Epidermis skin model with melanin and construction method and application of epidermis skin model

[0054] Example 1:

[0055] This embodiment introduces a melanin-containing epidermal skin model. The melanin-containing epidermal skin model has a stratum corneum, a granular layer, a spinous cell layer, and a basal layer from top to bottom. The melanin-containing epidermal skin model It contains melanocytes, and the melanin-containing epidermal skin model adopts a mixture of primary human epidermal keratinocytes and melanocytes through in vitro co-culture, sub-liquid culture and air-liquid surface culture. The melanin-containing epidermal skin model provided in this embodiment is constructed by applying the principles of cell biology and tissue engineering to a small number of seed cells after being expanded in vitro. Its structure is highly similar to the normal epidermal structure, with a top-down structure The stratum corneum, the granular layer, the spinous layer and the basal layer are in sequence. They have a complete epithelial structure and contain melanocytes, which can highly mimic the synthesis, secretion and transport of melanin in normal epidermis. The melanin-containing epidermal skin model has both the functions of an epidermal skin model and the response of normal melanocytes, and can be used for epidermal-related safety and efficacy testing of various drugs and cosmetics (skin irritation, barrier, moisturizing, anti-inflammatory Efficacy, etc.), and long-term in vitro testing of whitening efficacy.

[0056] In the process of culturing the epidermal skin model containing melanin, human epidermal keratinocytes and melanocytes are co-extracted, co-cultured, and simultaneously inoculated into the model culture chamber, which is compared with the traditional method of separately obtaining and culturing two kinds of cells This operation simplifies the operation process, shortens the overall model construction time, reduces production costs in terms of materials and time, and facilitates the uniform distribution of melanocytes and epidermal keratinocytes.

[0057] The morphological photos of the human original representative skin keratinocytes and melanocytes co-cultured using this method can be seen under the microscope figure 1 The keratinocytes and melanocytes shown in the picture have good cell conditions. The nuclei of melanocytes have good light transmittance, and the dendrites of the cells are less branched (dendritic volume <3), with good cell activity and biological functions.

[0058] Example 2

[0059] This embodiment focuses on an in vitro construction method of an epidermal skin model containing melanin, which includes the following steps:

[0060] Step 1. Obtain primary human epidermal keratinocytes and melanocytes:

[0061] Soak the isolated human skin tissue in ethanol with a mass fraction of 75% for 30 seconds, rinse with phosphate buffered saline PBS, spread the tissue epidermis face down in a petri dish, peel off the subcutaneous loose connective tissue with elbow scissors, and expose it to dense The connective tissues are then transferred to fresh PBS to soak and clean, then the tissues are immersed in 1.2U/mL Dispase enzyme digestion solution with a mass fraction of 1%, soaked at 37°C for 2 hours, and put into 37°C trypsin/EDTA digestion Digest the solution at 37°C for 10 minutes, add 15 mL of stop solution, gently pipette 100 times with an elbow pipette, filter with a 200-mesh sieve under aseptic conditions, centrifuge at 800 rpm for 6 minutes, discard the supernatant, and obtain the original A mixture of human epidermal cells and melanocytes for use.

[0062] The above 100 mL pancreatin/EDTA digestion solution contains 0.025 g pancreatin and 0.01 g EDTA. The stop solution is DMEM containing 10% serum.

[0063] Step 2. Co-culture of primary human epidermal keratinocytes and melanocytes:

[0064] In the mixture of primary human epidermal keratinocytes and melanocytes obtained in step 1, add the first medium, pipette to form a uniform cell suspension, and add 3.5×10 5 Inoculate to T75 culture flask at the density of /bottle and place in 5% CO 2 Incubate in a 37°C incubator for 5 days, change the medium every other day, and obtain expansion products of human epidermal keratinocytes and melanocytes. Among the expansion products of human epidermal keratinocytes and melanocytes, primary human epidermal keratinocytes and melanin The cell ratio is 30:1, spare.

[0065] The composition of the above-mentioned first medium is the human epidermal keratinocyte medium KC-Growth, with 10μL/10 6 Cells integrin α3β1, 10ng of 12-O-tetradecanoylphorbol-13-acetate (12-O-tetradecanoylphorbol-13-acetate).

[0066] Step 3. Submerged culture of epidermal skin model containing melanin:

[0067] Resuspend the amplified products of human epidermal keratinocytes and melanocytes obtained in step 2 in the second medium, and inoculate the bottom area of ​​0.5 cm 2 In the Transwell chamber, the inoculation density is 2.0×10 5 / Chamber, add the second medium inside and outside the chamber, keep the liquid volume in the cell chamber at 200μL, in 5% CO 2 Incubate in a cell incubator at 37°C for 1 day, change the medium every day to prepare a model cell layer, the model cell layer comprising a basal cell layer, and a layer of hemidesmosomal protein on the lower surface of the basal cell layer.

[0068] The composition of the second medium mentioned above is: in 500mL KC-Growth basic medium, add 0.5μg epidermal growth factor, 5mg adenine, 2.5μg transferrin, 1μg keratinocyte growth factor, 2μg insulin, hydrogenated Pine 10μg, bovine pituitary gland extract 10mg, integrin α3β1 is 10μL/10 6 Cells, 12-O-tetradecanoylphorbol-13-acetate is 10ng.

