Separation and culture method of plectropomus leopardus haematochrome cells

Abstract

The invention relates to the technical field of cell separation and culture, in particular to a method for separating and culturing haematochrome cells of plectropomus leopardus. The separation method of the plectropomus leopardus haematochrome cells is created for the first time, the EDTA digestive juice is used for pre-digesting epidermis cells on the outer layers of the plectropomus leopardus fins, then the collagenase digestive juice is used for digesting connective tissues (pigmented cells) of internal dermis, digestive enzymes such as trypsin and other mechanical methods which have great damage to the cells are not used, and the method is suitable for large-scale production of the plectropomus leopardus haematochrome cells. The plectropomus leopardus haematochrome cell can reduce cell damage, can be effectively separated to obtain the plectropomus leopardus haematochrome cell which is high in purity, good in activity and capable of being cultured in vitro, and lays a foundation for subsequent experiments such as single cell sequencing, cell in-vitro culture and molecular function verification.

Description

Technical Field

[0001] The invention relates to the technical field of cell separation and culture, in particular to a method for separating and culturing red pigment cells of leopard gill bass. Background Art

[0002] The body color of fish is very diverse due to the different types, distribution and arrangement of pigment particles of different pigment cells on the surface of fish. Therefore, the ability to isolate a single pigment cell is of great significance to the study of the biological mechanism of animal body color formation, which is conducive to exploring the molecular mechanism behind the formation of fish body color and laying the foundation for molecular breeding. The pigment cells of fish mainly include melanocytes, xanthophytes, erythrocytes, iridophytes and cyanophores. In 1982, researchers such as Eisinger successfully isolated human melanocytes in vitro for the first time. Since then, pigment cells of many mammals have been successfully isolated and cultured in vitro. At present, pigment cells of many fish have been successfully isolated, mainly melanocytes and xanthophytes. In 1987, Clark et al. successfully isolated the melanocytes of goldfish using a digestive solution containing collagenase III and adrenaline; in 2004, Sato et al. successfully isolated the red melanocytes of Nile tilapia by digesting the tail fin tissue with collagenase II; in 2019, Han et al. successfully isolated the yellow melanocytes and melanocytes of large yellow croaker in vitro by digesting the scales with collagenase solution. In 2021, Song Hongmei et al. successfully isolated the yellow melanocytes and melanocytes of the orange cichlid by digestion with trypsin and collagenase I, and successfully passaged them to establish a cell line.

[0003] To separate pigment cells from the dermis of fish, researchers usually use collagenase to digest the fins, skin, and scale tissues of fish to obtain a single-cell suspension containing pigment cells, and then use density gradient centrifugation to separate the pigment cells from other cells. At present, due to the diversity of fish species, there are relatively large differences in the separation methods for pigment cells of different fish, but they mainly use collagenase to digest the tissue. Collagenase is derived from Clostridium histolyticum and can break down collagen in connective tissue. The pigment cells of fish are usually present in the connective tissue of the dermis, so collagenase can break down the connective tissue and dissociate the pigment cells from the connective tissue.

[0004] Cadherins are a class of calcium-dependent cell adhesion molecules whose main function is to mediate adhesion between cells of the same type and maintain the structure and stability of tissues. They are type I transmembrane glycoproteins that rely on the presence of calcium ions (Ca²⁺) to maintain their structure and function. EDTA in EDTA digestion solution is a chelating agent that reduces the adhesion between cells and between cells and the matrix by chelating the calcium ions required by calcium-dependent adhesion molecules (such as cadherins) on the cell membrane, thereby promoting cell separation. This method is gentle and does not damage the cell membrane, and is suitable for cells that require gentle treatment. EDTA digestion solution has a good digestive effect on the epidermis, but has little effect on the connective tissue of the dermis.

[0005] Density gradient centrifugation is a method that uses centrifugal force to form a continuous or discontinuous density gradient medium in a centrifuge tube. The sample is centrifuged so that the different components of the sample are distributed in different positions according to their density differences, thereby achieving separation and purification. In density gradient centrifugation, the sample is placed on the top of a centrifuge tube containing a density gradient medium. Under the action of centrifugal force, the different components in the sample settle or float in the gradient medium according to their density differences, forming different layers. By collecting these layers, the sample components can be separated and purified. Commonly used media for density gradient centrifugation include sucrose, Percoll, and cesium chloride.

