WO2020173164A1

WO2020173164A1 - Lyophilized powder of mesenchymal stem cell and preparation method therefor

Info

Publication number: WO2020173164A1
Application number: PCT/CN2019/123715
Authority: WO
Inventors: 王淑玲; 王晓惠; 刘洋; 李志生
Original assignee: 京东方科技集团股份有限公司
Priority date: 2019-02-28
Filing date: 2019-12-06
Publication date: 2020-09-03
Also published as: CN109846722A

Abstract

Disclosed is lyophilized powder of a mesenchymal stem cell activity factor, comprising the mesenchymal stem cell activity factor and a lyophilized excipient. Also disclosed are a method for preparing the lyophilized powder of the mesenchymal stem cell activity factor, and a composition containing the lyophilized powder of the mesenchymal stem cell activity factor and a lyophilized solvent.

Description

Mesenchymal stem cell freeze-dried powder and preparation method thereof

This application claims the priority of the Chinese patent application filed on February 28, 2019 with the application number CN 201910149528.8 and the invention title "Mesenchymal stem cell freeze-dried powder and its preparation method", and the above Chinese patent application is quoted here in full The disclosed content is taken as a part of this application.

Technical field

The present disclosure belongs to the field of stem cell technology. Specifically, the present disclosure relates to a lyophilized powder of mesenchymal stem cell activity factor, which comprises a mesenchymal stem cell activity factor and a freezing excipient. The present disclosure also relates to a method for preparing the freeze-dried powder of mesenchymal stem cell active factor, a composition comprising the freeze-dried powder of mesenchymal stem cell active factor, and the use of the freeze-dried powder and composition.

technical background

Freeze-dried powder is a common dosage form in the field of medicine. The vacuum freeze-drying method of a freeze dryer is used to freeze the moisture in the medicinal solution, and then the frozen water in the medicinal solution is sublimated in a vacuum aseptic environment to obtain freezing Dried freeze-dried powder, so as to achieve the extraction of water in the traditional Chinese medicine liquid in a low temperature environment, and retain its original drug effect.

Currently, in the fields of cosmetics, beauty products, etc., in order to solve the problems of storage time and active ingredient activity, the form of freeze-dried powder has also begun to be used.

Summary of the invention

In one aspect, the present disclosure relates to a lyophilized powder of mesenchymal stem cell active factor, which includes mesenchymal stem cell active factor and a lyophilized excipient.

In some embodiments, the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, and bone marrow mesenchymal stem cells, and other types of mesenchymal stem cells, and combinations thereof . In a preferred embodiment, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

In some embodiments of the lyophilized powder of mesenchymal stem cell activity factor, the mesenchymal stem cell activity factor is derived from the culture supernatant of mesenchymal stem cells. In a preferred embodiment, the mesenchymal stem cell activity factor is derived from the culture supernatant of mesenchymal stem cells with passage numbers P3-P10.

In some embodiments of the lyophilized powder of mesenchymal stem cell activity factor, the lyophilized excipient includes fish collagen. In some embodiments, the concentration of the lyophilized excipient, such as fish collagen, in the solution before lyophilization in the lyophilized powder is 1-20 ng/ml, for example, 5-10 ng/ml.

In another aspect, the present disclosure relates to a method for preparing a lyophilized powder of mesenchymal stem cell active factors, the method comprising the following steps:

a. Provide mesenchymal stem cells;

b. Cultivation and passage of the mesenchymal stem cells;

c. Collect the culture supernatant of the mesenchymal stem cells and sterilize by filtration to obtain a concentrated solution of the culture supernatant;

d. Add freeze-drying excipients to the concentrated solution obtained in step c, and perform freeze-drying procedures,

Thereby, the lyophilized powder of mesenchymal stem cell active factor is obtained.

In some embodiments of the above method, the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, and other types of mesenchymal stem cells, And its combination.

In some embodiments, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, and step a includes obtaining umbilical cord mesenchymal stem cells from the umbilical cord.

In some embodiments, obtaining umbilical cord mesenchymal stem cells from the umbilical cord includes the following steps:

a1. Separate Wharton's glue from the umbilical cord;

a2. Cut the Wharton's glue into small tissue pieces, and culture the tissue pieces in a petri dish for a sufficient period of time to allow mesenchymal stem cells to crawl out of the tissue pieces;

a3. When the mesenchymal stem cells are grown to 60%-100% confluence, for example, when 70%-90% confluent, the tissue mass is removed to obtain umbilical cord mesenchymal stem cells.

