EP2925862A1 - Serum-free medium for human mesenchymal stem cells - Google Patents
Serum-free medium for human mesenchymal stem cellsInfo
- Publication number
- EP2925862A1 EP2925862A1 EP13795440.0A EP13795440A EP2925862A1 EP 2925862 A1 EP2925862 A1 EP 2925862A1 EP 13795440 A EP13795440 A EP 13795440A EP 2925862 A1 EP2925862 A1 EP 2925862A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- human
- components
- growth medium
- salt
- pdgf
- 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.)
- Withdrawn
Links
- 210000002901 mesenchymal stem cell Anatomy 0.000 title description 4
- 239000012679 serum free medium Substances 0.000 title description 2
- 239000001963 growth medium Substances 0.000 claims abstract description 60
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- 102000008100 Human Serum Albumin Human genes 0.000 claims abstract description 23
- YASAKCUCGLMORW-UHFFFAOYSA-N Rosiglitazone Chemical compound C=1C=CC=NC=1N(C)CCOC(C=C1)=CC=C1CC1SC(=O)NC1=O YASAKCUCGLMORW-UHFFFAOYSA-N 0.000 claims abstract description 18
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 claims abstract description 17
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims abstract description 17
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- 229910052711 selenium Inorganic materials 0.000 claims abstract description 17
- 239000011669 selenium Substances 0.000 claims abstract description 17
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- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims abstract description 10
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- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 13
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- MIJPAVRNWPDMOR-ZAFYKAAXSA-N L-ascorbic acid 2-phosphate Chemical compound OC[C@H](O)[C@H]1OC(=O)C(OP(O)(O)=O)=C1O MIJPAVRNWPDMOR-ZAFYKAAXSA-N 0.000 claims description 11
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- 102000009618 Transforming Growth Factors Human genes 0.000 claims description 8
- 108010009583 Transforming Growth Factors Proteins 0.000 claims description 8
- 108010081589 Becaplermin Proteins 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 150000002632 lipids Chemical class 0.000 claims description 7
- 230000002293 adipogenic effect Effects 0.000 claims description 6
- 230000002648 chondrogenic effect Effects 0.000 claims description 5
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- 108010049974 Bone Morphogenetic Protein 6 Proteins 0.000 claims description 4
- 102100022525 Bone morphogenetic protein 6 Human genes 0.000 claims description 4
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N Glutamine Chemical compound OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 4
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 claims description 4
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 150000000996 L-ascorbic acids Chemical class 0.000 claims description 3
- -1 PDGF Proteins 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000000034 method Methods 0.000 claims description 3
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 claims description 2
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 claims description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 2
- 229940054269 sodium pyruvate Drugs 0.000 claims description 2
- 159000000000 sodium salts Chemical group 0.000 claims description 2
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 claims 5
- 210000004027 cell Anatomy 0.000 description 23
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- 102000012422 Collagen Type I Human genes 0.000 description 2
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- 239000006143 cell culture medium Substances 0.000 description 2
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- MAZUXGSAOIEIIG-UHFFFAOYSA-N 2-hydroxyimino-1-pyridin-3-ylethanone Chemical compound ON=CC(=O)C1=CC=CN=C1 MAZUXGSAOIEIIG-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
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- 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
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- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/98—Xeno-free medium and culture conditions
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- C12N2501/10—Growth factors
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- C12N2501/15—Transforming growth factor beta (TGF-β)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2501/30—Hormones
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- C12N2501/395—Thyroid hormones
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
-
- 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
- C12N2533/54—Collagen; Gelatin
Definitions
- the present invention relates to the field of artificial substrates for the growth and maintenance of human cells.
- hMSCs human mesenchymal stem cells
- Cell culture media are widely used in the industrial production of biologically active products, such as hormones, enzymes, antigens, antibodies, etc.
- Cell growth media are usually a mixture of cell nutrients of organic and inorganic origin, dispersed or dissolved in a liquid or a semisolid system capable to support the growth of biological organisms.
- hMSCs Human mesenchymal stem cells
- hMSCs require especially adapted growth media: these must preserve/support the capacity of MSCs to differentiate into specific target tissues (vascular, neural, lipid, osseous, cartilage, etc.), and such capacity is quite sensitive to changes in the medium composition.
