U.S. patent application number 14/953132 was filed with the patent office on 2016-03-17 for medium for culturing stem cells.
This patent application is currently assigned to AJINOMOTO CO., INC.. The applicant listed for this patent is AJINOMOTO CO., INC.. Invention is credited to Yumi ANDO, Masayo DATE, Daisuke EJIMA, Takayoshi FUJII, Manabu KITAZAWA, Yoko KURIYAMA, Haruna SATO, Shou SENDA, Tomomi YOSHIDA.
Application Number | 20160075993 14/953132 |
Document ID | / |
Family ID | 51988956 |
Filed Date | 2016-03-17 |
United States Patent
Application |
20160075993 |
Kind Code |
A1 |
KURIYAMA; Yoko ; et
al. |
March 17, 2016 |
MEDIUM FOR CULTURING STEM CELLS
Abstract
Culture media, which contain albumin carrying a reduced amount
of fatty acid, are useful for culturing stem cells.
Inventors: |
KURIYAMA; Yoko;
(Kawasaki-shi, JP) ; SENDA; Shou; (Kawasaki-shi,
JP) ; ANDO; Yumi; (Kawasaki-shi, JP) ;
YOSHIDA; Tomomi; (Kawasaki-shi, JP) ; SATO;
Haruna; (Kawasaki-shi, JP) ; EJIMA; Daisuke;
(Kawasaki-shi, JP) ; FUJII; Takayoshi;
(Kawasaki-shi, JP) ; DATE; Masayo; (Kawasaki-shi,
JP) ; KITAZAWA; Manabu; (Kawasaki-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AJINOMOTO CO., INC. |
Tokyo |
|
JP |
|
|
Assignee: |
AJINOMOTO CO., INC.
Tokyo
JP
|
Family ID: |
51988956 |
Appl. No.: |
14/953132 |
Filed: |
November 27, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
PCT/JP2014/064497 |
May 30, 2014 |
|
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14953132 |
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Current U.S.
Class: |
435/15 ;
435/407 |
Current CPC
Class: |
C12N 2500/36 20130101;
C12N 5/0696 20130101; C12N 5/0018 20130101; C12N 5/0602 20130101;
C12N 2501/998 20130101 |
International
Class: |
C12N 5/071 20060101
C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
May 30, 2013 |
JP |
2013-114285 |
Claims
1. A medium for culturing stem cells, comprising an albumin which
comprises a reduced amount of fatty acid.
2. The medium according to claim 1, wherein said albumin which
comprises a reduced amount of fatty acid is obtained by subjecting
albumin to a fatty acid removal treatment.
3. The medium according to claim 2, wherein said fatty acid removal
treatment comprises treating albumin with activated carbon.
4. The medium according to claim 1, wherein said albumin comprises
fatty acid in an amount of 0.1 mg/g to 0.65 mg/g, based on the
total amount of said albumin.
5. A medium for culturing stem cells, which comprises fatty acid in
an amount of 0.1 mg to 0.65 mg per 1 g albumin in said medium.
6. The medium according to claim 1, wherein said fatty acid is a
long chain fatty acid.
7. The medium according to claim 1, wherein said fatty acid is at
least one member selected from the group consisting of oleic acid,
palmitic acid, stearic acid, linoleic acid, linolenic acid, and
arachidonic acid.
8. The medium according to claim 1, wherein said albumin is a human
serum-derived albumin.
9. A method of culturing stem cells, comprising cultivating stem
cells in a medium according to claim 1.
10. The method according to claim 9, wherein said stem cells are
pluripotent stem cells.
11. The method according to claim 10, wherein said pluripotent stem
cells are embryonic stem cells (ES cells) or induced pluripotent
stem cells (iPS cells).
12. A method of selecting an albumin suitable for addition to a
medium, comprising: measuring the amount of fatty acid comprised in
an albumin; and selecting an albumin which comprises a reduced
amount of fatty acid.
13. A method of producing a medium for culturing stem cells,
comprising: preparing an albumin comprising a reduced amount of
fatty acid by a fatty acid removal treatment; and adding said
albumin comprising a reduced amount of fatty acid to a medium.
14. The method according to claim 13, wherein said fatty acid
removal treatment comprises treating albumin with activated
carbon.
15. The method according to claim 14, wherein said albumin is
treated with 30 to 60 wt % of activated carbon, based on the weight
of said albumin.
16. The method according to claim 14, wherein said treating albumin
with activated carbon is conducted at a pH of 6.7 to 7.3.
17. The method according to claim 14, wherein said treating albumin
with activated carbon is conducted at a pH of 3.7 to 4.3.
18. The method according to claim 13, wherein said albumin
comprises fatty acid in an amount of 0.1 mg/g to 0.65 mg/g, based
on the amount of albumin.
19. The method according to claim 13, wherein said fatty acid is a
long chain fatty acid.
20. The method according to claim 13, wherein said fatty acid is at
least one member selected from the group consisting of oleic acid,
palmitic acid, stearic acid, linoleic acid, linolenic acid, and
arachidonic acid.
21. The method according to claim 13, wherein said albumin is human
serum-derived albumin.
22. The medium according to claim 2, wherein said albumin comprises
fatty acid in an amount of 0.1 mg/g to 0.65 mg/g, based on the
total amount of said albumin.
23. The medium according to claim 3, wherein said albumin comprises
fatty acid in an amount of 0.1 mg/g to 0.65 mg/g, based on the
total amount of said albumin.
24. A cell culture, comprising stem cells in a culture medium
according to claim 1.
25. The cell culture according to claim 24, wherein said stem cells
are pluripotent stem cells.
26. The cell culture according to claim 25, wherein said
pluripotent stem cells are embryonic stem cells (ES cells) or
induced pluripotent stem cells (iPS cells).
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of International Patent
Application No. PCT/JP2014/064497, filed on May 30, 2014, and
claims priority to Japanese Patent Application No. 2013-114285,
filed on May 30, 2013, both of which are incorporated herein by
reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to media for culturing stem
cells, method for producing such a medium, and the like.
[0004] 2. Discussion of the Background
[0005] Conventionally, the culturing of stem cells (embryonic stem
cells, induced pluripotent stem cells (iPS cell,) and the like) has
been conducted using a medium containing a serum. For example,
fetal bovine serum (FBS) and the like are widely used for cell
culture as an important additive for cell proliferation. However,
when stem cells after such culturing are used for medical purposes,
a xeno-derived component may become a source of infection with
blood-borne pathogen or a xenoantigen. In addition, culture results
may be inconsistent due to a difference between serum lots.
Therefore, it has become mainstream in recent years to use a medium
having a clear chemical composition (chemically-defined medium) for
culturing stem cells, and the development of a serum-free medium is
ongoing.
[0006] One of the highly important components for serum-free medium
is albumin. Addition of albumin is expected to provide an effect of
stably maintaining the medium property. Several kinds of albumin
are commercially available for culturing cells. However, not all
albumins provide an equivalent effect for cell culture,
particularly the culture of stem cells, and the quality of albumin
affects the culture results.
[0007] On the other hand, a treatment with activated carbon, ion
exchange, heat treatment, and the like are known purification
methods of albumin (see J. Biological Chemistry 1968, 212(2)
173-181; BioChim. Biophy. Acta 1970, 221, 376-378; Biologics 1997,
25, 391-401; and Brazilian journal of medicinal and biological
research 1998, 31, 1383-1388, all of which are incorporated herein
by reference in their entireties). Also, palmitic acid is reported
to induce apoptosis of mesenchymal stem cells (see Endocrinology
November 2012, 153(11), 5275-5284, which is incorporated herein by
reference in its entirety), and there is a report that addition of
a substance that inhibits oleic acid synthase in iPS cells results
in accumulation of palmitic acid, which induces ER stress-apoptosis
(see Cell Stem Cell 2013, 12, 167-179, which is incorporated herein
by reference in its entirety).
