Adipose-derived mesenchymal stem cell serum and preparation method therefor

Abstract

An adipose-derived mesenchymal stem cell serum and a preparation method therefor. The adipose-derived mesenchymal stem cell serum, in percentage ratios, comprises the following raw materials: 2-5% of an adipose-derived mesenchymal stem cell concentrate, 0.5-1% sodium pyrrolidone carboxylate, 2-5% glycerin, 1-3% propylene glycol, 0.1-0.5% glutathione, 0.1-0.5% allantoin, 0.2-1% ascorbic acid, and 1-5% sodium hyaluronate. The adipose-derived mesenchymal stem cell concentrate is made, via ultraconcentration, using a culturing solution from adipose-derived stem cells and a protein suspension obtained from lysed stem cells. The present invention is rich in cell growth factors, and once prepared into a serum product, the invention has cosmetic effects such as wrinkle reduction, spot reduction, and skin repair.

Description

Adipose-derived mesenchymal stem cell essence and preparation method thereof Technical Field

[0001] The invention relates to an adipose mesenchymal stem cell essence and a preparation method thereof, and belongs to the field of biological beauty. Background Art

[0002] Currently, cosmetics primarily consist of chemical ingredients, often supplemented with plant extracts or other deep-sea nutrients, to create beauty products that moisturize, whiten, and control oil. While these cosmetics contain nutrients, their effectiveness and availability are limited, making them far from effective in skin regeneration and repair.

[0003] Stem cells are a type of multipotent cell with the ability to self-replicate. Hailed as the "fountain of immortality," they hold tremendous promise for application in regenerative medicine. Stem cells can be categorized as embryonic stem cells and adult stem cells. Due to ethical considerations, adult stem cells currently hold the most promise for clinical application. As stem cells grow, they secrete a large number of cytokines, including various growth factors such as insulin, vascular endothelial growth factor, transforming growth factor, skin growth factor, fibroblast growth factor, and related collagen substances. These factors are involved in the mechanisms of various tissues and reactions.

[0004] The cell growth factors expressed and secreted by stem cells have excellent repair, regeneration, and whitening effects, and their combination with cosmetic physical therapy can produce even better results. The culture medium of adipose-derived stem cells and the protein suspension obtained by lysing adipose-derived stem cells are then super-concentrated to create a stem cell essence rich in cell growth factors with regenerative and repairing properties. These cell growth factors primarily include epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and stem cell factor (SCF). The cell growth factors secreted by stem cells can specifically improve skin microcirculation, promote the synthesis and secretion of extracellular macromolecules such as hyaluronic acid and glycoproteins, enhance the hydrophilicity of the skin, and maintain moisture in the skin; by promoting the growth and differentiation of various cells in the skin, a large number of new cells are produced to replace pigmented cells; promote the proliferation of fibroblasts in the dermis, repair aging collagen fibers and elastic fibers; improve skin microcirculation, provide a good nutritional environment, maintain a certain amount of skin fat, restore skin elasticity, make it even and firm, and reduce wrinkles; promote cell metabolism, promote cell proliferation and differentiation.

[0005] Adipose-derived mesenchymal stem cells have significant advantages as seed cells for stem cell anti-aging products. They do not involve ethical issues, are easy to isolate and culture, and have enormous in vitro expansion potential, meeting the necessary conditions for being excellent seed cells. When continuously cultured in serum-free medium, adipose-derived stem cells can naturally express and secrete a large number of cell growth factors. Adipose-derived mesenchymal stem cell factors have significant anti-aging effects on the skin, and have better cosmetic application prospects in inhibiting inflammation, reducing scar formation, and repairing acne scars. They can be used to prepare cosmetic products such as stem cell essences to combat skin aging and promote wound healing. Secondly, in this preparation method, adipose-derived mesenchymal stem cells have a high in vitro expansion efficiency, which can provide a sufficient amount of cell raw material for the preparation of adipose-derived mesenchymal stem cell essences. Better storage and timely use within the biological validity period will produce the best regenerative and repair effects on skin wrinkles, spots, and scars. Technical issues

