阅读说明：本技术 一种间充质干细胞制备抗衰去皱化妆品的方法 (Method for preparing anti-aging and wrinkle-removing cosmetic by mesenchymal stem cells ) 是由 熊学清 黄金花 于 2021-09-08 设计创作，主要内容包括：本发明提出了一种间充质干细胞制备抗衰去皱化妆品的方法,涉及细胞技术领域。本申请实施例提供一种间充质干细胞制备抗衰去皱化妆品的方法,包括以下步骤：获取间充质干细胞原代细胞；将间充质干细胞原代细胞进行传代培养,得到间充质干细胞；收集第3代以后的间充质干细胞的上清液,加入六胜肽、寡肽-1,寡肽-3,胶原蛋白、玻尿酸、绿藻提取物、绵毛多管藻提取物、凝结多糖和水,得到混合溶液,将混合溶液经冻干工艺后,得到抗衰去皱化妆品。本发明中采用间充质干细胞制备的化妆品针对皮肤松弛有皱纹的情况具有很好的抑制效果。(The invention provides a method for preparing an anti-aging and wrinkle-removing cosmetic by using mesenchymal stem cells, and relates to the technical field of cells. The embodiment of the application provides a method for preparing an anti-aging and wrinkle-removing cosmetic by using mesenchymal stem cells, which comprises the following steps: obtaining mesenchymal stem cell primary cells; carrying out subculture on the primary mesenchymal stem cell to obtain a mesenchymal stem cell; collecting the supernatant of the 3 rd generation and later mesenchymal stem cells, adding the hexapeptide, oligopeptide-1, oligopeptide-3, collagen, hyaluronic acid, green algae extract, Polysiphonia lanuginosa extract, curdlan and water to obtain a mixed solution, and freeze-drying the mixed solution to obtain the anti-aging and wrinkle-removing cosmetic. The cosmetics prepared by adopting the mesenchymal stem cells have good inhibition effect on the condition that the skin is loose and has wrinkles.)

1. A method for preparing an anti-aging and wrinkle-removing cosmetic by mesenchymal stem cells is characterized by comprising the following steps:

obtaining mesenchymal stem cell primary cells;

carrying out subculture on the primary mesenchymal stem cell to obtain a mesenchymal stem cell;

collecting the supernatant of the 3 rd generation and later mesenchymal stem cells, adding the hexapeptide, oligopeptide-1, oligopeptide-3, collagen, hyaluronic acid, green algae extract, Polysiphonia lanuginosa extract, curdlan and water to obtain a mixed solution, and freeze-drying the mixed solution to obtain the anti-aging and wrinkle-removing cosmetic.

2. The method for preparing the anti-aging and anti-wrinkle cosmetic from the mesenchymal stem cells according to claim 1, wherein the source of the mesenchymal stem cells primary cells comprises one of bone marrow, umbilical cord, placenta, fat and teeth of human or animals.

3. The method for preparing the anti-aging and anti-wrinkle cosmetic from mesenchymal stem cells according to claim 2, wherein the source of the mesenchymal stem cells is placenta.

4. The method for preparing the anti-aging and anti-wrinkle cosmetic from the mesenchymal stem cells according to claim 1, wherein the step of obtaining the mesenchymal stem cell primary cells comprises the following steps: washing and centrifuging sample cells for the first time, and then, mixing the sample cells according to a volume ratio of 1: (1-1.5) adding a cell separation solution, performing gradient centrifugation to obtain a mononuclear cell layer, performing centrifugation again, performing inoculation culture on the precipitate, changing the solution every 48-72 hours, and removing non-adherent cells to obtain the mesenchymal stem cells.

5. The method for preparing the anti-aging and wrinkle-removing cosmetic from the mesenchymal stem cells according to claim 4, wherein the washing is specifically to resuspend sample cells in a complete culture medium, the single cell suspension is prepared by repeatedly blowing and beating the sample cells with a pipette, the rotating speed of the first centrifugation is 400-600 rpm, the centrifugation time is 20-40 min, the cell separation solution is Percoll cell separation solution with the specific gravity of 1.07-1.09 g/ml, the gradient centrifugation is specifically to centrifuge at 1000-1100 rpm for 10-15 min, then centrifuge at 1150-1200 rpm for 10-15 min, and finally centrifuge at 1500-1600 rpm for 15-20 min.