[0008] WO 2013/006675, which is incorporated herein by reference in
its entirety, describes that octanoic acid is used in a medium to
stabilize albumin, and octanoic acid is harmful for culture of stem
cells. However, use of a long chain fatty acid for stabilization of
albumin is not described, nor are there any data or description
showing that a long chain fatty acid is more harmful for culture of
stem cells than octanoic acid which is a middle chain fatty acid,
and exerts an adverse influence on the maintenance of
undifferentiation potency. Also, WO 2013/134378, which is
incorporated herein by reference in its entirety, describes that
fatty acid-free albumin is contained in a medium for stem cells.
This document does not describe how much fatty acid to be removed
from albumin is sufficient for the cultivation of stem cells, and
does not consider complication of removal of fatty acid from
albumin since commercially available bovine serum-derived albumin
manufactured by Proliant is used as fatty acid-free albumin.
SUMMARY OF THE INVENTION
[0009] Accordingly, it is one object of the present invention to
provide novel novel media for culturing stem cells.
[0010] It is another object of the present invention to provide
novel methods for preparing such a medium.
[0011] It is another object of the present invention to provide
novel methods for culturing stem cells in such a medium.
[0012] It is another object of the present invention to elucidate
the mechanism of inconsistent culture results due to the difference
in the quality of albumin to be added to a medium.
[0013] It is another object of the present invention to provide
media for culturing stem cells which show good culture results.
[0014] It is another object of the present invention to provide a
method of producing media for culture.
[0015] It is another object of the present invention to provide a
method of selecting albumin suitable for addition to a medium for
culture.
[0016] It is another object of the present invention to provide a
medium additive for media for culturing stem cells.
[0017] It is another object of the present invention to provide a
culture system capable of maintaining stem cells in an
undifferentiated state.
[0018] These and other objects, which will become apparent during
the following detailed description, have been achieved by the
inventors' discoveries of a correlation between a fatty acid
carried by albumin to be added in culturing stem cells and cell
proliferation that culture results can be improved by purification
of albumin to remove fatty acid.
[0019] Accordingly, the present invention is as described
below.
[0020] (1) A medium for culturing stem cells, comprising an albumin
carrying a reduced amount of fatty acid.
[0021] (2) The medium of (1), wherein the reduction in the amount
of fatty acid to be carried is achieved by a fatty acid removal
treatment.
[0022] (3) The medium of (2), wherein the fatty acid removal
treatment is a treatment with activated carbon.
[0023] (4) The medium of any of (1) to (3), wherein the amount of
fatty acid carried by albumin is not more than 10 mg/g.
[0024] (5) The medium of any of (1) to (3), wherein the amount of
fatty acid carried by albumin is not more than 6 mg/g.
[0025] (6) The medium of any of (1) to (3), wherein the amount of
fatty acid carried by albumin is 0.1 mg/g to 0.65 mg/g.
[0026] (7) The medium of any of (1) to (6), wherein the content of
fatty acid in the medium is not more than 60 .mu.M.
[0027] (8) A medium for culturing stem cells, wherein the amount of
fatty acid to be carried is 0.1 mg to 0.65 mg per 1 g albumin.
[0028] (9) The medium of (8), comprising not more than 60 .mu.M of
fatty acid.
[0029] (10) The medium of any of (1) to (9), wherein the fatty acid
is a long chain fatty acid.
[0030] (11) The medium of any of (1) to (9), wherein the fatty acid
is at least one kind selected from the group consisting of oleic
acid, palmitic acid, stearic acid, linoleic acid, linolenic acid
and arachidonic acid.
[0031] (12) The medium of any of (1) to (11), wherein the albumin
is a human serum-derived albumin.
[0032] (13) The medium of any of (1) to (12), wherein the stem cell
is a pluripotent stem cell.
[0033] (14) The medium of (13), wherein the pluripotent stem cell
is an embryonic stem cell (ES cell) or an induced pluripotent stem
cell (iPS cell).
[0034] (15) A method of culturing stem cells, comprising a step of
cultivating in the medium of any of (1) to (12).
[0035] (16) The method of (15), wherein the stem cell is a
pluripotent stem cell.
[0036] (17) The method of (16), wherein the pluripotent stem cell
is an embryonic stem cell (ES cell) or an induced pluripotent stem
cell (iPS cell).
[0037] (18) A method of selecting an albumin suitable for addition
to a medium, comprising a step of measuring the amount of fatty
acid carried, and selecting an albumin carrying a reduced amount of
fatty acid.
[0038] (19) A method of producing a medium for culturing stem
cells, comprising preparing an albumin carrying a reduced amount of
fatty acid by a fatty acid removal treatment, and adding the
prepared albumin to a medium.
[0039] (20) The method of (19), wherein the fatty acid removal
treatment is a treatment with activated carbon.
[0040] (21) The method of (20), wherein the treatment with
activated carbon is performed using 30 to 60 wt % of activated
carbon relative to the weight of the albumin.
[0041] (22) The method of (20) or (21), wherein the treatment with
activated carbon is performed at pH 6.7 to 7.3.
[0042] (23) The method of (20) or (21), wherein the treatment with
activated carbon is performed at pH 3.7 to 4.3.
[0043] (24) The method of any of (19) to (23), wherein the amount
of fatty acid carried by albumin is not more than 10 mg/g
[0044] (25) The method of any of (19) to (23), wherein the amount
of fatty acid carried by albumin is not more than 6 mg/g.
[0045] (26) The method of any of (19) to (23), wherein the amount
of fatty acid carried by albumin is 0.1 mg/g to 0.65 mg/g.
[0046] (27) The method of any of (19) to (26), wherein the content
of fatty acid in the medium is not more than 60 .mu.M.
[0047] (28) The method of any of (19) to (27), wherein the fatty
acid is a long chain fatty acid.
[0048] (29) The method of any of (19) to (27), wherein the fatty
acid is at least one kind selected from the group consisting of
oleic acid, palmitic acid, stearic acid, linoleic acid, linolenic
acid and arachidonic acid.
[0049] (30) The method of any of (19) to (29), wherein the albumin
is human serum-derived albumin.
[0050] (31) The method of any of (19) to (30), wherein the stem
cell is a pluripotent stem cell.
[0051] (32) The method of (31), wherein the pluripotent stem cell
is an embryonic stem cell (ES cell) or an induced pluripotent stem
cell (iPS cell).
[0052] (33) An additive for a medium for culturing stem cells,
comprising an albumin carrying 0.1 mg/g to 0.65 mg/g of fatty
acid.
[0053] (34) The additive of (33), wherein the fatty acid is a long
chain fatty acid.
[0054] (35) The additive of (33), wherein the fatty acid is at
least one kind selected from the group consisting of oleic acid,
palmitic acid, stearic acid, linoleic acid, linolenic acid and
arachidonic acid.
[0055] (36) The additive of any of (33) to (35), wherein the
albumin is human serum-derived albumin.
[0056] (37) The additive of any of (33) to (36), wherein the stem
cell is a pluripotent stem cell.
[0057] (38) The additive of (37), wherein the pluripotent stem cell
is an embryonic stem cell (ES cell) or an induced pluripotent stem
cell (iPS cell).
[0058] (39) A culture system of a stem cell, comprising a step of
cultivating in a medium comprising an albumin carrying a reduced
amount of fatty acid, wherein the amount of the fatty acid to be
carried is so selected as to enable maintenance of the stem cell in
an undifferentiated state.
