CN110917111A - Preparation method of mesenchymal stem cell derived liquid and application of mesenchymal stem cell derived liquid in cosmetics - Google Patents
Preparation method of mesenchymal stem cell derived liquid and application of mesenchymal stem cell derived liquid in cosmetics Download PDFInfo
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Abstract
The invention discloses a preparation method of mesenchymal stem cell derived liquid and application thereof in cosmetics, wherein the preparation method of the mesenchymal stem cell derived liquid comprises the following steps: (1) culturing mesenchymal stem cells from umbilical cord tissues by using a mesenchymal stem cell culture medium, and collecting cell culture supernatant; (2) and (2) adding glutathione, coenzyme Q10, chitosan, seaweed collagen and sodium hyaluronate into the cell culture solution obtained in the step (1) to obtain the mesenchymal stem cell derived solution. The mesenchymal stem cell derived liquid prepared by the invention is used for cosmetics, has a deep moisturizing function, can obviously improve the brightness and smoothness of skin, can effectively improve the symptoms of skin relaxation, dullness, chloasma and the like, and has a very good repairing effect on damaged skin.
Description
Technical Field
The invention relates to skin care products, a preparation method and application thereof, in particular to a preparation method of mesenchymal stem cell derived liquid and application thereof in the field of cosmetics.
Background
The life cycle of a human is generally divided into three stages, a growth stage, a maturation stage and an aging stage. Generally, when the age reaches 25 years, various aging mechanisms of the body start to start, various indexes related to aging also start to decline, and people gradually enter an aging state. Skin aging is one of the most noticeable features of human aging. With age, the skin becomes gradually dull and dull, stains and wrinkles are generated, and sagging are caused, which seriously affect the quality of life and mental health of modern women, and 'aging resistance' has become an indispensable topic in their lives.
Modern women pay great attention to skin care in daily life, and a safe and effective skin care product is expected to be found, but the skin care products in the current market have poor quality and are difficult to identify by consumers. Some skin care products contain lead mercury, ethanol, colorant, mineral oil and other substances, which are not only not beneficial to skin but also may cause serious dermatitis, infection and other problems.
With the rapid development of biomedicine, scientists find that the aged cells can be rejuvenated by containing a lot of active substances in the mesenchymal stem cell culture supernatant, but cosmetics containing the bioactive substances cannot be put on the market due to the productivity problem caused by technical barriers. The cosmetics sold in the market at present are all prepared by directly extracting plant components or processing the plant components through a fine chemical manufacturing process, main effective components in the products are macromolecular compounds, the macromolecular compounds are not easy to permeate into the skin, the problem of skin aging cannot be substantially improved, and certain adverse reactions such as allergy and the like can be possibly caused.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a preparation method of mesenchymal stem cell derived liquid and application of the mesenchymal stem cell derived liquid in cosmetics. The mesenchymal stem cell derived liquid has a deep moisturizing function, can remarkably improve the brightness and smoothness of skin, effectively improves the symptoms of skin relaxation, dullness, chloasma and the like, and has a good repairing effect on damaged skin; the preparation method is simple and feasible, can be popularized in a large scale, and can realize industrialized production and processing.
The technical scheme of the invention is as follows:
a method for preparing a mesenchymal stem cell-derived liquid, the method comprising the steps of:
(1) culturing mesenchymal stem cells from umbilical cord tissues by using a mesenchymal stem cell culture medium, and collecting cell culture supernatant;
(2) and (2) adding glutathione, coenzyme Q10, chitosan, seaweed collagen and sodium hyaluronate into the cell culture supernatant obtained in the step (1) to obtain the mesenchymal stem cell derived liquid. Further preferred is: in the mesenchymal stem cell derived liquid, the concentration of glutathione is 3-6 mg/L; the concentration of the coenzyme Q10 is 2-5 mg/L; the concentration of the chitosan is 3-6 mg/L; 2-5 mg/L of seaweed collagen; the volume ratio of the sodium hyaluronate is 10-15%.
(3) Preferably: the mesenchymal stem cell culture medium is a culture medium suitable for culturing mesenchymal stem cells purchased from formal commercial sources, such as StemPro, a product name of Thermo Scientific corporation in the United statesTMMSCSFM XenoFree medium. The culture medium is used for growing and expanding human mesenchymal stem cells and adipose-derived stem cells under completely serum-free and xenogeneic (xenogeneic-free) conditions. The medium supports the expansion of human mesenchymal stem cells, and can maintain the phenotype of multiple differentiation potentials.
