CN110684718A - A kind of high-efficiency separation and culture method of primary hepatocytes of pufferfish with dark stripes - Google Patents

Abstract

The invention discloses a method for separating and culturing primary hepatocytes of puffer fish with dark stripes, comprising the following steps: (1) selecting juvenile puffer fish with dark stripes, aseptically dissecting the livers, washing and shredding them; The chopped liver was digested with collagenase II digestion solution, filtered, centrifuged, and the lysate was added to the precipitation to obtain a cell suspension, which was then added to the separation solution and centrifuged to remove impurities; Complete medium, mix well, supplement complete medium, and culture. As for the prior art, the method for separating and culturing primary hepatocytes of pufferfish with dark streaks of the present invention can solve the problem of difficulty in separating hepatocytes, and has high separation efficiency of hepatocytes. training requirements.

Description

A kind of high-efficiency separation and culture method of primary hepatocytes of pufferfish with dark stripes

technical field

The invention relates to a method for efficiently separating and culturing primary hepatocytes of pufferfish with dark stripes, and belongs to the technical field of separation and culture of primary liver cells of puffer fish with dark stripes.

Background technique

Takifugu fasciatus belongs to sea and freshwater migratory fish, mainly distributed in the middle and lower reaches of the Yangtze River, and is one of the three freshwater species of the Yangtze River. In recent years, the state and government have increased support for the puffer fish farming industry. The breeding technology of pufferfish has matured, and the output has increased year by year. However, with the rapid development of the industry, various problems in the breeding process have gradually emerged (such as the degradation of germplasm resources and the frequent occurrence of various diseases). Therefore, it is beneficial to the healthy and rapid development of its breeding industry to intensify the basic research on the pufferfish. However, there is still no breakthrough in the culture of primary hepatocytes in the basic research of pufferfish.

As the main metabolic organ of fish, liver plays an important role in life activities such as digestion and absorption, material metabolism, toxin decomposition and disease defense of fish. The primary cultured hepatocytes have a short in vitro time, and their biological properties have not changed significantly. They can better preserve and maintain the integrity and metabolic activity of hepatocytes, and to a certain extent, can more accurately reflect the metabolism in vivo. However, according to previous experiments, the fat content in the liver of pufferfish with dark stripes is over 75%, far exceeding the fat content in the liver of conventional fish. The accumulation of high-density fat in the liver brings great difficulties to the isolation of hepatocytes.

SUMMARY OF THE INVENTION

Purpose of the invention: In view of the above technical problems, the purpose of the present invention is to provide an efficient method for separating and culturing primary hepatocytes of pufferfish, which provides technical support for subsequent theoretical research at the cell level.

Technical scheme: in order to achieve the above-mentioned purpose of the invention, the present invention adopts the following technical scheme:

A method for separating and culturing primary hepatocytes of pufferfish with dark stripes, comprising the following steps:

(1) select the juvenile puffer fish with dark stripes, aseptically dissect the liver, clean it, and cut it into pieces;

(2) digesting the chopped liver with collagenase II digestion solution, filtering, centrifuging, adding lysing solution to the precipitation to obtain a cell suspension, then adding it to the separating solution, and centrifuging to remove impurities;

(3) Add pre-warmed complete medium to the precipitation, mix well, supplement the complete medium, and culture.

As a preference:

The dark-striped puffer fish juveniles described in the step (1) are juveniles with a body length of 2±0.5cm and a body weight of 2±0.3g without feed conversion and raised with red worms or Artemia, before taking the liver tissue. Raised in a water environment with salinity 1-3, starvation treatment.

In step (2), the concentration of the collagenase II digestion solution is 0.5-2 mg/ml, and the addition amount is 7-9 ml per gram of liver tissue, and digested at 25-28° C. for 1.5-3 hours.

In step (2), the erythrocyte lysate is added to the precipitation, and the method is as follows: adding the erythrocyte lysate to the precipitation, mixing uniformly, and centrifuging to obtain a cell precipitate, adding basal medium to the precipitation, and mixing to obtain a cell suspension .

