Formula and preparation method of mesenchymal stem cell conditioned medium freeze-dried ball for facial wrinkle resistance and compactness

Abstract

The invention discloses a formula and a preparation method of a mesenchymal stem cell conditioned culture medium freeze-dried ball for facial wrinkle resistance and firming, the prepared umbilical cord mesenchymal stem cell conditioned culture medium freeze-dried ball can effectively remove DPPH free radicals, has the wrinkle resistance and firming effects, can effectively improve the skin elasticity of a subject in a human body experiment, and can be used for preparing the umbilical cord mesenchymal stem cell conditioned culture medium freeze-dried ball for facial wrinkle resistance and firming. And wrinkles are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of cord blood stem cell freeze-dried powder, and particularly relates to a formula and a preparation method of freeze-dried balls of mesenchymal stem cell conditioned medium for facial anti-wrinkle and firming. Background Art

[0002] The skin is the outer shell on the body surface and the largest organ of the human body. On the one hand, it protects the body by reducing the harm of ultraviolet rays, pollution, infections and other irritants; on the other hand, it reflects the health and appearance status of the human body through skin characteristics. Wrinkles are the convex and concave stripes on the skin surface. From the aspect of skin appearance, wrinkles are its important features, and from the aspect of skin aging, wrinkles are the results of its occurrence. It has been found through research that wrinkles generally first appear on the human forehead at about 20 years old, and increase and deepen with age; at the same time, wrinkles begin to appear at the outer corners of the eyes and spread in a fan shape, and then radial wrinkles and crow's feet continue to appear around the eyelids; after about 45 years old, deep furrows of wrinkles develop in the area from the mouth to the jaw and extend around the mouth. As people age, wrinkles become increasingly severe and develop to the ear roots, neck and whole body. Skin wrinkles can reflect the aging degree of human skin. Developing cosmetics with anti-wrinkle effects helps to delay skin aging.

[0003] Stem cells are a unique cell population with self-renewal and differentiation characteristics, and can produce specific cell lines or cells. Umbilical cord mesenchymal stem cells (UC-MSCs) are isolated from Wharton's jelly of the umbilical cord. The serum-free conditioned medium (SFM) of UC-MSCs has anti-photoaging characteristics. The SFM prepared from UC-MSCs has an anti-photoaging effect after long-term ultraviolet irradiation, promotes the proliferation of HDF, and can reduce UVA-induced cell death. It has been found that topical application of UC-MSC SFM to mouse skin before ultraviolet irradiation blocks the inhibition of the activities of SOD and glutathione peroxidase (GSH-Px) and inhibits the up-regulation of MDA. Therefore, UC-MSC SFM can prevent photoaging caused by UVA and UVB radiation and is a promising skin anti-photoaging treatment method. Adding cytokines and active substances to beauty cosmetics not only has moisturizing and whitening effects, but also can repair damaged skin, eliminate skin wrinkles, shrink pores and improve complexion. Summary of the Invention

[0004] Since the original preparation method has low efficiency and is prone to cause a decrease in the activity of mesenchymal stem cell factors, for this reason, we propose a preparation method of freeze-dried balls of mesenchymal stem cell conditioned medium for facial anti-wrinkle and firming.

[0005] The specific technical solutions are as follows:

[0006] The first aspect of the present invention provides a method for preparing a mesenchymal stem cell conditioned medium for facial anti-wrinkle and firming, the preparation method comprising the following steps:

[0007] 1) Place the umbilical cord in 75% alcohol for disinfection, and then wash it repeatedly with saline until the umbilical cord tissue is bloodless;

[0008] 2) Cut the umbilical cord tissue in step 1 into tissue segments, then peel off the Wharton's Jelly from the tissue segments, place the Wharton's Jelly in a clean container, and cut it into blocks with tissue scissors. During the cutting process, add α-MEM culture medium to keep the tissue moist;