In some embodiments, in step a2, DMEM/F-12 medium supplemented with FBS is used to culture the tissue mass.

In some embodiments, DMEM/F-12 medium supplemented with FBS is used in step b to culture and passage the mesenchymal stem cells.

The culture medium used for culturing the umbilical cord tissue mass in step a2 and the medium used for culturing mesenchymal stem cells in step b may be the same or different. For example, in steps a2 and b, DMEM/F-12 medium supplemented with FBS of the same concentration, or DMEM/F-12 medium supplemented with FBS of different concentrations can be used.

In some embodiments of the method of the present disclosure, the culture supernatant of mesenchymal stem cells with passage numbers P3-P10 is collected in step c.

In some embodiments of the method of the present disclosure, the lyophilized excipient added in step d includes fish collagen. In some embodiments, the final concentration of the lyophilized excipient, such as fish collagen, is 1-20 ng/ml. In some embodiments, the final concentration of the lyophilized excipient, such as fish collagen, is 5-10 ng/ml.

In some embodiments of the method of the present disclosure, the freeze-drying procedure includes the following steps: pre-freezing, cold trap freezing, and dry freezing. For example, the freeze-drying procedure includes the following steps:

d1. Pre-freezing at normal pressure and a temperature of -60 to -80°C for more than 24 hours, for example, 24 hours to 5 days;

d2. Perform cold trap freezing in a freeze dryer at a temperature of -56 to -60°C for about 4 hours; and

d3. Perform freeze-drying in a freeze dryer at a pressure of about 1 Pa and a temperature of -56 to -60°C for about 12 hours.

The present disclosure also relates to a lyophilized powder of mesenchymal stem cell active factor prepared by the method of the present disclosure.

In another aspect, the present disclosure relates to a lyophilized vehicle used in combination with the lyophilized powder of mesenchymal stem cell active factor of the present disclosure.

In some embodiments, the lyophilization vehicle comprises at least three different molecular weight hyaluronic acids, such as at least one high molecular weight hyaluronic acid, at least one medium molecular weight hyaluronic acid, and at least one low molecular weight hyaluronic acid.

In some embodiments, the content ratio of the high molecular weight hyaluronic acid, the medium molecular weight hyaluronic acid, and the low molecular weight hyaluronic acid in the freeze-dried vehicle is about 1:1:1.

In some embodiments, in the lyophilized vehicle, the content of the high molecular weight hyaluronic acid, the medium molecular weight hyaluronic acid, and the low molecular weight hyaluronic acid is 0.05% to 1% each.

In some embodiments of the freeze-dried vehicle of the present disclosure, the freeze-dried vehicle contains saffron extract. In some embodiments, the content of the saffron extract in the freeze-dried vehicle is about 0.5%-5%.

In one aspect, the present disclosure relates to a composition comprising the lyophilized powder of mesenchymal stem cell activity factor of any aspect of the present disclosure, and the lyophilized vehicle of the present disclosure used in combination with the lyophilized powder.

In another aspect, the present disclosure relates to a stem cell cosmetic product, which comprises the mesenchymal stem cell active factor freeze-dried powder of any aspect of the present disclosure or the composition of the present disclosure.

In one aspect, the present disclosure relates to the use of the mesenchymal stem cell active factor lyophilized powder or composition of the present disclosure in stem cell beauty.

In another aspect, the present disclosure relates to the use of the mesenchymal stem cell active factor freeze-dried powder or composition of the present disclosure in the preparation of stem cell cosmetic products.

Description of the drawings

Figure 1 shows an exemplary photograph of mesenchymal stem cells excluded from umbilical cord tissue mass. Figure 1A shows a photograph of the tissue block after 72 hours of culture; Figure 1B shows a photograph of 5 days after culture; Figure 1C shows a photograph of 6 days after culture.

Figure 2 shows an exemplary photograph of cultured mesenchymal stem cells. Figure 2A shows a photo of mesenchymal stem cells with passage number P2; Figure 2B shows a photo of cells with passage number P6.

