Separation and in-vitro culture method of hepatopancreas blister cells of litopenaeus vannamei

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a litopenaeus vannamei hepatopancreas blister cell separation and in-vitro culture method which comprises the following steps: S1, obtaining hepatopancreas of litopenaeus vannamei; s2, mixing the hepatopancreas in the step S1 with trypsin, and performing sieving treatment to obtain a sieved solution; s3, performing precipitation resuspension and Percoll treatment on the sieved liquid obtained in the step S2 for multiple times to obtain a cell suspension; and S4, inoculating the cell suspension in the step S3 into a culture medium, sealing the culture medium, and placing the culture medium in an incubator. The hepatopancreas B cells of the litopenaeus vannamei obtained through separation and purification by a density gradient centrifugation optimization method are complete in morphology and relatively good in activity. Successful separation of the B cells is beneficial to research on change and immune response of the B cells in pathogen infection, and a technical support is provided for follow-up research on a molecular basis of immune function differences of different types of hepatocytes.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, in particular to a method for separating and culturing hepatopancreatic alveolar cells of Litopenaeus vannamei in vitro. Background Art

[0002] The hepatopancreas is a vital multifunctional organ in decapods. It is the largest organ in the digestive tract, the primary metabolic organ, and the primary source of immune molecules. Its health is crucial to the immune status and growth and development of individual shrimp. In 2022, the total production of Penaeus vannamei shrimp (L. vannamei) reached 2.099 million tons. In recent years, China's L. vannamei aquaculture technology has continuously improved, the aquaculture area has expanded, and the output value has increased annually, bringing significant economic benefits. However, the complex and changing aquaculture environment has led to the frequent occurrence of various parasitic, viral, and bacterial diseases, severely restricting the development of shrimp aquaculture. Major diseases such as white spot syndrome, enterococci, and iridovirus disease target the hepatopancreas as their primary invasion site. They proliferate extensively there, destroying the original hepatic ductule structure, causing hepatopancreas albinism, reducing its edible value, and causing significant economic losses to the aquaculture industry.

[0003] As an important nutrient accumulation and immune organ of shrimp, the hepatopancreas plays a vital role in its immune response. However, the functions and molecular mechanisms of different types of hepatopancreatic cells in the innate immune process of shrimp are still unclear.

[0004] Current research has determined that the hepatopancreas comprises four major cell types: ① Embryonic cells (E cells), located at the distal ends of the hepatopancreatic tubules, are undifferentiated embryonic cells with functions in regeneration, differentiation, and tissue stability. ② Resorptive cells (R cells), located primarily in the proximal and mid-regions of the hepatopancreatic tubules, are rich in lipid droplets and are the most abundant cell type in the tubules. They are responsible for nutrient absorption and metabolism. ③ Fibrillar cells (F cells), located primarily in the proximal and mid-regions of the hepatopancreatic tubules, have abundant rough endoplasmic reticulum and large Golgi apparatus, and possess a large nucleus with a prominent nucleolus. They synthesize and secrete digestive enzymes and contribute to immune defense. ④ Blister-like cells (B cells), located in the proximal and mid-regions of the hepatopancreatic tubules, contain a large digestive vacuole and numerous vacuoles. They are responsible for nutrient absorption and storage and also synthesize digestive enzymes. There is an urgent need for a method to isolate and culture hepatopancreatic B cells in vitro to address the lack of research on the immunological function of hepatopancreatic cells and the existing albinism problem. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for isolating and culturing hepatopancreatic alveolar cells of Litopenaeus vannamei in vitro, so as to solve the problem of lack of research on the immunological function of hepatopancreatic cells and the existing albinism problem in the prior art.