[0009] 3) Add 0.1% type II collagenase and 0.25% trypsin to the block tissue obtained in step 2 for digestion, collect the cell solution by filtration, and then centrifuge to remove the supernatant;

[0010] 4) Resuspend the cell pellet obtained in step 3 using a culture medium containing fetal bovine serum, place it in a container coated with fibronectin, add a sufficient amount of culture medium containing fetal bovine serum, gently shake the container to fully moisten the adherent surface, evenly spread it on the adherent surface of the container, and place it in a CO2 incubator for culture;

[0011] 5) The first medium change is performed 4 to 5 days after culture, and the medium is changed every 3 to 4 days thereafter. When the cell confluence reaches more than 70%, trypsin is used for digestion and then passage;

[0012] 6) Inoculate P5 umbilical cord mesenchymal stem cells into a culture bottle and add culture medium containing fetal bovine serum for culture;

[0013] 7) When the cells grow to 80% confluence, rinse the cell surface several times with saline, and then add DMEM / F12 medium for starvation culture;

[0014] 8) Collect the umbilical cord mesenchymal stem cell culture conditioned medium obtained in step 7 into a centrifuge tube, centrifuge, and discard the precipitate;

[0015] 9) Collect the conditioned medium obtained in step 8, and concentrate the stem cell conditioned medium using a 5KD dialysis device to obtain an effective stem cell secretory mixed factor with a molecular weight of more than 5KD;

[0016] 10) The concentrated conditioned medium obtained in step 9 is homogenously mixed with 50-60% water, 2-4.5% mannitol, 0.5-1% trehalose, 0.15-0.25% hydrolyzed collagen, 0.025-0.05% oligopeptide-1, 0.015-0.025% hydrolyzed sodium hyaluronate, and 0.015-0.025% carnosine.

[0017] Further, in step 1 of the preparation method, the umbilical cord used is from a full-term healthy fetus.

[0018] Further, the umbilical cord of the full-term healthy fetus is the umbilical cord with negative results in the detection of umbilical cord serum HbsAg, anti-HCV, HCV-RNA, anti-HDV, anti-HEV, anti-HIV-1 / 2, HIV-1-RNA, and CMV-DNA.

[0019] Further, the components of the medium containing fetal bovine serum used in the preparation method are protein, amino acids, and trace elements, and the usage amount is 40%.

[0020] Further, the density of passage 5 umbilical cord mesenchymal stem cells used in step 6 of the preparation method is 6000 cells / cm 2 .

[0021] In the present invention, the term "stem cell" refers to a class of pluripotent cells with the ability of self-renewal. Under certain conditions, stem cells can differentiate into various functional cells. The term "mesenchymal stem cell" refers to an important member of the stem cell family. It is derived from the mesoderm in the early stage of development, belongs to pluripotent stem cells, was initially discovered in the bone marrow, and has characteristics such as multi-directional differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation, and self-renewal. The term "umbilical cord mesenchymal stem cell" refers to a kind of multipotent stem cell existing in the umbilical cord tissue of newborns.

[0022] In the present invention, the term "mannitol" refers to sugar alcohol, that is, a polyol obtained by reducing sugars (such as by hydrogenation of mannose or fructose).

[0023] In the present invention, the term "trehalose", also known as rhaponticin and mycose, is a non-reducing disaccharide composed of two glucose molecules. Its structural formula is α-D-glucopyranosyl~α-D-glucopyranoside, and it often exists as a dihydrate, with the molecular formula C12H22O11·2H2O. Trehalose is a typical stress metabolite. It can form a unique protective film on the cell surface under harsh environmental conditions such as high temperature, high cold, high osmotic pressure, and dry water loss, effectively protecting the structure of biological molecules from being damaged, thereby maintaining the life process and biological characteristics of living organisms. It has 3 optical isomers, namely α,α-trehalose (mushroom sugar, Mushroom Sugar), α,β-trehalose (neotrehalose, Neotrehalose), and β,β-trehalose (isotrehalose, Isotrehalose).

