TWI558421B - Use of stem cell conditioned medium for preparing compositions to inhibit oxidation for anti-aging skin - Google Patents

Description

Stem cell conditioned medium for the preparation of a composition for inhibiting oxidation to achieve anti-aging of the skin

The invention relates to a use of a stem cell conditioned medium for preparing a composition for inhibiting oxidation to achieve skin anti-aging, in particular to a conditioned medium for human Whaiton's Jelly mesenchymal stem cells, which can be used to scavenge free radicals and increase skin cells. The survival rate under oxidative stress is used to improve the skin condition of the user to achieve anti-oxidation and anti-aging effects.

According to the hole in the ozone layer, the ultraviolet rays we face are more intense. One of the primary causes of UV oxidation is the rapid oxidation and aging of cells. Moreover, with the increase of age, the ability of the human body to repair free radicals also decreases. If the antioxidants are not added in time to maintain or repair the skin, the cell damage will become more and more obvious. In order to maintain or restore the appearance of youthfulness, not only women attach importance to beauty and maintenance, but even men are eagerly awaiting. Therefore, the beauty care products used by the human body have become the focus of manufacturers' development.

At present, natural substances such as vitamin C (L-ascorbic acid), vitamin E, beta carotene, kojic acid, and super dismutase have been confirmed by studies. It can prevent the damage of most free radicals; although these substances do have significant effects on antioxidants, they have limitations in their use, such as vitamin C in yang. Under light or contact with air, it is easily oxidized and not heat-resistant. When the concentration exceeds 5%, it will cause irritation and redness to the skin. The acidity is very unstable, it is easily oxidized, discoloration and skin allergies, and long-term excessive use of tannic acid. The product can cause cytotoxicity and pathological changes; in addition, some antioxidants act to directly destroy cells, which is easy to cause cell toxicity. Therefore, how to develop a safe and effective antioxidant component has become the direction that the inventors of the relevant field have thought.

Stem cell research has become a global trend in recent years. It can be divided into two major categories: embryo and adult stem cells. Mesenchymal Stem Cell (MSC) belongs to adult stem cells and has strong differentiation ability, except for differentiation. It is a cell tissue derived from mesodermal cells such as human bones, and can also be differentiated into liver cells such as endoderm, visceral cells such as pancreas, and nerve cells derived from ectoderm. Although mesenchymal stem cells are widely distributed in adults, they can be isolated from bone marrow and various organs. However, due to the small number of mesenchymal stem cells in each part, and the mesenchymal stem cells in adults, they gradually increase with age. Reduced, so how to obtain sufficient stem cell stem cells is very important. Bone marrow mesenchymal stem cells are mainly derived from adult bone marrow. However, invasive methods can cause donors to feel painful and uncomfortable. The umbilical cord contains abundant and young mesenchymal stem cells, which are convenient and strong. Potential, therefore, can be used as an important source of mesenchymal stem cells. In addition, studies have also indicated that mesenchymal stem cell-conditioned medium (MSC-CM) can increase the dorsal root ganglia cells exposed to hydrogen peroxide under oxidative stress. Cell viability ( PLoS One. 8(5): e62807, 2013) with neuroprotective properties.

Nowadays, the inventor is still in the spirit of tirelessness and rich in professional knowledge, in view of the fact that the existing antioxidant products still have multiple defects in practical use. With the help of years of practical experience, and improved, and based on this, the present invention was developed.

The main object of the present invention is to provide a dry stem cell conditioned medium for preparing a composition for inhibiting oxidation to achieve skin anti-aging, which is a conditioned medium for human Wharton's Jelly mesenchymal stem cells, which can be used to scavenge free radicals and increase skin cells. The survival rate under oxidative pressure, whereby the stem cell conditioned medium is applied to the cosmetic composition, can be used to improve the skin condition of the user to achieve antioxidant and anti-aging effects.

In order to achieve the above-mentioned object, the present invention relates to a stem cell conditioned medium for preparing a composition for inhibiting oxidation to achieve anti-aging of the skin, wherein the conditioned medium first grows the stem cells in a cell culture dish containing the complete medium, and the complete medium contains There are α-MEM, fetal bovine serum and human basic fibroblast growth factor, and the conditioned medium is obtained after subculture of stem cells in basal medium, which contains only α-MEM and human base. Fibroblast growth factor (excluding fetal bovine serum). Conditioned medium was obtained after subculture of stem cells for at least three generations.

