Method for separating and culturing red pigment cells of leopard bristletooth

Abstract

The present application relates to the technical field of cell separation and culture, in particular to a method for separating and culturing red pigment cells of Plectrypops leopardus.The present application first creates a method for separating red pigment cells of Plectrypops leopardus, and the present application uses EDTA digestion solution to pre-digest epidermal cells on the outer layer of the fin of Plectrypops leopardus, and then uses collagenase digestion solution to digest connective tissue (pigment cells) in the inner dermis, without using trypsin and other digestion enzymes which can cause great damage to cells and other mechanical methods, so that the damage to cells can be reduced, and high-purity red pigment cells of Plectrypops leopardus which are active and can be cultured in vitro can be effectively separated, thereby laying a foundation for subsequent single-cell sequencing, in-vitro cell culture and molecular function verification experiments.

Description

Technical Field

[0001] This invention relates to the field of cell isolation and culture technology, and in particular to a method for isolating and culturing red pigment cells of the leopard gill spiny perch. Background Technology

[0002] The diverse types, distributions, and arrangements of pigment cells on the body surface of fish contribute to their remarkable coloration. Therefore, isolating individual pigment cells is crucial for understanding the biological mechanisms underlying body color formation in animals, facilitating the exploration of the molecular mechanisms behind fish coloration, and laying the foundation for molecular breeding. Fish pigment cells primarily include melanocytes, xanthocyanins, erythrocytes, iridocyanins, and cyanocyanins. In 1982, Eisinger et al. successfully isolated human melanocytes in vitro for the first time. Since then, pigment cells from various mammals have been successfully isolated and cultured in vitro. Currently, pigment cells from various fish species have been successfully isolated, primarily melanocytes and xanthocyanins. In 1987, Clark et al. successfully isolated melanocytes from goldfish using a digestive solution containing collagenase III and adrenaline. In 2004, Sato et al. successfully isolated red cells from Nile tilapia by digesting caudal fin tissue with collagenase II. In 2019, Han et al. successfully isolated yellow and melanocytes from large yellow croaker in vitro by digesting scales with collagenase solution. In 2021, Song Hongmei et al. successfully isolated yellow and melanocytes from orange-crowned cichlids by digestion with trypsin and collagenase I, and successfully passaged them to establish a cell line.

[0003] For isolating pigment cells from the dermis of fish, researchers typically use collagenase to digest the fins, skin, and scales of the fish, obtaining a single-cell suspension containing pigment cells. Density gradient centrifugation is then used to separate the pigment cells from other cells. Currently, due to the diversity of fish species, the methods for isolating pigment cells vary considerably, but all primarily involve digesting the tissue with collagenase. Collagenase, derived from Clostridium histolyticum, can break down collagen in connective tissue. Since fish pigment cells are usually located in the connective tissue of the dermis, collagenase can break down the connective tissue, thus separating the pigment cells from it.

[0004] Cadherins are a class of calcium-dependent cell adhesion molecules whose main function is to mediate adhesion between cells of the same species, maintaining tissue structure and stability. They are type I transmembrane glycoproteins that depend 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, making it suitable for cells requiring gentle treatment. EDTA digestion solution has a good digestive effect on the epidermis, but has almost no effect on the connective tissue of the dermis.

[0005] Density gradient centrifugation is a method that utilizes centrifugal force to create a continuous or discontinuous density gradient medium within a centrifuge tube. By centrifuging, the different components of the sample are separated and purified according to their density differences. In density gradient centrifugation, the sample is placed at the top of a centrifuge tube containing the density gradient medium. Under the influence 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 sea bass, commonly known as the Eastern Star Grouper, is a prized commercial fish. Not only is its flesh delicate and delicious, but its skin is also rich in nutrients such as astaxanthin and β-cryptoxanthin, giving it high economic value. Furthermore, its vibrant coloration makes it highly valued for its ornamental qualities, making it a popular and important aquaculture species in southern coastal my country. The economic value of leopard-gill sea bass is related to its body color; red individuals command significantly higher prices than black ones. During aquaculture, increasing the red coloration of black individuals requires protection from direct sunlight and feeding them astaxanthin-rich feed, which significantly increases the farmers' costs. Moreover, even after the coloration process, the fish's coloration remains uneven, with some still retaining a darker hue.

