CN109161515B

CN109161515B - Isolation culture method of primary hepatocytes of megalobrama amblycephala

Info

Publication number: CN109161515B
Application number: CN201810886568.6A
Authority: CN
Inventors: 刘文斌; 曹秀飞; 蒋广震; 戴永军; 袁向阳; 王聪聪; 黄洋洋
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2018-08-06
Filing date: 2018-08-06
Publication date: 2021-12-03
Anticipated expiration: 2038-08-06
Also published as: CN109161515A

Abstract

The present invention discloses a method for separating and culturing primary hepatocytes of bream bream. Healthy juvenile bream with a weight of 30-50 g were selected, and the whole body was sterilized with 1% potassium permanganate. After blood was collected from the tail vein, the liver was aseptically dissected. After digestion with collagenase, the cell suspension was obtained by filtration through a 200-mesh cell sieve. The excess red blood cells in the hepatocytes were removed by red blood cell lysate, and cell debris was removed by gradient centrifugation. Add an appropriate amount of complete medium to the obtained cell pellet to suspend the cells, and then use a cell counter to calculate and adjust the cell concentration. After plating, put it into a cell incubator at 28 °C and 5% CO ₂ for culture, and observe the adherence situation after 48 hours. The invention innovatively combines the characteristics of the research species to separate and culture hepatocytes, and the obtained cell viability rate is as high as 90%, which meets the requirements of primary culture, and provides theoretical basis and technical support for further experiments related to primary hepatocytes of Tuantou bream .

Description

Isolation culture method of primary hepatocytes of megalobrama amblycephala

Technical Field

The invention belongs to the technical field of cell biology and biology, relates to a separation culture method of local tissue cells of fishes, and particularly relates to a separation culture method of primary hepatocytes of megalobrama amblycephala.

Background

With the development of aquaculture industry, the culture density is continuously improved, the feed formula is unreasonable, the environmental pollution and other problems cause serious damage to the liver, and the liver is the most important physiological and biochemical metabolic organ in the fish body, so that the method has practical significance in culturing the fish hepatic cells. In addition, culturing primary hepatocytes in vitro has many advantages that cannot be replaced by in vivo experiments. The primary hepatocytes can be obtained in a large amount in a short time, the experimental time is shortened, the pertinence is strong, and the original physiological functions of the liver are retained while the in vitro hepatocytes eliminate the influence of the interaction of other tissues in the body. Therefore, hepatocytes are widely used in fish nutrition, pharmacology and toxicology studies.

The megalobrama amblycephala belongs to the family Cyprinus, is a medium lake omnivorous freshwater fish mainly distributed in the middle and lower reaches of the Yangtze river, has the advantages of rapid growth, delicious meat quality, less interspinal stings and low cost, has the yield of the freshwater fish ranked in the sixth place of the country, and is popular with farmers and consumers in China. However, due to the physical and structural characteristics of the megalobrama amblycephala, the proportion of the head and the visceral mass in the whole body is small, so that the megalobrama amblycephala is not stress-resistant, and excessive fat accumulation can cause fatty liver like mammals. Therefore, the megalobrama amblycephala is better researched by separating and culturing the hepatic cells.

At present, the liver cell culture of some fishes is researched, but the obtained cell impurities are more, the cell viability is lower, and the methods are not suitable for the culture of the megalobrama amblycephala primary liver cells. The experiment combines the biological characteristics of the megalobrama amblycephala species, and establishes the isolation culture method of the megalobrama amblycephala primary hepatocyte by improving the defects of the prior culture method of the primary fish hepatocyte.

Disclosure of Invention

The invention aims to provide a method for separating and culturing primary hepatocytes of megalobrama amblycephala, which is used for obtaining hepatocytes with high purity and high activity so as to provide theoretical basis and technical support for further research on nutriology, immunology, toxicology, pharmacology, cell biology and the like related to fishes. The method improves the existing fish primary hepatocyte culture method, and the obtained cells have high purity and the survival rate is as high as 90%.

