TW202305116A - Method for producing cell-conditioned medium, and method for producing cosmetic product - Google Patents

Abstract

To solve the problem in which: culturing stem cells takes massive time and effort, such as frequent replacement of the medium; but poor growth of the cells produces a conditioned medium with a low concentration of active ingredients. The present invention discloses a method for producing a conditioned medium by removing cells from a culture medium, in which the cells were cultured. Using stem cells as starting materials, extract of placenta, umbilical cord, or amnion is added to a medium, producing a conditioned medium containing active ingredients in high concentration.

Description

Method for producing cell culture supernatant, and method for producing cosmetics

The present invention relates to a method for producing a culture supernatant obtained by removing cells from a culture solution obtained by culturing cells, and a method for producing a cosmetic product blended with the culture supernatant.

In the medical field, regenerative medicine using stem cells is attracting attention as an alternative therapy for diseases that are difficult to treat with medicines. As stem cells, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and somatic stem cells have been reported. As somatic stem cells, isolated from bone marrow, adipose tissue, skin, umbilical cord, etc. Leaf lineage stem cells (MSC) among various tissues such as , placenta, etc. In stem cell transplantation, it has been known that not only the stem cells themselves bring therapeutic effects, but also various physiologically active substances secreted by stem cells, such as cytokines and exosomes, also contribute greatly to the therapeutic effects. The so-called Cytokine is a general term for physiologically active substances secreted by cells and participating in the mutual response between cells, causing responses such as cell proliferation, differentiation, and functional performance in the receiving cells. In addition, the so-called exosome is a small cell secreted by cells, which contains various proteins or nucleic acids, and has been known to be responsible for the possibility of information transmission between cells. When stem cells are cultured artificially, these physiologically active substances are released from the cells into the culture medium, and thus the culture medium obtained by removing the cells can be recovered and effectively used as a culture supernatant.

Here, in Patent Document 1, a hair growth agent containing a culture supernatant derived from mesenchymal stem cells of the umbilical cord is disclosed. In addition, Patent Document 2 discloses a method for producing a stem cell culture supernatant, which includes a step of supplying a culture solution to the stem cells seeded on the inner surface of a permeable membrane hollow fiber, and culturing the stem cells by bringing the culture solution into contact with the stem cells. and the step of recovering the culture medium containing the components secreted by the stem cells. [Prior Art Literature] [Patent Document]

[Patent Document 1] Japanese Patent Laid-Open No. 2019-26573 [Patent Document 2] International Publication No. 2016/148230

[Problem to be Solved by the Invention]

Adherent cells such as stem cells adhere to the bottom surface of a culture container and proliferate in layers, so unlike cells capable of planktonic culture, the amount of culture solution increases relative to the amount of cells. In addition, the cultivation of stem cells requires a lot of labor and requires frequent replacement of the medium. If the growth of the cells is poor, a culture supernatant (culture supernatant) with a low concentration of useful components is produced. Therefore, an object of the present invention is to provide a culture supernatant containing an active ingredient at a high concentration, and a method for producing a cosmetic in which the culture supernatant is blended. [Means to solve the problem]

In the method for producing a cell culture supernatant corresponding to Claim 1, it is a method for producing a culture supernatant obtained by removing cells from a culture solution obtained by culturing cells, and is characterized in that stem cells are used as a starting point Substances, extracts of placenta, umbilical cord or amniotic membrane are added to the culture medium. According to the present invention described in Claim 1, it is possible to provide a culture supernatant containing an active ingredient at a high concentration.

The present invention described in claim 2 is characterized in that in the method for producing a cell culture supernatant described in claim 1, the stem cells are human-derived umbilical cord stem cells or umbilical cord blood stem cells. According to the present invention described in claim 2, the risk of allergies can be avoided, and in addition, a stem cell culture supernatant that contains cytokines in a good balance can be obtained.

The present invention described in claim 3 is characterized in that in the method for producing the cell culture supernatant described in claim 2, the stem cells are human-derived umbilical cord blood stem cells, and the extract added to the culture medium is horse or pig placenta of extracts. According to the present invention described in Claim 3, the concentrations of cytokines and extracellular bodies contained in the culture supernatant can be further increased.