(4) Preferably: the umbilical cord tissue-derived mesenchymal stem cells are obtained by (1) rinsing umbilical cord tissue with a physiological saline containing penicillin-streptomycin 5 times; (2) using a surgical scissors to cut the umbilical cord tissue into small pieces of 1cm 2; (3) paving a small tissue dermal fat layer of 1cm2 downwards in a culture dish, adding 0.5-1 mg/mL collagenase IV to immerse the tissue block, and sterilizing in a refrigerator at 4 ℃ for 12-16 h; (4) removing epidermis, cutting the tissue into 3 pieces with a diameter of 1mm by an ophthalmic scissors, inoculating into a culture bottle, adding a proper amount of culture medium, and culturing in a cell culture box to obtain the umbilical cord tissue-derived mesenchymal stem cells with high survival rate.
(5) Preferably: the culturing of the umbilical cord tissue-derived mesenchymal stem cells comprises primary culture and subculture to obtain more cell culture supernatants, wherein the primary culture and the subculture can adopt conventional methods in the prior art, for example, penicillin and streptomycin are added into a mesenchymal stem cell culture medium to ensure that the concentration of the penicillin and the streptomycin is 1 percent, the mesenchymal stem cell culture medium is placed in a 37 ℃ and 5 percent CO2 incubator for culturing, and a culture solution is replaced every 3 to 4 days; and when the cell fusion degree reaches 70-80%, passage can be performed. Digesting with 0.25% pancreatin at 37 ℃ for 3-5 minutes during passage; when the cells are coiled, namely the cells are about to be separated from the bottle wall, the pancreatin digestive juice is sucked away; collecting the cells with a proper amount of fresh culture solution and centrifuging; discarding the cleaning solution, resuspending the cells with 3-4 times of the original culture volume of fresh culture solution, then inoculating the cells into a culture bottle, and placing the culture bottle in an incubator with 37 ℃ and 5% CO2 for subculture, wherein the preferred subculture interval time is 7-10 days.
(6) Preferably, the cell culture supernatant collected in the step ⑴ is preferably collected from 3 rd to 8 th generations, when the cells grow to 3 th to 8 th generations, the peak value of the active protein molecules beneficial to the skin is reached, but when the generation exceeds 8 th generations, the content of the active protein molecules in the supernatant is reduced, and meanwhile, cell metabolites which are unfavorable to skin tissues are generated, so that the mesenchymal stem cell derivative liquid is preferably prepared from the culture supernatant of the 3 rd to 8 th generations of cells.
(7) Preferably: the umbilical cord tissue is preferably of human origin.
Preferably: can be used in anti-aging cosmetic.
The beneficial technical effects of the invention are as follows:
(1) the mesenchymal stem cell derived liquid adopts a 'cell engineering' method, collects culture supernatant containing active substances by culturing mesenchymal stem cells derived from human umbilical cord tissues, and takes the culture supernatant as a basic formula of the whole product. The active substance in the mesenchymal stem cell culture supernatant has a liposome structure which is the same as the structure of human skin, is beneficial to the absorption and utilization of the human skin, and can fundamentally improve the skin aging symptom.
(2) The preparation method of the mesenchymal stem cell derived liquid gets rid of the traditional plant component extraction and fine chemical production process, has no chemical additives such as sensitization toxins such as ethanol, plumbum and mercury, mineral oil and the like, and ensures the use safety of the product to the maximum extent;
(3) the mesenchymal stem cell derived liquid can promote division and proliferation of skin epithelial cells and increase the thickness of skin epidermis, thereby enhancing the barrier protection effect of the epidermis skin;
(4) the mesenchymal stem cell derived liquid can permeate into a deep structure of the skin, activate subcutaneous adipose-derived stem cells to regenerate the subcutaneous adipose-derived stem cells, thereby promoting the generation of collagen, cell adhesion protein, EGF (epidermal growth factor) and the like, remarkably reducing fine wrinkles, and increasing the elasticity and the smoothness of the skin.