In order to achieve the purpose of the invention, the technical scheme is as follows:

a method for separating and culturing primary hepatocytes of megalobrama amblycephala comprises the following steps:

(1) pretreatment of the culture plate: before plating, the mixture is mixed with 1-2 mu g/cm²Fibronectin-coated plates at concentration;

(2) liver isolation and digestion: picking megalobrama amblycephala liver, cleaning liver with DPBS, removing impurity, and cutting into pieces of 1-3mm³Collecting liver, placing into a sterile centrifuge tube, adding collagenase IV or collagenase II 5-6 times the volume of the liver, digesting at 28 deg.C for 30-50min, adding equal volume of complete culture medium to stop digestion, sieving the digestive juice with a 200 mesh cell sieve, centrifuging at 1000rmp for 10min, and removing supernatant to obtain cell precipitate;

(3) preparing and purifying cell suspension: b, adding erythrocyte lysate into the cell sediment obtained in the step b, repeatedly blowing and beating the cell sediment into suspension by using a pipette, standing for 5 minutes, centrifuging at 800rmp for 10 minutes to remove supernatant to obtain cell sediment, then adding a preheated complete culture medium, repeatedly blowing and beating the cell sediment into suspension by using the pipette to dilute the residual erythrocyte lysate, centrifuging at 500rmp for 10 minutes to remove supernatant to obtain cell sediment, and obtaining purified cells by using the erythrocyte lysate and gradient centrifugation;

(4) culturing megalobrama amblycephala hepatocytes: before plating, the cells were suspended in serum-free DMEM/F12 medium, 20. mu.l of the suspension was stained with an equal volume of trypan blue and counted by a cell counter and the cell viability was counted at 10⁶-10⁷Planting cells at cell/ml concentration to make megalobrama amblycephala liver cells at 28 deg.C and 5% CO₂Culturing under the condition, placing in an incubator for 30min, adding 15% fetal calf serum and 5% megalobrama amblycephala serum into the culture plate, and culturing cells with the formed complete culture medium to increase the adherence rate of hepatic cellsAnd finally obtaining the primary liver cells of the megalobrama amblycephala in vitro.

As the preferable separation and culture method of the megalobrama amblycephala primary hepatocytes, in the step (1), 1mg/ml of fibronectin is used in a Ca-free manner²⁺,Mg²⁺Diluting with ionic phosphate buffer solution by 100 times, adding into the culture plate, incubating at 37 deg.C for 1h or overnight in a refrigerator at 4 deg.C, and washing the culture plate with sterilized double distilled water for 2 times.

As the optimization of the primary hepatocyte isolation culture method for the megalobrama amblycephala, the megalobrama amblycephala is pretreated before the megalobrama amblycephala liver is extracted in the step (2): selecting healthy young megalobrama amblycephala fish with the weight of 30-50g, soaking and sterilizing the young megalobrama amblycephala fish with 1% potassium permanganate for 10min, then taking blood from tail vein with a sterile syringe until gill of the fish is slightly white, and wiping the fish body with alcohol cotton balls.

Preferably, in the step (2), sterilized ophthalmic scissors are used for cutting the anus of the megalobrama amblycephala, the left side body wall of the fish is cut along the lateral line lower fin, the lateral line and the gill cap edge, the abdominal cavity is opened, and the liver is picked up by using sterilized ophthalmic tweezers.

Preferably, the isolation and culture method of the megalobrama amblycephala primary hepatocytes comprises the steps of washing the liver obtained in the step (2) with DPBS for 4-5 times, and shearing the liver into pieces with the size of 1-3mm³Small pieces, the concentration of type IV collagenase or type II collagenase is 0.1% (mass volume ratio), the amount of the type IV collagenase or type II collagenase added is 5 times of the liver volume, the used cell sieve is 200 meshes, the centrifugal rotating speed is 1000rmp, and the time is 10 min.

Preferably, in the step (3), the centrifugation speed is 800rmp and the time is 10min after the erythrocyte lysate is added, and the centrifugation speed is 500rmp and the time is 10min after the complete culture medium is added.

As the optimization of the isolation and culture method of the megalobrama amblycephala primary hepatocytes, the basic culture medium used in the step (4) is DMEM/F12, and the final complete culture medium formula is as follows: DMEM/F12, 15% fetal bovine serum, 5% megalobrama amblycephala serum, 100IU/ml penicillin, 100. mu.g/ml streptomycin, hepatocyte growth factor (20ng/ml), bovine insulin (10. mu.g/ml) and glutamineAmide (2 mM). Cell plating density of 10⁶-10⁷cell/ml, cell culture temperature 28 ℃, CO₂The content is 5%.

