A method for obtaining pigment cells by in vitro culture of skin tissue of leopard perch

Abstract

The invention discloses a method for obtaining pigment cells by in vitro culture of skin tissue of leopard gill perch, comprising the following steps: step A: preparing primary cell culture fluid; step B: pre-treating skin tissue of leopard gill perch to obtain pre-treated fish skin tissue; step C-1: digesting the pre-treated fish skin tissue by enzymatic hydrolysis to obtain an explant; step C-2: culturing the explant in vitro by an explant method to obtain primary pigment cells of skin tissue of leopard gill perch. Leopard gill perch skin tissue is successfully separated and cultured by combining enzymatic hydrolysis with an explant method to obtain a primary pigment cell group of skin tissue of leopard gill perch, and co-existence culture of several pigment cells is achieved. Raw materials are provided to explore the function of pigment cells themselves and the gene regulation mechanism, and the in vitro verification experiment prerequisite is provided for further exploring the influence of pigment cells on body color. At the same time, it also lays a foundation for the breeding of fine varieties of leopard gill perch.

Description

Technical Field

[0001] The invention belongs to the technical field of marine fish cell culture, and particularly relates to a method for obtaining pigment cells by in vitro culture of leopard perch skin tissue. Background Art

[0002] Body color is one of the most important traits of fish, and it is also the result of long-term natural selection in the process of fish adapting to the environment. Fish body color and patterns such as stripes are formed by the types and numbers of pigment cells distributed in the skin, the state of the pigment particles contained in the pigment cells, and the reflective ability of the crystal reflective plates in the iris cells. At present, 6 types of pigment cells have been found in fish, which are concentrated in the dermis of skin tissue, namely melanin cells, erythrophores, xanthophores, leucophores, canophores and iridocytes.

[0003] Leopard gill perch (Plectropomus leopardus), also known as the eastern star grouper, belongs to the order Perciformes, family Serranidae, subfamily Epinephelinae, genus Plectropomus, and species of blue-spotted gill perch. It is distributed in tropical or subtropical waters of the Indian Ocean and Pacific Ocean, concentrated on the edges of coral reefs in the South China Sea, East China Sea and Western Pacific Ocean. It has a complex social structure and changing body colors such as black-brown, brown and red. As an important economic fish species in my country, the leopard gill perch has formed a unique body color in order to adapt to the living environment and achieve population exchanges in long-term natural selection: the fish body is bright red, and blue spots are distributed on the skin tissue except on the ventral side. Body color is an important economic trait of the leopard gill perch, and red has a higher economic value. The body color of the eastern star grouper is mainly related to the type, number and state of pigment cells distributed in the skin tissue. At present, it is known that there are four types of pigment cells in the skin of the eastern star grouper: red pigment cells, yellow pigment cells, melanocytes, and iridophores. However, the formation and regulation mechanism of the body color of the leopard gill perch is still unclear. The reason is partly because the research on the regulatory effect of pigment cells on body color is still in its early stages. The reproduction cycle of the eastern star grouper is long, and the embryo microinjection technology has not yet been broken through. It is very difficult to conduct research through technologies such as gene knockout and transgenic family construction. The body color is significantly correlated with the market price of the leopard gill perch, but the analysis of the body color regulation mechanism of the leopard gill perch is still in its early stages. Pigment cells are important units for the formation of body color. Because the pigment cells in the skin tissue of fish are highly differentiated cells with weak proliferation ability, and the fish skin tissue has a high density, the epidermis and dermis are closely combined, and the tissue cell structure is complex, it is difficult to obtain highly active primary pigment cells.

[0004] At present, the methods for isolating and culturing primary pigment cells generally have problems such as low cell yield and survival rate, high operating skills, easy contamination, and cells are prone to perfusion re-injury and have low viability. They have only been successful in some pigment cell types of a small number of fish species. Existing skin pigment separation mainly uses enzyme digestion and culture methods. The shredded skin tissue is digested with trypsin and collagenase to directly obtain pigment cells, and then the mixture is filtered and the obtained cell suspension is cultured. In the existing enzyme digestion and culture method, since the digestive enzyme mainly changes the cell morphology of the extracellular matrix, it may be due to the damage of some protein receptors on the cell membrane by enzyme digestion, resulting in damage and death of pigments, as well as changes in the phenotype and function of the separated cells.