The invention also provides a use of a stem cell conditioned medium for reducing oxidation, which is applied to a skin at a concentration of 10% by weight or more to remove free radicals and increase the survival rate of skin cells under oxidative stress.

In one embodiment of the invention, the stem cell line is a mesenchymal stem cell, and the best line is Wharton's Jelly mesenchymal stem cells from the human umbilical cord.

In one embodiment of the present invention, the complete medium comprises 10-20% fetal bovine serum and 2-6 ng/ml human basic fibroblast growth factor, calculated as a total weight percentage of 100% of the components. And the remaining weight percentage of Minimum Essential Medium Alpha (α-MEM); wherein the stem cells are best cultured for three generations.

In an embodiment of the present invention, the basal medium is calculated as a total weight percentage of 100% of the components, comprising 2-6 ng / ml of human basic fibroblast growth factor, and the remaining weight percentage of α-MEM; Among them, the best stem cells are subcultured for three generations.

First: Stem cell conditioned medium can effectively remove hydrogen peroxide.

Figure II-A: Schematic diagram of stem cell conditioned medium in ABTS free radical scavenging capacity.

Figure II-B: Stem cell conditioned medium has the effect of scavenging ABTS free radicals.

Figure III-A: Schematic diagram of stem cell conditioned medium in DPPH free radical scavenging capacity.

Figure 3 - B: Stem cell conditioned medium has the effect of scavenging DPPH free radicals.

Figure 4: Schematic diagram of cell viability under oxidative stress.

Figure 5: Stem cell conditioned medium increases the survival rate of cells under oxidative stress.

The object of the present invention and its structural and functional advantages will be explained in conjunction with the specific embodiments according to the structure shown in the following drawings, so that the reviewing committee can have a more in-depth and specific understanding of the present invention.

The invention relates to a stem cell conditioned medium for preparing a composition for inhibiting oxidation to achieve skin anti-aging, wherein the conditioned medium first grows stem cells in a cell culture dish containing complete growth medium; % of the total weight percentage of the constituents, which may include 10-20% (20% of the best) fetal blood Fetal bovine serum, 2-6 ng/ml (optimal line 4 ng/ml) human-basic fibroblast growth factor, and residual weight percentage of α-MEM; The conditioned medium is obtained by subculture of the above stem cells in basal medium for at least three generations, and the optimal system is three generations of culture; the basal medium is calculated by the total weight percentage of 100% of the components, and may include 2-6 ng. /ml (optimal line 4 ng/ml) of human basic fibroblast growth factor, and the remaining weight percent of alpha-MEM, but not fetal calf serum. Stem cell line mesenchymal stem cells, preferably human Wharton's Jelly mesenchymal stem cells.

Furthermore, the use of a stem cell conditioned medium for reducing oxidation in the present invention is to administer a conditioned medium having a concentration of 10% by weight or more to the skin to scavenge free radicals and increase the survival rate of skin cells under oxidative stress. The conditioned medium is first grown in a cell culture dish containing a complete medium containing α-MEM, fetal bovine serum, and human basic fibroblast growth factor, and the conditioned medium is ligated to the above stem cells. Subcultured in basal medium for at least three generations, the best line is cultured for three generations; the basal medium is calculated as 100% of the total weight percentage of components, which may include 2-6 ng/ml (the best system is 4 ng/ml) The human basic fibroblast growth factor, as well as the remaining weight percentage of alpha-MEM.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.

Experiment 1: Effect of stem cell conditioned medium on hydrogen peroxide scavenging ability

<Cell culture>

First, human foreskin fibroblasts (Hs68: BCRC 603800) were cultured. In a cell culture dish containing complete growth medium (BD Falcon/BD biosciences), complete medium containing DMEM (Gibco) plus 10% Fetal bovine serum (FBS) (Gibco); and human Wharton's Jelly mesenchymal stem cells (WJMSC) :BCRC H-WJ001) was cultured in a cell culture dish containing complete growth medium (BD Falcon/BD biosciences) containing α-MEM (Gibco) plus 20% fetal bovine serum (fetal bovine) Serum, FBS) (Gibco) and 4 ng/ml human-basic fibroblast growth factor (bFGF) (Peprotech). The above two cells were cultured in a cell incubator at a temperature of 37 ° C and containing 5% carbon dioxide. The cells were cultured for 3 days and then subjected to subculture.