[0006] Leopard gill perch, commonly known as grouper, is a precious commercial fish. It not only has delicate meat and delicious taste, but also has high economic value because its skin is rich in nutrients such as astaxanthin and β-cryptoxanthin. In addition, leopard gill perch has a certain ornamental value due to its bright body color. It is very popular among consumers and has become an important precious marine aquaculture species along the southern coast of my country. The economic value of leopard gill perch is related to its body color. The price of red individuals is significantly higher than that of black individuals. During the breeding process, the black individuals need to be isolated from direct sunlight and fed with astaxanthin-rich feed to make them redder. This greatly increases the breeding costs of farmers. After the reddening, the body color of the fish is uneven, and some are still black.

[0007] At present, the technologies that can separate a single type of cell population include serial dilution, mouth pipette technology, micromanipulation, flow cytometry (FACS), laser capture microdissection (LCM) and microfluidics. The serial dilution method is easy to operate, but the separation efficiency is low. Micromanipulation, FACS, LCM and microfluidics require certain instruments and equipment, and the cost is relatively high. The mouth pipette technology is low in cost, but requires the operator to have a certain level of proficiency. So far, there has been no research on the separation of red pigment cells in leopard gill perch. There are relatively few studies on fish pigment cells, and fish pigment cells are more difficult to separate than mammals. The inventor tried to digest the skin of leopard gill perch with trypsin, but trypsin has a weak digestion effect on the connective tissue of the dermis where pigment cells are located, and because trypsin has a broad-spectrum digestion effect on a variety of proteins, it will damage the cells and cause rupture. Therefore, isolating a single leopard gill perch's red pigment cell is conducive to exploring the molecular mechanism of leopard gill perch's body color formation, and can lay the foundation for subsequent molecular breeding work focusing on leopard gill perch's body color. Summary of the invention

[0008] The purpose of the present invention is to provide a method for separating and culturing erythrocytes of leopard gill perch, so as to solve the problems existing in the above-mentioned prior art.

[0009] To achieve the above object, the present invention provides the following solutions: The invention provides a method for separating erythrocytes of leopard gill perch, comprising the following steps: Taking the fin of the leopard gill perch, performing a first digestion, a second digestion and a density gradient centrifugation in sequence to obtain the leopard gill perch red pigment cell; The digestion solution used in the first digestion is EDTA digestion solution; the digestion solution used in the second digestion is collagenase digestion solution; and the medium used in the density gradient centrifugation is Percoll solution.

[0010] Preferably, the concentration of EDTA in the EDTA digestion solution is 0.04wt%; And / or, the collagenase digestion solution further comprises DNase I and bovine serum albumin; the concentration of collagenase in the collagenase digestion solution is 1 mg / mL, the concentration of DNase I is 0.1 mg / mL, and the concentration of bovine serum albumin is 10 mg / mL; and the collagenase is collagenase IV.

[0011] Preferably, the first digestion time is 20-30 min, the temperature is 25°C-28°C, and the rotation speed is 200 rpm; And / or, the second digestion time is 2 hours, the temperature is 25°C-28°C, and the rotation speed is 60 rpm.

[0012] Preferably, the density gradient centrifugation comprises the steps of sequentially adding a 20% by volume Percoll solution and a 30% by volume Percoll solution.

[0013] Preferably, the fish fins include one or more of a caudal fin, a dorsal fin and anal fin.

[0014] The inventors found that it was difficult to obtain pigment cells by digestion using the traditional trypsin digestion method, and the cells were severely damaged, so they improved the method. First, the epidermis of the fish fins was digested using EDTA digestion solution, which caused relatively little tissue damage, and then the connective tissue where the pigment cells were located was digested using collagenase digestion solution, and a single cell suspension containing red pigment cells could be successfully obtained. The cells were layered using Percoll reagent to obtain a single cell suspension rich in red pigment cells. Finally, red pigment cells were accurately sucked out under a microscope using a mouth pipette to obtain red pigment cells with extremely high purity.

