CN113041206A - Essence containing mesenchymal stem cell factor and preparation method thereof - Google Patents
Essence containing mesenchymal stem cell factor and preparation method thereof Download PDFInfo
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- CN113041206A CN113041206A CN201911269690.XA CN201911269690A CN113041206A CN 113041206 A CN113041206 A CN 113041206A CN 201911269690 A CN201911269690 A CN 201911269690A CN 113041206 A CN113041206 A CN 113041206A
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Abstract
The invention provides an essence containing a mesenchymal stem cell factor, which comprises the following components in parts by weight, based on 100% of the weight of the essence containing the mesenchymal stem cell factor: 3-8% of glycerin, 3-10% of butanediol, 2-7% of biological polysaccharide gum, 1-4% of trehalose, 0.5-1% of sodium hyaluronate, 0.1-1% of hydrolyzed collagen, 0.1-1% of vitamin A acetate, 0.1-1% of vitamin E acetate, 0.05-0.3% of carbomer, 0.5-1% of preservative, 0.5-2% of stem cell factor solution, 0.5-2% of stem cell exocrine and the balance of pure water. The essence containing the mesenchymal stem cell factor not only can strengthen the repair of the skin, but also can inhibit the reaction of excessive inflammation of the skin, and improve the repair effect and the anti-wrinkle ability.
Description
Technical Field
The invention relates to the field of beauty, in particular to essence containing mesenchymal stem cell factors and a preparation method thereof.
Background
Over time, the skin ages, which is affected by endogenous and exogenous factors. Endogenous factors include physiology, endocrine, and gene, also known as chronological aging. Extrinsic factors include environmental influences such as air pollution, sunlight and ultraviolet light, smoking, chemical exposure and malnutrition, and extrinsic aging is also known as photoaging. Photoaging causes thinning of the skin, degradation of collagen, deposition of denatured elastic fibers, manifesting as wrinkles and discoloration of yellow skin. In addition, photoaging generates free radicals, which activate metallomatrix proteases, which in turn degrade the extracellular matrix. When the dermis layer of the skin begins to become thin, the elasticity of the skin deteriorates, and wrinkles begin to develop gradually. When collagen in the dermis is oxidized and broken, the supporting effect on the epidermis is lost, thereby causing non-uniform collapse and wrinkles.
Many skin care products and cosmetics are currently on the market mainly for wrinkle removal, most of these products are supplemented with stem cells, and the application of stem cells is the fastest growing field of regenerative medicine, including regenerative medicine for the skin. For photoaging, non-invasive treatments and topical products are sought to reverse the appearance of wrinkles and photoaged skin.
The stem cell cosmetics have the functions of relieving the damage of exogenous factors and exploring the potential of endogenous factors. Effective stem cell cosmetics need to retain various growth factors, maintain their effects and enable them to penetrate the stratum corneum, improving the visual appearance of the skin in a short time without affecting the skin barrier.
Stem cell cosmetics may contain many stem cell extracts to promote skin renewal, regeneration and repair. The stem cell related product does not contain stem cells, but is rich in growth factors, cytokines, exosomes, proteins and the like secreted by the stem cells. These cell-derived growth factors can activate skin stem cells, inducing their differentiation into new skin. Dermal stem cells play an important role in wound repair, interact with keratinocytes, adipocytes and mast cells, and are also a source of other cytokines and molecules that are involved in supporting intercellular interactions, promoting wound healing and maintaining skin integrity and rejuvenation, and also promote fibroblast and keratinocyte proliferation, promoting extracellular matrix formation.
The renewal of fibroblasts, keratinocytes and melanocytes in aging skin is slowed down and the levels of secreted growth factors are reduced, and the local supplementation of these normal growth factors can promote the normal repair of the skin.
Many stem cell skin care products and cosmetics are available on the market at present, but the effects are not good, for example, the products prepared by stem cell culture supernatant have the safety problems of residual sugar in the supernatant, containing fetal calf serum and the like. Some products are prepared by freezing and concentrating the cell factor in the culture supernatant, but the activity of the cell factor is reduced in the concentration process, so that the repair effect is poor.
Disclosure of Invention
The invention aims to provide an essence containing mesenchymal stem cell factors and a preparation method thereof, and aims to solve the problems of poor repairing effect and low anti-wrinkle capability caused by the fact that the stem cell factors are added independently in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
the essence containing the mesenchymal stem cell factor comprises the following components in parts by weight, based on 100% of the weight of the essence containing the mesenchymal stem cell factor:
3-8% of glycerin, 3-10% of butanediol, 2-7% of biological polysaccharide gum, 1-4% of trehalose, 0.5-1% of sodium hyaluronate, 0.1-1% of hydrolyzed collagen, 0.1-1% of vitamin A acetate, 0.1-1% of vitamin E acetate, 0.05-0.3% of carbomer, 0.5-1% of preservative, 0.5-2% of stem cell factor solution, 0.5-2% of stem cell exocrine and the balance of pure water.
And a preparation method of the essence containing the mesenchymal stem cell factor, wherein the preparation method of the essence containing the mesenchymal stem cell factor comprises the following steps:
weighing the components according to the essence containing the mesenchymal stem cell factors;
adding the glycerol, the trehalose, the sodium hyaluronate, the carbomer and the preservative into the pure water, heating to 60-65 ℃, and mixing to obtain a first composition;
cooling the first composition to 20-25 ℃, adding the butanediol, the biological polysaccharide gum, the hydrolyzed collagen, the vitamin A acetate and the vitamin E acetate, and mixing to obtain a second composition;
and cooling the second composition to 2-8 ℃, adding the stem cell factor solution and the stem cell exudate, and mixing to obtain the essence containing the mesenchymal stem cell factor.
The essence containing the mesenchymal stem cell factor mainly comprises glycerin, butanediol, biological polysaccharide gum, trehalose, sodium hyaluronate, hydrolyzed collagen, vitamin A acetate, vitamin E acetate, carbomer, a preservative, a stem cell factor solution and a stem cell exudate; the essence containing the mesenchymal stem cell factor is added with a stem cell factor solution and stem cell exudate simultaneously, wherein the stem cell factor contains a large amount of growth factors such as Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), stem cell growth factor (SCF), Transforming Growth Factor (TGF) and other effective growth factors, and the effective growth factors can perform synergistic action with cell signaling proteins Wnt4, Wnt11, STAT3, miRNA-126, miRNA-130a, miRNA-125a and miRNA-31 in the stem cell exudate, further promote the regeneration of skin-related fibroblasts and keratinocytes, secrete collagen, elastin and matrix components, promote angiogenesis required by skin cell growth nutrition supply and achieve the function of repairing skin; meanwhile, micro RNA molecules of miRNA-124a, miRNA-125b, miRNA-21, miRNA-146a, miRNA-181c and miRNA-let-7b in stem cell external secretion can inhibit MAPK cascade reaction, reduce expression of TNF-alpha, IL-6 and IL-1 beta inflammatory factors, inhibit TLP-4, activate p-STAT3 and p-AKT to change the polarity of macrophages, and inhibit excessive inflammation reaction of skin, so that the prepared essence containing the mesenchymal stem cell factor not only can strengthen the repair of skin, but also can inhibit excessive inflammation reaction of the skin, and the repair effect and the anti-wrinkle capability are improved.