[0024] The "hydrolyzed collagen" used in this text, also known as "hydrolyzed collagen protein", is a mixture of short-chain amino acids derived from a collagen-containing starting material with natural (full-length) collagen, usually obtained through a hydrolysis step, including enzymatic hydrolysis (also known as enzymatic hydrolyzation). The degree of hydrolysis usually affects the average molecular weight of the final product. The molecular weight of hydrolyzed collagen is usually 1 to 10 kDa. The hydrolyzed collagen taught in the present invention may include hydrolyzed or partially hydrolyzed collagen. Hydrolyzed collagen can be produced from a collagen-containing starting material (such as animal connective tissue) in a one-step process or through an intermediate gelatin stage, where type A and / or type B gelatin can be used. Therefore, the hydrolyzed collagen used in this text may refer to hydrolyzed gelatin, which is obtained by hydrolyzing the gelatin obtained from collagen.

[0025] The term "oligopeptide-1" used in the present invention refers to a synthetic polypeptide composed of glycine, histidine, and lysine.

[0026] The term "sodium hyaluronate hydrolyzate" used in the present invention is low-molecular-weight sodium hyaluronate obtained by degrading high-molecular-weight hyaluronic acid through enzymatic hydrolysis technology. Its molecular weight is usually less than 10,000 daltons and has better permeability than high-molecular-weight hyaluronic acid, capable of penetrating deep into the dermis of the skin.

[0027] The term "carnosine" used in the present invention is intended to refer to L-carnosine, D-carnosine, or a racemic mixture thereof.

[0028] In some embodiments, the content of water used in the preparation method of the first aspect is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%.

[0029] In some embodiments, the content of the concentrated conditioned medium (which can also be called umbilical cord mesenchymal stem cell conditioned medium, specifically located in the examples) used in the preparation method of the first aspect is 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%.

[0030] In some embodiments, the content of mannitol used in the preparation method of the first aspect is 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%.

[0031] In some embodiments, the content of trehalose used in the preparation method of the first aspect is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%.

[0032] In some embodiments, the content of hydrolyzed collagen used in the preparation method of the first aspect is 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%.

[0033] In some embodiments, the content of oligopeptide-1 used in the preparation method of the first aspect is 0.025%, 0.026%, 0.027%, 0.028%, 0.029%, 0.030%, 0.031%, 0.032%, 0.033%, 0.034%, 0.035%, 0.036%, 0.037%, 0.038%, 0.039%, 0.040%, 0.041%, 0.042%, 0.043%, 0.044%, 0.045%, 0.046%, 0.047%, 0.048%, 0.049%, 0.050%.

[0034] In some embodiments, the content of hydrolyzed sodium hyaluronate used in the preparation method of the first aspect is 0.015%, 0.016%, 0.017%, 0.018%, 0.019%, 0.020%, 0.021%, 0.022%, 0.023%, 0.024%, 0.025%.

[0035] In some embodiments, the content of carnosine used in the preparation method of the first aspect is 0.015%, 0.016%, 0.017%, 0.018%, 0.019%, 0.020%, 0.021%, 0.022%, 0.023%, 0.024%, 0.025%.

[0036] The second aspect of the present invention provides a mesenchymal stem cell conditioned medium obtained according to the preparation method of the first aspect.

[0037] In some embodiments, the formulation of the mesenchymal stem cell conditioned medium is composed of 50 - 60% water, 15 - 30% concentrated conditioned medium prepared by the preparation method of the first aspect of the present invention, 2 - 4.5% mannitol, 0.5 - 1% trehalose, 0.15 - 0.25% hydrolyzed collagen, 0.025 - 0.05% oligopeptide-1, 0.015 - 0.025% hydrolyzed sodium hyaluronate, and 0.015 - 0.025% carnosine.