[0007] Currently, techniques for isolating single-species cell populations include serial dilution, pipette technique, micromanipulation, flow cytometry (FACS), laser capture microdissection (LCM), and microfluidics. Serial dilution is simple to operate but has low separation efficiency. Micromanipulation, FACS, LCM, and microfluidics require specific equipment and are relatively expensive. Pipette technique is less expensive but requires operators with a certain level of skill. To date, there has been no research on the isolation of red pigment cells from the leopard-gill sea bass. Research on pigment cells in fish is relatively limited, and fish pigment cells are more difficult to isolate than those in mammals. The inventors attempted to digest the skin of the leopard-gill sea bass with trypsin, but trypsin has a weak digestive effect on the connective tissue of the dermis where pigment cells are located, and because trypsin has a broad-spectrum digestive effect on various proteins, it can damage and rupture cells. Therefore, isolating a single red pigment cell of the leopard-gill spiny perch is beneficial for exploring the molecular mechanism of body color formation in the leopard-gill spiny perch, and can lay the foundation for subsequent molecular breeding work focused on body color. Summary of the Invention

[0008] The purpose of this invention is to provide a method for isolating and culturing red pigment cells of the leopard gill spiny perch, so as to solve the problems existing in the prior art.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] This invention provides a method for isolating red pigment cells from the leopard-gill spiny perch, comprising the following steps:

[0011] The fins of the leopard-gill spiny perch were taken and subjected to first digestion, second digestion and density gradient centrifugation in sequence to obtain the red pigment cells of the leopard-gill spiny perch.

[0012] The digestion solution used in the first digestion was EDTA digestion solution; the digestion solution used in the second digestion was collagenase digestion solution; and the medium used in the density gradient centrifugation was Percoll solution.

[0013] Preferably, the concentration of EDTA in the EDTA digestion solution is 0.04 wt%.

[0014] And / or, the collagenase digestion solution further includes 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; the collagenase is collagenase IV.

[0015] Preferably, the first digestion time is 20-30 minutes, the temperature is 25℃-28℃, and the rotation speed is 200 rpm;

[0016] And / or, the second digestion time is 2 hours, the temperature is 25℃-28℃, and the rotation speed is 60 rpm.

[0017] Preferably, the density gradient centrifugation includes the steps of sequentially adding a Percoll solution with a volume percentage of 20% and a Percoll solution with a volume percentage of 30%.

[0018] Preferably, the fins include one or more of the caudal fin, dorsal fin, and anal fin.

[0019] The inventors discovered that traditional trypsin digestion methods are ineffective in obtaining pigment cells and cause significant cell damage. Therefore, they improved the method. First, they used a less damaging EDTA digestion solution to digest the epidermis of the fish fins. Then, they used a collagenase digestion solution to digest the connective tissue containing pigment cells, successfully obtaining a single-cell suspension containing erythropoietin. Using Percoll reagent to separate the cells into layers yielded a single-cell suspension rich in erythropoietin. Finally, using a pipette under a microscope, they precisely aspirated the erythropoietin cells, obtaining highly pure erythropoietin cells.

[0020] This invention provides the application of red pigment cells of the leopard-gill spiny perch obtained by the above-described separation method in the study of the formation mechanism of body color of the leopard-gill spiny perch and / or in molecular breeding.

[0021] This invention provides the application of leopard-gill spiny perch red pigment cells obtained using the above-described separation method in studying the molecular mechanisms of leopard-gill spiny perch red pigment cell proliferation and differentiation.

[0022] This invention provides an in vitro culture method for leopard gill spiny perch red pigment cells, including the step of inoculating leopard gill spiny perch red pigment cells into a complete culture medium for culture.

[0023] Preferably, the complete culture medium is based on L-15 medium and further includes 20% fetal bovine serum by volume and 1% triantigen solution by volume; the triantigen solution contains penicillin, streptomycin and gentamicin; the concentration of the triantigen solution is 100×.

[0024] Preferably, the culture temperature is 28°C.

[0025] The present invention discloses the following technical effects:

[0026] Fish skin consists of an epidermis and a dermis. The epidermis is composed of multiple layers of epithelial cells, while the dermis, located beneath the epidermis, contains connective tissue, blood vessels, nerves, and glands. This invention is the first to establish a method for isolating leopard-gill spiny perch erythroblast cells. This method uses EDTA digestion solution to pre-digest the epidermal cells of the outer fin of the leopard-gill spiny perch, and then uses collagenase digestion solution to digest the connective tissue (pigment cells) of the inner dermis. This method avoids using trypsin or other highly damaging digestive enzymes and mechanical methods, thus minimizing cell damage. It effectively isolates high-purity, highly active leopard-gill spiny perch erythroblast cells suitable for in vitro culture, facilitating subsequent cell culture and single-cell sequencing experiments, and laying the foundation for subsequent single-cell sequencing, in vitro cell culture, and functional verification. Meanwhile, this separation method is simple to operate, time-saving, and low-cost, requiring no expensive equipment. The operation steps are flexible, allowing for the addition of digestion steps midway to increase the total number of cells obtained. Digestion is performed using EDTA and collagenase, which are less damaging to cells. The separated cells are in good condition and can be cultured in vitro. This provides technical support and a theoretical basis for studying the molecular mechanisms of red pigment cell proliferation and differentiation in leopard-gill spiny perch, and is also conducive to revealing the formation mechanism of body color in leopard-gill spiny perch, laying the foundation for subsequent molecular breeding work.