Has the advantages that:

in conclusion, the method is simple and feasible, the materials are convenient to obtain, and the cell survival rate is high. Sterilizing megalobrama amblycephala in vitro, taking out liver in sterile environment, obtaining suspension liver cells by collagenase digestion method, removing more than red blood cells with red blood cell lysate, removing other impurities by gradient centrifugation, diluting cells with complete culture medium containing 20% serum, and displaying cell density at 10 with cell counter⁶-10⁷cell/ml, cell viability 90% and above.

Drawings

FIG. 1 Primary megalobrama amblycephala cells, which were just isolated, were round spheres (magnification 10X 20).

FIG. 2 shows that the megalobrama amblycephala primary hepatocytes cultured for 12h begin to adhere to the wall (magnification 10X 20).

FIG. 3 shows that megalobrama amblycephala primary hepatocytes cultured for 24 hours are stretched and deformed into islands and firmly attached to the walls (magnification 10X 20).

FIG. 4 shows megalobrama amblycephala liver cell viability rate obtained by isolation culture in methods 1,2, 3 and 4

FIG. 5 shows that megalobrama amblycephala hepatocytes obtained by isolation culture in methods 1,2, 3 and 4 have adherence rate of 48h

FIG. 6 lactic dehydrogenase Activity in supernatants from hepatocyte isolation by methods 1,2, 3, and 4

FIG. 7 Albumin content in supernatants from isolation of cultured hepatocytes by methods 1,2, 3, and 4

FIG. 8 Urea Nitrogen content in supernatants from isolation of cultured hepatocytes by methods 1,2, 3, and 4

Detailed description of the invention

The present invention will be further described with reference to the following examples. It should be noted that variations and modifications can be made by those skilled in the art without departing from the principle of the present invention, and these should also be construed as being within the scope of the present invention.

Example 1 isolation and culture method of megalobrama amblycephala primary hepatocytes:

1, main materials, sources and formula:

1) fetal bovine serum was purchased from sigma, usa.

2) DMEM/F12, glutamine and diabody (penicillin and streptomycin) were purchased from the company gibco.

3) DPBS was purchased from HyClone.

4) Erythrocyte lysates and collagenase type iv were purchased from biosharp.

5) Trypan blue was purchased from south kyaky bio corporation.

6) The complete culture medium formula comprises: DMEM/F12, 15% fetal bovine serum, 5% megalobrama amblycephala serum, double antibody (100IU/ml penicillin and 100. mu.g/ml streptomycin), hepatocyte growth factor (20ng/ml), bovine insulin (10. mu.g/ml) and glutamine (2 mM).

7) Hepatocyte growth factor was purchased from R & D Systems.

8) Fibronectin was purchased from Sciencell.

9) Bovine insulin was purchased from Solarbio corporation.

2, operation steps:

a. pretreatment of the culture plate: before plating, 2 mu g/cm is needed²The culture plate is coated with fibronectin at a concentration of 1mg/ml and Ca-free fibronectin²⁺,Mg²⁺Diluting with ionic phosphate buffer solution by 100 times, adding into the culture plate, incubating at 37 deg.C for 1h or overnight in a refrigerator at 4 deg.C, and washing the culture plate with sterilized double distilled water for 2 times.

b. Pretreatment before separation of megalobrama amblycephala liver: selecting healthy young megalobrama amblycephala fish with the weight of 30-50g, soaking and sterilizing the young megalobrama amblycephala fish with 1% potassium permanganate for 10min, then taking blood from tail vein with a sterile syringe until gill of the fish is slightly white, and wiping the fish body with alcohol cotton balls.

c. Liver isolation and digestion: cutting from the anus of megalobrama amblycephala with sterilized ophthalmic scissors, cutting the left body wall of the megalobrama amblycephala along the lateral line lower fin, lateral line and gill cap edge, opening abdominal cavity, picking up liver with sterilized ophthalmic forceps, placing into a sterile culture dish containing pre-cooled DPBS, and placing in a super-clean workbenchCleaning liver with DPBS for 4-5 times, removing impurity part, and cutting liver into 1-3mm with ophthalmic scissors³Left and right size. Collecting liver, placing into a sterile centrifuge tube, adding collagenase IV 5 times the volume of the liver, shaking in a water bath at 28 deg.C for 30min, and adding equal volume of complete culture medium to stop digestion. Passing the digestive juice through 200 mesh cell sieve, centrifuging at 1000rmp for 10min, and removing supernatant to obtain cell precipitate.