[0015] The present invention provides the use of the leopard gill perch red pigment cells obtained by the separation method in studying the formation mechanism of the leopard gill perch body color and / or molecular breeding.

[0016] The present invention provides application of the leopard gill perch erythrocytic cells obtained by the separation method in studying the molecular mechanism of proliferation and differentiation of the leopard gill perch erythrocytic cells.

[0017] The invention provides an in vitro culture method of leopard gill perch erythrocytic cells, comprising the step of inoculating the leopard gill perch erythrocytic cells into a complete culture medium for culture.

[0018] Preferably, the complete culture medium uses L-15 culture medium as the basal culture medium and further includes 20% by volume of fetal bovine serum and 1% by volume of a triple antigen solution; the triple antibodies in the triple antigen solution include penicillin, streptomycin and gentamicin; and the concentration of the triple antigen solution is 100×.

[0019] Preferably, the culture temperature is 28°C.

[0020] The present invention discloses the following technical effects: The skin of fish is composed of epidermis and dermis. The epidermis is composed of multiple layers of epithelial cells, and the dermis is located below the epidermis, including connective tissue, blood vessels, nerves, glands, etc. The present invention has created for the first time a method for isolating leopard gill perch red pigment cells. The present invention uses EDTA digestion solution to pre-digest the epidermal cells of the outer layer of the fin of the leopard gill perch, and then uses collagenase digestion solution to digest the connective tissue (pigment cells) of the inner dermis, without using digestive enzymes such as trypsin that are highly damaging to cells and other mechanical methods. It can reduce cell damage and effectively separate leopard gill perch red pigment cells with high purity, good activity and can be cultured in vitro, which is conducive to the subsequent culture of cells and single-cell sequencing experiments, laying the foundation for subsequent single-cell sequencing and cell in vitro culture and functional verification. At the same time, the separation method has a simple operation process, short time consumption, low cost, does not require expensive instruments, and has flexible operation steps. The digestion step can be added in the middle to increase the total amount of cells obtained. EDTA and collagenase, which cause relatively little damage to the cells, are used for digestion. The separated cells are in good condition and can be cultured in vitro. This provides technical support and theoretical basis for studying the molecular mechanism of proliferation and differentiation of red pigment cells of leopard gill perch, and is conducive to revealing the formation mechanism of body color of leopard gill perch, laying the foundation for subsequent molecular breeding work.

[0021] Furthermore, the present invention uses collagenase IV for digestion, which has lower trypsin activity and can reduce damage to cells. The present invention also adds bovine serum albumin and DNase I to the collagenase digestion solution. Bovine serum albumin can stabilize other enzymes and slow down the degradation rate of the enzymes; DNase I can decompose the DNA released by the ruptured cells during the cell digestion process to prevent it from entangled on the tissue surface and inhibiting digestion. The present invention uses Percoll for density gradient centrifugation. By setting different medium concentrations, the erythrocytes can be preliminarily separated from other cells to obtain high-purity erythrocytes. The present invention uses a mouth pipette technique to further separate the erythrocytes obtained after density gradient centrifugation, which has low cost and simple operation, and can obtain a single pure erythrocyte for subsequent single-cell sequencing and cell in vitro culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is the experimental flow chart; Figure 2 This is a diagram of cell stratification after density gradient centrifugation; Figure 3 Figure 1 is a picture of erythrocytes cultured in vitro; Figures AD are pictures of erythrocytes cultured in vitro under different scale conditions; the scales of Figures AD are 750μm, 300μm, 75μm and 50μm respectively; Figure 4 The figure shows a comparison of the effects of the traditional method and the method provided by the present invention; wherein A is the traditional method; B is the method provided by the present invention; the arrows represent the red pigment cells; and the scale bar is 120 μm. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The experimental subjects and experimental reagents of the present invention are as follows: 1. Experimental Subjects The 2-year-old leopard gill bass comes from the Huangliu base of Hainan Blue Grain Technology Co., Ltd. The fish are temporarily raised and given normal light, dissolved oxygen and water temperature.

