Serum-free epidermal melanocyte culture medium and use thereof
By adding specific components to DMEM/F12 medium to construct a biomimetic culture system, the problems of low cell growth efficiency and insufficient melanin secretion in serum-free medium were solved, achieving safe and efficient melanocyte culture, which is suitable for the treatment of vitiligo.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HANGZHOU SINGCLEAN MEDICAL PROD
- Filing Date
- 2026-02-28
- Publication Date
- 2026-06-05
AI Technical Summary
Existing serum-free culture media have problems with unsatisfactory cell growth efficiency and insufficient melanin secretion when culturing epidermal melanocytes, and there are also safety and batch-to-batch variability risks associated with animal-derived components.
A biomimetic culture system was constructed using DMEM/F12 as the basal medium, supplemented with adenine, hepatocyte growth factor, glutamine, retinoic acid, vitamin E, niacin, sodium selenite, melanocyte-stimulating hormone, endothelin-3, transferrin, triiodothyronine, cholesterol, neuromodulatory protein 1, 3-isobutyl-1-methylpurine, dibutyrylcyclic adenosine monophosphate, cholera toxin, hydrocortisone, recombinant human insulin, sodium hyaluronate, and recombinant human type XVII collagen to simulate the extracellular matrix environment.
This study achieved the safety and stability of serum-free culture medium, promoted the proliferation and differentiation of melanocytes, improved the synthetic capacity of melanocytes, solved the problems of cell acquisition time and quality, and reduced the risk of viral contamination.
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Figure CN122146572A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell culture technology, specifically to an animal-free, serum-free epidermal melanocyte culture medium for in vitro culture of melanocytes and its application. Background Technology
[0002] Vitiligo remains one of the most challenging areas to treat in dermatology. Due to the diversity of immune responses and melanin-related genes, some patients exhibit tolerance to traditional treatments, necessitating new treatment strategies. In recent years, cell therapy has developed rapidly, and serum, a key component of cell culture media, promotes cell proliferation and migration, with fetal bovine serum (FBS) being the most widely used. However, serum demand is increasing annually, leading to supply and demand imbalances. Furthermore, the complex and unclear composition of serum, along with batch-to-batch variability, ethical controversies, and animal-derived safety risks, limits its application in cell cultures for human treatment. Therefore, developing animal-free serum alternatives has become an important direction for development in the field of cell therapy.
[0003] Currently, commercially available Melanocyte Growth Medium M3 is a serum-free culture medium that does not contain bovine pituitary extract (BPE) or serum. This type of medium primarily supports basic cell survival and proliferation, but its effect on highly functional cells (such as melanin secretion) is insufficient. Among existing related patents, there is a culture medium for co-culturing melanocytes and keratinocytes (CN107151648B). This patented medium can promote melanocyte adhesion to the basement membrane, thereby promoting melanocyte proliferation. However, this patented formulation contains bovine pituitary extract, an animal-derived component, posing potential safety and batch-to-batch variability risks. A serum-free culture medium for culturing tissue-engineered epidermis (CN115181719B) has clearly defined components, effectively eliminating the drawbacks of adding serum. It has a certain proliferative effect on melanocytes, but it has not demonstrated any effect on promoting melanin secretion by melanocytes or the differentiation of related cells into melanocytes.
[0004] Therefore, current serum-free culture media used for culturing epidermal melanocytes have several shortcomings. On the one hand, due to the unsatisfactory in vitro cell growth efficiency, many patients have to endure long waiting periods; furthermore, some patients cannot obtain sufficient quantities of qualified cells for transplantation due to poor cell expansion. On the other hand, even in cases of successful transplantation, poor pigment regeneration at the transplant site's edges is often a problem. Summary of the Invention
[0005] The purpose of this invention is to provide a serum-free epidermal melanocyte culture medium based on extracellular matrix biomimicry and its application, so as to solve the problems mentioned in the background art.
