CN118546878B - A serum-free culture medium for in vitro culture of hemolymphocytes of scallop - Google Patents

Abstract

The invention provides a serum-free culture medium for in-vitro culture of patinopecten yessoensis blood lymphocytes, and belongs to the technical field of culture mediums. The culture medium provided by the invention is an L-15 culture medium added with salt ions, lysine, proline and taurine. The culture medium can be used for in-vitro culture of blood cells, scallop blood lymphocytes can rapidly migrate in 12h of inoculation, a cell monolayer with the confluence of 60-80% is formed, and survival and growth of in-vitro cultured cells can be better maintained.

Description

A serum-free culture medium for in vitro culture of hemolymphocytes of scallop

Technical Field

The invention belongs to the technical field of culture medium of marine biological cell lines, and in particular relates to a culture medium of hemolymph cells of scallops and an application thereof.

Background Art

In recent years, the breakthrough and rapid development of high-throughput sequencing technology have greatly promoted research in the field of life sciences, enabling scientists to read genetic codes more quickly and accurately, and providing a basis for the analysis of key gene expression regulation mechanisms. At present, research on gene expression regulation mainly relies on model animal platforms and in vitro cell line research platforms, but there is still a lack of relevant basic research information on non-model animals (such as marine crown wheel groups) that occupy key nodes in the evolutionary process.

Molluscs, commonly known as shellfish, are the second largest category in the animal kingdom and the largest group of marine crown-wheel animals, with more than 100,000 existing species. In recent years, the rapid development of high-throughput sequencing technology has provided shellfish with an opportunity to decipher the genomes of different lineages. For example, Wang et al. (2017) revealed that shellfish have ancient chromosome karyotypes through in-depth analysis of the genome of Patinopecten yessoensis , and their unique evolutionary status can provide key clues for understanding the early origin and regulatory mechanisms of animal cells.

At present, a complete model animal gene function research platform has not been established in shellfish mollusks, which makes the in vitro cell gene function research platform particularly important. In previous studies, most of the in vitro cell cultures of shellfish mollusks used mammalian or other insect culture media, supplemented with autologous serum as a cell nutrient additive. Among them, L-15 culture medium has the best culture effect and is the most widely used. Takeuchi et al. (1999) cultured the podocytes of Mediterranean mussels in L-15 culture medium supplemented with 20% FBS; Odintsova et al. (2010) used 2% FBS, 1.75 mg/L vitamin E and 50 mg/L insulin as additives to L-15 basal culture medium to culture mussel larval cells; Yoon et al. (2022) achieved short-term in vitro culture of scallop heart cells and blood cells by adding salt ions and fetal bovine serum to L-15; Bai et al. (2023) found that adding mussel autologous serum to the mantle culture of freshwater mussels can promote cell growth. However, a large number of experiments have shown that shellfish cells can only maintain survival in vitro without proliferation ability, and model animal studies have shown that the addition of serum will promote cell differentiation, resulting in a decrease in cell proliferation ability. This has hindered the study of key signaling pathways and regulatory modules such as cell cycle and cell proliferation. Therefore, how to improve the proliferation ability of shellfish cells under serum-free conditions has become a research focus in shellfish cell culture.

Summary of the invention

The invention provides a scallop hemolymph cell culture medium and application thereof. The provided culture medium can better maintain the survival and growth of the scallop hemolymph cells in vitro, thereby solving the problem of culturing the scallop hemolymph cells in vitro and providing a basis for establishing a mollusk cell line.

The scallop blood lymphocyte culture medium provided by the present invention is an L-15 culture medium added with salt ions, lysine, proline and taurine;

Furthermore, the concentration of lysine added is 0.258 g/L; the concentration of proline added is 0.234 g/L; the concentration of taurine added is 0.1 g/L;

As a specific record of the embodiment, the components and added concentrations of the salt ions are as follows: 2g/L NaCl, 0.3g/L KCl, 1.2g/L CaCl ₂ , 2.76g/L MgSO ₄ , 3g/L MgCl ₂ , 0.19g/L Na ₂ HPO ₄ , 0.06g/L KH ₂ PO ₄ .

