U.S. patent application number 17/080128 was filed with the patent office on 2021-02-11 for method for manufacturing stem cell having appropriate size for intravascular administration.
This patent application is currently assigned to K-STEMCELL CO., LTD. The applicant listed for this patent is K-STEMCELL CO., LTD., JEONG CHAN RA. Invention is credited to Sung Keun KANG, Jeong Chan RA, Il Seob SHIN.
Application Number | 20210040453 17/080128 |
Document ID | / |
Family ID | 1000005170423 |
Filed Date | 2021-02-11 |
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United States Patent
Application |
20210040453 |
Kind Code |
A1 |
RA; Jeong Chan ; et
al. |
February 11, 2021 |
METHOD FOR MANUFACTURING STEM CELL HAVING APPROPRIATE SIZE FOR
INTRAVASCULAR ADMINISTRATION
Abstract
The present invention relates to a method for preparing stem
cells having a size suitable for intravascular administration, and
preferably to a method for preparing stem cells with a diameter
ranging from 11-16 .mu.m. Additionally, the present invention
relates to media composition for preparing stem cells having a size
suitable for intravascular administration. According to the present
invention, stem cells having a size suitable for intravascular
administration can be prepared such that stem cells administered
into a vein can stably reach a target tissue, and thus can more
efficiently increase efficacy displaying activity, thereby
innovatively enhancing the efficacy of cell therapy using stem
cells.
Inventors: |
RA; Jeong Chan; (Suwon-si,
KR) ; KANG; Sung Keun; (Seoul, KR) ; SHIN; Il
Seob; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
RA; JEONG CHAN
K-STEMCELL CO., LTD. |
SUWON-SI
Seoul |
|
KR
KR |
|
|
Assignee: |
K-STEMCELL CO., LTD
Seoul
KR
RA; JEONG CHAN
Suwon-si
KR
|
Family ID: |
1000005170423 |
Appl. No.: |
17/080128 |
Filed: |
October 26, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14395170 |
Oct 17, 2014 |
10815459 |
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PCT/KR2013/003251 |
Apr 17, 2013 |
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17080128 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/33 20130101;
C12N 2501/39 20130101; C12N 2501/115 20130101; C12N 2500/14
20130101; C12N 2500/32 20130101; C12N 5/0667 20130101; C12N 2500/38
20130101; C12N 2501/11 20130101 |
International
Class: |
C12N 5/0775 20060101
C12N005/0775 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 18, 2012 |
KR |
10-2012-0040488 |
Claims
1.-17. (canceled)
18. A medium composition for preparing stem cells having a size
suitable for intravascular administration, comprising K-SFM
containing N-acetyl-L-cysteine (NAC), insulin or insulin-like
factor, hydrocortisone, basic fibroblast growth factor (bFGF),
about 5% FBS (fetal bovine serum), calcium, EGF (epidermal growth
factor) and about 1 ng/ml of selenium.
19. The medium composition of claim 18, wherein the stem cells are
adult stem cells.
20. The method composition of claim 19, wherein the stem cells are
adipose tissue-derived mesenchymal stem cells.
Description
TECHNICAL FIELD
[0001] The present invention relates to a method for preparing stem
cells having a size suitable for intravascular administration, and
more particularly to a method for preparing stem cells having a
diameter of 1-16 .mu.m, which is suitable for intravascular
administration.
BACKGROUND ART
[0002] Stem cells refer to cells having not only self-replicating
ability but also the ability to differentiate into at least two
types of cells, and can be divided into totipotent stem cells,
pluripotent stem cells, and multipotent stem cells. Totipotent stem
cells are cells having totipotent properties capable of developing
into one perfect individual, and these properties are possessed by
cells up to the 8-cell stage after the fertilization of an oocyte
and a sperm. When these cells are isolated and transplanted into
the uterus, they can develop into one perfect individual.
Pluripotent stem cells, which are cells capable of developing into
various cells and tissues derived from the ectodermal, mesodermal
and endodermal layers, are derived from an inner cell mass located
inside of blastocysts generated 4-5 days after fertilization. These
cells are called "embryonic stem cells" and can differentiate into
various other tissue cells but not form new living organisms.
Multipotent stem cells, which are stem cells capable of
differentiating into only cells specific to tissues and organs
containing these cells, are involved not only in the growth and
development of various tissues and organs in the fetal, neonatal
and adult periods but also in the maintenance of homeostasis of
adult tissue and the function of inducing regeneration upon tissue
damage. Tissue-specific multipotent cells are collectively called
"adult stem cells".
