CN114053205A - Preparation method of anti-aging facial skin nutrient solution freeze-dried powder, product and application thereof - Google Patents
Preparation method of anti-aging facial skin nutrient solution freeze-dried powder, product and application thereof Download PDFInfo
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- CN114053205A CN114053205A CN202111492042.8A CN202111492042A CN114053205A CN 114053205 A CN114053205 A CN 114053205A CN 202111492042 A CN202111492042 A CN 202111492042A CN 114053205 A CN114053205 A CN 114053205A
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- aging
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Abstract
The invention provides a preparation method of anti-aging nutrient solution freeze-dried powder for facial skin, and a product and application thereof. The stem cell factor extracting solution is obtained by performing cell lysis on umbilical cord mesenchymal stem cells obtained by culture and centrifugally collecting supernatant, contains various cell factors, and can exert anti-aging, beautifying and skin-care effects. According to the invention, the animal source-free serum culture medium is used for collecting cell lysis substances after the umbilical cord mesenchymal stem cells are subjected to amplification culture, so that the safety of the product is improved on the premise of ensuring the effectiveness.
Description
Technical Field
The invention relates to the technical field of beauty medicine, in particular to a preparation method of freeze-dried powder of an anti-aging nutrient solution for facial skin, a product and application thereof, and the nutrient solution for the anti-aging of the facial skin.
Background
The skin is positioned on the outermost layer of the organism and directly contacts with the external environment, so that external stimulation such as microorganism invasion, chemical component harm, ultraviolet ray damage and the like can be prevented, and the skin is the first defense line of the organism. Due to the natural increase of age, inadequate daily care, improper or excessive use of cosmetics, transition fatigue, various accidents and other factors, the phenomenon of premature skin aging appears in many people. Especially facial skin, and symptoms comprise dry and rough skin, looseness, wrinkles, spots increase, thinned cutin layer, easy allergy and the like, however, effective anti-skin aging products are rarely available in the market at present.
Stem cells are a type of pluripotent cells having the ability to self-renew and proliferate and differentiate, and can be classified into embryonic stem cells and adult stem cells. The adult stem cells have wide application prospect in clinic, in particular to the mesenchymal stem cells from the umbilical cord. During the growth, proliferation and differentiation of stem cells, a large number of cytokines are secreted in the cells, including various growth factors, such as active substances of Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Keratinocyte Growth Factor (KGF), insulin-like growth factor (IGF), Vascular Endothelial Growth Factor (VEGF), transforming growth factor-beta (TGF-beta), related collagens, and the like, and all of the factors are involved in the mechanisms of different tissues and reactions.
Cytokines have a variety of functions and play an important role in promoting the metabolism of fibroblasts and the formation of collagen. The cell factor can promote the growth and reproduction of skin tissues; can promote the rapid growth and reproduction of cells related to skin injury, and regulate the synthesis, secretion and decomposition of intercellular matrix; can promote the regeneration of stratum corneum cells, accelerate the repair of stratum corneum and matrix layers of the skin and promote the growth of skin cells of a human body; can enhance the synthesis and cell metabolism of skin cells, delay skin cell aging, promote repair and growth of epidermal cells, and make skin smooth and plump.
The mesenchymal stem cells have wide sources, are easy to separate and culture, and have great advantages as seed cells of stem cell anti-aging products. However, most of the culture media used in the conventional mesenchymal stem cell culture methods contain animal serum, and unsafe factors exist in the use. The traditional mesenchymal stem cell culture method also has the defects of long passage period, low effective passage and the like, for example, the method utilizes a DMEM-F12 culture medium containing 10% FBS to culture umbilical cord mesenchymal stem cells, the passage period is 3-5 days, the passage ratio is 1:2 or 1:3, and the effective passage is about 15 passages. In addition, many cytokines are currently derived from stem cell culture media, and cell contents including other active ingredients such as cytokines cannot be efficiently released into the culture media. At present, anti-aging products taking cytokines as main active ingredients mostly exist in a liquid state or an emulsion form, are not beneficial to storage, and the development of a new dosage form suitable for long-term storage is urgently needed.
Disclosure of Invention
The invention aims to provide a preparation method of freeze-dried powder of an anti-aging nutrient solution for facial skin.
Yet another object of the present invention is to: provides a freeze-dried powder product of the anti-aging nutrient solution for the facial skin prepared by the method.
Yet another object of the present invention is to: provides an application of the product.