[0005] Therefore, it is necessary to establish a standardized method for isolating and culturing the pigment cells of leopard gill perch, in order to solve the problems of difficult dissociation, low cell survival rate, difficulty in cell adhesion, easy contamination and culture in the existing technology, so as to obtain pigment cells with high activity, provide raw materials for exploring the function of pigment cells themselves and gene regulation mechanism, and provide in vitro verification experimental prerequisites for further exploring the influence of pigment cells on body color. At the same time, it also lays the foundation for the breeding of leopard gill perch. Summary of the invention

[0006] The purpose of the present invention is to establish a standardized method for separating and culturing pigment cells from the skin tissue of leopard gill perch, in order to solve the technical problem:

[0007] A first aspect of the present invention provides a method for obtaining pigment cells by culturing skin tissue of leopard gill bass in vitro, comprising the following steps:

[0008] Step A: preparing primary cell culture medium;

[0009] Step B: pre-treating the skin tissue of the leopard gill perch to obtain pre-treated fish skin tissue;

[0010] Step C-1: digesting the pretreated fish skin tissue by enzymatic hydrolysis to obtain an explant;

[0011] Step C-2: The explants are cultured in vitro by the explant method to obtain primary pigment cells of the skin tissue of the leopard perch.

[0012] Furthermore, the primary cell culture medium includes: DMEM, FBS, triple antibody, TPA solution or BFGF solution.

[0013] Furthermore, FBS is 15% of the volume of the primary cell culture medium; the final concentration of the triple antibody is 3× the triple antibody; the final concentration of the TPA solution is 9.5-9.8 ng / mL, 9.8-10.0 ng / mL or 10.0-10.2 ng / mL; the final concentration of the BFGF solution is 19.5-19.8 ng / mL, 19.8-20.0 ng / mL or 20.0-20.2 ng / mL.

[0014] Furthermore, step B comprises: step B-1: removing skin tissue from the leopard gill perch; step B-2: rinsing the skin tissue with DPBS buffer with different antibiotic concentrations to obtain pretreated fish skin tissue.

[0015] Further, step B-1 is specifically: anesthetizing a 10-month-old, 11-month-old or 12-month-old leopard gill perch, disinfecting the fish body surface of the leopard gill perch with a disinfectant, dissecting and removing the outer membrane of the fish under sterile conditions to obtain skin tissue; step B-2 is specifically: rinsing the skin tissue twice in a DPBS buffer containing 5× triple antibodies, each rinsing time is 17-19 min, 19-21 min, or 21-23 min; rinsing once with a DPBS buffer containing 3× triple antibodies, rinsing for 13-14 min, 14-15 min or 15-16 min, then placing in a culture dish, and cutting the skin tissue into 2 mm 2 The chopped skin tissue was repeatedly rinsed three to four times with DPBS buffer containing 3× tertiary antibodies, each rinse for 18-20 min or 20-22 min, to obtain pretreated fish skin tissue.

[0016] Furthermore, the DPBS buffer containing 5× triple antibodies includes: 500 IU / mL penicillin, 500 μg / mL streptomycin and 1.25 μg / mL amphotericin B; the DPBS buffer containing 3× triple antibodies includes: 300 IU / mL penicillin, 300 μg / mL streptomycin and 750 ng / mL amphotericin B.

[0017] Furthermore, step C-1 is specifically as follows: using the first digestion solution to perform a first digestion reaction on the pretreated fish skin tissue by an enzymatic method to obtain a first precipitate; repeatedly washing the first precipitate three times with a DPBS buffer solution to obtain a fourth precipitate; using the second digestion solution to digest the fourth precipitate is a second digestion reaction to obtain a second digestion reaction suspension; placing the second digestion reaction suspension in a cell filter screen for filtration and centrifugation to obtain an explant and a fifth precipitate; step C-2 is specifically as follows: placing the explant in a PDL-coated cell culture bottle by an explant method for inverted adherent culture; adding 1.4-1.5 mL, 1.5-1.6 mL or 1.6-1.8 mL of primary cell culture fluid to the inverted adherent culture cell culture bottle for upright adherent culture to obtain primary pigment cells of the skin tissue of leopard gill perch.