6. The method for preparing the anti-aging and anti-wrinkle cosmetic from the mesenchymal stem cells according to claim 1, wherein the subculture specifically comprises: when the fusion degree of the cells reaches 80-90%, adding a trypsin-EDTA solution for digestion, stopping the digestion by adopting fetal calf serum, centrifuging, discarding the supernatant, re-suspending with a complete culture medium, and performing 1: (3-5) subculturing.

7. The method for preparing the anti-aging and anti-wrinkle cosmetic from the mesenchymal stem cells according to claim 6, wherein the concentration of trypsin in the trypsin-EDTA solution is 0.2-0.3 mg/ml, and the concentration of EDTA is 0.02-0.1 mg/ml.

8. The method for preparing the anti-aging and anti-wrinkle cosmetic from the mesenchymal stem cells according to claim 5 or 6, wherein the culture conditions are 37 ℃ and 4-6% CO₂And (4) saturated humidity.

9. The method for preparing the anti-aging and anti-wrinkle cosmetic from the mesenchymal stem cells according to claim 1, wherein the mixed solution comprises the following raw materials in parts by weight: 30-40 parts of mesenchymal stem cell fluid, 1-3 parts of hexapeptide, 0.5-1 part of oligopeptide-1, 0.5-1 part of oligopeptide-3, 3-5 parts of collagen, 3-5 parts of hyaluronic acid, 1-3 parts of green algae extract, 0.5-2 parts of pipewort extract, 2-5 parts of curdlan and 35-50 parts of water.

10. The method for preparing the anti-aging and anti-wrinkle cosmetic from the mesenchymal stem cells according to claim 1, wherein the lyophilization process specifically comprises: the mixed solution is pre-frozen for 10 to 12 hours at the temperature of between 50 ℃ below zero and 40 ℃ below zero, is pre-frozen for 2 to 4 hours at the temperature of between 60 ℃ below zero and 50 ℃ below zero, and is finally vacuum-frozen for 20 to 40 hours at the temperature of between 0.2Pa and 0.6Pa and between 48 ℃ below zero and 42 ℃ below zero.

Technical Field

The invention relates to the technical field of stem cells, in particular to a method for preparing an anti-aging and wrinkle-removing cosmetic by using mesenchymal stem cells.

Background

Mesenchymal stem cells are derived from mesoderm tissues, belong to multipotent stem cells, are characterized by wide sources, low immunogenicity, certain differentiation potential, self-renewal capacity and immunity regulation capacity, and are mainly divided into human umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, placenta mesenchymal stem cells and the like according to the sources.

The mesenchymal stem cell is a cell with multidirectional differentiation potential, and can be directionally induced into a plurality of cells such as islet-like cells or chondrocytes. The mesenchymal stem cells have the advantages of wide source, easy collection, no ethical problem and the like, can be greatly expanded in vitro, and can be induced and differentiated into the islet-like cells in vivo in a large scale. In addition, mesenchymal stem cells are also capable of supporting hematopoiesis and suppressing immune responses. Based on the advantages, the method becomes an important seed cell in the aspects of tissue engineering, cell therapy, stem cell technology and the like, and has wide application prospect.

In the prior art, mesenchymal stem cells can be used in the application research of the alternative treatment of various diseases such as blood system diseases, cardiovascular diseases (such as myocardial infarction), cirrhosis, cartilage and bone injury repair and the like. Meanwhile, the mesenchymal stem cells have important application prospect in the aspect of repairing central nervous system diseases such as spinal cord injury and treating intractable diseases such as neurodegenerative diseases such as brain atrophy, Parkinson's disease, Alzheimer's syndrome and the like. The mesenchymal stem cells can also be used as a gene vector, applied to the treatment of genetic diseases caused by gene defects and used for solving the problems of certain autoimmune diseases. In addition, mesenchymal stem cells are combined with biomaterials to repair defects in various tissues such as bone, cartilage, and tendon, which is a new field in tissue engineering, and studies on the use of mesenchymal stem cells in the fields of cosmetics and cosmetics are rare.

