NZ616977B - Culture medium for human mesenchymal stem cells - Google Patents
Culture medium for human mesenchymal stem cellsInfo
- Publication number
- NZ616977B NZ616977B NZ616977A NZ61697713A NZ616977B NZ 616977 B NZ616977 B NZ 616977B NZ 616977 A NZ616977 A NZ 616977A NZ 61697713 A NZ61697713 A NZ 61697713A NZ 616977 B NZ616977 B NZ 616977B
- Authority
- NZ
- New Zealand
- Prior art keywords
- culture medium
- stem cells
- mesenchymal stem
- human mesenchymal
- human
- Prior art date
Links
- 239000001963 growth media Substances 0.000 title claims abstract description 66
- 210000002901 Mesenchymal Stem Cells Anatomy 0.000 title claims abstract description 27
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 20
- 102000003974 Fibroblast Growth Factor 2 Human genes 0.000 claims abstract description 15
- 108090000379 Fibroblast Growth Factor 2 Proteins 0.000 claims abstract description 15
- 210000001772 Blood Platelets Anatomy 0.000 claims abstract description 11
- 210000002865 immune cell Anatomy 0.000 claims abstract description 11
- 102000004877 Insulin Human genes 0.000 claims abstract description 10
- 108090001061 Insulin Proteins 0.000 claims abstract description 10
- 239000007640 basal medium Substances 0.000 claims abstract description 10
- HZAXFHJVJLSVMW-UHFFFAOYSA-N ethanolamine Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000006166 lysate Substances 0.000 claims abstract description 9
- 210000002381 Plasma Anatomy 0.000 claims abstract description 8
- BVTBRVFYZUCAKH-UHFFFAOYSA-L Sodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims abstract description 8
- 239000012592 cell culture supplement Substances 0.000 claims abstract description 7
- 229960001471 sodium selenite Drugs 0.000 claims abstract description 7
- 239000011781 sodium selenite Substances 0.000 claims abstract description 7
- 235000015921 sodium selenite Nutrition 0.000 claims abstract description 7
- 239000012091 fetal bovine serum Substances 0.000 claims abstract description 4
- 239000002609 media Substances 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 7
- 210000002966 Serum Anatomy 0.000 claims description 6
- 230000003115 biocidal Effects 0.000 claims description 6
- 241000283690 Bos taurus Species 0.000 claims description 4
- 229940076788 Pyruvate Drugs 0.000 claims description 4
- 238000004113 cell culture Methods 0.000 claims description 4
- LCTONWCANYUPML-UHFFFAOYSA-M pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 claims description 4
- 238000000338 in vitro Methods 0.000 claims description 3
- 239000007759 RPMI Media 1640 Substances 0.000 claims description 2
- 239000003242 anti bacterial agent Substances 0.000 claims description 2
- 210000004271 bone marrow stromal cells Anatomy 0.000 claims description 2
- 210000000130 stem cell Anatomy 0.000 claims 2
- 229940009444 Amphotericin Drugs 0.000 claims 1
- APKFDSVGJQXUKY-INPOYWNPSA-N BRL-49594 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims 1
- 239000006143 cell culture media Substances 0.000 claims 1
- 210000004027 cells Anatomy 0.000 abstract description 21
- 230000012010 growth Effects 0.000 description 5
- 230000010261 cell growth Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 210000000577 Adipose Tissue Anatomy 0.000 description 2
- 210000004369 Blood Anatomy 0.000 description 2
- 101700033006 EGF Proteins 0.000 description 2
- 102100010813 EGF Human genes 0.000 description 2
- 229940116977 Epidermal Growth Factor Drugs 0.000 description 2
- 229940091258 Selenium supplements Drugs 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- BUGBHKTXTAQXES-UHFFFAOYSA-N selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 2
- 239000011669 selenium Substances 0.000 description 2
- 229910052711 selenium Inorganic materials 0.000 description 2
- 210000001519 tissues Anatomy 0.000 description 2
- 206010003816 Autoimmune disease Diseases 0.000 description 1
- 210000001185 Bone Marrow Anatomy 0.000 description 1
- 210000000988 Bone and Bones Anatomy 0.000 description 1
- 210000000845 Cartilage Anatomy 0.000 description 1
- 210000000170 Cell Membrane Anatomy 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010018651 Graft versus host disease Diseases 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 210000000987 Immune System Anatomy 0.000 description 1
- 210000004698 Lymphocytes Anatomy 0.000 description 1
- 210000003205 Muscles Anatomy 0.000 description 1
- 210000001672 Ovary Anatomy 0.000 description 1
- 210000002435 Tendons Anatomy 0.000 description 1