[0018] Furthermore, the first digestion solution includes: trypsin with a final concentration of 2.0-2.2 mg / mL, 2.2-2.4 mg / mL or 2.4-2.6 mg / mL, EDTA with a final concentration of 0.7-0.9 mM / L, 0.9-1.0 mM / L or 1.0-1.2 mM / L; the temperature of the first digestion reaction is: 25-26°C, 26-27°C or 27-28°C; the time of the first digestion reaction is: 18-19 min, 19-20 min or 20-21 min.

[0019] Furthermore, the second digestion solution includes: collagenase I with a final concentration of 0.08-0.09 mg / mL, 0.09-0.11 mg / mL or 0.11-0.13 mg / mL, DNase I with a final concentration of 11.5-12.0 μg / mL, 12.0-12.5 μg / mL or 12.5-13.0 μg / mL; the temperature of the second digestion reaction is: 25-26°C, 26-27°C or 27-28°C; the time of the second digestion reaction is: 4.5-5.0 h, 5.0-5.5 h or 5.5-6.2 h.

[0020] Furthermore, the temperature of the inverted adherent culture is 27.5-27.8°C, 27.8-28.2°C or 28.2-28.5°C; and the time of the inverted adherent culture is 4.5-4.8 h, 4.8-5.2 h or 5.2-5.5 h.

[0021] A second aspect of the present invention provides a pigment cell group cultured by the above method, wherein the pigment cell group includes melanocytes, erythrocytes, xanthophytes, iridophores and / or neochromatocytes.

[0022] Furthermore, melanocytes, erythrocytes, xanthochromocytes, iridophores and neochromocytes can be co-cultured.

[0023] The present invention uses the skin tissue of leopard gill perch to carry out in vitro culture by an optimized cell dissociation method, that is, an enzymatic method and an explant method are combined to successfully separate the primary pigment cell group of leopard gill perch and realize co-culture. The present invention optimizes the primary pigment cell culture method to realize the culture of leopard gill perch pigment cells, that is, the obtained pigment cells are placed in an optimized primary culture solution for culture and proliferation, and when the number of cells reaches the target value, they are digested by trypsin until the cells become round, and then a culture medium is added to stop digestion and an explant is obtained by centrifugation, thereby realizing the separation and culture of leopard gill perch primary cells. At the same time, the present invention also detects an unknown pigment cell that has not been reported yet, which is defined as a new pigment cell in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above content and the following specific embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. It should be noted that the accompanying drawings are only examples of the technical solutions claimed for protection.

[0025] Figure 1 This is a cell status diagram of the primary culture of skin pigment cells of leopard perch on the 4th day;

[0026] Figure 2 This is the cell status diagram of the primary culture of skin pigment cells of leopard gill bass on the 15th day;

[0027] Figure 3 This is a picture of the skin pigment cell culture of the leopard gill perch (in which, the black arrow in A indicates the melanocyte; the purple arrow in B indicates the iris pigment cell; the red arrow in C indicates the red pigment cell; the yellow arrow in D indicates the yellow pigment cell; the scale bar is 50 µm);

[0028] Figure 4 This is a picture of the co-existence culture of the skin pigment cell group of the leopard gill perch (wherein, the black arrow indicates the melanocyte; the yellow arrow indicates the yellow pigment cell; the purple arrow indicates the iris pigment cell; the red arrow indicates the red pigment cell; the green arrow marks the new pigment cell, and the scale bar is 50 µm);

[0029] Figure 5 This is a diagram showing the primary culture results of skin pigment cells of leopard perch using a simple explant method in comparative example 1;

[0030] Figure 6 This is the result of primary culture of skin pigment cells of leopard gill perch by simple enzymatic hydrolysis in comparative example 2;

[0031] Figure 7 This is a graph showing the results of culturing skin pigment cells of Leopard gill perch using ordinary complete culture medium in Comparative Example 3. DETAILED DESCRIPTION

[0032] The detailed features and advantages of the present invention are described in detail in the specific implementation modes below, and the contents are sufficient to enable any person skilled in the art to understand the technical contents of the present invention and implement them accordingly. Moreover, according to the description, claims and drawings disclosed in this specification, those skilled in the art can easily understand the relevant objects and advantages of the present invention.