- the fine tuning of the media is also important in view of the fact that MSCs are used in clinical trials and will be used in the future as advanced cell therapy products (ACTPs), for delicate therapeutic/cosmetic applications, such preparation of cell suspensions /tissues/organs to be implanted in patients.
- ACTPs advanced cell therapy products
- the MSC growth media should also be free of toxic components which could contaminate the stem cell culture and be transferred to the patient: in particular, the growth media should be free of ingredients of animal origin (often present in standard cell growth media) such as serum.
- Serum is a universal nutrient for the growth and maintenance of mammalian cells; growth media usually contain up to about 10% of animal serum, such as fetal bovine serum; although widely used, serum has the drawback of containing possible contaminants, e.g. virus such as BSE (Bovine Spongiform Encephalopathy), a transmissible neurodegenerative disease of cattle with a long latency or incubation period; this has raised regulatory concerns about using animal-derived sera in the production of biologically active products.
- BSE Bovine Spongiform Encephalopathy
- the present inventors have now obtained a group of highly effective serum-free growth media, especially adapted for MSCs and their subtypes, characterized by comprising, dissolved and/or suspended in a basis cell growth medium:
- additives selected from: a strontium salt, vitamin D, rosiglitazone, a
- the invention extends to the method of preparing the above medium, and its use in culturing human MSCs. DESCRIPTION OF THE FIGURES
- Figure 1 microscope images of human MSCs cultured in a growth medium in accordance with the present invention. Cells were photographed at two different confluences, with 4X, 10X or 20X magnifications.
- FIG. 2 Example of seeded and cultured SVF cells. A) 72 hours after seeding adherent cells can be observed. B) After washing the same cells reach the semi-confluence. C) Re-seeded cells arrange themselves forming so called pseudo-islands. D) In some instances cells can form spheroids or atypical groupings.
- the basis cell growth medium is preferably a liquid system, i.e. a solution and/or suspension of different components; semi-solid media like gels and similar can also be used.
- the basis cell growth medium can be widely chosen among known standard cell culture media; these contain mixtures of nutrients such as inorganic salts, aminoacids, vitamins; preferred basis media are the commercially available products DMEM (Low- or High Glucose Dulbecco Modified Eagle Medium), HAM's F-12, IMDM (Iscove's Modified Dulbecco's Medium), and mixtures thereof preferaby in 1 :1 ratio; among the mixtures, especially preferred are: HAM's F12/IMDM and HAM's F12/DMEM.
- the human albumin is preferably an injectable human albumin;
- the ascorbic acid salt is preferably a phosphate salt, e.g ascorbic acid-2-phosphate salt;
- the pyruvic acid salt is preferably a sodium salt.
- the human growth factors are preferably selected from bFGF (basic fibroblast growth factor), PDGF (platelet-derived growth factor, e.g. PDGF-AB, PDGF-BB), TGF (transforming growth factor, e.g. TGF- ⁇ , TGF- 3), EGF (epidermal growth factor).
- bFGF basic fibroblast growth factor
- PDGF platelet-derived growth factor, e.g. PDGF-AB, PDGF-BB
- TGF transforming growth factor, e.g. TGF- ⁇ , TGF- 3
- EGF epidermal growth factor
- the prostaglandin is preferably selected from PGE 2 ;
- the BMP protein is preferably BMP-6.
- MSCs of lipid tissue source or for adipogenic, osteogenic or chondrogenic induction
- growth media adapted for MSCs of lipid tissue source should preferably comprise the components (a) combined with the human growth factors (b').
- growth media adapted for MSCs of lipid tissue source preferably include: bFGF, PDGF, TGF.
- a particularly preferred composition for MSCs of lipid tissue source is the following:
- the composition comprises the following.
- a growth medium comprising, in a HAM's F12/IMDM 1 :1 basic growth medium, the following:
- TGF- ⁇ (0.6 ng/ml).
- a particularly preferred composition is the following:
- 2-Phospho-L-ascorbic acid trisodium salt (Sigma 49752), MW 322.05 g/mol. Dissolve 160 mg in 10 ml of culture medium.
- a passage of the cells shall be carried out: remove the surnatant and wash with PBS, apply a minimum dose of trypsin (TrypLE) and incubate at 37°C for 5 min; check cell detachment; dilute in SF and re-seed the cells at 15 ⁇ 00 cells/cm2 in 50% SF and 50% centrifuged surnatant. 24 hours after re-seeding the cells will show an homogeneous appearance and will form so called pseudo-islands (fig. 2C).