Disclosure of Invention

The invention aims to provide a method for preparing an anti-aging and anti-wrinkle cosmetic by using mesenchymal stem cells, and the method can be used for preparing the cosmetic with good anti-aging and anti-wrinkle effects.

The technical problem to be solved by the invention is realized by adopting the following technical scheme.

The embodiment of the application provides a method for preparing an anti-aging and wrinkle-removing cosmetic by using mesenchymal stem cells, which comprises the following steps:

obtaining mesenchymal stem cell primary cells;

carrying out subculture on the primary mesenchymal stem cell to obtain a mesenchymal stem cell;

collecting the supernatant of the 3 rd generation and later mesenchymal stem cells, adding the hexapeptide, oligopeptide-1, oligopeptide-3, collagen, hyaluronic acid, green algae extract, Polysiphonia lanuginosa extract, curdlan and water to obtain a mixed solution, and freeze-drying the mixed solution to obtain the anti-aging and wrinkle-removing cosmetic.

Compared with the prior art, the embodiment of the invention has at least the following advantages or beneficial effects:

the method comprises the steps of separating bone marrow, umbilical cord, placenta or fat cells of human or animals to obtain primary mesenchymal stem cell, subculturing to obtain mesenchymal stem cell, adding hexapeptide, oligopeptide-1, oligopeptide-3, collagen, hyaluronic acid, green algae extract, multitubular ophyta lanuginosa extract, curdlan and water into the mesenchymal stem cell after the 3 rd generation, and preparing freeze-dried powder for skin anti-aging and anti-wrinkle through a freeze-drying process, so that the mesenchymal stem cell is applied to the field of cosmetics, and has innovative significance. The polypeptide-3 can stimulate the activity of mesenchymal stem cells, the oligopeptide-1 can promote the growth of cell molecules and increase the metabolic capacity of skin, hyaluronic acid and collagen have the moisturizing capacity, green alga polysaccharide in a green alga extract can promote the growth of collagen and react with water molecules to generate hydrogen bonds to improve the water content of the skin, and the polypeptide-1 can be matched with a Polysiphonia lansium extract to form a protective film on the surface of the skin to further lock water and be matched with curdlan to promote the water absorption performance of cells, so that the long-acting moisturizing effect is achieved on the skin, and the effect of delaying wrinkles is further achieved.

The cosmetics prepared by adopting the mesenchymal stem cells have a good inhibition effect on the condition that the skin is loose and has wrinkles, and particularly, the placenta-derived mesenchymal stem cells have a good effect on improving the water content of the skin, smoothing the skin and inhibiting the wrinkles.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.

A method for preparing an anti-aging and wrinkle-removing cosmetic by mesenchymal stem cells comprises the following steps:

obtaining mesenchymal stem cell primary cells;

carrying out subculture on the primary mesenchymal stem cell to obtain a mesenchymal stem cell;

collecting the supernatant of the 3 rd generation and later mesenchymal stem cells, adding the hexapeptide, oligopeptide-1, oligopeptide-3, collagen, hyaluronic acid, green algae extract, Polysiphonia lanuginosa extract, curdlan and water to obtain a mixed solution, and freeze-drying the mixed solution to obtain the anti-aging and wrinkle-removing cosmetic.

In some embodiments of the present invention, the source of the above-mentioned mesenchymal stem cell primary cell comprises one of bone marrow, umbilical cord, placenta, fat and tooth of human or animal.

In some embodiments of the invention, the source of the primary mesenchymal stem cell is placenta. Compared with mesenchymal stem cells from other sources, the mesenchymal stem cells from placenta have better effects on skin aging resistance and wrinkle resistance.

In some embodiments of the present invention, the step of obtaining the mesenchymal stem cell primary cell specifically comprises: washing and centrifuging sample cells for the first time, and then, mixing the sample cells according to a volume ratio of 1: (1-1.5) adding a cell separation solution, performing gradient centrifugation to obtain a mononuclear cell layer, performing centrifugation again, performing inoculation culture on the precipitate, changing the solution every 48-72 hours, and removing non-adherent cells to obtain the mesenchymal stem cells.