[0033] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

[0034] In order to make the purpose, technical scheme and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0035] (1) Source of sample materials

[0036] In the present embodiment, the leopard spiny sea bass was purchased from Chenhai Aquatic Products Co., Ltd., Dongfang City, Hainan Province.

[0037] (2) Sources of reagents and consumables

[0038] Table 1 Sources and item numbers of reagents and consumables required for the experiment

[0039] name Manufacturer Part Number DMEM Gibco 12491015 FBS Sigma F8687 Triple Antibiotic (Antimycotic Solution) Merck A5955 TPA Sigma P8139 DMSO Sigma D8418 BFGF Abmole M11108 MS-222 Abmole M14788 Trypsin Sigma T7409 EDTA Abmole M9878 1× DPBS Gibco 14190144 Collagenase I Sigma C1-28 DNase I MERCK 10104159001 PDL MERCK P6407-5mg Cell culture flasks NEST 707003[T25 vent cover]TC 70μm pore cell filter Shanghai Jingan J0070

[0040] (3) Source of instruments and equipment

[0041] Table 2 Sources and models of instruments and equipment required for the experiment

[0042] name Manufacturer model Biochemical incubator Bluepard BPN-80RHP Fluorescence inverted microscope LEICA DMi8

[0043] Example

[0044] A method for obtaining pigment cells by in vitro culture of skin tissue of leopard perch, comprising the following steps:

[0045] S1. Preparation of primary cell culture medium. The specific steps are as follows:

[0046] FBS (fetal bovine serum) with a volume ratio of 15% of primary cell culture medium, 3× tertiary antibodies (final concentrations: 300 IU / mL penicillin, 300 μg / mL streptomycin and 750 ng / mL amphotericin B), TPA solution with a final concentration of 10 ng / mL (phorbol 12-myristate 13-acetate; preparation of TPA solution: take 100 μg of TPA powder, add 1 mL of DMSO (dimethyl sulfoxide) and stir evenly to obtain a TPA solution with a concentration of 100 μg / mL), BFGF solution with a final concentration of 20 ng / mL (recombinant rat FGF-basic; preparation of BFGF solution: take 100 μg of BFGF powder, add 1 mL of sterile water and stir evenly to obtain a BFGF solution with a concentration of 100 μg / mL), and DMEM (Dulbecco's Modified Eagle's medium) were added. Medium) to the final volume of primary cell culture medium.

[0047] Take the preparation of 1 L of primary cell culture medium as an example: add 150 mL of FBS, 30 mL of 3× triple antibody, 100 μL of 100 μg / mL TPA solution, 200 μL of 100 μg / mL BFGF solution, add DMEM to make up to 1L, and mix to obtain the primary culture medium.

[0048] S2. Pretreatment of skin tissue of leopard gill perch. The specific reference steps are as follows:

[0049] (1) After 12-month-old leopard gill perch was anesthetized with MS-222, the surface of the fish was repeatedly wiped with 75% alcohol, and the skin tissue of the leopard gill perch was removed using sterile scissors and sterile tweezers in a sterile environment.

[0050] (2) After removing the outer membrane of the skin tissue of the leopard gill perch, rinse it twice in DPBS buffer (Dulbecco's Phosphate-Buffered Saline; containing 500 IU / mL penicillin, 500 μg / mL streptomycin and 1.25 μg / mL amphotericin B) containing 5× tertiary antibodies, each rinse for 20 min, and then rinse it once in 3× tertiary antibodies DPBS buffer (containing 300 IU / mL penicillin, 300 μg / mL streptomycin and 750 ng / mL amphotericin B) for 15 min. After rinsing for 15 min, transfer the fish skin tissue to a sterile 90 mm culture dish and cut the fish skin tissue into 2 mm pieces using sterile scissors. 2The pieces were divided into pieces of different sizes, and then DPBS buffer containing 3× tertiary antibodies (containing 300 IU / mL penicillin, 300 μg / mL streptomycin and 750 ng / mL amphotericin B) was added and rinsed repeatedly for 3 times, each time for 20 min, to obtain the pretreated fish skin tissue of leopard gill perch.