[0006] In order to achieve the above object, the present invention aims to provide a method for isolating and culturing hepatopancreatic alveolar cells of Litopenaeus vannamei in vitro, the method comprising the following steps:

[0007] S1: Obtain the hepatopancreas of Litopenaeus vannamei;

[0008] S2: mixing the hepatopancreas and trypsin described in step S1 and sieving the mixture to obtain a sieved solution;

[0009] S3: The sieved liquid in step S2 is subjected to multiple precipitation, resuspending, and Percoll treatment to obtain a cell suspension;

[0010] S4: The cell suspension described in step S3 is inoculated into the culture medium, sealed and placed in an incubator.

[0011] Preferably, the mixing volume ratio of the hepatopancreas and trypsin in step S2 is 5:1.

[0012] Preferably, the mixing of the hepatopancreas and trypsin and the sieving process in step S2 comprises:

[0013] The hepatopancreas obtained in step S1 was minced, and trypsin was added. After standing in an incubator at 28°C, fetal bovine serum was added and mixed by pipetting. The sieved liquid was filtered through a 100-mesh sieve and a 200-mesh sieve in sequence to obtain the sieved liquid.

[0014] Preferably, the sieved liquid in step S3 is subjected to multiple precipitation and resuspension treatments, including:

[0015] S31: Collect the sieved liquid obtained in step S2, centrifuge at 4°C and 1000 rpm for 5 minutes, take the precipitate, and resuspend the precipitate in HEPES buffer;

[0016] S32: centrifuge the precipitate resuspended in S31 at 4°C and 800 rpm for 5 min, take the precipitate, and resuspend it in HEPES buffer;

[0017] S33: centrifuging the resuspended precipitate at 4°C and 500 rpm for 5 min, taking out the precipitate, resuspending the precipitate in HEPES buffer, and passing the precipitate through a 200-mesh sieve to obtain a cell filtrate;

[0018] S34: adding the cell filtrate described in S33 to a Percoll solution gradient, centrifuging, collecting the precipitates in each layer of the band and the precipitate at the bottom of the centrifuge tube, resuspending the precipitate with HEPES buffer, centrifuging again, and collecting the precipitate;

[0019] S35: Resuspend the precipitate described in S34 with HEPES buffer, centrifuge again, take the precipitate and add culture medium to dilute and mix to obtain a cell suspension.

[0020] Preferably, the HEPES buffer is prepared as follows: 500 mL DNase / RNase-Free Water (enzyme-free water) + 7 mL HEPES + 18 g NaCl.

[0021] Preferably, the culture medium is M199, and the preparation of M199 is: 9 mL M199 + 1 mL fetal bovine serum + 100 ul amphotericin B + 1 g glucose.

[0022] Preferably, the temperature of the incubator in step S4 is 28°C.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention uses Litopenaeus vannamei as the research subject. By optimizing density gradient centrifugation, a hepatopancreatic B cell layer of Litopenaeus vannamei is precisely isolated and purified from a mixed cell suspension of multiple cell types. The B cells isolated using the optimized density gradient centrifugation method of the present invention are verified to be morphologically intact and highly viable. This highly purified B cell layer provides a pure sample foundation for subsequent B cell research, avoiding potential interference from other cell types, resulting in more accurate and reliable experimental results that truly reflect the characteristics and functions of B cells.

[0025] (2) The isolation method and long-term culture of B cells of the present invention are helpful for studying the changes and immune responses of B cells during pathogen infection, providing technical support for subsequent exploration of the molecular basis of differences in immune functions of different types of hepatopancreatic cells, and providing important information for further exploration of the functional differentiation of different types of liver cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0027] Figure 1 Schematic diagram of the Percoll separation results of hepatopancreatic cells.

[0028] Figure 2 Schematic diagram of the results of hepatopancreatic B cell isolation.

[0029] Figure 3 Schematic diagram of flow cytometer analysis results; the left picture is the mixed cell flow cytometer analysis picture; the right picture is the B cell flow cytometer analysis picture.

[0030] Figure 4 Schematic diagram of the Percoll separation results of hepatopancreatic cells.