The present invention described in claim 4 is characterized in that in the method for producing the cell culture supernatant described in claim 2, the stem cells are human-derived umbilical cord blood stem cells, and the extract added to the culture medium is horse or pig umbilical cord of extracts. According to the present invention described in claim 4, the concentrations of cytokines and extracellular bodies contained in the culture supernatant can be further increased.

The present invention described in Claim 5 is characterized in that in the method for producing the cell culture supernatant described in Claim 2, the stem cells are human-derived umbilical cord blood stem cells, and the extract added to the culture medium is horse amniotic membrane extract things. According to the present invention described in Claim 5, the concentrations of cytokines and extracellular bodies contained in the culture supernatant can be further increased.

In the method for producing a cosmetic corresponding to Claim 6, it is characterized in that the cell culture supernatant obtained by the method for producing a cell culture liquid as described in any one of Claims 1 to 5 is blended. According to the present invention described in Claim 6, it is possible to obtain a cosmetic that further suppresses the reduction in the lifespan of stem cells and produces new cells. [Invention effect]

According to the present invention, a culture supernatant containing an active ingredient at a high concentration can be provided.

Hereinafter, a method for producing a cell culture supernatant and a cell culture supernatant according to an embodiment of the present invention will be described.

The steps for producing the stem cell culture supernatant are not particularly limited, and generally used conditions can be applied as they are. In addition, the steps of isolating and selecting stem cells are appropriately adjusted according to the type of stem cells. The so-called stem cells are defined as unspecialized cells that possess self-replication and differentiation capabilities. Specifically, cells that possess the "differentiation ability" that can differentiate into various tissues The cycle also maintains "self-replicating ability" as a characteristic cell in an undifferentiated state. In mammals, there are various types of stem cells depending on the location of the stem cells in the body, including pancreatic stem cells, liver stem cells, hematopoietic stem cells, vascular stem cells, mesenchymal stem cells, neural stem cells, omental stem cells, etc. Among these, epithelial stem cells, hair follicle stem cells, fat stem cells, bone marrow stem cells, umbilical cord stem cells, placental stem cells, dental pulp stem cells, skeletal muscle stem cells, etc. are known as examples of mesenchymal stem cells.

In the method of producing a cell culture supernatant, it is necessary to collect tissue from a donor. The stem cells to be cultured in this embodiment are umbilical cord blood stem cells derived from humans. In addition, the stem cells to be cultured may also be human-derived umbilical cord stem cells. The so-called umbilical cord is a tissue that connects the placenta and the fetus with a total length of 30 to 70 cm. It is disposed of as medical waste after delivery during childbirth. With the consent of the donor, the umbilical cord was recovered, and cord blood was collected from the vein, and anticoagulated treatment was performed if necessary. Store at around 5°C until the next working step, and perform cell separation within 24 hours.

Next, the umbilical cord blood stem cells were isolated. After the umbilical cord blood was diluted with 2mM EDTA-PBS and other buffers, mononuclear cells were obtained by Ficoll density gradient centrifugation. The cells were seeded in a culture dish at a cell density of 1×10 ⁶ /cm ² , and cultured in Dulbecco's Modified Eagle's Medium-Low Glucose (DMEM-LG) medium supplemented with antibiotics as needed. After 12-48 hours, non-adherent cells were removed, and the obtained adherent cells were stored in a humidified environment containing 5% CO ₂ at 37°C in Dulbecco's modified Eagle's medium-low glucose (DMEM-LG) containing 10% MSCGS. ) for cultivation. The culture medium was changed every other day. Cells were harvested at subconfluent using trypsin and passaged as desired. After the cells were grown to 1.0×10 ⁷ /mL, they were recovered to obtain cord blood stem cells. Next, preparation of the stem cell culture supernatant was carried out, and in addition, the cells were stored in liquid nitrogen as needed and thawed as appropriate for use.