[0006] To achieve the above objectives, the present invention provides the following technical solution:
[0007] This invention discloses a serum-free epidermal melanocyte culture medium, which consists of a basal culture medium and additives; the basal culture medium is DMEM / F12; the additives consist of adenine, hepatocyte growth factor (HGF), glutamine, retinoic acid, vitamin E, niacin, sodium selenite, melanocyte stimulating factor, endothelin-3, transferrin, triiodothyronine, cholesterol, neuromodulatory protein 1 (NRG1), 3-isobutyl-1-methylpurine (IBMX), dibutyryl cyclic adenosine monophosphate (db-cAMP), cholera toxin, hydrocortisone, recombinant human insulin, sodium hyaluronate, and recombinant human type XVII collagen.
[0008] As a further improvement, the concentration of each component in the additive of the present invention is adenine: 0-1.8 x 10⁻⁶. -4 M, Hepatocyte Growth Factor (HGF): 10-100 ng / mL, Glutamine: 0.01-2 mM, Retinoic Acid: 0.01-0.07 mg / L, Vitamin E: 0.001-0.005 mg / L, Niacin: 0-0.0125 mg / L, Sodium Selenite: 0.1-5 ng / mL, Melanocyte-Stimulating Factor: 0.01-1 µM, Endothelin-3: 0-200 ng / mL, Transferrin: 5-100 µg / mL, Triiodothyronine: 1-50 nM, Cholesterol: 0.1-5 µg / mL, Neuroregulatory Protein 1 (NRG1): 0-25 ng / mL, 3-Isobutyl-1-methylpurine (IBMX): 5-100 µg / mL, Dibutyrylcyclic Adenosine Monophosphate (db-cAMP): 5-100 µg / mL, Cholera Toxin: 0-100 ng / mL, hydrocortisone: 0.01-10 µg / mL, recombinant human insulin: 5-100 µg / mL, sodium hyaluronate 1-10 µg / mL, recombinant human type XVII collagen 0.5-4 mg / mL.
[0009] As a further improvement, the concentration of the additive described in this invention is adenine: 1.8 x 10⁻⁶. -4M, Hepatocyte Growth Factor (HGF): 100 ng / mL, Glutamine: 2 mM, Retinoic Acid: 0.07 mg / L, Vitamin E: 0.005 mg / L, Niacin: 0.0125 mg / L, Sodium Selenite: 5 ng / mL, Melanocyte-Stimulating Factor: 0.01 µM, Endothelin-3: 50 ng / mL, Transferrin: 10 µg / mL, Triiodothyronine: 10 nM, Cholesterol: 0.1 µg / mL, Neuroregulatory Protein 1 (NRG1): 0 ng / mL, 3-Isobutyl-1-methylpurine (IBMX): 5 µg / mL, Dibutyrylcyclic Adenosine Monophosphate (db-cAMP): 100 µg / mL, Cholera Toxin: 0 ng / mL, Hydrocortisone: 10 µg / mL, Recombinant Human Insulin: 10 µg / mL, Sodium Hyaluronate 5 µg / mL, recombinant human type XVII collagen 0.5 mg / mL.
[0010] Serum-free epidermal melanocyte culture medium can be used for the preparation of cells for vitiligo treatment, induction of MSC differentiation into melanocytes, and cell therapy.
[0011] Compared with the prior art, the present invention has the following beneficial effects:
[0012] 1. The culture medium of the present invention is a serum-free culture medium for in vitro culture of human epidermal melanocytes, which eliminates the risk of contamination from the source by pathogens such as prions and exogenous viruses that may be carried by serum, while avoiding batch-to-batch differences caused by complex and ambiguous components in serum.
[0013] 2. Most existing serum-free culture media (including commercially available products) only remove serum, and are essentially still simple "nutrient solutions" with a single component. This invention uses recombinant type XVII collagen as the main ECM structural protein mimicking factor, sodium hyaluronate to simulate the hydration network formed by natural GAG polysaccharides, and other culture medium components to construct a functional culture system that integrates basic nutrition, active biochemical signaling, and physical biomimicry.
[0014] 3. Compared with existing serum-free culture media, the culture medium of the present invention not only ensures basic cell proliferation, but also promotes melanin synthesis and the differentiation of related cells into melanocytes, which has a key advantage in obtaining functional melanocytes with high activity and therapeutic potential. Attached Figure Description
[0015] Figure 1 The graph shows the CCK-8 assay results for each culture medium.