The purpose of the invention is realized by the following scheme: a preparation method of freeze-dried powder of an anti-aging nutrient solution for facial skin comprises the following steps:
(1) carrying out in-vitro culture on umbilical cord-derived mesenchymal stem cells;
(2) collecting umbilical cord mesenchymal stem cells obtained by culture, and performing cell lysis to obtain cell lysate; centrifuging the cell lysate to obtain supernatant as stem cell factor extracting solution;
(3) mixing the stem cell factor extracting solution and the nutrient solution in proportion to obtain a stock solution;
(4) mixing the stock solution and the freeze-drying protective agent in proportion, and freeze-drying;
(5) the nutrient solution is as follows: vitamin C10-50ug/ml, sodium hyaluronate 1-5mg/ml and human transferrin 1-10 ug/ml; the freeze-drying protective agent is as follows: 50-150g/L of mannitol, 1-10g/L of trehalose and 405-15 g/L of dextran.
Wherein, the cell culture medium used in the step (1) is: DMEM medium containing recombinant human insulin 1-100ug/ml, human transferrin 1-50ug/ml and selenious acid 1-80 ug/ml; the culture conditions were: 36.5-37.5 ℃, 5% CO 2; carrying out adherent culture on the umbilical cord mesenchymal stem cells, digesting and collecting the cells when the cells grow to 85-90%; the umbilical cord mesenchymal stem cells are selected from at least one of the generations P3-P20.
The step (2) comprises the following steps: collecting umbilical cord mesenchymal stem cells obtained by culturing, adding 0.1-1mL of physiological saline into 1000 ten thousand IU of cells for resuspension, placing the cells in a centrifugal tube, placing the tubes in a refrigerator with the temperature of minus 60 ℃ to minus 80 ℃ for repeated freeze thawing, then ultrasonically breaking the cells, filtering the obtained cell lysate by a screen, centrifuging the obtained filtrate at the temperature of 4 ℃ and 10000rpm for 15min, and collecting supernatant; preferably, the ultrasonic power is 400-650W, the frequency is 20-25KHz, and the ultrasonic treatment is carried out for 5 times in 5-10s each time; and/or the aperture of the screen mesh is 20-40 um.
And (3) mixing the stem cell factor extracting solution with the nutrient solution according to the volume ratio of 2-3: 2-3.
Step (4), mixing the stock solution and the freeze-drying protective agent according to the volume ratio of 0.8-1.2: 8.5-9.5; the temperature of freeze drying is-60 ℃ to-80 ℃, the freeze dryer is pre-frozen for 4h to 8h, and then vacuum freezing is carried out for 48h to 72 h.
The step (2) also comprises the step of carrying out stem cell marker identification on the umbilical cord mesenchymal stem cells obtained by culture; and (2) detecting the cell factors in the stem cell factor extracting solution.
The invention provides face skin anti-aging nutrient solution freeze-dried powder which is prepared according to any one of the methods.
The invention provides a freeze-dried powder of an anti-aging nutrient solution for facial skin, which is dissolved in 0.9 percent of normal saline to prepare the anti-aging nutrient solution.
The invention provides an anti-aging nutrient solution which is characterized by being introduced into skin through nanocrystalline or nanoneedles.
The cell culture medium used in step (1) was: DMEM medium containing recombinant human insulin 1-100ug/ml, human transferrin 1-50ug/ml and selenious acid 1-80 ug/ml. Preferably, the cell culture medium used is: DMEM + recombinant human insulin 5ug/ml + human transferrin 5ug/ml + selenious acid 7 ug/ml.
The culture conditions were: 36.5-37.5 deg.c (preferably 37.0 deg.c) and 5% CO 2.
The umbilical cord mesenchymal stem cells are cultured in an adherent way, and when the cells grow to 85-90% (preferably about 90%), the cells are digested and collected.
The umbilical cord mesenchymal stem cells are selected from at least one of the generations P3-P20 (preferably the generations P8-P12, and more preferably the generations P9).
The step (2) comprises the following steps: collecting the umbilical cord mesenchymal stem cells obtained by culture, adding 0.5-1mL of physiological saline into 1000 ten thousand IU of cells for resuspension, and placing the cells in a centrifugal tube. Freezing and thawing in a refrigerator at-80 deg.C repeatedly (freezing for 1 hr, thawing in a 37 deg.C constant temperature water bath, repeating for 3 times), ultrasonically crushing cells, filtering the obtained cell lysate with a screen (mesh diameter of 20-40um), centrifuging the filtrate at 4 deg.C and 10000rpm for 15min, and collecting supernatant.
Preferably, the step (2) further comprises performing stem cell marker identification on the umbilical cord mesenchymal stem cells obtained by the culture by using flow cytometry. The stem cell markers include positive markers: CD105, CD73 and CD90, and the negative markers CD45, CD34 and CD14, and the like.