In some embodiments of the present invention, the cleaning specifically includes resuspending the sample cells in a complete medium, repeatedly blowing and beating the sample cells with a pipette to obtain a single cell suspension, wherein the rotation speed of the first centrifugation is 400-600 rpm, the centrifugation time is 20-40 min, the cell separation solution is a Percoll cell separation solution with a specific gravity of 1.07-1.09 g/ml, and the gradient centrifugation specifically includes centrifugation at 1000-1100 rpm for 10-15 min, centrifugation at 1150-1200 rpm for 10-15 min, and centrifugation at 1500-1600 rpm for 15-20 min.

In some embodiments of the present invention, the subculture is specifically: when the fusion degree of the cells reaches 80-90%, adding a trypsin-EDTA solution for digestion, stopping the digestion by adopting fetal calf serum, centrifuging, discarding the supernatant, re-suspending with a complete culture medium, and performing 1: (3-5) subculturing.

In some embodiments of the present invention, the concentration of trypsin in the trypsin-EDTA solution is 0.2-0.3 mg/ml, and the concentration of EDTA is 0.02-0.1 mg/ml.

In some embodiments of the present invention, the culturing is performed at 37 ℃ and 4-6% CO₂And (4) saturated humidity.

In some embodiments of the present invention, the weight parts of the raw materials in the mixed solution are: 30-40 parts of mesenchymal stem cell fluid, 1-3 parts of hexapeptide, 0.5-1 part of oligopeptide-1, 0.5-1 part of oligopeptide-3, 3-5 parts of collagen, 3-5 parts of hyaluronic acid, 1-3 parts of green algae extract, 0.5-2 parts of pipewort extract, 2-5 parts of curdlan and 35-50 parts of water.

The mesenchymal stem cells have paracrine effect, have the characteristics of fast growth and strong activity, and can secrete a large amount of anti-apoptosis factors and cytokines in the metabolic process, including: EGF (epidermal growth factor), HGF (hepatocyte growth factor), IGF-1 (insulin growth factor No. 1), PDFG (platelet derived growth factor), TGF-beta (transforming growth factor), EGF (vascular endothelial growth factor) and bFGF (fibroblast growth factor), wherein the anti-apoptosis factors and cytokines can promote endogenous repair, once the mesenchymal stem cells are activated and work in vivo, the number of the cytokines in the internal environment of the body is increased, thus being beneficial to the damage repair and regeneration of other cells and leading the body to regain vitality.

The hexapeptide can reduce the force of muscle contraction, relax muscles, reduce the occurrence of dynamic lines, eliminate fine lines, reorganize collagen elasticity, increase the activity of elastin, relax facial lines, smooth wrinkles and improve relaxation. Oligopeptide-1 can promote cell growth, improve cell activity, and repair damaged skin. Repairing collagen fibers and elastic fibers. Oligopeptide-3 can induce stem cell differentiation, promote tissue repair, and repair basal layer fibroblasts. Hyaluronic acid has strong moisturizing capability, can not only lock moisture in skin, but also catch water molecules in external environment, can continuously keep the skin moist, enables the skin to be tender and compact, and prevents the skin from being loose and wrinkles. The collagen is extracted from animal skin, which contains proteoglycan such as hyaluronic acid and chondroitin sulfate besides collagen, and the proteoglycan contains a large amount of polar groups, is a moisturizing factor and has the function of preventing tyrosine in the skin from being converted into melanin, so the collagen has the functions of purely natural moisturizing, whitening, crease resistance, freckle removal and the like. In the invention, the hexapeptide is used for preventing wrinkles, the oligopeptide-3 stimulates the activity of mesenchymal stem cells, the oligopeptide-1 promotes the growth of cell molecules, the metabolic capacity of skin is increased, and hyaluronic acid and collagen play a role in moisturizing.

The green alga polysaccharide in the green alga extract can promote the growth of collagen, reacts with water molecules to generate hydrogen bonds, improves the water content of the skin, can be matched with the Polysiphonia lanuginosa extract to form a protective film on the surface of the skin to further lock water, can be matched with the curdlan to promote the water absorption performance of cells, has a long-acting moisturizing effect on the skin, and further achieves the effect of delaying wrinkles.

In some embodiments of the present invention, the above-mentioned lyophilization process specifically comprises: the mixed solution is pre-frozen for 10 to 12 hours at the temperature of between 50 ℃ below zero and 40 ℃ below zero, is pre-frozen for 2 to 4 hours at the temperature of between 60 ℃ below zero and 50 ℃ below zero, and is finally vacuum-frozen for 20 to 40 hours at the temperature of between 0.2Pa and 0.6Pa and between 48 ℃ below zero and 42 ℃ below zero.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

A method for preparing an anti-aging and wrinkle-removing cosmetic from mesenchymal stem cells comprises the following steps:

washing the extracted bone marrow cells with sterile water, then suspending the bone marrow cells in a complete culture medium, and repeatedly blowing and beating the bone marrow cells with a straw to obtain a single cell suspension; the single cell suspension was first centrifuged at 500rpm for 20min at room temperature, the fat layer was discarded, and then the mixture was centrifuged at a volume ratio of 1: 1 adding Percoll cell separation liquid with specific gravity of 1.075g/ml, centrifuging at 1000rpm, 10min, 1200rpm, 10min, 1500rpm, 15min, discarding the upper mixed solution, collecting the nebulous mononuclear cell layer at the liquid interface, centrifuging at 500rpm for 5min, removing the supernatant, washing the precipitate with sterile water, and concentrating to obtain the final product with density of 1 × 10⁷Individual cell/cm²Inoculating into cell culture bottle, wherein the culture medium is DMEM/F12 containing 6.5% fetal calf serum, and the culture conditions are 37 deg.C and 5% CO₂Changing the solution every 72h at saturated humidity, and removing non-adherent cells to finally obtain the mesenchymal stem cells;

when the fusion degree of the cells reaches 80-90%, digesting by using a trypsin-EDTA solution with the concentration of 0.25mg/ml and the concentration of EDTA being 0.04mg/ml, stopping digestion by adding fetal calf serum when cell protrusions are found to shrink and deform into a circle by a microscope and mostly float in a cell culture bottle, then centrifuging at 1200rpm for 8min, discarding the supernatant, re-suspending with a complete culture medium, and performing the steps of 1: 5, subculturing;

collecting the supernatant of the mesenchymal stem cells of the cells with the passage number of 3, and filtering and sterilizing to obtain mesenchymal stem cell liquid; adding 1 weight part of hexapeptide, 0.5 weight part of oligopeptide-1, 1 weight part of oligopeptide-3, 3 weight parts of collagen, 4 weight parts of hyaluronic acid, 3 weight parts of green algae extract, 1 weight part of Polysiphonia lanuginosa extract, 2 weight parts of coagulated polysaccharide and 35 weight parts of water into 30 weight parts of mesenchymal stem cell liquid to obtain a mixed solution, pre-freezing the mixed solution at-48 ℃ for 12 hours, then freezing at-60 ℃ for 2 hours, and finally freezing at 0.2Pa and-48 ℃ in vacuum for 28 hours to obtain the anti-aging cosmetic wrinkle prepared from the mesenchymal stem cell source.

Example 2

A method for preparing an anti-aging and wrinkle-removing cosmetic from umbilical cord mesenchymal stem cells comprises the following steps:

cutting the umbilical cord tissue into 3-5 mm³The fragments are washed by sterile water and then are resuspended in a complete culture medium, and a single cell suspension is prepared by repeatedly blowing and beating the fragments by a pipette; the single cell suspension was first centrifuged at 600rpm for 30min at room temperature, the fat layer was discarded, and then the mixture was mixed in a volume ratio of 1: 1.5 adding Percoll cell separation liquid with specific gravity of 1.08g/ml, centrifuging at 1100rpm, 10min, 1150rpm, 15min, 1600rpm, 15min, discarding the upper mixed solution, collecting the nebulous mononuclear cell layer at the liquid interface, centrifuging at 600rpm for 3min, removing the supernatant, washing the precipitate with sterile water, and concentrating at a density of 1 × 10⁷Individual cell/cm²Inoculating into cell culture bottle, wherein the culture medium is DMEM/F12 containing 6% fetal calf serum, and the culture conditions are 37 deg.C and 6% CO₂Changing the solution every 48h at saturated humidity, and removing non-adherent cells to finally obtain umbilical cord mesenchymal stem cells;

when the fusion degree of the cells reaches 80-90%, digesting by using a trypsin-EDTA solution with the concentration of 0.2mg/ml and the concentration of EDTA being 0.08mg/ml, adding fetal calf serum to stop digestion when cell protrusions are found to shrink and deform into a circle by a microscope and mostly float in a cell culture bottle, then centrifuging at 1100rpm for 10min, discarding supernatant, re-suspending with complete culture medium, and performing the following steps of 1: 3, subculturing;

collecting supernatant of the umbilical cord mesenchymal stem cells with the passage number of 3, and filtering and sterilizing to obtain bone marrow mesenchymal stem cell liquid; adding 2 parts by weight of hexapeptide, 1 part by weight of oligopeptide-1, 0.5 part by weight of oligopeptide-3, 5 parts by weight of collagen, 3 parts by weight of hyaluronic acid, 2 parts by weight of green algae extract, 0.5 part by weight of Polysiphonia lanuginosa extract, 5 parts by weight of curdlan and 50 parts by weight of water into 40 parts by weight of mesenchymal stem cell liquid to obtain a mixed solution, pre-freezing the mixed solution at-45 ℃ for 10h, then freezing at-55 ℃ for 3h, and finally freezing under vacuum at 0.5Pa and-45 ℃ for 24h to obtain the anti-aging and wrinkle-wrinkle cosmetic prepared from the umbilical cord mesenchymal stem cell source.

Example 3

A method for preparing an anti-aging and wrinkle-removing cosmetic from placenta mesenchymal stem cells comprises the following steps:

cutting the placenta tissue into 3-5 mm³The fragments are washed by sterile water and then are resuspended in a complete culture medium, and a single cell suspension is prepared by repeatedly blowing and beating the fragments by a pipette; the single cell suspension was first centrifuged at 400rpm for 40min at room temperature, the fat layer was discarded, and then the cell suspension was centrifuged at a volume ratio of 1: 1 adding Percoll cell separation liquid with specific gravity of 1.083g/ml, centrifuging at 1100rpm, 15min, 1200rpm, 12min, 1600rpm, and 20min in gradient manner, discarding the upper mixed solution, collecting the nebulous mononuclear cell layer at the liquid interface, centrifuging at 600rpm for 3min, removing the supernatant, washing the precipitate with sterile water, and concentrating to obtain the final product with density of 1 × 10⁶Individual cell/cm²Inoculating into cell culture bottle, wherein the culture medium is DMEM/F12 containing 6% fetal calf serum, and the culture conditions are 37 deg.C and 6% CO₂Changing the solution once every 60h at saturated humidity, and removing non-adherent cells to finally obtain the placenta mesenchymal stem cells;

when the fusion degree of the cells reaches 80-90%, digesting by using a trypsin-EDTA solution with the concentration of 0.26mg/ml and the concentration of EDTA being 0.1mg/ml, adding fetal calf serum to stop digestion when the cell protrusions are found to shrink and deform into a circle by a microscope and mostly float in a cell culture bottle, then centrifuging at 1200rpm for 8min, discarding the supernatant, re-suspending with a complete culture medium, and performing the following steps of 1: 4, subculturing;

collecting supernatant of the cell placenta mesenchymal stem cells with the passage number of 3, and filtering and sterilizing to obtain bone marrow mesenchymal stem cell liquid; adding 1 weight part of hexapeptide, 2 weight parts of oligopeptide-1, 0.8 weight part of oligopeptide-3, 4 weight parts of collagen, 4 weight parts of hyaluronic acid, 3 weight parts of green algae extract, 2 weight parts of Polysiphonia lanigera extract, 4 weight parts of curdlan and 40 weight parts of water into 34 weight parts of mesenchymal stem cell liquid to obtain a mixed solution, pre-freezing the mixed solution at-48 ℃ for 10h, then freezing at-50 ℃ for 3h, and finally freezing at 0.6Pa and-40 ℃ in vacuum for 20h to obtain the anti-aging cosmetic wrinkle prepared from the source of the mesenchymal stem cells of placenta.