[0016] Figure 2 Image showing the cultured state of human epidermal melanocytes;
[0017] Figure 3 Image showing the melanin content of human epidermal melanocytes;
[0018] Figure 4 The figure shows the results of an experiment on the differentiation of human umbilical cord mesenchymal stem cells using serum-free epidermal melanocyte culture medium. Detailed Implementation
[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0020] Example 1
[0021] The specific implementation method of this embodiment is as follows:
[0022] Serum-free epidermal melanocyte culture medium consists of basal medium and additives. The basal medium is DMEM / F12 medium, and the preparation method of the additives is as follows:
[0023] ① Adenine: Weigh 1.22 mg of adenine powder, dissolve it in 1 mL of DMEM / F12 medium, and use sonication to accelerate its dissolution. After complete dissolution, add 1 mL of adenine solution to 30 mL of DMEM / F12 medium and use this as solution 1.
[0024] ② Hepatocyte growth factor (HGF): Dissolve 50 μg of HGF in 1 mL of PBS (use this as the stock solution), add 100 μL of the stock solution to solution 1, and use this as solution 2. (30 mL of DMEM / F12 medium containing adenine)
[0025] ③ Glutamine: Dissolve 0.5 mL of glutamine in solution 2 and use this as solution 3.
[0026] ④ Retinoic acid: Dissolve 1 mg of retinoic acid in 1 mL of DMSO (ultrasonic treatment and heating to 60°C) (this is the stock solution 1). Dilute the stock solution 1 to make the stock solution 2 (10 μL stock solution 1 + 990 μL DMEM / F12 medium). Take 350 μL of the stock solution 2 and add it to the solution 3. This is the solution 4.
[0027] ⑤ Vitamin E: Dissolve 10.5 μL of vitamin E solution in 9.99 mL of anhydrous ethanol, add 10 μL to 9.99 mL of PBS, and finally add 250 μL to solution 4. This is solution 5.
[0028] ⑥ Nicotinic acid: Dissolve 1 mg of nicotinic acid in 1 mL of PBS (sonication is required) (this is the stock solution 1). Dilute stock solution 1 100 times (10 μL stock solution 1 + 990 μL PBS). Add 62.5 μL of the solution to solution 5. This is the solution 6.
[0029] Adjust the volume of solution 6 to 50 mL and name it the initial culture medium. To dissolve the additives other than the six additives mentioned above, prepare stock solutions of the additives other than the six additives mentioned above according to the table below.
[0030]
[0031] Add the stock solution as shown in the table below:
[0032]
[0033] The concentrations of each component in the culture medium in this example are as follows:
[0034]
[0035] Example 2
[0036] The specific implementation method of this embodiment refers to the steps in Embodiment 1.
[0037] The concentrations of each component in the culture medium in this example are as follows:
[0038]
[0039] Example 3
[0040] The specific implementation method of this embodiment refers to the steps in Embodiment 1.
[0041] The concentrations of each component in the culture medium in this example are as follows:
[0042]
[0043] Comparative Example 1
[0044] Unlike Example 1, this culture medium did not contain recombinant human type XVII collagen and sodium hyaluronate.
[0045] Comparative Example 2
[0046] Unlike Example 1, this culture medium did not contain sodium hyaluronate.
[0047] Comparative Example 3
[0048] Unlike Example 1, this culture medium did not contain recombinant human type XVII collagen.
[0049] Example 4
[0050] Cellular CCK-8 assay
[0051] The specific implementation method of this embodiment is as follows:
[0052] Digest human epidermal melanocytes, count them, add 100 μL of 2000 cells to each well, and perform three replicates per group;
[0053] After culturing for 24 hours, observe the cell status. If the cell status is good, proceed to the next experiment.
[0054] Discard the culture medium in the 96-well plate and add the prepared experimental sample (Examples 1-6) and control sample (Melanocyte Growth Medium M3) culture medium, 100 μL / well;
[0055] The cells were cultured in an incubator for 72 hours.