- Trypsin Trypsin
- Growth media for adipogenic induction of MSCs preferably comprise the components (a) combined with EGF as a component (b') and with rosiglitazone as a component (b").
- a particularly preferred composition for adipogenic induction of MSCs is the following:
- composition for adipogenic induction of MSCs is the following:
- Growth media adapted for osteogenic induction of MSCs should preferably comprise the components (a) in combination with a strontium salt and/or vitamin D as a component (b").
- the components (b') are not present in this case.
- a particularly preferred composition for osteogenic induction of MSCs is the following:
- Vitamin D (10-200 nM)
- composition for osteogenetic induction of MSCs is the following:
- Vitamin D (10 nM) Growth media adapted for chondrogenic induction of MSCs should preferably comprise the components (a) (including the pyruvic acid salt and the ascorbic acid salt), in combination TGF and bFGF as components (b') and a BMP protein and a prostaglandin as components (b").
- a particularly preferred composition for chondrogenic induction of MSCs is the following:
- All the above growth media can be used as a solution/suspension, or can be coated onto a solid support (plate), as well-known in the art.
- the solution/suspension of the growth medium is supplemented with a suitable thickening agent, making the medium capable to stably adhere to the support, and then the obtained mixture is coated onto the support.
- the support is preventively coated with the thickening agent, and then the solution/suspension of the growth medium is applied onto the thus treated support; in a further alternative, the solution/suspension of the growth medium is applied to the support, and then the thickening agent is applied onto the thus treated support.
- the thickening agent is preferably collagen, more preferably human collagen, in particular human collagen I; it is preferably used in form of solution or suspension.
- an exemplified composition in accordance with the present invention was thickened with human collagen I and coated onto a solid support. After incubation, the plates were observed under the microscope to assess the growth and viability of the MSCs. The microscope pictures at different magnifications ( Figure 1 ) showed highly viable MSCs and a widespread diffusion denoting an extended growth.
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EP13795440.0A EP2925862A1 (en) | 2012-11-30 | 2013-10-30 | Serum-free medium for human mesenchymal stem cells |
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PCT/EP2012/074167 WO2014082685A1 (en) | 2012-11-30 | 2012-11-30 | Serum-free medium for human mesenchymal stem cells |
PCT/EP2013/061118 WO2014082759A2 (en) | 2012-11-30 | 2013-05-29 | Serum-free medium for human mesenchymal stem cells |
PCT/EP2013/072738 WO2014082814A1 (en) | 2012-11-30 | 2013-10-30 | Serum-free medium for human mesenchymal stem cells |
EP13795440.0A EP2925862A1 (en) | 2012-11-30 | 2013-10-30 | Serum-free medium for human mesenchymal stem cells |
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Citations (1)
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US20100015710A1 (en) * | 2008-04-25 | 2010-01-21 | Sunghoon Jung | Methods and Compositions for Isolating, Maintaining and Serially Expanding Human Mesenchymal Stem Cells |
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US20100015710A1 (en) * | 2008-04-25 | 2010-01-21 | Sunghoon Jung | Methods and Compositions for Isolating, Maintaining and Serially Expanding Human Mesenchymal Stem Cells |
Non-Patent Citations (4)
Title |
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HAACK-SORENSEN M ET AL: "Comparison of different culture conditions for human mesenchymal stromal cells for clinical stem cell therapy.", SCANDINAVIAN JOURNAL OF CLINICAL AND LABORATORY INVESTIGATION 2008, vol. 68, no. 3, 2008, pages 192 - 203, ISSN: 0036-5513 * |
PARKER A M ET AL: "Low serum and serum-free culture of multipotential human adipose stem cells.", CYTOTHERAPY 2007, vol. 9, no. 7, 2007, pages 637 - 646, ISSN: 1465-3249 * |
SCHÄFFLER ANDREAS ET AL: "Concise review: adipose tissue-derived stromal cells--basic and clinical implications for novel cell-based therapies.", STEM CELLS (DAYTON, OHIO) APR 2007, vol. 25, no. 4, April 2007 (2007-04-01), pages 818 - 827, XP009133278, ISSN: 1066-5099, DOI: doi:10.1634/stemcells.2006-0589 * |
See also references of WO2014082814A1 * |
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