Example 4

A method for preparing an anti-aging and anti-wrinkle cosmetic from adipose mesenchymal stem cells comprises the following steps:

washing adipose tissues with sterile water, resuspending in a complete culture medium, and repeatedly blowing and beating with a straw to obtain a single cell suspension; the single cell suspension was first centrifuged at 450rpm for 20min at room temperature, the fat layer was discarded, and then the mixture was mixed in a volume ratio of 1: 1 adding Percoll cell separation liquid with specific gravity of 1.09g/ml, centrifuging at 1000rpm, 12min, 1200rpm, 15min, 1600rpm, 15min, discarding the upper mixed solution, collecting the nebulous mononuclear cell layer at the liquid interface, centrifuging at 600rpm for 5min, removing the supernatant, washing the precipitate with sterile water, and concentrating to obtain the final product with density of 1 × 10⁵Individual cell/cm²Inoculating into cell culture bottle, wherein the culture medium is DMEM/F12 containing 6% fetal calf serum, and the culture conditions are 37 deg.C and 4% CO₂Changing the solution every 72h at saturated humidity, and removing non-adherent cells to finally obtain the adipose-derived mesenchymal stem cells;

when the fusion degree of the cells reaches 80-90%, digesting by using a trypsin-EDTA solution with the concentration of 0.28mg/ml and the concentration of EDTA being 0.07mg/ml, when the cell protrusions are found to shrink and deform to be round by a microscope and mostly float in a cell culture bottle, adding fetal calf serum to stop digestion, then centrifuging at 1200rpm for 8min, discarding the supernatant, re-suspending with a complete culture medium, and performing the following steps of 1: 3, subculturing;

collecting supernatant of adipose-derived mesenchymal stem cells with the passage number of 3, and filtering and sterilizing to obtain bone marrow mesenchymal stem cell liquid; adding 2.5 parts by weight of hexapeptide, 0.6 part by weight of oligopeptide-1, 0.9 part by weight of oligopeptide-3, 4 parts by weight of collagen, 4 parts by weight of hyaluronic acid, 2.5 parts by weight of green algae extract, 1 part by weight of multicladium lanosum extract, 3 parts by weight of curdlan and 38 parts by weight of water into 36 parts by weight of mesenchymal stem cell fluid to obtain a mixed solution, pre-freezing the mixed solution at-40 ℃ for 10 hours, then freezing at-60 ℃ for 2 hours, and finally freezing at 0.5Pa and-40 ℃ in vacuum for 30 hours to obtain the anti-aging cosmetic wrinkle prepared from the adipose mesenchymal stem cell source.

Example 5

A method for preparing an anti-aging and wrinkle-removing cosmetic from placenta mesenchymal stem cells comprises the following steps:

cutting the placenta tissue into pieces of 3-5 mm3, then washing with sterile water, re-suspending in a complete culture medium, and repeatedly blowing and beating with a straw to obtain a single cell suspension; the single cell suspension was first centrifuged at 550rpm for 20min at room temperature, the fat layer was discarded, and then the mixture was mixed in a volume ratio of 1: 1.3 adding Percoll cell separation liquid with specific gravity of 1.075g/ml, centrifuging at 1000rpm, 11min, 1150rpm, 15min, 1550rpm and 20min, discarding the upper mixed solution, collecting the nebulous mononuclear cell layer at the liquid interface, centrifuging at 500rpm for 4min, removing the supernatant, washing the precipitate with sterile water, and concentrating to obtain the final product with density of 1 × 10⁶Individual cell/cm²Inoculating into cell culture bottle, wherein the culture medium is DMEM/F12 containing 6% fetal calf serum, and the culture conditions are 37 deg.C and 5% CO₂Changing the solution once every 60h at saturated humidity, and removing non-adherent cells to finally obtain the placenta mesenchymal stem cells;