The subculture steps were as follows: the original cell culture medium was removed, and then the attached cells were moistened with phosphate buffered saline (PBS) (Roche), and the supernatant was removed and reused with 0.05% Trypsin-EDTA (Life Technologies). The cell incubator was allowed to act for 5 minutes to remove the cells from the top of the dish. After the medium was added, the cells were dispersed, centrifuged at 1,200 rpm for 3 minutes, the supernatant was removed, and the remaining cell pellets were dispersed in the medium and cultured in a cell culture incubator at 5% CO ₂ at 37 ° C. Inside. After subculture to the third generation, cell conditioned medium was collected.

Cell Conditioning Medium

Human Wharton's Jelly mesenchymal stem cells were seeded in cell culture dishes at a density of 5 X 10 ⁴ cells/cm ² . After 1 day of cell culture, the attached cells were treated with PBS three times, and the basal medium (containing only α-MEM and 4 ng) was added. /ml human basic fibroblast growth factor), after 48 hours, the medium was collected in a 50 ml centrifuge tube, centrifuged at 2,000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm filter bowl (BD Falcon/BD biosciences). That is, a mesenchymal stem cell conditioned medium can be obtained. This medium can be stored in a -20 ° C refrigerator.

<Hydrogen peroxide (H ₂ O ₂ ) removal test>

This experiment was based on the method developed by Ruch et al. in 1989. Hydrogen peroxide will have maximum absorption at 230 nm. Therefore, the hydrogen peroxide removal rate can be detected using the absorbance value. Hydrogen peroxide was formulated into 2 mg/L using phosphate-buffered saline (PBS, pH 7.4), and 1 ml sample (100% conditioned medium, 50% conditioned medium, and control group 200 mg/L of vitamin C (L-ascorbic acid) , L-AA)) Add 0.6 ml of hydrogen peroxide, react for 10 minutes, and finally analyze with a wavelength of 230 nm using a multi-functional microplate analyzer. Hydrogen peroxide scavenging effects (%) = [(Control group at 230 nm absorbance (A ₂₃₀ of control) - sample at 230 nm absorbance (A ₂₃₀ of sample)) / control group absorbance at 230 nm (A ₂₃₀ of control)] × 100%. This experiment was performed at least three replicates, and the values in the graph are mean ± mean standard error (SEM). *** P < 0.005.

Experiment 2: Effect of stem cell conditioned medium on ABTS free radical scavenging ability

<ABTS Free Radical Scavenging Test>

In this experiment, an antioxidant test kit (Cayman) was used to analyze the antioxidant capacity, and 10 μl of the sample (1, 0.5, 0.25, 0.125, 0.0625 mM vitamin C or 100%, according to the method of the kit). 50%, 25%, 12.5%, 6.25% stem cell conditioned medium) 10 μl of metmyoglobin, 150 μl of chromogen and 40 μl of hydrogen peroxide were mixed. Since the stem cell conditioned medium (CM) is a composite component, the concentration of the antioxidant component is not known, so the percentage is used as a unit. After reacting for 5 minutes at room temperature, the analysis was carried out using a wavelength of 750 nm of a multifunctional microplate analyzer.

Experiment 3: Effect of stem cell conditioned medium on DPPH free radical scavenging ability

<DPPH free radical scavenging test>

1 ml of sample (100%, 50%, 10% and 1% stem cell conditioned medium) was mixed with 1 ml of freshly prepared 0.1 mM DPPH dissolved in 95% ethanol (EOH). After reacting for 30 minutes at room temperature, the analysis was carried out using a wavelength of 517 nm of a multifunctional microplate analyzer. This experiment was performed at least three replicates, and the values in the graph are mean ± mean standard error (SEM). *P<0.05; ***P<0.005.