[0006] The purpose of the present invention is to provide a culture medium derived from adipose-derived stem cells and a protein suspension obtained by lysing stem cells, which are ultra-concentrated and mixed with other components to produce an adipose-derived mesenchymal stem cell essence, which has an optimal regenerative and repairing effect on skin wrinkles, spots, scars, etc. Technical Solutions

[0007] In order to achieve the above objectives, the first object of the present invention is to provide an adipose-derived mesenchymal stem cell essence.

[0008] A fat-derived mesenchymal stem cell essence comprises the following raw materials in percentage: 2-5% fat-derived mesenchymal stem cell concentrate, 0.5-1% sodium pyrrolidonecarboxylate, 2-5% glycerol, 1-3% propylene glycol, 0.1-0.5% glutathione, 0.1-0.5% allantoin, 0.2-1% ascorbic acid, and 1-5% sodium hyaluronate.

[0009] Preferably, the percentage of the adipose-derived mesenchymal stem cell concentrate is 3-4%.

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned adipose-derived mesenchymal stem cell essence, and the technical solution is as follows:

[0011] The preparation is prepared by thoroughly mixing the following raw materials: 2-5% adipose-derived mesenchymal stem cell concentrate, 0.5-1% sodium pyrrolidonecarboxylate, 2-5% glycerol, 1-3% propylene glycol, 0.1-0.5% glutathione, 0.1-0.5% allantoin, 0.2-1% ascorbic acid, and 1-5% sodium hyaluronate.

[0012] Furthermore, the method for preparing the adipose-derived mesenchymal stem cell concentrate comprises the following steps:

[0013] (1) Isolation, culture, and identification of adipose-derived mesenchymal stem cells: The fat was washed thoroughly with physiological saline, digested with trypsin, and then cultured in a sterile culture flask. After the culture, flow cytometry was used for identification.

[0014] (2) Rapid expansion of adipose-derived mesenchymal stem cells: The 4th generation umbilical cord mesenchymal stem cells with good growth status were cultured, digested, centrifuged, washed, and the cells were collected;

[0015] (3) Lysis and centrifugation of adipose-derived mesenchymal stem cells: 1.5×10 9 Human mesenchymal stem cells were gradually added with 1× PBS and protease inhibitors, placed on ice, and disrupted in an ultrasonic disruptor for 7 minutes. The supernatant was centrifuged at 15,000 rpm for 20 minutes, and added to the culture medium of human mesenchymal stem cells.

[0016] (4) The culture medium containing the human mesenchymal stem cell lysate obtained in step (3) is ultrafiltered and concentrated using a 500 molecular weight cellulose acetate membrane to obtain the adipose mesenchymal stem cell concentrate.

[0017] Furthermore, in the preparation method of the adipose-derived mesenchymal stem cell concentrate, the sterile culture conditions in step (1) are 37° C., 5% CO 2 , and a saturated humidity sterile incubator, and the culture time is 1 to 2 hours.

[0018] Furthermore, in the method for preparing the adipose-derived mesenchymal stem cell concentrate, in step (1), the culture is stopped when the cell clones grow to 80% to 90% fusion.

[0019] Furthermore, in the method for preparing the adipose-derived mesenchymal stem cell concentrate, the method for rapidly expanding adipose-derived mesenchymal stem cells in step (2) is to take the 4th generation umbilical cord mesenchymal stem cells with good growth status and add 6×10 4 / cm 2 The density of inoculation was 75cm 2 The cells were placed in a sterile culture bottle with 15 mL of α-MEM medium containing 10% fetal bovine serum and cultured in a 37°C, 5% CO2, saturated humidity incubator for 5 days.

[0020] Furthermore, in the method for preparing the adipose-derived mesenchymal stem cell concentrate, the amount of 1×PBS added in step (3) is 10 ml, and the amount of protease inhibitor added is 0.8 ug.