Figure 3 shows the staining results after induced differentiation of mesenchymal stem cells. Figure 3A shows the result of staining cells with Alizarin Red after osteogenic differentiation. Figure 3B shows the result of staining cells with Oil Red O after adipogenic induction and differentiation.

Figure 4 shows the results of flow cytometry using corresponding antibodies to stain cells with positive and negative markers of mesenchymal stem cells.

Figure 5 shows the results of detecting IL-6 in the mesenchymal stem cell active factor concentrate by the ELISA method. The IL-6 standard was used to establish a standard curve to quantify the IL-6 in the mesenchymal stem cell active factor concentrate.

detailed description

The present disclosure will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present disclosure will become clearer with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present disclosure. Those skilled in the art should understand that the details and forms of the technical solutions of the present disclosure can be modified or replaced without departing from the spirit and scope of the present disclosure, but these modifications and substitutions fall within the protection scope of the present disclosure.

In the following examples, the hyaluronic acid used is an acid mucopolysaccharide, and with its unique molecular structure and physical and chemical properties, it exhibits a variety of important physiological functions in the body, such as lubricating joints and regulating the permeability of blood vessel walls. Sex, regulate protein, water and electrolyte diffusion and operation, promote wound healing, etc. More importantly, hyaluronic acid has a special water retention effect and is known as an ideal natural moisturizing factor. Hyaluronic acid is a multifunctional matrix, which is widely distributed in various parts of the human body. The skin also contains a lot of hyaluronic acid. The maturation and aging process of human skin also changes with the content and metabolism of hyaluronic acid. It can improve skin nutrition metabolism, make skin soft, smooth, wrinkle-free, increase elasticity, and prevent aging. It is also a good transparency while moisturizing. Skin absorption enhancer. Used in conjunction with other nutrients, can play a more ideal effect of promoting nutrient absorption.

According to the range of molecular weight, hyaluronic acid can be divided into three types: high molecular weight, medium molecular weight, and low molecular weight. For example, the molecular weight range can be ≥500kDa, 100-400kDa, ≤10kDa, etc.

In the following examples, without special exclusion, in industrial production, sodium hyaluronate can be used as a substitute for hyaluronic acid, or hyaluronic acid and sodium hyaluronate can be used together.

In the following examples, the saffron extract is an effective ingredient separated and extracted from the dried flowers of the natural Comoositae plant Carthamus (tinctoriusl). Safflower contains safflower yellow pigment and safflower glycosides. Safflower glycoside is hydrolyzed to produce glucose and carthamin, and it also contains 15α-20β-dihydroxy △4-pregneno-3-one, vitamin E, uronic acid, etc. When added to the freeze-dried vehicle of the present disclosure and used in combination with the freeze-dried powder of mesenchymal stem cell activity factor, it has multiple beneficial effects. Saffron plays a role in calming, soothing, and cooling the skin. The essence extracted from saffron acts on the skin to quickly calm allergies, improve allergic reactions, and condition and strengthen the skin. For example, when it is used as a beauty product, it has a unique effect of activating the skin and delaying skin aging.

Example 1. Obtaining mesenchymal stem cells

The umbilical cord mesenchymal stem cells are obtained by separating Wharton's Jelly from the umbilical cord and using the tissue block attachment method. The specific steps are as follows:

Take the umbilical cord of a healthy full-term newborn about 10 cm from the end of the baby and place it in a petri dish and cut it into small sections, and rinse with phosphate buffered saline (PBS) repeatedly until there is no obvious blood clot;

Cut the umbilical cord longitudinally, remove the blood vessels and peel off the Wharton's glue. Use ophthalmic scissors to cut Wharton's glue into tissue pieces;

Inoculate the tissue block in a petri dish at an appropriate density and place it in a 37°C, 5% CO ₂ incubator for 2-3 hours to make it adhere to the wall;

Add 3ml of DMEM/F-12 medium supplemented with FBS and place it in an incubator at 37°C and 5% CO ₂ for culture;

After culturing for about 3-5 days, cell colonies can be seen around the tissue mass (Figure 1A and Figure 1B). The cells are in the shape of a long spindle with an obvious swirling morphology, which is a typical umbilical cord mesenchymal cell-like morphology;

After culturing for about 5-15 days (Figure 1C), the cell density reaches close to saturation and can be passaged.