[0051] S3. Isolation and culture of primary pigment cells from skin tissue of leopard gill perch. The specific reference steps are as follows:

[0052] (1) The pretreated skin tissue of leopard gill perch was added to a mixture of 2.5 mg / mL trypsin and 1 mM / L EDTA (i.e., the first digestion solution) with a volume of 10 times that of the pretreated skin tissue of leopard gill perch for the first digestion reaction. The temperature of the first digestion reaction was 25-28°C and the time of the first digestion reaction was 20 min. After the first digestion reaction was completed, the mixture was centrifuged at 1000 r / min for 5 min, and the supernatant was discarded to obtain the first precipitate. Then, the first precipitate was resuspended with DPBS buffer, centrifuged at 1000 r / min for 5 min, and the washing was repeated 3 times to obtain the fourth precipitate. The fourth precipitate was subjected to the second digestion reaction using collagenase I with a final concentration of 0.1 mg / mL and DNase I with a final concentration of 12.5 μg / mL (i.e., the second digestion solution). The temperature of the second digestion reaction was 25-28°C and the time of the second digestion reaction was 6 h to obtain the second digestion reaction suspension. The second digestion reaction suspension was transferred into a 70 μm stomatal cell filter and allowed to stand for 5 min, then centrifuged at 1200 r / min for 5 min. The tissue remaining on the 70 μm stomatal cell filter was the explant. The supernatant in the centrifuge tube was discarded to obtain the fifth precipitate in the centrifuge tube, which was resuspended in 2 mL of primary cell culture medium.

[0053] (2) Place the explants in a cell culture flask pre-coated with PDL (Poly-D-lysine) and place it upside down in a biochemical incubator. Culture it at 28°C for 5 h. Slowly add 1.5 mL of primary cell culture medium and place the cell culture flask upright. Continue primary cell culture at 28°C. On the fourth day of primary cell culture, cells begin to migrate from the edge of the tissue block. Add 2 mL of primary cell culture medium (such as Figure 1 As shown); On the 15th day of primary cell culture, the cells that migrated out of the tissue block have basically covered the bottom of the cell culture flask, thus forming a monolayer of cells (as shown Figure 2 as shown).

[0054] The formed monolayer cells were placed under an inverted fluorescence microscope for examination. Figure 3As shown in the figure, different types of pigment granules can be observed, indicating the existence of different types of pigment cells. There are obvious black, red, yellow and iris pigment granules, which represent the successful separation of primary pigment cells of Leopard gill perch: melanocytes, red pigment cells, yellow pigment cells and iris cells.

[0055] like Figure 4 As shown, the attached pigment cells are intact, not broken or damaged, growing well, and the pigment particles are aggregated (e.g. Figure 4 melanocytes indicated by black arrows) or diffuse (e.g. Figure 4 The pigment cells are densely distributed, indicating that the number of cells after separation is large and suitable for subsequent experiments. In addition, the present invention also discovered a new pigment cell (such as Figure 4 The new pigment cell indicated by the green arrow in the middle), and can coexist with the above-mentioned melanocytes, red pigment cells, yellow pigment cells and iris cells, forming the primary pigment cell group of the skin tissue of leopard gill perch.

[0056] Comparative Example 1

[0057] In step S3 of the embodiment of the present invention, the primary cell culture method is a combination of an enzymatic hydrolysis method and an explant method to obtain primary pigment cells.

[0058] Compared with the embodiment, the difference between this comparative example 1 is that the primary cell culture method is different. This comparative example 1 adopts a simple explant method, that is, the leopard gill perch pretreated fish skin tissue obtained by step S2 is directly placed as an explant in a cell culture bottle pre-coated with PDL for adherent culture.

[0059] The experimental results found that Figure 5 As shown, the primary cell culture using the simple explant method of Comparative Example 1 is difficult for pigment cells to migrate out due to the dense skin tissue, resulting in no cell migration or muscle cell migration and proliferation.

[0060] Comparative Example 2

[0061] In step S3 of the embodiment of the present invention, the primary cell culture method is a combination of an enzymatic hydrolysis method and an explant method to obtain primary pigment cells.

