Preparation method and application of leukocyte-inducing extract

Abstract

The invention discloses a preparation method and application of a leukocyte-inducing extract. The preparation method comprises the following steps: acquiring mesenchymal stem cells; inducing the mesenchymal stem cells to differentiate to generate dendritic cells; splitting the dendritic cells, and separating to obtain a cell extract; the differentiation comprises the steps of inducing the mesenchymal stem cells to differentiate into immature dendritic cells through the first induction composition and inducing the immature dendritic cells to mature through the second induction composition. The preparation method provided by the embodiment of the invention finds a new way, starts from the mesenchymal stem cells, and utilizes the characteristic of wide distribution of the mesenchymal stem cells to solve the problems of single source and difficult acquisition of leukocytes in the production process of the leukocyte extract; and moreover, the mesenchymal stem cells are differentiated, so that the situation that monocytes obtained by directly separating the albuginea layer are mixed into inflammatory cells is avoided.

Description

Technical Field

[0001] The present application relates to the technical field of cell biology, and in particular to a preparation method and application of an induced leukocyte extract. Background Art

[0002] Leukocyte extract (LE) is a complex secreted by leukocytes and possesses numerous cosmetic benefits, including antioxidant properties (scavenging free radicals and protecting the skin from oxidative damage); anti-inflammatory properties (reducing skin inflammation and promoting skin repair); repair properties (stimulating skin cell metabolism and regeneration, accelerating wound healing, and improving skin barrier function); and whitening properties (inhibiting melanin production and reducing pigmentation and dark spots). Currently, the most common method for extracting LE is to use neonatal umbilical cord blood (or adult peripheral blood) as the raw material, separate the plasma and buffy coat, and then obtain mononuclear cells (PBMCs) from the buffy coat. These cells are then cultured with inactivated plasma, centrifuged, and the supernatant is then ultrafiltered to yield the LE. However, the monocytes isolated in this manner are often mixed with inflammatory cells such as neutrophils, whose secretions can strongly stimulate inflammatory responses, hindering the LE's ability to exert its anti-inflammatory, antioxidant, and free radical scavenging properties. Summary of the Invention

[0003] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a method for preparing an induced leukocyte extract and its application. The cell extract obtained by this method does not affect the anti-inflammatory, antioxidant, and free radical scavenging effects of inflammatory cell secretions and can serve as an effective alternative to leukocyte extracts.

[0004] In a first aspect of the present application, a method for preparing a cell extract is provided, comprising the following steps:

[0005] S100: Obtain mesenchymal stem cells;

[0006] S200: Induce mesenchymal stem cells to differentiate into dendritic cells;

[0007] S300: lyse dendritic cells and separate cell extracts;

[0008] Among them, S200 includes:

[0009] S210: inducing mesenchymal stem cells to differentiate into immature dendritic cells using a first induction composition,

[0010] S220: inducing maturation of immature dendritic cells using a second induction composition;

[0011] The first induction composition includes IL-4 and GM-CSF, and the second induction composition includes the first induction composition, TNF-α, carcinoembryonic antigen CEA, cancer antigen 125, MAGE-A1, NY-ESO-1, HER2 / neu and EGFR.

[0012] The preparation method according to the embodiment of the present application has at least the following beneficial effects:

[0013] The preparation method provided in the embodiments of the present application takes a different approach. Starting from mesenchymal stem cells, it utilizes their widely distributed characteristics to solve the problem of a single source of leukocytes and their difficulty in obtaining them during the production of leukocyte extracts. Differentiation from mesenchymal stem cells also avoids the situation where monocytes obtained by directly separating the buffy coat layer are mixed with inflammatory cells. In addition, through the specific first induction composition and the second induction composition, the differentiated dendritic cells have the immune regulation function of immune cells and the self-renewal function of stem cells, and the various functional protein components secreted are stable, fully covering the functions required by leukocyte extracts, such as antioxidant, free radical scavenging, anti-inflammatory, skin wound repair, tissue regeneration, and skin barrier improvement.

[0014] In some embodiments of the present application, the final concentration of IL-4 in the first induction composition is 80-150 ng / mL, for example, it can be 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, or 150 ng / mL.

[0015] In some embodiments of the present application, the final concentration of GM-CSF in the first induction composition is 300-800 ng / mL, for example, 300 ng / mL, 350 ng / mL, 400 ng / mL, 450 ng / mL, 500 ng / mL, 550 ng / mL, 600 ng / mL, 650 ng / mL, 700 ng / mL, 750 ng / mL, or 800 ng / mL.

[0016] In some embodiments of the present application, the final concentration of IL-4 in the first induction composition is 80-150 ng / mL, and the final concentration of GM-CSF is 300-800 ng / mL.

[0017] In some embodiments of the present application, the final concentration of TNF-α in the second induction composition is 10-30 ng / mL, for example, it can be 10 ng / mL, 12 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 18 ng / mL, 20 ng / mL, 22 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 28 ng / mL, or 30 ng / mL.

[0018] In some embodiments of the present application, the final concentration of carcinoembryonic antigen CEA in the second induction composition is 100-300 ng / mL, for example, it can be 100 ng / mL, 120 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 180 ng / mL, 200 ng / mL, 220 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 280 ng / mL, or 300 ng / mL.

[0019] In some embodiments of the present application, the final concentration of cancer antigen 125 in the second induction composition is 100-300 ng / mL, for example, it can be 100 ng / mL, 120 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 180 ng / mL, 200 ng / mL, 220 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 280 ng / mL, or 300 ng / mL.

[0020] In some embodiments of the present application, the final concentration of MAGE-A1 in the second inducing composition is 100-300 ng / mL, for example, it can be 100 ng / mL, 120 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 180 ng / mL, 200 ng / mL, 220 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 280 ng / mL, or 300 ng / mL.

[0021] In some embodiments of the present application, the final concentration of NY-ESO-1 in the second inducing composition is 100-300 ng / mL, for example, 100 ng / mL, 120 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 180 ng / mL, 200 ng / mL, 220 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 280 ng / mL, or 300 ng / mL.