Example 2. Culture and passage of mesenchymal stem cells

The cultivation and passage of mesenchymal stem cells are as follows:

When the cells reach about 70-90% confluence, gently rinse the cells in the culture flask with PBS for 1-2 times, and add 3-5ml of digestion solution containing 0.25% trypsin;

Place the culture flask at 37°C for 1-3 minutes, observe that the cells are close to a circle under the microscope, tap gently to stop the digestion;

Transfer the cells to a centrifuge tube and mix well, sample and count them, and inoculate them in T75 culture flasks at a density of about 13000 cells/cm ² for passage. The cells are recorded as generation P0;

After 2-3 days of culture, when the cell density reaches about 70-90%, repeat the above digestion and passaging operations to obtain a sufficient amount of mesenchymal stem cells and mesenchymal stem cell culture supernatant.

For the cultivation of mesenchymal stem cells, DMEM/F-12 medium containing FBS is used. Starting from the P2 generation, the FBS content in the medium gradually decreased to 5%-3%. Fig. 2A and Fig. 2B show exemplary photographs of mesenchymal stem cells with passage numbers P2 and P6, respectively.

Example 3. Characterization of Mesenchymal Stem Cells

Mesenchymal stem cells have the potential for adipogenic, osteogenic, and chondrogenic differentiation. In this example, the osteogenic and adipogenic differentiation capabilities of the isolated mesenchymal stem cells were tested. Specifically, when the cells to be tested for osteogenic induction grow to 50-90% confluence, and when the cells for adipose induction test grow to more than 90% confluence, osteogenic and adipogenic induction media are added respectively. The cells were tested after 7 days of induction culture. Osteoblast induced cells were stained with Alizarin Red, and adipogenic cells were stained with Oil Red O. The staining results are shown in Figures 3A and 3B. From the results, it can be seen that the mesenchymal stem cells isolated and cultured by the method of the present disclosure have differentiation potential and can efficiently differentiate into bone and adipocytes.

In addition, the mesenchymal stem cell markers are detected by flow cytometry to further characterize mesenchymal stem cells. The flow cytometry markers used for mesenchymal stem cells include positive expression of CD73, CD90, and CD105, while negative expression of HLA-DR, CD11b, CD19, CD34, and CD45. In this example, the expression of CD105, CD34, CD31 and CD117 was detected by flow cytometry. The results are shown in Figure 4 and summarized as follows:

CD105/CD34 99.64%/0.02%;

CD105/CD31 99.04%/0.00%;

CD105/CD117 95.53%/0.51%.

The above results indicate that the cells isolated and cultured by the method of the present disclosure highly express positive markers for mesenchymal stem cells, but basically do not express negative markers for mesenchymal stem cells, further confirming the identity of their mesenchymal stem cells.

Example 4. Preparation of lyophilized powder of mesenchymal stem cell active factor

Generally, the culture supernatant of mesenchymal stem cells with passage numbers P3-P10 is used to prepare freeze-dried powder. The specific steps include:

When the cells are passaged, the cell culture supernatant is collected in a liquid storage bottle, and the supernatants collected multiple times are mixed and stored in a -80°C refrigerator. The storage solution is used as the cell supernatant stock solution or stored for later use, and the mesenchymal stem cell active factor lyophilized powder is prepared in the subsequent steps;

The cryopreserved culture supernatant was thawed at room temperature. After thoroughly mixing, filter and sterilize with a 0.22μm filter membrane to obtain a mesenchymal stem cell culture supernatant concentrate, that is, a mesenchymal stem cell active factor concentrate;

A freeze-dried excipient is added to the obtained mesenchymal stem cell active factor concentrate, and the amount is divided into a vial of 1.5ml/vessel. In this example, the freeze-dried excipient used is 5-10ng/ml fish bone protein.

The sample is freeze-dried according to a predetermined freeze-drying procedure, and the mesenchymal stem cell active factor freeze-dried powder is obtained by pressing and sealing.

The specific freeze drying procedure is as follows:

Pre-freezing: place the vial under normal pressure, half-stopped, and pre-freeze at a temperature of -60 to -80°C for 24 hours to 5 days;

Cold trap freezing: Place the vial in a freeze dryer, turn on the freeze desiccant, and reduce the temperature of the cold trap to the range of -56 to -60°C, and freeze for about 4 hours;

Freeze-drying: Turn on the vacuum pump to reduce the air pressure to about 1Pa, maintain the temperature of the cold trap in the range of -56 to -60°C, and continue freeze-drying for 12 hours under this condition;

The amount of the freeze-dried protective agent added is 0.25-0.5%.