[0056] CCK-8 assay was performed after 72 hours of cell culture. The test solution was prepared at a ratio of CCK-8 stock solution to culture medium of 1:10. The culture medium was discarded, and 100 μL of the test solution was added to each well. The cells were incubated for 2-4 hours, and the absorbance was measured at 450 nm. Figure 1 As shown, the T-test results for Example 1 and Comparative Example 1 were 0.001098, which is much less than 0.01. The T-test results for Example 1 and Comparative Examples 2 and 3 were 0.006989 and 0.002901, respectively, which are also less than 0.01. These results indicate that when recombinant human type XVII collagen and sodium hyaluronate are added to the preferred culture medium of the present invention, they have a certain promoting effect on the proliferation of human epidermal melanocytes. When recombinant human type XVII collagen and sodium hyaluronate are added simultaneously, the promoting effect on the proliferation of human epidermal melanocytes is more obvious, and even better than the currently commercially available M3 culture medium.
[0057] Example 5
[0058] Human epidermal melanocyte culture
[0059] To observe the cell status of human epidermal melanocytes revived and cultured using the culture medium of this patent, after switching to MelanocyteGrowth Medium M3 serum-free medium, which can be further passaged.
[0060] The specific implementation method of this embodiment is as follows:
[0061] Human epidermal melanocytes were revived using the culture medium from Example 1 in the table below and seeded in 25cm culture media. 2The cells were placed in culture flasks. The flasks were then placed in a CO2 incubator at 37°C (95% air, 5% CO2), with the medium changed every 3 days. Melanocytes generally fused after 2 weeks, at which point photographs were taken for observation. After cell fusion, the cells were detached with trypsin-EDTA solution, centrifuged, diluted, and passaged in Melanocyte Growth Medium M3 serum-free medium for two weeks, with photographs taken at each stage. Results are as follows: Figure 2 As shown, after changing to M3 medium, the cell state was not significantly different from that of cells cultured using the medium from Example 1.
[0062]
[0063] Example 6
[0064] Melanin content detection
[0065] The specific implementation method of this embodiment is as follows:
[0066] ① Digest the cells cultured in the T25 culture flask;
[0067] ② Count, in units of 5 × 10 5 The number of cells / well was transferred to 6-well plates, with 2 replicates per group;
[0068] ③ After culturing in serum-free medium of Examples 3, 6 and Melanocyte Growth Medium M3 for 3 days, the cells were digested with EDTA digestive enzyme and transferred to 15 mL centrifuge tubes, and collected by centrifugation.
[0069] ④ Add 50 μL of lysis buffer (containing a mixed solution of 10% and 1M NaOH) to each 15 mL centrifuge tube.
[0070] ⑤ Place in an 80℃ water bath for 1.5 hours for pyrolysis, shaking continuously during the process;
[0071] ⑥ After pyrolysis, transfer to a 96-well plate;
[0072] ⑦ Measure the absorbance at 405 nm using an ELISA reader; the results are as follows. Figure 3 As shown, the melanin content of human epidermal melanocytes cultured in the medium of Example 1 (serum-free melanin medium containing recombinant human type XVII collagen and sodium hyaluronate) exceeded that of human epidermal melanocytes cultured in the medium of Comparative Example 1 (serum-free melanin medium without recombinant human type XVII collagen and sodium hyaluronate) by 24.4%, and even exceeded that of the currently commercially available M3 medium by 37.6%. This indicates that recombinant human type XVII collagen and sodium hyaluronate have a certain promoting effect on the melanin accumulation capacity of human epidermal melanocytes.
[0073] Example 10
[0074] Serum-free epidermal melanocyte culture medium promotes human umbilical cord mesenchymal stem cell differentiation assay
[0075] The specific implementation method of this embodiment is as follows:
[0076] Human umbilical cord mesenchymal stem cells were resuscitated and cultured using DMEM complete medium. When the cell confluence reached approximately 80%, they were digested with 0.05% trypsin / EDTA. The cells were then passaged using the medium from Example 1 of this patent, and cell status was observed after 72 hours. Results are as follows: Figure 4 As shown, after 72 hours of culture, human umbilical cord mesenchymal stem cells began to show obvious dendrites and differentiate into melanocytes. This indicates that the culture medium selected in this patent has a certain promoting effect on the differentiation of human umbilical cord mesenchymal stem cells into human epidermal melanocytes, providing a method for treating vitiligo using cell transplantation technology in the future.