[0031] Figure 5 Schematic diagram of CCK8 activity detection results. DETAILED DESCRIPTION

[0032] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, the production processes, experimental methods or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art, and their names and / or abbreviations are conventional names in the field and are very clear and unambiguous in the relevant fields of use. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment to implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0034] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources and are all conventional products that can be purchased through regular commercial channels or prepared according to conventional methods well known to those skilled in the art.

[0035] Litopenaeus vannamei is a commercially available product;

[0036] WaterNuclease-Free water was provided by Savier Biotechnology Co., Ltd.

[0037] HEPES solution was provided by Sevier Biotechnology Co., Ltd.;

[0038] Sodium chloride (NaCI) was provided by Sinopharm Chemical Reagent Co., Ltd.;

[0039] M199 medium was provided by Thermo Fisher Scientific;

[0040] Anhydrous glucose was provided by Sinopharm Chemical Reagent Co., Ltd.;

[0041] Fetal bovine serum was provided by Lanjieke Technology Co., Ltd.;

[0042] Penicillin-Streptomycin dual antibody solution (Penicillin-Streptomycin 100×) was provided by Beijing Quanshijin Biotechnology Co., Ltd.;

[0043] Trypsin was provided by Sangon Biotech (Shanghai) Co., Ltd.;

[0044] Percoll separation medium was provided by Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0045] Example 1 Isolation of Hepatopancreatic B Cells from Litopenaeus vannamei

[0046] Test method:

[0047] S1: Obtain the hepatopancreas of Litopenaeus vannamei;

[0048] 100 g of hepatopancreas of Litopenaeus vannamei was extracted and mixed, washed 2-3 times with HEPES buffer and then placed in 10 mL of HEPES buffer.

[0049] S2: mixing the hepatopancreas and trypsin described in step S1, and sieving the mixture to obtain a sieved solution;

[0050] Cut the hepatopancreas into pieces, add 0.25% trypsin (0.25 g of trypsin per 100 ml of solution) at a ratio of 5:1, and let it stand in a 28°C incubator for 10 minutes. Then take it out and add it to a centrifuge tube. Add 1 mL of fetal bovine serum to each tube, mix it by pipetting, and filter it into a new centrifuge tube using a 100-mesh sieve and a 200-mesh sieve in sequence to obtain the sieved liquid.

[0051] S3: The sieved liquid in step S2 is subjected to multiple precipitation, resuspending, and Percoll treatment to obtain a cell suspension;

[0052] The sieved liquid obtained in step S2 was collected and centrifuged at 4°C and 1000 rpm for 5 min;

[0053] The supernatant was discarded, the precipitate was taken, resuspended in HEPES buffer, and centrifuged again at 4°C, 800 rpm for 5 min.

[0054] The supernatant was discarded, the pellet was taken, and resuspended in HEPES buffer. The cell filtrate was centrifuged again at 4°C, 500 rpm for 5 min, the supernatant was discarded, the pellet was taken, and resuspended in HEPES buffer. The cell filtrate was passed through a 200-mesh sieve to obtain the cell filtrate.

[0055] The cell filtrate obtained above was added to the Percoll solution for gradient centrifugation, and the Percoll solution gradient was prepared using HEPES buffer (operated on ice):

[0056] 100% Percoll: 9 parts Percoll stock solution + 1 part HEPES buffer;

[0057] 35% and 5% Percoll were prepared using 100% Percoll and HEPES buffer;

[0058] Insert a 2.5mL syringe with a long needle into the bottom of a 10mL centrifuge tube and slowly inject the solutions from low to high concentration, layering 5% Percoll and then 35% Percoll, 3mL per layer. You should now see a stable distribution of Percoll concentrations within the tube. (Note: The layering speed should be slow and there should be no bubbles in the syringe.)

[0059] Mix the filtrate by pipetting. Slowly add 2 ml of the filtrate to the top of the gradient using a syringe with a needle, ensuring that the boundaries between the gradients are still clearly visible. Centrifuge the layers in a vertical centrifuge at 500 rpm, 4°C, for 45 minutes. After centrifugation, observe the cells as Percoll bands in the tube under natural light.