Furthermore, antibiotics are added to the culture medium;

As a specific record of the embodiment, the types and added concentrations of the antibiotics are as follows: penicillin 60 mg/L, streptomycin 100 mg/L, gentamicin 40 mg/L;

Furthermore, a pH buffer is added to the culture medium.

As a specific description of the embodiment, the pH buffer is HEPES.

The pH of the culture medium is 7.1, and the osmotic pressure is 900-1100 mOsm/kg.

The culture medium provided by the present invention can be used for culturing hemolymphocytes of the scallop.

Compared with the prior art, the present invention has the following technical effects:

(1) The present invention provides a scallop blood lymphocyte culture medium, which can be used for in vitro culture of blood cells. Scallop blood lymphocytes can migrate rapidly within 12 hours of inoculation to form a cell monolayer with a confluence of 60%-80%, and can better maintain the survival and growth of cells cultured in vitro;

(2) The culture medium of the present invention contains some salt ions and some scallop serum free amino acid mixture. The scallop cells cultured with the culture medium break through the cell mitotic arrest phase and show some proliferation potential.

(3) The present invention also provides a method for using a scallop cell culture medium in culturing scallop blood lymphocytes. The method is simple to operate, and the cultured scallop blood lymphocytes have a high cell density, good morphology, and high wall adhesion.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Photographs comparing the culture effects of three basic culture media for Yezo cells based on free amino acids from Yezo serum and L-15 culture medium: PYM-1, PYM-2, and PYM-3 represent culture media with osmotic pressure adjusted by NaCl, culture media with osmotic pressure adjusted by adding NaCl and other salt ions, and culture media with osmotic pressure adjusted by adding NaCl and other salt ions and additional nutrients, respectively.

Figure 2: Bar graph of cell adhesion rate of scallop blood lymphocytes cultured in commercial L-15 basal medium, PYM-1, PYM-2, and PYM-3, with significant differences p<0.05.

Figure 3: Cell live-death staining and significant difference analysis of scallop blood lymphocytes cultured in commercial L-15 basal medium, PYM-1, PYM-2, and PYM-3, p<0.05.

Figure 4: Schematic diagram of the cell proliferation results of scallop blood lymphocytes cultured in the optimized optimal culture medium and commercial L-15 culture medium after 7 days of culture.

DETAILED DESCRIPTION

The present invention, based on the analysis of free amino acid components of the serum of the Yesso scallop, uses L-15 as a basic culture medium and adds additional salt ions and nutrient components to establish a serum-free optimized culture medium for in vitro cell culture suitable for the Yesso scallop, thereby laying a foundation for establishing an in vitro cell manipulation platform for molluscs and studying the function regulation of key genes.

The preparation method of the hemolymphocytes of the scallop used in the embodiments of the present invention is as follows:

Take a 1mL disposable syringe, draw 0.1mL of anticoagulant, then gently pierce the blood cavity from the disinfected striated muscle of the Yesso scallop, and extract the hemolymph of the Yesso scallop at a uniform speed; shake the syringe slightly up and down to ensure that the anticoagulant and hemolymph are evenly mixed to prevent blood coagulation; remove the syringe needle, filter the Yesso scallop hemolymph cells with a 40μm filter membrane, and transfer them to a 15mL centrifuge tube; centrifuge at 800×g for 5min; discard the supernatant, and add L-15 basal culture medium to make a cell suspension.

The preparation method of the anticoagulant is as follows: 30 nmol/L PBS phosphate buffer, 1.5% EDTA, 2.5% NaCl. After fully dissolved, filter and sterilize, and then store at 4°C.

However, the present invention can also adopt other existing methods to prepare the hemolymph cells of the scallop.

The present invention will be further described below in conjunction with embodiments and drawings.

Example 1: Determination of free amino acid types and contents, osmotic pressure and pH in scallop serum

The filtered seawater was boiled, placed in a culture frame and cooled to 10°C, and the air pump was used to continuously inflate the scallops for 24 hours. During the experiment, the shell surface of the scallops was wiped clean with 75% alcohol, the scallop shells were pried open, the seawater and other impurities in the body cavity were cleaned, and the scallop column striated muscle was wiped with a cotton ball soaked in iodine solution for disinfection.