[0022] In some embodiments of the present application, the final concentration of HER2 / neu in the second induction composition is 100-300 ng / mL, for example, it can be 100 ng / mL, 120 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 180 ng / mL, 200 ng / mL, 220 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 280 ng / mL, or 300 ng / mL.

[0023] In some embodiments of the present application, the final concentration of EGFR in the second induction composition is 100-300 ng / mL, for example, it can be 100 ng / mL, 120 ng / mL, 140 ng / mL, 150 ng / mL, 160 ng / mL, 180 ng / mL, 200 ng / mL, 220 ng / mL, 240 ng / mL, 250 ng / mL, 260 ng / mL, 280 ng / mL, or 300 ng / mL.

[0024] In some embodiments of the present application, S200 is 3D induction culture.

[0025] In some embodiments of the present application, 3D induction culture includes 3D induction culture without a support and 3D induction culture with a support.

[0026] In some embodiments of the present application, the support-free 3D induction culture includes any one or a combination of several of the hanging drop method, the low adsorption spherical culture plate method, and the magnetic levitation method.

[0027] In some embodiments of the present application, the 3D induced culture with a support includes using at least one of a hydrogel and a microcarrier as a support for 3D induced culture.

[0028] In some embodiments of the present application, the 3D induction culture with a support includes attaching cells to at least one support such as a hydrogel, a microcarrier, etc., and culturing the cells under rotation conditions using a first induction composition and a second induction composition.

[0029] In some embodiments of the present application, S200 includes:

[0030] Mesenchymal stem cells are inoculated onto a support and cultured. After the mesenchymal stem cells grow and completely cover the surface of the support, the mesenchymal stem cells are induced to differentiate into immature dendritic cells using a first induction composition, and the immature dendritic cells are induced to mature using a second induction composition.

[0031] In some embodiments of the present application, the inoculation amount of mesenchymal stem cells on the support is 1 million to 3 million cells per 0.3 to 1 g of the support.

[0032] In some embodiments of the present application, after the mesenchymal stem cells are seeded on the support and cultured for 4 to 6 days, the mesenchymal stem cells grow and completely cover the surface of the support.

[0033] In some embodiments of the present application, S210 includes culturing the mesenchymal stem cells in a mixed manner with a first culture medium containing a first induction composition, so as to differentiate the mesenchymal stem cells into immature dendritic cells.

[0034] In some embodiments of the present application, S210 includes mixing and culturing the mesenchymal stem cells with a first culture medium containing the first induction composition for 2 to 4 days, and then adding the first culture medium containing the first induction composition and continuing to mix and culture for 2 to 4 days.

[0035] In some embodiments of the present application, S220 includes separating the first culture medium from the differentiated immature dendritic cells, and re-culturing the immature dendritic cells with a second culture medium containing a second induction composition to induce maturation of the immature dendritic cells.

[0036] In some embodiments of the present application, after the dendritic cells are differentiated and generated in S200, the cells are cultured continuously, and the culture supernatant is collected every 3 to 5 days. The culture supernatant can be used to separate and purify the dendritic cell extract.

[0037] In some embodiments of the present application, after the dendritic cells are differentiated and generated in S200, the culture is continued, and the 3D bottles are transferred once every 8 to 12 days. The carriers covered with dendritic cells are divided into flasks for culture. One bottle can be divided into 3 to 5 bottles for further culture, and the supernatant is continued to be collected to separate the white blood cell extract.

[0038] In some embodiments of the present application, the mesenchymal stem cells are derived from at least one of bone marrow, periosteum, synovium, skeletal muscle, dental pulp, umbilical cord, umbilical cord blood, placenta, peripheral blood, fat, and foreskin.

[0039] In some embodiments of the present application, mesenchymal stem cells are derived from foreskin. Experimental findings indicate that foreskin, discarded after circumcision surgery, can be used to culture mesenchymal stem cells with stronger immune function and proliferation capacity than umbilical cord mesenchymal stem cells. Therefore, foreskin-derived mesenchymal stem cells are used as basal cells for subsequent culture.

[0040] In some embodiments of the present application, the foreskin is foreskin dermis tissue.

[0041] In some embodiments of the present application, the method for obtaining foreskin dermis tissue is: the foreskin after circumcision is cleaned and set aside; the foreskin is longitudinally sliced to spread the tissue, separate the epidermis and dermis, and then the dermis is cut into small pieces to form a tissue homogenate.

[0042] In some embodiments of the present application, the foreskin cleaning process includes sequentially soaking in iodine, disinfecting with alcohol, and cleaning with phosphate buffered saline.

[0043] In some embodiments of the present application, S100 includes: mixing and culturing multiple tissues from different individuals with culture medium to obtain mesenchymal stem cells. Mesenchymal stem cells from different individual sources vary significantly. To address the issue of functional homogeneity of mesenchymal stem cells, a mixed stem cell culture method is employed. This not only addresses these differences in mesenchymal stem cells from different individuals, but also demonstrates significantly greater immunomodulatory and proliferation capacity than mesenchymal stem cells obtained from a single foreskin culture.

[0044] In some embodiments of the present application, S300 includes:

[0045] Lysis: Repeated freezing and thawing of dendritic cells to obtain cell lysate;

[0046] Separation: Centrifuge the cell lysate, collect the supernatant, and filter to obtain the cell extract.

[0047] In some embodiments of the present application, the dendritic cells are repeatedly frozen and thawed 3 to 5 times.

[0048] In some embodiments of the present application, the centrifugation procedure is 5000 g to 20000 g, and the centrifugation time is 5 to 30 min.

[0049] In some embodiments of the present application, the filtering comprises filtering using a filter with a diameter of 0.22 μm.

[0050] In a second aspect of the present application, a cell extract is provided, which is prepared by any of the aforementioned preparation methods.

