A culture medium for promoting proliferation of primary human epidermal melanocytes and a preparation method and application thereof

Abstract

The present application belongs to the field of cell and tissue culture, and particularly relates to a culture medium for promoting proliferation of primary human epidermal melanocytes, a preparation method and application thereof. The present application innovatively uses epidermal concentrate factor to promote proliferation of melanocytes. The epidermal concentrate factor is obtained by collecting and concentrating the culture medium of keratinocytes. By simulating the in-vivo microenvironment, the epidermal concentrate factor prepared by the present application can effectively activate the proliferation signal pathway of melanocytes. The concentrate factor contains various growth factors and cytokines secreted by keratinocytes, which can synergistically enhance the division ability of melanocytes. Compared with the traditional culture method, after adding the epidermal concentrate factor, the population doubling time of primary melanocytes is significantly shortened, and the stable pigment production function is maintained.

Description

Technical Field

[0001] This invention belongs to the field of cell and tissue culture, specifically relating to a culture medium for promoting the proliferation of primary human epidermal melanocytes, its preparation method, and its application. Background Technology

[0002] Melanocytes are key cells in the skin, primarily responsible for synthesizing and secreting melanin, playing a crucial role in skin pigmentation, UV protection, and immune regulation. Melanocytes do not differentiate from epidermal cells but originate from embryonic neural crest cells. These cells are pluripotent stem cells that can develop not only into melanocytes but also into other cell types, such as neurons, glial cells, osteocytes, smooth muscle cells, cardiomyocytes, adrenal medullary cells, and chondrocytes. Melanocytes are predominantly dendritic and synthesize melanosomes (the form in which melanin exists within melanocytes). They are mainly distributed in the epidermis and hair follicles, exhibiting a slender, flattened shape and extending long, dendritic processes into surrounding keratinocytes, enabling them to transport melanosomes to other cells.

[0003] However, melanocytes constitute only 3%-7% of the human epidermis, and in primary cell culture, they are easily competitively grown by keratinocytes and fibroblasts. Furthermore, melanocytes have an extremely slow division rate and readily differentiate into mature melanocytes, ceasing proliferation. These problems severely restrict the widespread use of primary human epidermal melanocytes in basic research and clinical applications. Therefore, developing a dedicated melanocyte culture medium with clearly defined components, high efficiency, safety, and suitability for large-scale culture has become an important research direction. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a culture medium that can promote the rapid proliferation of primary melanocytes.

[0005] The present invention also provides a method for preparing the above-mentioned culture medium.

[0006] Another object of the present invention is to provide the application of the above-mentioned culture medium in promoting the rapid proliferation of primary melanocytes.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a culture medium that can promote the rapid proliferation of primary melanocytes, comprising: basal culture medium, fetal bovine serum 5%-10%, bis(butyryl) cyclic adenosine monophosphate 0.1-1mM, 3-isobutyl-1-methylxanthine 0.1-1mM, sodium orthovanadate 1-5μM, phorbol 12-tetradecanoate 13-acetate 10-50ng / ml, bovine pituitary extract 20-30μg / ml, basic fibroblast growth factor 1-5ng / ml, hydrocortisone 0.1-0.5μg / ml, insulin 0.01-0.1μg / ml, bovine transferrin 1-5μg / ml, epidermal concentrating factor 50-100μg / ml, cholera toxin 1-10ng / ml, and endothelin-1 1-10nM.

[0008] The basal culture medium used in the culture medium provided by this invention is F12.

[0009] Preferably, the epidermal concentration factor is prepared by the following method: cryopreserved keratinocytes are revived, subjected to salt stress, added to epidermal culture medium, and cultured at a constant temperature. After the culture is completed, the epidermal culture medium is collected, centrifuged through an ultrafiltration concentration tube, and the supernatant is concentrated to obtain the product.

[0010] Preferably, the specific steps of the salt stress treatment are as follows: add the revived keratinocytes to the salt stress culture medium, culture for 3 hours, then increase the salt concentration and continue to culture for 2 hours, discard the salt stress treatment solution, then wash with preheated calcium- and magnesium-free PBS culture medium, and resuspend.

[0011] Preferably, the salt stress culture medium consists of basal culture medium F12, 5% FBS, 50 U / mL penicillin, 80 μg / mL streptomycin, and 50 mM NaCl; the salt concentration is increased to 80 mM based on the NaCl in the salt stress culture medium.

[0012] Preferably, 1.5-2.0 × 10⁻⁶ mg / mL of the epidermal culture medium is added to each 15 mL of the culture medium. 6 Keratinocytes.

[0013] Preferably, the components of the epidermal culture medium are: DMEM, epidermal growth factor 1-5 ng / ml, insulin 1-5 μg / ml, transferrin 1-5 μg / ml, and hydrocortisone 0.1-0.5 μg / ml; the filtration is performed using a 3 kDa MWCO; the centrifugation speed is 4500 rpm; and the concentrate is concentrated to a 100 mg / ml stock solution.