Firstly, scallop serum was prepared, and the osmotic pressure and pH value of the scallop serum were determined using an osmometer and a pH meter respectively. The types and contents of free amino acids in the serum were determined using an amino acid analyzer. According to the determination results, the nutritional supplements of the scallop optimized culture medium were designed, and the osmotic pressure and pH of the culture medium were adjusted to make it more suitable for the growth of Yesso scallop cells.

1) The preparation method of scallop serum is as follows:

Use a 1mL disposable syringe to gently pierce the blood cavity from the striated muscle of the Yesso scallop and extract the hemolymph of the Yesso scallop at a uniform speed; remove the syringe needle and transfer the hemolymph of the Yesso scallop to a 15mL centrifuge tube; let it stand in a 4℃ refrigerator for one hour, wait for it to coagulate, and the collected supernatant is the Yesso scallop serum.

2) Take some of the serum of the Yesso scallop and use an osmometer and a pH meter to measure the osmotic pressure and pH value of the serum of the Yesso scallop respectively. It was found that the osmotic pressure of the serum of the Yesso scallop was 900~1100 mOsm/kg, and the pH value was 7.0-7.2.

3) The method for determining the types and contents of free amino acids in serum is as follows:

The obtained scallop serum was thoroughly mixed with 0.25 times the volume of sulfosalicylic acid, placed in a 4°C refrigerator for 60 minutes, and centrifuged at 2000×g for 15 minutes; the supernatant was collected and centrifuged once; the supernatant was aspirated and sterilized by filtration with a 0.22 μm filter membrane under sterile conditions, and the content and type of free amino acids in the scallop serum were determined with an A300 amino acid analyzer. The results are shown in Table 1.

Among them, the free amino acid data measured by the serum of the Japanese safari animal showed that the content of two free amino acid components in the commercial L-15 was insufficient, namely lysine and proline. Therefore, the present invention intends to add the missing free amino acid components to L-15 to optimize the culture medium. In addition, the present invention also adds taurine.

Table 1: Analysis results of free amino acids and their contents in serum of scallop

Example 2: Optimization of scallop blood lymphocyte basal culture medium

The optimization of the basic culture medium for Yezo was based on L-15 culture medium (Hyclone). Combined with the types, composition and content of free amino acids in Yezo serum (Table 1), the optimal osmotic pressure and pH, an optimized culture medium (PYM) was designed and prepared to compare their ability to support the growth and survival of Yezo hemolymph cells.

Among them, the culture medium numbered PYM-1 is a culture medium prepared by adding 18g/L NaCl to L-15 basic culture medium to adjust the osmotic pressure; PYM-2 is a culture medium prepared by adding 12g/L NaCl, 0.3g/L KCl, 1.2g/LCaCl ₂ , 2.76g/L MgSO ₄ , 3g/L MgCl ₂ , 0.19g/L Na ₂ HPO ₄ , and 0.06g/L KH ₂ PO ₄ to L-15 basic culture medium; PYM-3 is a culture medium prepared by adding amino acid components to PYM-2, with final concentrations of 0.258g/L lysine, 0.234g/L Proline; Taurine final concentration is 0.1g/L; Penicillin-Streptomycin-Gentamicin antibiotics and HEPES buffer are added to PYM1-3 medium and L-15 basal medium, with final concentrations of: Penicillin 60mg/L, Streptomycin 100mg/L, Gentomycin 40mg/L; HEPES 2.38g/L, pH of the medium is 7.1. The osmotic pressure of the above PYM-1, PYM-2, and PYM-3 medium is 900~1100mOsm/kg.