- 102000009618 Transforming Growth Factors Human genes 0.000 description 1
- 108010009583 Transforming Growth Factors Proteins 0.000 description 1
- 210000003954 Umbilical Cord Anatomy 0.000 description 1
- 229940029983 VITAMINS Drugs 0.000 description 1
- 229940021016 Vitamin IV solution additives Drugs 0.000 description 1
- 230000003110 anti-inflammatory Effects 0.000 description 1
- 230000001028 anti-proliferant Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001419 dependent Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000002519 immonomodulatory Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000006956 minimum essential medium Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 230000001613 neoplastic Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000056 organs Anatomy 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000012090 serum-supplement Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamins Natural products 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
-
- 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
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2501/30—Hormones
- C12N2501/33—Insulin
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
- C12N2502/115—Platelets, megakaryocytes
-
- 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
-
- 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
- C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
-
- 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
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
-
- 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
- C12N5/0668—Mesenchymal stem cells from other natural sources
Abstract
Disclosed is a culture medium for human mesenchymal cells, comprising: a) basal medium suitable for the culture of mesenchymal stem cells; b) human leucocyte/platelet coat lysate (buffy coat); c) insulin; d) sodium selenite; e) ethanolamine; f) basic fibroblast growth factor (bFGF); and either g) fetal bovine serum; or h) cell culture supplement from human plasma (CCS); or both g) and h). er g) fetal bovine serum; or h) cell culture supplement from human plasma (CCS); or both g) and h).
Description
CULTURE MEDIUM FOR HUMAN MESENCHYMAL STEM CELLS
DESCRIPTION
This invention relates to a new culture medium
for human mesenchymal stem cells (hMSC). More
particularly this invention relates to a culture medium
in which hMSC lines can grow, including those which do
not grow in the culture media normally used for cells of
this type because they have adapted to specific culture
media.
hMSC are multipotent cells fundamentally
characterised by their ability to differentiate into
various mesenchymal tissues such as bone, cartilage,
tendon, muscle and adipose tissue, among others. hMSC
can be isolated from various tissues in adults,
including those which are to be found in bone marrow,
adipose tissue and the blood of the umbilical cord.
The anti-proliferative, immunomodulating and
anti-inflammatory effect of hMSC has centred attention
on these cells as potential therapeutic agents in
diseases caused by the immune system, including graft
versus host disease, rejection following the transplant
of solid organs and autoimmune diseases.
It is known that for the in vitro culture of
hMSC it is imperative to preserve their pluripotential
capacity, and for this the addition of exogenous factors
such as for example basic fibroblast growth factor
(bFGF), epidermal growth factor (EGF) and transforming
growth factor (TGFβ-1) to a normal culture media is
required. However most hMSC are dependent on a specific
culture medium and also cannot be cultured on the media
which have been conventionally used for the culture of
mesenchymal stem cells such as DMEM/F12 medium
supplemented with bovine foetal serum qualified for
mesenchymal stem cells(MSCQ).
It is an object of the present invention to go
some way towards overcoming these problems and/or to
provide the public with a useful choice.
Accordingly, this invention relates to a novel
culture medium for hMSC on which hMSC that do not grow
in other culture medium formulations can grow. The
culture medium according to this invention comprises:
a) mesenchymal stem cell basal medium;
b) human leucocyte/platelet coat (buffy
coat) lysate;
c) insulin;
d) sodium selenite;
e) ethanolamine;
f) basic fibroblast growth factor (bFGF); and
either
g) fetal bovine serum; or
h) cell culture supplement derived from human
plasma (CCS); or both g) and h).