[0051] In some embodiments of the present application, the cell extract contains CCL2 (Chemokine Ligand 2), CXCL1 (CXC Motif Chemokine Ligand 1), CXCL5 (CXC Motif Chemokine Ligand 5), CXCL6 (CXC Motif Chemokine Ligand 6), CXCL8 (CXC Motif Chemokine Ligand 8), IL6 (Interleukin 6), IL11 (Interleukin 11), LIF (Leukemia inhibitory factor), IL1RN (Interleukin 1 Receptor Antagonist), TGFβ1 (Transforming Growth Factor Beta 1), and IL-6. 1) and at least one of TGFβ2 (Transforming Growth Factor Beta 2).

[0052] In some embodiments of the present application, the cell extract contains CCL2, CXCL1, CXCL5, CXCL6, CXCL8, IL6, IL11, LIF, IL1RN, TGFβ1 and TGFβ2.

[0053] In some embodiments of the present application, the cell extract contains proteins with antioxidant and free radical scavenging functions. In some embodiments, the proteins with antioxidant and free radical scavenging functions include at least one of glutathione peroxidase 1, glutathione peroxidase 3, glutathione peroxidase 4, glutathione peroxidase 7, selenoprotein P, SOD enzyme 1, SOD enzyme 2, SOD enzyme 3, glutathione reductase, CXC motif chemokine 5, and interleukin 6.

[0054] In some embodiments of the present application, the cell extract contains a protein with anti-inflammatory function. In some embodiments, the protein with anti-inflammatory function includes at least one of interleukin-1α, interleukin-6, interleukin-11, transforming growth factor β1, transforming growth factor β2, transforming growth factor β3, chemokine ligand 2, and leukemia inhibitory factor.

[0055] In some embodiments of the present application, the cell extract contains a protein with whitening function. In some embodiments, the protein with whitening function contains at least one of platelet-derived growth factor subunit A, platelet-derived growth factor subunit C, platelet-derived growth factor subunit D, keratinocyte growth factor, hepatocyte growth factor, insulin-like growth factor 2, interleukin 6, and fibronectin 1.

[0056] In some embodiments of the present application, the cell extract contains proteins with repair and wound healing functions. In some embodiments of the present application, the proteins with repair and wound healing functions include at least one of fibronectin 1, chemokine ligand 2, CXC motif chemokine 1, CXC motif chemokine 6, CXC motif chemokine 8, interleukin 1, interleukin 6, interleukin 11, leukemia inhibitory factor, and transforming growth factor β1.

[0057] In some embodiments of the present application, the cell extract contains a protein having a proliferation-promoting function. In some embodiments, the protein having a proliferation-promoting function comprises at least one of vascular endothelial growth factor A, vascular endothelial growth factor C, interleukin 6, hepatocyte growth factor, transforming growth factor β1, transforming growth factor β2, and transforming growth factor β3.

[0058] In a third aspect of the present application, a composition is provided, which comprises the aforementioned cell extract.

[0059] In some embodiments of the present application, the composition includes an active ingredient, and the active ingredient includes the aforementioned cell extract.

[0060] In some embodiments of the present application, the active ingredient further includes other ingredients having at least one of the functions of anti-oxidation, free radical scavenging, anti-inflammatory, whitening, repairing, wound healing, and promoting proliferation.

[0061] In some embodiments of the present application, the composition further comprises at least one of a carrier, a diluent, a stabilizer, a filler, a wetting agent, a colorant, a solvent, a co-solvent, a film former, a spontaneity promoter, an emulsifier, a dispersant, a preservative, an antifreeze agent, a thickener, and an adjuvant.

[0062] In a fourth aspect of the present application, a cosmetic product is provided, which comprises the aforementioned cell extract, or the aforementioned composition.

[0063] In some embodiments of the present application, the dosage form of the cosmetic product includes any one of an aqueous solution, an oil, a cream, an emulsion, an essence, a gel, a mask, a spray, and the like.

[0064] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The following are the morphological observation results of mesenchymal stem cells during different time periods of induction culture in one embodiment of the present application.

[0066] Figure 2 The results of the test on T cell immunoregulatory function of dendritic cell extracts in one embodiment of the present application are shown in Figure 1. A is the activated group and B is the dendritic cell extract group.

[0067] Figure 3 The results of the regulatory function test of dendritic cell extract on macrophages in one embodiment of the present application are shown in Figure 1. A is the flow cytometry result, and B is the quantitative statistical result of the M1 phenotype.

[0068] Figure 4 The results of an endothelial cell scratch test using a dendritic cell extract in one embodiment of the present application are shown in Figure 1. A shows the results of microscopic observation, and B shows the comparison of endothelial cell migration rates.

[0069] Figure 5 The results of an endothelial cell tube formation experiment using dendritic cell extracts in one embodiment of the present application are shown in Figure 1. A represents the microscopic observation result, and B represents the comparison of vascular density.

[0070] Figure 6 In one embodiment of the present application, the skin condition of a user is compared over a period of time.

[0071] Figure 7 In one embodiment of the present application, the skin condition of another user using the introduction method over a period of time is compared. DETAILED DESCRIPTION

[0072] The following will clearly and completely describe the concept and technical effects of this application in conjunction with the embodiments to fully understand the purpose, features and effects of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of this application.

[0073] The embodiments of the present application are described in detail below. The described embodiments are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0074] In the description of this application, "several" means more than one, "plurality" means more than two, "greater than," "less than," and "exceed" are understood to exclude the number itself, while "above," "below," and "within" are understood to include the number itself. The use of "first" and "second" in the description is solely for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, implicitly specifying the number of the indicated technical features, or implicitly specifying the order of the indicated technical features.

[0075] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art to which this application belongs. The terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application.

[0076] In the description of this application, reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples.

[0077] Example 1: Preparation of Children's Foreskin Mesenchymal Stem Cells

[0078] This embodiment provides a method for preparing children's foreskin mesenchymal stem cells, the specific steps are as follows:

[0079] (1) Foreskin samples (FSK) from three different children discarded after circumcision were collected into sterile sample containers containing sterile phosphate-buffered saline (PBS, Lonza) supplemented with 50 U / mL penicillin / streptomycin (Lonza).

[0080] (2) Upon arrival at the laboratory, the foreskin samples were soaked in iodine for 1 minute, cleaned with 75% alcohol for 30 seconds, and then washed with sterile PBS and transferred to a sterile culture dish.