[0003] Adult stem cells are obtained by taking cells from various
human organs and developing the cells into stem cells and are
characterized in that they differentiate into only specific
tissues. However, recently, experiments for differentiating adult
stem cells into various tissues, including liver cells, were
dramatically successful, which comes into spotlight. In particular,
efforts have been made in the field of regenerative medicine for
regenerating biological tissues and organs and recovering their
functions that were lost due to illness or accident and the like by
using cells. Methods which are frequently used in this field of
regenerative medicine comprise the steps of: collecting stem cells,
blood-derived mononuclear cells or marrow-derived mononuclear cells
from a patient; inducing the proliferation and/or differentiation
of the cells by tube culture; and introducing the selected
undifferentiated (stem cells and/or progenitor cells) and/or
differentiated cells into the patient's body by transplantation.
Accordingly, existing classical methods for treating diseases by
medication or surgery are expected to be replaced with cell/tissue
replacement therapy which replaces a damage cell, tissue or organ
with healthy one, and thus the utility of stem cells will further
increase.
[0004] Thus, the various functions of stem cells are currently
being studied. Particularly, since cell therapy technology using
mesenchymal stem cells started to receive attention, technology for
improving mesenchymal stem cells so as to be suitable for
therapeutic purposes have been developed (WO 2006/019357, Korean
Patent No. 0795708, and Korean Patent No. 0818214).
[0005] However, technology related to a method for preparing stem
cells suitable for intravascular administration has not yet been
sufficiently studied.
[0006] Accordingly, the present inventors have found that, when
stem cells are cultured in a medium containing a basal medium and
at least two components selected from the group consisting of
N-acetyl-L-cysteine (NAC), insulin or insulin-like factor,
hydrocortisone, basic fibroblast growth factor (bFGF), and
antioxidant, stem cells having a size suitable for intracellular
administration can be prepared, thereby completing the present
invention.
DISCLOSURE OF INVENTION
Technical Problem
[0007] It is an object of the present invention to provide a method
for preparing stem cells having a size suitable for intravascular
administration.
[0008] Another object of the present invention is to provide a
medium composition for preparing stem cells having a size suitable
for intravascular administration.
Technical Solution
[0009] To achieve the above objects, the present invention provides
a method for preparing stem cells having a size suitable for
intravascular administration, the method comprising the step of
culturing stem cells in a medium containing a basal medium; and at
least two components selected from the group consisting of
N-acetyl-L-cysteine (NAC), insulin or insulin-like factor,
hydrocortisone, basic fibroblast growth factor (bFGF), and
antioxidant.
[0010] The present invention also provides a medium composition for
preparing stem cells having a size suitable for intravascular
administration, the medium composition containing a basal medium;
and at least two components selected from the group consisting of
N-acetyl-L-cysteine (NAC), insulin or insulin-like factor,
hydrocortisone, basic fibroblast growth factor (bFGF), and
antioxidant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a graphic diagram showing the size of
adipose-derived mesenchymal stem cells according to medium and
passage number.
[0012] FIG. 2 is a graphic diagram showing the marker expression of
adipose-derived mesenchymal stem cells according to medium.
BEST MODE FOR CARRYING OUT THE INVENTION
[0013] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains.
Generally, the nomenclature used herein and the experiment methods
are those well known and commonly employed in the art.
[0014] As used herein, the term "stem cells" refer to cells having
not only self-replicating ability but also the ability to
differentiate into at least two types of cells. "Adult stem cells"
refer to stem cells that appear either in the stage in which each
organ of an embryo is formed after the developmental process or in
the adult stage.
[0015] As used herein, the term "mesenchymal stem cells" refers to
undifferentiated stem cells that are isolated from human or
mammalian tissue and may be derived from various tissues.
Particularly, the mesenchymal stem cells may be umbilical
cord-derived mesenchymal stem cells, umbilical cord blood-derived
mesenchymal stem cells, bone marrow-derived mesenchymal stem cells,
adipose-derived mesenchymal stem cells, muscle-derived mesenchymal
stem cells, nerve-derived mesenchymal stem cells, skin-derived
mesenchymal stem cells, amnion-derived mesenchymal stem cells, or
placenta-derived mesenchymal stem cells. Technology for isolating
stem cells from each tissue is already known in the art.