[0030] 2. Experimental Reagents Bovine serum albumin (BSA), deoxyribonuclease I (DNase I), 0.04wt% EDTA digestion solution, 1× PBS buffer and 10× PBS buffer were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; 100× triple antigen solution (penicillin, streptomycin and gentamicin) and collagenase IV were purchased from Jinclone (Beijing) Biotechnology Co., Ltd.; trypsin-EDTA (0.05%) digestion solution and Percoll were purchased from General Electric; fetal bovine serum (FBS) and L-15 medium were purchased from Gibco.

[0031] Collagenase digestion solution: 1 mg / mL collagenase IV, 0.1 mg / mL DNase I, 10 mg / mL bovine serum albumin, dissolved in PBS, and sterilized by filtration using a 0.22 μm filter membrane.

[0032] 100% Percoll solution: Mix Percoll and 10× PBS buffer in a volume ratio of 1:9.

[0033] 4% BSA solution: add 2 g BSA to 50 mL PBS and filter through a 0.22 μm filter to sterilize.

[0034] Complete medium: 10 mL FBS, 500 μL 100× triple antigen solution, add L-15 medium and make up to 50 mL.

[0035] Example 1 The test process of this embodiment is as follows Figure 1 As shown, the details are as follows: 1) Pre-mix 1× PBS buffer and 100× triple antigen solution at a volume ratio of 9:1 to prepare PBS with 10× triple antibody concentration. Anesthetize the leopard gill perch in an ice bath, wipe off the mucus on the body surface with a clean paper towel, cut the dorsal fin, caudal fin and anal fin of the fish with pre-sterilized scissors, and put them in PBS with 10× triple antibody concentration.

[0036] 2) Rinse with PBS five times, place the tissue in 0.04wt% EDTA digestion solution, and digest on a horizontal shaker at 28°C and 200 rpm for 30 min.

[0037] 3) Remove the 0.04wt% EDTA digestion solution and rinse with PBS five times to remove the epidermis and bacteria.

[0038] 4) Transfer the tissue into 20 mL collagenase digestion solution and digest the tissue in the digestion solution at 28°C and 60 rpm on a horizontal shaker for 2 h.

[0039] 5) The digestion solution is passed through a 40μm cell sieve and centrifuged at 200g for 8 minutes in a centrifuge. If more cells are needed, an additional portion of collagenase digestion solution can be prepared during centrifugation to continue digestion (28°C, horizontal shaker 60rpm digestion). After each 10-30 minutes of digestion, the centrifugation step is repeated to collect the cells, and the supernatant containing collagenase obtained from the previous centrifugation is poured back into the tissue to continue digestion (28°C, horizontal shaker 60rpm digestion for 10-30 minutes), and repeated 5-10 times. The centrifuged cell pellet is resuspended in PBS with 4% BSA and stored at 4°C.

[0040] 6) After centrifugation (200g for 8 minutes) of all the cells collected in the previous step, resuspend and wash three times with 4% BSA in PBS, and finally resuspend with 5mL of 4% BSA. Use Percoll to prepare density gradients and perform gradient centrifugation. Add 100% Percoll solution to PBS to prepare 20% and 30% Percoll solutions. In a 15 or 50mL centrifuge tube, use a syringe to add 2-3mL of 30% Percoll solution and 2-3mL of 20% Percoll solution in sequence, add single cell suspension to the top layer, and centrifuge at 400g and 4°C in a centrifuge for 30 minutes to stratify the cells.

[0041] 7) After density gradient centrifugation, the erythrocytes are at the interface of 20% Percoll solution and 30% Percoll solution ( Figure 2 ), carefully aspirate this layer of cells with a pipette, centrifuge at 200g for 8min, resuspend in 4% BSA, and repeat the centrifugation 3 times (200g for 8min) to completely remove Percoll.

[0042] 8) Resuspend the obtained pigment cells in 4% BSA, dilute the cells with 4% BSA to an appropriate concentration, and aspirate some cells to observe in a culture dish, with 8-10 cells between cells. Observe under a microscope, add 10μL PBS to a 200μL centrifuge tube, use a mouth pipette (Yoshida Bio) to aspirate a single red pigment cell, and transfer it to a centrifuge tube. The obtained cells can be used for single-cell sequencing and cell culture in vitro.

