Cosmetic agent or Skin regeneration promoter comprising a culture sup of
non-human stem cells, and a method for introducing a protein
Technical Field
[0001]
The present invention relates to a cosmetic including culture sup of a stem
cell selected from the group consisting of non-human dental pulp stem cell, bone
marrow stem cell, and adipose stem cell.
Background Art
[0002]
It is known that aged people have more blotches and wrinkles, which is
caused by increasing amount of exposure to ultraviolet B (UVB) with advancing age.
It is considered that UVB increases production amount of collagenase by human
dermal fibroblast in dermis (HDF) so that it accelerates to collagen lysis in dermis
and induces the deposition of degenerated elastic tissue, resulting the wrinkles and
yellowing the skin.
Since ancient times, cosmetics have been produced by using plant derived
ingredients as raw materials and used. In order to prevent to weaken the skin by
aging, for example, such as wrinkle and fine lines formation and to improve
moisture-retaining property, recently, the cosmetic combined with animal derived
ingredients such as placenta, squalane, collagen and the like became commercially
available widely.
Also, in order to achieve the same purposes, the cosmetics including
culture sup of cells has been developed. It is proposed to culture smooth muscle
cells of human lung, epidermal cells, and fibroblast cells, and homogenates thereof
or culture supernatant to use them as components for the cosmetics (see JP 2005 -
336188 A, herein below, it is referred to as the prior art 1 ).
[0003]
On the other hand, it is known a method to use stem cells from human
bone marrow, dental pulp and the like, not but the culture sup, as a composition for
treating neurological disease (WO 02/086108, herein below, it is referred to as the
prior art 2).
2
Alternatively, it is proposed that the culture sup of human stem cells from
exfoliated deciduous tooth (stem cells from exfoliated deciduous tooth; SHED) is
used as the composition for treating lesion site (international publication WO
2011/118795, herein below, it is referred to as the prior art 3).
Prior art documents
Patent documents
[0004]
Patent document 1.: JP 2005-336188 A
Patent document 2.: International publication WO 02/086108
Patent document 3.: International publication WO 2011/118795
Summary of the Invention
Problem to be solved by the Invention
[0005]
In the prior art 1, a technique for combining leukemia inhibitory factor
(lymphoma inhibition factor: LIF), LIF analogs, or LIF mimic (herein below, they are
collectively referred to as “LIFs”) with the components for cosmetics is proposed.
The prior art 1 is excellent from the view point that the high quality epidermis is
formed by adding the culture sup containing LIF to cryopreserved epidermis in
vitro.
However, it is not promised that of experimental results in vivo is reflected
to that in vivo. Therefore, there is no proof for the cosmetic to have the same
effect in vitro and in vivo.
Also, the prior art 2 is excellent from the view point that the stem cell
having higher growth ability is used to utilize biologic factors produced by the stem
cells.
[0006]
However, since it uses the human-derived stem cell, there are problems
such as: (a) number of the stem cell to be obtained is decreasing by aging; (b)
safety for transplantation of the stem cell is not always secured, because gene
mutations caused by the aging are accumulated; (c) the stem cell has low cellular
proliferative potential, although it is the stem cell, because the number, the
proliferative potential, and differential potential of the bone marrow cell (BMSC)
become lower by the aging; (d) it is heavily invasive and dangerous to obtain the
3
stem cell through bone marrow puncture. Also, use of human cells causes
troubles that it is difficult to provide enough amounts of stem cell source, and also
in ethical aspects.
[0007]
The prior art 3 uses the culture sup of the human dental pulp stem cells.
By this, it is excellent that it is safe that the use of the cell by themselves.
However, as the same as the prior art 2, it uses the human cells, the
exfoliated tooth, even though they are referred to as medical waste. Therefore, it
has the problem in the ethical aspect, and also has the difficulty to provide enough
amounts of the stem cell resources.
[0008]
On the other hand, it is thought that a pluripotent stem cell may
differentiate into many tissues such as dermal tissue, bone tissue, muscle tissue
and others. At present, since a ratio of aging population becomes high, there is
strong need to maintain the dermis in healthy condition by preventing to dermal
lesion caused by the aging, namely, to prevent to form spots and blotches, wrinkles
and the like. Also, there is the need to solve the problem related to hair, such as
thinning hair, fallen hair and the like, regardless of age.
Also, there is the strong need to the cosmetic having the effects to reduce
or recover damages from UVB.
Problems to be solved by the invention
[0009]
Under such circumstances, the inventors of the present invention have
continued to study to solve the above-mentioned problems. Then, they have
completed the present invention.
The first aspect of the present invention is a cosmetic comprising any one
of powdery culture sup of stem cell selected form the group consisting of a
non-human mammal dental pulp stem cell, a non-human bone marrow stem cell and
a non-human adipose stem cell as a main ingredient. Here, the non-human stem
cell is preferably obtained from any one of tissue selected from the group
consisting of a swine dental pulp of exfoliated deciduous teeth, a swine bone
marrow, and a swine adipose. Also, the powdery culture sup is preferably
prepared from a culture sup selected from the group consisting of the swine dental
pulp of the exfoliated deciduous teeth, the swine bone marrow, and the swine
4
adipose tissue by freeze-drying after alcohol condensation.
[0010]
The powdery culture sup preferably comprises at least one of a growth
factor selected from the group consisting of platelet derived growth factor (PDGF),
vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF),
keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and
transforming growth factor (TGF).
Furthermore, the cosmetic has preferably any one of form selected from
the group consisting of a liquid, a cream, an ointment, a gel, an emulsion, and a
cataplasm.
The cosmetic is preferably applied for skin including scalp or hair.
[0011]
The second aspect of the present invention is a cosmetic stored in a
container comprising: a first compartment, a second compartment, a bar member to
form a through hole on a partition between said first and said second compartment;
wherein, said first compartment includes a solvent; said second compartment
includes an active ingredient composition comprising a carrier and any one of
powdery culture sup selected from the group consisting of that of a swine dental
pulp exfoliated deciduous teeth, that of a swine bone marrow, and that of a swine
adipose tissue; and said bar member formed said through hole in said partition to
solve said active ingredient composition into said solvent immediately before to use
said cosmetic. The solvent is preferably any one of liquid selected from the group
consisting of a liquid to which a minus ion, anion, is charged, saline and phosphate
buffered saline.
The cosmetic is preferably applied for skin including scalp and hair.
[0012]
The third aspect of the present invention is an iontophoresis method for a
protein comprising the steps of: placing a sheet form of a moisture-retaining
member containing said cosmetic as described above on a predetermine site
through which said cosmetic is absorbed; attaching a positively-charged electrode
to predetermined site; and attaching a negatively- charged rod-like electrode to
rotate and move on said sheet from.
The predetermined site is preferably any one selected from the group
consisting of an arm, a hand, a palm, a leg, and a ham, and a bottom of foot.
[0013]
5
The fourth aspect of the present invention is a dermal formation promoting
agent comprising a powdery culture sup of a stem sell selected from the group
consisting of that of a dental pulp stem cell, a bone marrow stem cell, and an
adipose stem cell of non-human mammal. Here, the stem cell of the non-human
mammal is preferably obtained from any one of tissue selected from the group
consisting of the swine dental pulp tissue of exfoliated deciduous teeth, the swine
bone marrow tissue, and the swine adipose tissue. The powdery culture sup is
preferably prepared from the culture sup selected from the group consisting of the
dental pulp of the exfoliated deciduous teeth, the bone marrow, and the adipose
tissue by freeze-drying after alcohol condensation.
[0014]
The powdery culture sup preferably comprises at least one of a growth
factor selected from the group consisting of platelet-derived growth factor (PDGF),
vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF),
keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and
transforming growth factor (TGF).
The cosmetic preferably has any one of the form selected from the group
consisting of a liquid, a cream, an ointment, a gel, an emulsion, and a cataplasm.
Advantageous Effect of the Invention
[0015]
According to the present invention, the cosmetic having advantageous
effects such as line smoothing, whitening, new hair growing, making hair thick and
the like.
Brief description of the drawings
[0016]
Fig. 1 is a schematic drawing showing the dermal regeneration effect by
using the culture sup of the swine stem cell to skin aging (dermal aging by light) by
UV irradiation.
Fig. 2 is a graph showing the ratio of the bromodeoxyuridine (BrdU) uptake
cells, when BrdU incorporation into the swine bone marrow stem cells (pBMSC), the
swine dental pulp stem cells (pDPSC) and the swine exfoliate deciduous teeth stem
cells (pSHED) are allowed. In Fig. 2, * shows p < 0.05.