(5) The invention adds glutathione, coenzyme Q10, chitosan, seaweed collagen and sodium hyaluronate into the mesenchymal stem cell derived liquid, can obviously improve the capacities of resisting oxidation and radiation and removing free radicals of skin tissues, can resist skin aging phenomenon induced by illumination, and has obvious whitening and spot-lightening effects after long-term use;
(6) the mesenchymal stem cell derived liquid can activate the regeneration of subcutaneous fat stem cells, secrete a large amount of epidermal growth factors, promote the proliferation of dermal histiocytes and fibroblasts, and has excellent repairing effect on physically damaged skin.
(7) According to the invention, chitosan and seaweed collagen are added into the mesenchymal stem cell derived liquid, and the two components can promote the generation of collagen and elastic fiber in the skin, so that the deep moisturizing and anti-wrinkling functions are achieved; the components of various formulas in the mesenchymal stem cell derived liquid are mutually cooperated, so that the optimal skin care effect is realized;
(8) the preparation method of the mesenchymal stem cell derived liquid is simple and feasible, can be popularized in a large scale, and can realize industrialized production and processing.
Drawings
Fig. 1 is a photograph of a whitening effect test scan according to example 4 of the present invention;
FIG. 2 is a photograph of a speckle reduction effect test scan according to example 4 of the present invention;
FIG. 3 is a photograph showing a cell scratch repair test in example 4 of the present invention.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples.
A preparation method of mesenchymal stem cell derived liquid and application thereof in cosmetics comprise the following steps:
(1) flushing the umbilical cord tissue 5 times with physiological saline containing penicillin-streptomycin;
(2) using a surgical scissors to cut the umbilical cord tissue into small pieces of 1cm 2;
(3) spreading 1cm2 small piece of tissue with dermal fat layer facing downwards in a culture dish, adding 0.5mg/mL collagenase IV to immerse the tissue piece, and digesting in a refrigerator at 4 deg.C for 16 h;
(4) removing epidermis, cutting the tissue into 3 pieces with a diameter of 1mm by an ophthalmic scissors, inoculating the pieces into a culture bottle, and culturing by using a proper amount of culture medium to obtain the mesenchymal stem cells with high survival rate from the umbilical cord tissue. The culture medium is a product name StemPro produced by Thermoscientific corporation of AmericaTMMSC SFM XenoFree medium;
(5) replacing the mesenchymal stem cell culture solution every 3-4 days; passaging is performed when the cells grow to 80% confluent state;
(6) digesting with 0.25% pancreatin at 37 ℃ for 3-5 minutes during passage, and sucking and removing pancreatin digestive juice when cells are coiled and are about to separate from the bottle wall;
(7) collecting the cells with a proper amount of fresh culture solution and centrifuging;
(8) discarding the supernatant, resuspending the cells with 3-4 times volume of fresh culture solution, then inoculating the cells into a culture flask, and placing the culture flask in a 5% CO2 incubator at 37 ℃ for subculture, wherein the preferable subculture interval time is 7-10 days.
Example 1
(1) Collecting the culture supernatant of the 3 rd generation mesenchymal stem cells.
(2) Adding 3mg/L glutathione, 2mg/L coenzyme Q10 and 3mg/L chitosan into 1L mesenchymal stem cell culture supernatant collected in the step (1); 2mg/L of seaweed collagen and sodium hyaluronate with the volume ratio of 10 percent to obtain the mesenchymal stem cell derived liquid.
Example 2
(1) Collecting the 5 th generation mesenchymal stem cell culture supernatant.
(2) Adding 6mg/L glutathione, 5mg/L coenzyme Q10 and 6mg/L chitosan into 1L mesenchymal stem cell culture supernatant collected in the step (1); 5mg/L of seaweed collagen and 15 percent of sodium hyaluronate by volume ratio to obtain the mesenchymal stem cell derived liquid.
Example 3
(1) Collecting the culture supernatant of the mesenchymal stem cells of the generation 8.
(2) Adding 4.5mg/L glutathione, 3.5mg/L coenzyme Q10 and 4.5mg/L chitosan into 1L of mesenchymal stem cell culture supernatant collected in the step (9); 3.5mg/L of seaweed collagen and sodium hyaluronate with the volume ratio of 12.5 percent to obtain the mesenchymal stem cell derived liquid.