[0043] 9) After centrifugation in step 7, add 5 mL of complete medium to resuspend the pellet and place in a 25 cm 2 The cells were cultured in a 28°C incubator and the complete culture medium was replaced every 2 days. After 3 days of culture, observation was performed. The results were as follows: Figure 3The results show that: the method of the present invention can successfully separate erythroblasts, and the separated erythroblasts can be cultured in vitro, and the separated erythroblasts have high purity and good activity.

[0044] Example 2 Comparison of the effects of the conventional method and the method provided by the present invention The method provided by the present invention is the same as step 1) to step 5) of Example 1); Traditional method: trypsin digestion, that is, digestion with pancreatic enzymes, the steps are as follows: 1) Pre-mix 1× PBS buffer and 100× triple antigen solution at a volume ratio of 9:1 to prepare PBS with 10× triple antibody concentration. Anesthetize the leopard gill perch in an ice bath, wipe off the mucus on the body surface with a clean paper towel, cut the dorsal fin, caudal fin and anal fin of the fish with pre-sterilized scissors, and put them in PBS with 10× triple antibody concentration.

[0045] 2) Rinse with PBS five times, cut the tissue into 3-4 mm wide pieces with scissors, put into 20 mL of trypsin-EDTA (0.05%) digestion solution, digest on a horizontal shaker at 28°C and 200 rpm for 30 min, and then use a sterile Pasteur pipette to blow for 5 min.

[0046] 3) The digestion solution was passed through a 40 μm cell sieve and centrifuged at 200 g for 8 min. Afterwards, the digestion solution and the cell pellet obtained by centrifugation in step 5) of Example 1 were resuspended in 4% BSA PBS, and 20 μL of each cell suspension was dripped onto a glass slide to observe the type of cells obtained under a microscope.

[0047] The results are as follows Figure 4 The results show that the method provided by the present invention can successfully prepare a single cell suspension containing red pigment cells ( Figure 4 B in the figure), while the traditional method of digestion using trypsin cannot digest red pigment cells from tissues ( Figure 4 A in ).

[0048] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for isolating erythrocytes of leopard gill perch, characterized in that: The following steps are involved: Taking the fin of the leopard gill perch, performing a first digestion, a second digestion and a density gradient centrifugation in sequence to obtain the leopard gill perch red pigment cell; The digestion solution used in the first digestion is EDTA digestion solution; the digestion solution used in the second digestion is collagenase digestion solution; and the medium used in the density gradient centrifugation is Percoll solution.

2. The separation method according to claim 1, characterized in that The concentration of EDTA in the EDTA digestion solution is 0.04wt%; And / or, the collagenase digestion solution further comprises DNase I and bovine serum albumin; the concentration of collagenase in the collagenase digestion solution is 1 mg / mL, the concentration of DNase I is 0.1 mg / mL, and the concentration of bovine serum albumin is 10 mg / mL; and the collagenase is collagenase IV.

3. The separation method according to claim 1, characterized in that The first digestion time is 20-30 min, the temperature is 25°C-28°C, and the rotation speed is 200 rpm; And / or, the second digestion time is 2 hours, the temperature is 25°C-28°C, and the rotation speed is 60 rpm.

4. The separation method according to claim 1, characterized in that The density gradient centrifugation comprises the steps of sequentially adding a Percoll solution with a volume percentage of 20% and a Percoll solution with a volume percentage of 30%.

5. The separation method according to claim 1, characterized in that The fish fins include one or more of a caudal fin, a dorsal fin and anal fin.

6. Application of the erythrocytes of the leopard perch obtained by the separation method described in any one of claims 1 to 5 in studying the formation mechanism of the leopard perch body color and / or molecular breeding.

7. Application of the erythrocytic cells of the leopard perch obtained by the separation method according to any one of claims 1 to 5 in studying the molecular mechanism of erythrocytic cell proliferation and differentiation of the leopard perch.

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