[0059] (40) The culture system of (39), wherein the reduction in
the amount of fatty acid is achieved by a fatty acid removal
treatment.
[0060] (41) The culture system of (40), wherein the fatty acid
removal treatment is a treatment with activated carbon.
[0061] (42) The culture system of any of (39) to (41), wherein the
amount of fatty acid carried by albumin is not more than 10
mg/g.
[0062] (43) The culture system of any of (39) to (41), wherein the
amount of fatty acid carried by albumin is not more than 6
mg/g.
[0063] (44) The culture system of any of (39) to (41), wherein the
amount of fatty acid carried by albumin is 0.1 mg/g-0.65 mg/g.
[0064] (45) The culture system of any of (39) to (44), wherein the
content of fatty acid in the medium is not more than 60 .mu.M.
[0065] (46) The culture system of any of (39) to (45), wherein the
fatty acid is a long chain fatty acid.
[0066] (47) The culture system of any of (39) to (45), wherein the
fatty acid is at least one kind selected from the group consisting
of oleic acid, palmitic acid, stearic acid, linoleic acid,
linolenic acid and arachidonic acid.
[0067] (48) The culture system of any of (39) to (47), wherein the
albumin is human serum-derived albumin.
[0068] (49) The culture system of any of (39) to (48), wherein the
stem cell is a pluripotent stem cell.
[0069] (50) The culture system of (49), wherein the pluripotent
stem cell is an embryonic stem cell (ES cell) or an induced
pluripotent stem cell (iPS cell).
Effect of the Invention
[0070] Using the medium of the present invention, a stem cell can
be grown while maintaining an undifferentiated state. Furthermore,
using the medium of the present invention, a stem cell can be
efficiently grown. Consequently, the frequency of medium exchange
during culture can be reduced, and the cost of stem cell culture
can be decreased.
BRIEF DESCRIPTION OF THE DRAWINGS
[0071] A more complete appreciation of the invention and many of
the attendant advantages thereof will be readily obtained as the
same become better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein:
[0072] FIG. 1 shows culture results of stem cells in an
albumin-added medium. The left Figure shows one embodiment of a
culture dish marked with culture results + in Table 1, and the
right Figure shows one embodiment of a culture dish marked with
culture results -.
[0073] FIG. 2 shows measurement results of viable cell number when
stem cells are cultured in a medium added with albumin adsorbing
fatty acid.
[0074] FIG. 3 shows an influence of human serum-derived albumin at
varying purification degrees on the growth of iPS cells.
[0075] FIG. 4 shows an influence of human serum-derived albumin
re-carrying a different kind of fatty acid, on the growth of iPS
cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0076] In the present invention, the term "stem cell" means an
immature cell having self-renewal capacity and
differentiation/proliferation capacity. The stem cell includes
subpopulations such as pluripotent stem cell, multipotent stem
cell, unipotent stem cell, and the like, according to the
differentiation potency. The term pluripotent stem cell means a
cell capable of differentiating into any tissue or cell
constituting living organisms. The term multipotent stem cell means
a cell capable of differentiating into plural, though not all,
kinds of tissues and cells. The term unipotent stem cell means a
cell capable of differentiating into particular tissues and
cells.
[0077] Examples of pluripotent stem cells include embryonic stem
cells (ES cell), embryonic germ cell (EG cell), induced pluripotent
stem cell (iPS cell) and the like. It is preferably embryonic stem
cells (ES cell) or an induced pluripotent stem cell (iPS cell). A
stem cell established by cultivating an early embryo generated by
nuclear transplantation of the nucleus of a somatic cell is also
preferable as the pluripotent stem cell (see 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); and Nature Genetics,
24, 109 (2000), all of which are incorporated herein by reference
in their entireties). In addition, a pluripotent stem cell induced
and selected by stress and stimulation on the cell is an example of
the pluripotent stem cell.
[0078] Examples of multipotent stem cells include somatic stem
cells such as mesenchymal stem cell, hematopoietic stem cell,
neural stem cell, myeloid stem cell, germ line stem cell and the
like, and the like. The multipotent stem cell is preferably a
mesenchymal stem cell, more preferably a bone marrow mesenchymal
stem cell. The mesenchymal stem cell broadly means a population of
stem cells or progenitor cells thereof, which can differentiate
into all or some of the mesenchymal cells such as osteoblast,
chondroblast, lipoblast and the like.
[0079] As the basal medium to be used in the present invention, one
known per se can be used depending on the kind of the stem cells,
and is not particularly limited as long as it does not inhibit
proliferation of the stem cells. Examples thereof include DMEM,
EMEM, IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's
MEM), RPMI-1640, .alpha.-MEM, Ham's Medium F-12, Ham's Medium F-10,
Ham's Medium F12K, Medium 199, ATCC-CRCM30, DM-160, DM-201, BME,
Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107,
MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's MB752/1,
CMRL-1066, Williams' medium E, Brinster's BMOC-3 Medium, E8 medium
(Nature Methods, 2011, 8, 424-429), ReproFF2 medium (ReproCELL
Inc), a mixed medium thereof and the like. In addition, a medium
altered for culture of stem cells, a mixture of the above-mentioned
basal medium and other medium, and the like may also be used.
[0080] The medium to be used in the present invention can contain
an additive substance known per se. The additive substance is not
particularly limited as long as it does not inhibit proliferation
of stem cells. Examples thereof include growth factor (e.g.,
insulin etc.), iron source (e.g., transferrin etc.), polyamines
(e.g., putrescine etc.), mineral (e.g., sodium selenate etc.),
saccharides (e.g., glucose etc.), organic acid (e.g., pyruvic acid,
lactic acid etc.), amino acid (e.g., L-glutamine), reducing agent
(e.g., 2-mercaptoethanol), vitamins (e.g., ascorbic acid, d-biotin
etc.), steroid (e.g., .beta.-estradiol, progesterone etc.),
antibiotic (e.g., streptomycin, penicillin, gentamicin etc.),
buffering agent (e.g., HEPES etc.) and the like. In addition,
additives that have been conventionally used for culturing stem
cells can be contained as appropriate. The additive is preferably
contained within a concentration range known per se.
[0081] The medium to be used in the present invention may contain a
serum. Serum is not particularly limited as long as it is derived
from an animal and does not inhibit the growth of stem cells.
Preferred is a mammal-derived serum (e.g., fetal bovine serum,
human serum etc.). The concentration of the serum may be any as
long as it is within a concentration range known per se. However, a
lower content of serum is more preferable, and the absence of serum
is most preferable, since it is known that serum components also
contain a differentiation factor of human ES cell, and the like,
and the culture results may be inconsistent due to a difference
between serum lots. Furthermore, when a stem cell after culture is
used for medical purposes, a xeno-derived component may become an
infection source of blood-mediated pathogen or a xenoantigen.
Therefore, the absence of serum is preferable. When serum is not
contained, a replacement additive of serum (e.g., Knockout Serum
Replacement (KSR) (Invitrogen), Chemically-defined Lipid
concentrated (Gibco), Glutamax (Gibco) etc.) may also be used.
[0082] The present invention provides a medium for culturing stem
cells, which characteristically has a decreased fatty acid content
(hereinafter to be also referred to as the medium of the present
invention).
1. Medium of the Present Invention.
[0083] While the medium of the present invention can be preferably
used for proliferation of any stem cells, it is preferably used for
proliferation of an embryonic stem cell or an induced pluripotent
stem cell.