Experiment 4: Determination of cell viability of stem cell conditioned medium under oxidative stress

<Cellular Antioxidant Test>

The fibroblasts are cultured in a cell culture dish at a density of 2 X 10 ⁴ cells/cm ² for one day, the original medium is removed, and the basal medium (containing only DMEM), the stem cell complete medium plus different concentrations are added. H ₂ O ₂ (0, 0.0125, and 0.025 mM) were incubated together, and after 24 hours, photographs were taken using an optical microscope (Leica). The original cell culture medium was first removed, and then the attached cells were moistened with PBS (Roche), and the supernatant was removed, and then the cells were cultured for 5 minutes using 0.05% Trypsin-EDTA (Life Technologies) in a cell culture incubator. Remove the dish above. After the medium was added, the cells were dispersed, centrifuged at 1,200 rpm for 3 minutes, the supernatant was removed, and the remaining cell pellet was dispersed by adding 0.2 ml of PBS, followed by 0.2 ml trypan blue (Invitrogen). (PBS: trypan blue = 1:1), staining. The number of cells can be calculated using the trypan blue exclusion test in conjunction with a hemocytometer. Three replicates are performed for each set of samples.

result

Result 1: Stem cell conditioned medium can effectively remove hydrogen peroxide

Please refer to the first figure. According to the student t- test analysis, after treatment with 200mg/L of vitamin C (L-AA), 88.18%±1.06% of hydrogen peroxide can be removed; after stem cell conditioned medium (CM) treatment, it can be removed. 61.69% ± 1.88% of hydrogen peroxide, 50% stem cell conditioned medium treatment can remove 27.77% ± 1.32% of hydrogen peroxide. The data for this experiment was defined as 100% with sterile water plus hydrogen peroxide treatment. This experiment was performed at least three replicates and the values in the figure are mean ± mean standard error (SEM). *** P <0.005. From this result, it is understood that the stem cell conditioned medium of the present invention can effectively remove hydrogen peroxide.

Result 2: Stem cell conditioned medium has the effect of scavenging ABTS free radicals

Please refer to Figure II-A for a schematic diagram of the free radical scavenging capacity of stem cell conditioned medium in ABTS. Different concentrations of vitamin C (L-AA) and stem cell conditioned medium (CM) are reacted with an appropriate amount of ABTS and an oxidizing agent. Since ABTS has the maximum absorbance at OD 750 nm when catalyzed by an appropriate amount of oxidant, the ability of the sample to resist oxidation can be inferred by the value of the absorbance. The results are shown in Figure II-B. After statistical analysis, the semi-inhibitory concentration (IC ₅₀ ) of vitamin C (L-AA) is 0.8059 mM, which is equivalent to the antioxidant capacity of 119.45% stem cell conditioned medium. This experiment demonstrates that stem cell conditioned medium has the ability to scavenge ABTS free radicals, although not as strong as L-AA, but still has good antioxidant capacity.

Result 3: Stem cell conditioned medium has the effect of scavenging DPPH free radicals

Please refer to the third figure-A, which is a schematic diagram of the DPPH free radical scavenging ability of stem cell conditioned medium, and different concentrations of stem cell conditioned medium (CM) are reacted with an appropriate amount of DPPH. With DPPH, the maximum absorbance at OD 517nm is changed when the antioxidant reduces DPPH. Therefore, the ability of the sample to resist oxidation can be inferred by the level of absorbance. For the results, please refer to the third figure-B. After statistical analysis, more than 10% of the stem cell conditioned medium (CM) mixed has DPPH inhibition ability (inhibition). The results show that the stem cell conditioned medium can It is used to effectively remove DPPH free radicals and has antioxidant capacity.

Results 4: Stem cell conditioned medium can increase the survival rate of cells under oxidative stress

Please refer to the fourth figure for the cell viability under oxidative stress. The fibroblasts are subjected to normal medium (DMEM), DMEM + 0.025 mM H ₂ O ₂ , DMEM + 0.0125 mM H ₂ O ₂ , stem cell conditions. Under different treatment conditions of medium (CM), CM+0.025 mM H ₂ O ₂ and CM+0.0125 mM H ₂ O ₂ cells, stem cell conditioned medium can significantly increase cell survival rate. Please refer to the fifth figure. After the Student t- test analysis, adding 0.025 or 0.0125 mM H ₂ O ₂ in general conditioned medium (DMEM) will cause total fibroblast death; stem cell conditioned medium plus 0.025 After treatment with mM H ₂ O ₂ , 28.57% ± 8.25% of cells survived, and 85.71% ± 8.25% of cells survived after treatment with stem cell conditioned medium plus 0.0125 mM H ₂ O ₂ . The data in the figure is defined as 100% after treatment with normal medium (DMEM), and the experiment is performed at least three replicates, and the values in the figure are mean ± mean standard error (SEM). * P <0.05; ** P <0.001; *** P < 0.005. It can be seen that the stem cell conditioned medium can significantly increase the survival rate of the cells under oxidative stress, and thus has good anti-cell oxidizing ability.

