results are shown in Tables 8-13.
  • the absolute value of the zeta potential is preferably 10 or less, more preferably 5 or less. In the case of hydroxyl group-containing polymers such as VA and HPA, examples of lower values were observed. The results are shown in Tables 8-13.
  • Example 5 Measurement of Critical Micelle Concentration (CMC)
  • CMC Critical Micelle Concentration
  • a micelle is formed by a certain number of molecules gathering in an aqueous solution by orienting a hydrophilic group portion toward the aqueous phase and a hydrophobic group portion facing inward. It is an aggregate of water-soluble polymer molecules.
  • the “micelle” includes colloidal aggregates called pseudo-micelles (eg, Revue Roumaine de Chimie, 54, 577-581, 2009) in addition to such narrowly defined micelles. Is done.
  • CMC is too low, there is a concern that surfactants contained in the medium may be agglomerated due to its extremely high micelle formation ability, and if it is too high, it is necessary to add a large amount for micelle formation. In this case, there is a concern that the viscosity or osmotic pressure of the medium increases and affects the cultured cells (becomes cytotoxic).
  • Measurement of CMC can be carried out using a method commonly used in this field or a method equivalent thereto, for example, using an electric conduction method, a viscosity method, a dye method, a surface tension method, a light scattering method, or the like. Can be measured.
  • a method of calculating by measuring fluorescence intensity using a fluorescent substance pyrene is also used.
  • the adsorption capability of a hydrophobic substance was measured by applying a critical micelle concentration (CMC) measurement method by the method described in Journal of controlled release 2010, 143, 201-206 using a fluorescent substance pyrene.
  • CMC critical micelle concentration
  • Example 6 Example of cell evaluation (screening on different scaffolds)
  • iPS cell 201B7 strain purchased from iPS Academia Japan was used.
  • vitronectin Life Technologies
  • Matrigel Becton Dickinson
  • iMatrix Nippi coated culture vessel
  • An aqueous test compound solution was added to Essential8 medium (manufactured by Inbiogen) or StemFit AK02N medium (manufactured by Ajinomoto Co., Inc.) to a predetermined concentration to prepare a test medium.
  • Essential8 medium manufactured by Inbiogen
  • StemFit AK02N medium manufactured by Ajinomoto Co., Inc.
  • Example 7 Cell evaluation example (7-day culture evaluation) As the iPS cell, 201B7 strain purchased from iPS Academia Japan was used. Cell culture, using vitronectin culture vessel coated with (Life Technologies) (Japan vector Bok emissions Dickinson 6-well cell culture plate) was carried out under the conditions of 5% CO 2/37 °C. A test compound aqueous solution was added to Essential8 medium (manufactured by Invitrogen) so as to have a predetermined concentration to obtain a test medium, and the effect was examined by using it in culture. Y-27632 was added to the medium used at the time of seeding (final concentration 10 ⁇ M, manufactured by Nacalai Tesque: 08945-84).
  • Essential8 medium manufactured by Invitrogen
  • the cells were cultured in a test medium to which Y-27632 was not added.
  • the medium was changed every 2-3 days, and the number of cells was measured after 6 days of culture.
  • the number of cells was measured by the method described in “Revised Cell Culture Introduction No. ⁇ , pp. 77-83, 2010, Yodosha”, and the number of cells obtained when the same culture was performed without adding the test compound. Compared with. The results are shown in FIG. When the polymer of the present invention was added, a significant cell growth promoting effect was observed.
  • the cell growth promoting action of the polymer of the present invention may depend on chelate formation with an inorganic salt in the medium.
  • stem cells can be efficiently propagated. Therefore, the frequency of medium exchange during culture can be lowered, and the culture cost of stem cells can be reduced.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)

Abstract

La présente invention concerne : une composition stimulant la croissance cellulaire dans laquelle le principe actif est un polymère aléatoire comprenant un monomère neutre, un monomère anionique et un monomère cationique ; et un milieu de croissance cellulaire qui contient le polymère aléatoire. La présente invention permet d'obtenir un milieu destiné à la culture de cellules bien établies, en particulier de cellules souches pluripotentes telles que des cellules iPS, à l'aide de matériaux qui sont moins chers et qui peuvent être fournis en grandes quantités, et permet également d'obtenir une composition stimulant la croissance cellulaire pour produire le milieu de culture.
PCT/JP2019/014022 2018-03-30 2019-03-29 Composition pour croissance cellulaire Ceased WO2019189769A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2018-069850 2018-03-30
JP2018069850 2018-03-30

Publications (1)

Publication Number Publication Date
WO2019189769A1 true WO2019189769A1 (fr) 2019-10-03

Family

ID=68061973

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/014022 Ceased WO2019189769A1 (fr) 2018-03-30 2019-03-29 Composition pour croissance cellulaire