when the fusion degree of the cells reaches 80-90%, digesting by using a trypsin-EDTA solution with the concentration of 0.25mg/ml and the concentration of EDTA being 0.07mg/ml, when the cell protrusions are found to shrink and deform to be round by a microscope and mostly float in a cell culture bottle, adding fetal calf serum to stop digestion, then centrifuging at 1100rpm for 8min, discarding the supernatant, re-suspending with a complete culture medium, and performing the following steps of 1: 3, subculturing;

collecting supernatant of the cell placenta mesenchymal stem cells with the passage number of 3, and filtering and sterilizing to obtain bone marrow mesenchymal stem cell liquid; adding 1 weight part of hexapeptide, 0.7 weight part of oligopeptide-1, 0.5 weight part of oligopeptide-3, 3 weight parts of collagen, 4 weight parts of hyaluronic acid, 2.5 weight parts of green algae extract, 1.5 weight parts of Polysiphonia lanuginosa extract, 5 weight parts of coagulated polysaccharide and 40 weight parts of water into 32 weight parts of mesenchymal stem cell fluid to obtain a mixed solution, pre-freezing the mixed solution at-50 ℃ for 12 hours, then freezing at-60 ℃ for 3 hours, and finally freezing in vacuum at 0.6Pa and-40 ℃ for 25 hours to obtain the anti-aging cosmetic wrinkle prepared from the mesenchymal stem cell source of placenta.

Comparative example 1

The difference between the comparative example and the example 1 is that in the comparative example, the mesenchymal stem cell fluid is directly freeze-dried into freeze-dried powder to be used as a cosmetic without adding the hexapeptide, the oligopeptide-1, the oligopeptide-3, the collagen, the hyaluronic acid, the green algae extract, the multitubular peronospora lanuginosa extract, the curdlan and water.

Comparative example 2

The comparative example is different from example 1 in that hexapeptide, oligopeptide-1, oligopeptide-3, collagen, hyaluronic acid, green algae extract, Polysiphonia lanuginosa extract, curdlan and water are mixed and then lyophilized into lyophilized powder for use as a cosmetic.

Examples of the experiments

160 women with relaxed and wrinkled skin of 35-67 years old are invited to serve as experimenters and are divided into 68 young groups (age 35-50 years old) and 92 old groups (age 51-67 years old) and are randomly divided into 7 groups, the cosmetics prepared in the embodiments 1-5 and the comparative examples 1-2 are respectively used, the using time is 3 months, the water content of the skin of the women is measured before and after the cosmetics are used, the growth rate is counted, visia photographing record is adopted, the effect is improved by more than 50% through visia analysis and is significant, the effect is improved by 20-50% and is ineffective, and the effect is improved by less than 20%. The results of the young and old groups are shown in tables 1 and 2, respectively.

TABLE 1

TABLE 2

As can be seen from tables 1 and 2, compared with experimental group 6 and experimental group 7, the experimenter who used the cosmetics prepared in embodiments 1 to 5 of the present invention had an obvious effect on skin improvement, which indicates that the mesenchymal stem cells had better effects than the hexapeptide, oligopeptide-1, oligopeptide-3, collagen and hyaluronic acid commonly used in the existing cosmetics. Particularly, the effect of the placenta mesenchymal stem cells in the experimental group 3 and the experimental group 5 is more prominent for the aged patients with serious skin problems, which shows that the placenta mesenchymal stem cells have the best effect on resisting aging and wrinkles.

In summary, the embodiment of the invention provides a method for preparing an anti-aging and wrinkle-removing cosmetic by using mesenchymal stem cells. The method comprises the steps of separating bone marrow, umbilical cord, placenta or fat cells of human or animals to obtain primary mesenchymal stem cell, subculturing to obtain mesenchymal stem cell, adding melatonin, green algae extract, multitubular ophyta lanuginosa extract, human serum albumin, curdlan and water into the 3 rd generation of mesenchymal stem cell, and freeze-drying to obtain the freeze-dried powder for resisting skin aging and wrinkles, so that the mesenchymal stem cell is applied to the field of cosmetics, and has innovative significance.

The cosmetics prepared by adopting the mesenchymal stem cells have a good inhibition effect on the condition that the skin is loose and has wrinkles, and particularly, the placenta-derived mesenchymal stem cells have a good effect on improving the water content of the skin, smoothing the skin and inhibiting the wrinkles.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.