As used in this document the term "culture
medium" relates to a nutrient solution for the
culturing, growth or proliferation of cells. The term
"cell culture" refers to cells which are maintained,
cultivated or grown in an artificial in vitro
environment.
The term “comprising” as used in this
specification and claims means “consisting at least in
part of”. When interpreting statements in this
specification, and claims which include the term
“comprising”, it is to be understood that other features
that are additional to the features prefaced by this
term in each statement or claim may also be present.
Related terms such as “comprise” and “comprised” are to
be interpreted in similar manner.
The term "basal media" or “basal medium” used
in this document refers to any medium in which human
mesenchymal cells are capable of growing when
supplemented with different supplements, which may or
may not contain serum. Basal media provide standard
inorganic salts such as zinc, iron, magnesium, calcium
and potassium, together with vitamins, glucose, a buffer
system and essential amino acids.
Preferably the basal medium normally used for
the culture of mesenchymal cells in the culture medium
according to this invention is DMEM/F12 + pyruvate,
DMEM/F12 medium being a mixture of equal parts of
Dubelcco's Modified Eagle Medium (DMEM), which is a
modification of the minimum essential medium developed
by Harry Eagle, and Ham's F12 medium, developed by Ham
for the growth of CHO (Chinese Hamster Ovary) cells,
Ham’s F10, Ham’s F12, MCDB 131, a medium developed by
Knedler and Ham as a medium with reduced serum
supplement for the growth of human cells, or RPMI 1640,
the name of which derives from the initials of the
Roswell Park Memorial Institute where it was developed
by Moore and co-workers for the culture of normal and
neoplastic lymphocytes, although when adequately
supplemented it can grow other different types of cells.
The formulation and composition of these media is widely
known and can be obtained from any producer or supplier,
such as for example Gibco (Life Technologies). More
preferably the medium is DMEM/F12 + pyruvate.
One of the components of the culture medium
for human mesenchymal cells according to this invention
is leucocyte/platelet coat lysate. The leucocyte/
platelet coat, also known as "buffy coat", is the coat
which is observed between the plasma and the red cells
after the centrifuging or sedimentation of whole blood.
It basically comprises leucocytes and platelets. The
leucocyte/platelet coat lysate (buffy coat) is present
in the culture medium according to this invention in a
quantity of between 5 and 20% (v/v), more preferably
between 7 and 15% (v/v), and most preferably 10% (v/v).
Another of the components of the culture
medium according to this invention is insulin. The
beneficial effect of insulin on cell growth is known
(Cartwright, T. y Shah, G.P Culture media. Basic cell
culture, 2nd edition, Davis, J.M. ed. 2002. Oxford
University Press, New York, USA). In the culture medium
according to this invention insulin is preferably
present in a quantity of between 2 and 20 mg/L, more
preferably between 5 and 15 mg/L and most preferably 10
mg/L.
Also another component of the culture medium
according to this invention is selenium in the form of
sodium selenite. Selenium is a trace element which is
normally provided to culture medium by serum and which
may be essential for it. Sodium selenite is present in
the culture medium in a quantity of between 0.005 and
1.730 mg/L.
Furthermore the culture medium for human
mesenchymal cells according to this invention comprises
ethanolamine. It is known that ethanolamine encourages
the construction of cell membranes. The ethanolamine in
the culture medium according to this invention is
present in a quantity of between 1 and 8 mg/L,
preferably between 2 and 5 mg/L.
Finally the culture medium for mesenchymal
cells according to this invention also comprises bFGF.
It is known that bFGF has been recommended for culturing
mesenchymal cells (Sato, J. et al. Specific cell types
and their requirements. Basic cell culture, 2nd edition,
Davis, J.M. ed. 2002. Oxford University Press, New York,
USA). The bFGF is present in the culture medium
according to this invention in a quantity of between 5
and 25 ng/mL.
Surprisingly, with the combination of
components in the culture medium according to this
invention it is possible to cultivate human mesenchymal
cell lines, including those adapted to other culture
media that are generally commercially available, which
do not grow in the culture media normally used for this
type of cells, such as for example the human mesenchymal
cells offered by Lonza or PromoCell.
