Preparation method of umbilical cord mesenchymal stem cell supernatant coating

Abstract

The invention discloses a preparation method of an umbilical cord mesenchymal stem cell supernatant coating, which comprises the following steps: collecting umbilical cord mesenchymal stem cells cultured for 2-4 days and a culture solution, separating supernatant and cell precipitate, preserving 90% of the supernatant at low temperature, repeatedly freezing and thawing the remaining 10% of the cell supernatant and the cell precipitate to break the cells, and uniformly mixing with the supernatant to obtain the umbilical cord mesenchymal stem cell supernatant coating. After high-speed centrifugation, clear and transparent umbilical cord mesenchymal stem cell supernatant is obtained; weighing macromolecular sodium hyaluronate powder, dissolving with water, and sterilizing for later use; weighing nicotinamide powder, dissolving with water, filtering and sterilizing for later use; uniformly mixing a sodium hyaluronate solution, a nicotinamide solution and the umbilical cord mesenchymal stem cell supernatant, and fully dissolving to obtain the umbilical cord mesenchymal stem cell supernatant coating. The effects of the umbilical cord mesenchymal stem cells and the culture solution are fully exerted, waste of the culture solution is avoided, meanwhile, the cells and growth factors in culture are collected to the maximum extent, preparation is easy, and the continuous use effect is obvious.

Description

Technical Field

[0001] The invention relates to a method for preparing an umbilical cord mesenchymal stem cell supernatant coating. Background Art

[0002] With the continuous improvement of living standards, people pay more attention to skin and body care than ever before, and their pursuit of beauty is getting higher and higher. Umbilical cord mesenchymal stem cells are a type of matrix cells separated from the umbilical cord Wharton's jelly. They are rich in sources, have a high proliferation rate, have self-renewal and multidirectional differentiation potentials, and also have strong repair and regeneration capabilities. They are basically non-immunogenic, have high biosafety, and show strong therapeutic potential. They have gradually become a research hotspot in plastic surgery in recent years. Studies have found that umbilical cord mesenchymal stem cells can differentiate and regenerate new cells and new blood vessels when skin wounds occur, promote skin repair and reconstruction, and improve skin damage. The reason is that they can secrete factors such as epidermal growth factor (EGF), fibroblast growth factor (FGFs), platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), and cystine knot growth factor (VEGF), which can affect the proliferation, differentiation, and growth of new skin cells, especially basal cells, promote the formation of intercellular matrix, secrete collagen, enhance skin elasticity and gloss, and at the same time promote the proliferation and division of vascular endothelial cells, induce the formation of skin microvessels, thereby improving skin cell metabolism, delaying aging, and promoting tissue repair.

[0003] During the culture process of umbilical cord mesenchymal stem cells, a large amount of cytokines can be secreted, which exist in both cells and culture supernatant. During the culture process, the culture supernatant is generally disposed of and not used. In view of the above shortcomings, the present invention collects the umbilical cord mesenchymal stem cell culture fluid, and at the same time, the umbilical cord mesenchymal stem cells are lysed to release the growth factors in the cells, which are evenly mixed with the culture fluid, and then sodium hyaluronate is added to facilitate factor absorption, and niacinamide solution is added to prepare the umbilical cord mesenchymal stem cell supernatant coating, which is applied to the surface of the skin, so that the culture fluid and the growth factors in the cells can better penetrate into the skin base, improve the skin condition, promote collagen production, and enhance the elasticity and glossiness of the skin. Summary of the invention

[0004] The purpose of the invention is to provide a method for preparing an umbilical cord mesenchymal stem cell supernatant coating.

[0005] Specifically, a method for preparing an umbilical cord mesenchymal stem cell supernatant coating comprises the following steps:

[0006] (1) Obtaining the supernatant of umbilical cord mesenchymal stem cells: collecting umbilical cord mesenchymal stem cells and culture medium after culturing for 2-4 days, centrifuging, separating the supernatant and the cell pellet, and storing the supernatant at low temperature. 90% of the supernatant is stored at low temperature, and the remaining 10% of the cell supernatant and the cell pellet are repeatedly frozen and thawed to break the cells. The cell suspension after repeated freezing and thawing is evenly mixed with the supernatant, and after high-speed centrifugation, the excess cell fragments are removed to obtain a clear and transparent umbilical cord mesenchymal stem cell supernatant;

[0007] (2) Obtaining sodium hyaluronate solution: Weigh the macromolecular sodium hyaluronate powder, dissolve the powder with water for injection, and sterilize for later use;

[0008] (3) Obtaining nicotinamide solution: Weigh nicotinamide powder, dissolve the powder with water for injection, and filter and sterilize for later use;

[0009] (4) Preparation of umbilical cord mesenchymal stem cell coating: The sterilized sodium hyaluronate solution obtained in step (2), the sterilized nicotinamide solution obtained in step (3), and the umbilical cord mesenchymal stem cell supernatant obtained in step (1) are mixed evenly and fully dissolved to obtain an umbilical cord mesenchymal stem cell supernatant coating.

[0010] (5) Use of the umbilical cord mesenchymal stem cell coating: directly apply the supernatant coating obtained in step (4) to the face, or apply it wetly to the face using a medical dressing.

[0011] Preferably, in step (2), the volume ratio of the sodium hyaluronate solution to the supernatant of the umbilical cord mesenchymal stem cells is 1:16.

[0012] Preferably, in step (3), the volume ratio of the nicotinamide solution to the supernatant of umbilical cord mesenchymal stem cells is 1:100.

[0013] Preferably, in step (4), the prepared umbilical cord mesenchymal stem cell supernatant coating is filled into a container and stored at -20°C for a long time.