Example 4 comparative experiment with reference to FIGS. 1 to 3
1. Water-locking and moisturizing efficacy test
(1) Recruiting 30 healthy, dry-skin volunteers 25-60 years old, 12 men and 18 women;
(2) subjects were uniformly cleaned of the inside of the forearm by laboratory test personnel. The subject sits still in a laboratory at a temperature of 21 + -1 deg.C and 50 + -5% RH for 30 minutes, during which time water and drink cannot be taken, the forearm is exposed and remains relaxed;
(3) selecting and marking 2 areas on the inner side of the double-arm as a product testing area and a comparison area, wherein the area of each area is 3cm multiplied by 3 cm;
(4) the basal value of the moisture content of the horny layer of each area of the forearm of the subject is tested by a British bio-x AF-200 skin moisture loss tester: the average moisture content of the skin in the test area of the product was 39.7%, and the average moisture content of the skin in the control area was 40.6%.
(5) Using the test sample in the product test area according to the product use requirement; the test sample is prepared by adding 90% sterile pure water into 10% mesenchymal stem cell derived liquid prepared in example 1, uniformly mixing, subpackaging 3mL each, sealing and packaging, using up within 24 hours after uncovering, and refrigerating; the control sample is sterile pure water containing 10% sodium hyaluronate stock solution and needs to be refrigerated; the using method comprises the following steps: cleaning the skin of the divided area with clear water, respectively smearing corresponding samples, respectively one time in the morning and at night, and massaging for 1 minute, so as to help the skin to absorb; continuously smearing for 14 days;
(6) referring to steps (2) and (4), detecting the stratum corneum water content value of each area of the forearm of the subject: the average moisture content of the skin in the test area of the product was 48.3%, and the average moisture content of the skin in the control area was 47.2%.
(7) Continuing to use the test sample for 14 days with reference to step (5);
(8) referring to steps (2) and (4), detecting the stratum corneum water content value of each area of the forearm of the subject: the average moisture content of the skin in the test area of the product was 69.8%, and the average moisture content of the skin in the control area was 50.6%. (ii) a
(9) Comparing and analyzing the stratum corneum water content values of the forearm of the subjects before, 14 days and 28 days, it can be known that the mesenchymal stem cell derived liquid in example 1 has a long-acting moisturizing effect, and the moisturizing effect is significantly better than that of the control group sample.
2. Skin elasticity improvement effect test
(1) Recruiting 30 volunteers 25-60 years old with healthy and dry skin, wherein 10 men and 20 women;
(2) skin elasticity of the volunteers was tested using a skin elasticity tester MPA580 of CK company, Germany, the test areas were two areas inside the left and right arms, each area was 3cm x 3cm, and the test results were used as blank values for this test;
(3) using the test sample in the product area according to the product use requirement; the test sample is prepared by adding 90% sterile pure water into 10% mesenchymal stem cell derived liquid prepared in example 2, uniformly mixing, subpackaging 3mL each, sealing and packaging, using up 24 hours after uncovering, and needing to be refrigerated; the control sample is sterile pure water containing 15% sodium hyaluronate stock solution and needs to be refrigerated; the using method comprises the following steps: cleaning the skin of the divided area with clear water, respectively smearing corresponding samples, respectively one time in the morning and at night, and massaging for 1 minute, so as to help the skin to absorb; continuously smearing for 14 days;
(4) referring to step (2), the increase rate of the skin elasticity value of each area of the forearm of the subject is detected: the average increase rate of the skin elasticity of the product test area is 5.8 percent, and the average increase rate of the skin elasticity of the control area is 2.7 percent;
(5) continuing to use the test sample for 14 days with reference to step (3);
(6) referring to step (2), the increase rate of the skin elasticity value of each area of the forearm of the subject is detected: the average increase rate of the skin elasticity of the product test area is 20.2 percent, and the average increase rate of the skin elasticity of the control area is 4.3 percent;
(7) comparing and analyzing the skin elasticity increasing rate of each area of the left arm and the right arm of the subject before, 14 days and 28 days, it can be known that the mesenchymal stem cell derived liquid in the example 2 can significantly improve the skin elasticity increasing rate, and the improving effect is significantly better than that of the control group sample.