Optionally the culture medium according to
this invention comprises an antibiotic. Any antibiotic
normally used in the culturing of this type of cells may
be used. Preferably this antibiotic is a mixture of
gentamycin-amphotericin. The quantity of antibiotic that
has to be used in the culture medium may easily be
determined by a person skilled in the art.
For the growth of mesenchymal stem cells it is
essential that the culture medium according to this
invention be supplemented, for example by foetal bovine
serum (FBS) or cell culture supplement derived from
human plasma (CCS), obtained, for example, as described
in US patent 8,252,590 B2. If FBS is used, a quantity of
between 10 and 20% (v/v) is considered to be sufficient.
On the other hand if CCS is used to supplement the
culture medium according to this invention, a quantity
of between 10 and 40% (v/v) may be considered
sufficient.
The culture medium for human mesenchymal stem
cells according to this invention may be in dry form or
in liquid form. For use in dry form its components may
be dissolved in a suitable liquid for the culture of
mesenchymal cells. The type of liquid in which the
components of the culture medium according to this
invention may be dissolved is known to those skilled in
the art, such as for example sterile distilled water.
This invention is described in further detail
below with reference to a number of embodiments. These
examples are however not intended to limit the scope of
this invention.
EXAMPLES
Example 1.
A culture medium according to this invention
containing DMEM/F12 + pyruvate as basal medium, 10%
(v/v) human leucocyte/platelet coat lysate (buffy coat),
10 mg/L of insulin, 0.0067 mg/L of sodium selenite, 2
mg/L of ethanolamine and 20 ng/mL of bFGF was prepared.
This medium was referred to as LV2. Medium LV2 was used
alone or supplemented with 10% (v/v) of foetal bovine
serum (FBS) qualified for mesenchymal stem cells (MSCQ)
or with 15% (v/v) of cell culture supplement derived
from human plasma (CCS). hMSC commercially available
from Lonza (Basel, Switzerland) or PromoCell
(Heidelberg, Germany) were cultured. The culture media
recommended by the manufacturer were used as a positive
control. Just one commercially available basal medium
was used as the negative control. The results are shown
in Table 1.
Table 1. Experimental results from the culturing of Lonza and
PromoCell hMSC.
Cell Growth
Culture Medium
Lonza hMSC PromoCell hMSC
Recommended by the
(+) (+)
manufacturer
DMEM/F12 (-) (-)
DMEM/F12 + FBS (MSCQ) (-) (-)
DMEM/F12 + CCS (-) (-)
LV2 (-) (-)
LV2 + FBS (MSCQ) (+) (+)
LV2 + CCS (+) (+)
As may be seen in Table 1, Lonza hMSC are no
longer able to grow when only DMEM/12 basal medium is
used as the culture medium, including when that basal
medium is supplemented with FBS or CCS. They also do not
grow when LV2 medium according to this invention is used
alone. However they were capable of growing in the
culture medium according to this invention when it was
supplemented with FBS or CCS, and in the culture medium
recommended by the manufacturer.
Example 2.
In order to determine the effect of combining
all the components of the culture medium according to
this invention an LV2 culture medium was prepared as
described in Example 1 and in addition to this various
media which lacked at least one component of the culture
medium according to this invention were also prepared.
The media prepared, and the results obtained, are shown
in Table 2.
Table 2. Effect of leaving out components from the culture
medium on the growth of Lonza and PromoCell hMSC.
Cell growth
Culture medium
Lonza PromoCell
hMSC hMSC
Recommended by the manufacturer (+) (+)
DMEM/F12 (-) (-)
LV2 + CCS (+) (+)
LV2 + CCS without insulin, sodium
(-) (-)
selenite and ethanolamine
LV2 + CCS without leucocyte/platelet
(-) (-)
lysate(buffy coat)
LV2 + CCS without bFGF (-) (-)
LV2 + CCS without LBC/bFGF (-) (-)
As may be seen from Table 2, Lonza and
PromoCell hMSC only grew in the culture medium
recommended by the manufacturer and in the culture
medium according to this invention supplemented with
CCS. When at least one of the components of the culture
medium according to this invention was left out the hMSC
were no longer capable of growing in it.