Fig. 3 is a microscopic image showing the effects of the swine growth
6
factor (pGF) by the photoaged skin. Fig. 3(A) shows the microscopic observation
image of a skin replica formed from a control skin fragment to which pGF was not
administrated; and Fig. 3(B) is that of a sample skin fragment to which pGF was
administrated.
Fig. 4 is the graph showing skin aging status of the control group to which
the culture sup of the stem cell was not administrated; the group to which the
culture sup of the swine exfoliate deciduous teeth stem cells (pSHED-CM) is
administrated; and the group to which the swine exfoliate deciduous teeth stem
cells (pSHED) is administrated.
[0017]
Fig. 5 is a histologically-stained image showing the skin status of the each
group mice shown in Fig. 4. In the Figure, upper arrow shows the epidermis and
the lower arrow shows dermis, respectively.
Fig. 6 is the graph respectively showing the cutaneous cells without UV
irradiation (negative control), with UV was irradiation (UVS, photoaged cutaneous
cells), and with both of photoaging and subsequent the swine growth factor (PGF)
administration. In Fig. 6, ** means p < 0.01.
Fig. 7 is the microscopic image respectively showing the status of the cells
the cultured swine bone marrow stem cells (pBMSC) and the swine dental pulp
stem cells (pSHED) on day 3 and day 7.
[0018]
Fig. 8 is a photograph showing the status of the blotches and the spots.
Fig. 8(A) shows the status of the blotches and the spots before test starting, and
Fig 8(B) shows those of after the test finishing (6 month later).
Fig. 9 is the photograph showing the wrinkle on a forehead before and after
the test. Fig. 9(A) shows the status of the wrinkle before test starting, and Fig
9(B) shows that of after the test finishing (12 month later).
Fig. 10 is the microscopic images of the replicas made of the skin prepared
as the same as those in Fig. 3 before and after the test. Fig. 10(A) shows the
status before the test starting, and Fig. 10(B) shows that after the test finishing.
Fig. 11 is the photograph showing the status of the hair before the test
starting.
Fig. 12 is the photograph showing the status of the hair after 14 days of the
test starting.
[0019]
7
Fig. 13 is the photograph of the scalp before the test starting.
Fig. 14 is the photograph of the scalp on day 3 from the test starting.
Fig. 15 is the photograph of the scalp on day 5 from the test starting.
Fig. 16 is the photograph of the scalp on day 7 from the test starting.
Mode for carrying out the invention
[0020]
The present invention is explained more detail in below.
The powdery culture sup of the swine dental pulp stem cell used for the
cosmetic of the present invention is used as follows.
Firstly, as a swine jaw, it is preferably to use a lower jaw with tooth
immediately after slaughtering for meat. Here, since obtained cells are fresh, the
jaw from the swine immediately after slaughtering is preferable. Also, the jaw of
the swine for meat is preferable: because their dental pulps may be easily
obtained; they are highly safe without infections to viruses or bacteria; and is low in
cost. It is also preferably used the swine of 3 to 12 month old from the view point
of stem cell number, and is more preferably used that of 5 to 6 month old.
[0021]
Firstly, the lower jawbone with tooth of the swine for meat immediately
after slaughtering are refrigerated from the temperature range of -40 to -30 ºC to be
transported. For example, an icebox including refrigerants (-30 ºC) and the like
may be used.
Next, in order to sterilize the entire surface of the lower jaw, for example,
disinfection is performed by using an antiseptic agent such as Isodine (registered
trademark) and the like. Then, a crown of the tooth is cut in horizontal direction
by using, for example, a diamond point for a dentist and the like. Subsequently, it
is cut in vertical direction along with a pulp space. By these cuttings, the
overcanopy of the tooth may be smoothly removed. Then, the dental pulp is
collected both from the dental crown and dental root portions treated as mentioned
above by using, for example, a hand scaler for the dentist, a hand file for the
dentist and the like.
[0022]
Obtained dental pulps are chopped by using an ophthalmic knife to be
suspended in a basal medium containing predetermined serum and antibiotics.
As the basal medium used here, Dulbecco’s modified eagle’s MEM containing 5 to
8
20% (v/v) of bovine serum, and 1 % (v/v) of 5 x 103 to 5 x 104 U/mL of penicillin and
1 % of 5 to 50 mg/mL of streptomycin, and the like.
Next, an enzyme solution containing predetermined concentration of both
collagenase and disperse is prepared to separate dental pulp cells into single cells.
For example, obtained cells are suspended in the enzyme solution containing both
of 1 to 5 mg/mL of collagenase and disperse to be treated at 35 to 38 ºC for 30
minutes to 1.5 hr in a thermostatic chamber.
After that, they are centrifuged for 2 to 5 minutes, preferably, for 3 minutes,
(for example, about 1,500 rpm (about 1,000 x g)); the isolated dental pulp cells by
the enzyme treatment are collected. In this time, it is preferable to avoid the use
of a cell strainer for cell sorting, because it decreases the recovery ratio of a neural
stem cell fraction for SHED or DPSC.
[0023]
Also, the adipose cell may be obtained as follows. Since the cells are
fresh and easily obtained, the adipose tissue in the swine mesentery is used. The
transport conditions are the same as those used for the swine lower jaw with tooth
for meat. Firstly, the adipose tissue of the swine mesentery for meat immediately
after slaughtering is excised and collected. Next, other tissues attached to the
obtained adipose tissues are removed, and then the adipose tissue is minced by
using surgical knife or scissors.
Next, the minced tissue is suspended in the basal medium containing the
predetermined serum, antibiotics and the like. The basal medium used here is as
mentioned above. Subsequently, as the same as the preparation of the dental
pulp stem cells, the enzyme solution containing both of collagenase and disperse at
the predetermined concentrations are prepared as mentioned above to be used to
obtain the adipose stem cells.
[0024]
Next, each of the isolated dental pulp stem cells, bone marrow stem cells
or adipose cells are cultured by using predetermined media to obtain the culture
sups thereof. For example, the obtained cells are re-suspended in 2 to 8 mL of
the basal media to be plated to suitable size of dishes for adherent cell cultures.
For example, the suspension is plated into the dishes with 6 cm diameter, and the
culture medium (for example, 10%FCS containing DMEM (Dulbecco's Modified
Eagle's Medium)) is added into the dishes. After that, they are cultured for about
10 to 20 days in the presence of 5% CO2 in an incubator adjusted at 35 to 38 ºC.
9
After removal of the medium, the cells are washed from 1 to several times
with PBS and the like. Instead of above-mentioned procedure (the removal of the
medium and wash of the cells), the adherent cells which formed colonies may be
collected. In this case, for example, the cells are treated by using a solution
including both of 0.01 to 0.1 % trypsin and 2 mM EDTA for 5 minutes at 37 ºC to be
detached from the dish. By collecting the detached cells, adherent stem cells
may be obtained.
[0025]
Subsequently, selected adherent cells are cultured. For example, the
cells are plated to similar dishes for the adherent cell culture, cultured under the
similar conditions, and passed depending on the necessity. For example, when
the cells become sub-confluent (about 70 % of the bottom area of the culture
vessel is covered by the adherent cells) or confluent by macroscopic observation,
the cells are detached from the culture vessel by using the same treatment as
described above to be collected. Then, they are again plated in the culture vessel
including the fresh medium having the same composition. Passage may be
performed repeatedly until the cell number becomes sufficient. For example,
when the passage culture is repeated 1 to 8 times, the cell number reaches about 1
x 107 cells /mL.
After the above-mentioned cultivation, the cells may be collected, and, for
example, stored in liquid nitrogen.
[0026]
In the initial stage of culture, the medium is replaced as necessary to reach
sub-confluent, for example, 2 to 3 times a week. The sup of the sub-confluent
flask is removed by using an aspirator and the like; then, Hepes buffer including
0.01 to 0.1 % (v/v) of trypsin is poured into the flask, the cells are detached from
the bottom of the flask. Fresh medium is added into the flask in a suitable amount
to suspend the detached cells to transfer into a sterilized centrifuge tube. Next,
the tube is centrifuged at 800 to 1,200 x g (about 1,500 rpm) at room temperature
for 2 to 5 minutes, preferably for 3 minutes. For example, Hepes buffer including
0.05 % of trypsin is prepared, and add the flask, form which the medium was
removed, in a small amount so as to penetrate entire of the bottom of the flask.