In step (2), the separation solution is 60% percoll separation solution, and the volume ratio of the cell suspension to the percoll separation solution is 3:(4-6).

The complete culture medium composition described in the step (3) includes: adding fetal bovine serum and pufferfish serum in the DMEM basal medium, and the volume ratio of the DMEM basal medium, fetal bovine serum and pufferfish serum is 12-15: 4-5: 1-3, and add the double antibody solution and glutamine solution containing penicillin and streptomycin, and adjust the pH to be between 7.6 and 7.8.

Preferably, 110 μl of double antibody solution and 0.5-1.5 ml of L-glutamine solution are added to every 100 ml of complete medium, the double antibody solution contains 100 Units/ml of penicillin and 100 μg/ml of streptomycin, and the concentration of glutamine solution is 20 mmol /L

The applicant of the present invention has carried out repeated experiments using the reported methods for separating and culturing hepatocytes from fish and mammals. For the pufferfish, only a small amount of hepatocytes can be obtained, and the viability is weak, which is difficult to apply to experimental research. Based on this, the applicant of the present invention, after years of research, provides the technical solution of the present invention, which solves the problem of difficult separation of hepatocytes of pufferfish, and obtains a better primary culture method. This lays a foundation for the research on the theoretical basis of pufferfish, and also provides a reference for the establishment of other high-fat tissue cell isolation and culture methods.

Technical effect: Compared with the prior art, the method for separating and culturing primary hepatocytes of the dark-striped pufferfish of the present invention can solve the problem of difficulty in separating hepatocytes, and has high separation efficiency of hepatocytes, and the cultured cells grow well and have a stable physiological state. , in line with the requirements of primary culture.

Description of drawings

Figure 1 is the liver tissue of the dark-patterned pufferfish.

Figure 2 is the primary hepatocytes (400×) of pufferfish with dark streaks after digestion.

Figure 3 shows the primary hepatocytes (400×) of pufferfish with dark streaks after 1d culture.

Figure 4 is a comparison of pictures (400×) of the liver cells of the dark-striped pufferfish obtained after culturing for 1 d in Example 1, Comparative Example 1, and Comparative Example 2.

Figure 5 shows the relative number of hepatocytes of the pufferfish with dark streaks isolated in Example 1, Comparative Example 1 and Comparative Example 2 (the relative number of hepatocytes refers to a microscope field of 400×).

Figure 6 shows the viability of hepatocytes of the pufferfish with dark streaks isolated from Example 1, Comparative Example 1, and Comparative Example 2.

Detailed ways

For a clearer understanding of the technical solutions of the present invention, the present invention is further described in detail herein.

Example 1

1. A method for efficiently separating and culturing primary hepatocytes of puffer fish with dark streaks, the concrete operation steps are:

(1) Select the juvenile fish that are 2 ± 0.5 cm in length and 2 ± 0.3 g in body weight, and are reared with red worms or Artemia (raised at a salinity of 1- 3 in the water environment for one week, starvation for 48h), use 70% alcohol cotton balls to wipe the fish body, aseptically dissect, and wash with Hank's solution several times to remove impurities, and the obtained liver tissue is shown in Figure 1;

(2) Put the cleaned liver into the prepared collagenase type II (1mg/mL) digestive solution (the dosage is 8ml per gram of liver tissue), and cut the liver into pieces with high temperature sterilized ophthalmic scissors About 1-3mm ³ size. The liver fragments and collagenase II digestion solution were transferred to a 50 ml sterile centrifuge tube, and digested at 28°C on a constant-temperature shaking shaker for 3 hours.