[0084] Also, the medium of the present invention can be preferably
used for proliferation of stem cells derived from any animals. The
stem cells cultured by using the medium of the present invention
are, for example, pluripotent stem cells derived from rodents such
as mouse, rat, hamster, guinea pig and the like, Lagomorpha such as
rabbit and the like, Ungulata such as swine, bovine, goat, horse,
sheep and the like, Carnivora such as dog, cat and the like,
primates such as human, monkey, Macaca mulatta, marmoset,
orangutan, chimpanzee and the like. Preferred are stem cells
derived from human.
[0085] The medium of the present invention characteristically has a
decreased fatty acid content. In one embodiment thereof, the medium
of the present invention characteristically contains an albumin
carrying a reduced amount of fatty acid. The albumin to be used in
the present invention is not particularly limited as long as it is
used for cell culture, and is directly added to a medium when the
amount of fatty acid to be carried it already reduced, or added
after a fatty acid removal treatment when the amount of fatty acid
to be carried has not been reduced.
[0086] In the present specification, a fatty acid removal treatment
of albumin is sometimes expressed as "purification" of albumin.
[0087] Examples of the fatty acid include saturated fatty acid
having 8 to 20 carbon atoms (e.g., palmitic acid, stearic acid) and
unsaturated fatty acid having 16 to 20 carbon atoms (e.g., oleic
acid, linoleic acid, linolenic acid, arachidonic acid).
[0088] The present inventors studied to determine whether an
influence on the growth of stem cells varies depending on the kind
of fatty acid to be carried by albumin. As a result, they have
found that long chain fatty acid shows a higher growth inhibitory
action on stem cells than middle chain fatty acid. Therefore, in
the medium of the present invention, long chain fatty acid is
preferably reduced.
[0089] In the present specification, the term "long chain fatty
acid" means a fatty acid having 12 or more carbon number. The long
chain fatty acid to be reduced in the medium of the present
invention is not particularly limited as long as it has an action
to inhibit growth of stem cells. Specific examples of the long
chain fatty acid to be reduced include oleic acid, stearic acid,
palmitic acid, linoleic acid, linolenic acid and arachidonic acid.
Preferable examples of the long chain fatty acid to be reduced
include stearic acid, palmitic acid, linoleic acid, linolenic acid
and arachidonic acid.
[0090] Specific examples of albumin include naturally-derived
albumin such as ovalbumin, swine-derived albumin, bovine-derived
albumin, human-derived albumin and the like, gene recombinant
albumin such as bovine type, swine type, human type and the like,
and the like. Particularly preferable examples thereof include
serum-derived albumin and human type gene recombinant albumin
(recombinant human albumin (rHSA)). Of these, human serum-derived
albumin is particularly preferable.
[0091] Albumin is a protein having a high ability to bind to
various substances, and binds to trace elements such as calcium,
zinc and the like, fatty acid, enzyme, hormone and the like. For
example, serum-derived albumin binds to various substances
contained in serum. In the case of fatty acid, 1 molecule of
albumin generally has an ability to bind to 2 molecules of fatty
acid.
[0092] The fatty acid removal treatment of albumin is not
particularly limited as long as it can reduce the amount of fatty
acid carried by albumin, and a treatment with activated carbon (see
J. Biological Chemistry 1968, 212(2), 173-181), an ion exchange
treatment (see BioChim. Biophy. Acta 1970, 221, 376-378 and
Biologics 1997, 25, 391-401), a heat treatment (see Brazilian
journal of medicinal and biological research 1998, 31, 1383-1388)
and the like can be mentioned. From the aspects of economic
efficiency, convenience and the like, a treatment with activated
carbon is preferable. The amount of fatty acid carried by albumin
can be measured by a method generally practiced in the pertinent
field, or a method analogous thereto. Examples thereof include
methyl esterification of free fatty acid followed by detection by
GC-MS, quantification by infrared spectroscopy and extraction
method of Duncombe, ACS-ACOD method using acyl-CoA synthase (ACS)
and acyl-CoA oxydase (ACOD) and the like. A commercially available
measurement kit can be utilized for any of these.
[0093] The treatment with activated carbon may be performed under
any conditions as long as a desired effect can be obtained. In one
embodiment, the treatment with activated carbon can be performed
using 30 to 60 wt %, preferably 40 to 50 wt %, of activated carbon
per weight of albumin. While the pH of the treatment with activated
carbon is not particularly limited as long as a desired effect can
be obtained, it is pH 3 to 8, preferably pH 3.7 to 7.3, more
preferably pH 3.7 to 4.3 or pH 6.7 to 7.3.
[0094] In one embodiment, a fatty acid removal treatment of albumin
can also be preferably performed by ion exchange chromatography. A
specific method thereof includes dialyzing an albumin solution
against 40 mM sodium phosphate buffer adjusted to near neutral pH,
subjecting same to anion exchange chromatography column (e.g., DEAE
sepharose FF (GE Healthcare Japan)) previously equilibrated with
the same buffer, then applying a linear concentration gradient to
the 80 mM sodium phosphate adjusted to near neutral pH in a 10-fold
volume of the column volume, whereby albumin fraction not bound to
fatty acid can be recovered.
[0095] The amount of fatty acid carried by albumin in the present
invention is reduced to a level at which stem cells can grow well.
The amount of fatty acid carried by albumin when a fatty acid
removal treatment is not performed is, for example, about 14 mg/g.
The amount of fatty acid carried by albumin used in the present
invention is preferably reduced to not more than 10 mg/g, more
preferably not more than 6 mg/g, further more preferably
substantially free of fatty acid. As used herein, "substantially
free of fatty acid" means that albumin does not at all bind to
fatty acid, or even if it does, it is less than the detection limit
of the measurement method or measurement kit used for measuring the
amount of fatty acid being carried.
[0096] When two or more kinds of fatty acids are bound to albumin,
the total amount thereof is preferably reduced to fall within the
above-mentioned range.
[0097] In one embodiment, the amount of fatty acid carried by
albumin to be used in the medium of the present invention can be
not more than 10 mg/g, more preferably not more than 6 mg/g and not
less than 0.1 mg/g, based on the total weight of albumin.
Specifically, the amount of fatty acid carried can be 0.1 mg/g to
0.8 mg/g, preferably 0.1 mg/g to 0.65 mg/g or 0.2 mg/g to 0.8 mg/g,
more preferably 0.2 mg/g to 0.65 mg/g, most preferably 0.29 mg/g to
0.65 mg/g, based on the total weight of albumin. The amount of
fatty acid to be carried is, for example, the amount of fatty acid
carried by albumin which can be achieved by a fatty acid removal
treatment of albumin performed by a convenient method such as a
treatment with activated carbon and the like. When the amount of
fatty acid to be carried is within the aforementioned range, the
growth of stem cells can be promoted as compared to the use of a
medium containing albumin without a fatty acid removal treatment.
Therefore, the time, labor, cost and the like necessary for
preparing a medium can be reduced, and the amount of fatty acid to
be carried, which is within said range, can be preferably adopted
for a convenient and large-scale production of the medium of the
present invention.
[0098] While an amount of less than 0.1 mg/g of fatty acid carried
by albumin can afford a desired effect, the amount of less than 0.1
mg/g of fatty acid to be carried requires, for example, a further
fatty acid removal treatment by a method such as ion exchange
chromatography and the like, thus requiring time, labor, cost and
the like as compared to convenient methods such as a treatment with
activated carbon and the like. Therefore, an amount of less than
0.1 mg/g of fatty acid carried by albumin is disadvantageous for a
large-scale production of the medium of the present invention at an
industrial level.
[0099] When two or more kinds of fatty acids are bound to albumin,
the total amount thereof is preferably reduced to fall within the
above-mentioned range.