In summary, human Wharton's Jelly mesenchymal stem cells (WJMSC) secrete many growth factors into the medium to form a stem cell conditioned medium (WJMSC-CM); this stem cell conditioned medium can be cultured by removing H ₂ O ₂ and clearing ABTS. It removes DPPH free radicals and increases the survival rate of skin cells under oxidative stress to achieve antioxidant effects. Thereby, the stem cell conditioned medium of the present invention can be further applied and added to the cosmetic material composition to improve the skin condition of the user and achieve the effect of slowing down the skin aging.

It can be seen from the above description that the present invention has the following advantages compared with the prior art:

1. The leaf stem cell conditioned medium of the present invention contains a large amount of growth factors, which can effectively reduce the oxidation effect and achieve the anti-aging effect by removing free radicals and increasing the antioxidant capacity of the cells, thereby improving skin cells. Damage and other issues.

2. The umbilical cord Wharton's jelly mesenchymal stem cell line used in the present invention is taken from an umbilical cord which is not required after the baby is born, and does not cause pain and avoids ethical problems as compared with the bone marrow; and the stem cells are in the umbilical cord. The content is higher, so it is easier to obtain.

3. Bone marrow mesenchymal stem cells belong to more advanced cells, which can differentiate into fewer cell types and are more susceptible to donor age; while umbilical cord Wharton's jelly mesenchymal stem cells belong to earlier groups and can differentiate into more cell types. The conditioned medium has a better protein factor, which is effective for slowing down the oxidation, repairing the skin and improving the skin's freezing age.

In summary, the use of the stem cell conditioned medium of the present invention for preparing a composition for inhibiting oxidation to achieve anti-aging of the skin can indeed achieve the intended efficacy by the above-disclosed examples, and the present invention has not Before being disclosed to the application, Cheng has fully complied with the requirements and requirements of the Patent Law.爰Issuing an application for a patent for invention in accordance with the law, and asking for a review, and granting a patent, is truly sensible.

The illustrations and descriptions of the present invention are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention; those skilled in the art, which are characterized by the scope of the present invention, Equivalent variations or modifications are considered to be within the scope of the design of the invention.

Claims (5)

A stem cell conditioned medium for preparing a composition for inhibiting oxidation to achieve skin anti-aging, wherein the conditioned medium is first grown by growing stem cells in a cell culture dish containing a complete growth medium for 3 days. For culture, the stem cell conditioned medium is obtained by subculture of the stem cells in a basal medium, which is collected by centrifugation and filtration, and the complete medium contains α-MEM, Fetal bovine serum, and Human basic fibroblast growth factor, which comprises α-MEM and human basic fibroblast growth factor; wherein the stem cell line is human Wharton's Jelly mesenchymal stem cells. The stem cell conditioned medium according to claim 1 is for use in the preparation of a composition for inhibiting oxidation to achieve anti-aging of the skin, wherein the stem cell line is subcultured for at least three generations. The stem cell conditioned medium according to claim 2 of the patent application is for use in the preparation of a composition for inhibiting oxidation to achieve anti-aging of the skin, wherein the stem cell line is subcultured for three generations. The use of a stem cell conditioned medium according to claim 1 for the preparation of a composition for inhibiting oxidation to achieve anti-aging of the skin, wherein the complete medium is calculated as a total weight percentage of 100% of the constituent components, and comprises 10-20 % fetal calf serum, 2-6 ng/ml of human basic fibroblast growth factor, and a remaining percentage by weight of alpha-MEM, and the basal medium is calculated as a total weight percentage of 100% of the constituents, including 2- 6 ng/ml of human basic fibroblast growth factor and residual weight percent alpha-MEM. The stem cell conditioned medium according to claim 1 is for use in the preparation of a composition for inhibiting oxidation to the skin against aging, wherein the conditioned medium is further used as a cosmetic or anti-aging cosmetic material composition.