When the cells are detached from the bottom, about 5 to 8 mL of fresh medium is
added. Then, the medium including the cells may be centrifuged at 1,500 rpm for
3 minutes at room temperature to be concentrated. The cell concentration in the
10
condensed solution per mL is obtained by using viable count method; then, 10 to 40
mL of them are added into the flask including about 10 to 40 mL of fresh medium to
perform passage culture to obtain the exfoliated deciduous teeth dental pulp stem
cell (pSHED), the bone marrow stem cell or adipose cells of swine.
[0027]
Also, a hole is formed in a lingual side of a lower anterior tooth of the
swine lower jaw bone body by using, for example, the diamond point for the dentist
and the like. For example, a syringe with a needle of 18G is inserted into the hole
to aspirate. The obtained bone marrow is cultured until it becomes sub-confluent
in the culture flask as mentioned above. Then, the sub-confluent cells are
detached from the bottom of the culture flask, and separated by the centrifugation.
The separated cells are passed to obtain the swine bone marrow cells (pBMSC).
[0028]
The stem cells obtained as described above are the deciduous teeth dental
pulp stem cells, the bone marrow stem cells or adipose stem cells, which are
mesenchymal-derived somatic stem cells. In the culture sups of these stem cells,
cytokines such as vascular endothelial growth factor(VEGF), hepatocyte growth
factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF),
transforming growth factor -beta (TGF-β)-1 and -3, TGF-α, KGF, HBEGF, SPARC,
and others are secreted. Therefore, these stem cells are preferably employed.
[0029]
The stem cell for obtaining the culture sup of the stem cell used in the
present invention is preferably obtained from the swine for meat; because the cells
themselves are highly safe, they neither have any surgical invasive matters caused
by removal from the human tissues; nor ethical matters for use thereof. Therefore,
there are great advantages.
Also, the culture sup of the stem cell (pSHED-CM) preferably comprises a
combination of at least two factors selected from the group consisting of VEGF,
HGF, IGF, PDGF and TGF-β as mentioned above, because of the excellent
collagen regeneration ability.
Note that the stem cell culture sup may be used by the addition of at least
one known corresponding cytokine.
[0030]
The stem cell culture sup is obtained by culturing the stem cell under the
predetermined conditions, and removing the cells from the medium with the
11
centrifugation and the like. Thus obtained culture sup is subjected to several
treatments such as, for example, the centrifugation, condensing, replacing the
solvent, dialysis, lyophilizing, desalting and others, and the treated culture sup may
be used. For example, lyophilized powder of the culture sup is obtained by
freezing the culture sup in dry ice-acetone and then drying.
[0031]
As the medium for the stem cell culture, either the sole basal media or the
basal media supplemented with serum and the like may be used. As the basal
media, the following media may be used: DMEM, Iscove’s Modifed Dulbecco‘s Medium
(IMDM; GIBCO, etc.), Ham’s F12 medium, HamF12; SIGMA, GIBCO, etc.),
RPMI1640 medium, and the like. Also, a mixed media comprising at least two
media may be used. As an example of the mixed medium, there is mentioned that
including IMDM and HamF12 in equal amount (for example, it is commercially
available under the product name: IMDM/HamF12 (GIBCO)).
[0032]
As the components to be added to the media, there are mentioned, for
example, serum (fetal calf serum, fetal equine serum, human serum, and sheep
serum, etc.), serum replacement (Knockout serum replacement (KSR), etc.), bovine
serum albumin (BSA), antibiotics, vitamins, minerals, and the like.
Note that serum-free media are preferably used through entire steps of the
stem cell culture, or in several passage cultures, at least in the last passage
culture, for obtaining “the stem cell culture sup” without serum as described above.
The stem cell culture may be performed by applying the generally employed
conditions.
[0033]
As mentioned above, pSHED included in the cosmetic of the present
invention comprises a variety of growth factors. Therefore, such growth factors
may be absorbed transdermally by applying it onto the skin. As the components
to be added to the cosmetic of the present invention, there are mentioned, for
example, organic bioabsorbable materials such as hyaluronic acid, collagen,
fibrinogen (for example, BOLHEAL (registered trademark)) and the like; highly
biocompatible gelation materials such as hyaluronic acid, collagen, fibrin glue, and the
like. Alternatively, pharmaceutically available other components (for example, a
carrier, an excipient, a disintegrator, a buffering agent, an emulsifier, a suspending
agent, a soothing agent, a stabilizer, a preservatives, an antiseptic, physiological
12
saline, etc.) may be added.
[0034]
As collagen used here, soluble collagen such as acid-soluble collagen,
alkaline-soluble collagen, enzyme-soluble collagen and the like may be preferably
used, because of their compatibility to the cosmetic of the present invention.
As the excipient, for example, lactose, starch, sorbitol, D-mannitol, sucrose
and the like may be used. As the disintegrator, carboxymethyl cellulose, calcium
carbonate and the like may be used. As the buffering agent, phosphate, citrate,
acetate and the like may be used. As the emulsifier, gum arabic, sodium alginate,
tragacanth gum and the like may be used. As the suspending agent, glyceryl
monostearate, aluminium monostearate, methyl cellulose, carboxymethyl cellulose,
hydroxymethyl cellulose, sodium dodecyl laurate and the like may be used.
[0035]
As the stabilizer, propylene glycol, ascorbic acid and the like may be used.
As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol,
metylparaben, and the like may be used. As the antiseptic, benzalkonium chloride,
para-hydroxybenzonate, chlorobutanol and the like may be used. Other than those,
pH control chemicals may be included.
The final cosmetic form of the present invention is not particularly limited,
and they may be the skin toning lotion, milky lotion, lotion, gel, cream and the like.
Also, the cosmetic is preferably applied to the skin including scalp and
hair.
[0036]
The preparation method of the cosmetic as mentioned above is not
particularly limited. For example, when the swine lower jay is used as the raw
material, it is preferably prepared as describe below.
Firstly, the dental pulp stem cells are obtained from the swine lower jaw
obtained as described above. The swine adipose tissue is used instead of the
swine lower jaw; the adipose stem cells are obtained as described above. Next,
for example, the dental pulp stem cell is cultured under the above-mentioned
conditions by using the serum-free medium, and then their culture sup is collected
by using, for example, a syringe, a pipette and the like. The collected culture sup
may be used as is, or used after at least one of the following treatment such as the
centrifugation, condensation, dialysis, lyophilization, dilution, and desalting.
Note that as shown in the following examples, the culture sup of the stem
13
cell shows predetermined actions without highly purification. This means that the
composition used for the cosmetic of the present invention may be prepared
conveniently and speedy. Therefore, there is a merit that it is avoidable to
decrease the component activities during the purification.
[0037]
The culture sup obtained as mentioned above is firstly determined its
protein contents. Then, its collagen purification activity is determined to obtain
specific activity. By using the specific activity as an indicator, the quality of the
product may be maintained constant.
If the specific activity is low, the sup may be condensed by using a spin
column condensation or ethanol precipitation. In the spin column condensation,
the maximum volume of the culture sup for the predetermined size of the spin
column is applied to the column; then the column is subjected to the centrifugation
at 3,000 x g to 5,000 x g for about 30 to 90 minutes. The serum-free medium is
added into the tube in the equal volume to the obtained condensed culture sup
volume; then, the tube is subjected to the centrifugation at 3,000 x g to 5,000 x g
for about 30 to 90 minutes. By this operation, the media of the culture sup is
replaced to the serum-free media.
For example, Amicon Ultra Centrifugal Filter Units-10K (Millipore) is used
as the spin column; the maximum volume to be loaded on the column is 15mL.
Therefore, about 15mL of the culture sup is loaded to the column, and then column
is centrifuged in 4,000 x g for about 60 minutes at 4 °C. By this operation, it
gives up to 200 μL of the condensed culture sup. The same volume of the
sterilized PBS as that of the culture sup, which is condensed up to 200 μL, is added,
and again the tube is centrifuged 4,000 x g for about 60 minutes at 4 °C to replace
the solvent of the culture sup to PBS. The obtained solution is collected into a
micro test tube, and it is used as the condensed stem cell culture sup. If the
finally obtained volume is about 200 μL, the enrichment of the sup is 75 fold.
[0038]
For example, ethanol precipitation is performed as follows. Firstly, 9
times volume of 100 % ethanol is added to a certain amount of the culture sup in a
tube to mix, and then the mixture is stood at about -10 to 30 °C for 30 to 90
minutes. Next, the tube is centrifuged at about 4 °C in 10,000 to 20,000 x g for
about 10 to 20 minutes. The sup is removed, and then 1/5 volume of 90 % of
ethanol is added, and then agitated well. Subsequently, it is centrifuged at about
14
4 °C, in 10,000 to 20,000 x g for 3 to 10 minutes. The sup is removed, and the
obtained pellet is suspended in the predetermined volume of the sterilized water,
for example, 500 μL of the sterilized water to be collected into the micro test tube.