[0100] In the present invention, while the amount of an albumin
carrying a reduced amount of fatty acid to be added to a medium is
not particularly limited as long as it is an amount generally added
to a medium for cell culture, it is added to a basal medium for
stem cell culture to a final concentration of 0 to 50 mg/mL,
preferably 0.01 to 30 mg/mL, more preferably 0.05 to 10 mg/mL,
further preferably 0.5 to 5 mg/mL, based on the total volume of the
medium.
[0101] In another embodiment, the medium of the present invention
has a reduced fatty acid content of the medium as a whole. As used
herein, the "reduced fatty acid content of the medium as a whole"
is intended to mean that not only the amount of fatty acid bound to
albumin but also the amount of free fatty acid are reduced. In a
preferable embodiment, the amount of fatty acid bound to albumin is
reduced. The content of fatty acid in the medium is preferably not
more than 60 .mu.M, more preferably not more than 30 .mu.M, further
preferably not more than 10 .mu.M, further more preferably not more
than 9, 8, 7, 6, 5, 4, 3, 2 or 1 .mu.M, based on the total volume
of the medium, most preferably a concentration that does not exert
a great influence on the concentration of fatty acid contained in
the basal medium for culturing stem cells. The concentration of
fatty acid contained in the basal medium for culturing stem cells
is, for example, about 0.3 .mu.M and 0.15 .mu.M in commercially
available Ham's Medium F-12 and DMEM/Ham's Medium F-12. Not
exerting a great influence means remaining within 10-fold
concentration change from the concentration of fatty acid contained
in the basal medium for culturing stem cells. That is, not less
than 0 .mu.M, a concentration exceeding 0 .mu.M, not less than 0.15
.mu.M, not less than 0.3 .mu.M, not less than 0.5 .mu.M of fatty
acid can be contained as long as it does not go beyond a 10-fold
amount of the concentration of fatty acid contained in the basal
medium for stem cell culture. The fatty acid content can be
measured according to the above-mentioned method for measuring the
amount of fatty acid bound to albumin, and a measurement kit is
also commercially available.
[0102] When two or more kinds of fatty acids are contained in the
medium, the total amount thereof is preferably reduced to fall
within the above-mentioned range.
[0103] Examples of the fatty acid include saturated fatty acid
having 8 to 20 carbon atoms (e.g., palmitic acid, stearic acid) and
unsaturated fatty acid having 16 to 20 carbon atoms (e.g., oleic
acid, linoleic acid, linolenic acid, arachidonic acid).
[0104] Based on the finding by the present inventors that long
chain fatty acid shows a higher stem cell proliferation inhibitory
action than middle chain fatty acid, in one embodiment, the fatty
acid to be reduced is long chain fatty acid (fatty acid having 12
or more carbon atoms), and specific examples thereof include oleic
acid, stearic acid, palmitic acid, linoleic acid, linolenic acid
and arachidonic acid. Specific preferable examples include stearic
acid, palmitic acid, linoleic acid, linolenic acid and arachidonic
acid.
[0105] A medium for culturing stem cells, containing 0.1 mg to 0.65
mg of fatty acid to be carried per 1 g albumin is preferable.
Furthermore, a medium for culturing stem cells, having a content of
fatty acid in the medium of not more than 60 .mu.M is
preferable.
[0106] The present invention provides a method of producing a
medium for culturing stem cells which characteristically has a
reduced content of fatty acid (hereinafter to be also referred to
as the production method of the present invention).
2. Production Method of the Present Invention.
[0107] In one embodiment, the production method of the present
invention includes preparing an albumin carrying a reduced amount
of fatty acid by a fatty acid removal treatment (step 1), and
adding the prepared albumin to a medium (step 2).
Step 1. Step of Preparing an Albumin Carrying a Reduced Amount of
Fatty Acid by a Fatty Acid Removal Treatment.
[0108] It can be performed according to the above-mentioned section
of "the medium of the present invention". In brief, human-derived
serum albumin and rHSA are subjected to a fatty acid removal
treatment such as a treatment with activated carbon, an ion
exchange treatment, a heat treatment and the like (preferably
treatment with activated carbon) to prepare an albumin carrying a
reduced amount of fatty acid (preferably the amount of fatty acid
to be carried is not more than 10 mg/g, more preferably not more
than 6 mg/g, based on the total amount of albumin, and further
preferably, substantially no fatty acid is carried). Reduction of
the amount of fatty acid carried can be confirmed by measuring the
amount of fatty acid bound to albumin by a method generally
performed in the pertinent field or a method analogous thereto, and
a commercially available measurement kit can be utilized.
[0109] In one embodiment, the amount of the fatty acid carried by
albumin can be not more than 10 mg/g, more preferably not more than
6 mg/g, and not less than 0.1 mg/g, based on the total amount of
albumin. Specifically, the amount of the fatty acid to be carried
can be 0.1 mg/g to 0.8 mg/g, preferably 0.1 mg/g to 0.65 mg/g or
0.2 mg/g to 0.8 mg/g, more preferably 0.2 mg/g to 0.65 mg/g, most
preferably 0.29 mg/g to 0.65 mg/g, based on the total amount of
albumin.
Step 2. Step of Adding the Albumin Carrying a Reduced Amount of
Fatty Acid Obtained in the Above-Mentioned Step 1 to a Medium.
[0110] It can be performed according to the above-mentioned section
of "the medium of the present invention". An albumin carrying a
reduced amount of fatty acid is added to a medium (basal medium) to
a final concentration of 0 to 50 mg/mL, preferably 0.01 to 30
mg/mL, more preferably 0.05 to 10 mg/mL, further preferably 0.5 to
5 mg/mL, based on the total volume of the medium. As the basal
medium, those exemplified in the above-mentioned section of "the
medium of the present invention" can be similarly used.
[0111] In this way, a medium for culturing stem cells can be
produced.
[0112] The present invention provides a method of selecting an
albumin suitable for addition to a medium (hereinafter to be also
referred to as the selection method of the present invention).
3. Selection Method of the Present Invention.
[0113] In one embodiment, the selection method of the present
invention is a method including measuring the amount of fatty acid
carried by albumin (step 1), and selecting an albumin carrying a
reduced amount of fatty acid (step 2).
Step 1. Step of Measuring the Amount of Fatty Acid Carried by
Albumin.
[0114] It can be performed according to the above-mentioned section
of "the medium of the present invention". In brief, it can be
performed by a method generally performed in the pertinent field or
a method analogous thereto. Examples thereof include an extraction
method of Duncombe, ACS-ACOD method using acyl-CoA synthase (ACS)
and acyl-CoA oxydase (ACOD) and the like. A commercially available
measurement kit can be utilized for any of these.
Step 2. Step of Selecting an Albumin Carrying a Reduced Amount of
Fatty Acid.
[0115] Based on the measurement results obtained in the
above-mentioned step 1, an albumin carrying a reduced amount of
fatty acid is selected. The "albumin carrying a reduced amount of
fatty acid" is an albumin preferably carrying not more than 10
mg/g, more preferably not more than 6 mg/g, of fatty acid, based on
the total amount of albumin, further preferably substantially no
fatty acid carried.
[0116] In one embodiment, the amount of fatty acid carried by
albumin can be not more than 10 mg/g, more preferably not more than
6 mg/g, and not less than 0.1 mg/g, based on the total amount of
albumin. Specifically, the amount of fatty acid to be carried can
be 0.1 mg/g to 0.8 mg/g, preferably 0.1 mg/g to 0.65 mg/g or 0.2
mg/g to 0.8 mg/g, more preferably 0.2 mg/g to 0.65 mg/g, most
preferably 0.29 mg/g to 0.65 mg/g, based on the total amount of
albumin.
[0117] The thus-selected "albumin carrying a reduced amount of
fatty acid" is preferably added to a medium for culturing stem
cells.