[0038] The third aspect of the present invention is a method for preparing freeze-dried balls of mesenchymal stem cell conditioned medium for facial anti-wrinkle and firming, and the preparation method includes the step of freeze-drying the mesenchymal stem cell conditioned medium described in the second aspect at -196°C.

[0039] The fourth aspect of the present invention provides freeze-dried balls of mesenchymal stem cell conditioned medium prepared by the preparation method described in the third aspect.

[0040] The present invention uses the terms "freeze-dried balls" or "freeze-dried beads" to refer to droplets of a liquid material that have been frozen and dried. The liquid material can be any material in a liquid state or suspended in a liquid, including but not limited to solutions, suspensions, emulsions, foams, sols, gels, semi-solids, melts, or mixtures thereof.

[0041] In some embodiments, the freeze-dried balls are obtained using freeze-drying technology, which is also known as lyophilization technology. It is a drying method that removes moisture from a substance under low-temperature and vacuum conditions. Freeze-drying technology utilizes the phase change of water. At low temperature, the moisture in the substance is frozen into a solid state, and then under vacuum conditions, the solid water directly sublimes into a gaseous state, thereby removing the moisture. The freeze-drying process requires precise control of parameters such as temperature, pressure, and time, and has high requirements for technicians.

[0042] The fifth aspect of the present invention provides the application of the preparation method described in the third aspect above, or the freeze-dried balls of mesenchymal stem cell conditioned medium described in the fourth aspect, in the preparation of products for facial anti-wrinkle and firming.

[0043] In some embodiments, the products of the present invention include one or more of tablets, capsules, powders, microparticles, solutions, lozenges, jellies, cream preparations, spirits, suspensions, tinctures, poultices, liniments, lotions, and aerosols, etc. They can be prepared using commonly known preparation techniques, and appropriate stabilizers can be added to maintain the stability and effectiveness of the products.

[0044] Furthermore, the facial anti-wrinkle and firming improve the facial skin elasticity index, reduce oxidation, and whiten.

[0045] In some embodiments, the products can be selected from one or more forms such as facial creams, serums, facial masks, applied gels, lotions, or skin boosters, etc.

[0046] The present invention uses the term "stabilizer" to refer to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacturing, storage, and application. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols, and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or its salts (such as arginine hydrochloride), glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opines, alanylaminopine, octopine, strombine, and N-oxide of trimethylamine (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAaseA. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play a role in controlling osmotic pressure. The stabilizers specifically used in the present invention are selected from one or more of polyols, amino acids, salts, and sugars. The preferred salt is sodium chloride, the preferred sugars are sucrose and trehalose, the preferred polyols are sorbitol and mannitol. The preferred amino acids are arginine or its salts (such as arginine hydrochloride), glycine, and proline. The preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose.

[0047] The sixth invention of the present invention provides the use of the preparation method described in the third aspect or the freeze-dried ball of the mesenchymal stem cell conditioned medium described in the fourth aspect in the preparation of a product for scavenging DPPH radicals.

[0048] The present invention uses the term "DPPH radical" as a stable radical. DPPH can stably exist in an organic solvent. Its alcohol solution is purple and needs to be stored at low temperature in the dark. It has a single electron, so it can accept an electron or a hydrogen ion and has a maximum absorption at a wavelength of 517 nm. In the presence of a radical scavenger, the single electron of DPPH is captured, making its color lighter, and the absorbance value at the maximum light absorption wavelength decreases. Moreover, the degree of decrease shows a linear relationship. The decrease in the absorbance level indicates an increase in antioxidant activity, thereby evaluating the antioxidant capacity of the test sample. This antioxidant capacity is expressed by the inhibition rate. The greater the inhibition rate, the stronger the antioxidant activity.