[0016] As used herein, "adipose tissue-derived mesenchymal stem
cells" are undifferentiated adult stem cells isolated from adipose
tissue and are also referred to herein as "adipose-derived adult
stem cells", "adipose stem cells", or "adipose-derived stem cells".
These cells can be obtained according to any conventional method
known in the art. A method for isolating adipose tissue-derived
mesenchymal stem cells may be, for example, as follows. That is,
adipose-derived mesenchymal stem cells can be isolated by culturing
an adipose-containing suspension (in physiological saline) obtained
by liposuction, and then either collecting a stem cell layer,
attached to a culture container such as a flask, by trypsin
treatment, or directly collecting those suspended in a small amount
of physiological saline by rubbing with a scraper.
[0017] In the present invention, "stem cells having a size suitable
for intravascular administration" refers to stem cells that have a
diameter of preferably 11-16 .mu.m, which is smaller than the
diameter of veins or capillary vessels, such that the stem cells,
when administered intravascularally, can easily migrate to their
target tissue without interfering with blood flow or circulation
and can exhibit their activity in the target tissue.
[0018] Stem cells can be administered into the body by various
routes, for example, intravenously, intra-arterially or
intraperitoneally. Among such administration routes, intravenous
administration is preferred, because it enables a disease to be
treated in a convenient and safe manner without surgical operation.
In order for intravenously administered stem cells to securely
reach the target site and to exhibit a desired therapeutic effect,
various requirements should be satisfied. First, stem cells should
have a size suitable for intravascular administration such that
these stem cells, when administered intravascularly, neither reduce
blood flow velocity nor form thrombi. The degree of proliferation
of mesenchymal stem cells that can be derived from various tissues
differs between patients and varies depending on their origin,
culture condition or method, and the size thereof also varies from
about 10 to 300 .mu.m in diameter. However, human post-capillary
venules have a diameter of about 10-50 .mu.m, arterioles have a
diameter of 8-30 .mu.m (Schmidt GT, 1989), and capillary vessels
have a diameter of about 8 .mu.m (Schmidt GT, 1989; Chien, 1975;
John Ross, 1991; Herbert et al., 1989; Arthur and Guyton, 1997;
Renkin, 1989; Gaehtgens, 1980; Row 1979), which are smaller than
the diameter of general mesenchymal stem cells. Thus, when
mesenchymal stem cells having a relatively large size are
administered intravascularly, they can influence intravascular
activity. Specifically, they can reduce blood flow velocity and
also interfere with blood circulation to cause blood flow stoppage,
thrombus formation and vascular obstruction, even leading to death.
In connection with this, it was reported that, when mesenchymal
stem cells having a diameter of about 16-53 .mu.m administered
intravenously to mice, blood flow velocity was reduced and the
induction of myocardial infarction and thrombus formation was
observed (D. Furlani et al. Microvasular Research 77 (2009)
370-376). Thus, it is important to administer stem cells having a
suitable size by an intracellular route. Moreover, stem cells
should not be disrupted or aggregated before intravascular
administration, and should securely reach their target site as
single cells without disruption or aggregation after intravascular
administration. In addition, stem cells should be administered at a
certain concentration or higher concentration such that they
exhibit a desired therapeutic effect after they reached the target
site. In view of several requirements as described above, the
present invention is intended to provide stem cells that have a
size suitable for intravascular administration so as to securely
exhibit a therapeutic effect without reducing blood flow velocity
or forming thrombi after intravascular administration.
[0019] In one aspect, the present invention is directed to a method
for preparing stem cells having a size suitable for intravascular
administration, the method comprising the step of culturing stem
cells in a medium containing a basal medium; and at least two
components selected from the group consisting of
N-acetyl-L-cysteine (NAC), insulin or insulin-like factor,
hydrocortisone, basic fibroblast growth factor (bFGF), and
antioxidant.
[0020] The basal medium used in the present invention refers to a
typical medium having a simple composition known as being suitable
for the culture of stem cells. Examples of the basal medium
generally used to culture the stem cells include MEM (Minimal
Essential Medium), DMEM (Dulbecco modified Eagle Medium), RPMI
(Roswell Park Memorial Institute Medium), and K-SFM (Keratinocyte
Serum Free Medium). As the basal medium used in the present
invention, any mediums can be used without any limitation as long
as they are used in the art. Preferably, the basal medium may be
selected from the group consisting of M199/F12 (mixture)(GIBCO),
MEM-alpha medium (GIBCO), low-concentration glucose-containing DMEM
medium (Welgene), MCDB 131 medium (Welgene), IMEM medium (GIBCO),
K-SFM, DMEM/F12 medium, PCM medium, and MSC expansion medium
(Chemicon). Particularly, among them, K-SFM may be preferably
used.