[0118] The present invention provides a method of culturing stem
cells (hereinafter to be also referred to as the culture method of
the present invention).
4. Culture Method of the Present Invention.
[0119] The culture method of the present invention includes a step
of cultivating stem cells in the medium of the present
invention.
[0120] While a culture container to be used for the culture of stem
cell is not particularly limited as long as stem cells can be
cultured, a flask, tissue culture flask, dish, petri dish, tissue
culture dish, multidish, microplate, microwell plate, multiplate,
multiwell plate, microslide, chamber slide, Schale, tube, tray,
culture bag, and roller bottle can be mentioned.
[0121] The culture container may be cell adhesive or cell
non-adhesive, and is appropriately selected according to the
object. A cell adhesive culture container may be coated with any
cell supporting substrate such as extracellular matrix (ECM) and
the like, in an attempt to improve the adhesiveness of the culture
container surface to a cell. The cell supporting substrate may be
any substance aiming at adhesion of stem cell or feeder cell (when
used).
[0122] Other culture conditions can be appropriately determined.
For example, while the culture temperature is not particularly
limited, it can be about 30 to 40.degree. C., preferably about
37.degree. C. The CO.sub.2 concentration can be about 1 to 10%,
preferably about 2 to 5%. The oxygen partial pressure can be 1 to
10%.
5. Additive of the Present Invention.
[0123] The present invention also provides an additive for a medium
for culturing stem cells, which contains an albumin carrying a
reduced amount of fatty acid (in the present specification,
sometimes to be referred to as "the additive of the present
invention").
[0124] The amount of fatty acid carried by albumin to be contained
in the additive of the present invention is not more than 10 mg/g,
more preferably not more than 6 mg/g, based on the total amount of
albumin, and further preferably, substantially no fatty acid is
contained. In one embodiment, the amount of fatty acid carried by
albumin can be not more than 10 mg/g, more preferably not more than
6 mg/g, and not less than 0.1 mg/g, based on the total amount of
albumin. Specifically, the amount of fatty acid to be carried can
be 0.1 mg/g to 0.8 mg/g, preferably 0.1 mg/g to 0.65 mg/g, or 0.2
mg/g to 0.8 mg/g, more preferably 0.2 mg/g to 0.65 mg/g, most
preferably 0.29 mg/g to 0.65 mg/g, based on the total amount of
albumin.
[0125] The additive of the present invention can further contain an
additive substance other than albumin as long as the desired effect
is not impaired. The additive substance is not particularly limited
as long as it does not inhibit proliferation of stem cells.
Examples thereof include growth factor (e.g., insulin etc.), iron
source (e.g., transferrin etc.), polyamines (e.g., putrescine
etc.), mineral (e.g., sodium selenate etc.), saccharides (e.g.,
glucose etc.), organic acid (e.g., pyruvic acid, lactic acid etc.),
amino acid (e.g., L-glutamine), reducing agent (e.g.,
2-mercaptoethanol), vitamins (e.g., ascorbic acid, d-biotin etc.),
steroid (e.g., .beta.-estradiol, progesterone etc.), antibiotic
(e.g., streptomycin, penicillin, gentamicin etc.), buffering agent
(e.g., HEPES etc.) and the like. In addition, additive substance(s)
that have been conventionally used for culturing stem cells can be
contained as appropriate. The additive substance is preferably
contained within a concentration range known per se.
[0126] The additive of the present invention may take any dosage
form as long as the desired effect is obtained and, for example,
solution, solid, powder and the like can be mentioned. When it is a
solid or powder, it is dissolved in an appropriate buffer and the
like to a desired concentration, and can be used.
[0127] The content of albumin carrying a reduced amount of fatty
acid in the additive of the present invention is not particularly
limited as long as the desired effect is obtained and, examples
thereof include 0.05 to 250 mg/mL, preferably 0.05 to 150 mg/mL,
more preferably 0.25 to 50 mg/mL, further preferably 2.5 to 25
mg/mL. The additive is added such that the final concentration of
albumin in the medium is 0.01 to 50 mg/mL, preferably 0.01 to 30
mg/mL, more preferably 0.05 to 10 mg/mL, further preferably 0.5 to
5 mg/mL, based on the total volume of the medium, and can be
preferably used for culturing stem cells.
[0128] The fatty acid that can be reduced in the additive of the
present invention is as mentioned above, and the definition of stem
cell in the additive of the present invention is also as mentioned
above.
6. Culture System of the Present Invention.
[0129] The present invention also provides a culture system of a
stem cell, comprising a step of cultivating in a medium comprising
an albumin carrying a reduced amount of fatty acid, wherein the
amount of the fatty acid to be carried is so selected as to enable
maintenance of the stem cell in an undifferentiated state (in the
present specification, sometimes to be described as "the culture
system of the present invention").
[0130] Being "so selected as to enable maintenance of the stem cell
in an undifferentiated state" means that an amount of fatty acid to
be carried which is within the range permitting growth of stem cell
in an undifferentiated state is selected. In the culture system of
the present invention, an amount of fatty acid to be carried which
is within the range permitting that, preferably, the proportion of
differentiated cells does not increase in the culture period, and
stem cells are cultured while said proportion is maintained within
about 10% at most, and more preferably, differentiated cells are
not substantially mixed, stem cells are cultured in an
undifferentiated state, and permitting growth of undifferentiated
cells can be selected. The undifferentiated state of stem cells can
be confirmed by a method known per se, and examples thereof include
methods such as confirmation by alkaline phosphatase staining,
confirmation of undifferentiated marker protein positive rate by
FACS, confirmation of colony by a microscope and the like. In
addition, cells judged by the method to be not in an
undifferentiated state can be identified as differentiated
cells.
[0131] The amount of fatty acid carried by albumin to be used for
the culture system of the present invention is not particularly
limited as long as it is reduced to an amount of fatty acid which
is so selected as to enable maintenance of stem cells in an
undifferentiated state. Examples thereof include an albumin
carrying not more than 10 mg/g, more preferably not more than 6
mg/g, of fatty acid, based on the total amount of albumin, and
still more preferably, an albumin carrying substantially no fatty
acid. In one embodiment, the amount of fatty acid carried by
albumin can be not more than 10 mg/g, more preferably not more than
6 mg/g, and not less than 0.1 mg/g, based on the total amount of
albumin. Specifically, the amount of fatty acid to be carried can
be 0.1 mg/g to 0.8 mg/g, preferably 0.1 mg/g to 0.65 mg/g or 0.2
mg/g to 0.8 mg/g, more preferably 0.2 mg/g to 0.65 mg/g, most
preferably 0.29 mg/g to 0.65 mg/g, based on the total amount of
albumin.
[0132] The fatty acid that can be reduced in the culture system of
the present invention is as mentioned above, and the definition of
stem cell in the culture system of the present invention is also as
mentioned above.
[0133] Other features of the invention will become apparent in the
course of the following descriptions of exemplary embodiments which
are given for illustration of the invention and are not intended to
be limiting thereof.
Examples
Materials and Methods
1. Purification of Albumin (Treatment with Activated Carbon)
[0134] To a solution (40 ml, 25%) of human serum albumin in saline
was added phosphate buffer (pH 7.2, 40 ml), and the mixture was
added to a phosphate buffer (20 ml) suspension of activated carbon
(5 g, manufactured by Wako Pure Chemical Industries, Ltd.)
previously heated at 200.degree. C. for 30 minutes. After stirring
at 4.degree. C. for 3 hours, the mixture was centrifuged at
4.degree. C., 11,900 rpm for 20 minutes. The activated carbon
sediment was removed by decantation, and the reaction mixture was
filtered through a 0.22 .mu.m syringe filter. The filtered solution
was diluted 100-fold, the absorbance was measured at wavelengths of
260 nm, 280 nm, 320 nm by UV absorption measuring apparatus UV1800
(manufactured by Shimadzu Corporation), and the concentration was
calculated by calculation formula (A280-A320)/0.55.times.100.