[0069] Step 4. Air-liquid surface culture of epidermal skin model containing melanin:

[0070] Replace the second culture medium in the model cell layer with the third culture medium, carry out gas-liquid surface culture, 2 , 37℃, 95% relative humidity, incubate for 6 days, change the medium every day to prepare a melanin-containing epidermal skin model with a complete epithelial structure.

[0071] The above-mentioned third culture medium is: in 500mL KC-Growth basic solution, add 0.5μg epidermal growth factor, 5mg adenine, 2.5μg transferrin, keratinocyte growth factor 1μg, insulin 2μg, hydrocortisone 50μg, bovine Pituitary gland extract 10mg, integrin α3β1 is 30μL/10 6 cells, 12-O-tetradecanoylphorbol-13-acetate is 50ng, CaCl 2 It is 50mg.

[0072] The morphological photos of the human original representative skin keratinocytes and melanocytes co-cultured using this method can be seen under the microscope figure 1 The keratinocytes and melanocytes shown in the picture have good cell conditions. The nuclei of melanocytes have good light transmittance, and the dendrites of the cells are less branched (dendritic volume <3), with good cell activity and biological functions.

[0073] Example 3:

[0074] This embodiment focuses on an in vitro construction method of an epidermal skin model containing melanin, which includes the following steps:

[0075] Step 1. Obtain primary human epidermal keratinocytes and melanocytes:

[0076] Soak the isolated human skin tissue in ethanol with a mass fraction of 75% for 30 seconds, rinse with phosphate buffered saline PBS, spread the tissue epidermis face down in a petri dish, peel off the subcutaneous loose connective tissue with elbow scissors, and expose it to dense The connective tissue is then transferred to fresh PBS and soaked and cleaned. Then the tissue is immersed in 1.2U/mL Dispase digestion solution with a mass fraction of 1%, soaked at 37°C for 2 hours, and put into 37°C trypsin/EDTA digestion Digest the solution at 37°C for 12 minutes, add 15 mL stop solution, gently pipette 150 times with an elbow pipette, filter with a 200-mesh sieve under aseptic conditions, centrifuge at 800 rpm for 6 minutes, discard the supernatant, and obtain the original A mixture of human epidermal cells and melanocytes for use.

[0077] The above 100 mL pancreatin/EDTA digestion solution contains 0.03 g pancreatin and 0.01 g EDTA. The stop solution is DMEM containing 10% serum.

[0078] Step 2. Co-culture of primary human epidermal keratinocytes and melanocytes:

[0079] In the mixture of primary human epidermal keratinocytes and melanocytes obtained in step 1, add the first medium, pipette into a uniform cell suspension, and measure the volume of 4.5×10 5 Inoculate to T75 culture flask at the density of /bottle and place in 5% CO 2 Incubate in a 37°C incubator for 7 days, change the medium every other day, and obtain expansion products of human epidermal keratinocytes and melanocytes. Among the expansion products of human epidermal keratinocytes and melanocytes, primary human epidermal keratinocytes and melanin The cell ratio is 20:1, spare.

[0080] The above-mentioned first medium is human epidermal keratinocyte medium KC-Growth with 20μL/10 6 Cell integrin α3β1, 30ng of 12-O-tetradecanoylphorbol-13-acetate (12-O-tetradecanoylphorbol-13-acetate).

[0081] Step 3. Submerged culture of epidermal skin model containing melanin:

[0082] The expansion products of the human epidermal keratinocytes and melanocytes were resuspended in the second medium and seeded on the bottom area of ​​0.5 cm 2 In the Transwell chamber, the inoculation density is 2.5×10 5 / Chamber, add the second medium inside and outside the chamber, keep the liquid volume in the cell chamber at 200μL, in 5% CO 2 Incubate in a cell incubator at 37°C for 2 days, change the medium every day to prepare a model cell layer. The model cell layer includes a basal cell layer, and the lower surface of the basal cell layer has a layer of hemidesmosomal protein.

[0083] The above-mentioned second medium is: in 500mL KC-Growth basic medium, add epidermal growth factor 2μg, adenine 15mg, transferrin 4μg, keratinocyte growth factor 2.5μg, insulin 4μg, hydrocortisone 20μg, bovine Pituitary gland extract 30mg, integrin α3β1 is 20μL/10 6 Cells, 12-O-tetradecanoylphorbol-13-acetate is 30ng.

[0084] Step 4. Air-liquid surface culture of epidermal skin model containing melanin:

[0085] Discard the culture medium in the model cell chamber obtained in step 3, and replace the second medium in the culture dish with the third medium, and perform gas-liquid surface culture at 5% CO 2 , 37℃, 95% relative humidity, incubate for 8 days, change the medium every day, and prepare a melanin-containing epidermal skin model with a complete epithelial structure.

[0086] The above-mentioned third culture medium is: in 500mL KC-Growth basal solution, add epidermal growth factor 2μg, adenine 15mg, transferrin 4μg, keratinocyte growth factor 2.5μg, insulin 4μg, hydrocortisone 50μg, bovine brain Pituitary extract 30mg, integrin α3β1 is 40μL/10 6 cells, 12-O-tetradecanoylphorbol-13-acetate is 70ng, CaCl 2 For 50mg.

[0087] The epidermal skin model with melanin constructed by this method is photographed figure 2 , See the H&E stained photo of the histological section of the model image 3. The results show that the constructed epidermal skin model containing melanin contains basal layer, spinous cell layer, granular layer and stratum corneum structure. It has a complete epithelial structure, and most of the melanin is at the bottom of the model, which is close to the melanin in the natural skin structure. Positioning.