Next, a cord blood stem cell culture supernatant was prepared. The term "culture supernatant" means a supernatant obtained by culturing cells using an appropriate medium for cell culture under conditions that allow cell growth, and removing cells from the culture medium after culture. Specifically, the term "medium" refers to a liquid for cell culture that has been adjusted to contain components necessary for cell culture in advance, and which does not come into contact with cells. The liquid obtained as a result of culturing in a state where the medium and cells are in contact is called "culture solution", regardless of the presence or absence of cells. The product obtained by specifically removing cells from the "culture solution" is called "culture supernatant", and the removal of cells from the culture solution can be achieved, for example, by appropriately performing centrifugation, dialysis, or membrane separation. A commercially available medium can be used for the production of the stem cell culture supernatant. For example, DMEM, Ham F-12, MEMα, EMEM, IMDM, RPMI-1640, etc. are used as basal medium, and various vitamins, minerals, etc. are added and appropriately prepared. In addition, in order to improve safety, the stem cell culture supernatant is preferably free of animal serum. As a method for this purpose, multiple subcultures are carried out in the culture of stem cells. By using a serum-free medium in all steps or the last several subcultures, a stem cell culture without animal serum can be obtained. Serum. In addition, serum can also be removed from the stem cell culture supernatant by dialysis or solvent replacement.

In this embodiment, serum is added as needed to basic medium such as DMEM, and the umbilical cord blood stem cells are suitably subcultured in a humidified environment containing 5% CO ₂ at 37°C and cultured for 120-240 hours at the same time. 0.01 to 1% of umbilical cord extract sterilized in advance is added to the culture medium collected as cord blood stem cell culture supernatant and cultured. Umbilical cord extract is used as a raw material for cosmetics and health food, and it is supplied from horses or pigs. The umbilical cord extract is to wash the umbilical cord, add water and enzymes to hydrolyze it, filter it, and add preservatives to make cosmetic raw materials. In addition, it is dried to make health food raw materials. The obtained exchanged medium was collected, and a separation membrane through which cells could not pass was used to obtain a cord blood stem cell culture supernatant (a cord blood-derived stem cell culture supernatant). In addition, instead of the umbilical cord extract, a placenta extract or an amnion extract may be added to the culture medium. Furthermore, at least two of umbilical cord extract, placenta extract or amnion extract may be added to the culture medium.

The culture supernatant obtained by using stem cells is "stem cell culture supernatant", and those with a specific source name indicate the use of stem cells isolated from the tissue. "Umbilical cord blood-derived stem cell culture supernatant" means the Culture supernatant obtained from cord blood stem cells. As an example of the stem cells used to prepare the stem cell culture supernatant, the above-mentioned various stem cells can be used. Stem cell lines include natural (primary) ones taken from donors, strained ones by genetic modification, etc., embryogenic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and the like. As the source animal species of stem cells, from the viewpoint of avoiding the risk of allergies and compatibility, it is most ideal to be derived from humans. In addition, pigs, cows, sheep, and horses can be used as those with high genetic similarity or low risk of infection.

Stem cell lines are known to produce epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), transforming growth factor-α (TGF- α), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), proliferation and differentiation factor-11 (GDF-11) and other cytokines are secreted into the culture medium. In addition, the concentrations of these cytokines can be easily measured using a commercially available ELISA kit for each cytokine. In addition, it is known that the content and balance of physiologically active substances such as cytokines contained in the stem cell culture supernatant vary depending on the source of the stem cell line. It is generally believed that stem cell lines obtained from umbilical cord blood, umbilical cord, placenta, amniotic membrane, etc. collected from the accessory organs of newborns rather than adult tissues are particularly useful.

The recovered umbilical cord blood stem cell culture supernatant can be used as it is as a raw material for cosmetics, or it can also be used after appropriate adjustments such as concentration, solvent replacement, freezing, drying, freeze-drying, dilution, and desalination. Raw materials for cosmetics, etc. In addition, lecithin, cholesterol, etc. may be added to the cord blood stem cell culture supernatant to form liposomes. The culture supernatant is sometimes referred to as "Hue culture medium" or "conditioned medium" in terms of cosmetic ingredients, and is synonymous with "culture supernatant" here. In addition, when it is called "culture solution", it is also synonymous with "culture supernatant" when it can be clearly confirmed that cells have been removed. Since the culture supernatant does not contain the cells themselves or cell components, there is no doubt about immune rejection, even if the culture supernatant is made from the stem cells of a donor that is not the user's own, there is no doubt about it. Use without problems. In addition, since it is not the cells themselves, it is easy to manage or store, and the pre-prepared culture supernatant can be stored and used immediately when needed.