3. Test of whitening and spot-lightening effect
(1) 15 female volunteers with healthy bodies and speckled faces of 30-50 years old are recruited and randomly divided into 2 groups, wherein the experimental group comprises 10 persons, and the control group comprises 5 persons;
(2) after the volunteers cleaned facial skin by themselves, the experimenter performed facial top and deep skin scans of the volunteers using the SyltonObserv professional skin image analysis system, a dutch import device, with scan time labeled D0.
(3) The experimental group used the test sample, the control group used the control sample, the samples were used according to the product use requirements: the test sample is prepared by adding 90% sterile pure water into 10% mesenchymal stem cell derived liquid prepared in example 3, uniformly mixing, subpackaging 5mL each, sealing and packaging, using up 24 hours after uncovering, and needing to be refrigerated; the control sample is sterile pure water containing 10% sodium hyaluronate stock solution and needs to be refrigerated; the using method comprises the following steps: cleaning facial skin with facial cleanser, smearing corresponding samples, and massaging for 1 min in the morning and evening respectively to help skin absorption; continuously smearing for 14 days;
(4) referring to step (2), the subject receives the scan of the superficial skin and the deep skin of the face, and the scan time is marked as D14;
(5) continuing to use the test and control samples for 14 days with reference to step (3);
(6) referring to step (2), the subject receives the scan of the superficial skin and the deep skin of the face, and the scan time is marked as D28;
(7) comparing the photographs of the top skin and the deep skin of the face of the subjects D0, D14 and D28 before and after application, it can be seen that the mesenchymal stem cell-derived liquid of example 3 has significant skin lightening and spot removing effects, while the control samples have no significant difference before and after application.
4. Anti-wrinkle Effect test
(1) 30 male and female mixed volunteers of 50-60 years old and healthy were recruited, wherein 14 male and 16 female were enrolled. Randomly dividing the test group into a test group and a control group, wherein each group contains 15 persons;
(2) volunteers were allowed to self-clean facial skin and sit still in a laboratory at 21 + -1 deg.C and 50 + -5% RH for 30 minutes during which time no skin care product was available. The experimenter uses a skin rapid optical imaging system DermaTOP to carry out deep scanning on wrinkles around eyes of the volunteer, the change of the skin texture degree is directly quantified through the skin roughness Rt, and the quantification result is recorded as a test blank value.
(3) According to the use requirement of the product, the test sample is used by the experimental group, and the control sample is used by the control group; the test sample is prepared by adding 90% sterile pure water into 10% mesenchymal stem cell derived liquid prepared in example 3, uniformly mixing, subpackaging 3mL each, sealing and packaging, using up within 24 hours after uncovering, and refrigerating; the control sample is a blank gel and needs to be refrigerated; the using method comprises the following steps: cleaning facial skin with facial cleanser, applying appropriate amount of the facial cleanser around eyes, gently looping until the eye is completely absorbed, applying the facial cleanser once in the morning and at night for 14 days;
(4) referring to step (2), the subject receives the skin around the eye to be scanned, and the scanning result is as follows: the Rt value of the test group is reduced by 4.1 percent on average, and the Rt value of the control zone is reduced by 2.3 percent on average;
(5) continuing to use the test and control samples for 28 days with reference to step (3);
(6) referring to step (2), the subject receives the skin around the eye to be scanned, and the scanning result is as follows: the Rt value of the test group is reduced by 12.3 percent on average, and the Rt value of the control zone is reduced by 2.7 percent on average;
(7) the results of analyzing the average value of Rt of the ocular skin of the subject before, 14 days and 42 days show that the mesenchymal stem cell-derived solution of example 3 has significant wrinkle-removing effect, and the control group sample has no significant wrinkle-removing effect.
5. Detection of cell scratch repair effect
(1) The mesenchymal stem cell derived solution obtained in example 1 was diluted in MEM medium containing 2% FCS at 2-fold gradient concentrations of 2%, 4% and 8%, respectively; the control group used complete medium, i.e., 10% FCS in MEM.
(2) Hacat cells were cultured in T175 flasks using 10% FCS MEM medium until the cells were confluent, digested with 0.05% trypsin, and centrifuged at 1000rpm/min for 10 min. Resuspend seeded 6-well plates, 3 × 105 cells/well.