[0021] A basal medium that is used to obtain the cultured
mesenchymal stem cells may be supplemented with additives in the
art, which promote the proliferation of mesenchymal stem cells in
an undifferentiated state while inhibiting the differentiation
thereof. Also, the medium may contain a neutral buffer (such as
phosphate and/or high-concentration bicarbonate) in isotonic
solution, and a protein nutrient (e.g., serum such as FBS, FCS
(fetal calf serum) or horse serum, serum replacement, albumin, or
essential or non-essential amino acid such as glutamine or
L-glutamine). Furthermore, it may contain lipids (fatty acids,
cholesterol, an HDL or LDL extract of serum) and other ingredients
found in most stock media of this kind (such as insulin or
transferrin, nucleosides or nucleotides, pyruvate, a sugar source
such as glucose, selenium in any ionized form or salt, a
glucocorticoid such as hydrocortisone and/or a reducing agent such
as .beta.-mercaptoethanol).
[0022] Also, for the purpose of preventing cells from adhering to
each other, adhering to a container wall, or forming too large
clusters, the medium may advantageously contain an anti-clumping
agent, such as one sold by Invitrogen (Cat #0010057AE).
[0023] Among them, one or more of the following additional
additives may advantageously be used: [0024] stem cell factor (SCF,
Steel factor), other ligands or antibodies that dimerize c-kit, and
other activators of the same signaling pathway [0025] ligands for
other tyrosine kinase related receptors, such as the receptor for
platelet-derived growth factor (PDGF), macrophage
colony-stimulating factor, Flt-3 ligand and vascular endothelial
growth factor (VEGF) [0026] factors that elevate cyclic AMP levels,
such as forskolin [0027] factors that induce gp130 such as LIF or
Oncostatin-M [0028] hematopoietic growth factors such as
thrombopoietin (TPO) [0029] transforming growth factors such as
TGF.beta.1 [0030] neurotrophins such as CNTF [0031] antibiotics
such as gentamicin, penicillin or streptomycin.
[0032] The medium that is used in the present invention may
contain, in addition to the basal medium, at least two components
selected from the group consisting of N-acetyl-L-cysteine (NAC),
insulin or insulin-like factor, hydrocortisone, basic fibroblast
growth factor (bFGF), and antioxidant.
[0033] Specifically, the medium may contain insulin-like factor as
insulin replacement, which functions to promote cell growth by
enhancing glucose metabolism and protein metabolism. Particularly,
recombinant IGF-1 (insulin-like growth factor-1) is preferably
used. The preferred content of insulin-like factor is 10-50 ng/ml.
If the content of insulin-like factor is less than 10 ng/ml,
apoptosis will occur, and if the content is more than 50 ng/ml, it
will increase the cytotoxicity and cost of the medium.
[0034] The medium may contain basic fibroblast growth factor (bFGF)
that can induce various types of cell proliferation in vivo.
Preferably, recombinant bFGF protein is used. The preferred content
of bFGF is 1-100 ng/ml.
[0035] Examples of an antioxidant that may be used in the present
invention include selenium, ascorbic acid, vitamin E, catechin,
lycopene, .beta.-carotene, coenzyme Q-10, EPA (eicosapentaenoic
acid), DHA (docosahexanoic acid) and the like. Preferably, selenium
may be used. In an example of the present invention, selenium was
used as an antioxidant. The content of selenium in the medium is
preferably 0.5-10 ng/ml. If the content of selenium is less than
0.5 ng/ml, the medium will be sensitive to oxygen toxicity, and if
the content is more than 10 ng/ml, it will cause severe
cytotoxicity.
[0036] The medium that is used in the present invention may
additionally contain a component selected from the group consisting
of FBS (fetal bovine serum), calcium and EGF. Epidermal growth
factor (EGF) can induce various types of cell proliferation in
vivo, and recombinant EGF protein is preferably used. The preferred
content of epidermal growth factor is 10-50 ng/ml. If the content
of epidermal growth factor in the medium is less than 10 ng/ml, it
will have no particular effect, and if the content is more than 50
ng/ml, it will be toxic to cells.
[0037] Stem cells cultured in the medium according to the present
invention preferably have a diameter of 11-16 .mu.m, and thus are
suitable for intravascular administration.
[0038] Thus, in another aspect, the present invention is directed
to a medium composition for preparing stem cells having a size
suitable for intravascular administration, the medium composition
containing a basal medium; and at least two components selected
from the group consisting of N-acetyl-L-cysteine (NAC), insulin or
insulin-like factor, hydrocortisone, basic fibroblast growth factor
(bFGF), and antioxidant.
[0039] In an example of the present invention, adipose-derived
mesenchymal stem cells were cultured in the medium of the present
invention. Adipose-derived mesenchymal stem cells can be obtained
in the following manner. First, human adipose tissue obtained from
the abdomen by liposuction or the like is isolated and washed with
PBS, after which the tissue is cut finely and degraded using DMEM
medium containing collagenase. The degraded tissue is washed with
PBS and centrifuged at 1000 rpm for 5 minutes. The supernatant is
removed, and the pellet remaining at the bottom is washed with PBS,
and then centrifuged at 1000 rpm for 5 minutes. The resulting cells
are filtered through a 100-mesh filter to remove the debris, and
then washed with PBS. The cells are cultured overnight in DMEM
medium (10% FBS, 2 mM NAC, 0.2 mM ascorbic acid), and then the
cells that did not adhere to the bottom of the culture container
were washed out with PBS, and the cells are subcultured while the
medium was replaced with K-SFM medium containing NAC, ascorbic
acid, calcium, rEGF, insulin and hydrocortisone at 2-day intervals,
thereby obtaining adipose-derived mesenchymal stem cells. In
addition to this method, any method known in the art may also be
used to obtain mesenchymal stem cells.
[0040] The preparation method of the present invention may further
comprise the step of treating the stem cells, cultured in the
medium of the present invention, with trypsin. When the cultured
stem cells are treated with trypsin, stem cells in the form of
single cells can be obtained. Herein, trypsin functions to inhibit
intercellular aggregation such that the cells are maintained as
single cells. Any substance capable of inhibiting intercellular
aggregation may also be used in place of trypsin.
[0041] The preparation method of the present invention may further
comprise a step of suspending the stem cells, cultured in the
medium of the present invention, in an aspirin-containing solution.
When the cultured stem cells are suspended in an aspirin-containing
solution, the stem cells can effectively be prevented from being
disrupted or aggregated during transport or storage. Thus, the stem
cells that are used for intravascular administration may be used
after they are suspended in an aspirin-containing physiological
saline. The aspirin-containing solution refers to a solution
containing an aspirin compound. The solvent of the solution may be
physiological saline. In addition to physiological saline, any
base, such as Hartman-D solution or PBS (phosphate buffered
saline), which is generally used in the art, may be used without
limitation. As the aspirin, not only a commercially available
aspirin formulation, but also an aspirin-like compound may be used.
The amount of aspirin added is preferably 0.0001-0.01 mg/ml. If the
amount of aspirin added is larger than the upper limit of the above
range, cell viability can decrease, and if the amount is smaller
than the lower limit of the above range, the effect of inhibiting
cell aggregation can be insufficient.
EXAMPLES
[0042] Hereinafter, the present invention will be described in
further detail with reference to examples. It will be obvious to a
person having ordinary skill in the art that these examples are
illustrative purposes only and are not to be construed to limit the
scope of the present invention.
Example 1: Isolation of Human Adipose Tissue-Derived Mesenchymal
Stem Cells
[0043] Adipose tissue isolated from abdominal tissue by liposuction
was washed with PBS and cut finely, after which the tissue was
digested in DMEM media supplemented with collagenase type 1 (1
mg/ml) at 37.degree. C. for 2 hours. The collagenase-treated tissue
was washed with PBS and centrifuged at 1000 rpm for 5 minutes. The
supernatant was removed, and the pellet was washed with PBS and
then centrifuged at 1000 rpm for 5 minutes. The resulting cells
were filtered through a 100-mesh filter to remove debris, after
which the cells were washed with PBS and cultured overnight in DMEM
medium containing 10% FBS, 2 mM NAC (N-acetyl-L-cysteine) and 0.2
mM ascorbic acid.