(3) The digested tissue suspension was added with an equal volume of complete medium to terminate the digestion, filtered through an 80-mesh sieve, and the filtrate was put into a new centrifuge tube, 4° C. 1000 rpm for 10 min, and the supernatant was discarded;

(4) Add erythrocyte lysate to the pellet, add 3-6ml to a 10ml centrifuge tube, mix by pipetting, centrifuge at 800 rpm at 4°C for 5 minutes to obtain a cell pellet, add 6ml of basal medium to the pellet, and repeat with the pipette. Mix by pipetting, slowly add the cell suspension to the top of the 60% percoll separation solution, make the volume ratio of the cell suspension to the percoll separation solution 3:5, and centrifuge at 3000 rpm at 4°C for 5 min to obtain cell pellets. The preparation method of the 60% percoll separation solution used is: mix 90ml of percoll stock solution with 10ml of 10×PBS (pH 7.4, osmotic pressure 335mOsm) to make a buffered percoll solution; take 30ml of buffered percoll solution and mix it with 20ml of 1×PBS, Centrifuge at 5000 r/min for 3 min at 4°C to obtain a 60% percoll gradient separation solution with a density of 1.05-1.06 g/ml.

(5) Add 3-5ml of complete culture medium to the precipitate, mix by pipetting and beating repeatedly in a centrifuge tube, transfer the liquid to a cell culture flask (7ml, 25cm ² ), make up the complete culture medium to 8ml, and keep the cell density at 1 Between ×10 ³ /ml and 1 × 10 ⁵ /ml, observe the cells under an inverted microscope as shown in Figure 2. It can be seen from the figure that the cells are evenly dispersed and in good condition, suitable for subsequent culture. After observation, put them into cell culture at 28°C Incubate for 1-2 days;

The preparation method of the complete culture solution: the DMEM basal medium is added with fetal bovine serum, puffer fish serum, the volume ratio of DMEM basal medium to fetal bovine serum and pufferfish serum is 7:2:1, and then every 100ml Add 110 μl of double antibody solution (100 Units/ml of penicillin, 100 μg/ml of streptomycin) and 1.5 ml of L-glutamine solution (20 mmol/L) to the culture medium, and use 3.7% NaHCO ₃ solution to adjust the pH to 7.6-7.8 between.

(6) After 1-2 days, pour out the culture solution in the original culture bottle, add about 5ml of DEME basal medium (serum-free), wash twice, pour out the wash solution, add 8ml of new complete culture solution, and continue to expand the culture.

(7) The primary hepatocytes of the dark striped pufferfish cultured after 1 day are shown in Figure 3 and Figure 4. It can be seen from the figures that the morphology of most of the hepatocytes has changed and the state is stable.

2. Trypan blue staining and cell counting of primary liver cells of puffer fish with dark stripes

Randomly aspirate the cell suspension in the culture flask, add trypan blue for staining (the cell suspension is mixed with 0.4% trypan blue solution at a ratio of 9:1), and within 3 minutes, use a hemocytometer to count live cells and dead cells respectively. (Under the microscope, the dead cells were stained with obvious blue color, while the live cells were colorless and transparent without staining), and finally the cell viability was counted.

Viable cell rate (%) = total number of live cells / (total number of live cells + total number of dead cells) × 100%

The cell count results show that the primary hepatocytes of the dark striped pufferfish isolated and cultured by the method of the present invention have a stable viable cell rate of more than 85%, as shown in Figure 5 and Figure 6, and the cells are in a stable physiological state and good cell shape. The requirement of primary cell culture is met, and the experiment proves that the method of the invention is feasible.

As a supplementary description of Example 1, on the basis of the same basic experimental operation, two groups of experiments were carried out with different experimental parameters. The time was 25 °C for 3 h, and the ratio of the complete medium was DMEM basal medium, fetal bovine serum, and pufferfish serum in a volume ratio of 12:5:3; (b) the concentration of type II collagenase was 2 mg/day for liver digestion. ml, the digestion treatment time was 28°C for 1.5h, and the volume ratio of the complete medium was DMEM basal medium, fetal bovine serum and pufferfish serum, the volume ratio was 15:4:1; the results were basically the same as in Example 1, and the cells were dispersed. Good, the viability of trypan blue-stained cells reaches more than 85%, which proves that the selection range is reliable.