[0049] Advantages and beneficial effects of the present invention:

[0050] The freeze-dried balls of conditioned medium containing umbilical cord mesenchymal stem cells prepared by this patent can effectively scavenge DPPH free radicals, have the efficacy of anti-wrinkle and firming. Secondly, in human experiments, it can effectively improve the skin elasticity of the subjects and reduce wrinkles. The freeze-dried balls of conditioned medium containing umbilical cord mesenchymal stem cells prepared by this patent have good anti-wrinkle and firming effects. The freeze-dried balls of conditioned medium containing umbilical cord mesenchymal stem cells prepared by this patent adopt the ultra-low temperature biological dormancy technique at -196 °C, which can quickly lock the activity of the conditioned medium of umbilical cord mesenchymal stem cells. Description of the Drawings

[0051] Figure 1 It is the result chart of the DPPH free radical scavenging rate of the freeze-dried balls of conditioned medium containing umbilical cord mesenchymal stem cells, **p<0.05;***p<0.005.

[0052] Figure 2 It is the result chart of the right facial skin elasticity index after using the freeze-dried balls of conditioned medium containing umbilical cord mesenchymal stem cells, ***p<0.005.

[0053] Figure 3 It is the result chart of the right facial wrinkle feature index after using the freeze-dried balls of conditioned medium containing umbilical cord mesenchymal stem cells, **p<0.05;***p<0.005.

[0054] Figure 4 It is the result chart of the left facial skin elasticity index after using the freeze-dried balls of conditioned medium containing umbilical cord mesenchymal stem cells, ***p<0.005.

[0055] Figure 5 It is the result chart of the left facial wrinkle feature index after using the freeze-dried balls of conditioned medium containing umbilical cord mesenchymal stem cells, **p<0.05;***p<0.005.

[0056] Figure 6 It is the comparison chart of the outer canthus situation of the subjects after using the freeze-dried balls of conditioned medium containing umbilical cord mesenchymal stem cells. Detailed Embodiments

[0057] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0058] Example 1

[0059] 1. Experimental Materials

[0060] Formulation composition: water 50 - 60%, conditioned medium of umbilical cord mesenchymal stem cells 15 - 30%, mannitol 2 - 4.5%, trehalose 0.5 - 1%, hydrolyzed collagen 0.15 - 0.25%, oligopeptide - 1 0.025 - 0.05%, sodium hyaluronate 0.015 - 0.025%, carnosine 0.015 - 0.025%.

[0061] 2. Preparation method of conditioned medium of umbilical cord mesenchymal stem cells

[0062] (1) Take the umbilical cord of a full - term healthy fetus, and use the colloidal gold method to detect HbsAg, anti - HCV, HCV - RNA, anti - HDV, anti - HEV, anti - HIV - 1 / 2, HIV - 1 - RNA, CMV - DNA in umbilical cord serum. Only the umbilical cords with negative test results can be used;

[0063] (2) Put the umbilical cord into a culture dish containing 75% alcohol for disinfection, and then wash it repeatedly with physiological saline until the umbilical cord tissue has no blood color;

[0064] (3) Cut the umbilical cord tissue obtained in step (2) into tissue segments, then peel off the Wharton's jelly of the tissue segments, place the Wharton's jelly in a clean container, and cut it into pieces with tissue scissors. During the cutting process, drop α - MEM culture medium to keep the tissue moist;

[0065] (4) In the obtained block - shaped tissue in step (3), add 0.1% type II collagenase and 0.25% trypsin for digestion successively, filter the cell solution with a sieve, and then centrifuge to remove the supernatant;

[0066] (5) Resuspend the cell precipitate obtained in step (4) with a medium containing fetal bovine serum, put it into a culture flask coated with fibronectin, add a sufficient amount of medium containing fetal bovine serum, gently shake the culture flask to fully moisten the adherent surface, evenly spread it on the adherent surface of the culture flask, and place it in a CO2 incubator for culture;

[0067] (6) Conduct the first medium change on the 4th - 5th day after culture, and then change the medium every 3 - 4 days. After the cell confluence reaches more than 70%, digest it with trypsin and then passage;