[0014] Preferably, during use in step (5), the coating can be massaged with the fingertips until it is absorbed, or wet-applied with a medical dressing for 10-15 minutes.

[0015] The method of the present invention is simple to operate, gives full play to the role of umbilical cord mesenchymal stem cells and culture fluid, avoids the waste of culture fluid, and collects cells and growth factors in culture to the maximum extent. The preparation method is simple, low-cost, and no expensive instruments and reagents are used. The finished coating component is safe and non-irritating, has high stability, and has simple storage conditions. After application, the skin condition can be significantly improved, the formation of skin collagen can be promoted, and the glossiness of recovery can be enhanced. Long-term application is beneficial to the wound repair of the skin surface and the lightening of fine lines and scars. The effect is particularly obvious when used continuously. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Photo of the finished product of coating the supernatant for umbilical cord mesenchymal stem cells.

[0017] Figure 2 This is a photo of the effect of using the umbilical cord mesenchymal stem cell supernatant coating.

[0018] Figure 3 This is a photo of the effect of using the umbilical cord mesenchymal stem cell supernatant coating.

[0019] Figure 4 This is a photo of the effect of using the umbilical cord mesenchymal stem cell supernatant coating. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with embodiments, but the description of the embodiments does not impose any limitation on the protection scope of the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0022] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0023] Example 1

[0024] The method for preparing the umbilical cord mesenchymal stem cell supernatant coating comprises the following steps:

[0025] 1. Obtaining the supernatant of umbilical cord mesenchymal stem cells: Umbilical cord mesenchymal stem cells were cultured in serum-free medium. After 2-4 days of culture, the supernatant and cells were collected by digestion method. After low-speed centrifugation, 90% of the supernatant was placed in a sterile container and stored at low temperature. The remaining 10% of the supernatant and the cell pellet were repeatedly frozen and thawed to rupture the cell membrane and release the growth factors in the cells. The cryopreserved supernatant was melted and mixed evenly with the repeatedly frozen and thawed cell suspension. After centrifugation at 1000g for 10 minutes, the excess cell debris was removed to obtain a clear and transparent umbilical cord mesenchymal stem cell supernatant.

[0026] 2. Obtaining sodium hyaluronate solution: Weigh the macromolecular sodium hyaluronate powder, dissolve the powder with water for injection, the content of sodium hyaluronate after dissolution is 5-10%, sterilize and set aside;

[0027] 3. Obtaining nicotinamide solution: Weigh nicotinamide powder and dissolve it with water for injection. The nicotinamide content after dissolution is 0.5-2%. Filter and sterilize for later use.

[0028] 4. Preparation of umbilical cord mesenchymal stem cell supernatant coating: the sterilized sodium hyaluronate solution obtained in step 2, the sterilized nicotinamide solution obtained in step 3, and the umbilical cord mesenchymal stem cell supernatant obtained in step 1 are mixed evenly, with the ratio of sodium hyaluronate: umbilical cord mesenchymal stem cell supernatant = 1:16, and the ratio of nicotinamide: umbilical cord mesenchymal stem cell supernatant = 1:50, and fully dissolved;

[0029] 5. Filling: Fill the prepared coating into the vial. Figure 1 Store at -20°C.

[0030] 6. Usage: After melting the paint, apply it directly to the face and massage until absorbed, or use medical dressing to wet compress for 10-15 minutes. The effect after use is as follows: Figure 2-4 As shown, the usage frequency is once a month. Figure 2-4 The date of the first picture is the first use date. It can be seen that it has firming, brightening, repairing and other effects after use.

[0031] The umbilical cord used in the present invention has a clear source, the donor has signed an informed consent form, and provided a corresponding physical examination report, thus avoiding the risk of genetic diseases and infectious diseases that may lead to failure of primary acquisition.

[0032] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a supernatant coating of umbilical cord mesenchymal stem cells, characterized in that The steps include: (1) Obtaining the supernatant of umbilical cord mesenchymal stem cells: collecting umbilical cord mesenchymal stem cells and culture medium after culturing for 2-4 days, centrifuging, separating the supernatant and the cell pellet, and storing the supernatant at low temperature. 90% of the supernatant is stored at low temperature, and the remaining 10% of the cell supernatant and the cell pellet are repeatedly frozen and thawed to break the cells. The cell suspension after repeated freezing and thawing is evenly mixed with the supernatant, and after high-speed centrifugation, the excess cell fragments are removed to obtain a clear and transparent umbilical cord mesenchymal stem cell supernatant; (2) Obtaining sodium hyaluronate solution: Weigh the macromolecular sodium hyaluronate powder, dissolve the powder with water for injection, and sterilize for later use; (3) Obtaining nicotinamide solution: Weigh nicotinamide powder, dissolve the powder with water for injection, and filter and sterilize for later use; (4) Preparation of umbilical cord mesenchymal stem cell coating: The sterilized sodium hyaluronate solution obtained in step (2), the sterilized nicotinamide solution obtained in step (3) and the umbilical cord mesenchymal stem cell supernatant obtained in step (1) are mixed evenly and fully dissolved to obtain an umbilical cord mesenchymal stem cell supernatant coating.

2. The preparation method according to claim 1, characterized in that In step (2), the volume ratio of the sodium hyaluronate solution to the supernatant of the umbilical cord mesenchymal stem cells is 1:10 to 1:

20.

3. The preparation method according to claim 1, characterized in that In step (3), the volume ratio of the nicotinamide solution to the supernatant of the umbilical cord mesenchymal stem cells is 1:50 to 1:

100.

4. The preparation method according to claim 1, characterized in that The method further comprises step (4): filling the prepared umbilical cord mesenchymal stem cell supernatant coating into a container and storing the container at -20°C.

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