[0044] Then, non-adherent cells were removed by washing with PBS,
and the remaining cells were cultured for 3 passages while the
medium was replaced with K-SFM (keratinocyte serum free medium)
containing 5% FBS, 2 mM NAC, 0.2 mM ascorbic acid, 0.09 mM calcium,
5 ng/ml rEGF, 5 ng/ml insulin, 10 ng bFGF and 74 ng/ml
hydrocortisone and 1 ng/ml selenium) at 2-day intervals, thereby
isolating adipose-derived mesenchymal stem cells.
[0045] The adipose-derived mesenchymal stem cells obtained by
culture for 3 passages in Example 1 were seeded in each of the
following media and were cultured for 5 days. At 5 days of culture,
the size and characteristics of the cells were measured.
[0046] Medium Composition
[0047] 1) KSFM+additive: K-SFM+FBS+NAC+ascorbic
acid+calcium+rEGF+insulin+bFGF+hydrocortisone+selenium;
[0048] 2) DMEM+FGF: DMEM (low-glucose)+10% FBS+5 ng/ml FGF;
[0049] 3) .alpha.-MEM+FGF: .alpha.-MEM+10% FBS+5 ng/ml FGF.
TABLE-US-00001 TABLE 1 Cell size (.mu.m) according to medium
composition Passage 2 Passage 3 Passage 4 KSFM + additive 14 .+-.
2.0 13.8 .+-. 2.2 14.1 .+-. 2.4 DMEM + FGF 20 .+-. 1.7 21.8 .+-.
1.4 22.1 .+-. 1.8 .alpha.-MEM + FGF 20.3 .+-. 2.1.sup. 19.2 .+-.
1.8 22.0 .+-. 1.9
[0050] The cell size of the cells of the `KSFM+additive` culture
group was the smallest, and the change in the cell size of the
group cultured using .alpha.-MEM as the basal medium was greater
than that of the DEME group. The cells of the `KSFM+additive` group
maintained a size of 11-16 .mu.m, whereas other medium groups
showed a cell size larger than 20 .mu.m as the number of passages
increased (see FIG. 1).
[0051] The characteristics of the passage-4 adipose-derived
mesenchymal stem cells obtained using the above-described culture
media were analyzed by FACS.
TABLE-US-00002 TABLE 2 KSFM + additive DMEM + FGF .alpha.-MEM + FGF
CD 29 99.0% 99.4% 99.9% CD 90 99.7% 99.6% 99.6% CD 31 0.1% 4.2%
0.5% CD 34 0.0% 2.3% 0.2% CD 45 0.1% 4.2% 2.1%
[0052] The characteristics of the adipose-derived mesenchymal stem
cells obtained using the above-described media were analyzed, and
as a result, it was shown that the media had no effect on the
expression of positive markers of the stem cells, but the
expression level of negative markers was slightly higher in the
cells of the DMEM+FGF medium group than in the cells of the
`KSFM+additive` medium group (see FIG. 2).
[0053] The stem cells isolated in Example 1 were observed by SEM
(scanning electron microscopy), and as a result, it could be seen
that the stem cells mostly had a diameter of about 11-16 .mu.m and
that an aggregate of the cells was not formed.
Example 2: Investigation of Medium Components for Preparation of
Small-Size Stem Cells
[0054] Media free of each of FBS, NAC, ascorbic acid, calcium,
rEGF, 5 ng/ml insulin, bFGF, hydrocortisone and selenium, which are
active ingredients added to the K-SFM medium used in Example 1,
were prepared, and adipose-derived stem cells were cultured in the
`KSFM+additive` medium group and the media free of each of the
active ingredients. After the cells were cultured in each of the
media for 3 passages, the cells were treated with trypsin, and then
the diameter of the cells was measured with a confocal microscope.
As a result, it could be seen that the size of the adipose-derived
stem cells cultured in the selenium-containing medium was 11.6-16.5
.mu.m, but the size of the stem cells cultured in the selenium-free
medium was 18.1-23.9 .mu.m.
INDUSTRIAL APPLICABILITY
[0055] According to the present invention, stem cells having a
diameter of 11-16 .mu.m can be prepared, which easily migrate to
their target tissue and have high stability. Thus, the effect of
intravascular administration of the stem cells on cell therapy can
be significantly increased.
[0056] Although the present invention has been described in detail
with reference to the specific features, it will be apparent to
those skilled in the art that this description is only for a
preferred embodiment and does not limit the scope of the present
invention. Thus, the substantial scope of the present invention
will be defined by the appended claims and equivalents thereof.
* * * * *