Thus, the condensed sup of the stem cell may be obtained.
For example, 45 mL of 100 % ethanol is added to 5 mL of the culture sup in
a tube to be mixed, and then it is stood at -20 °C for 60 minutes. Then, it is
centrifuged at 4 °C in 15,000 x g for 15 minutes, and then the sup is removed.
After that, 10 mL of 90 % ethanol is added and agitated well. Again, it is
centrifuged at 4 °C in 15,000 x g for 5 minutes, and the sup is removed. The
obtained pellet is suspended in 500 μL of the sterilized water to be collected into
the micro test tube. Thus, the condensed culture sup is obtained. In this case,
the enrichment of the sup is 10 fold, if the first volume of the sup used is 5 mL.
[0039]
The culture sup of the stem cell used to the cosmetic of the present
invention may be lyophilized product as follows. Firstly, the culture sup or the
condensed one obtained as mentioned above is dispensed into each container with
lid of which volume is about 50 to 150 mL in a fixed amount. Then, the lid of the
sample tube is tightly sealed, and the tube is frozen at about -100 °C to about
-60 °C for 2 hr to half day. After the sup is frozen, the tube is opened to be set in
a lyophilizer. Next, the sample is lyophilized for 1 to 2 days to obtain the
lyophilized stem cell culture sup, which may be stored at about - 100 °C to -60 °C.
For example, the culture sup is respectively dispensed into 8 vials with
rubber stopper, of which volume is 50 mL, by 20 mL. Then, the vials are sealed
with the rubber stoppers and frozen at about -30 °C over a period of hours. Next,
the rubber stoppers are removed, and the vials are set in the lyophilizer (Yamato
Science Corp., DC41A). Next, the lyophilization is continued to dry up the sample.
By this, the lyophilized culture sup may be obtained.
According to the above-mentioned procedures, the lyophilized products
having excellent preservation stability is produced.
[0040]
When the cosmetic is introduced through the skin by using the
iontophoresis, the cosmetic is absorbed in the sheet formed moisture-retaining
member, and the member is located on the site through which the cosmetic is
introduced, for example, face or scalp. Then, the positively-charged electrode is
attached to the predetermined portion, and negatively-charged rod-shaped
15
electrode is rotated and moved on the sheet member.
Here, the predetermined site is not particularly limited. However, the site
is preferably selected from the group, for example, consisting of arm, hand, palm,
leg, ham, and foot bottom, because the positively-charged electrode may be stably
attached to such sites.
As the sheet formed moisture-retaining member, a commercially available
sheet for pack made of unwoven cloth, gauze, cosmetic cotton, and the like may be
used. If the cosmetic is a liquid form or milky lotion form, they may be used to
impregnate the sheet formed moisture-retaining member. Alternatively, if the
cosmetic is cream or gel, it may be applied to the skin and covered by the sheet
formed moisture-retaining member.
[0041]
In the following, the present invention is explained as the example, when it
is the skin toning lotion. Firstly, the powdery culture sup is dissolved in proper
amount of the saline to prepare a powder-dissolved solution. The
moisture-retaining member is impregnated into that so as that the solution is
dripped. Next, the moisture-retaining member is attached to the predetermined
portion of the body, for example, entire of the face, and a part of the body is
attached to the positively-charged electrode. For example, the positively-charged
electrode is grabbed by one hand.
Next, the negatively-charged electrode in placed on the moisture-retaining
member, and then slowly rotated and moved on the member. It is not necessary
to apply pressure. Since all of the growth factors included in the solution are
negatively charged, they are repelling to the negative charge on the roller, and
transfers to the positively-charged electrode. By this transfer, the growth factors
are absorbed from the skin.
[0042]
Alternatively, the cosmetic may be stored in a bottles made of plastic or
glass, the bottles made of plastic or glass with a dropper, a container having thin
outlet made of plastic and the like to be used. When it is used as the cosmetic for
scalp or hair, the container having thin outlet made of plastic is preferably used,
because the cosmetic is attached to the scalp.
The cosmetic is attached to the scalp and hair to be coated evenly. After
that, it is introduced by iontophoresis method through the skin as described above.
Then, entire of the hair is wrapped by using a plastic film, for example,
16
polyethylene film and the like to be packed.
For both case described above, a steam-generating unit is placed to a
predetermined distance from the head so as to spray the steam, it provides high
introduction efficiency.
[0043]
Herein below, the example of the present invention is explained; however,
the present invention is not limited to them. Also, the term, “%” in the example is
weight (mass) basis, unless otherwise noted.
The present invention is not limited to the description of the
above-mentioned embodiment or the examples. If they are fallen in the scope of
the claims, and various modifications of them are predictable by the person skilled
in the art, they are contained in the present invention.
Example 1
[0044]
Production and quality evaluation of the growth factors derived from the
swine SHED/BM/adipose tissue

(1) Fractionation and culture of the cells
From Shokuniku Kosha Co. Ltd (Minato-ku, Nagoya city, Japan), the jaw
(the lower jaw with tooth) and mesentery of 5 to 6 month of swine were obtained.
Those immediate after slaughtering were transported in the ice box including the
thermal gel (-30 °C).
From the swine tooth and the lower jaw, the swine dental pulp stem cells
(SHED) were obtained according to the following procedure.
[0045]
The transferred swine tooth and the lower jaw were sterilized with Isodine.
Then, the crowns of the tooth in the lower jaw were cut in horizontal direction by
using the diamond point for the dentist, and cut in the vertical direction along with
the pulp space to delete the overcanopy. The dental pulps were collected from
the crowns and roots of the tooth treated as described above by the scaler for the
dentist.
The obtained dental pulps were chopped by using the ophthalmic knife to
be suspended in 2 mg/L of collagenase solution. The solution was placed in the
incubator at 37 ºC for 1hr, and the cells were separated. In order to obtain the
17
cells for passage, the separated cells were preliminarily cultured in Dulbecco’s
modified Eagle’s MEM (DMEM; SIGMA, St. Louis, MO) supplemented 10 % FBS
and 1 % Anti-Anti (Invitrogen, Carlsbad, CA) under the conditions at 37 °C and 5 %
CO2.
[0046]
Firstly, in the initial stage of the culture, the cells were cultured until
sub-confluent, replacing the medium 2 to 3 times per week. The sub-confluent
cells were detached from the flask by using Hepes solution including 0.05% trypsin,
and then, the cells were collected by the centrifugation in 1,500 rpm for 3 minutes
at room temperature. The obtained cells were transferred into the fresh medium
and the entire of the cells were used to the passage culture under the same
conditions as described above.
The swine bone marrow (BM) was obtained from the cortical bone of the
lower jaw body. Firstly, the hole was formed in the lingual side of the lower
anterior tooth of the swine lower jaw bone by using the diamond point for the
dentist. The 5 mL of the syringe with the needle 18G was inserted into the hole to
aspirate the bone marrow to collect. The collected bone marrow was transferred
into the DMEM supplemented with 10 % bovine serum, 100 U/mL of penicillin and
100 μg/mL of streptomycin; then they were preliminarily cultured under the same
conditions. Then, they were cultured under the conditions at 37 ºC and 5% CO2,
until they became sub-confluent. All of the supplemented amounts as described
were shown at the final concentrations.
[0047]
Similarly to the swine SHED, the cells were detached by using 0.05 %
trypsin solution to be centrifuged for 3 minutes in 1,500 rpm to be subjected to the
passage culture.
From the swine mesentery, the adipose tissues were excised by using the
dissecting scissors and knife to remove unnecessary tissue parts, and washed out
the blood in the phosphate buffered saline. Except those, the adipose cells were
separated into the single cell as described above to obtain the adipose stem cells.
[0048]
(2) Analysis of the cell growth
According to the instruction attached BrdU staining kit (Invitrogen,
Carlsbad, CA), bromodeoxyuridine (BrdU) was incorporated into SHED or BM
obtained as described above. Then, the growth speed for 12 hr of each cell was
18
evaluated. For the sample of each group, three slide glasses were prepared for
one sample to evaluate in triplicates. The experiments were repeated 5 times.
After one-way analysis of variance, Turkey-Kramer test (Tukey-Kramer test) was
employed to perform significance test.