Fig. 1 is a graph comparing the contents of cytokines contained in stem cell culture supernatants. Fig. 1(a) is obtained by using human umbilical cord blood stem cells as the starting material and adding 0.1% The data of the umbilical cord blood stem cell culture supernatant (embodiment 1) obtained by culturing the extract of horse umbilical cord (equine umbilical cord extract) is shown in Fig. 1(b) as a comparative example, by Data on the culture supernatant of adipose-derived stem cells (Comparative Example 1) obtained by culturing human adipose-derived stem cells as a starting material without adding extracts such as umbilical cord extract to the medium. In addition, for easy visibility, the indicated upper limit of the content is set at 700 [pg/ml], and the actual content is described above each data. From FIG. 1 , it can be seen that compared with the adipose-derived stem cell culture supernatant of Comparative Example 1, the umbilical cord blood stem cell culture supernatant of Embodiment 1 contains more GDF-11. GDF-11 is a factor that functions to inhibit stem cells from differentiating into normal cells and maintain stem cells. That is, umbilical cord blood stem cells can maintain stem cells while promoting cell proliferation compared to fat-derived stem cells. Therefore, if the culture supernatant of umbilical cord blood stem cells is used as a raw material for cosmetics, the reduction in the lifespan of stem cells can be suppressed, and new cells can be produced at the same time, which can keep the skin youthful for a longer period of time. On the other hand, using only stem cells or growth factors as raw materials for cosmetics will produce new cells, and on the contrary, it will shorten the lifespan of stem cells. In addition, as shown in FIG. 1 , in the umbilical cord blood stem cell culture supernatant of Embodiment 1, in addition to GDF-11, EGF, FGF, HGF, and KGF are also contained in a good balance. Accordingly, by blending the umbilical cord blood stem cell culture supernatant containing well-balanced cytokines into cosmetics, it is possible to further suppress the reduction in the lifespan of stem cells while producing new cells.

由表1，瞭解到與屬於比較例2之製品1~3的臍帶血幹細胞培養上清液(無添加培養)相比，實施形態1的臍帶血幹細胞培養上清液(馬臍帶提取物添加培養)亦包含較多的GDF-11及其以外之細胞介素。由此，可推斷藉由將臍帶提取物添加至培養基中，便會引起幹細胞的活化。 依此，由於使培養液中含有使用於化妝品或健康食品的原料之臍帶提取物並供予細胞培養，而可更增加每個細胞所釋放之生理活性物質，達成高濃度地包含有效成分之培養上清液的回收。藉此，可提供高濃度地包含有效成分之培養上清液。此外，由於實施形態1的臍帶血幹細胞培養上清液與比較例2之情況相比含有較多的EGF，而可進一步促進細胞的增進。此外，由於實施形態1的臍帶血幹細胞培養上清液與比較例2之情況相比含有較多的被稱為回春因子之GDF-11，而可促進膠原蛋白、彈性蛋白等的增殖，進一步期待皮膚的再生或老化護理作用。 The contents of the cytokines contained in the umbilical cord blood stem cell culture supernatant (Embodiment 1) of this embodiment obtained by adding horse umbilical cord extract to the culture medium, and the content of cytokines not contained in the medium as a comparative example The contents of the cytokines contained in three kinds of umbilical cord blood stem cell culture supernatants (Comparative Example 2) of other companies manufactured by adding extracts such as umbilical cord extracts are shown in Table 1 below.

From Table 1, it can be understood that the cord blood stem cell culture supernatant of embodiment 1 (horse umbilical cord extract added culture ) also contains more GDF-11 and other cytokines. From this, it can be inferred that the addition of the umbilical cord extract to the medium induces the activation of stem cells. According to this, since the umbilical cord extract used as a raw material for cosmetics or health food is included in the culture solution and provided for cell culture, the physiologically active substance released by each cell can be increased, and a culture containing active ingredients at a high concentration can be achieved. Recovery of supernatant. Thereby, a culture supernatant containing an active ingredient at a high concentration can be provided. In addition, since the umbilical cord blood stem cell culture supernatant of Embodiment 1 contains more EGF than that of Comparative Example 2, cell proliferation can be further promoted. In addition, since the umbilical cord blood stem cell culture supernatant of Embodiment 1 contains more GDF-11 called a rejuvenation factor than that of Comparative Example 2, it can promote the proliferation of collagen, elastin, etc., and it is further expected Skin regeneration or aging care.