For example, the lyoprotectant is selected from one or a mixture of several of trehalose, sucrose, lactose, glucose, raffinose, dextran, mannitol, sorbitol, or xylitol in any ratio.

In some embodiments, after the mesenchymal stem cell active factor concentrate is obtained, the active factor can be tested. Taking IL-6 as an example, take 1 ml of mesenchymal stem cell active factor concentrate, and use a recombinant human antibody-coated enzyme-linked immunoassay kit to determine the IL-6 content according to the manufacturer's instructions. The result is shown in Figure 5. The results indicate that the mesenchymal stem cell active factor concentrate obtained by the method of the present disclosure has a high concentration of active factors.

Example 5. Preparation of lyophilized powder solvent

In addition, a lyophilized powder solvent used in combination with the mesenchymal stem cell lyophilized powder of the present disclosure is formulated. In an exemplary embodiment, the human mesenchymal stem cell activity factor freeze-dried powder 1.5ml/dominant 3ml/freeze-dried powder solvent is used directly, and one 3ml freeze-dried powder solvent is made of high molecular weight hyaluronic acid of cosmetics. The molecular weight hyaluronic acid and the small molecular weight hyaluronic acid are mixed in deionized water in a ratio of 1:1:1, and the final concentration of the three hyaluronic acids is 0.05% to 1%.

In addition, the saffron extract is used as a component of the lyophilized powder solvent, and its content is 0.5-5%. Saffron extract is an effective ingredient isolated from the dried flowers of the natural Compositae plant safflower. Safflower contains safflower yellow pigment and safflower glycoside. Safflower glycoside is hydrolyzed to produce glucose and carthamin, and it also contains 15α-20β-dihydroxy △4-pregneno-3-one, vitamin E, uronic acid, etc. When added to the freeze-dried vehicle of the present disclosure and used in combination with the freeze-dried powder of mesenchymal stem cell activity factor, it has multiple beneficial effects. Saffron plays a role in calming, soothing, and cooling the skin. The essence extracted from saffron acts on the skin to quickly calm allergies, improve allergic reactions, and condition and strengthen the skin. For example, when it is used as a beauty product, it has a unique effect of activating the skin and delaying skin aging.

The cytokines in the freeze-dried powder plus saffron and hyaluronic acid can produce significant beneficial technical effects when applied, including skin lightening, wrinkle removal, anti-allergic and anti-inflammatory effects when used as a beauty product.

Example 6. Detection of freeze-dried powder products

Various indicators of the freeze-dried powder of the present disclosure were tested, including bacteria, fungi, mycoplasma, and endotoxins. The results show that the lyophilized powder of the present disclosure has nine conventional chemicals (lead, arsenic, mercury, cadmium, Staphylococcus aureus, heat-resistant coliforms, Pseudomonas aeruginosa, total number of molds and yeasts, total number of colonies) All qualified.

In addition, multiple application to allergy irritation tests have been performed, and the results show that the freeze-dried powder of the present disclosure is non-irritating.

Claims

A lyophilized powder of mesenchymal stem cell activity factor, which comprises mesenchymal stem cell activity factor and a lyophilized excipient, wherein the mesenchymal stem cell activity factor is derived from the culture supernatant of mesenchymal stem cells, and The lyophilized excipient includes fish bone collagen.
The mesenchymal stem cell activity factor freeze-dried powder of claim 1, wherein the mesenchymal stem cells are selected from the group consisting of umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, And its combination.
The mesenchymal stem cell active factor freeze-dried powder of claim 2, wherein the mesenchymal stem cells are umbilical cord mesenchymal stem cells.
The mesenchymal stem cell active factor freeze-dried powder according to any one of claims 1 to 3, wherein the mesenchymal stem cell active factor is derived from the culture supernatant of mesenchymal stem cells with passage numbers P3-P10.
The lyophilized powder of mesenchymal stem cell activity factor according to any one of claims 1 to 4, wherein the concentration of the lyophilized excipient in the solution before lyophilization is 1-20ng/ml.
The mesenchymal stem cell active factor lyophilized powder of claim 5, wherein the concentration of the lyophilized excipient in the solution before lyophilization is 5-10 ng/ml.
A method for preparing a freeze-dried powder of mesenchymal stem cell active factors, the method comprising the following steps:

a. Provide mesenchymal stem cells;

b. Cultivation and passage of the mesenchymal stem cells;

c. Collect the culture supernatant of the mesenchymal stem cells and sterilize by filtration to obtain a concentrated solution of the culture supernatant;

d. Add freeze-drying excipients to the concentrated solution obtained in step c, and perform freeze-drying procedures,