[0049]
In order to perform STRO-1 immunofluorescence staining, the swine SHED
or the swine BM was fixed by using 3 % paraformaldehyde. After that, they were
rinsed twice with the phosphate buffered saline (PBS), treated with 100 mM glycine
solution for 20 minutes. Next, these cells were permeabilized with 0.2 % Triton-X
(Sigma-Aldrich, St. Louis, MO) solution for 30 minutes at room temperature. Then,
PBS including 5 % Equus asinus serum and 0.5 % bovine serum albumin was
added to the cells, which were incubated at 37 ºC for 20 minutes in the solution.
[0050]
Next, the primary antibody, mouse anti-human STRO-1 antibody (R&D,
Minneapolis, MN) and cells were mixed at the ratio of 1: 100, and incubated for 1 hr
at room temperature in PBS. Next, PBS was removed by the aspiration, the
secondary antibody, goat anti-mouse immunoglobulin M-FITC antibody (Southern
Biotech, Birmingham, AL) and the cells were mixed at the ratio of 1: 500, and
incubated for 30 minutes at 37 ºC. Then, they were mounted by using the vector
shield and DAPI (Vector Laboratories Inc, Burlingame, CA).
[0051]
(3) Animal experiment (see Fig. 1)
Five week age of female hairless mice (Hos; HR-1) were provided by SLC
(SLC Inc., Shizuoka, Japan). The all of the mice were placed under the
conditions controlled temperature, humidity (22 ± 1 ºC, 50 % humidity) and 12 hr
light dark cycle. The animals could freely take water and solid feed, and they
could freely move in the cage during the radiation exposure. Observation was
carried out every day. With UVB irradiation device RMX-3W (Handok Biotech,
Seoul, Korea), UVB was irradiated onto the mouse back for 10 weeks at 5 times a
week, and the distance from the lump to the animal back was 89 cm. The
irradiation was performed by using an array composed of 10 SE lump (Toshiba)
without filtering for UVB. Peak irradiation wave length was about 312 nm. Also,
the irradiation amount was set to that the radiation ray having the wavelength of
290 to 320 nm occupies 55 % of whole amount of UVB.
19
[0052]
The irradiation amount was set to 1 MED (minimal erythema dose; 60
mJ/cm2) for the first 2 weeks, 2 MED (120mJ/cm2) for 3rd week, 3 MED (180
mJ/cm2) for 4th week, 4 MED (240 mJ/cm2) for 5th to 8th weeks. Total UVB
radiation dose was about 115 MED (6.9J/cm2) to induce the wrinkle formation.
At five weeks after the wrinkle induction, pSHED-CM (100 %) was
administrated in s.c. to the limited area of the mouse back. As the positive
control, the swine SHED (4 x 105) suspended in PBS was directly injected into the
dermis, and as the negative control, PBS was solely directly injected into the
dermis.
[0053]
(4) Preparation of the swine SH-CM
The swine SHED (4 x 105 cells) was cultured in DMEM/F12
(Invitrogen-Gibco-BRL, Grand Island, NY) serum free medium at 37 °C, under 5%
CO2. After 72 hr, the culture sup of the swine SHED was collected to be
centrifuged in 300 x g for 5 minutes at room temperature. Then, it was filtered by
using a filter having the pore size of 0.22 mm (Millipore).
[0054]
(5) Analysis of skin replica and its image
At wrinkle induction and after 1 week from the above-mentioned injections,
a negative type replica was prepared by using a silicone rubber impression material
(Flextime 1; Heraeus Kulzer, New York, NY) from the skin surface of the mouse
back. In order to obtain a wrinkle replica from the same area of the skin, a
permanent marker was used to mark up the area.
[0055]
After 5 weeks from the final injection of either the swine SH-CM or SHED to
the skin, the impression was obtained from the marked up area. For easy
measurement, all replicas were cut into 1 cm on four sides, and the back side of
the replica was processed to be flat face by using the same impression material.
Shedding the light at an angle of 208 °, the images of the replicas were captured by
using a CCD camera.
The image of the negative type replica was observed by using the wrinkle
analyzer, Skin visiometer (skin visiometer) SV 600 (Courage & Khazaka, Cologne,
Germany). Parameters used for the evaluation of the skin wrinkle were the
number and the depth of the skin wrinkle per unit area, and the area of the wrinkle.
20
[0056]
(6) Histological analysis
The back skin (1 cm x 1 cm) was fixed in the neutral buffer including 10 %
formalin. Then, the fixed sample was embedded into the polyester wax to prepare
a section having 6 mm thickness. The section was exposed to hematoxylin &
eosin (H & E) to perform Masson trichrome staining.
Firstly, hematoxylin & eosin (herein below, it is referred to as “H&E
staining”) was carried out. The section was subjected to deparaffinization by
using 3 batches of Xylene (Resomoll). Then, it was subjected to dehydration by
using 4 batches of absolute ethanol, and washed with running water for 3 minutes.
Next, it was immersed in Mayer's hematoxylin solution for 5 minutes to stain the
nucleus, and then washed with running water of about 45°C for 3 minutes.
[0057]
Next, it was immersed in the eosin solution for 5 minutes. Then, the
tissue was dehydrated by using 4 batches of absolute ethanol, and treated 4 timed
with xylene to softly mounted to remove the colorant stayed in other positions,
which was not to be stained.
Subsequently, it was subjected to Masson trichrome staining. The
section was subjected to deparaffinized as the same as that for hematoxylin &
eosin staining, and then washed. The first mordanting was performed by using
the mixed solution of equal volume of 10 % trichloroacetate (Wako Pure Chemicals,
special grade) solution and 10 % potassium dichromate (Wako Pure Chemicals,
special grade) solution. Next, the section was washed with water for 3 minutes
and then distilled water for 1 minute.
A solution including 1.0 g of hematoxylin (MERCK) and 100 mL of 95 %
ethanol and B solution including 2 g of ferric chloride (Wako Pure Chemical,
Special grade, in both), 1 mL of hydrochloride and 95 mL of distilled water were
mixed when using to prepare Weigert's iron hematoxylin to be used for immersing
the section for 10 minutes. Next, it was washed with water for 10 minutes.
[0058]
Equal volume solutions of 2.5% phosphotungstic acid (Wako Pure
Chemicals, special grade) and 2.5% phosphomolybdic acid (Wako Pure Chemicals,
special grade) were mixed, and the section was immersed for 1 minute therein to
perform the second mordanting. Next, the section was immersed in 0.75%
21
Orange G (Wako Pure Chemicals, 1st grade) solution for 2 minutes. Then, it was
subjected to treatment with 2 batches of 1 % acetic acid solution.
Next, ponceau de xylidine - acid fuchsin solution was prepared by mixing 2
volumes of 1% of ponceau de xylidine (CHROMA) in 1 % acetic acid solution and 1
volume of 1 % acid fuchsin (Wako Pure Chemicals, chemical grade) in 1 % acetic
acid solution. Then, the section was immersed in the solution for 20 minutes.
Then, it was subjected to treatment with 2 batches of 1 % acetic acid solution.
Subsequently, the section was immersed in 2.5 % phosphotungstic acid for
7 minutes, and then it was subjected to treatment with 2 batches of 1 % acetic acid
solution. Next, it was stained by using aniline blue and subjected to treatment
with 2 batches of 1 % acetic acid solution. Then, it was immersed in isopropanol
one by one to perform to remove the colorant stayed in other positions, which was
not to be stained as the same as those done in hematoxylin & eosin staining.
[0059]
(7) The culture of dermis fibroblast (HDF) in human dermis and irradiation amount
of UVB
HDF was cultured in DMEM supplemented with 10 % fetal bovine serum,
100 U/mL of penicillin, and 100 mg/mL of streptomycin under 5 % CO2 at 37 °C.
The cells in the culture flask were cultured in the serum-free medium for 24 hours.
After that, they were washed with PBS, and subjected to UVB exposure with 3 to 4
drops of PBS, wherein the irradiation amount of cells were between the ranges of
50 to 250 mJ/cm2. UVB irradiation was performed by using the above-mentioned
UV source (Waldmann, Schwenningen, Germany). Immediately after the
irradiation, PBS was aspirated, and was replaced with the complete medium.
UVB irradiation amount was finally fixed to 70 mJ/cm2, and it was used for following
experiments.
[0060]
(8) Cell growth assay
HDF was plated at 5 x 103cells /well in a 96 well plate, and their growth
rate was measured by using CCK-8 kit (Dojindo, Gaithersburg, MD). They were
cultured in the serum-free medium for 24 hours; subsequently, they were continue
to culture further 24 hours with or without the swine SH-CM. Then, they were
exposed to UVB (70mJ/cm2) for 90 seconds.