由表2，瞭解到本實施形態2~5的臍帶血幹細胞培養上清液相比於比較例1、2而言包含較多的CD9及CD63，相比於比較例3而言包含較多的CD9及CD63中之至少任一者。由此，可推斷藉由將豬或馬的胎盤提取物，或者馬的臍帶提取物或羊膜提取物添加至培養基中，便會引起幹細胞的活化。另外，雖然在表2中未加以記載，但在培養基中添加豬臍帶提取物並培養人類的臍帶血幹細胞所獲得之臍帶血幹細胞培養上清液亦獲得同樣的效果。 依此，藉由在培養基中添加提取物並進行培養，便可更增加每個細胞所釋放之生理活性物質，達成高濃度地包含有效成分之培養上清液的回收。藉此，可獲得高濃度地包含有效成分之培養上清液。特定而言，由於實施形態3及實施形態5，即，使用馬臍帶提取物或馬羊膜提取物作為添加至培養基中之提取物，而可獲得相比於屬於無添加培養之比較例3而言含有較多的CD9及CD63兩者之臍帶血幹細胞培養上清液。 Table 2 below shows the quantitative results of extracellular bodies contained in the cord blood stem cell culture supernatant (embodiments 2 to 5) of the present embodiment obtained by adding 1% extract to the medium and culturing the stem cells. The extract added to the culture medium is an extract of pig placenta (pig placenta extract) in embodiment 2, an extract of horse umbilical cord (equine umbilical cord extract) in embodiment 3, and an extract of horse umbilical cord in embodiment 4. It is an extract of horse placenta (horse placenta extract), and in Embodiment 5, it is an extract of horse amnion (horse amnion extract). In addition, the stem cells cultured in embodiments 2-5 are human umbilical cord blood stem cells. In addition, the value of the umbilical cord blood stem cell culture supernatant (Comparative Example 3) obtained by culturing human umbilical cord blood stem cells without adding extracts such as umbilical cord extract to the medium was expressed as 100. Moreover, the data of the product 3 of the said comparative example 1 and the comparative example 2 are also published as a comparative object.

From Table 2, it is understood that the umbilical cord blood stem cell culture supernatants of Embodiments 2 to 5 contain more CD9 and CD63 than Comparative Examples 1 and 2, and more CD9 and CD63 than Comparative Example 3. At least any one of CD9 and CD63. From this, it can be inferred that by adding pig or horse placenta extract, or horse umbilical cord extract or amniotic membrane extract to the culture medium, stem cells are activated. In addition, although it is not described in Table 2, the same effect was also obtained in the umbilical cord blood stem cell culture supernatant obtained by adding porcine umbilical cord extract to the medium and culturing human umbilical cord blood stem cells. Accordingly, by adding the extract to the medium and culturing, the physiologically active substance released by each cell can be further increased, and the recovery of the culture supernatant containing the active ingredient at a high concentration can be achieved. Thereby, a culture supernatant containing an active ingredient at a high concentration can be obtained. Specifically, due to Embodiment 3 and Embodiment 5, that is, using horse umbilical cord extract or horse amniotic membrane extract as the extract added to the medium, compared with Comparative Example 3, which belongs to no-addition culture, it is possible to obtain The umbilical cord blood stem cell culture supernatant containing more CD9 and CD63.

由表3，瞭解到添加提取物並進行培養而得之實施形態2~5的幹細胞培養中之細胞生存率超過未添加提取物並進行培養而得之比較例3的幹細胞培養中之細胞生存率，經添加提取物之培養不會對幹細胞造成妨礙。另外，在經由FCM(流式細胞儀)之幹細胞標記確認中，亦無添加提取物所引發之不良影響。 The cell viability in the stem cell culture of Embodiments 2 to 5 is shown in Table 3 below. In addition, the value of the comparative example 3 was shown as 100.

From Table 3, it can be seen that the cell survival rate in the stem cell culture of Embodiments 2 to 5 obtained by adding the extract and culturing is higher than the cell survival rate in the stem cell culture of Comparative Example 3 obtained by culturing without adding the extract , The culture by adding the extract will not hinder the stem cells. In addition, in the confirmation of stem cell markers by FCM (flow cytometry), there is no adverse effect caused by adding the extract.