Although the invention has been described in
relation to examples of preferred embodiments, these
must not be regarded as restricting the invention, which
is defined by the broadest interpretation of the
following claims.
In this specification where reference has been made
to patent specifications, other external documents, or
other sources of information, this is generally for the
purpose of providing a context for discussing the
features of the invention. Unless specifically stated
otherwise, reference to such external documents is not
to be construed as an admission that such documents, or
such sources of information, in any jurisdiction, are
prior art, or form part of the common general knowledge
in the art.
In the description in this specification reference
may be made to subject matter that is not within the
scope of the claims of the current application. That
subject matter should be readily identifiable by a
person skilled in the art and may assist in putting into
practice the invention as defined in the claims of this
application.
Claims (19)
1. A culture medium for human mesenchymal stem cells, comprising: a) basal medium suitable for the culture of 5 mesenchymal stem cells; b) human leucocyte/platelet coat (buffy coat) lysate; c) insulin; d) sodium selenite; 10 e) ethanolamine; f) basic fibroblast growth factor (bFGF); and either g) fetal bovine serum; or h) cell culture supplement non human plasma 15 (CCS); or both g) and h).
2. The culture medium for human mesenchymal stem cells according to claim 1, wherein the mesenchymal stem cell basal medium is DMEM/F12 + pyruvate, Ham’s F10, Ham’s 20 F12, MCDB 131 or RPMI 1640.
3. The culture medium for human mesenchymal stem cells according to claim 1 or 2, wherein the human leucocyte and platelet coat (buffy coat) lysate is present in 25 the culture medium in a quantity of 5 to 20% (v/v).
4. The culture medium for human mesenchymal stem cells according to claim 3, wherein the human leucocyte and platelet coat 30 (buffy coat) lysate is present in the culture medium in a quantity of 7 to 15% (v/v).
5. The culture medium for human mesenchymal stem cells according to any one of the previous claims, wherein the insulin is present in a quantity of 2 to 20 mg/L. 5
6. The culture medium for human mesenchymal stem cells according to claim 5, wherein the insulin is present in a quantity of 5 to 15 mg/L.
7. The culture medium for human mesenchymal 10 stem cells according to any one of the previous claims, wherein the sodium selenite is present in a quantity of 0.005 to 1.730 mg/L.
8. The culture medium for human mesenchymal 15 stem cells according to any one of the previous claims, wherein the ethanolamine is present in a quantity of 1 to 8 mg/L.
9. The culture medium for human mesenchymal stem cells according to claim 8, wherein 20 the ethanolamine is present in a quantity of 2 to 5 mg/L.
10. The culture medium for human mesenchymal stem cells according to any one of the previous claims, wherein the bFGF is 25 present in a quantity of 5 to 25 ng/mL.
11. The culture medium for human mesenchymal stem cells according to any one of the previous claims, further comprising an antibiotic. 30
12. The culture medium for human mesenchymal stem cells according to claim 11, wherein the antibiotic is a mixture of gentamycin and amphotericin.
13. The culture medium for human mesenchymal stem cells according to any one of the 5 previous claims, wherein said medium is supplemented with foetal bovine serum (FBS) in a quantity of 10% to 20% (v/v).
14. The culture medium for human mesenchymal stem cells according to any one of claims 10 1 to 12, wherein said medium is supplemented with cell culture supplement derived from human plasma (CCS) in a quantity of 10% to 40% (v/v).
15. A method of cell culture comprising 15 adding human mesenchymal stem cells to a culture medium of any one of claims 1-14 and culturing in vitro.
16. Human mesenchymal stem cells cultured according to the method of claim 15. 20
17. A cell culture medium as claimed in claim 1, substantially as herein described or exemplified with reference to any example thereof.
18. A method as claimed in claim 15, 25 substantially as herein described or exemplified with reference to any example thereof.
19. Human mesenchymal stem cells as claimed in claim 16, substantially as herein 30 described or exemplified with reference to any example thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES201231753 | 2012-11-13 | ||
| ES201231753 | 2012-11-13 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| NZ616977A NZ616977A (en) | 2015-05-29 |
| NZ616977B true NZ616977B (en) | 2015-09-01 |
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