[0021] Preferably, in the method for preparing the adipose-derived mesenchymal stem cell concentrate, the volume is concentrated 5 times in step (4). Beneficial effects

[0022] The beneficial effects of the present invention are as follows: the culture medium containing stem cell lysate obtained by the method of the present invention is rich in cell growth factors after concentration, and the essence product prepared from the culture medium has cosmetic effects on skin such as wrinkle removal, freckle removal and repair. Best Mode for Carrying Out the Invention

[0023] A method for preparing adipose-derived mesenchymal stem cell essence, comprising:

[0024] (1) Isolation, culture and identification of adipose-derived mesenchymal stem cells

[0025] According to the conventional operation method in the field, the fat was fully washed with physiological saline in a sterile operating table, digested with trypsin, and placed in a 75cm 2Place the cells in a sterile culture bottle at 37°C, 5% CO2, and saturated humidity incubator. After about 1 to 2 hours, when the tissue block is slightly dry and can be firmly attached to the wall of the culture bottle, slowly add α-MEM culture medium containing 10% fetal bovine serum to allow the culture medium to soak the tissue block. Continue to culture, changing the medium every 3 to 4 days. When the cell clones grow to 80%-90% fusion, use trypsin substitute to digest and subculture them into new sterile culture bottles.

[0026] The flow cytometry identification method is as follows: the 4th generation umbilical cord mesenchymal stem cells with good growth status are taken, washed, filtered, fixed, labeled with fluorescent antibodies, washed again, loaded onto the flow cytometer, and the expression rates of surface markers CD19, HLA-DR, CD34, CD11b, CD45, CD90, CD105 and CD73 are detected.

[0027] The test results showed that CD73, CD90, and CD105 were all strongly expressed, with an expression rate of no less than 98%. CD19, CD11b, CD34, CD45, and HLA-DR were all weakly expressed, with an expression rate of less than 2%. The results of inverted microscopy and flow cytometry were consistent with the characteristics of human umbilical cord mesenchymal stem cells.

[0028] (2) Rapid expansion of adipose-derived mesenchymal stem cells

[0029] The 4th generation umbilical cord mesenchymal stem cells with good growth status were collected and 6×10 4 / cm 2 The density of inoculation was 75cm 2 The cells were cultured in a sterile culture flask with 15 mL of α-MEM medium containing 10% fetal bovine serum in a 37°C, 5% CO2, and saturated humidity incubator for 5 days, digested, centrifuged, washed, and collected.

[0030] (3) Lysis and centrifugation of adipose-derived mesenchymal stem cells

[0031] After the expanded adipose-derived mesenchymal stem cells were collected, 1.5×10 9 Human mesenchymal stem cells were added to 10 ml of 1×PBS0, mixed thoroughly, and 0.8 μg of protease inhibitor was added. The cells were placed on ice and disrupted in an ultrasonic disruptor for 7 minutes. The cells were centrifuged at 15,000 rpm for 20 minutes, and the supernatant was added to the culture medium containing human mesenchymal stem cells. The culture medium containing the human mesenchymal stem cell lysate was concentrated by ultrafiltration using a 500 molecular weight cellulose acetate membrane, with the volume concentrated 5-fold. This produced the adipose-derived mesenchymal stem cell concentrate.

[0032] (4) Preparation of adipose-derived mesenchymal stem cell essence

[0033] The components are obtained according to the following percentages: 4% adipose-derived mesenchymal stem cell concentrate; 0.7% sodium pyrrolidonecarboxylate; 3% glycerol; 2% propylene glycol; 0.5% glutathione; 0.4% allantoin; 0.8% ascorbic acid; and 4% sodium hyaluronate. After uniform mixing, an essence containing adipose-derived mesenchymal stem cells is obtained. Industrial Applicability

[0034] The adipose-derived mesenchymal stem cell concentrate of the present invention is made by ultra-concentrating a culture medium derived from adipose-derived stem cells and a protein suspension obtained by lysing stem cells. It is rich in cell growth factors and can be prepared into an essence product. It has cosmetic effects on the skin, such as wrinkle removal, freckle removal, and repair, and can be widely used in the field of biological beauty.