[0014] Preferably, the culture is carried out at a constant temperature of 37°C and with 5% carbon dioxide for 4-5 days.

[0015] Another object of the present invention is to provide the application of the above-mentioned culture medium in promoting the rapid proliferation of primary melanocytes.

[0016] This invention innovatively utilizes epidermal condensation factor to promote melanocyte proliferation. Epidermal condensation factor is obtained by concentrating cultured keratinocyte culture medium. By mimicking the in vivo microenvironment, the epidermal condensation factor prepared in this invention can effectively activate the melanocyte proliferation signaling pathway. This condensation factor contains various growth factors and cytokines secreted by keratinocytes, which synergistically enhance the melanocyte division capacity. Compared with traditional culture methods, the addition of epidermal condensation factor significantly shortens the population doubling time of primary melanocytes while maintaining stable pigment production function.

[0017] The beneficial effects of this invention are as follows: (1) This invention combines purification culture medium and proliferation culture medium into one culture medium for the first time. The culture medium of this invention can not only purify melanocytes without contamination by keratinocytes and fibroblasts, but also promote the rapid proliferation of melanocytes, reaching 90% confluence in 20 days.

[0018] (2) The epidermal concentration factor prepared by the present invention achieves efficient enrichment and activity retention of active ingredients such as growth factors through optimized preparation process. It has the advantages of high efficiency and controllability, safety and low toxicity. Moreover, the prepared epidermal concentration factor has high activity and purity, and has important application value and industrialization prospects in multiple fields such as 3D model construction and tissue repair. Attached Figure Description

[0019] Figure 1 Cell morphology diagram after culture according to the method provided in Example 1; Figure 2 Cell morphology diagram after cell culture provided in Comparative Example 1; Figure 3 Cell morphology diagram after method culture provided for Comparative Example 2; Figure 4 MITF green immunofluorescence identification of melanocytes (10X); Figure 5 The vimentin of fibroblasts is identified by red immunofluorescence (10X). Detailed Implementation

[0020] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0021] The sources of the raw materials used in this invention are shown in Table 1.

[0022] Table 1 Example 1 (1) Add 1.5 million cells to each T75 flask of cryopreserved keratinocytes in liquid nitrogen tank to salt stress medium (basal medium F12, FBS 5%, 50 U / mL penicillin, 80 μg / mL streptomycin, 50 mM NaCl), and culture at 37°C with 5% carbon dioxide for 3 h. Then increase the salt concentration to 80 mM and continue culturing for 2 h. Discard the salt stress treatment medium, wash with preheated PBS buffer, and resuspend. Add epidermal culture medium (15 ml of medium per T75 flask, change the medium twice a week) and culture at constant temperature. After the culture is completed, collect the epidermal culture medium, place it in an Amicon® Ultra (3 kDa MWCO) filter, centrifuge at 4500 rpm, and concentrate to a stock solution of 100 mg / ml.

[0023] Epidermal culture medium components: DMEM, epidermal growth factor 5ng / ml, insulin 3μg / ml, transferrin 5μg / ml, hydrocortisone 0.25μg / ml; (2) A culture medium that can promote the rapid proliferation of primary melanocytes. The raw material composition is as follows: basal culture medium F12, fetal bovine serum 10%, bis(butyryl) cyclic adenosine monophosphate 0.5M, 3-isobutyl-1-methylxanthine 1mM, sodium orthovanadate 3μM, phorbol 12-tetradecanoate 13-acetate 50ng / ml, bovine pituitary extract 25μg / ml, basic fibroblast growth factor 4ng / ml, hydrocortisone 0.3μg / ml, insulin 0.1μg / ml, bovine transferrin 3μg / ml, epidermal concentrating factor 100μg / ml, cholera toxin 8ng / ml, endothelin-1 10nM.

[0024] (3) Culture medium preparation: Sterilize with UV lamp in the laminar flow hood for 30 minutes and ventilate for 10 minutes; equilibrate all reagents at room temperature for 30 minutes to avoid pH fluctuations caused by temperature differences; mix the raw materials and stir at low speed; use a 0.22 μm PVDF filter membrane for vacuum filtration and sterilization. The entire filtration process is completed in the laminar flow hood, and the filtrate is collected into a sterile reagent bottle; the culture medium is obtained and stored at 4℃ in the dark and used within 72 hours.

[0025] Comparative Example 1 Steps (1) and (3) are the same as in Example 1, except that: (2) A culture medium that can promote the rapid proliferation of primary melanocytes. The raw material composition is as follows: basal culture medium F12, fetal bovine serum 10%, bis(butyryl) cyclic adenosine monophosphate 0.5M, 3-isobutyl-1-methylxanthine 1mM, sodium orthovanadate 3μM, phorbol 12-tetradecanoate 13-acetate 50ng / ml, bovine pituitary extract 25μg / ml, basic fibroblast growth factor 4ng / ml, hydrocortisone 0.3μg / ml, insulin 0.1μg / ml, bovine transferrin 3μg / ml, cholera toxin 8ng / ml, endothelin-1 10nM.