Thereby, a lyophilized powder of mesenchymal stem cell active factor is obtained, wherein the lyophilized excipient includes fish bone collagen.
The method of claim 7, wherein the mesenchymal stem cells are selected from the group consisting of umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, and combinations thereof.
8. The method of claim 8, wherein the mesenchymal stem cells are umbilical cord mesenchymal stem cells, and step a includes obtaining umbilical cord mesenchymal stem cells from the umbilical cord.
9. The method of claim 9, wherein obtaining umbilical cord mesenchymal stem cells from the umbilical cord comprises the following steps:

a1. Separate Wharton's glue from the umbilical cord;

a2. Cut the Wharton's glue into small tissue pieces, and culture the tissue pieces in a petri dish for a sufficient period of time to allow mesenchymal stem cells to crawl out of the tissue pieces;

a3. When the mesenchymal stem cells grow to 60%-100% confluence, remove the tissue mass to obtain umbilical cord mesenchymal stem cells.
The method according to claim 10, wherein in step a2, a DMEM/F-12 medium supplemented with FBS is used to culture the tissue mass.
The method of claim 10 or 11, wherein the tissue mass is removed when the mesenchymal stem cells grow to 70%-90% confluence in step a3.
The method according to any one of claims 7-12, wherein in step b, DMEM/F-12 medium supplemented with FBS is used for culturing the mesenchymal stem cells.
The method according to any one of claims 7-13, wherein the culture supernatant of mesenchymal stem cells with passage numbers P3-P10 is collected in step c.
The method according to any one of claims 7-14, wherein the final concentration of the lyophilized excipient added in step d is 1-20 ng/ml.
The method of claim 15, wherein the final concentration of the lyophilized excipient added in step d is 5-10 ng/ml.
The method according to any one of claims 7-16, wherein the freeze-drying procedure comprises the following steps:

d1. Pre-freezing for 24 hours to 5 days under normal pressure and a temperature of -60 to -80°C;

d2. Perform cold trap freezing in a freeze dryer at a temperature of -56 to -60°C for about 4 hours; and

d3. Perform freeze-drying in a freeze dryer at a pressure of about 1 Pa and a temperature of -56 to -60°C for about 12 hours.
A lyophilized powder of mesenchymal stem cell active factor prepared by the method of any one of claims 7-17.
A composition comprising the lyophilized powder of mesenchymal stem cell active factor according to any one of claims 1-6 and 18 and a lyophilized vehicle used in combination with the lyophilized powder.
The composition of claim 19, wherein the freeze-dried vehicle comprises at least three different molecular weight hyaluronic acids, and the at least three different molecular weight hyaluronic acids comprise at least one high molecular weight hyaluronic acid, at least one A medium molecular weight hyaluronic acid and at least one low molecular weight hyaluronic acid.
22. The composition of claim 20, wherein the content ratio of the high molecular weight hyaluronic acid, the medium molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in the freeze-dried vehicle is about 1:1:1.
The composition according to claim 20 or 21, wherein the content of the high molecular weight hyaluronic acid, the medium molecular weight hyaluronic acid and the low molecular weight hyaluronic acid in the lyophilized solvent is 0.05% to 1% each.
The composition according to any one of claims 19-22, wherein the lyophilized vehicle comprises saffron extract.
22. The composition of claim 23, wherein the content of the saffron extract in the freeze-dried vehicle is 0.5%-5%.
A stem cell cosmetic product comprising the mesenchymal stem cell active factor freeze-dried powder according to any one of claims 1-6 and 18 or the composition according to any one of claims 19-24.
Use of the mesenchymal stem cell active factor freeze-dried powder according to any one of claims 1-6 and 18 or the composition according to any one of claims 19-24 in stem cell beauty.