Preferably, the step (2) further comprises detecting (controlling) the cytokine in the stem cell factor extracting solution.
When in use, 0.9% physiological saline is used for dissolving the anti-aging composition freeze-dried powder, the solution is uniformly dripped on the skin, the nanocrystalline or the nanoneedle is used for conducting the introduction operation, and the redundant residual liquid can be continuously massaged until the residual liquid is completely absorbed.
The anti-aging nutrient solution freeze-dried powder is prepared by compounding an umbilical cord-derived mesenchymal stem cell factor extracting solution and nutrient components and then freeze-drying the mixture. The stem cell factor extracting solution is obtained by performing cell lysis on umbilical cord mesenchymal stem cells obtained by culture and centrifugally collecting supernatant, contains various cell factors, and can exert anti-aging, beautifying and skin-care effects. According to the invention, the animal source-free serum culture medium is used for collecting cell lysis substances after the umbilical cord mesenchymal stem cells are subjected to amplification culture, so that the safety of the product is improved on the premise of ensuring the effectiveness.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention uses the animal source-free serum culture medium to perform amplification culture on the umbilical cord mesenchymal stem cells, and then collects cell lysis substances for a facial rejuvenation product, thereby ensuring the use safety of the product.
And (II) by adopting the mesenchymal stem cell culture method, the passage period is 2-3 days, the passage ratio is 1:10, the generation is effectively carried out for 20 generations, and the generation efficiency is high.
And (III) the umbilical cord mesenchymal stem cells are subjected to freeze thawing and ultrasonic crushing to obtain a cell lysis stock solution, the types of the contents are rich, and the operation is simple and efficient.
Drawings
FIG. 1 is a morphological diagram showing normal growth of umbilical cord-derived mesenchymal stem cells;
figure 2 shows the results of the mesenchymal stem cell marker identification using flow cytometry.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
The embodiment provides an anti-aging composition, which is prepared by the following steps of culturing umbilical cord-derived mesenchymal stem cells, preparing a freeze-dried powder stock solution and preparing freeze-dried powder:
(1) culturing umbilical cord mesenchymal stem cells: the mesenchymal stem cells from the umbilical cord are 5000-10000/cm2Inoculating the umbilical cord mesenchymal stem cells at the density of (1: 10) and performing conventional in-vitro cell culture, wherein the passage ratio of each time is 1:10, the umbilical cord mesenchymal stem cells are at least one of the generations P3-P20, and the temperature is 36.5-37.5 ℃, and the CO content is 5 percent2Cell cultureCarrying out adherent culture in a culture medium, digesting and collecting cells when the cells grow to 85-90%;
wherein, the cell culture medium is: DMEM medium containing recombinant human insulin 1-100ug/ml, human transferrin 1-50ug/ml and selenious acid 1-80 ug/ml; identifying stem cell markers of the umbilical cord mesenchymal stem cells, as shown in a morphological schematic diagram in the growth process of the source mesenchymal stem cells in figure 1, and displaying the result that the characteristics of the original umbilical cord mesenchymal stem cells are retained; morphological identification is carried out on the umbilical cord mesenchymal stem cells, and the result shows that the morphology of the stem cells is basically unchanged;
as shown in figure 1, the umbilical cord-derived mesenchymal stem cells grow in a fusiform or irregular triangle shape under the observation of a microscope, an oval nucleus is arranged in the center of the cells, 2-3 protrusions with different lengths extend outwards from cytoplasm, the shape is normal, and the state is good.