(3) After 1 day the cells were approximately 100% long and scored in each well using a 200. mu.L tip. Washing cells with 1 × PBS (prewarmed at 37 deg.C) for 3 times, adding 2% FCS MEM to obtain gradient concentration sample to be tested and control group;
(4) after 36h, the scar was observed to heal significantly, the medium was aspirated, the cells were washed 3 times with pre-warmed 1 × PBS at 37 deg.C, 1.5mL of cold methanol was added, and incubated at 4 deg.C.
(5) After 5h, the cold methanol was aspirated off and 1.5mL of 1 × Geimsa stain was added.
(6) After 45min, the dye liquor is sucked off, washed with running water and dried.
(7) The scratch was photographed and the Image was analyzed using Image J, with the results shown: the mesenchymal stem cell-derived fluid obtained in example 1 can significantly promote the wound healing capacity of Hacat cells, and the promotion effect of the complete culture fluid on the wound healing capacity is not obvious. The result shows that the stem cell derived liquid can promote the proliferation and migration capacity of Hacat cells and has a remarkable effect on repairing scarred damaged cells.
Claims (9)
1. A preparation method of mesenchymal stem cell derived liquid is characterized by comprising the following steps:
(1) culturing mesenchymal stem cells from umbilical cord tissues by using a mesenchymal stem cell culture medium, and collecting cell culture supernatant;
(2) and (2) adding glutathione, sodium hyaluronate, coenzyme Q10, chitosan and seaweed collagen into the cell culture supernatant obtained in the step (1) to obtain the mesenchymal stem cell derived liquid.
2. The method for preparing a mesenchymal stem cell-derived liquid according to claim 1, wherein in the mesenchymal stem cell-derived liquid obtained in the step (2): the concentration of the glutathione is 3-6 mg/L; the concentration of the coenzyme Q10 is 2-5 mg/L; the concentration of the chitosan is 3-6 mg/L; 2-5 mg/L of seaweed collagen; the volume ratio of the sodium hyaluronate is 10-15%.
3. The method for preparing mesenchymal stem cell-derived liquid according to claim 1, wherein the mesenchymal stem cell culture medium used in step (1) is StemPro, a product name of Thermo Scientific, USATMMSC SFM XenoFree medium.
4. The method for preparing mesenchymal stem cell-derived liquid according to claim 1, wherein the umbilical cord tissue-derived mesenchymal stem cells of step (1) are obtained by:
(1) flushing the umbilical cord tissue 5 times with physiological saline containing penicillin-streptomycin;
(2) cutting umbilical cord tissue to 1cm with surgical scissors2Small blocks;
(3) mixing 1cm2Paving the small tissue dermis fat layer downwards in a culture dish, adding 0.5-1 mg/mL collagenase IV to immerse the small tissue dermis fat layer into the tissue mass, and digesting the tissue mass in a refrigerator at the temperature of 4 ℃ for 12-16 hours;
(4) removing epidermis, and cutting tissue into 1mm with ophthalmic scissors3Small pieces, inoculating into culture flaskAnd adding a proper amount of culture medium, and culturing in a cell culture box to obtain the umbilical cord tissue-derived mesenchymal stem cells with high survival rate.
5. The method for preparing a mesenchymal stem cell-derived liquid according to claim 1, wherein the culturing of umbilical cord tissue-derived mesenchymal stem cells in step (1) comprises primary culture and subculture.
6. The method for preparing mesenchymal stem cell-derived liquid according to claim 5, wherein the primary culture and the subculture are performed by conventional methods in the prior art.
7. The method for preparing mesenchymal stem cell-derived liquid according to claim 1, wherein the collecting of the cell culture supernatant in step (1) is preferably a collection of cell culture supernatant of passage 3 to 8.
8. The method for preparing mesenchymal stem cell-derived liquid according to claim 1, wherein the umbilical cord tissue in the step (1) is preferably of human origin.
9. The method for preparing mesenchymal stem cell-derived liquid according to claim 1, wherein the mesenchymal stem cell-derived liquid obtained in the step (2) is used in an anti-aging cosmetic.
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| CN115089698A (en) * | 2022-06-16 | 2022-09-23 | 北京绎源生物科技有限公司 | Application of active peptide and stem cell exosome for improving skin in medicines or cosmetics |
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