2. Cell Evaluation
[0135] Proliferative effect of various test compounds on induced
pluripotent stem cell (iPS cell) was evaluated. As the iPS cell,
201B7 strain purchased from iPS Academia Japan, Inc. was used. Cell
culture was performed under conditions of 5% CO.sub.2/37.degree. C.
and using a culture vessel (Nippon Becton Dickinson Company, Ltd.,
Falconculture petri dish or Falconculture plate) coated with a
basal membrane matrix.
[0136] Various test compounds were added at given concentrations to
a medium of "E8" composition (disclosed in Nature Methods, 2011, 8,
424-429, which is incorporated herein by reference in its entirety)
currently considered to be the minimum composition for cultivating
human pluripotent stem cells, and used for culture, from which the
effect was studied.
3. Measurement of the Amount of Fatty Acid Carried
[0137] A solution or powder corresponding to 10 mg of albumin was
prepared to 200 .mu.l with 1% brine, methanol (400 .mu.l) and
chloroform (200 .mu.l) were added, and the mixture was shaken for
10 minutes. Chloroform (200 .mu.l) and 1% brine (200 .mu.l) were
added, the mixture was shaken for 10 min and centrifuged at 10,000
rpm for 2 minutes, and the bottom layer was collected. The solvent
in the obtained chloroform layer was dried to solidness to give a
sample for assay. The sample was dissolved in 2-propanol, and the
amount of fatty acid was quantified based on oleic acid, by an
analytical curve method and using a fatty acid measurement kit
(LabAssay.TM. NEFA, manufactured by Wako Pure Chemical Industries,
Ltd.). The absorbance was measured using SH900 (CORONA ELECTRIC
Co., Ltd.).
Example 1
Influence of Various Albumins on Cell Culture
[0138] Human serum-derived albumin was added to a medium to a final
concentration of 2.6 g/L, and the culture results of each albumin
were compared. Several kinds of albumins were subjected to a
purification treatment (fatty acid removal treatment) to remove
lipid. The culture period was 1 week. 13,000 viable cells were
seeded as single-cell per well of a 6-well plate. As a basal
membrane matrix, a fragment containing an active domain of laminin
511, which was purchased from Osaka University, was applied at 5
.mu.g/well. Y-27632 was added (final concentration 10 .mu.M,
NACALAI TESQUE, INC.: 08945-84) to a medium to be used for seeding.
From the next day, the cells were cultured in a medium free of
Y-27632.
[0139] The experiment was performed in triplicate for each medium
and the results are shown in Table 1.
[0140] After the culture for 1 week, alkaline phosphatase (ALP)
staining was performed to confirm maintenance of undifferentiation
potency. For staining, alkaline phosphatase staining kit
(Sigma-Aldrich Co. LLC.: 86-R) was used. FIG. 1 shows the state
after culture of the cells marked with + or - in the column of
culture results. When albumin obtained from each of Nova Biologics,
Sigma, Biocell Laboratories was added to the medium, the cells did
not proliferate except a part thereof. However, when the albumin
after a purification treatment was added to the medium, the cells
proliferated. In addition, the amount of fatty acid carried (fatty
acid content) of all these albumins was measured to find the
results shown in Table 1. From the above results, it was found that
fatty acid bound to albumin inhibits normal cell proliferation.
TABLE-US-00001 TABLE 1 culture amount of fatty albumin derivation
purification results acid carried A human serum - + 0.5 B human
serum - - 14.9 B human serum + + 1.4 C human serum - - 14.6 C human
serum + + 2.2 purification: treatment with activated carbon amount
of fatty acid carried (mg/g)
Example 2
Influence of Fatty Acid Re-Addition on Purified Human Serum-Derived
Albumin
Fatty Acid Addition.
[0141] Oleic acid (12.6 .mu.l) was charged in a 15 ml falcon tube,
and a solution of purified human serum albumin B (5.9 ml, 8.8%) was
added. The solution was shaken at 37.degree. C. for 3 hours,
allowed to cool and filtered with a 0.22 .mu.m syringe filter.
[0142] In this way, albumin adsorbed with oleic acid was obtained.
Experiment was performed using a medium containing the obtained
albumin adsorbed with oleic acid at a final concentration of 2.6
g/L (concentration of oleic acid in the medium 196 .mu.M).
[0143] As for palmitic acid, stearic acid, linoleic acid, linolenic
acid and arachidonic acid, a similar treatment was performed using
the fatty acids at a dose described in Table 2 and purified human
serum albumin to give albumin adsorbed with various fatty acids,
and media added with albumin adsorbed with various fatty acids were
prepared.
[0144] The amount of fatty acid carried by albumin was not measured
after the above-mentioned adsorption operation.
Measurement of Fatty Acid Amount.
[0145] A solution or powder corresponding to 10 mg of albumin was
prepared to 200 .mu.l with 1% brine, methanol (400 .mu.l) and
chloroform (200 .mu.l) were added, and the mixture was shaken for
10 minutes. Chloroform (200 .mu.l) and 1% brine (200 .mu.l) were
added, the mixture was shaken for 10 minutes and centrifuged at
10,000 rpm for 2 minutes, and the bottom layer was collected. The
solvent in the obtained chloroform layer was dried to solidness to
give a sample for assay. The sample was dissolved in 2-propanol,
and the amount of fatty acid was quantified based on oleic acid, by
an analytical curve method and using a fatty acid measurement kit
(LabAssay.TM. NEFA, manufactured by Wako Pure Chemical Industries,
Ltd.). The absorbance was measured using SH900 (CORONA ELECTRIC
Co., Ltd.).
[0146] The fatty acids added to the media, concentration of fatty
acids added, and the content of fatty acid per 1 g albumin (amount
of fatty acid carried) are shown in Table 2.
TABLE-US-00002 TABLE 2 addition amount of fatty albumin fatty acid
added concentration (.mu.M) acid carried B oleic acid 196 18.6 B
oleic acid 59 6.8 B oleic acid 20 2.8 B oleic acid 60 6.1 B oleic
acid 200 10.9 B palmitic acid 20 1.3 B palmitic acid 60 5.5 B
palmitic acid 200 12.1 B stearic acid 200 12.1 B linoleic acid 200
15.3 B linolenic acid 200 23.6 B arachidonic acid 200 14 amount of
fatty acid carried (mg/g)
Cell Evaluation.
[0147] Using a medium containing, at the concentration indicated in
Table 2, an albumin adsorbed with fatty acid, stem cells were
cultured. The culture period was 1 week. 13,000 viable cells were
single-cell seeded per well of a 6-well plate. As a basal membrane
matrix, a fragment containing an active domain of laminin 511,
which was purchased from Osaka University, was applied at 5
.mu.g/well. Y-27632 was added (final concentration 10 .mu.M) to a
medium to be used for seeding. From the next day, the cells were
cultured in a medium free of Y-27632.
[0148] The cells were detached from each well by TrypLE Select
(Life Technologies: 12563-011), and the number of viable cells in
the well was measured. The results are shown in FIG. 2. Addition of
oleic acid and palmitic acid was found to suppress cell
proliferation. The suppressive effect was in proportion to the
concentration of fatty acid added, and addition of a high
concentration of fatty acid resulted in stronger suppression of
cell proliferation. From the above-mentioned results, it was found
that cell culture efficiency decreases in a manner dependent on the
concentration of fatty acid adsorbed to albumin.