[0081] (3) The foreskin was sliced longitudinally to spread the tissue and separate the epidermis and dermis. The dermis was then cut into small pieces of 0.5 mm to 1 mm to obtain tissue homogenate. The tissue homogenates of the foreskin samples from three different children were then mixed.

[0082] (4) The mixed tissue homogenate was evenly inoculated into a T75 culture flask. The culture flask was placed flat so that the tissue blocks were as evenly distributed as possible on the entire bottom surface. Serum-free mesenchymal stem cell culture medium (Lonza) supplemented with 50 U / mL penicillin / streptomycin (Lonza) was added, and then the flask was placed in a constant temperature and humidity incubator at 37°C and 5% CO2 for primary culture until cells crawled out around the tissue blocks.

[0083] (5) The medium was changed once every 5 days during primary culture, and non-adherent cells were removed during the medium change. When the adherent cells reached subconfluence (80-90%), they were digested with 2.5% trypsin (Gibco, Life Technologies) and passaged. The cells were repeatedly passaged to the desired stage (e.g., P2, P3, or P4) to establish a cell bank.

[0084] During the culture process, under phase contrast microscopy, FSK-MSCs showed typical fibroblast-like appearance, with elongated, adherent and spindle-shaped morphology. Flow cytometry identification showed that the cultured cells were CD73 + CD90 + CD105 + CD45 - CD34 - CD14 - CD19 - , and the human leukocyte antigen HLA-DR was also negative, indicating that FSK-MSCs had the phenotype of MSCs and met the standards of the International Society for Cellular Therapy (ISCT).

[0085] Example 2: 3D induced differentiation of mesenchymal stem cells

[0086] This embodiment provides a method for 3D induced differentiation of mesenchymal stem cells, comprising the following steps:

[0087] (1) Sterilized microcarriers (Cytodex 1, Corning, 0.3 g / L) were added to a spinner flask culture system. 500 mL of serum-free mesenchymal stem cell culture medium (Lonza) supplemented with 50 U / mL penicillin / streptomycin (Lonza) was then added. Finally, the FSK-MSCs described in Example 1 were added. The inoculum size was 1 million cells per 0.3 g of microcarrier. The spinner flask was set to 30 rpm and cultured in a constant temperature and humidity incubator at 37°C and 5% CO2 for 4 to 6 days until the FSK-MSCs completely covered the microcarrier surface.

[0088] (2) Induced differentiation:

[0089] Day 0, when FSK-MSCs completely covered the microcarrier surface, was designated as day 0. The original culture medium was discarded, and 250 mL of Stage 1 DC Induction Medium was added to the spinner flask. On day 3, a fresh 250 mL of Stage 1 DC Induction Medium was added to bring the total volume to 500 mL. During this process, the spinner flask was maintained at 30 rpm in a constant temperature and humidity incubator at 37°C and 5% CO2.

[0090] On the fifth day, the flask was removed from the incubator and placed in a biosafety cabinet. It was allowed to stand for 20 minutes to allow the microcarriers and cells to sink to the bottom. The upper culture medium was then carefully aspirated with a 50 mL pipette. 250 mL of the second stage DC induction medium was then added and the flask was returned to a constant temperature and humidity incubator at 37°C and 5% CO2 and cultured at 30 rpm.

[0091] On day 8, remove the spinner flask from the incubator and place it in a biosafety cabinet. Allow the microcarriers and cells to settle to the bottom for 20 minutes. Carefully aspirate the upper layer of culture medium with a 50mL pipette. Remove the remaining cells and microcarriers from the bottom of the culture medium and transfer them to three to five 50mL centrifuge tubes. Add 10mL of 4°C pre-chilled PBS-EDTA to each tube and place in a 4°C refrigerator for 15 minutes. Repeatedly pipette the cell-microcarrier suspension to dissociate the cells from the microcarriers. Rinse thoroughly four times and collect in a 50mL centrifuge tube. Fill the tube to 45mL with saline, centrifuge at 1200 rpm for 10 minutes, and discard the supernatant. The cell pellet represents DCs derived from foreskin mesenchymal stem cells after 3D induction culture. Collected DCs are used for testing and obtaining cell extracts.

[0092] The first stage DC induction culture medium consists of serum-free culture medium (KBM 581 Corning) supplemented with IL-4 at a final concentration of 100 ng / mL and GM-CSF at a final concentration of 500 ng / ml.

[0093] The second-stage DC induction medium consisted of KBM 581 serum-free medium (Corning) supplemented with IL-4 at a final concentration of 100 ng / mL, GM-CSF at a final concentration of 500 ng / mL, TNF-α at a final concentration of 20 ng / mL, and antigenic peptides at a final concentration of 100 ng / mL. The antigenic peptides included carcinoembryonic antigen CEA (Novoprotein, CM95), cancer antigen 125 (CA-125) (Novoprotein, CW57), MAGE-A1, NY-ESO-1 (Novoprotein, C10A), HER2 / neu (human epidermal growth factor receptor 2) (Novoprotein, CP69), and EGFR (epidermal growth factor receptor) (Novoprotein, Cl61), all at a final concentration of 100 ng / mL.

[0094] In addition to cytokines such as IL-4, this induced culture system also adds multiple types of tumor antigen peptides. These antigen peptides can stimulate the immune system to produce an immune response against tumor cells, thereby stimulating the maturation of DC cells and the expression of specific proteins.

[0095] Example 3: 2D induced differentiation of mesenchymal stem cells

[0096] This embodiment provides a method for 2D induced differentiation of mesenchymal stem cells, comprising the following steps:

[0097] (1) 2D culture

[0098] The foreskin mesenchymal stem cells passaged to P3-P5 in Example 1 were taken and 1.5×10 6 The inoculum volume of cells / flask is inoculated to an area of 75 cm 2 Add 10 mL of serum-free mesenchymal stem cell medium (Lonza) supplemented with 50 U / mL penicillin / streptomycin (Lonza) to each T75 culture flask. After shaking, place the cells in a 37°C, 5% CO2 constant temperature and humidity incubator. Cultivate for approximately 2-3 days. When the cells reach approximately 90% confluence, induce the culture.