由表4，瞭解到與比較例3的臍帶血幹細胞培養上清液(無添加培養)相比，實施形態2~5的臍帶血幹細胞培養上清液(提取物添加培養)亦包含較多的GDF-11、EGF及其以外之細胞介素。 依此，由於使培養液中含有源自豬或馬之萃取物並供予細胞培養，而可更增加每個細胞所釋放之生理活性物質，達成高濃度地包含有效成分之培養上清液的回收。藉此，可獲得高濃度地包含有效成分之培養上清液。此外，實施形態2~5的臍帶血幹細胞培養上清液與比較例3之情況相比含有較多的EGF及GDF-11，故可進一步促進細胞的增進，同時可促進膠原蛋白、彈性蛋白等的增殖，進一步期待皮膚的再生或老化護理作用。 The quantitative results of cytokines in the stem cell culture supernatants of Embodiments 2 to 5 are shown in Table 4 below.

From Table 4, it is understood that compared with the cord blood stem cell culture supernatant (no supplement culture) of Comparative Example 3, the umbilical cord blood stem cell culture supernatant (extract addition culture) of embodiments 2 to 5 also contains more GDF-11, EGF and other cytokines. According to this, since the culture solution contains extracts derived from pigs or horses and is used for cell culture, the physiologically active substances released by each cell can be increased, and a high concentration of the culture supernatant containing active ingredients can be achieved. Recycle. Thereby, a culture supernatant containing an active ingredient at a high concentration can be obtained. In addition, the umbilical cord blood stem cell culture supernatants of Embodiments 2 to 5 contain more EGF and GDF-11 than that of Comparative Example 3, so it can further promote the growth of cells, and at the same time promote collagen, elastin, etc. Proliferation of the skin is further expected for skin regeneration or aging care.

Fig. 2 is a graph obtained by comparing the moisturizing effects. Under the conditions of a temperature of 27°C and a humidity of 35%, each sample was applied to a human arm according to the following test method to measure the moisture content of the skin. The samples were the umbilical cord blood stem cell culture supernatant of Embodiment 1 (equine umbilical cord extract supplemented culture), the umbilical cord blood stem cell culture supernatant of Comparative Example 3 (no additive culture) and the water of the comparative example (comparative example 4). three kinds. [experiment method] 1. Use an oil-based pen to mark a square frame with a length of 3.0 cm and a width of 2.0 cm on the measurement point on the inner side of the lower arm. 2. Wash the measuring point with soap and spend 30 minutes quietly. 3. Measure the moisture content of the skin at the measurement point in normal times (before application of each sample). This measurement was performed 5 times, and the average value was calculated|required. 4. Take 0.1 g of the sample to the measurement site, and use fingers to spread the sample evenly vertically and horizontally 10 times until the second joint part. 5. After applying the sample, measure the moisture content of the skin 10, 30, and 90 minutes later using a skin surface stratum corneum moisture content measuring device (SKICON-200EX manufactured by Yayoi Co., Ltd.). [result] The lines connecting "×" in Fig. 2 represent the data of the culture supernatant of cord blood stem cells (equine umbilical cord extract supplemented culture) in Embodiment 1, and the lines connecting "■" represent the culture supernatant of cord blood stem cells in Comparative Example 3 The data of the liquid (no addition culture) and the line connecting "◆" show the data of the water of Comparative Example 4. In addition, the moisture content of the skin at normal time was set to zero. From Fig. 2, it can be seen that after 90 minutes of application, the amount of skin moisture in the case of applying the umbilical cord blood stem cell culture supernatant (equine umbilical cord extract supplemented culture) of Embodiment 1 is compared with that of Comparative Examples 3 and 4 is higher.