Example 3: Application of scallop cell culture medium in culturing scallop cells

1) Wipe the surface of the scallop shells with 75% alcohol and place them in a clean bench for UV sterilization for 30 minutes;

2) Use a 1 mL disposable sterile syringe to extract 2-3 mL of hemolymph from the scallop column;

3) Filter the hemolymph obtained in step 2) using a 40 μm filter membrane;

4) Centrifuge the blood lymphocytes filtered in step 3) at 800 rpm for 5 min, and discard the upper serum;

5) Use L-15, PYM-1, PYM-2, and PYM_3 basal culture media with penicillin-streptomycin-gentamicin antibiotics to resuspend the blood lymphocytes of the Japanese scallop (dilution concentration is 1.5×10 ⁵ /mL), and inoculate them into 96-well plates for culture. The culture conditions are: O ₂ content is 5%, temperature is 20℃, no additional CO ₂ and humidity conditions are required, and static culture is carried out. Change the medium appropriately according to the cell growth status.

The growth status of scallop blood lymphocytes after culturing L-15, PYM-1, PYM-2, and PYM-3 cells in Example 3 for 1 Day, 3 Days, and 7 Days were observed respectively.

Example 4: Culture effect of culture medium

1. The cell adhesion test was used to detect the adhesion rate of the hemolymph cells of the scallop in Example 3 after 3 days of culture.

After culturing the cells in Example 3 for 72 hours, the culture medium was discarded, the cells were rinsed three times with 3×PBS, new culture medium was added, and 100 μL of CCK-8 (Beyotime) solution was added to each well. After culturing in an incubator for 3 hours, the absorbance was measured with a microplate reader.

2. The activity of hemolymphocytes of the scallops in Example 3 was detected by Calcein-AM/PI staining, and the specific steps were as follows:

First, 1mM Calcein-AM stock solution was diluted into 1μM Calcein-AM working solution with 3×PBS; the culture medium in the well plate was discarded, 3×PBS solution was added, and it was gently shaken and discarded; then 200μL of the prepared Calcein-AM solution was added to the 24-well plate, and the cells were transferred to a 20℃ incubator and incubated for 30min; then 5mM PI stock solution was diluted into 5μM with 3×PBS, added to the cells, incubated in the dark for 5min, and then washed twice with 3×PBS solution, and observed and photographed with an inverted fluorescence microscope.

3. The proliferation capacity of scallop hemolymphocytes in Example 3 was detected by EdU staining, and the specific method is as follows:

⑴ Labeling of blood lymphocytes of scallop

After the cells of Example 3 were cultured overnight to a stable state, the EdU stock solution (10 mM) was diluted with PYM-3 medium and L-15 medium (containing antibiotics) to prepare a 2×EdU working solution (10 μM), which was then preheated in a 20°C water bath. The culture medium in the 24-well cell culture plate (250 μL/well) was replaced, and then 250 μL of 2×EdU working solution was added in an equal volume, mixed, and incubated in a 20°C incubator for 2 days.

⑵Fix and permeabilize cells

After the cells have been incubated as above, the culture medium is aspirated, and 200 μL of 3×PBS solution containing 4% formaldehyde is added to the 24-well plate. Incubate at room temperature in the dark for 15 minutes, and aspirate the fixative. Wash the cells twice with washing solution (3×PBS solution containing 3% bovine serum albumin). Aspirate the washing solution, add 500 μL of permeabilization solution (0.5 mL of TritonX-100 per 100 mL of 3×PBS solution) to each well, and permeabilize for 20 minutes. After removing the above-mentioned additives, wash at least twice.

(3) Incubation with Edu binding solution

First, prepare the EdU binding solution (430 μL Click Reaction Buffer, 20 μL CuSO ₄ , 1 μL Zie 488, 50 μL Click Additive Solution per well). The binding solution should be prepared and used immediately. After adding the binding solution, gently shake the plate to ensure that the binding solution is evenly distributed. Incubate at room temperature in the dark for 30 minutes. Then remove the EdU binding solution and wash the cell monolayer twice with washing solution, 5 minutes each time.

⑷ Nuclear staining

First, dilute the staining solution according to the ratio of Hoechst33258:3×PBS=1μL:1mL. Then add 500μL of the diluted Hoechst33258 solution to the cell culture well, cover with tin foil, incubate at room temperature in the dark for 10 minutes, and then aspirate the Hoechst33258 solution. Finally, wash the cells three times with 3×PBS, each time for 5 minutes, and aspirate the 3×PBS solution. Take pictures and record with LSM900 confocal microscope.