Comparative Example 1

Basically the same as Example 1, the difference is only the individual size difference, as follows:

The juvenile puffer fish with a body length of 10 ± 2 cm and a body weight of 45 ± 8 g were selected for domestication with normal feed.

The results showed that the cells were sparsely scattered and few in number observed under the inverted microscope, as shown in Figure 4 and Figure 5. Trypan blue staining showed lower cell viability, as shown in Figure 6. It shows that the selection of young individuals without transfer can greatly enhance the cell separation effect.

Comparative Example 2

Embodiment 1 is basically the same, and the difference is only in the difference in the separation method of hepatocytes, as follows:

The trypsin digestion solution used for routine separation of animal cells was used in the experiment, and the liver fragments were put into a centrifuge tube containing 0.25% trypsin digestion solution. The preparation method of 0.25% trypsin is as follows: stir the trypsin powder and D-Hank's balanced salt solution (about pH 7.2) in proportion, mix well, and filter and sterilize. The results showed that the cells were well dispersed and the number was small, as shown in Figure 4 and Figure 5. The results of trypan blue staining showed that the cell viability was low, only 64%. As shown in FIG. 6 , compared with Example 1, it shows that the collagenase digestion in the present invention has less damage to the cells.

Claims (7)

1. a method for separating and culturing primary hepatocytes of pufferfish with dark streaks, is characterized in that, comprises the following steps: (1) select the juvenile puffer fish with dark stripes, aseptically dissect the liver, clean it, and cut it into pieces; (2) digesting the chopped liver with collagenase II digestion solution, filtering, centrifuging, adding lysing solution to the precipitation to obtain a cell suspension, then adding it to the separating solution, and centrifuging to remove impurities; (3) Add pre-warmed complete medium to the precipitation, mix well, supplement the complete medium, and culture. 2. primary hepatocyte separation and culture method of puffer fish with dark stripes according to claim 1, is characterized in that, described in step (1), juvenile puffer fish with dark stripes is that body length is 2 ± 0.5cm, body weight The juveniles of 2 ± 0.3 g without feed conversion and raised with red worms or artemia, were raised in a water environment with a salinity of 1-3 before liver tissue was harvested, and were starved. 3. the method for separating and culturing primary hepatocytes of pufferfish according to claim 1, is characterized in that, the concentration of collagenase II digestion solution described in step (2) is 0.5-2mg/ml, and the add-on is Add 7-9ml per gram of liver tissue, digest at 25-28°C for 1.5-3h. 4. the method for separating and culturing primary hepatocytes of pufferfish according to claim 1, is characterized in that, in the precipitation described in step (2), add erythrocyte lysate to process, and the method is: add erythrocyte lysis in the precipitation solution, mix well, centrifuge to get cell pellet, add basal medium to the pellet, mix well to get cell suspension. 5. The method for separating and culturing primary hepatocytes of pufferfish according to claim 1, characterized in that, the separating solution described in step (2) is a 60% percoll separating solution, and the mixture of cell suspension and percoll separating solution The volume ratio is 3:(4-6). 6. dark streak puffer primary hepatocyte separation and culturing method according to claim 1, is characterized in that, described in step (3), complete medium composition comprises: in DMEM basal medium, add fetal bovine serum and dark The volume ratio of puffer fish serum, DMEM basal medium, fetal bovine serum and puffer fish serum is 12-15:4-5:1-3, and the double antibody solution containing penicillin and streptomycin and glutamine are added The pH of the solution was adjusted to between 7.6 and 7.8. 7. The method for separating and culturing primary hepatocytes of pufferfish according to claim 6, characterized in that, in every 100ml complete culture medium, add 110 μl double antibody solution and 0.5～1.5ml L-glutamine solution, The anti-solution contained 100 Units/ml of penicillin and 100 μg/ml of streptomycin, and the concentration of glutamine solution was 20 mmol/L.

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