[0099] (2) 2D induction culture

[0100] On day 0, the serum-free medium in the culture flask was discarded and replaced with 10 mL of the first stage DC induction medium in Example 2. The cells were cultured in a constant temperature and humidity incubator at 37°C and 5% CO2. On day 3, 10 mL of fresh first stage DC induction medium was replaced and culture continued.

[0101] On the fifth day, 10 mL of the second-stage DC induction medium in Example 2 was added to the existing culture medium and culture was continued.

[0102] On day 8, collect the culture medium and cells from the T75 flask into a 50 mL centrifuge tube. Add 10 mL of 4°C pre-cooled saline and rinse vigorously four times. Centrifuge the saline solution after each rinse and collect it in a 50 mL centrifuge tube. Add 10 mL of PBS-EDTA to the centrifuge tube and place it in a 4°C refrigerator for 10 minutes. Then, add 10 mL of 4°C pre-cooled saline and rinse vigorously four times. Centrifuge the saline solution after each rinse and collect it in a 50 mL centrifuge tube. Fill the centrifuge tube to 45 mL with saline, centrifuge at 1200 rpm for 10 minutes, and discard the supernatant. The collected cells are used for testing and obtaining cell extracts.

[0103] Example 4: Identification of FSK-MSCs-derived dendritic cells

[0104] 1. Cell Morphology Identification

[0105] Take the cells in Example 2 at different time periods and observe the morphological changes under the microscope. The results are as follows: Figure 1 As shown in the figure, it can be seen that the cells gradually shrink, become smaller and aggregate from the slender spindle shape of foreskin mesenchymal stem cells, and transform into a polygonal shape similar to dendritic cells.

[0106] 2. Flow Cytometry

[0107] The dendritic cells prepared in Example 2 and Example 3 were tested by flow cytometry. The positive rates of different cells under different indicators are shown in Table 1:

[0108] Table 1. Flow cytometry results of cell positive rate

[0109] Detection indicators Example 3 Example 2 HLA-DR+ 92.4％ 98.1％ CD11c+ 18.1％ 23.6％ CD141+ 43.5％ 61.7％ HLA-DR+CD11c+ 16.8％ 19.4％ HLA-DR+141+ 39.2％ 54.1％

[0110] As can be seen from the table, the method of Example 2 for inducing foreskin mesenchymal stem cells into dendritic cells in a 3D culture state has a higher positive rate.

[0111] In addition, 3D culture bottles can use 500 to 5000 mL of 3D culture system at a time, and fermentation tanks can induce 20 to 50 liters of 3D culture system at a time, obtaining a large number of chemically induced dendritic cells for isolation and purification of cell extracts, indicating that this method has higher efficiency.

[0112] Example 5: Isolation and purification of dendritic cell extracts

[0113] After obtaining the DC cells induced by foreskin mesenchymal stem cells in Example 2 or Example 2, the collected DC cells are lysed and ultrafiltration is performed to obtain a leukocyte extract, as follows:

[0114] 1) Add 10 mL of ice-cold PBS solution to the DC cell pellet collected in a 50 mL centrifuge tube and resuspend to obtain a DC cell suspension.

[0115] 2) Place the centrifuge tube of DC cell suspension in a -80°C freezer for 30 minutes;

[0116] 3) Immediately remove the frozen DCs and thaw them quickly in a 37°C water bath;

[0117] 4) After the DC cells are completely thawed, return the centrifuge tube to the -80°C freezer and freeze for 30 minutes;

[0118] 5) Repeat steps 3) to 4) three times, rapidly freezing and thawing the DC cells between ultra-low temperature and 37°C to rupture all DC cells and release proteins, nucleic acids, lipids, carbohydrates, and other metabolites in the DC cells to obtain cell lysate.

[0119] 6) Transfer the cell lysate to a new 50 mL centrifuge tube, centrifuge at 12,000 × g for 15 min at 4°C, collect the supernatant, and filter through a 0.22 μm filter to obtain a dendritic cell extract.

[0120] Example 6: Identification of components of dendritic cell extracts

[0121] The isolated and purified leukocyte extract was transported on dry ice to Shanghai Zhongke New Life Biotechnology Co., Ltd. for protein analysis using the 4D-label-free quantitative proteomics analysis method. The specific steps are as follows:

[0122] (1) Protein extraction and peptide enzymatic hydrolysis

[0123] Protein was extracted using the SDT (4 w / v% SDS, 100 mM Tris-HCl, 0.1 M DTT, pH 7.6) lysis method, and protein quantification was performed using the BCA assay. An appropriate amount of protein from each sample was trypsinized using the Filter-aided proteome preparation (FASP) method. Peptides were desalted using a C18 cartridge, lyophilized, and reconstituted in 40 μL of 0.1% formic acid solution. Peptides were quantified (OD280).

[0124] (2) LC-MS / MS data acquisition

[0125] Each sample was separated using an Easy nLC HPLC system operating at a nanoliter flow rate. Buffer A consisted of 0.1% formic acid in water, and buffer B consisted of 0.1% formic acid in acetonitrile (84% acetonitrile). The column was equilibrated with 95% buffer A. Samples were loaded onto a loading column (Thermo Scientific Acclaim PepMap100, 100 μm x 2 cm, nanoViper C18) via an autosampler and separated on an analytical column (Thermo Scientific EASY column, 10 cm, ID 75 μm, 3 μm, C18-A2) at a flow rate of 300 nL / min. Following chromatographic separation, samples were analyzed by mass spectrometry using a timsTOF Pro mass spectrometer. Detection was positive ionization, with an ion source voltage set at 1.5 kV. Both MS and MSMS were performed using a TOF detector and analysis system. The mass spectrometer scan range was set to 100–1700 m / z.