In addition, the preparation method of the essence containing the mesenchymal stem cell factor is only required to mix the components according to the proportion, the preparation method adopts a distribution mixing method, the components can be uniformly dispersed, different components are uniformly heated and mixed at different temperatures, the components can keep good performance, and the components can achieve the beneficial effects and are endowed with stability. In addition, the preparation method has the advantages of simple process, controllable conditions, low equipment requirements and convenient and quick preparation.
Drawings
Fig. 1 is a growth state of umbilical cord mesenchymal primary stem cells provided in example 1 of the present invention on day 10.
FIG. 2 is the growth state of umbilical cord mesenchymal primary stem cells provided in example 1 of the present invention on day 15.
FIG. 3 shows the growth state of the umbilical cord mesenchymal passaged cells provided in example 1 of the present invention.
FIG. 4 shows the results of Western blot analysis of the expression levels of CD63 and Alix in the stem cell exosomes cultured in different culture modes in the examples of the present invention.
FIG. 5 is a graph showing the effect of the addition of an RNA activity protectant on stem cell exosomes in an example of the present invention.
Detailed Description
In order to make the objects, technical solutions and technical effects 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, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention.
In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
The invention provides an essence containing a mesenchymal stem cell factor, which comprises the following components in parts by weight, based on 100% of the weight of the essence containing the mesenchymal stem cell factor:
3-8% of glycerin, 3-10% of butanediol, 2-7% of biological polysaccharide gum, 1-4% of trehalose, 0.5-1% of sodium hyaluronate, 0.1-1% of hydrolyzed collagen, 0.1-1% of vitamin A acetate, 0.1-1% of vitamin E acetate, 0.05-0.3% of carbomer, 0.5-1% of preservative, 0.5-2% of stem cell factor solution, 0.5-2% of stem cell exocrine and the balance of pure water.
The essence containing the mesenchymal stem cell factor mainly comprises glycerin, butanediol, biological polysaccharide gum, trehalose, sodium hyaluronate, hydrolyzed collagen, vitamin A acetate, vitamin E acetate, carbomer, a preservative, a stem cell factor solution and a stem cell exudate; the essence containing the mesenchymal stem cell factor is added with a stem cell factor solution and stem cell exudate simultaneously, wherein the stem cell factor contains a large amount of growth factors such as Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), stem cell growth factor (SCF), Transforming Growth Factor (TGF) and other effective growth factors, and the effective growth factors can perform synergistic action with cell signaling proteins Wnt4, Wnt11, STAT3, miRNA-126, miRNA-130a, miRNA-125a and miRNA-31 in the stem cell exudate, further promote the regeneration of skin-related fibroblasts and keratinocytes, secrete collagen, elastin and matrix components, promote angiogenesis required by skin cell growth nutrition supply and achieve the function of repairing skin; meanwhile, micro RNA molecules of miRNA-124a, miRNA-125b, miRNA-21, miRNA-146a, miRNA-181c and miRNA-let-7b in stem cell external secretion can inhibit MAPK cascade reaction, reduce expression of TNF-alpha, IL-6 and IL-1 beta inflammatory factors, inhibit TLP-4, activate p-STAT3 and p-AKT to change the polarity of macrophages, and inhibit excessive inflammation reaction of skin, so that the prepared essence containing the mesenchymal stem cell factor not only can strengthen the repair of skin, but also can inhibit excessive inflammation reaction of the skin, and the repair effect and the anti-wrinkle capability are improved.
Specifically, the essence containing the mesenchymal stem cell factor comprises 0.5-2% of stem cell factor solution by taking the weight of the essence containing the mesenchymal stem cell factor as 100%. If the addition amount of the plurality of cytokine solutions is too low, the content of various effective growth factors in the prepared essence containing the mesenchymal stem cell factors is too small, the effective growth factors cannot perform a synergistic effect with effective components of stem cell external secretion, the generation of skin-related cells cannot be promoted, and the function of repairing the skin cannot be well achieved. If the addition amount is too large, the stability of a skin system is easily influenced, the synergistic effect with stem cell exudate is poor, the expression of each factor of the stem cell exudate is inhibited, and the action effect of the essence containing the mesenchymal stem cell factor is influenced.
Preferably, the concentration of the stem cell factor in the stem cell factor solution is 1ug/ml to 10 ug/ml.
If the concentration of the stem cell factor solution is too low, the content of various effective growth factors in the prepared essence containing the mesenchymal stem cell factor is too low, the effective growth factors cannot perform a synergistic effect with effective components of stem cell external secretion, the generation of skin-related cells cannot be promoted, and the function of repairing the skin cannot be well achieved; if the concentration of the stem cell factor solution is too high, excessive active ingredients easily influence the stability of a skin system, and meanwhile, the synergistic effect with stem cell external secretion is poor, so that the expression of various factors of the stem cell external secretion can be inhibited, and the effect of the essence containing the mesenchymal stem cell factor is influenced.
Specifically, the essence containing the mesenchymal stem cell factor comprises 0.5-2% of stem cell exocrine fluid by taking the weight of the essence containing the mesenchymal stem cell factor as 100%. If the addition amount of the stem cell exocrine is too low, the prepared essence containing the mesenchymal stem cell factors has less content of exosome core factors, cannot perform synergistic effect with effective factors of a stem cell factor solution, cannot promote the generation of skin-related cells and inhibit the generation of excessive skin inflammation, and cannot well achieve the function of repairing the skin. If the addition amount is too large, the stability of a skin system is easily influenced, and meanwhile, the synergistic effect with the stem cell factor solution is poor, the expression of each effective factor of the stem cell factor solution is inhibited, and the action effect of the essence containing the mesenchymal stem cell factor is influenced.
Preferably, the concentration of the stem cell exosomes in the stem cell exudate solution is 1ug/ml to 10 ug/ml. If the concentration of the stem cell exocrine fluid is too low, the prepared essence containing the mesenchymal stem cell factors has less content of exosome core factors, cannot perform synergistic action with effective factors of a stem cell factor solution, cannot promote the generation of skin-related cells and inhibit the generation of excessive skin inflammation, and cannot well achieve the function of repairing the skin. If the addition amount is too large, the stability of a skin system is easily influenced, and meanwhile, the synergistic effect with the stem cell factor solution is poor, the expression of each effective factor of the stem cell factor solution is inhibited, and the action effect of the essence containing the mesenchymal stem cell factor is influenced.
Specifically, the essence containing the mesenchymal stem cell factor comprises, by weight of 100%, 3-8% of glycerol, 3-10% of butanediol, and 2-7% of biological polysaccharide gum; the moisturizing effect of the essence containing the mesenchymal stem cell factor can be improved by mixing the three components with the contents, wherein the added glycerol and the added butanediol are both micromolecular moisturizing components, so that the absorption of the essence containing the mesenchymal stem cell factor can be facilitated, and the moisturizing effect of the essence containing the mesenchymal stem cell factor can be improved; meanwhile, the biological polysaccharide gum contains a large amount of fucose molecules, has a stronger moisturizing capability under the synergistic action of the fucose molecules, the glycerol and the butanediol, and can further improve the moisturizing effect of the essence containing the mesenchymal stem cell factor. In a preferred embodiment of the present invention, the glycerol is added in an amount of 3%, 5%, 6%, 8% based on 100% by weight of the essence containing mesenchymal stem cell factor. In a preferred embodiment of the invention, the addition amount of the butanediol is 3%, 4%, 5%, 8% based on 100% of the essence containing the mesenchymal stem cell factor. In a preferred embodiment of the present invention, the addition amount of the biological polysaccharide gum is 3%, 4%, 5%, 7% based on 100% by weight of the essence containing mesenchymal stem cell factor.
Specifically, the essence containing the mesenchymal stem cell factor comprises 1-4% of trehalose, 0.5-1% of sodium hyaluronate and 0.1-1% of hydrolyzed collagen, wherein the weight of the essence containing the mesenchymal stem cell factor is 100%; when the cells are seriously dehydrated, the trehalose in the essence containing the mesenchymal stem cell factor can replace the water in the cells, can interact with hyaluronic acid and hydrolyzed collagen, improves the moisturizing effect, can keep tissues, skins and cosmetics fresh all the time, and also plays a role in moisturizing. In a preferred embodiment of the present invention, the trehalose is added in an amount of 2%, 3%, 4% based on 100% by weight of the essence containing mesenchymal stem cell factor. In a preferred embodiment of the present invention, the amount of the sodium hyaluronate added is 0.5%, 0.8% or 1% based on 100% by weight of the essence containing mesenchymal stem cell factor. In a preferred embodiment of the present invention, the hydrolyzed collagen is added in an amount of 0.3%, 0.5%, 0.6%, 1% based on 100% by weight of the essence containing mesenchymal stem cell factor.
Specifically, the essence containing the mesenchymal stem cell factor comprises 0.1-1% of vitamin A acetate and 0.1-1% of vitamin E acetate, and the vitamin A acetate and the vitamin E acetate are added, so that the synergistic effect of the vitamin A acetate and the vitamin E acetate can regulate the growth and health factors of epithelial cells, thin the rough and aged skin surface, promote the normalization of cell metabolism and have obvious wrinkle removing effect. In a preferred embodiment of the invention, the addition amount of the vitamin a acetate is 0.2%, 0.3%, 0.7% and 1% based on 100% by weight of the essence containing the mesenchymal stem cell factor. In a preferred embodiment of the invention, the addition amount of the vitamin E acetate is 0.2%, 0.3% and 1% based on 100% of the essence containing the mesenchymal stem cell factor.
Specifically, the essence containing the mesenchymal stem cell factor comprises 0.05-0.3% of carbomer and 0.5-1% of preservative, wherein the weight of the essence containing the mesenchymal stem cell factor is 100%. The carbomer is acrylic acid crosslinked resin obtained by crosslinking pentaerythritol and the like with acrylic acid, and mainly plays roles in thickening and suspending, so that the prepared essence containing the mesenchymal stem cell factor is good in stability. The preservative is added to mainly prevent the essence containing the mesenchymal stem cell factor from deteriorating. In a preferred embodiment of the invention, the addition amount of the carbomer is 0.08%, 0.1%, 0.2% and 0.3% based on 100% by weight of the essence containing the mesenchymal stem cell factor. In a preferred embodiment of the invention, the carbomer is added in an amount of 0.5%, 0.7%, 0.8%, 1.0% by weight of the preservative, based on 100% by weight of the preservative.
Preferably, the storage temperature of the essence containing the mesenchymal stem cell factor is 2-4 ℃. Because the stem cell factor solution and the stem cell external secretion are added into the essence containing the mesenchymal stem cell factor, if the storage temperature is too high, effective factors of the stem cell factor solution and the stem cell external secretion in the prepared essence containing the mesenchymal stem cell factor are inactivated, and proteins are decomposed, so that the effect of the essence containing the mesenchymal stem cell factor is poor; if the storage temperature is too low, the essence containing the mesenchymal stem cell factor is crystallized, and the biological activity of each effective factor is also influenced, so that the effect of the essence containing the mesenchymal stem cell factor is further influenced.
The essence containing the mesenchymal stem cell factor is prepared by the following preparation method of the essence containing the mesenchymal stem cell factor.
Correspondingly, the embodiment of the invention also provides a preparation method of the essence containing the mesenchymal stem cell factor. The method comprises the following steps:
s01, weighing each component according to the essence containing the mesenchymal stem cell factor;
s02, adding the glycerol, the trehalose, the sodium hyaluronate, the carbomer and the preservative into the pure water, heating to 60-65 ℃, and mixing to obtain a first composition;
s03, cooling the first composition to 20-25 ℃, adding the butanediol, the biological polysaccharide gum, the hydrolyzed collagen, the vitamin A acetate and the vitamin E acetate, and mixing to obtain a second composition;
s04, cooling the second composition to 2-8 ℃, adding the stem cell factor solution and the stem cell exocrine liquid, and mixing to obtain the essence containing the mesenchymal stem cell factors.
Specifically, the preferable content and kind of each component of the serum containing the mesenchymal stem cell factor in the step S01 are as described above, and are not described herein again for brevity.
Specifically, in the step S02, the glycerin, the trehalose, the sodium hyaluronate, the carbomer and the preservative are added into the pure water, heated to 60-65 ℃, and mixed to obtain the first composition. The heating temperature is controlled to be 60-65 ℃, if the heating temperature is too low, the glycerin, the trehalose, the sodium hyaluronate, the carbomer and the preservative cannot be completely dissolved in water, and if the heating temperature is too high, resource waste is caused, and the reaction risk coefficient is increased.
Preferably, the mixing time is 30 to 60 minutes. If the mixing time is too short, the glycerin, the trehalose, the sodium hyaluronate, the carbomer and the preservative cannot be completely dissolved in water, the reaction is incomplete, and if the mixing time is too long, the properties of the components of each substance are affected.
Preferably, the mixing is carried out in a conventional manner, such as stirring, so long as the components are uniformly mixed. In a preferred embodiment of the present invention, a magnetic stirrer is used for stirring and mixing.
Specifically, in the step S03, the first composition is cooled to 20 to 25 ℃, and the butylene glycol, the polysaccharide gum, the hydrolyzed collagen, the vitamin a acetate, and the vitamin E acetate are added and mixed to obtain the second composition. Cooling the first composition to 20-25 ℃, adding the components, and if the temperature is too high, easily decomposing the butanediol, the biological polysaccharide gum, the hydrolyzed collagen, the vitamin A acetate and the vitamin E acetate in the mixing process; if the temperature is too low, the above components are not uniformly dissolved.
Preferably, the mixing time is 5 to 10 minutes, and if the mixing time is too short, the butylene glycol, the polysaccharide gum, the hydrolyzed collagen, the vitamin a acetate, and the vitamin E acetate may not be completely dissolved in water, and the reaction may not be complete, and if the mixing time is too long, the properties of the components may be affected.