[0026] Comparative Example 2 (1) Add 1.5 million cells to each T75 bottle of keratinocytes frozen in liquid nitrogen tank, add epidermal culture medium (15 ml of culture medium per T75 bottle, change the medium twice a week), and culture at constant temperature. After the culture is completed, collect the epidermal culture medium, place it in an Amicon® Ultra (3 kDa MWCO) filter, centrifuge at 4500 rpm, and concentrate it into a stock solution of 100 mg / ml.

[0027] The other steps are the same as in Example 1.

[0028] Example 1 Primary melanocytes were placed in the culture media prepared in Example 1 and Comparative Examples 1-2 and cultured in a 37°C incubator (5% CO2). The medium was changed 2-3 times per week. The confluence of cells in the culture medium prepared in Example 1 reached 90% after 20 days of culture. However, the confluence of cells in the culture medium prepared in Comparative Example 1 was only 73% after 20 days of culture, and the confluence of cells in the culture medium prepared in Comparative Example 2 was only 83% after 20 days of culture.

[0029] Example 1: Primary melanocytes after culture, as shown Figure 1 As shown. From Figure 1 As can be seen, the cells are densely distributed, exhibiting an overall state of vigorous cell proliferation. Comparative Example 1: Primary melanocytes after culture... Figure 2 As shown. From Figure 2 As can be seen, the cell density is lower than that of the culture medium provided in Example 1. Primary melanocytes cultured using the method provided in Comparative Example 2, as shown... Figure 3 As shown.

[0030] MITF protein, also known as microphthalmia-associated transcription factor, is a highly specific marker for melanocytes. This was confirmed by immunofluorescence analysis, and the cells cultured in Example 1 were identified as melanocytes. Figure 4 Green fluorescence can be detected by fluorescence microscopy, and there is no fibroblast contamination. Figure 5 No red fluorescence was captured when photographed with a fluorescence microscope.

Claims

1. A culture medium that can promote the rapid proliferation of primary melanocytes, characterized in that, include: The basal culture medium consisted of 5%-10% fetal bovine serum, 0.1-1 mM bis(butyl)-cyclic adenosine monophosphate (Bis(2-butyl)-1-methylxanthine) (BMP), 0.1-1 mM 3-isobutyl-1-methylxanthine (BMP), 1-5 μM sodium orthovanadate (PB), 10-50 ng / ml phorbol 12-tetradecanoate 13-acetate (PGA), 20-30 μg / ml bovine pituitary extract (BMP), 1-5 ng / ml basic fibroblast growth factor (BGF), 0.1-0.5 μg / ml hydrocortisone (HPC), 0.01-0.1 μg / ml insulin (PI), 1-5 μg / ml bovine transferrin (BLT), 50-100 μg / ml epidermal concentrating factor (ECF), 1-10 ng / ml cholera toxin (CVT), and 1-10 nM endothelin-1 (EH).

2. The culture medium according to claim 1, characterized in that, The basal culture medium is F12.

3. The culture medium according to claim 1 or 2, characterized in that, The epidermal concentration factor is prepared by the following method: frozen keratinocytes are revived, subjected to salt stress, added to epidermal culture medium, and cultured at a constant temperature. After the culture is completed, the epidermal culture medium is collected, centrifuged through an ultrafiltration concentration tube, and the supernatant is concentrated to obtain the product.

4. The culture medium according to claim 3, characterized in that, The specific steps of the salt stress treatment are as follows: add the revived keratinocytes to the salt stress culture medium and culture for 3 hours. Then increase the salt concentration and continue to culture for 2 hours. Discard the salt stress treatment solution, wash with preheated calcium- and magnesium-free PBS medium, and resuspend.

5. The culture medium according to claim 4, characterized in that, The salt stress culture medium consists of basal medium F12, 5% FBS, 50 U / mL penicillin, 80 μg / mL streptomycin, and 50 mM NaCl; the salt concentration is increased to 80 mM based on the NaCl in the salt stress culture medium.

6. The culture medium according to claim 3, characterized in that, Add 1.5-2.0 × 10 to each 15 mL of epidermal culture medium. 6 Keratinocytes.

7. The culture medium according to claim 3, characterized in that, The components of the epidermal culture medium are: DMEM, epidermal growth factor 1-5 ng / ml, insulin 1-5 μg / ml, transferrin 1-5 μg / ml, and hydrocortisone 0.1-0.5 μg / ml; the filtration is performed using a 3 kDa MWCO; the centrifugation speed is 4500 rpm; and the concentrate is concentrated to a 100 mg / ml stock solution.

8. The culture medium according to any one of claims 3-7, characterized in that, The culture was carried out at a constant temperature of 37℃ and with 5% carbon dioxide for 4-5 days.

9. The use of a culture medium as described in any one of claims 1-8 in promoting the rapid proliferation of primary melanocytes.

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