[0068] (7) Inoculate the P5 - generation umbilical cord mesenchymal stem cells into the culture flask at a density of 6000 cells / cm 2 and add a medium containing fetal bovine serum for culture;

[0069] (8) When the cells grow to 80% confluence, wash the cell surface with physiological saline multiple times, and then add DMEM / F12 medium for starvation culture;

[0070] (9) Collect the conditioned medium of umbilical cord mesenchymal stem cells obtained in step (8) into a centrifuge tube, centrifuge, and discard the precipitate;

[0071] (10) Collect the conditioned medium obtained in step (9), and concentrate the stem cell conditioned medium using a 5KD dialysis bag to obtain an effective stem cell secreted mixed factor with a molecular weight above 5KD, obtaining a concentrated conditioned medium.

[0072] 3. Preparation method of freeze-dried balls of mesenchymal stem cell conditioned medium

[0073] Homogeneously mix the concentrated conditioned medium in 2 with 50 - 60% water, 2 - 4.5% mannitol, 0.5 - 1% trehalose, 0.15 - 0.25% hydrolyzed collagen, 0.025 - 0.05% oligopeptide-1, 0.015 - 0.025% hydrolyzed sodium hyaluronate, and 0.015 - 0.025% carnosine. After thoroughly mixing the composition, place it at -196 °C for freeze-drying treatment.

[0074] 4. Experimental results

[0075] Conduct DPPH free radical scavenging detection and human anti-wrinkle efficacy detection on the prepared freeze-dried balls.

[0076] After collecting the umbilical cord mesenchymal stem cell conditioned medium, add the composition components in proportion, and conduct freeze-drying treatment at -196 °C. The DPPH free radical scavenging rate is shown in Table 1 and Figure 1 as shown. The freeze-dried balls containing umbilical cord mesenchymal stem cell conditioned medium are significantly higher than those without mesenchymal stem cell conditioned medium, indicating that the umbilical cord mesenchymal stem cell conditioned medium prepared by this invention has anti-wrinkle and firming effects.

[0077] Table 1 DPPH free radical scavenging rate of freeze-dried balls of umbilical cord mesenchymal stem cell conditioned medium

[0078]

[0079] In addition, through comparison of -196 °C liquid nitrogen freeze-drying and -55 °C freeze-dryer freeze-drying of the composition containing umbilical cord mesenchymal stem cell conditioned medium, it is found that the -196 °C liquid nitrogen freeze-drying method adopted in this study can better preserve the biological activity of umbilical cord mesenchymal stem cell conditioned medium and has a better antioxidant effect.

[0080] To further prove the anti-wrinkle and firming effects of the freeze-dried balls containing umbilical cord mesenchymal stem cell conditioned medium, this invention recruited 33 subjects and carried out a 28-day human efficacy detection. The results are as Figures 2 - 6 shown, among which, Figure 2 is the right facial skin elasticity index after using the freeze-dried balls of umbilical cord mesenchymal stem cell conditioned medium, Figure 3is the right facial wrinkle characteristic index after the use of freeze-dried balls of umbilical cord mesenchymal stem cell conditioned medium, Figure 4 is the left facial skin elasticity index after the use of freeze-dried balls of umbilical cord mesenchymal stem cell conditioned medium, Figure 5 is the left facial wrinkle characteristic index after the use of freeze-dried balls of umbilical cord mesenchymal stem cell conditioned medium, Figure 6 is the comparison chart of the outer canthus of the subjects after using the freeze-dried balls of umbilical cord mesenchymal stem cell conditioned medium. The results show that the wrinkle characteristic index decreased by 13.55% and the skin elasticity increased by 12.19% 14 days after using the freeze-dried balls of umbilical cord mesenchymal stem cell conditioned medium; after 28 days of use, the wrinkle characteristic index decreased by 18.67% and the skin elasticity increased by 16.81%.