[0062] Compared with the embodiment, the difference between this comparative example 2 is that the primary cell culture method is different, and this comparative example 2 adopts a simple enzymatic hydrolysis method. That is, the reaction time of the second digestion reaction in step S3 is prolonged until the pigment cells on the tissue block are completely digested and fall off into the second digestion solution, and the skin tissue and supernatant filtered through the 70 μm pore cell filter are discarded after centrifugation, and the fifth precipitate is resuspended in 2 mL of primary cell culture solution, and the resuspended solution of the fifth precipitate is inoculated in a cell culture bottle and cultured in the primary culture solution.

[0063] The experimental results found that Figure 6 As shown, the primary cells were cultured by the simple enzymatic hydrolysis method of Comparative Example 2. Because the skin tissue is dense, the enzymatic hydrolysis method resulted in that only pigment cell fragments were collected after the dissociation.

[0064] Comparative Example 3

[0065] In steps S1 and S3 of the embodiment of the present invention, the primary cell culture medium is: FBS with a volume ratio of 15% of the primary cell culture medium, 3× triple antibodies (final concentrations of 300 IU / mL penicillin, 300 μg / mL streptomycin and 750 ng / mL amphotericin B), TPA solution with a final concentration of 10 ng / mL, and BFGF solution with a final concentration of 20 ng / mL are added, and DMEM is added to make up to the final volume of the primary cell culture medium.

[0066] Compared with the embodiment, the difference between this comparative example 3 is that the culture medium formula for primary cell culture is different. The culture medium formula used in this comparative example 3 is a common complete culture medium: 449.5mL of DMEM stock solution, 50mL of FBS, and 500uL of double antibiotic solution.

[0067] The experimental results found that Figure 7 As shown, the use of the primary culture medium formula of Comparative Example 3 for cell culture results in that the pigment cells are not easy to adhere to the wall or do not proliferate.

[0068] It can be concluded that the present invention uses the skin tissue of the leopard gill perch through an optimized cell dissociation method, that is, the enzymatic method and the explant method are combined for in vitro culture, and the primary pigment cells of the leopard gill perch are successfully isolated. The present invention optimizes the primary pigment cell culture method to achieve the culture of the leopard gill perch pigment cells, that is, the obtained pigment cells are placed in an optimized primary culture medium for culture and proliferation. If there is a fixed requirement for the number of cells, when the number of cells reaches the target value, the cells are digested by trypsin until they become round, and then the culture medium is added to stop the digestion and the cell suspension is obtained by centrifugal resuspension, thereby achieving the separation and culture of the primary cells of the leopard gill perch. At the same time, the present invention also detects an unknown type of pigment cell, which may be another type of iris cell, or it may be a yellow pigment and iris donor cell.

[0069] The terms and expressions used herein are for descriptive purposes only and the present invention should not be limited to these terms and expressions. The use of these terms and expressions does not mean to exclude any equivalent features of the illustrations and descriptions (or parts thereof), and it should be recognized that various modifications that may exist should also be included in the scope of the claims. Other modifications, changes and substitutions may also exist. Accordingly, the claims should be deemed to cover all such equivalents.

[0070] Similarly, it should be pointed out that although the present invention has been described with reference to the current specific embodiments, ordinary technicians in this technical field should realize that the above embodiments are only used to illustrate the present invention, and various equivalent changes or substitutions may be made without departing from the spirit of the present invention. Therefore, as long as the changes and modifications to the above embodiments are within the scope of the essential spirit of the present invention, they will fall within the scope of the claims of the present invention.