[0126] The proteomics results of dendritic cell extracts showed that a total of 3045 proteins were detected. Functional analysis of these 3045 proteins was performed. The results of GO functional analysis are shown in Table 2. There are 10 major categories of functional proteins:

[0127] Table 2. GO functional analysis results

[0128]

[0129] The proteomic results of dendritic cell extracts were compared with those of the "Catalogue of Used Cosmetic Raw Materials (2021 Edition)" and Front Cell Dev Biol. 2021Apr 7:9:647012. The main functional proteins contained therein were classified according to their functions, including the following categories:

[0130] ① Antioxidation and free radical scavenging: glutathione peroxidase 1, 3, 4, 7 (GPX1 / 3 / 4 / 7), selenoprotein P (SELENOP), SOD enzyme 1, 2, 3 (SOD1 / 2 / 3), glutathione reductase (GSR), CXC motif chemokine 5 (CXCL5), interleukin 6 (IL6).

[0131] ② Anti-inflammatory: interleukin 1α, 6, 11 (interleukin 1α, 6, 11), transforming growth factor β1, 2, 3 (TGFβ1, TGFβ2, TGFβ3), chemokine ligand 2 (CCL2), leukemia inhibitory factor (LIF).

[0132] ③ Whitening: Platelet-derived growth factor subunits A, C, D (PDGFA / PDGFC / PDGFD), keratinocyte growth factor (FGF7), hepatocyte growth factor (HGF), insulin-like growth factor 2 (IGF2), interleukin 6 (IL6), fibronectin 1 (FN1)

[0133] ④ Repair and wound healing: fibronectin 1 (FN1), chemokine ligand 2 (CCL2), CXC motif chemokines 1, 6, 8 (CXCL1, CXCL6, CXCL8), interleukins 1, 6, 11 (IL1, 6, 11), leukemia inhibitory factor (LIF), transforming growth factor β1 (TGFβ1).

[0134] Promote proliferation: vascular endothelial growth factor A, C (VEGFA / VEGFC), interleukin 6 (IL6), hepatocyte growth factor (HGF), transforming growth factor β1, 2, 3 (TGFβ1, TGFβ2, TGFβ3).

[0135] The acquisition of the above-mentioned different functional proteins proves that the method provided in the embodiment can obtain extracts by chemically inducing foreskin mesenchymal stem cells into dendritic cells, which can meet the requirements of the national cosmetics standards for the function of leukocyte extracts.

[0136] The children's foreskin mesenchymal stem cells prepared in Example 1 were lysed according to the method of Example 5 to obtain a cell lysate. Proteomic analysis was then performed using the method of this Example. The proteome of the dendritic cell extract was compared with the proteome of the children's foreskin mesenchymal stem cell extract. The results showed that compared with the FSK-MSCs extract, a total of 243 proteins were significantly upregulated in the dendritic cell extract. The upregulated proteins were mainly correlated with cosmetic functions, among which the cosmetic function proteins mainly included:

[0137] CCL2: anti-inflammatory (PMID: 18802011), skin repair / wound healing (PMID: 32060374);

[0138] CXCL1: skin repair / wound healing (PMID: 36613652);

[0139] CXCL5: Antioxidant (PMID: 35696764), Skin Repair / Wound Healing (PMID: 21734176);

[0140] CXCL6: skin repair / wound healing (PMID: 30605352);

[0141] CXCL8: skin repair / wound healing (PMID: 36725964);

[0142] IL6: Antioxidant (PMID: 35290918), Skin Repair / Wound Healing (PMID: 15191552);

[0143] IL11: Skin repair / wound healing (PMID: 12704372);

[0144] LIF: anti-inflammatory (PMID: 9651226), skin repair / wound healing (PMID: 14871222);

[0145] IL1RN: anti-inflammatory (PMID: 25738940), skin repair / wound healing (PMID: 29553981);

[0146] TGFB1: anti-inflammatory (PMID: 29269050), skin repair / wound healing (PMID: 32012802);

[0147] TGFB2: anti-inflammatory (PMID: 20875417), skin repair / wound healing (PMID: 23110169).

[0148] Example 7: Detection of regulatory function of T cells

[0149] PBMCs were isolated from the peripheral blood of healthy donors using density gradient centrifugation. CD3+ T cells were isolated from PBMCs using a CD3+ T cell isolation magnetic bead kit. The isolated CD3+ T cells were suspended in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin, counted, and adjusted to a concentration of 1×10 6 10 μM CFSE was added to the isolated T cell suspension and incubated at 37°C for 10 minutes. The reaction was terminated by adding 10 volumes of RPMI-1640 medium containing 10% FBS and incubating at 37°C for 5 minutes. The cells were washed three times with PBS to remove unbound CFSE.

[0150] The isolated and CFSE-stained T cells were divided into the following groups: control group (untreated T cells), activated group (cells treated with CD3 / CD28 only), and dendritic cell extract group, with at least 3 replicates per group. Dynabeads were added to the activated T cells. TM Human T-Activator CD3 / CD28 was stimulated for 96 hours according to the manufacturer's recommended ratio (1:1). An equal amount of Dynabeads was added to the leukocyte extraction group. TM Human T-Activator CD3 / CD28 and dendritic cell extract at a final concentration of 50 μg / mL were used for continuous stimulation for 96 hours. Cells from each group were harvested and analyzed by flow cytometry to assess T cell activation and proliferation. FITC signal was used to detect CFSE signal and to assess T cell proliferation.

[0151] The results are as follows Figure 2 As shown in the figure, the red part on the right is the fluorescence distribution of T cells without proliferation, and the blue part on the left is the fluorescence distribution of T cells after proliferation. Compared with the activated group ( Figure 2 A), after the extracts in the dendritic cell extract group were co-cultured with activated T cells, the blue fluorescence decreased, but the decrease was smaller than that in the activation group ( Figure 2 B), indicating that the dendritic cell extract prepared in the examples of the present application can effectively inhibit the proliferation of activated T cells.