Claims

1. A method for preparing a mesenchymal stem cell conditioned medium for facial anti-wrinkle and firming, the preparation method comprising the following steps: 1) Place the umbilical cord in 75% alcohol for disinfection, and then wash it repeatedly with saline until the umbilical cord tissue is bloodless; 2) Cut the umbilical cord tissue in step 1 into tissue segments, then peel off the Wharton's Jelly from the tissue segments, place the Wharton's Jelly in a clean container, and cut it into blocks with tissue scissors. During the cutting process, add α-MEM culture medium to keep the tissue moist; 3) Add 0.1% type II collagenase and 0.25% trypsin to the block tissue obtained in step 2 for digestion, collect the cell solution by filtration, and then centrifuge to remove the supernatant; 4) Resuspend the cell pellet obtained in step 3 using a culture medium containing fetal bovine serum, place it in a container coated with fibronectin, add a sufficient amount of culture medium containing fetal bovine serum, gently shake the container to fully moisten the adherent surface, evenly spread it on the adherent surface of the container, and place it in a CO2 incubator for culture; 5) The first medium change is performed 4 to 5 days after culture, and the medium is changed every 3 to 4 days thereafter. When the cell confluence reaches more than 70%, trypsin is used for digestion and then passage; 6) Inoculate P5 umbilical cord mesenchymal stem cells into a culture bottle and add culture medium containing fetal bovine serum for culture; 7) When the cells grow to 80% confluence, rinse the cell surface several times with saline, and then add DMEM / F12 medium for starvation culture; 8) Collect the umbilical cord mesenchymal stem cell culture conditioned medium obtained in step 7 into a centrifuge tube, centrifuge, and discard the precipitate; 9) Collect the conditioned medium obtained in step 8, and concentrate the stem cell conditioned medium using a 5KD dialysis device to obtain an effective stem cell secretory mixed factor with a molecular weight of more than 5KD; 10) The concentrated conditioned medium obtained in step 9 is homogenously mixed with 50-60% water, 2-4.5% mannitol, 0.5-1% trehalose, 0.15-0.25% hydrolyzed collagen, 0.025-0.05% oligopeptide-1, 0.015-0.025% hydrolyzed sodium hyaluronate, and 0.015-0.025% carnosine.

2. The preparation method according to claim 1, wherein the umbilical cord used in step 1 of the preparation method is the umbilical cord of a full-term healthy fetus.

3. The preparation method according to claim 2, wherein the full-term healthy fetal umbilical cord is an umbilical cord whose umbilical cord serum HbsAg, anti-HCV, HCV-RNA, anti-HDV, anti-HEV, anti-HIV-1 / 2, HIV-1-RNA, and CMV-DNA results are all negative.

4. The preparation method according to claim 1, wherein the density of the passage 5 umbilical cord mesenchymal stem cells used in step 6 of the preparation method is 6000 cells / cm 2 .

5. The mesenchymal stem cell conditioned medium obtained according to the preparation method according to claims 1-4.

6. A method for preparing freeze-dried pellets of mesenchymal stem cell conditioned medium for facial anti-wrinkle and firming, the method comprising the step of freeze-drying the mesenchymal stem cell conditioned medium according to claim 5 at -196°C.

7. A mesenchymal stem cell conditioned medium freeze-dried pellet prepared by the preparation method according to claim 6.

8. Use of the preparation method according to claim 6, or the freeze-dried ball of the mesenchymal stem cell conditioned medium according to claim 7, in the preparation of a product for facial anti-wrinkle and firming.

9. The use according to claim 8, wherein the facial anti-wrinkle and firming improves the facial skin elasticity index, reduces oxidation, and whitens.

10. Use of the preparation method according to claim 6, or the freeze-dried ball of the mesenchymal stem cell conditioned medium according to claim 7, in the preparation of a product for scavenging DPPH free radicals.

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