Country Status (1)

Country Link
WO (1) WO2019189769A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6379587A (ja) * 1986-09-22 1988-04-09 Saburo Senoo 細胞培養用基材
JPH06335382A (ja) * 1993-03-31 1994-12-06 Iatron Lab Inc 細胞培養基材及び細胞培養方法
US6103528A (en) * 1998-04-17 2000-08-15 Battelle Memorial Institute Reversible gelling culture media for in-vitro cell culture in three-dimensional matrices
JP2002541113A (ja) * 1999-04-02 2002-12-03 ザ・ブリガーム・アンド・ウーメンズ・ホスピタル・インコーポレーテッド 免疫調節ポリマー
WO2012172291A1 (fr) * 2011-06-14 2012-12-20 The University Court Of The University Of Edinburgh Croissance de cellules
JP2014180255A (ja) * 2013-03-21 2014-09-29 Kinki Univ 細胞処理用基板

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6379587A (ja) * 1986-09-22 1988-04-09 Saburo Senoo 細胞培養用基材
JPH06335382A (ja) * 1993-03-31 1994-12-06 Iatron Lab Inc 細胞培養基材及び細胞培養方法
US6103528A (en) * 1998-04-17 2000-08-15 Battelle Memorial Institute Reversible gelling culture media for in-vitro cell culture in three-dimensional matrices
JP2002541113A (ja) * 1999-04-02 2002-12-03 ザ・ブリガーム・アンド・ウーメンズ・ホスピタル・インコーポレーテッド 免疫調節ポリマー
WO2012172291A1 (fr) * 2011-06-14 2012-12-20 The University Court Of The University Of Edinburgh Croissance de cellules
JP2014180255A (ja) * 2013-03-21 2014-09-29 Kinki Univ 細胞処理用基板

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SCHARNAGL N. ET AL.: "Behaviour of fibroblasts on water born acrylonitrile-based copolymers containing different cationic and anionic moieties", CLIN. HEMORHEOL. MICROCIRC., vol. 52, no. 2-4, 2012, pages 295 - 311, XP055638480, Retrieved from the Internet <URL:https://content.iospress.com/articles/clinical-hemorheology-and-microcirculation/ch1606> *

Similar Documents

Publication Publication Date Title
Yin et al. Comparison of senescence-related changes between three-and two-dimensional cultured adipose-derived mesenchymal stem cells
JP6596381B2 (ja) 幹細胞由来心筋細胞を培養するための合成表面
US8426176B2 (en) Synthetic microcarriers for culturing cells
EP2391709B1 (fr) Surfaces de (méth)acrylate pouvant gonfler pour la culture de cellules dans des milieux chimiquement définis
EP2247747B1 (fr) Méthode pour la fabrication d&#39;un article pour culture cellulaire
CN104640972B (zh) 释放介质
Oda et al. Efficient differentiation of stem cells encapsulated in a cytocompatible phospholipid polymer hydrogel with tunable physical properties
US10457911B2 (en) Medium for stem cell use
Zhao et al. Sphingosine-1-phosphate promotes the differentiation of human umbilical cord mesenchymal stem cells into cardiomyocytes under the designated culturing conditions
US20210355424A1 (en) Cell culture substrate, method for producing cell culture substrate, and method for producing spheroids
EP3541928A1 (fr) Induction d&#39;hépatocytes par différenciation de cellules souches avec l&#39;arn
EP2958987B1 (fr) Prodédé de stockage ou de conservation de cellules souches humaines vivantes en stase
WO2019189769A1 (fr) Composition pour croissance cellulaire
JP2022008203A (ja) 細胞用培地添加剤およびその利用
Ulker et al. Thermoresponsive Poly (N, N′-dimethylacrylamide)-based diblock copolymer worm gels via RAFT solution polymerization: synthesis, characterization, and cell biology applications
WO2018094114A2 (fr) Induction de cellules bêta pancréatiques par différenciation de cellules souches avec de l&#39;arn
CN115558634B (zh) 动态交联水凝胶培养细胞的应用
JP6286947B2 (ja) 細胞凝集塊の製造方法、及び薬剤のスクリーニング方法
US12378341B2 (en) Tuneable cell substrates
US20170191026A1 (en) Cell culture substrate
JP7226634B1 (ja) 生体適合性ポリマー組成物及びその用途
JP6183070B2 (ja) 細胞凝集塊の製造方法、及び薬剤のスクリーニング方法
JP6286948B2 (ja) 細胞凝集塊の製造方法、及び薬剤のスクリーニング方法
Samsudin et al. Proliferation of rat amniotic stem cell (AFSC) on modified surface microcarrier
US20230113074A1 (en) Derivation of hepatocytes and hematopoietic progenitors from human embryonic stem cells

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: 19777563

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 19777563

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP