Culture medium and culture method of fibroblast stem cells and application of fibroblast stem cells in facial beautification

Abstract

The invention provides a culture method of fibroblast stem cells and application of the fibroblast stem cells in facial beautification, and relates to the technical field of cell biology. The culture medium provided by the invention comprises a basic culture medium, a growth factor, an immunomodulatory factor, an antioxidant protective agent and amino acid, the growth factors comprise bFGF, TGF-beta and EGF (Epidermal Growth Factor); the immunoregulation factors comprise IL-6 (Interleukin-6) and IL-10 (Interleukin-10); the antioxidant protective agent comprises vitamin C and glutathione. Through scientific combination of the growth factor, the immune factor and the antioxidant, the culture medium and the culture method significantly improve the proliferation efficiency, the passage frequency and the functional stability of the fibroblast stem cells, and efficient and safe cell products are obtained.

Description

Technical Field

[0001] The invention belongs to the field of cell biology, and in particular relates to a method for culturing fibroblast stem cells and an application thereof in facial beauty. Background Art

[0002] Fibroblast stem cells are a type of multipotent stem cells that exist in a variety of tissues. They have the ability to self-renew in the body and can differentiate into a variety of cell types, including but not limited to adipocytes, bone cells, chondrocytes, tendon cells, ligament cells, and fibroblast stem cells. Fibroblast stem cells are mainly found in a variety of tissues, including bone marrow, adipose tissue, umbilical cord, placenta, dental pulp, etc.

[0003] Fibroblast stem cells have many application potentials in the fields of regenerative medicine and cosmetic medicine. They not only have the potential to differentiate into multiple cell types, but when the body is injured or affected by disease, these stem cells can be activated and differentiated into corresponding types of cells to repair damaged tissues and promote wound healing. They can also secrete a variety of growth factors and cytokines to promote tissue repair and regeneration.

[0004] In cosmetic medicine, fibroblast stem cells can be used for skin regeneration and anti-aging treatment by promoting collagen synthesis and extracellular matrix remodeling, improving skin texture and elasticity. In addition, fibroblast stem cells also have immunomodulatory functions, can inhibit inflammatory responses, help maintain tissue homeostasis, and reduce scar formation.

[0005] Fibroblast stem cells have shown great potential in tissue engineering, regenerative medicine, drug screening, disease model construction, etc., and are currently one of the hot topics in the field of life science and medical research. However, the problem of insufficient passage times and differentiation potential of fibroblast stem cells has gradually become prominent.

[0006] Traditional culture methods often fail to completely simulate the natural growth environment of cells in vivo, resulting in limited cell activity and differentiation ability. Factors such as the culture medium, additives, and culture conditions used in the culture process may also affect the number of passages and differentiation potential of fibroblast stem cells.

[0007] The prior art CN103911339B provides a fibroblast stem cell culture medium, comprising the following components: triiodothyronine, hydrocortisone, dexamethasone, adenine, sodium selenite, 2-mercaptoethanol, VE, VC, lipid concentrate, glutamine, ethanolamine, bFGF, EGF, TGF-β, PDGF, insulin or insulin-like growth factor and transferrin, which solves the problem that the serum-free culture medium has a small number of passages and the cell proliferation rate slows down after 10 generations of culture. However, when using this culture medium, the primary cells can reach a confluence of 60% in about 10 days, and the cell growth rate is still slow for passage culture. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a culture medium and a culture method for fibroblast stem cells and application thereof in facial beauty.

[0009] In one aspect, the present invention provides a culture medium for fibroblast stem cells.

[0010] Specifically, the culture medium includes the following components: basic culture medium, growth factors, immunomodulatory factors, antioxidant protective agents and amino acids;

[0011] The growth factors include: bFGF;

[0012] The immunomodulatory factors include: IL-10;

[0013] The antioxidant protective agent includes: vitamin C.

[0014] More specifically, the basic culture medium includes: DMEM / F12, RPMI 1640, MEM, MEMα and Ham's F12 culture medium.

[0015] Preferably, the basal culture medium is: DMEM / F12.

[0016] More specifically, the growth factor also includes but is not limited to at least one of EFG, TGF-β, IGF-1 and / or PDGF.

[0017] Preferably, the final concentrations of the growth factors in the culture medium are: bFGF: 5-15 ng / mL; TGF-β: -8 ng / mL; EGF: 3-8 ng / mL.

[0018] More specifically, the immunomodulatory factors also include but are not limited to at least one of IL-6, Wnt5a, IDO1, CXCL1-3, CXCL9-11, LGALS9 and / or IL-1β.

[0019] Preferably, the final concentration of the immunomodulatory factors in the culture medium is: IL-6: 4-8 ng / mL; IL-10: 5-15 ng / mL.

[0020] More specifically, the antioxidant protective agent also includes, but is not limited to, at least one of glutathione, vitamin E, anthocyanidin, resveratrol, niacinamide and / or cinnamic acid.

[0021] Preferably, the final concentration of the antioxidant protective agent in the culture medium is: vitamin C: 100-300 μM; glutathione: 0.5-1.5 mM.

[0022] More specifically, the amino acids include, but are not limited to, at least one of L-glutamine, arginine, alanine, arginine, aspartic acid, asparagine, cysteine, cystine, glutamic acid or glycine.

[0023] Preferably, the final concentrations of the amino acids in the culture medium are: L-glutamine: 3-8 mM; arginine: 3-8 mM.

[0024] In another aspect, the present invention provides use of the above culture medium in culturing fibroblast stem cells.

[0025] In yet another aspect, the present invention provides a method for culturing fibroblast stem cells.

[0026] Specifically, the culture method comprises the following steps:

[0027] (1) Preparation of culture medium: Add 5-15 ng / mL bFGF, 3-8 ng / mL TGF-β, 3-8 ng / mL EGF, 4-8 ng / mL IL-6, 5-15 ng / mL IL-10, 100-300 μM vitamin C, 0.5-1.5 mM glutathione, 3-8 mM L-glutamine, and 3-8 mM arginine to DMEM / F12 culture medium and continue stirring until dissolved evenly;

[0028] (2) Sterilization;

[0029] (3) Inoculation and cultivation.

[0030] More specifically, in step (2), the sterilization method is: filtering the culture medium with a 0.22 μm filter membrane.

[0031] More specifically, in step (3), the inoculation density is 1-2.5×10 4 cells; the culture conditions are: shaking speed 50-60rpm, changing the culture medium every 46-50 hours, and performing subculture operation when the cell confluence reaches 80%-90%.

[0032] More specifically, the culture method may further include a freezing step.

[0033] Preferably, the freezing step comprises:

[0034] S1: prepare freezing solution: by volume, including 5-15% DMSO, 10-20% fetal bovine serum, 100-300 μM vitamin C, 25-75 μM vitamin E and 0.5-1.5% human albumin;

[0035] S2: Fibroblast stem cells cultured to 80%-90% confluence were treated with trypsin to prepare a single cell suspension with a concentration of 0.8-1.2×10 6 Pieces / mL;

[0036] S3: Mix the cell suspension with the freezing solution at a ratio of 1:0.8-1.2, aliquot and freeze.

[0037] In another aspect, the present invention provides the use of fibroblast stem cells cultured by the above-mentioned culture method and / or their culture products in the preparation of facial beauty and anti-aging products.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] (1) The culture medium provided by the present invention can increase the cell proliferation rate by 50% compared with the traditional method, and shorten the culture period to 5-7 days.

[0040] (2) After the culture medium provided by the present invention is passaged 10 times, the pluripotency and differentiation potential of the cells can still be maintained at more than 80%.

[0041] (3) The fibroblast stem cells cultured in the present invention are stored in the cryopreservation solution provided by the present invention, and the cell recovery survival rate is increased to more than 90%.

[0042] (4) The synthesis and secretion level of collagen in the cultured fibroblast stem cells of the present invention is significantly increased, and can be used for the preparation of facial beauty and / or anti-aging products. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0044] Table 1 Reagents

[0045]

[0046] Basic Example

[0047] 1. Culture medium preparation process:

[0048] In a sterile environment, add all ingredients to DMEM / F12 medium and continue stirring until dissolved evenly. Adjust the pH value to 7.4 and filter with a 0.22 μm filter membrane to ensure sterility. Dispense the filtered medium into sterile containers and store at 4°C. The shelf life is 7 days.

[0049] 2. Cell culture and subculture operation:

[0050] (1) Cell acquisition, inoculation and initial culture:

[0051] Take a 2cm×2cm full-thickness skin and soak it in a sterile phosphate buffered saline (PBS) containing 250U / mL penicillin and 250mg / L streptomycin. Gently scrape off the epidermal cells and subcutaneous tissue in the clean bench, retain the dermis, and cut it into small pieces of 1cm×1cm with ophthalmic scissors. Transfer it to a 50mL centrifuge tube with a pipette, inject sterile double-antibody PBS solution, repeat washing 3 times, discard the washing solution, and store the centrifuge tube on ice;

[0052] PBS was used to prepare 0.1% type I collagenase solution, which was sterilized by filtering with a 0.22 μm filter membrane. The shredded skin tissue was transferred to the centrifuge tube and digested in a 37°C water bath for 40 min. The cells were shaken every 10 min during digestion. The cell suspension after digestion was filtered with a 40-mesh and a 100-mesh filter membrane, respectively. The supernatant was discarded after centrifugation at 1200 r / min for 5 min. The cells in the centrifuge tube were blown evenly and incubated at 37°C and 5% CO. 2 The cells were cultured in a cell culture incubator for 1-2 days. During this period, the cell adhesion was observed under a microscope every 2 days. When the adherent cells grew to 90%, the cells were purified and washed with PBS for 3 times. 0.25% trypsin was added and the cells were digested in a water bath at 37°C. The cells were repeatedly blown until the rounded fibroblasts were completely and evenly mixed. The liquid was transferred to a 15mL centrifuge tube and centrifuged at 1200r / min for 2min. The supernatant was discarded and the cells were resuspended.

[0053] The cells were diluted and inoculated into culture flasks at a density of 2×10 per square centimeter. 4 cells, 37°C, 5% CO 2 , maintain humidity above 95%, place the culture bottle on a shaker, and adjust the rotation speed to 50 rpm for cultivation.

[0054] (2) Culture medium replacement:

[0055] The culture medium was replaced every 48 hours by aspirating the old culture medium and adding an equal amount of fresh culture medium to ensure that the cells were always in the best growth environment.

[0056] (3) Subculture operation:

[0057] When the cell fusion degree reaches 80%-90%, add 0.25% trypsin by volume and let it act at room temperature for 2 minutes. Observe the cell morphology, tap the bottle wall to make the cells completely fall off, and immediately add culture medium containing fetal bovine serum to neutralize the action of trypsin. Adjust the separated cells to the appropriate concentration and re-inoculate them into a new culture bottle.

[0058] 3. Scale up and cultivate:

[0059] Use a 500 mL shake flask or bioreactor and add the optimized culture medium to the device at an inoculum density of 1 × 10 per mL. 4 cells, 60 rpm, 37°C, 5% CO 2 When the cell density reached 1×10 6 / mL, centrifugation was used for scale-up and culture expansion.

[0060] 4. Cryopreservation

[0061] (1) Cryopreservation solution components: by volume, including 10% DMSO, 15% fetal bovine serum, 200 μM vitamin C, 50 μM vitamin E and 1% human albumin.

[0062] (2) Cell preparation: Fibroblast stem cells cultured to 80%-90% confluence were treated with trypsin and prepared into a single-cell suspension. The cell concentration was adjusted to 1×10 cells / mL.

[0063] (3) Mixing cryopreservation solution: Mix the cell suspension and cryopreservation solution in a 1:1 ratio, gently invert to mix, place in a programmed cooling device after packaging, and freeze to -80°C at a rate of 1°C per minute. Transfer the cryopreservation tubes to a liquid nitrogen tank (-196°C) for long-term storage.

[0064] 5. Recovery

[0065] (1) Rapid thawing: Remove the cryotube from liquid nitrogen and immediately place it in a 37°C water bath and shake gently. It will be completely thawed after 1-2 minutes.

[0066] (2) Removal of cryopreservation solution:

[0067] The thawed cell suspension was transferred into a centrifuge tube, 10 mL of preheated culture medium was added, and the tube was centrifuged at 1000 rpm for 5 minutes to remove the freezing solution.

[0068] (3) Recovery culture: Resuspend the cells in culture medium, inoculate them into culture bottles, and place them in an incubator for culture and observation.

[0069] Examples 1-3

[0070] Prepare the culture medium according to the components in Table 2.

[0071] Table 2

[0072] Components Example 1 Example 2 Example 3 bFGF (ng / mL) 10 5 15 TGF-β(ng / mL) 5 8 3 EGF (ng / mL) 20 8 3 IL-6 (ng / mL) 6 8 4 IL-10 (ng / mL) 10 5 15 Vitamin C (μM) 200 100 300 Glutathione (mM) 1 1.5 0.5 L-Glutamine (mM) 5 8 3 Arginine (mM) 5 8 3

[0073] Comparative Examples 1-5

[0074] The culture medium was prepared according to Table 3.

[0075] Table 3

[0076] Components Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 bFGF (ng / mL) - 10 10 - TGF-β(ng / mL) 5 5 5 5 EGF (ng / mL) 20 20 20 20 IL-6 (ng / mL) 6 6 6 6 IL-10 (ng / mL) 10 - 10 - Vitamin C (μM) 200 200 - - Glutathione (mM) 1 1 1 1 L-Glutamine (mM) 5 5 5 5 Arginine (mM) 5 5 5 5

[0077] Experimental Example 1

[0078] Proliferation rate assay:

[0079] Take according to the basic example and dilute to 2×10 4 / mL of primary fibroblast stem cells were inoculated in a 96-well culture plate, 100 μL / well, and cultured under standard conditions for 24 h. After synchronization, the liquid in the wells and the non-adherent cells were discarded, and the cells were washed once with sterile PBS. The cells were randomly divided into 4 wells in each group, and 100 μL / well of the culture medium prepared in each example or comparative example was added, and the culture medium was incubated at 37°C, 5% CO 2 At 1, 3, 5, and 7 days, the growth state of the cells was observed under a microscope, and the cell confluence was recorded. When the cells grew to a confluence of 80%, the number of culture days was recorded as the culture cycle. The experimental results are shown in Table 4.

[0080] Table 4

[0081] Cultivation period (days) Example 1 4 Example 2 5 Example 3 5 Comparative Example 1 9 Comparative Example 2 10 Comparative Example 3 8 Comparative Example 4 14

[0082] It can be seen from Table 4 that the use of the optimized culture medium provided by the present invention increases the proliferation rate of primary fibroblast stem cells by about 50% compared with the control example, and the culture period is shortened to 5 days.

[0083] Experimental Example 2

[0084] Differentiation potential detection method:

[0085] Take the 10th generation fibroblast stem cells that have grown well, and adjust the cell density to 2×10 in DMEM culture medium containing 2% FBS. 4 Cells / well were seeded in 6-well plates, and after the cells adhered to the plate and cultured for 24 hours, the culture medium was discarded.

[0086] Adipogenic differentiation potential detection: Add adipogenic differentiation induction medium according to the kit instructions and continue culturing. After the culture is completed, fix with 4% paraformaldehyde for 15 minutes. Wash with PBS for 3 times. Add 0.2% Oil Red O staining solution and stain at 37℃ for 30 minutes. Aspirate the staining solution and wash with PBS for 3 times. Observe under a microscope and take pictures.

[0087] Osteogenic differentiation ability test: Add osteogenic differentiation induction medium according to the instructions of the kit and continue culturing. After the culture, fix with 4% paraformaldehyde at room temperature for 20 minutes. Discard the paraformaldehyde, wash twice with PBS, add 2% alizarin red (pH 4.2, filtered before use) and stain for 30 minutes at room temperature. Discard the alizarin red, wash with PBS until the supernatant is colorless or light-colored, and finally add PBS, observe under a microscope and take pictures.

[0088] The culture medium of the present invention still has osteogenic and adipogenic differentiation potential after being passaged 10 times.

[0089] Experimental Example 3

[0090] Cryopreservation survival rate

[0091] The fifth generation of fibroblast stem cells with good growth were taken and cryopreserved and revived according to the basic example. After revivification, the cell density was adjusted to 2×10 4 / mL, inoculated in a 96-well culture plate, 100 μL / well, and cultured under standard conditions for 24 h. After synchronization, the liquid in the wells and the non-adherent cells were discarded, and washed once with sterile PBS. The wells were randomly divided into 4 wells in each group, and 100 μL / well of the culture medium prepared in Example 1 was added and incubated at 37°C, 5% CO 2 After culturing for 3-4 days, 10 μL of MTT (5 g / L) was added to each well, and the culture was continued for 8 hours. Then, 10 μL of SDS (200 g / L) was added, and the culture was continued overnight. The absorbance value of each group was read using a microplate reader at 570 nm for colorimetry. The freezing solution used in the present invention increased the cell recovery survival rate from 85% to more than 90%.

[0092] Experimental Example 4

[0093] The fifth generation of fibroblast stem cells with good growth were taken and the cell density was adjusted to 2×10 4 / mL, inoculated in a 96-well culture plate, 100 μL / well, and cultured for 24 hours under standard conditions. After synchronization, the liquid in the wells and the non-adherent cells were discarded, and washed once with sterile PBS. The cells were randomly divided into groups, each with 4 wells, and 100 μL / well of the culture medium prepared in each example or comparative example was added, and the culture medium was incubated at 37°C, 5% CO 2 After culturing for 48 h, an equal amount of cell culture supernatant was collected from each group and the expression of collagen was detected using an ELISA kit. The test results are shown in Table 5.

[0094] Table 5

[0095] Medium components Collagen content (μg / mL) Example 1 565±25.65 Example 2 512±34.12 Example 3 548±32.45 Comparative Example 1 202±24.35*** Comparative Example 2 235±30.46*** Comparative Example 3 283±22.98*** Comparative Example 4 150±10.21***

[0096] Note: Compared with Example 1, "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001.

[0097] According to the experimental results, it can be concluded that the culture medium provided by the present invention can promote the synthesis and secretion of collagen by fibroblast stem cells.

[0098] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A culture medium for fibroblast stem cells, characterized in that: The composition includes the following components: basic culture medium, growth factors, immunomodulatory factors, antioxidant protective agents and amino acids; The growth factors include: bFGF; The immunomodulatory factors include: IL-10; The antioxidant protective agent includes: vitamin C.

2. The culture medium according to claim 1, characterized in that The basic culture medium includes: DMEM / F12, RPMI1640, MEM, MEMα and Ham's F12 culture medium.

3. The culture medium according to claim 1, characterized in that The growth factors also include: TGF-β and EGF, and the final concentration of the growth factors in the culture medium is: bFGF: 5-15 ng / mL; TGF-β: 3-8 ng / mL; EGF: 3-8ng / mL.

4. The culture medium according to claim 1, characterized in that The immunomodulatory factor also includes IL-6, and the final concentration of the immunomodulatory factor in the culture medium is: IL-6: 4-8 ng / mL; IL-10: 5-15ng / mL.

5. The culture medium according to claim 1, characterized in that The antioxidant protective agent also includes glutathione, and the final concentration of the antioxidant protective agent in the culture medium is: Vitamin C: 100-300 μM; Glutathione: 0.5-1.5mM.

6. The culture medium according to claim 1, characterized in that The amino acids include: L-glutamine, arginine, alanine, arginine, aspartic acid, asparagine, cysteine, cystine, glutamic acid or glycine, and the final concentration of the amino acids in the culture medium is: L-glutamine: 3-8 mM; Arginine: 3-8mM.

7. Use of the culture medium according to any one of claims 1 to 6 in culturing fibroblast stem cells.

8. A method for culturing fibroblast stem cells, characterized in that: The following steps are involved: (1) Preparation of culture medium: Add 5-15 ng / mL bFGF, 3-8 ng / mL TGF-β, 3-8 ng / mL EGF, 4-8 ng / mL IL-6, 5-15 ng / mL IL-10, 100-300 μM vitamin C, 0.5-1.5 mM glutathione, 3-8 mM L-glutamine, and 3-8 mM arginine to DMEM / F12 culture medium and continue stirring until dissolved evenly; (2) Sterilization; (3) Inoculation and cultivation.

9. The culture method according to claim 8, characterized in that: The following steps are also included: (4) Cryopreservation; The freezing step comprises: S1: prepare freezing solution: by volume, including 5-15% DMSO, 10-20% fetal bovine serum, 100-300 μM vitamin C, 25-75 μM vitamin E and 0.5-1.5% human albumin; S2: Fibroblast stem cells cultured to 80%-90% confluence were treated with trypsin to prepare a single cell suspension with a concentration of 0.8-1.2×10 6 Pieces / mL; S3: Mix the cell suspension with the freezing solution at a ratio of 1:0.8-1.2, aliquot and freeze.

10. Use of fibroblast stem cells cultured by the culture method according to any one of claims 8 to 9 and / or their culture products in the preparation of facial beauty and anti-aging products.

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