[0060] Carefully pipette each layer of strips into each centrifuge tube, add HEPES buffer to resuspend, and incubate at 500 rpm, 4°C, for 5 min.

[0061] Discard the supernatant, collect the pellet, resuspend in HEPES buffer, mix thoroughly by pipetting, and centrifuge again at 500 rpm, 4°C, for 5 minutes to wash away the Percoll. Remove the centrifuge tube, discard the supernatant, collect the pellet, and add the appropriately prepared culture medium to dilute each cell suspension to the appropriate concentration. Mix thoroughly by pipetting to obtain a cell suspension.

[0062] S4: The cell suspension described in step S3 is inoculated into the culture medium, sealed with a membrane and placed in an incubator.

[0063] The clean bench and experimental supplies and reagents that can be irradiated by ultraviolet should be sterilized in advance using the clean bench for 1 hour.

[0064] Using a 24-well plate, pipette 600 μl of each prepared cell suspension into each well. Then, add 1 mL of prepared M199 culture medium to each well. The M199 medium is prepared as follows: 9 mL M199 + 1 mL fetal bovine serum + 100 μl amphotericin B + 1 g glucose. Wrap an appropriately sized piece of sealing film around the plate and seal. Gently shake the plate horizontally to mix the culture medium and cell suspension. Incubate in a cell culture incubator at 28°C. Perform all procedures in a clean bench to avoid contamination.

[0065] The preparation of HEPES buffer is: 500 mL DNase / RNase-Free Water (enzyme-free water) + 7 mL HEPES + 18 g NaCl.

[0066] Test results:

[0067] The results of hepatopancreatic B cell isolation were as follows Figure 2 As shown;

[0068] The results of hepatopancreatic B cell isolation and culture were as follows Figure 4 As shown;

[0069] Result analysis:

[0070] After separating hepatopancreatic cells through different concentration gradients of Percoll, a single B cell layer was obtained. The B cells were cultured in isolation in vitro and exhibited good growth after 48 hours of culture. Microscopic observation revealed regular cell morphology, intact cell membranes with clear boundaries, and no obvious cell swelling, shrinkage, or rupture. The cells were round or oval, uniform in size, with diameters within the normal range. The nuclei were clearly visible, and no apoptotic features such as nuclear condensation or nuclear fragmentation were observed. This demonstrates that this method can be used to isolate and culture hepatopancreatic B cells in isolation in vitro.

[0071] Example 2 Verification

[0072] The B cell layer separated in Example 1 and other cell layers were observed under an optical microscope and analyzed by flow cytometry.

[0073] Percoll separation results of hepatopancreatic cells: Figure 1 Schematic diagram of hepatopancreatic B cell isolation results: Figure 2 shown.

[0074] Results analysis: After Percoll centrifugation, the hepatopancreatic cells of Penaeus vannamei can be divided into three layers. The hepatopancreatic cells of different layers were collected by centrifugation and observed under a microscope. It was found that: the upper layer (5% Percoll upper layer) was mostly R cells, the middle layer (5% Percoll and 35% Percoll interface layer) was mostly F cells and the lower layer (35% Percoll precipitation) was a single B cell.

[0075] Flow cytometry analysis results, such as Figure 3 As shown;

[0076] Result analysis: Figure 3 The left picture is a mixed cell flow cytometry analysis. The sample is a cell suspension that has not been treated with Percoll. The hepatopancreatic cells can be divided into three types by flow cytometry analysis. The permeability of the three types of cells is from low to high, namely F cells, B cells, and R cells. Figure 3The right figure shows the results of flow cytometry analysis of a suspension containing only B cells after Percoll treatment, which is consistent with the permeability of B cells in mixed cell flow cytometry analysis.