(2) Preparation of lyophilized powder stock solution
Adding 0.5mL of physiological saline into 1000 ten thousand IU of cells for resuspension, putting the cells into a 1.5mL centrifugal tube, putting the tubes into a refrigerator at minus 80 ℃ for repeated freeze thawing for 3 times, putting the tubes into a constant-temperature water bath kettle for thawing at 37 ℃ each time, then treating the cells for 5 times under the ultrasonic condition, wherein the ultrasonic time is 5-10s each time, and ultrasonic lysis is carried out for multiple times, the ultrasonic power is 600W, and the frequency is: filtering with 20KHz sieve with aperture of 20um, and collecting liquid to obtain cell lysate;
placing the collected cell lysate in a high-speed freezing centrifuge, centrifuging for 15min at 4 ℃ and 10000rpm, collecting supernatant to obtain stem cell factor extracting solution (liquid A), and storing the stem cell factor extracting solution in an environment at-20 ℃; detecting the components of the stem cell factor extracting solution, wherein the result shows that the extracting solution is rich in various growth factors, including Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Keratinocyte Growth Factor (KGF), insulin-like growth factor (IGF), Vascular Endothelial Growth Factor (VEGF), transforming growth factor-beta (TGF-beta) and the like;
③ adding 4mg of vitamin C, 3g of sodium hyaluronate and 0.8mg of human transferrin into 100ml of normal saline to obtain nutrient liquid B;
(3) mixing the liquid A and the liquid B to obtain a freeze-dried powder stock solution, namely the anti-aging nutrient solution;
(4) mixing the stock solution with a freeze-drying protective agent, and freeze-drying to obtain the face skin anti-aging nutrient solution freeze-dried powder, wherein the freeze-drying protective agent is as follows: weighing 70g of mannitol, 4g of trehalose and 405 g of dextran, dissolving the mannitol, the trehalose and the dextran in 500mL of deionized water until the mixture is clear, adding the freeze-dried powder stock solution, and finally fixing the volume to 1L;
wherein, the solution obtained in the step (i) is filtered in a filter with the diameter of 0.22 mu m and collected in a sterile container;
performing aseptic operation on the aseptic liquid, subpackaging the aseptic liquid into 5ml penicillin bottles (the filling amount is 3ml), and performing half-pressing;
pre-freezing for 4h with a freeze dryer, vacuum freezing for 48h to obtain anti-aging nutritional liquid lyophilized powder, and storing at-20 deg.C.
The temperature of freeze drying is-60 ℃ to-80 ℃, the freeze dryer is pre-frozen for 4h to 8h, and then vacuum freezing is carried out for 48h to 72 h.
Fig. 2 shows that the cultured mesenchymal stem cells are collected, and the stem cell marker identification is performed on the umbilical cord mesenchymal stem cells obtained by culture by adopting flow cytometry. Positive markers including CD105, CD73, and CD90 were identified as positive, and negative markers including CD45, CD34, and CD14 were identified as negative, indicating that the resulting cells were indeed mesenchymal stem cells.
The obtained face skin anti-aging nutrient solution freeze-dried powder is dissolved in 0.9% physiological saline to prepare the anti-aging nutrient solution.
The obtained anti-aging nutrient solution is introduced into skin through nanocrystalline or nanoneedle.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be apparent to those skilled in the art that various modifications can be made to the above embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and variations made without departing from the scope of the present invention should be within the scope of the present invention.
Claims (10)
1. A preparation method of freeze-dried powder of an anti-aging nutrient solution for facial skin is characterized by comprising the following steps:
(1) carrying out in-vitro culture on umbilical cord-derived mesenchymal stem cells, wherein the umbilical cord mesenchymal stem cells are at least one of P3-P20 generations at 36.5-37.5 ℃ and 5% CO2Carrying out adherent culture in a cell culture medium, digesting and collecting cells when the cells grow to 85-90%;
(2) carrying out cell lysis on the collected umbilical cord mesenchymal stem cells to obtain cell lysate; centrifuging the cell lysate to obtain supernatant as stem cell factor extracting solution;
(3) mixing the stem cell factor extracting solution and the nutrient solution according to the volume ratio of (2-3) to obtain a stock solution;
(4) mixing the stock solution with a freeze-drying protective agent according to the volume ratio of (0.8-1.2) to (8.5-9.5), and freeze-drying;
(5) the nutrient solution is as follows: vitamin C10-50ug/ml, sodium hyaluronate 1-5mg/ml and human transferrin 1-10 ug/ml; the freeze-drying protective agent is as follows: 50-150g/L of mannitol, 1-10g/L of trehalose and 405-15 g/L of dextran.
2. The method for preparing the facial skin anti-aging nutrient solution freeze-dried powder according to claim 1, wherein the cell culture medium in the step (1) is: DMEM medium containing recombinant human insulin 1-100ug/ml, human transferrin 1-50ug/ml and selenious acid 1-80 ug/ml.
3. The preparation method of the facial skin anti-aging nutrient solution freeze-dried powder according to claim 1, wherein the cell lysis step in the step (2) is as follows: collecting the umbilical cord mesenchymal stem cells obtained by culture, adding 0.1-1mL of physiological saline into 1000 ten thousand IU of cells for resuspension, placing the cells in a centrifugal tube, placing the tubes in a refrigerator with the temperature of minus 60 ℃ to minus 80 ℃ for repeated freeze thawing, then ultrasonically breaking the cells, filtering the obtained cell lysate by a screen, centrifuging the obtained filtrate at the temperature of 4 ℃ and 10000rpm for 15min, and collecting the supernatant.