Claims

1. A fat mesenchymal stem cell essence, characterized in that, It contains raw materials with the following percentage contents: 2-5% of adipose mesenchymal stem cell concentrate, 0.5-1% of sodium pyrrolidone carboxylate, 2-5% of glycerol, 1-3% of propylene glycol, 0.1-0.5% of glutathione, 0.1-0.5% of allantoin, 0.2-1% of ascorbic acid, and 1-5% of sodium hyaluronate.

2. The adipose mesenchymal stem cell essence according to claim 1, characterized in that The percentage content of the adipose mesenchymal stem cell concentrate is preferably 3-4%.

3. The preparation method of a fat mesenchymal stem cell essence according to any one of claims 1 to 2, characterized in that It is obtained by uniformly mixing the above raw materials of each component.

4. The preparation method of a fat mesenchymal stem cell essence according to any one of claims 1 to 2, characterized in that, The preparation method of the adipose mesenchymal stem cell concentrate includes the following steps: (1) Isolation, culture and identification of adipose mesenchymal stem cells: Wash the adipose tissue thoroughly with physiological saline, digest it with trypsin, and then culture it in a sterile culture flask. After the culture is completed, identify it with a flow cytometer; (2) Rapid amplification of adipose mesenchymal stem cells: Take the 4th generation of umbilical cord mesenchymal stem cells in good growth state for culture. After completion, digest, centrifuge and wash, and collect the cells; (3) Lysis and centrifugation of adipose mesenchymal stem cells: Take the 1.5×10 9 human mesenchymal stem cells obtained in step (2), gradually add 1×PBS and protease inhibitor, place on ice, break in an ultrasonic crusher for 7 minutes, centrifuge at a high speed of 15,000 rpm for 20 minutes, take the supernatant and add it to the culture medium that has cultured human mesenchymal stem cells; (4) Ultrafilter and concentrate the culture solution containing human mesenchymal stem cell lysate obtained in step (3) using a cellulose acetate membrane with a molecular weight cut-off of 500 to obtain the adipose mesenchymal stem cell concentrate.

5. The preparation method of a fat mesenchymal stem cell essence according to claim 4, characterized in that, In the preparation method of the adipose mesenchymal stem cell concentrate, the sterile culture conditions in step (1) are a sterile incubator at 37°C, 5% CO2, and saturated humidity, and the culture time is 1-2 h.

6. The preparation method of a fat mesenchymal stem cell essence according to claim 4, characterized in that, In the preparation method of the adipose mesenchymal stem cell concentrate, stop culturing when the cell clones grow to 80%-90% confluence in step (1).

7. The preparation method of a fat mesenchymal stem cell essence according to claim 4, characterized in that, The preparation method of the adipose mesenchymal stem cell concentrate. In step (2), the rapid amplification method of adipose mesenchymal stem cells is to take the 4th generation umbilical cord mesenchymal stem cells in good growth state and inoculate them at a density of 6×10 4 / cm 2 into a 75 cm 2 sterile culture flask. The culture medium is 15 mL of α-MEM medium containing 10% fetal bovine serum, and it is cultured in an incubator at 37 °C, 5% CO2, and saturated humidity for 5 days.

8. The preparation method of a fat mesenchymal stem cell essence according to claim 4, characterized in that In the preparation method of the adipose mesenchymal stem cell concentrate, the addition amount of 1×PBS in step (3) is 10 ml, and the addition amount of the protease inhibitor is 0.8 μg.

9. The preparation method of a fat mesenchymal stem cell essence according to claim 4, characterized in that, In the preparation method of the adipose mesenchymal stem cell concentrate, the volume is concentrated 5 times in step (4).