22
Subsequently, UVB-irradiated cells were cultured for 24 hours in the
complete medium to be collect. HDF-F was poured into 10mL of CCK-8 solution,
and then incubated for 3 hours. By using the microtiter plate reader (TECAN,
Gro¨ dig, Austria), absorbance at 450 nm was measured.
On the basis of the calibration curve prepared by using the control group,
cell number was obtained from OD values of each well.
[0061]
(9) Western blot analysis
HDF (2 x 104 cells/well) were plated in a 24 well plate, and they were the
pretreated as described above. Next, the cells were lysed in RIPA buffer (50mM
Tris-HCl (pH 7.4) including 0.15M NaCl, 1 mM EDTA, 1% Triton X-100, 1 % SDS,
50mM NaF, 1 mM Na3VO4, 5 mM dithiothreitol, 1 mg/mL leupeptin, and 20 mg/mL
PMSF). The cell-lysed solution was determined by using Biuret reagent, and
50μg of protein was subjected to 8 % SDS-polyacrylamide gel electrophoresis
(SDS-PAGE). After finishing SDS-PAGE, the gel electrophoresis pattern was
transferred to PVDF membrane.
The membrane was incubated for 15 minutes at room temperature together
with and anti-type I collagen antibody (Santa Cruz, Saint Louis, MO) and an
anti-matrix metalloproteinase 1 (MMP-1) antibody (Calbiochem, Darmstadt,
Germany). Next, the PDF membrane was washed by using PBS, and then it was
incubated for 15 minutes at room temperature together with anti-goat IgG conjugate
with horse radish peroxydase (1: 100,000、Santa Cruz, Saint Louis, MO). After
that, it was blotted with immunoglobulin Western reagent. X-ray film was laid
over the membrane to be exposed.
[0062]

(1) Feature analysis pSHED and pBMSC
Both pSHED and pDPSC showed the same fibroblast shapes as those of
pBMSC. From the immunofluorescence analysis, it was demonstrated that both
pSHED and pBMSC contain STRO-1 positive cells. Also, it was shown that the
growth rate of pSHED was significantly higher than that of pBMSC (see Fig. 2).
[0063]
(2) Reduce of UV induced wrinkle with the swine SHED-CM
During UV exposure, fine wrinkling on the mouse skin were observed. In
the treatment, it was observed that both pSHED-CM treatment group and pSHED
23
injection group had less wrinkling than those of the PBS administration group to
which PBS was solely injected (see Fig. 3 (A) and (B), in each group n=8).
Both in Figs. 3(A) and (B) showed that the wrinkling was reduced by the
repetitive administration of pSHED-CM (in the figure, pSHED-CM repetitive
administration group was shown as “pGF”.). The pSHED injection group showed
the same trend as that of pSHED-CM group.
As shown in Fig. 4, the wrinkle parameters were determined by using the
skin visiometer SV600. All of the wrinkle parameters were significantly reduced,
when the natural level of pSHED-CM (100 %) was injected. Also, the pSHED
treatment showed that higher availability than pSHED-CM treatment.
[0064]
(3) Tissue observation
Since the UVB irradiated hairless mouse showed large change in the
appendage of the skin, the effect of pSHED-CM against the thickness of the dermis
in the UVB irradiated hairless mouse was investigated.
Fig. 5 shows the histological staining results of the dermis thickness of the
hairless mouse skin with the hematoxylin-eosin staining (H & E staining). As
shown in Figs. 5 (A) to (C), the amount of collagen fibers (the part sandwiched by
the up and down arrows in the figure) significantly increased compared to the
control group in the pSHED-CM treatment group (Fig. 5 (B)) and the pSHED
injection group (Fig. 5 (C)).
Also, the measurement of the dermis thickness showed that the dermis
thickness significantly increased both in the pSHED injection group and the
pSHED-CM treatment group (Fig. 5 (B) and (C)). Furthermore, it was observed
the significant increase of the bundle of collagenous fibers in both of the groups, but it
was not observed in the control group (Fig. 5 (A)).
[0065]
(4) HDF growth promotion by pSHED-CM
In order to study a paracrine mechanism related to wrinkle amelioration of
the skin with the swine SHED, the cell growth assay was performed by using HDF
which was primarily cultured with pSHED-CM under the same conditions as
described above. As shown in Fig. 6, UVB irradiation significantly reduced the
HDF growth (in Fig.6, it was shown as “UVS”). However, the pre-treatment by the
swine SHED-CM diminished the growth declining width (In Fig. 6, it was shown as
“pGF”.). This means that the pSHED-CM has the protection effect to HDF.
24
Since the pSHED-CM comprised a variety of growth factors, it suggested
that the growth promotion by the swine SHED-CM was mediated by the growth
factors secreted from pSHED.
[0066]
(5) Expression of type I collagen and MMP1
In the hairless mouse treated with the swine SHED-CM, the collagen
content in the dermis significantly increased. Therefore, we studied the both
protein expression of the type I collagen and MMP1 in HDF after the pSHED-CM
treatment. Due to the UVB irradiation, the type I collagen expression amount
clearly decreased, but the expression of MMP1 increased by the induction.
Also, the expression amount of the type I collagen significantly increased
compared to after the pSHED-CM pre-treatment. In contrast, the expression
amount of MMP1 decreased after the pSHED-CM pre-treatment. As a result, it
was shown that the increase of the collagen content in the hairless mouse dermis
with the pSHED-CM treatment was caused by stimulating the collagen synthesis in
the dermis fibroblast and inhibiting the collagen lysis.
EXAMPLE 2
[0067]
Production of the growth factor cocktail derived from the swine SHED/BM/
adipose stem cells and evaluation of quality thereof
(1) Preparation of the growth factor cocktail (powder)
The stem cells of the swine SHED, swine BM or swine dental pulp were
used to prepare the cocktail of the swine growth factor (pGF). By using DMEM
containing 10 % FCS, the stem cells of the swine SHED, BM or dental pulp were
cultured in the dish from 2 to 8 passages. When the cells in the dish became
80 % confluent, 10 mL of sup (culture medium: CM) was sampled. Nine volume of
ethanol was added to the 10 mL of the sample to prepare ethanol diluted CM, which
was incubated at -20 ºC for 60 minutes. Then, it was transferred into the spin
column of 50 mL volume and centrifuged at 4 ºC for 15 minutes in 15,000 rpm to
obtain precipitates. The precipitates were washed with 90 % ethanol at -20 ºC,
and then it was applied on another spin column and again centrifuges as described
above.
The precipitates condensed as mentioned above were lyophilized to obtain
a powder including the growth factors.
25
[0068]
(2) Growth factors in the powder
The growth factors in the powder were analyzed by using Western blotting
method as described above. Detected growth factors were PDGF, VEGF, IGF,
KGF, HGF, and TGF.
(3) Quality evaluation
The results during the cell culture with the culture sup of the swine
cultured-stem cells or the swine bone marrow stem cells are shown in Figs. 7(A) to
(D). It was observed that both of the culture sup promoted the cell growth rates
compared to the rate without the culture sup.
EXAMPLE 3
[0069]
Results by the culture sup of the dental pulp stem cells derived from the
non-human animal against the skin
(1) Preparation of the culture sup of the dental pulp stem cells derived from the
non-human animal
The lyophilized powder was prepared by taking 1 mL of the culture sup
obtained from the non-human animal (swine) in the example 2 and lyophilizing of it.
The lyophilized powder was dissolved in 30 mL of the physiological saline
to be transferred into a washing bottle, and prepared the sample No. 1 for the skin
to test its effect to the skin.
[0070]
(2) Test conditions for the skin
The test for the skin was carried out according to the following procedures.
Firstly, the skin condition of the test subject was checked, and performed an
interview how they consider their skin conditions. Next, the entire face conditions
of the subjects were pictured by a camera before starting the test (see, Fig. 8 (A)),
and their skin conditions were pictured by using the microscope (x 200) (see, Fig.
10 (A)).
Next, whole amount of the sample No.1 was perfused in the non-woven
fabric for the face pack (Fuji Paper Chemical Company, Facemask). The
non-woven fabric was placed on the face of the subject, and the iontophoresis was
performed under the conditions for 20 minutes per time (an arm, a hand, a palm, a
leg, and a ham, and a bottom of foot) and 1 or 2 times/week at 0.2 to 1 mA.
26
During the iontophoresis, the steamer (Takara Belmont, Noage) was placed
at the position, 0.3 to 0.5 m from the face for providing the steam on the entire of
the face. After that, the facepack was removed from the face, and the face was
treated with a cream and the like to finish. The test subjects were 7 women and
their ages were 20’s to 70’s, average age 41.