Claims

1. A method for obtaining pigment cells by in vitro culture of skin tissue of leopard gill perch, characterized in that: The following steps are involved: Step A: preparing a primary cell culture medium, wherein the primary cell culture medium comprises DMEM, FBS, a tertiary antibody, a TPA solution and a BFGF solution; the FBS is 14.0-14.5%, 14.5-15.0% or 15.0-15.5% of the volume of the primary cell culture medium; the tertiary antibody is a 3×tertiary antibody; the 3×tertiary antibody is composed of penicillin at a final concentration of 300 IU / mL, streptomycin at a final concentration of 300 μg / mL and amphotericin B at a final concentration of 750 ng / mL; the final concentration of the TPA solution is 9.5-9.8 ng / mL, 9.8-10.0 ng / mL or 10.0-10.2 ng / mL; the final concentration of the BFGF solution is 19.5-19.8 ng / mL, 19.8-20.0 ng / mL or 20.0-20.2 ng / mL; Step B: pre-treating the skin tissue of the leopard gill perch to obtain pre-treated fish skin tissue; The step B comprises: step B-1: removing skin tissue from the leopard gill perch; step B-2: rinsing the skin tissue with DPBS buffer with different antibiotic concentrations to obtain the pretreated fish skin tissue; Step C-1: digesting the pretreated fish skin tissue by enzymatic hydrolysis to obtain an explant; subjecting the pretreated fish skin tissue to a first digestion reaction using a first digestion solution and then centrifuging to obtain a first precipitate; repeatedly washing and centrifuging the first precipitate three times using a DPBS buffer solution to obtain a fourth precipitate, wherein the centrifugation condition is centrifugation at a speed of 1000 r / min for 5 minutes; digesting the fourth precipitate using a second digestion solution to obtain a second digestion reaction suspension; filtering the second digestion reaction suspension in a 70 μm cell filter sieve and centrifuging at a speed of 1200 r / min for 5 minutes to obtain the explant and the fifth precipitate; The first digestion solution comprises: trypsin with a final concentration of 2.0-2.2 mg / mL, 2.2-2.4 mg / mL or 2.4-2.6 mg / mL, and EDTA with a final concentration of 0.7-0.9 mM, 0.9-1.0 mM or 1.0-1.2 mM; the temperature of the first digestion reaction is: 25-26°C, 26-27°C or 27-28°C; the time of the first digestion reaction is: 18-19 min, 19-20 min or 20-21 min; The second digestion solution comprises: collagenase I with a final concentration of 0.08-0.09 mg / mL, 0.09-0.11 mg / mL or 0.11-0.13 mg / mL, and DNase I with a final concentration of 11.5-12.0 μg / mL, 12.0-12.5 μg / mL or 12.5-13.0 μg / mL; the temperature of the second digestion reaction is: 25-26°C, 26-27°C or 27-28°C; the time of the second digestion reaction is: 4.5-5.0 h, 5.0-5.5 h or 5.5-6.2 h; Step C-2: The explant is cultured in vitro by the explant method to obtain primary pigment cells of the skin tissue of leopard gill perch; the explant is placed in a poly-lysine-coated cell culture bottle for inverted adherent culture; the temperature of the inverted adherent culture is 27.5-27.8°C, 27.8-28.2°C or 28.2-28.5°C; the time of the inverted adherent culture is 4.5-4.8 h, 4.8-5.2 h or 5.2-5.5 h; 1.4-1.5 mL, 1.5-1.6 mL or 1.6-1.8 mL of the primary cell culture solution is added to the inverted adherent culture cell culture bottle for upright adherent culture to obtain the primary pigment cells of the skin tissue of leopard gill perch.

2. The method for obtaining pigment cells according to claim 1, characterized in that: The step B-1 specifically comprises: anesthetizing the 10-month-old, 11-month-old or 12-month-old leopard gill perch, disinfecting the fish body surface of the leopard gill perch with a disinfectant, dissecting and removing the outer layer of the fish body membrane under aseptic conditions to obtain the skin tissue; The step B-2 specifically comprises: rinsing the skin tissue twice in a DPBS buffer solution containing 5× triple antibodies, each rinsing time being 17-19 min, 19-21 min, or 21-23 min; rinsing once in a DPBS buffer solution containing 3× triple antibodies, rinsing for 13-14 min, 14-15 min, or 15-16 min, then placing in a culture dish, and cutting the skin tissue into pieces of 2 mm 2 The cut pieces were cut into pieces of different sizes, and the DPBS buffer containing 3× tertiary antibodies was added to repeatedly rinse the cut skin tissue three to four times, with each rinsing for 18-20 min or 20-22 min to obtain the pretreated fish skin tissue.

3. The method for obtaining pigment cells according to claim 2, characterized in that: The DPBS buffer containing 5× tertiary antibodies includes: 500 IU / mL penicillin, 500 μg / mL streptomycin, and 1.25 μg / mL amphotericin B; The DPBS buffer containing 3× tertiary antibodies includes: 300 IU / mL penicillin, 300 μg / mL streptomycin and 750 ng / mL amphotericin B.

Images