Example 3
Influence of Human Serum-Derived Albumin with Varying Purification
Level on iPS Cell (FIG. 3)
[0149] Human serum albumins with the following five purification
levels were prepared.
[0150] Group (I): Human serum albumin (Sigma) treated by a fatty
acid removal treatment. The amount of fatty acid carried is
considered to be not more than 0.07 mg/g.
[0151] Group (II): Human serum-derived albumin (NOVA Biologics,
INC.) was used after purifying with 50 wt % of activated carbon
relative to the weight of albumin at pH 4. The measurement result
of the amount of fatty acid carried was 0.29 mg/g.
[0152] Group (III): Human serum-derived albumin (NOVA Biologics,
INC.) was used after purifying with 50 wt % of activated carbon
relative to the weight of albumin at pH 7. The measurement result
of the amount of fatty acid carried was 0.65 mg/g.
[0153] Group (IV): Human serum-derived albumin (NOVA Biologics,
INC.) was used after purifying with 25 wt % of activated carbon
relative to the weight of albumin at pH 7. The measurement result
of the amount of fatty acid carried was 0.92 mg/g.
[0154] Group (V): Human serum-derived albumin (NOVA Biologics,
INC.) was used after purifying with 13 wt % of activated carbon
relative to the weight of albumin at pH 7. The measurement result
of the amount of fatty acid carried was 1.86 mg/g.
[0155] Each above-mentioned group of human serum albumin was added
to a medium at a final concentration of 2.6 g/L, and iPS cells were
cultured. A 6-well plate coated with a fragment containing an
active domain of laminin 511 at 5 .mu.g/well (iMatrix-511 (Nippi,
Incorporated)) as a basal membrane matrix was used. iPS cells were
single-cell seeded at 13,000 cells/well and cultured for 1 week.
Y-27632 (NACALAI TESQUE, INC.: 08945-84) was added at a final
concentration of 10 .mu.M only to a medium to be used for
seeding.
[0156] The cells were detached from each well by TrypLE Select
(Life Technologies: 12563-011), and the number of viable cells was
measured. FIG. 3 shows mean of three independent experiments for
each group. When medium performance is free of problems, the number
of viable iPS cells becomes not less than about 3.0.times.10.sup.5
cells when cultured for one week by this culture method. As shown
in FIG. 3, groups (I) to (III) showed good cell proliferation with
a cell count exceeding 3.0.times.10.sup.5. On the other hand,
groups (IV) and (V) showed a clear growth inhibitory action.
Therefrom it is clear that iPS cells can proliferate well at least
when the amount of fatty acid carried by albumin is not more than
0.65 mg/g.
[0157] Groups (I) to (III) were further cultured for 1 week after
the above-mentioned culture, and the differentiation rate was
measured by alkaliphosphatase (ALP) staining. The differentiation
rate (%) was calculated by ALP negative colony number/total colony
number.times.100 of each well. The differentiation rates of groups
(I), (II), and (III) were 0.4%, 1.5% and 2.6%, respectively. These
numerical values can be said to be sufficiently low, which has
demonstrated that all conditions are suitable for proliferating iPS
cells while maintaining an undifferentiated state thereof. Similar
results were also obtained by observation of colony under a
microscope, and it was confirmed that a higher purification level
of albumin leads to the maintenance of undifferentiated state of
iPS cells at a higher rate.
Example 4
Influences of 7 Kinds of Purified Human Serum-Derived Albumins
Re-Carrying Fatty Acid (FIG. 4)
[0158] Octanoic acid (18.3 .mu.L) was charged in a 50 mL falcone
tube, and a human serum albumin solution B (15 mL, 10%, Sigma)
after a fatty acid removal treatment was added. The solution was
shaken at 37.degree. C. for 7 hours, left standing at 4.degree. C.
overnight, and filtered with a 0.22 .mu.m syringe filter. In this
way, an albumin re-adsorbed with octanoic acid was obtained. The
amount of fatty acid carried by albumin re-adsorbed with octanoic
acid was measured, and the albumin after re-adsorption with
octanoic acid and a purified albumin before re-adsorption and after
a fatty acid removal treatment were appropriately mixed while
adjusting the ratio such that the final concentration of octanoic
acid in the medium was 28 .mu.M or 57 .mu.M. The mixture was added
to a medium such that the final concentration of albumin was 2.6
g/L.
[0159] As for oleic acid, stearic acid, palmitic acid, linoleic
acid, linolenic acid and arachidonic acid, re-adsorption of fatty
acid was performed in the same manner. The final concentration in
and the amount of addition to the medium are also the same.
[0160] Using the medium produced as mentioned above, the influence
of each fatty acid on the proliferation of iPS cells was studied. A
6-well plate coated with a fragment containing an active domain of
laminin 511 at 5 .mu.g/well (iMatrix-511 (Nippi, Incorporated)) as
a basal membrane matrix was used. iPS cells were single-cell seeded
at 13,000 cells/well and cultured for 1 week. Y-27632 (NACALAI
TESQUE, INC.: 08945-84) was added at a final concentration of 10
.mu.M only to a medium to be used for seeding. As a positive
control, IFS cells were cultured using an albumin free of
re-adsorption of fatty acid, i.e., albumin after a fatty acid
removal treatment.
[0161] The cells were detached from each well by TrypLE Select
(Life Technologies: 12563-011), and the number of the viable cells
was measured. FIG. 4 shows mean of three independent experiments
for each group.
[0162] When stearic acid, palmitic acid and arachidonic acid were
re-adsorbed, the cells died at both 28 .mu.M and 57 .mu.M, and
viable cells could not be obtained. Linoleic acid and linolenic
acid had a strong proliferation inhibitory action on iPS cells, and
the cells died by re-adsorption at 57 .mu.M, and the proliferation
of iPS cells was markedly suppressed even by re-adsorption at 28
.mu.M, as compared to the positive control. Even when oleic acid
was re-adsorbed, a concentration-dependent cell proliferation
inhibitory action was found, and the number of viable cells was
smaller than that of the positive control for both 28 .mu.M and 57
.mu.M. In the case of octanoic acid, however, a certain cell
proliferation inhibitory action was found by re-adsorption at 57
.mu.M, but the number of viable cells was equivalent to that of the
positive control by re-adsorption at 28 .mu.M. From these studies,
it was demonstrated that when oleic acid, stearic acid, palmitic
acid, linoleic acid, linolenic acid or arachidonic acid is
re-adsorbed to albumin, the albumin shows an inhibitory action on
the proliferation of iPS cells, irrespective of the concentration
of addition. When octanoic acid is re-adsorbed, it was demonstrated
that the number of viable cells is equivalent to that of the
positive control depending on the concentration of re-adsorption,
and the inhibitory action thereof is low.
[0163] From the above, it was shown that long chain fatty acids
such as oleic acid and the like have strong toxicity to iPS cells
and a high proliferation inhibitory action as compared to middle
fatty acids such as octanoic acid and the like.
INDUSTRIAL APPLICABILITY
[0164] Using the medium of the present invention, stem cells can be
proliferated while maintaining an undifferentiated state.
Furthermore, using the medium of the present invention, stem cells
can be efficiently proliferated, the frequency of exchange of
culture medium can be reduced, and the culture cost of stem cells
can be decreased.
[0165] Where a numerical limit or range is stated herein, the
endpoints are included. Also, all values and subranges within a
numerical limit or range are specifically included as if explicitly
written out.
[0166] As used herein the words "a" and "an" and the like carry the
meaning of "one or more."
[0167] Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that, within the scope of the
appended claims, the invention may be practiced otherwise than as
specifically described herein.
[0168] All patents and other references mentioned above are
incorporated in full herein by this reference, the same as if set
forth at length.
* * * * *