[0152] Example 8: Detection of regulatory function of macrophages

[0153] Macrophages (RAW264.7 cells) were seeded in 6-well plates and used as a control group (untreated macrophages), an M1-activated group (cells treated only with LPS and IFN-γ), and a dendritic cell extract group. Macrophages in the M1-activated and dendritic cell extract groups, intended to be stimulated to the M1 phenotype (proinflammatory macrophages), were stimulated for 24 hours with lipopolysaccharide (LPS) at a final concentration of 500 μg / mL and interferon-γ at a final concentration of 20 ng / mL. Subsequently, the dendritic cell extract group was treated with dendritic cell extract at a final concentration of 50 μg / mL for an additional 24 hours. The M1 (CD86-positive) phenotype was assessed by flow cytometry.

[0154] The results are as follows Figure 3 As shown, the qualitative and quantitative analysis results showed that the CD86 signal of cells treated with leukocyte extracts was reduced compared with the cells in the activated group, indicating that the leukocyte extracts induced a decrease in the expression of pro-inflammatory macrophages and had an anti-inflammatory effect.

[0155] Example 9: Regeneration and repair function

[0156] (1) Endothelial cell scratch test

[0157] 2×10 5 Human vascular endothelial cells (HUVECs) were suspended in DMEM (2 mL, 2% FBS) and seeded into wells of a 6-well plate. After culture at 37°C until 90% confluence, the cells were streaked orthogonally using a sterile micropipette (200 μL) tip. Subsequently, the HUVECs were gently washed twice with PBS to remove debris and incubated in fresh DMEM for 24 hours. In this experiment, the dendritic cell extract from Example 5 was added to fresh DMEM at a final concentration of 50 μg / mL, while the extract was replaced with PBS in the control group. Finally, the cells were washed twice with PBS and observed under an inverted microscope (Olympus IX80) at a magnification of 100.

[0158] The results are as follows Figure 4 As shown, compared with the control group, the migration rate of endothelial cells in the experimental group was higher after HUVEC cells were co-incubated with dendritic cell extracts for 24 hours, indicating that dendritic cell extracts enable endothelial cells to have higher migration and proliferation abilities.

[0159] (2) Endothelial cell tube formation experiment

[0160] The day before the experiment, place the Matrigel in an ice box and place it in a 4°C refrigerator to allow the gel to slowly melt overnight. At the same time, prepare a 4°C pre-cooled pipette tip for sucking up the Matrigel. Cover the lid of the ibidi angiogenesis slide. Prepare a 10cm culture dish and place a paper towel soaked in water to make a wet box. Place the ibidi angiogenesis slide in the culture dish and cover it with the culture dish lid. Place the entire culture dish in the incubator and let it stand for 30 minutes to wait for the gel to solidify. While waiting, prepare a 2×10 5 The HUVEC cell suspension was prepared at a concentration of 50 μg / mL and mixed thoroughly. The ibidi angiogenesis slide with solidified gel was removed from the wet chamber and 50 μL of HUVEC cell suspension was added to each well (the control group was treated with untreated HUVEC original cell suspension, and the experimental group was treated with HUVEC cell suspension after 48 hours of treatment with a final concentration of 50 μg / mL extract). Images were collected regularly according to the cell growth rate, and the tube length, coverage area, number of rings, and number of nodes were measured and recorded, and statistical analysis was performed.

[0161] The results are as follows Figure 5 As shown, compared with the control group, the number of endothelial cell lumens in the experimental group was significantly increased and more tubular structures were induced to form after being treated with dendritic cell extracts, indicating that dendritic cell extracts have good potential to promote angiogenesis.

[0162] The above results indicate that the dendritic cell extract provided in the examples exhibits a powerful repair and regeneration function by promoting skin cell growth and angiogenesis.

[0163] Example 10: Verification of cosmetic effects

[0164] Skin patch test results showed that dendritic cell extract had no adverse reaction on the skin.

[0165] Verification of the cosmetic effects of dendritic cell extract: The skin was beautified using the Konka beauty instrument (KMTY-4003-T). 2 mL of dendritic cell extract (50 μg / mL) from Example 5 was applied to the facial skin within 30 minutes. The extract was used once a day for 4 consecutive weeks to verify its anti-wrinkle, anti-inflammatory (anti-allergic), whitening, and freckle removal effects. The results are shown in the figure below. Figure 6 and Figure 7 The specific change process is as follows:

[0166] ① Week 1: From the second day, the redness of sensitive skin is relieved, dark circles are significantly reduced, pigmentation begins to ease from the fourth day, and the pores begin to become smaller on the sixth or seventh day.

[0167] ② Second week: Starting from the seventh day, the redness of sensitive skin disappears, the sensitivity decreases, and the stress response is no longer caused by slight friction. The dark circles almost disappear, the pigmentation is significantly improved, the skin tone begins to brighten, the pores become noticeably smaller, and the skin begins to become delicate.

[0168] ③ Week 3: Skin becomes more resistant to allergies, redness disappears, dark circles disappear, skin feels firmer and more elastic, and the tear troughs around the eyes become noticeably shallower. Fine lines and wrinkles begin to improve, and spots begin to fade.

[0169] ④ Week 4: The overall facial pigmentation problem is greatly improved, the skin color returns to normal, the skin barrier recovers well, no sensitivity problems occur during the use period, some fine lines and wrinkles are smoothed, minor fine lines, wrinkles and spots disappear, large spots begin to dissipate, and deeper wrinkles show signs of lightening.

[0170] The key and difficulty of the current method of obtaining leukocyte extracts is to obtain blood mononuclear cells from neonatal umbilical cord blood or adult peripheral blood, but this source has many limitations, difficulties and shortcomings:

[0171] ① Large-scale collection (or collection) of umbilical cord blood and peripheral blood as a source of white blood cells to produce white blood cell extracts. The collection is governed by the National Blood Donation Law, has significant policy and legal restrictions, and the quantity of each extract is limited.

[0172] ② Blood is collected and only white blood cells are centrifuged to be used as the raw material for the white blood cell extract. The main component of the blood, red blood cells, cannot be comprehensively utilized, which is a huge waste of precious blood components.

[0173] ③ As an accessory after a mother gives birth, the ownership of umbilical cord blood theoretically belongs to the child and its parents. The hospital cannot dispose of it on its own. The compliance requirement for its source requires the signed consent of the parents of each umbilical cord blood sample before it can be collected by the manufacturer, which makes large-scale collection very difficult.

[0174] ④ The buffy coat layer obtained by centrifugation of umbilical cord blood and peripheral blood is a mixture of all white blood cells in the blood, including not only monocytes but also lymphocytes and neutrophils. The white blood cell extract obtained by ultrafiltration after culture has complex components, including not only proteins that inhibit inflammation, anti-oxidation, and anti-free radicals, but also a large number of proteins secreted by neutrophils that promote inflammation.

[0175] ⑤ The amount of umbilical cord blood obtained is generally 50-100 ml, and the mononuclear cells obtained are limited. The HLA of white blood cells in blood from different individuals is different and cannot be mixed and cultured. As a result, the white blood cell extracts derived from each blood mononuclear cell are different, and the uniformity of different batches of products cannot be achieved.

[0176] The applicant has found through research that leukocyte extracts can be obtained by centrifuging umbilical cord blood or adult peripheral blood to separate buffy coat mononuclear cells (PBMCs), inactivating plasma, and then amplifying and culturing them with monocytes. Although the cells obtained here are called monocytes, they are actually a mixture of leukocytes, including neutrophils that promote inflammation. Monocytes are actually a group of cells called the mononuclear phagocyte system (MPS), which includes monocytes (Mo), macrophages (macrophages, ) and dendritic cells (DCs), which have strong phagocytic ability and are important components of the body's innate immune system; they are also a major type of antigen-presenting cells, playing a key role in the induction and regulation of specific immune responses.

[0177] Humans already have functional dendritic cells at the 13th week of pregnancy, much earlier than monocytes and macrophages. They are a type of monocyte that has both the immune regulatory function of leukocytes and the characteristics of stem cells. Dendritic cell extracts have stronger antioxidant properties, can inhibit skin inflammation, and promote skin repair and regeneration.

[0178] To this end, the present application intends to propose a leukocyte (monocyte) extract that can avoid violating blood transfusion laws, has no ethical controversy, can be purified to high purity, has industrial production, and has uniform quality.

[0179] The white blood cells used are dendritic cells that have both the immune regulatory function of white blood cells and the characteristics of stem cells. The extract has stronger anti-inflammatory, antioxidant and regeneration-promoting abilities. The white blood cells (dendritic cells) used can be obtained by mixed culture of children's foreskin mesenchymal stem cells, which avoids the biological differences of different individual cells, eliminates the batch differences of cell culture, avoids the promiscuity of white coat cells separated from a single umbilical cord blood or peripheral blood by centrifugation, and meets the requirements of industrial production uniformity. The induction source of white blood cells (dendritic cells) is an induction system containing a variety of antigens and cytokine components, and the white blood cells (dendritic cells) finally obtained have better proliferation and immune activity. This scheme can separate and purify white blood cell extracts from white blood cells (dendritic cells). Due to the uniformity of the cell source and the standardized process of induction, separation and purification, the obtained white blood cell extracts can meet the requirements of industrial production uniformity. Proteomic studies on cell extracts isolated and purified from leukocytes (dendritic cells) have confirmed the presence of more than 3,000 protein components, covering all the functions that leukocyte extracts are required to possess as cosmetic raw materials: antioxidant, anti-inflammatory, tissue repair, tissue regeneration, whitening and other protein functional groups, and have been proven to have excellent freckle removal, wrinkle removal, whitening and skin modification functions.

[0180] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Various modifications can be made within the scope of knowledge possessed by a person of ordinary skill in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features of the embodiments can be combined with each other unless there is a conflict.

Claims

1. A method for preparing a cell extract, characterized in that: The following steps are involved: S100: Obtain mesenchymal stem cells; S200: inducing the mesenchymal stem cells to differentiate into dendritic cells; S300: Lysing the dendritic cells to separate and obtain the cell extract; Among them, S200 includes: S210: inducing the mesenchymal stem cells to differentiate into immature dendritic cells using a first induction composition, S220: inducing maturation of immature dendritic cells using a second induction composition; The first induction composition includes IL-4 and GM-CSF, and the second induction composition includes the first induction composition, TNF-α, carcinoembryonic antigen CEA, cancer antigen 125, MAGE-A1, NY-ESO-1, HER2 / neu and EGFR.

2. The preparation method according to claim 1, characterized in that The final concentration of IL-4 in the first induction composition is 80-150 ng / mL, and / or the final concentration of GM-CSF in the first induction composition is 300-800 ng / mL.

3. The preparation method according to claim 1, characterized in that The final concentration of TNF-α in the second induction composition is 10-30 ng / mL, the final concentration of carcinoembryonic antigen CEA is 100-300 ng / ml, the final concentration of cancer antigen 125 is 100-300 ng / mL, the final concentration of MAGE-A1 is 100-300 ng / mL, the final concentration of NY-ESO-1 is 100-300 ng / mL, the final concentration of HER2 / neu is 100-300 ng / mL, and the final concentration of EGFR is 100-300 ng / mL.

4. The preparation method according to claim 1, characterized in that The S200 is a 3D induction culture.

5. The preparation method according to claim 1, characterized in that The mesenchymal stem cells are derived from at least one of bone marrow, periosteum, synovium, skeletal muscle, dental pulp, umbilical cord, umbilical cord blood, placenta, peripheral blood, fat, and foreskin.

6. The preparation method according to claim 5, characterized in that The S100 includes: mixing and culturing a plurality of tissues from different individual sources with a culture medium to obtain the mesenchymal stem cells.

7. The preparation method according to claim 1, characterized in that The S300 includes: Lysis: repeatedly freezing and thawing the dendritic cells to obtain cell lysate; Separation: centrifuging the cell lysate, collecting the supernatant, and filtering to obtain the cell extract.

8. A cell extract, characterized in that The method is prepared according to any one of claims 1 to 7.

9. A composition, characterized in that The method comprises the cell extract according to claim 8.

10. A beauty product, characterized in that The method comprises the cell extract according to claim 8, or the composition according to claim 9.

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