[0071]
(3) Effects
The conditions after the test, 14 times iontophoresis, were confirmed by
using the microscope as the same as that before the test start. Compared to the
skin conditions before and after the tests, the skin color of the subjects became
totally whiter than before the test start. Also, the spots were faded (Figs. 8 (A)
and (B)), and the wrinkles were reduced (Figs. 9 (A) and (B)).
When the skin conditions were observed by using the microscope, there
were clear changes to reduce the depth of the wrinkle dent (Figs. 10 (A) and (B)).
It was demonstrated that the cosmetic including the culture sup of the
swine dental pulp stem cells has the effects to reduce the depth of the wrinkle and
lightening the skin color.
EXAMPLE 4
[0072]
Study for growing hair and making hair thick
(1) Preparation of the culture sup derived from the human adipose stem cells
The lyophilized powder was prepared by taking 1 mL of the culture sup
obtained from the non-human adipose cells (swine) in the example 2 and
lyophilizing of it.
The lyophilized powder was dissolved in 30 mL of the physiological saline
to be transferred into a washing bottle, and prepared the sample No. 2 for the
growing hair and making hair thick.
(2) Test conditions for growing hair and making hair thick
The test for the growing hair and making hair thick was carried out
according to the following procedures. Firstly, the hair and the scalp conditions
of the test subject was checked, and performed an interview how they consider
their hair and the scalp conditions. Next, the hair conditions of the subjects were
pictured by a camera before starting the test, and their scalp conditions were
27
pictured by using the microscope (x 20 to 200). Figs. 13 to 16 were pictured with
x 200.
[0073]
Next, appropriate volume of cleansing gel (National total Product, Pleasure
Cleansing Gel) was poured in the palm to be applied onto the scalp. Then, the
scalp was massaged by hands to release pore-clogging debris or those around the
pore. Subsequently, the release debris was washed out by using the
commercially available shampoo (Cosmenist, Pleasure C Shampoo). When the
debris amounts were much, shampoo was performed twice.
After that, the sample No. 2 prepared as described above was attached to
the scalp by contacting the nozzle of the washing bottle to the scalp. Also, the
sample No. 2 was applied on the hair.
The iontophoresis induction was performed under the conditions for 15
minutes per time (an arm, a hand, a palm, a leg, and a ham, and a bottom of foot)
and 1 or 2 times/week at 0.2 to 1 mA. Subsequently, the entire of the scalp and
the hair were wrapped by using polyethylene film. It was further covered by using
the towel like a turban to perform pack for 10 to 20 minutes. After that, the towel
and the polyethylene film were removed, and then the hair was finished with towel
dry or by using a dryer. The test subjects were 8 adults, including 6 men and 2
women (Ages: 30’s to 50’s, average 38).
[0074]
After the iontophoresis as described above, the subject by themselves
were applied to the sample No. 1 or 2 to washed and clean scalp (skin). They
applied the sample directly on their scalp by contacting the nozzle of the container,
and then massaged their scalp for a couple of minutes so as to rub therein. Also,
they applied the sample on their hair.
(3) The effects for growing the hair and making the hair thick
The changes of the situation of male subject before and after the test were
shown in the Figs. 11 to 16.
Fig. 11 shows the hair condition at the test starting. That of the 7 days from
the test start was shown in Fig. 12. Comparison of Figs, 11 and 12 clearly shows
the increase of the hair in macroscopic.
The changes after the test start were shown in Fig. 13 (before test start),
Fig. 14 (after 3 days), Fig. 15 (after 5 days), and Fig. 16 (after 7 days). As the
results of these observations, the hair growing has been confirmed at 3 days after
28
the test start (see, the arrow in Fig. 14). It was confirmed that plural hairs were
growing from one pore after 7 days (see the arrow in Fig. 16).
[0075]
In all of the 8 subjects, the effects for the hair growing and making the hair
thick were observed. Specifically, newly grown hair was black, even when the
hair of the subject has partially white hair. Also, in all of the subjects, the newly
grown hair was grown quickly and thick. Therefore, the volume on the top of the
head increased.
The subject felt the hair growing in the earliest stage was the next day of
the test start (1 day after).
Accordingly, it was confirmed that the cosmetic including the culture sup of
the swine adipose stem cells has the remarkable effects for growing the hair and
making the hair thick.
Example 5
[0076]
Preparations of the cosmetic comprising the growth factor derived from
pSHED/pBMSC are shown in below.
[0077]
[Table 4.]
3GF skin lotion (Trial No.11K-614)
Components
Component
code Name of Classification Indication Name Content amount
(wt%)
1370 Purified water Water Remains
100040 1,3- butylene glycol BG 5 - 10
1075 Ethanol Ethanol 5 - 10
522119 p-Hydroxybenzoate Methylparaben 0.1 - 0.5
105359 Polyoxyethylene hardened
castor oil
PEG- 60 castor oil
hydrogenated 0.01 - 0.1
110759 Edetate disodium EDTA-2Na 0.01 - 0.1
520894 Sodium hyaluronate (2) Hyaluronate Na 0.01 - 0.1
520095 Aloe extract (2) Aloe vera leaf extract 0.01 - 0.1
523147 Tea extract (1) Tea leaf extract 0.01 - 0.1
503038 Chamomilla extract (2) Chamomile flower
Extract 0.01 - 0.1
Culture sup 0.01 - 0.1
29
109336 Perfume Perfume Arbitrarily
Properties
Items Input
Appearance Liquid
Color tone Colorless, clear
Fragrance note No
pH 4.50 - 5.50
Viscosity No
[0078]
[Table 5.]
(Trial No. 11K-641)
Components
Component
code
Name of Classification Indication Name Content
amount
(wt%)
1370 Purified water Water Remains
104368 Myristic acid Myristic acid 10 - 20
1224 Dense glycerin Glycerin 10 - 20
2220 Stearic acid Stearic acid 5 - 10
1346 Potassium hydroxide Potassium hydroxide 5 - 10
103809 Lauric acid Lauric acid 1 - 5
105288 Polyethylene 1540 PEG-32 1 - 5
108556 Polyethylene 300 PEG-6 1 - 5
105370 Polyoxyethylene
laurylether
Laures-7 1 - 5
501064 Ethylene glycol
distearate
Glycol distearate 0.5 - 1.0
102544 Hydrophile-type glyceryl
monostearate
Glyceryl stearate
0.5 - 1.0
100040 1,3-butylene glycol BG 0.5 - 1.0
1191 Olive oil Olive oil 0.1 - 0.5
523096 High melting point
powdered polyethylene
Polyethylene 0.01 - 0.1
520388 Ultramarine pink Ultramarine 0.01 - 0.1
2303 Hydroxy propyl
Cellulose
Hydroxy propyl
Cellulose
0.01 - 0.1
102258 Ethyl cellulose Ethyl cellulose 0.01 - 0.1
8206 Hydroxy propyl methyl
cellulose
Hydroxy propyl
methyl cellulose
0.01 - 0.1
Powdered culture
sup
0.01 - 0.1
30
109336 Perfume Perfume Arbitrarily
Properties
Items Input
Appearance Cream
Color tone White
Fragrance Note No
pH 9.50 - 11.00
Viscosity >80,000 mPa·S
(Spindle No.4-6rpm,1min)
[0079]
[Table 6.]
3GF Cream (Trial No. 11K-642)
Components
Component
code Name of Classification Indication Name
Content
amount
(wt%)
1370 Purified water Water Remains
521058 Polyoxypropylene stearyl
ether PPG-15 Stearyl 1 - 5
1224 Dense glycerin Glycerin 1 - 5
520576 Vegetable squalane Squalane 1 - 5
105420
Polyoxyethylene stearyl
ether
Steareth-2 1 - 5
2220 Stearic acid Stearic acid 1 - 5
105420 Polyoxyethylene stearyl
ether Steareth-21 1 - 5
109250 Behenyl alcohol Behenyl alcohol 1 - 5
104210 Methyl polysiloxane Dimethicone 0.5 - 1.0
555103 Dipolyhydroxy stearic
acid PEG-30 0.5 - 1.0
522119 p-Hydroxybenzoate Methylparaben 0.1 - 0.5
522119 p-Hydroxybenzoate Propylparaben 0.1 - 0.5
520894 Sodium hyaluronate (2) Hyaluronate Na 0.01 - 0.1
506029 Magnesium L-ascorbyl
phosphate
Magnesium ascorbyl
phosphate 0.01 - 0.1
1501 Retinol palmitate Retinol palmitate 0.01 - 0.1
500129 Dipottasium
glyctrrhizinate
Dipottasium
glyctrrhizinate 0.01 - 0.1
104457 Natural vitamin E Tocopherol 0.01 - 0.1
31
1390 Soybean oil Soybean oil 0.01 - 0.1
3610 L-Arginine Arginine 0.01 - 0.1
520095 Aloe extract (2) Aloe vera leaf extract 0.01 - 0.1
523147 Tea extract (1) Tea leaf extract 0.01 - 0.1
503038 Chamomilla extract (2) Chamomile flower
Extract 0.01 - 0.1
523096 High melting point
powdered polyethylene Polyethylene 0.01 - 0.1
520388 Stearyc acid Stearyc acid 0.01 - 0.1
2303 Hydroxy propyl Cellulose Hydroxy propyl
Cellulose 0.01 - 0.1
102258 Ethylcellulose Ethylcellulose 0.01 - 0.1
100040 1, 3-butylene glycol BG 0.01 - 0.1
1076 Absolute ethanol Ethanol 0.01 - 0.1
5217 Dibutylhydroxytoluene BHT 0.01 - 0.1
Powdered culture sup 0.01 - 0.1
109336 Perfume Perfume Arbitrarily
Properties
Items Input
Appearance Cream
Color tone White
Fragrance Note Slightly raw material smell
pH 5.00 - 6.00
Viscosity 10,000 - 50,000 mPa·S
(Spindle No.4-6rpm, 1min)
32
[0080]
[Table 7.]