[0027] Furthermore, this invention uses collagenase IV for digestion, which has low trypsin activity, reducing cell damage. This invention also adds bovine serum albumin and DNase I to the collagenase digestion solution. Bovine serum albumin stabilizes other enzymes and slows down their degradation; DNase I breaks down DNA released from ruptured cells during digestion, preventing it from tangling on the tissue surface and inhibiting digestion. This invention uses Percoll density gradient centrifugation; by setting different medium concentrations, erythroblasts can be initially separated from other cells to obtain highly pure erythroblasts. This invention employs a pipette technique to further separate the erythroblasts obtained after density gradient centrifugation, which is low-cost, simple to operate, and yields single, pure erythroblasts for subsequent single-cell sequencing and in vitro cell culture. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a flowchart of the experiment.

[0030] Figure 2This is a layered diagram of cells after density gradient centrifugation;

[0031] Figure 3 Image of erythroblasts cultured in vitro; where AD represents images of erythroblasts cultured in vitro under different scale conditions; the scale bars of AD are 750μm, 300μm, 75μm and 50μm, respectively;

[0032] Figure 4 This is a comparison of the effects of the conventional method and the method provided by the present invention; where A represents the conventional method; B represents the method provided by the present invention; the arrows represent red pigment cells; the scale bar is 120 μm. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0036] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0037] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0038] The experimental subjects and reagents of this invention are as follows:

[0039] I. Experimental Subjects

[0040] The 2-year-old leopard gill sea bass came from the Huangliu base of Hainan Lanliang Technology Co., Ltd. The fish were temporarily held in a normal environment with normal lighting, dissolved oxygen, and water temperature.

[0041] II. Experimental Reagents

[0042] Bovine serum albumin (BSA), deoxyribonuclease I (DNase I), 0.04 wt% EDTA digestion solution, 1×PBS buffer, and 10×PBS buffer were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; 100× triple antigen solution (penicillin, streptomycin, and gentamicin) and collagenase IV were purchased from Kingclone (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.

[0043] Collagenase digestion solution: 1 mg / mL collagenase IV, 0.1 mg / mL DNase I, 10 mg / mL bovine serum albumin, dissolved in PBS and then filtered through a 0.22 μm filter membrane for sterilization.

[0044] 100% Percoll solution: Mix Percoll with 10×PBS buffer at a volume ratio of 1:9.

[0045] 4% BSA solution: Add 2g BSA to 50mL PBS and filter through a 0.22μm filter membrane for sterilization.

[0046] Complete culture medium: 10 mL FBS, 500 μL 100× triple antigen solution, add L-15 culture medium, and bring the volume to 50 mL.

[0047] Example 1

[0048] The test procedure in this embodiment is as follows: Figure 1 As shown, the details are as follows:

[0049] 1) Prepare PBS with a 10× triple antibody concentration by mixing 1×PBS buffer and 100× triple antibody solution at a volume ratio of 9:1. Anesthetize the leopard gill spiny perch with an ice bath, wipe off the mucus on the body surface with a clean paper towel, and cut off the dorsal fin, caudal fin, and anal fin with pre-sterilized scissors and place them in PBS with a 10× triple antibody concentration.

[0050] 2) Rinse 5 times with PBS, place the tissue in 0.04wt% EDTA digestion solution, and digest for 30 min at 28℃ and 200rpm on a horizontal shaker.

[0051] 3) Remove 0.04wt% EDTA digestion solution and rinse 5 times with PBS to remove the epidermis and bacteria.

[0052] 4) Transfer the tissue into 20 mL of collagenase digestion solution and digest the tissue in the digestion solution at 28 °C and 60 rpm on a horizontal shaker for 2 h.

[0053] 5) Pass the digestion solution through a 40μm cell sieve and centrifuge at 200g for 8 minutes. If more cells are needed, prepare an additional batch of collagenase digestion solution for further digestion (28℃, horizontal shaker at 60 rpm). Repeat the centrifugation step every 10-30 minutes to collect cells. Pour the supernatant containing collagenase obtained from the previous centrifugation back into the tissue and continue digestion (28℃, horizontal shaker at 60 rpm for 10-30 minutes), repeating 5-10 times. Resuspend the centrifuged cell pellet in PBS with 4% BSA and store temporarily at 4℃.

