EP2925862A1 - Serumfreies medium für menschliche mesenchymale stammzellen - Google Patents

Serumfreies medium für menschliche mesenchymale stammzellen

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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Withdrawn
Application number
EP13795440.0A
Other languages
English (en)
French (fr)
Inventor
Gianni Soldati
Tiziano Tallone
Luca MARIOTTA
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.)
SWISS STEM CELL FOUNDATION
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SWISS STEM CELL FOUNDATION
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
Priority claimed from PCT/EP2012/074167 external-priority patent/WO2014082685A1/en
Application filed by SWISS STEM CELL FOUNDATION filed Critical SWISS STEM CELL FOUNDATION
Priority to EP13795440.0A priority Critical patent/EP2925862A1/de
Publication of EP2925862A1 publication Critical patent/EP2925862A1/de
Withdrawn legal-status Critical Current

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0652Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662Stem cells
    • C12N5/0667Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00Specific components of cell culture medium
    • C12N2500/05Inorganic components
    • C12N2500/10Metals; Metal chelators
    • C12N2500/12Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • CCHEMISTRY; METALLURGY
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    • C12N2500/00Specific components of cell culture medium
    • C12N2500/05Inorganic components
    • C12N2500/10Metals; Metal chelators
    • C12N2500/20Transition metals
    • C12N2500/24Iron; Fe chelators; Transferrin
    • C12N2500/25Insulin-transferrin; Insulin-transferrin-selenium
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    • C12N2500/00Specific components of cell culture medium
    • C12N2500/30Organic components
    • C12N2500/38Vitamins
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00Specific components of cell culture medium
    • C12N2500/90Serum-free medium, which may still contain naturally-sourced components
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00Specific components of cell culture medium
    • C12N2500/98Xeno-free medium and culture conditions
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/02Compounds of the arachidonic acid pathway, e.g. prostaglandins, leukotrienes
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10Growth factors
    • C12N2501/11Epidermal growth factor [EGF]
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10Growth factors
    • C12N2501/115Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10Growth factors
    • C12N2501/135Platelet-derived growth factor [PDGF]
    • CCHEMISTRY; METALLURGY
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10Growth factors
    • C12N2501/15Transforming growth factor beta (TGF-β)
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30Hormones
    • C12N2501/33Insulin
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30Hormones
    • C12N2501/38Hormones with nuclear receptors
    • C12N2501/395Thyroid hormones
    • CCHEMISTRY; METALLURGY
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    • C12N2501/00Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998Proteins not provided for elsewhere
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00Supports or coatings for cell culture, characterised by material
    • C12N2533/50Proteins
    • C12N2533/54Collagen; 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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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Rheumatology (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)
EP13795440.0A 2012-11-30 2013-10-30 Serumfreies medium für menschliche mesenchymale stammzellen Withdrawn EP2925862A1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP13795440.0A EP2925862A1 (de) 2012-11-30 2013-10-30 Serumfreies medium für menschliche mesenchymale stammzellen

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
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 (de) 2012-11-30 2013-10-30 Serumfreies medium für menschliche mesenchymale stammzellen

Publications (1)

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EP2925862A1 true EP2925862A1 (de) 2015-10-07

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EP13795440.0A Withdrawn EP2925862A1 (de) 2012-11-30 2013-10-30 Serumfreies medium für menschliche mesenchymale stammzellen

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EP (1) EP2925862A1 (de)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100015710A1 (en) * 2008-04-25 2010-01-21 Sunghoon Jung Methods and Compositions for Isolating, Maintaining and Serially Expanding Human Mesenchymal Stem Cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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)

* Cited by examiner, † Cited by third party
Title
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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