[0077] The above description of the embodiments is provided to enable those skilled in the art to understand and apply the present invention. Those skilled in the art can readily make various modifications to the above embodiments and apply the general principles described herein to other embodiments without creative effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made to the present invention by those skilled in the art based on the disclosure thereof should be within the scope of protection of the present invention.
Claims
1. A serum-free epidermal melanocyte culture medium, characterized in that, It consists of a basal culture medium and additives; the basal culture medium is DMEM / F12; the additives consist of adenine, hepatocyte growth factor (HGF), glutamine, retinoic acid, vitamin E, niacin, sodium selenite, melanocyte-stimulating hormone, endothelin-3, transferrin, triiodothyronine, cholesterol, neuromodulatory protein 1 (NRG1), 3-isobutyl-1-methylpurine (IBMX), dibutyryl cyclic adenosine monophosphate (db-cAMP), cholera toxin, hydrocortisone, recombinant human insulin, sodium hyaluronate, and recombinant human type XVII collagen.
2. The serum-free epidermal melanocyte culture medium according to claim 1, characterized in that, The concentrations of each component in the additive are as follows: adenine: 0-1.8 x 10⁻⁶ -4 M, Hepatocyte Growth Factor (HGF): 10-100 ng / mL, Glutamine: 0.01-2 mM, Retinoic Acid: 0.01-0.07 mg / L, Vitamin E: 0.001-0.005 mg / L, Niacin: 0-0.0125 mg / L, Sodium Selenite: 0.1-5 ng / mL, Melanocyte-Stimulating Factor: 0.01-1 µM, Endothelin-3: 0-200 ng / mL, Transferrin: 5-100 µg / mL, Triiodothyronine: 1-50 nM, Cholesterol: 0.1-5 µg / mL, Neuroregulatory Protein 1 (NRG1): 0-25 ng / mL, 3-Isobutyl-1-methylpurine (IBMX): 5-100 µg / mL, Dibutyrylcyclic Adenosine Monophosphate (db-cAMP): 5-100 µg / mL, Cholera Toxin: 0-100 ng / mL, Hydrocortisone: 0.01-10 µg / mL, Recombinant Human Insulin: 5-100 µg / mL, Sodium Hyaluronate 1-10 µg / mL, Recombinant Human Type XVII Collagen 0.5-4 mg / mL.
3. The serum-free epidermal melanocyte culture medium according to claim 2, characterized in that, The concentration of the additive is adenine: 1.8 x 10⁻⁶. -4 M, Hepatocyte Growth Factor (HGF): 100 ng / mL, Glutamine: 2 mM, Retinoic Acid: 0.07 mg / L, Vitamin E: 0.005 mg / L, Niacin: 0.0125 mg / L, Sodium Selenite: 5 ng / mL, Melanocyte-Stimulating Factor: 0.01 µM, Endothelin-3: 50 ng / mL, Transferrin: 10 µg / mL, Triiodothyronine: 10 nM, Cholesterol: 0.1 µg / mL, Neuroregulatory Protein 1 (NRG1): 0 ng / mL, 3-Isobutyl-1-methylpurine (IBMX): 5 µg / mL, Dibutyrylcyclic Adenosine Monophosphate (db-cAMP): 100 µg / mL, Cholera Toxin: 0 ng / mL, Hydrocortisone: 10 µg / mL, Recombinant Human Insulin: 10 µg / mL, Sodium Hyaluronate 5 µg / mL, recombinant human type XVII collagen 0.5 mg / mL.
4. The serum-free epidermal melanocyte culture medium according to claim 1, 2 or 3 can be used for the preparation of cells for vitiligo treatment, induction of MSC differentiation into melanocytes and cell therapy.