3GF Serum ( Trial No. 11K-643)
Component
Component
code Name of Classification Indication Name Content amount
(wt%)
1370 Purified water Water Remains
100040 1,3-butylene glycol BG 5 - 10
1075 Ethanol Ethanol 5 - 10
1224 Dense glycerin Glycerin 1 - 5
1446 Triethanolamine TEA 0.5 - 1
101243 Carboxyvinylpolymer Carbomer 0.1 - 0.5
3015 Allantoin Allantoin 0.1 - 0.5
522119 p-Hydroxybenzoate Methylparaben 0.1 - 0.5
110759 Edetate disodium EDTA-2Na 0.01 - 0.1
520894 Sodium hyaluronate (2) HyaluronateNa 0.01 - 0.1
520095 Aloe extract (2) Aloe vera leaf extract 0.01 - 0.1
523147 Tea extract (1) Tea leaf extract 0.01 - 0.1
503038 Chamomilla extract (2) Chamomile flower
Extract 0.01 - 0.1
523096 High melting point
powdered polyethylene Polyethylene 0.01 - 0.1
520388 Ultramarine pink Ultramarine 0.01 - 0.1
2303 Hydroxy propyl
Cellulose
Hydroxy propyl
Cellulose 0.01 - 0.1
102258 Ethylcellulose Ethylcellulose 0.01 - 0.1
1075 Ethanol Ethanol 0.01 - 0.1
Powdered culture sup 0.01 - 0.1
109336 Perfume Perfume Arbitrarily
Properties
Items Input
Appearance Gel
Color tone Slightly white
Fragrance note None
pH 6.00 to 7.00
Viscosity 10,000 to 20,000 MPa·S
(Spindle No.4 - 6rpm, 1min)
33
[0081]
[Table 8.]
3GF Peeling gel (Trial No. 11K-644)
Component
Component
code Names of class Indication Name
Content
amount
(wt%)
1370 Purified Water Water Remains
500263 Sorbit solution Sorbitol 1 - 5
101243 Carboxyvinylpolymer Carbomer 1 - 5
500066 Octadearyl dimethyl
ammonium chloride
Stearyl trimonium
chloride 1 - 5
1075 Ethanol Ethanol 0.5 - 1.0
522119 methyl
parahydroxybenzoate methylparaben 0.5 - 1.0
100040 1, 3-butylene glycol BG 0.5 - 1.0
520286 hydrolyzed silk powder hydrolyzed silk 0.01 - 0.1
560620 hydrolyzed hyaluronic
acid 0.01 - 0.1
520894 Sodium hyaluronate Hyaluronate Na 0.01 - 0.1
520095 aloe extract (2) Aloe vera leaf extract 0.01 - 0.1
523147 tea extract (1) tea leaf extract 0.01 - 0.1
503038 Chamomilla extract (2) Chamomile flower
Extract 0.01 - 0.1
523096 High melting point
powdered polyethylene Polyethylene 0.01 - 0.1
520388 Ultramarine pink Ultramarine 0.01 - 0.1
2303 Hydroxypropyl cellulose Hydroxypropyl
cellulose 0.01 - 0.1
102258 Ethyl cellulose Ethyl cellulose 0.01 - 0.1
powdered culture sup 0.01 - 0.1
109336 perfume Perfume Arbitrarily
Properties
Item Input
Appearance Gel form
Colors Pale white
Flagrance No
pH 2.00 to 3.00
Viscosity 7,000 to 15,000 MPa·S
(Spindle No.4-6rpm,1min)
34
Industrial Applicability
[0082]
The present invention is useful in the cosmetic field.
35

Claims
1. A cosmetic comprising any one of powdery culture sup including at least two
growth factors selected from the group consisting of platelet-derived growth factor
(PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor
(IGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and
transforming growth factor (TGF) obtained by culturing a stem cell selected form
the group consisting of mammal dental pulp of exfoliated deciduous teeth a stem
cell of a swine from 3 to 12 moth old, a swine bone marrow stem cell and a swine
adipose stem cell as a main ingredient.
2. The cosmetic according to the claim 1, said powdery culture sup is prepared from
a culture sup selected from the group consisting of said swine dental pulp of said
exfoliated deciduous teeth, said swine bone marrow, and said swine adipose tissue
of the swine by freeze-drying after alcohol condensation.
3. A cosmetic according to any one of the claims 1 or 2, said cosmetic form is
anyone selected from the group consisting of a liquid, a cream, an ointment, a gel,
an emulsion, and a cataplasm.
4. A cosmetic according to the claim 3, said cosmetic is applied for skin including
scalp or hair.
5. A cosmetic stored in a container comprising:
a first compartment;
a second compartment;
a bar member to form a through hole on a partition between said first and
said second compartment; and
a thin outlet for attaching said preparation dissolved in a solution to a scalp;
wherein,
said first compartment includes a solvent; said second compartment
includes an active ingredient composition comprising a carrier and any one of
powdery culture sup including at least two growth factors selected from the group
consisting of platelet-derived growth factor (PDGF), vascular endothelial growth
36
factor (VEGF), insulin-like growth factor (IGF), keratinocyte growth factor (KGF),
hepatocyte growth factor (HGF), and transforming growth factor (TGF) obtained by
culturing selected from the group consisting of that of a swine dental pulp
exfoliated deciduous teeth from 3 to 12 month age, that of a swine bone marrow,
and that of a swine adipose tissue; and said bar member formed said through hole in
said partition to solve said active ingredient composition into said solvent
immediately before to use said cosmetic.
6. The cosmetic according to the claim 5, said solvent is any one of selected from
the group consisting of a liquid to which an ion is charged, saline and phosphate
buffered saline.
7. The cosmetic according to any one of the claims 5 and 6, which is applied for
skin including scalp and hair.
8. A an iontophoresis method for a protein comprising the steps of:
placing a sheet form of a moisture-retaining member containing said
cosmetic according to either the claim 1 or 5 on a predetermine site through which
said cosmetic is absorbed;
attaching a positively-charged electrode to another predetermined site,
different from the site to be placing the sheet form of a moisture-retaining
member containing said cosmetic; and
attaching a negatively- charged rod-like electrode to rotate and move on
said sheet from.
9. The method according to the claim 8, wherein said predetermined site is any one
of selected from the group consisting of an arm, a hand, a palm, a leg, and a ham,
and a bottom of foot.
10. A dermal formation promoting agent comprising a powdery culture sup
including at least two growth factors selected from the group consisting of
platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF),
insulin-like growth factor (IGF), keratinocyte growth factor (KGF), hepatocyte
growth factor (HGF), and transforming growth factor (TGF) obtained by culturing
of a stem cell of a swine from 3 to 12 month age selected from the group consisting
37
of that of a dental pulp stem cell, that of a bone marrow stem cell, and that of an
adipose stem cell.
11. The cosmetic according to the claim 10, said powdery culture sup is prepared
from said culture sup selected from the group consisting of said dental pulp of said
exfoliated deciduous teeth, said bone marrow, and said adipose tissue by
freeze-drying after alcohol condensation.
12. A cosmetic according to either the claim 10 or 11, said cosmetic has any one of
the form selected from the group consisting of a liquid, a cream, an ointment, a gel,
an emulsion, and a cataplasm.