4. The method for preparing the facial skin anti-aging nutrient solution freeze-dried powder as claimed in claim 3, wherein in the step (2), 400-650W ultrasonic power is adopted for ultrasonic cell disruption, the frequency is 20-25KHz, 5-10s of ultrasonic treatment is carried out for 5 times; and/or the aperture of the screen mesh is 20-40 um.
5. The preparation method of the facial skin anti-aging nutrient solution freeze-dried powder according to claim 1, wherein in the step (4), the freeze-drying temperature is-60 ℃ to-80 ℃, and the freeze-dryer is pre-frozen for 4h to 8h and then vacuum-frozen for 48h to 72 h.
6. The preparation method of the facial skin anti-aging nutrient solution freeze-dried powder according to claim 1 or 3, characterized in that in the step (2), stem cell marker identification is carried out on umbilical cord mesenchymal stem cells obtained by culture; and detecting the cell factors in the stem cell factor extracting solution.
7. The preparation method of the freeze-dried powder of the facial skin anti-aging nutrient solution according to any one of claims 1 to 5, which is characterized by comprising the following steps:
(1) culturing umbilical cord mesenchymal stem cells: the mesenchymal stem cells from the umbilical cord are 5000-10000/cm2Inoculating the umbilical cord mesenchymal stem cells at the density of (1: 10) and performing conventional in-vitro cell culture, wherein the passage ratio of each time is 1:10, the umbilical cord mesenchymal stem cells are at least one of the generations P3-P20, and the temperature is 36.5-37.5 ℃, and the CO content is 5 percent2Carrying out adherent culture in a cell culture medium, digesting and collecting cells when the cells grow to 85-90%;
wherein, the cell culture medium is: DMEM medium containing recombinant human insulin 1-100ug/ml, human transferrin 1-50ug/ml and selenious acid 1-80 ug/ml; performing morphological identification on the stem cell marker of the umbilical cord mesenchymal stem cells, wherein the result shows that the morphology of the stem cells is basically unchanged;
(2) preparation of lyophilized powder stock solution
Adding 0.5mL of physiological saline into 1000 ten thousand IU of cells for resuspension, putting the cells into a 1.5mL centrifugal tube, putting the tubes into a refrigerator at minus 80 ℃ for repeated freeze thawing for 3 times, putting the tubes into a constant-temperature water bath kettle for thawing at 37 ℃ each time, then treating the cells for 5 times under the ultrasonic condition, wherein the ultrasonic time is 5-10s each time, and ultrasonic lysis is carried out for multiple times, the ultrasonic power is 600W, and the frequency is: filtering the obtained solution in a filter with the diameter of 0.22 mu m at the frequency of 20KHz, and collecting the solution in a sterile container to obtain cell lysate;
placing the collected cell lysate in a high-speed freezing centrifuge, centrifuging at 4 ℃ and 10000rpm for 15min, collecting supernatant to obtain stem cell factor extracting solution serving as liquid A, and storing in an environment at-20 ℃; detecting the components of the stem cell factor extracting solution, wherein the extracting solution is rich in growth factors including Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Keratinocyte Growth Factor (KGF), insulin-like growth factor (IGF), Vascular Endothelial Growth Factor (VEGF) and transforming growth factor-beta (TGF-beta);
③ adding 4mg of vitamin C, 3g of sodium hyaluronate and 0.8mg of human transferrin into 100ml of normal saline to obtain nutrient liquid B;
(3) mixing the liquid A and the liquid B to obtain a freeze-dried powder stock solution;
(4) mixing the stock solution with a freeze-drying protective agent, freeze-drying at the temperature of-60 to-80 ℃, pre-freezing for 4 hours by a freeze dryer, and then freezing for 48 hours in vacuum to obtain the face skin anti-aging nutrient solution freeze-dried powder, and storing at-20 ℃, wherein the freeze-drying protective agent is as follows: weighing 70g of mannitol, 4g of trehalose and 405 g of dextran, dissolving the mannitol, the trehalose and the dextran in 500mL of deionized water until the mixture is clear, adding the freeze-dried powder stock solution, and finally fixing the volume to 1L.
8. Facial skin anti-aging nutrient solution freeze-dried powder characterized by being prepared according to the method of any one of claims 1 to 7.
9. The facial skin anti-aging nutrient solution lyophilized powder of claim 8 dissolved in 0.9% physiological saline to prepare an anti-aging nutrient solution.
10. The anti-aging nutritional liquid according to claim 8, wherein the anti-aging nutritional liquid is introduced into the skin through a nanocrystal or a nanoneedle.
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