Fig. 3 is a graph comparing the texture improvement effects. Using the umbilical cord blood stem cell culture supernatant of Embodiment 1 (with the addition of horse umbilical cord extract) and the umbilical cord blood stem cell culture supernatant of Comparative Example 3 (without addition of culture) as samples, the temperature was 25°C and the humidity was 35%. Next, take 2 drops (about 0.1g) of the sample on your hand after washing your face, and gently apply it to the corner of the eye with your fingertips to confirm the texture improvement effect. Fig. 3 (a) shows the result of the umbilical cord blood stem cell culture supernatant (horse umbilical cord extract addition culture) of Embodiment 1, Fig. 3 (b) shows the result of the umbilical cord blood stem cell culture supernatant of Comparative Example 3 (no addition result of cultivation). In Figure 3(a) and (b), the left side is the microscope image, the right side is after analysis, the upper row is before the sample was applied, and the lower row is the sample after 60 minutes of application. In addition, the microscopic images before coating and 60 minutes after coating are taken of the same site, and the same site is indicated by a circle mark or a solid line. Analyze the microscope image, and compare the texture of the two samples before and after coating visually. If the state of the texture is poor, the texture will change slowly, and there will be no drop between the piqiu and the pigou. When the texture is in good condition, the Pichu will bulge and the network will be latticed. As shown in FIG. 3 , the state of the texture before application was poor, but a clear texture was confirmed for both samples 60 minutes after application, and the texture was improved no matter which culture supernatant was applied. Furthermore, in the case of the umbilical cord blood stem cell culture supernatant (equine umbilical cord extract supplemented culture) of Embodiment 1, the gap between the pituitary and furrow was more clearly confirmed and became tighter. Therefore, it can be said that the umbilical cord blood stem cell culture supernatant (cultured with horse umbilical cord extract) in Embodiment 1 is more effective in improving texture than the umbilical cord blood stem cell culture supernatant (no supplemented culture) in Comparative Example 3.

FIG. 4 is a photograph showing an overall image of a cell migration image, and FIG. 5 is a photograph showing an enlarged image of a gap region of a cell migration image. Using normal human epidermal keratinocytes, the umbilical cord blood stem cell culture supernatant (equine umbilical cord extract supplemented culture) of Embodiment 1 was concentrated 10 times and used as a test substance (n=5), and observed what kind of Gap areas that are areas where cells are not present are filled to an extent. As a result, it was confirmed that when the test substance was added at 0.3% (corresponding to 3%), the interstitial region remarkably migrated.

In addition, the fluorescence intensity in the gap region was measured with respect to the images in Fig. 4 and Fig. 5, and the migration rate of the cells was quantified. FIG. 6 is a table showing the results of quantification of cell mobility, and FIG. 7 is a graph showing the results of quantification of cell mobility. Statistical analysis was carried out, and as a result, a significant increase in mobility was confirmed when the added concentration of the test substance was 0.3% (equivalent to 3%) or more. Accordingly, the umbilical cord blood stem cell culture supernatant (equine umbilical cord extract supplemented culture) of Embodiment 1 can promote the migration of keratinocytes, and can be expected to fill the area where the cells do not exist, that is, For example, the healing effect caused by the migration of damaged parts, etc.

[Figure 1] Graph comparing the content of cytokines contained in the stem cell culture supernatant [Figure 2] Graph comparing moisturizing effects [Figure 3] Comparison of texture improvement effects [Fig. 4] Photograph showing the overall image of the cell migration image [Fig. 5] A photograph showing an enlarged image of the gap region of the image of cell migration [Fig. 6] A table showing the quantitative results of cell mobility [Fig. 7] Graph showing the quantitative results of cell mobility

Claims (6)

A method for producing a cell culture supernatant, which is a method for producing a culture supernatant obtained by removing the aforementioned cells from a culture solution obtained by culturing cells, It is characterized in that stem cells are used as the starting material, and extracts of placenta, umbilical cord or amniotic membrane are added to the culture medium. The method for producing a cell culture supernatant according to claim 1, wherein the aforementioned stem cells are human-derived umbilical cord stem cells or umbilical cord blood stem cells. The method for producing a cell culture supernatant according to claim 2, wherein the stem cells are the umbilical cord blood stem cells derived from humans, and the extract added to the medium is the extract of the placenta of horse or pig. The method for producing a cell culture supernatant according to claim 2, wherein the stem cells are the umbilical cord blood stem cells derived from humans, and the extract added to the medium is the extract of the umbilical cord of horse or pig. The method for producing a cell culture supernatant according to claim 2, wherein the stem cells are the umbilical cord blood stem cells derived from humans, and the extract added to the medium is the extract of the amniotic membrane of a horse. A method for producing a cosmetic, characterized by blending the cell culture supernatant obtained by the method for producing a cell culture solution according to any one of claims 1 to 5.

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