As shown in Figure 1, L-15 and the three new PYM basal mediums can support the attachment and survival of the blood lymphocytes of the scallop in the cell culture plate. After 24 hours of inoculation, the growth state of the blood lymphocytes in the three PYM basal mediums is similar and can all attach to the cell plate, but the attachment rate of PYM-3 is significantly higher than that of PYM-1 and PYM-2, and PYM-3 can form a continuous cell monolayer; the blood lymphocytes of the scallop in the L-15 basal medium did not attach to the cell plate; and the dead cells aggregated in a floating state. After 72 hours of inoculation, the PYM-1 cells began to fall off, the PYM-2 cells aggregated, and the PYM-3 cells did not aggregate and had no obvious shrinkage phenomenon; after 7 days of inoculation, the cells in the L-15 basal medium, PYM-1, and PYM-2 showed large-scale rupture and death, and the blood lymphocytes of the scallop in PYM-3 had the best in vitro growth and survival. Compared with other culture media, the cells in this culture medium are healthier, the cell monolayer is dense, and there are fewer cell fragments. This indicates that PYM-3 can better maintain the survival and growth of scallop blood lymphocytes cultured in vitro.

As shown in Figure 2, after 3 days of L-15 culture, the average cell adhesion rate was about 17%, the PYM-1 cell adhesion rate was about 20%, the PYM-2 cell adhesion rate was about 45%, and the PYM-3 cell adhesion rate reached about 75% after culture. The optimized PYM-3 cell adhesion rate was greatly improved.

As shown in Figure 3, compared with L-15, PYM-1, and PYM-2, the number of dead cells in PYM-3 medium is greatly reduced, which shows that the scallop cell culture medium PYM-3 of the present invention can better culture scallop blood lymphocytes in vitro, so that the cells can survive and grow better in vitro. The fluorescent statistical signal also shows that the fluorescence signal intensity of dead cells per unit area of PYM-3 is significantly decreased, and the fluorescence signal intensity of live cells is significantly increased, indicating that the cells cultured in PYM-3 medium are in the best state.

As shown in Figure 4, the Edu staining results showed that compared with L-15 medium, scallop blood lymphocytes cultured in PYM-3 were able to break the mitotic arrest phase of scallop blood lymphocytes, and some cells showed proliferation.

The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims (10)

1. A serum-free culture medium for in-vitro culture of patinopecten yessoensis blood lymphocytes is characterized in that the serum-free culture medium is an L-15 culture medium added with salt ions, lysine, proline and taurine.

2. The serum-free medium of claim 1, wherein the lysine is added at a concentration of 0.258g/L; the addition concentration of the proline is 0.234g/L; the concentration of taurine added is 0.1g/L.

3. The serum-free medium according to claim 1, wherein the components and the addition concentrations of the salt ions are as follows ：2g/L NaCl、0.3g/L KCl、1.2g/L CaCl₂、2.76g/L MgSO₄、3g/L MgCl₂、0.19g/L Na₂HPO₄、0.06g/L KH₂PO₄.

4. The serum-free medium of claim 1, wherein an antibiotic is further added to the serum-free medium.

5. The serum-free medium according to claim 4, wherein the antibiotics are of the following types and added concentrations: penicillin 60mg/L, streptomycin 100mg/L and gentamicin 40mg/L.

6. The serum-free medium of claim 1, wherein a pH buffer is further added to the serum-free medium.

7. The serum-free medium of claim 6, wherein the pH buffer is HEPES.

8. The serum-free medium of claim 1, wherein the serum-free medium has a pH of 7.1 and an osmotic pressure of 900 to 1100 mOsm/kg.

9. Use of the serum-free medium of claim 1 for culturing patinopecten yessoensis blood lymphocytes.

10. A method for culturing patinopecten yessoensis blood lymphocytes is characterized in that, the method is to culture patinopecten yessoensis blood lymphocytes by using the serum-free culture medium according to claim 1.