[0077] Example 3 CCK8 activity detection

[0078] The mixed cells and B cells were cultured in vitro, and CCK8 activity was detected at six time points: 0h, 6h, 12h, 24h, 36h, and 48h. Figure 5 As can be seen, the trends of the activity of the two groups of cells (B cells and mixed cells) at different time points (measured by CCK-8 method) are as follows:

[0079] B cell group:

[0080] At 0 hours, the vitality value was approximately 0.7. Over time, the vitality value gradually increased. At 12 hours, the vitality value approached 1.0, indicating a good recovery and growth trend. At 24 hours, the vitality value decreased slightly, but remained around 1.0. From 24 to 48 hours, the vitality value increased significantly, ultimately reaching approximately 1.4, demonstrating that B cells have a good ability to maintain vitality and increase over a long period of time.

[0081] Mixed cell group:

[0082] At 0 h, the viability value was approximately 0.9. From 0 h to 48 h, the viability value continued to rise, eventually reaching approximately 1.7, indicating that the viability of the mixed cells increased significantly throughout the experiment.

[0083] The above analysis shows that the B cell group showed a stable and sustained increase in activity throughout the experiment, especially between 24 and 48 hours, showing strong ability to recover and maintain activity. This indicates that B cells have good survival and proliferation potential under the experimental conditions.

[0084] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for isolating and culturing hepatopancreatic alveolar cells of Litopenaeus vannamei in vitro, characterized in that: The method comprises the following steps: S1: Obtain the hepatopancreas of Litopenaeus vannamei; S2: mixing the hepatopancreas and trypsin described in step S1 and sieving the mixture to obtain a sieved solution; S3: The sieved liquid in step S2 is subjected to multiple precipitation, resuspending, and Percoll treatment to obtain a cell suspension; S4: The cell suspension described in step S3 is inoculated into the culture medium, sealed and placed in an incubator.

2. The method according to claim 1, characterized in that In step S2, the volume ratio of the hepatopancreas to trypsin is 5:

1.

3. The method according to claim 1, characterized in that The mixing of the hepatopancreas and trypsin and the sieving process in step S2 includes: The hepatopancreas obtained in step S1 was minced, and trypsin was added. After standing in an incubator at 28°C, fetal bovine serum was added and mixed by pipetting. The sieved liquid was filtered through a 100-mesh sieve and a 200-mesh sieve in sequence to obtain the sieved liquid.

4. The method according to claim 1, wherein The sieved liquid in step S3 is subjected to multiple precipitation and resuspension processes, including: S31: Collect the sieved liquid obtained in step S2, centrifuge at 4°C and 1000 rpm for 5 minutes, take the precipitate, and resuspend the precipitate in HEPES buffer; S32: centrifuge the resuspended precipitate at 4°C and 800 rpm for 5 min, discard the supernatant, and resuspend the precipitate in HEPES buffer. S33: centrifuging the resuspended precipitate at 4°C and 500 rpm for 5 min, taking the precipitate, resuspending the precipitate in HEPES buffer, and passing the precipitate through a 200-mesh sieve to obtain a cell filtrate; S34: adding the cell filtrate described in S33 to a Percoll solution gradient, centrifuging, collecting the precipitates in each layer of the band and the precipitate at the bottom of the centrifuge tube, resuspending the precipitate with HEPES buffer, centrifuging again, and collecting the precipitate; S35: Resuspend the precipitate described in S34 with HEPES buffer, centrifuge again, take the precipitate and add culture medium to dilute and mix to obtain a cell suspension.

5. The method according to claim 4, characterized in that The HEPES buffer solution is prepared as follows: 500 mL DNase / RNase-Free Water (enzyme-free water) + 7 mL HEPES + 18 g NaCl.

6. The method according to claim 4, characterized in that The culture medium is M199, and the preparation of M199 is: 9 mL M199+1 mL fetal bovine serum+100 ul amphotericin B+1 g glucose.

7. The method according to claim 1, characterized in that The temperature of the incubator in step S4 is 28°C.

Images