Preferably, the mixing is carried out in a conventional manner, such as stirring, so long as the components are uniformly mixed.
Specifically, in the step S04, the second composition is cooled to 2 to 8 ℃, the stem cell factor solution and the stem cell exudate are added, and the mixture is mixed to obtain the essence containing the mesenchymal stem cell factor. Cooling the second composition to 2-8 ℃, adding a stem cell factor solution and the stem cell exudate, and if the temperature is too high, degrading effective factors of the stem cell factor solution and the stem cell exudate to cause that the prepared essence containing the mesenchymal stem cell factor does not have a good anti-wrinkle effect; if the temperature attitude is high, the essence containing the mesenchymal stem cell factor is crystallized, and the stem cell factor solution and the stem cell exudate cannot be well mixed with other components, and the activity of effective factors of the stem cell factor solution and the stem cell exudate is influenced.
Preferably, the mixing time is 5-10 minutes, if the mixing time is too short, the stem cell factor solution and the stem cell exudate cannot be completely dissolved in water, the reaction is incomplete, and if the mixing time is too long, the solution generates heat, and the properties of each substance component are affected.
Preferably, the mixing is carried out in a conventional manner, such as stirring, so long as the components are uniformly mixed.
Preferably, in the essence containing the mesenchymal stem cell factor, the preparation method of the stem cell factor solution comprises the following steps:
G01. providing umbilical cord mesenchymal stem cells for culturing to obtain a first culture system, separating the first culture system to obtain cell bodies, and dissolving the cell bodies to prepare cell body suspension;
G02. and (3) freezing and thawing the cell body suspension by adopting liquid nitrogen to prepare a stem cell factor solution.
Specifically, in the step G01, the umbilical cord mesenchymal stem cells have a strong immunoregulatory effect, and at the same time, can promote cell differentiation and repair damaged tissues and organs.
In a preferred embodiment of the present invention, the method for preparing umbilical cord mesenchymal stem cells comprises the steps of:
G011. providing umbilical cord mesenchymal stem cell primary cells;
G012. when the umbilical cord mesenchymal stem cell primary cells are cultured until the cells reach 80% fusion state, digesting and centrifuging to obtain a first cell sediment, and adding a culture medium into the first cell sediment for subculture;
G013. and when the cells are subcultured until the tenth generation of cells reach a 90% fusion state, digesting and centrifuging to obtain a second cell precipitate, and adding a cell cryopreservation solution into the second cell precipitate to obtain the umbilical cord mesenchymal stem cells.
Specifically, in the step G011, the steps are all operated in an ultra-clean benchThe method is characterized by comprising the following steps of operating in an ultra-clean workbench, preferably, taking a sterile fresh umbilical cord, washing the fresh umbilical cord by using normal saline containing gentamicin to remove blood attached to the surface of the fresh umbilical cord, stripping blood vessels on the umbilical cord, washing umbilical cord matrix tissues stripped of the blood vessels by using normal saline containing gentamicin, washing until the liquid flowing through a screen is not sticky, and soaking in culture solution for culturing. Preferably, the umbilical cord matrix tissue is cut into pieces of about 3-5 mm3The purpose of the tissue blocks is to make the umbilical cord matrix tissue spread more uniformly, to provide good growth space for cells during cell culture, to make the growth of the cells unlimited, and to ensure that the number of cells cultured is sufficient. If the obtained block is too large, the number of cells cultured in the culture dish is too small, and the culture medium resource is wasted. Preferably, in the step of culturing by soaking in a culture solution, the addition amount of the culture solution is 5-8 mL, and sufficient nutrients can be provided for the culture solution by adding 5-8 mL of serum-free culture solution, so that the growth of cells is promoted; more preferably, the cultivation is carried out in an incubator at 37 ℃ and a saturated humidity incubator containing 5% by volume of carbon dioxide.
Preferably, the umbilical cord mesenchymal stem cell tissue block is cultured, 2.5mL of culture solution is supplemented into each culture dish every day, and then the culture solution is replaced completely every even number of days for culture, wherein the replacement of the culture solution is aimed at providing enough nutrition for the growth of the tissue and promoting the growth of cells around the tissue block.
Preferably, when the umbilical cord mesenchymal stem cell tissue mass culture reaches the 10 th generation, the umbilical cord mesenchymal stem cell tissue mass culture solution of the 10 th generation is digested and centrifuged to obtain the umbilical cord mesenchymal stem cell primary cells. Preferably, the method for digesting the umbilical cord mesenchymal stem cell tissue block culture solution of the 10 th generation comprises the following steps: removing tissue blocks in the 10 th generation of umbilical cord mesenchymal stem cell tissue block culture solution, adding 5mL of DPBS washing solution into the 10 th generation of umbilical cord mesenchymal stem cell tissue block culture solution for washing, and removing the DPBS solution; and adding 1mL of 0.25% pancreatin solution, shaking uniformly, digesting in an incubator for 3min to obtain a mixed solution of the umbilical cord mesenchymal stem cell tissue blocks after digestion treatment, adding 2mL of a culture medium into the mixed solution of the umbilical cord mesenchymal stem cell tissue blocks after digestion treatment, and shaking uniformly to terminate digestion. Preferably, the mixed solution after the digestion treatment is centrifuged for 5min at the rotating speed of 1000rpm, the supernatant is discarded, and 50ml of culture medium is added for heavy suspension to obtain the umbilical cord mesenchymal stem cell primary cells.
Specifically, in the step G012, the umbilical cord mesenchymal stem cell primary cells are cultured to reach a state where the cells are fused to 80%, and when the state of the cells is fused to 80%, the cells are digested in a proper state, and if the degree of fusion is too high, the digestion reaction is not easy to occur, and if the digestion effect is not good, the subsequent cell culture is affected, and meanwhile, if the degree of fusion is too high, the cells compete with each other to compete for nutrient components and growth space of the culture medium; if the fusion degree is too low, the number of cells obtained by digestion is too small, and the subsequent cell collection is influenced.
Specifically, when the cells are cultured to reach a state of 80% confluence, the digestion treatment step comprises the following steps: discarding the old culture solution, taking a DPBS10 ml/bottle to wash the old culture medium, and removing the DPBS solution; adding 3ml of 0.25% pancreatin, digesting in an incubator for 3min to obtain the primary cell liquid of the umbilical cord mesenchymal stem cells after digestion treatment, adding 6ml of culture medium into the primary cell liquid of the umbilical cord mesenchymal stem cells after digestion treatment to terminate the digestion, and shaking uniformly to terminate the digestion. Preferably, the mixture after digestion is centrifuged for 5min at 1000rpm, the supernatant is discarded to obtain a first cell pellet, and 50ml of culture medium is added to the first cell pellet for subculture.
Preferably, the final volume of the medium per flask per passage is determined to be 50ml during the subculture, and more preferably, the subculture is passage 10.
Specifically, in the above step G013, when the cells were subcultured until the tenth generation reached a 90% confluent state, digestion was performedThe processing method comprises the following steps: discarding the old culture solution, taking a DPBS10 ml/bottle to wash the old culture medium, and removing the DPBS solution; adding 3ml of 0.25% pancreatin, digesting in an incubator for 3min to obtain a digested subculture cell liquid, adding 6ml of culture medium into the digested subculture cell liquid after digestion, and shaking up to terminate digestion. Preferably, the mixed solution after the digestion treatment is centrifuged for 5min at the rotation speed of 1000rpm, the supernatant is discarded to obtain a second cell precipitate, and the cell cryopreservation solution is added into the second cell precipitate to perform cell cryopreservation to obtain the umbilical cord mesenchymal stem cell. Preferably, the density of the umbilical cord mesenchymal stem cells is 1 × 107/mL。
Specifically, in the step G01, umbilical cord mesenchymal stem cells are provided and cultured to obtain a first culture system, and the first culture system is subjected to a separation treatment to obtain a cell body, preferably, the separation treatment method includes the steps of centrifuging the first culture system to collect a cell body of the first culture system, washing the cell body of the first culture system with a PBS solution, and centrifuging the cell body at 1000rpm for 5min to collect a cell precipitate. Preferably, the washing step is repeated for 2-3 times to remove impurities in the culture medium on the cell surface.
Further, dissolving the cell body to prepare a cell body suspension; preferably, physiological saline is selected for dissolving; preferably, the cell body suspension has a density of 5 × 107/mL。
Specifically, in the step G02, the cell body suspension is frozen and thawed by using liquid nitrogen to prepare the stem cell factor solution. Preferably, the method for freezing and thawing by using liquid nitrogen comprises the following steps: placing the cell body suspension in a liquid nitrogen atmosphere for freezing and storing for 5 minutes; and quickly placing the frozen cell body suspension into a water bath kettle at 37 ℃, shaking back and forth, and unfreezing for 3 minutes. Preferably, the step of freezing and thawing by liquid nitrogen is repeated for 6-8 times. Preferably, the cell body suspension obtained by freezing and thawing with liquid nitrogen is placed into a low-temperature high-speed centrifuge, centrifuged for 5min at 12000rpm and 4 ℃, and the supernatant is collected to prepare the stem cell factor solution.
Preferably, the preparation method of the stem cell exudate comprises the following steps:
D01. providing umbilical cord mesenchymal stem cells for culturing to obtain a first culture system, collecting supernatant of the first culture system, carrying out first centrifugal treatment on the supernatant to obtain first supernatant, and carrying out second centrifugal treatment on the first supernatant to obtain a crude precipitate of a stem cell exosome;
D02. dissolving the crude stem cell exosome precipitate, performing third centrifugal treatment on mixed solution of the crude stem cell exosome precipitate obtained by dissolving, collecting the stem cell exosome precipitate, and dissolving the stem cell exosome precipitate to obtain the stem cell exosome.
In the step D01, umbilical cord mesenchymal stem cells are provided and cultured to obtain a first culture system, wherein the umbilical cord mesenchymal stem cells are prepared as described above and will not be described herein again for brevity.
Specifically, umbilical cord mesenchymal stem cells are cultured to obtain a first culture system, and in a preferred embodiment of the invention, the umbilical cord mesenchymal stem cells are cultured by using a hollow fiber cell culture system. Wherein, a hollow fiber cell culture system is adopted for culture. Can improve the enrichment of exosome and the content of exosome. Because the exosome can not permeate the fiber, the exosome in the circulating culture medium can not enter a cell growth space, so that the exosome with high purity and high concentration can be obtained, the cell debris is obviously reduced, the exosome can be continuously harvested for a long time, and the activity is kept unchanged.
Specifically, the supernatant of the first culture system is collected, and the supernatant is subjected to a first centrifugation treatment to obtain a first supernatant. Preferably, the first centrifugation treatment method comprises: centrifuging the supernatant for 10 to 12 minutes at the rotating speed of 300 to 500g, and collecting the supernatant as a first crude product; centrifuging the first crude product for 10-12 minutes at a rotating speed of 2000-2500 g, and collecting supernatant as a second crude product; and centrifuging the second crude product for 30-35 minutes under the condition that the rotating speed is 8000-10000 g, and collecting supernatant as first supernatant.
Further, performing second centrifugation treatment on the first supernatant to obtain a crude stem cell exosome precipitate; preferably, the second centrifugation treatment method comprises: and (3) placing the first supernatant into a condition with the rotating speed of 120000-130000 g for centrifugation for 70-75 minutes, and collecting the precipitate to obtain the crude precipitate of the stem cell exosome. In the step D02, the crude stem cell exosome precipitate is lysed, and the crude stem cell exosome precipitate mixture obtained by lysis is subjected to a third centrifugation treatment to collect the crude stem cell exosome precipitate. Preferably, the third centrifugation treatment method comprises: and (3) placing the mixed solution of the crude stem cell exosome precipitate obtained by dissolution in a condition of the rotating speed of 120000-130000 g for centrifugation for 70-75 minutes, and collecting the precipitate as the stem cell exosome precipitate. Further, dissolving the stem cell exosome precipitate to obtain the stem cell exosome. Preferably, the stem cell exosome precipitate is dissolved by purified water to obtain the stem cell exosome. Preferably, the exosomes collected per 100ml of cell culture supernatant are solubilized with 10ml of purified water.
Preferably, an RNA activity protective agent is added to at least one of the first culture system, the first supernatant, the mixed solution of the stem cell exosome crude precipitate and the stem cell exosome, and the RNA activity protective agent is mainly added to protect an exosome activity function. Preferably, the RNA activity protecting agent is selected from Trizol. More preferably, the addition amount of the RNA activity protecting agent is 5% to 6% based on 100% of the volume of the solution to which the RNA activity protecting agent is added. In conclusion, the preparation method of the essence containing the mesenchymal stem cell factor only needs to mix the components according to the proportion, the preparation method adopts a distribution mixing method, so that the components can be uniformly dispersed, different components are uniformly heated and mixed at different temperatures, so that the components can keep good performance, and the components can realize the beneficial effects and are endowed with stability. In addition, the preparation method has the advantages of simple process, controllable conditions, low equipment requirements and convenient and quick preparation.
Now, the content of each component of the essence containing the mesenchymal stem cell factor and the preparation method are taken as examples to further explain the invention in detail.
Example 1
Preparation of umbilical cord mesenchymal Stem cells
The preparation method of the umbilical cord mesenchymal stem cells comprises the following steps:
(1) providing umbilical cord mesenchymal stem cell primary cells; the specific operation is as follows: the method comprises the steps of operating in an ultra-clean workbench, taking a sterile fresh umbilical cord, flushing the fresh umbilical cord with normal saline containing gentamicin, removing blood attached to the surface of the fresh umbilical cord, stripping blood vessels on the umbilical cord, flushing umbilical cord matrix tissues stripped of the blood vessels with normal saline containing gentamicin, and soaking in a culture solution for culture after flushing until the liquid flowing through a screen is not sticky. Cutting the umbilical cord matrix tissue to about 3-5 mm3And adding 5-8 mL of serum-free culture solution into the tissue blocks with the sizes, and culturing in an incubator with carbon dioxide and saturated humidity, wherein the volume fraction of the carbon dioxide is 5%.
Culturing the umbilical cord mesenchymal stem cell tissue blocks, supplementing 2.5mL of culture solution into each culture dish every day, and then completely replacing the culture solution every even number of days for culturing, when the umbilical cord mesenchymal stem cell tissue blocks are cultured to reach the 10 th generation, removing the tissue blocks in the umbilical cord mesenchymal stem cell tissue block culture solution of the 10 th generation, adding 5mL of DPBS (double priming detergent) for washing, and removing the DPBS solution; adding 1mL of 0.25% pancreatin solution, shaking, digesting in incubator for 3min, adding 2mL of culture medium, and shaking to stop digestion. And further, centrifuging the mixture after the digestion treatment for 5min at the rotating speed of 1000rpm, discarding the supernatant, and adding 50ml of culture medium for heavy suspension to obtain the umbilical cord mesenchymal stem cell primary cells.
(2) When the umbilical cord mesenchymal stem cell primary cells are cultured until the cells reach 80% fusion state, removing old culture solution, taking 10 ml/bottle of DPBS (platelet-rich plasma-dependent stem cell) to wash the old culture medium, and removing the DPBS solution; adding 3ml of 0.25% pancreatin, digesting in an incubator for 3min, adding 6ml of culture medium, and shaking to terminate digestion. After the termination, the mixture was put into a 50ml centrifuge tube and centrifuged at 1000rpm for 5 min. The supernatant was discarded, the medium was added and resuspended, and passaging was carried out according to the standard of P0 passage (1 flask) → P1 passage (3 flasks) → P2 passage (10 flasks), and the final volume of the medium per flask for each passage was 50 ml.
(3) And when the cells are subcultured until the tenth generation of cells reach a 90% fusion state, removing the old culture medium, washing the old culture medium by using a DPBS10 ml/bottle, removing DPBS liquid, adding 3ml of 0.25% pancreatin, digesting for 3min in an incubator, adding 6ml of the culture medium, and shaking uniformly to terminate digestion. And transferring the umbilical cord mesenchymal stem cells into a 50ml centrifuge tube, sampling and counting, centrifuging at 1000rpm for 5min, removing supernatant, adding cell cryopreservation liquid for cell cryopreservation, and obtaining the umbilical cord mesenchymal stem cells. The density of the umbilical cord mesenchymal stem cells is 1 multiplied by 107/mL。
Example 2
Preparation of Stem cell factor solution
The preparation method of the stem cell factor solution comprises the following steps:
(1) providing umbilical cord mesenchymal stem cells for culturing to obtain a first culture system, centrifuging the first culture system to collect a first culture system cell body, washing the first culture system cell body by adopting a PBS (phosphate buffer solution) solution, and centrifuging at 1000rpm for 5min to collect cell precipitates. Repeating the washing step for 2-3 times to obtain a cell body, and dissolving the cell body by using normal saline to prepare a cell body suspension; wherein the cell body suspension has a density of 5 × 107/mL。
(2) The cell body suspension is prepared into a stem cell factor solution by adopting a liquid nitrogen freeze-thawing method, and the method for freeze-thawing by adopting liquid nitrogen comprises the following steps: placing the cell body suspension in a liquid nitrogen atmosphere for freezing and storing for 5 minutes; and quickly placing the frozen cell body suspension into a water bath kettle at 37 ℃, shaking back and forth, and unfreezing for 3 minutes. Preferably, the step of freezing and thawing by liquid nitrogen is repeated for 6-8 times. Preferably, the cell body suspension obtained by freezing and thawing with liquid nitrogen is placed into a low-temperature high-speed centrifuge, centrifuged for 5min at 12000rpm and 4 ℃, and the supernatant is collected to prepare the stem cell factor solution.
Example 3
Preparation of stem cell exudate
The preparation method of the stem cell external secretion comprises the following steps:
(1) culturing the umbilical cord mesenchymal stem cells by adopting a hollow fiber cell culture system: sterilizing a hollow fiber cell culture system, preparing a needle cylinder containing cell concentrated solution, screwing the hollow needle cylinder on a right outer opening of the system, screwing the needle cylinder with the cell concentrated solution on a left outer opening, pumping the cell concentrated solution from the left outer opening, flushing partial cell concentrated solution and culture medium into the right outer hollow needle cylinder by body pressure at the moment, flushing the left end and the right end back and forth until the turbidity of the liquid in the needle cylinders at the two ends is consistent, after reserving about equal amount of liquid in the needle cylinders at the two ends, closing the right outer valve, opening the right inner valve, loosening a liquid storage bottle opening to balance the air pressure in a pipeline, pumping the liquid into the left outer needle cylinder, and enabling the culture medium to flow into a liquid storage bottle from the right inner pipeline at the moment; the left outer valve was closed for culture.
Collecting the supernatant of the first culture system, centrifuging the supernatant for 10 minutes under the condition of the rotating speed of 300g, and collecting the supernatant as a first crude product; centrifuging the first crude product at a rotating speed of 2000g for 10 minutes, and collecting supernatant as a second crude product; and placing the second crude product at the rotation speed of 10000g for centrifuging for 30 minutes, collecting the supernatant as a first supernatant, placing the first supernatant at the rotation speed of 120,000g for centrifuging for 70 minutes, and collecting the precipitate to obtain the crude precipitate of the stem cell exosome.
(2) Dissolving the crude stem cell exosome precipitate by adopting a PBS (phosphate buffer solution), placing the mixed solution of the crude stem cell exosome precipitate obtained by dissolving in a condition of a rotating speed of 120,000g for centrifugation for 70 minutes, and collecting the precipitate as the stem cell exosome precipitate; dissolving the stem cell exosome precipitate by using purified water to obtain the stem cell exosome solution, wherein 10ml of purified water is used for dissolving exosomes collected per 100ml of cell culture solution supernatant.
Example 4
Component of essence containing mesenchymal stem cell factor and preparation method thereof
The essence containing the mesenchymal stem cell factor comprises the following components in parts by weight, wherein the weight of the essence containing the mesenchymal stem cell factor is 100 percent:
3% of glycerin, 8% of butanediol, 3% of biological polysaccharide gum, 4% of trehalose, 0.5% of sodium hyaluronate, 0.3% of hydrolyzed collagen, 0.2% of vitamin A acetate, 0.2% of vitamin E acetate, 0.08% of carbomer, 0.7% of preservative, 0.8% of stem cell factor solution, 0.8% of stem cell external secretion and the balance of pure water.
The preparation method of the essence containing the mesenchymal stem cell factor comprises the following steps:
weighing the components according to the essence containing the mesenchymal stem cell factor;
adding the glycerol, the trehalose, the sodium hyaluronate, the carbomer and the preservative into the pure water, heating to 60 ℃, and mixing to obtain a first composition;
cooling the first composition to 20 ℃, adding the butanediol, the biological polysaccharide gum, the hydrolyzed collagen, the vitamin A acetate and the vitamin E acetate, and mixing to obtain a second composition;
and cooling the second composition to 2-8 ℃, adding the stem cell factor solution and the stem cell exudate, and mixing to obtain the essence containing the mesenchymal stem cell factor.
Example 5
Component of essence containing mesenchymal stem cell factor and preparation method thereof
The essence containing the mesenchymal stem cell factor comprises the following components in parts by weight, wherein the weight of the essence containing the mesenchymal stem cell factor is 100 percent:
5% of glycerin, 5% of butanediol, 5% of biological polysaccharide gum, 2% of trehalose, 1% of sodium hyaluronate, 0.5% of hydrolyzed collagen, 0.3% of vitamin A acetate, 0.3% of vitamin E acetate, 0.3% of carbomer, 0.8% of preservative, 1% of stem cell factor solution, 1% of stem cell exudate and the balance of pure water.
The preparation method of the essence containing the mesenchymal stem cell factor comprises the following steps:
weighing the components according to the essence containing the mesenchymal stem cell factor;
adding the glycerol, the trehalose, the sodium hyaluronate, the carbomer and the preservative into the pure water, heating to 60 ℃, and mixing to obtain a first composition;
cooling the first composition to 20 ℃, adding the butanediol, the biological polysaccharide gum, the hydrolyzed collagen, the vitamin A acetate and the vitamin E acetate, and mixing to obtain a second composition;
and cooling the second composition to 2-8 ℃, adding the stem cell factor solution and the stem cell exudate, and mixing to obtain the essence containing the mesenchymal stem cell factor.
Example 6
Component of essence containing mesenchymal stem cell factor and preparation method thereof
The essence containing the mesenchymal stem cell factor comprises the following components in parts by weight, wherein the weight of the essence containing the mesenchymal stem cell factor is 100 percent:
glycerol 6%, butanediol 4%, biological polysaccharide gum 4%, trehalose 2%, sodium hyaluronate 0.8%, hydrolyzed collagen 0.6%, vitamin A acetate 1%, vitamin E acetate 1%, carbomer 0.3%, preservative 0.8%, stem cell factor solution 1.5%, stem cell exudate 1.5%, and the balance of pure water.
The preparation method of the essence containing the mesenchymal stem cell factor comprises the following steps:
weighing the components according to the essence containing the mesenchymal stem cell factor;
adding the glycerol, the trehalose, the sodium hyaluronate, the carbomer and the preservative into the pure water, heating to 60 ℃, and mixing to obtain a first composition;
cooling the first composition to 20 ℃, adding the butanediol, the biological polysaccharide gum, the hydrolyzed collagen, the vitamin A acetate and the vitamin E acetate, and mixing to obtain a second composition;
and cooling the second composition to 2-8 ℃, adding the stem cell factor solution and the stem cell exudate, and mixing to obtain the essence containing the mesenchymal stem cell factor.
Example 7
Component of essence containing mesenchymal stem cell factor and preparation method thereof
The essence containing the mesenchymal stem cell factor comprises the following components in parts by weight, wherein the weight of the essence containing the mesenchymal stem cell factor is 100 percent:
8% of glycerin, 3% of butanediol, 7% of biological polysaccharide gum, 3% of trehalose, 0.5% of sodium hyaluronate, 1% of hydrolyzed collagen, 0.7% of vitamin A acetate, 0.7% of vitamin E acetate, 0.2% of carbomer, 1% of preservative, 2% of stem cell factor solution, 2% of stem cell exocrine and the balance of pure water.
The preparation method of the essence containing the mesenchymal stem cell factor comprises the following steps:
weighing the components according to the essence containing the mesenchymal stem cell factor;
adding the glycerol, the trehalose, the sodium hyaluronate, the carbomer and the preservative into the pure water, heating to 60 ℃, and mixing to obtain a first composition;
cooling the first composition to 20 ℃, adding the butanediol, the biological polysaccharide gum, the hydrolyzed collagen, the vitamin A acetate and the vitamin E acetate, and mixing to obtain a second composition;
and cooling the second composition to 2-8 ℃, adding the stem cell factor solution and the stem cell exudate, and mixing to obtain the essence containing the mesenchymal stem cell factor.
Comparative example 1
Preparation of stem cell exudate
The preparation method of the stem cell external secretion comprises the following steps:
(1) culturing umbilical cord mesenchymal stem cells by using a culture bottle, collecting supernatant of the first culture system, centrifuging the supernatant for 10 minutes under the condition of a rotating speed of 300g, and collecting the supernatant as a first crude product; centrifuging the first crude product at a rotating speed of 2000g for 10 minutes, and collecting supernatant as a second crude product; and placing the second crude product at the rotation speed of 10000g for centrifuging for 30 minutes, collecting the supernatant as a first supernatant, placing the first supernatant at the rotation speed of 120,000g for centrifuging for 70 minutes, and collecting the precipitate to obtain the crude precipitate of the stem cell exosome.
(2) Dissolving the crude stem cell exosome precipitate by adopting a PBS (phosphate buffer solution), placing the mixed solution of the crude stem cell exosome precipitate obtained by dissolving in a condition of a rotating speed of 120,000g for centrifugation for 70 minutes, and collecting the precipitate as the stem cell exosome precipitate; dissolving the stem cell exosome precipitate by using purified water to obtain the stem cell exosome solution, wherein 10ml of purified water is used for dissolving exosomes collected per 100ml of cell culture solution supernatant.
Test experiments:
test run (one): observation and analysis of cells at each stage of umbilical cord mesenchymal stem cells
Selecting passage cells obtained by subculturing umbilical cord mesenchymal stem cell primary cells on the 10 th day and the 15 th day and umbilical cord mesenchymal stem cell primary cells for cell morphology analysis.
Test run (ii): verifying the influence of different culture methods on the expression amounts of CD63 and Alix in the secretion of stem cells
Western blot detection is used for detecting the protein contents of exosomes CD63 and Alix in stem cell exudate obtained by different culture methods in example 3 and comparative example 1 respectively.
Test run (iii): verification of protective Effect of RNA Activity protective Agents
Taking two groups of 1mL of stem cell exosomes prepared in example 3 as a first test group and a second test group, selecting Trizol as an RNA activity protective agent, adding no Trizol in the first test group, adding Trizol in the second test group, reacting for 48 hours, and detecting the protein content of the exosomes CD63 by using Western blot detection.
Test run (d): anti-aging effect of essence containing mesenchymal stem cell factor
Human body trial experiments show that the anti-aging effect of the essence containing the umbilical cord mesenchymal stem cell factor and the exosome and containing the mesenchymal stem cell factor is as follows: the essence containing the umbilical cord mesenchymal stem cell factor and exosome and containing the mesenchymal stem cell factor prepared in example 4 is selected.
Selecting test objects: half of each man and woman, one age group of 30-70 years old and one age group of ten years old are divided into four age groups, and each man and woman in each age group is healthy and has no dermatosis and allergic history.
The number of the tested persons is as follows: 80 people, one group of 20 people, are people of the same age and sex.
Evaluation indexes are as follows: and (5) wrinkling.
Test area: face, including the canthus, under the eye, cheek area.
And (3) testing period: before use and after 15 days of use.
And (4) analyzing results:
test run (one): observation and analysis of cells at each stage of umbilical cord mesenchymal stem cells
The analysis of the steps in example 1 is carried out, wherein the growth states of the umbilical cord mesenchymal stem cell primary cell on the 10 th day and the 15 th day are respectively shown in fig. 1 and fig. 2, and it can be seen that the growth potential of the umbilical cord mesenchymal stem cell primary cell is better. When the growth state of the passage cell obtained by subculturing the umbilical cord mesenchymal stem cell primary cell is shown in fig. 3, it can be seen that the growth of the passage cell is faster and the cell volume is larger.
Test run (ii): verifying the influence of different culture methods on the expression amounts of CD63 and Alix in the secretion of stem cells
The stem cell exudate of example 3 and comparative example 1 were analyzed, and the expression levels of CD63 and Alix in the stem cell exudate obtained by different culture methods of example 3 and comparative example 1 were measured by Western blot analysis, and as shown in fig. 4, the expression levels of CD63 and Alix were 8.2 times and 3.4 times higher than those of the culture flask when cultured in the hollow microsystem, which indicates that the content of exudate can be increased by the hollow fiber cell culture method of example 3.
Test run (iii): verification of protective Effect of RNA Activity protective Agents
The stem cell exudate of example 3 and comparative example 2 were analyzed, and the expression level of CD63 in the stem cell exudate of test group (i) and test group (ii) was measured by Western blot, and the results are shown in fig. 5, which shows that degradation of exosomes was prevented by adding Trizol.
Test run (d): verification of anti-aging effect of essence containing mesenchymal stem cell factor
The effect test is carried out on the essence containing the mesenchymal stem cell factor prepared in the example 4, the test is carried out, the whole cheek from the lower part of the eye to the corner of the eye is selected as an analysis area, the visual sense of wrinkles before and after the experiment person uses the essence containing the umbilical cord mesenchymal stem cell factor and the exosome and containing the mesenchymal stem cell factor is examined, the anti-aging effect of the cosmetic is evaluated, the elimination of the wrinkles is effective through visual observation, the result is shown in the following table 1, and the visual observation result of the wrinkles shows that the product of the invention obtains better effect on the large-scale trial experiment of men and women, and the total effective rate of the anti-aging of the facial skin reaches 92.5%.
TABLE 1
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The essence containing the mesenchymal stem cell factor is characterized by comprising the following components in parts by weight, based on 100% by weight of the essence containing the mesenchymal stem cell factor:
3-8% of glycerin, 3-10% of butanediol, 2-7% of biological polysaccharide gum, 1-4% of trehalose, 0.5-1% of sodium hyaluronate, 0.1-1% of hydrolyzed collagen, 0.1-1% of vitamin A acetate, 0.1-1% of vitamin E acetate, 0.05-0.3% of carbomer, 0.5-1% of preservative, 0.5-2% of stem cell factor solution, 0.5-2% of stem cell exocrine and the balance of pure water.
2. The essence containing mesenchymal stem cell factor according to claim 1, wherein the concentration of stem cell factor in the stem cell factor solution is 1ug/ml to 10 ug/ml.
3. The essence containing mesenchymal stem cell factor according to claim 1, wherein the concentration of stem cell exosomes in the stem cell exosome secretion is 1ug/ml to 10 ug/ml.
4. The essence containing mesenchymal stem cell factor according to any one of claims 1 to 3, wherein the storage temperature of the essence containing mesenchymal stem cell factor is 2 to 4 ℃.
5. The preparation method of the essence containing the mesenchymal stem cell factor is characterized by comprising the following steps of:
respectively weighing each component according to the essence containing the mesenchymal stem cell factor of any one of claims 1 to 4;
adding the glycerol, the trehalose, the sodium hyaluronate, the carbomer and the preservative into the pure water, heating to 60-65 ℃, and mixing to obtain a first composition;
cooling the first composition to 20-25 ℃, adding the butanediol, the biological polysaccharide gum, the hydrolyzed collagen, the vitamin A acetate and the vitamin E acetate, and mixing to obtain a second composition;
and cooling the second composition to 2-8 ℃, adding the stem cell factor solution and the stem cell exudate, and mixing to obtain the essence containing the mesenchymal stem cell factor.
6. The method for preparing essence containing mesenchymal stem cell factor according to claim 5, wherein the method for preparing the stem cell factor solution comprises the following steps:
providing umbilical cord mesenchymal stem cells for culturing to obtain a first culture system, separating the first culture system to obtain cell bodies, and dissolving the cell bodies to prepare cell body suspension;
and (3) freezing and thawing the cell body suspension by adopting liquid nitrogen to prepare a stem cell factor solution.
7. The method for preparing essence containing mesenchymal stem cell factor according to claim 5, wherein the method for preparing stem cell exudate comprises the following steps:
providing umbilical cord mesenchymal stem cells for culturing to obtain a first culture system, collecting supernatant of the first culture system, carrying out first centrifugal treatment on the supernatant to obtain first supernatant, and carrying out second centrifugal treatment on the first supernatant to obtain a crude precipitate of a stem cell exosome;
dissolving the crude stem cell exosome precipitate, performing third centrifugal treatment on mixed solution of the crude stem cell exosome precipitate obtained by dissolving, collecting the stem cell exosome precipitate, and dissolving the stem cell exosome precipitate to obtain the stem cell exosome.
8. The method for preparing essence containing mesenchymal stem cell factor according to claim 7, wherein the first centrifugation treatment method comprises: centrifuging the supernatant for 10 to 12 minutes at the rotating speed of 300 to 500g, and collecting the supernatant as a first crude product; centrifuging the first crude product for 10-12 minutes at a rotating speed of 2000-2500 g, and collecting supernatant as a second crude product; centrifuging the second crude product for 30-35 minutes at the rotation speed of 8000-10000 g, and collecting supernatant as first supernatant; and/or the presence of a gas in the gas,
the second centrifugal treatment method comprises the following steps: placing the first supernatant into a centrifuge at the rotating speed of 120000-130000 g for 70-75 minutes, and collecting the precipitate to obtain a crude precipitate of the stem cell exosome; and/or the presence of a gas in the gas,
the third centrifugal treatment method comprises the following steps: and (3) placing the mixed solution of the crude stem cell exosome precipitate obtained by dissolution in a condition of the rotating speed of 120000-130000 g for centrifugation for 70-75 minutes, and collecting the precipitate as the stem cell exosome precipitate.
9. The method for preparing essence containing mesenchymal stem cell factor according to claim 7, wherein an RNA activity protectant is added to at least one of the first culture system, the first supernatant, the mixed solution of the crude stem cell exosome precipitate and the stem cell exosome secretion solution.
10. The method for preparing essence containing mesenchymal stem cell factor according to claim 9, wherein the RNA activity protecting agent is added in an amount of 5-6% based on 100% by volume of the solution to which the RNA activity protecting agent is added.
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| CN113925825A (en) * | 2021-11-18 | 2022-01-14 | 张�杰 | Facial essence and preparation method thereof |
| CN114681387A (en) * | 2022-03-16 | 2022-07-01 | 和携科技有限公司 | Animal-derived exosome composition and application thereof in skin wound repair |
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