[0054] 6) After centrifuging all cells collected in the previous step (200g for 8 min), wash three times with 4% BSA in PBS, and finally resuspend in 5 mL of 4% BSA. Configure a density gradient using Percoll and perform gradient centrifugation. Add 100% Percoll solution to PBS to prepare 20% and 30% Percoll solutions. In 15 or 50 mL centrifuge tubes, add 2-3 mL of 30% Percoll solution and 2-3 mL of 20% Percoll solution sequentially using a syringe. Add a single-cell suspension on top. Centrifuge at 400g, 4℃ for 30 min to separate the cells into layers.

[0055] 7) After density gradient centrifugation, the erythroblasts were located at the interface between 20% Percoll solution and 30% Percoll solution. Figure 2 Carefully aspirate this layer of cells with a pipette, centrifuge at 200g for 8 minutes, resuspend in 4% BSA, and repeat centrifugation 3 times (200g for 8 minutes) to completely remove Percoll.

[0056] 8) Resuspend the obtained pigment cells in 4% BSA. Dilute the cells to an appropriate concentration with 4% BSA, and aspirate a portion of the cells into a culture dish for observation, ensuring an 8-10 cell gap between cells. Observe under a microscope. Add 10 μL of PBS to a 200 μL centrifuge tube, and use a pipette (Yoshida Biotechnology) to aspirate a single red pigment cell into the centrifuge tube. The obtained cells can be used for single-cell sequencing and in vitro cell culture.

[0057] 9) After centrifugation in step 7, add 5 mL of complete culture medium to resuspend the precipitate in the pigment cells, and place them in a 25 cm... 2Cells were cultured in flasks at 28°C, with the complete culture medium replaced every 2 days. After 3 days of culture, the results were observed as follows: Figure 3 As shown in the figure. The results show that erythroblasts can be successfully isolated using the method of the present invention, and the isolated erythroblasts can be cultured in vitro. Furthermore, the isolated erythroblasts have high purity and good activity.

[0058] Example 2: Comparison of the effects of the conventional method and the method provided by the present invention

[0059] The method provided by this invention is the same as steps 1) to 5) of Embodiment 1.

[0060] Traditional method: Trypsin digestion, which uses pancreatic enzymes for digestion, the steps are as follows:

[0061] 1) Prepare PBS with a 10× triple antibody concentration by mixing 1×PBS buffer and 100× triple antibody solution at a volume ratio of 9:1. Anesthetize the leopard gill spiny perch with an ice bath, wipe off the mucus on the body surface with a clean paper towel, and cut off the dorsal fin, caudal fin, and anal fin with pre-sterilized scissors and place them in PBS with a 10× triple antibody concentration.

[0062] 2) Rinse 5 times with PBS, cut the tissue into 3-4 mm wide pieces with scissors, put them 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 blow with a sterile Pasteur pipette for 5 min.

[0063] 3) Pass the digestion solution through a 40μm cell sieve and centrifuge at 200g for 8 minutes. Then, resuspend the cell pellet obtained by centrifugation in step 5) of Example 1 in PBS with 4% BSA. Take 20μL of each cell suspension and add it to a glass slide. Observe the types of cells obtained under a microscope.

[0064] The results are as follows Figure 4 As shown. The results show that the method provided by this invention can successfully prepare a single-cell suspension containing erythroblasts (as shown). Figure 4 (B in the text), while traditional methods using trypsin digestion cannot digest erythroblasts from tissues ( Figure 4 (A in the middle).

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for isolating red pigment cells from the leopard-gill spiny perch, characterized in that, It consists of the following steps: The fins of the leopard-gill spiny perch were taken and subjected to first digestion, second digestion and density gradient centrifugation in sequence to obtain the red pigment cells of the leopard-gill spiny perch. The digestion solution used in the first digestion was 0.04 wt% EDTA digestion solution; the digestion solution used in the second digestion was collagenase digestion solution; the collagenase digestion solution was prepared by dissolving 1 mg / mL collagenase IV, 0.1 mg / mL DNase I and 10 mg / mL bovine serum albumin in PBS; The density gradient centrifugation was performed using Percoll solution as the medium. The first digestion time is 20-30 minutes, the temperature is 25℃-28℃, and the rotation speed is 200 rpm; The second digestion time is 2 hours, the temperature is 25℃-28℃, and the rotation speed is 60 rpm; The density gradient centrifugation involves sequentially adding a 20% (v / v) Percoll solution and a 30% (v / v) Percoll solution. After density gradient centrifugation, the red pigment cells were located at the interface between a 20% (v / v) Percoll solution and a 30% (v / v) Percoll solution. The fins are one or more of the caudal fin, dorsal fin, and anal fin.

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