d. Preparing and purifying cell suspension: and (c) adding 5ml of erythrocyte lysate into the cell sediment obtained in the step (b), repeatedly blowing and beating the cell sediment into suspension by using a pipette gun, standing the suspension for 5 minutes, and centrifuging the suspension for 10 minutes at 800rmp to remove supernatant so as to obtain the cell sediment. Adding pre-warmed complete culture medium, repeatedly blowing and beating into suspension with a pipette to dilute the rest erythrocyte lysate, centrifuging at 500rmp for 10min, removing supernatant to obtain cell precipitate, and performing erythrocyte lysate and gradient centrifugation to obtain purified cells.

e. Culturing megalobrama amblycephala hepatocytes: before plating, the cells were suspended in DMEM/F12 medium without serum, 20. mu.l of the suspension was stained with an equal volume of trypan blue and counted by a cell counter and the cell viability was counted at 10⁶-10⁷Planting cells at cell/ml concentration to make megalobrama amblycephala liver cells at 28 deg.C and 5% CO₂Culturing under the condition, placing the culture box for 30min, adding 15% fetal calf serum and 5% megalobrama amblycephala serum into the culture plate, performing cell culture by using the formed complete culture medium to increase the adherence rate of the hepatocytes, finally obtaining the primary hepatocytes of the megalobrama amblycephala in vitro, and observing the adherence condition after 48 h.

Test analysis

1. Cell counting and survival rate detection: the cell density is 10 by using trypan blue staining method, and then counting and determining the viability in a cell counter⁶-10⁷cell/ml, and the activity rate reaches 90 percent or more.

2. And (3) morphological observation: observing the cell morphology obtained by separation and culture in an inverted microscope, wherein the hepatocyte obtained just after separation is spherical (0h, as shown in figure 1 below), and the cell state is good; after 24h of culture, the cells began to deform and adherence occurred (24h, as shown in FIG. 2 below); after 48h of culture, the cells were deformed, fibrillar and firmly attached (48h, see FIG. 3 below).

3. Comparing primary hepatocytes of megalobrama amblycephala cultured by different methods: separately culturing Cyprinus Carpio^[1]Crucian carp^[2]Jian carp^[3]The primary hepatocyte method is used for separating and culturing the primary hepatocytes of the megalobrama amblycephala, and the cell survival rate, the anchorage rate after 48 hours and the cell metabolic state are compared. Methods 1,2, 3 and 4 represent methods for culturing carp, crucian and jian carp hepatocytes, respectively, and methods of the present invention. As can be seen from FIG. 4, the megalobrama amblycephala obtained by separation culture by the method has a significantly higher survival rate than other three groups, and the survival rate of the megalobrama amblycephala obtained by the method is up to 91.93%. As can be seen from FIG. 5, after the hepatocytes are cultured for 48h, the method 4 obtains the highest adherence rate, and the other three methods have lower adherence rates, which is not favorable for the development of subsequent experiments. As can be seen from fig. 6, the activity of lactate dehydrogenase in the supernatant obtained by isolating the cultured hepatocytes by method 4 was significantly lower than that of the other three methods. Lactate dehydrogenase exists in liver cells, and when the permeability of damaged cell membranes of the liver cells is increased, the lactate dehydrogenase leaks out from the cells to the extracellular environment, so that the damaged degree of the liver cells can be known by detecting the activity of the lactate dehydrogenase in culture medium supernatant. Therefore, the other three separation methods have great damage to the liver cells. As can be seen from FIGS. 7 and 8, the content of albumin and urea nitrogen in the supernatant of method 4 was significantly higher than that of the other three groups after 48 hours of cell culture. Since urea nitrogen is produced in a large amount when cells are actively metabolized, urea nitrogen can be used as an index for determining cell survival. The liver is the only place for synthesizing albumin, and whether the synthesis and secretion functions of the liver cells are normal can be judged by detecting the content of albumin in the culture supernatant, and the albumin can be used as an early indicator of liver cell injury. From the results obtained, megalobrama amblycephala obtained by the method 4 was vigorous in growth and normal in synthesis and secretion functions.

Compared with the prior fish primary hepatocyte culture method, the method is more suitable for separating and culturing megalobrama amblycephala hepatocytes. The invention combines the characteristics of the research species and innovatively improves some defects in the separation culture step on the basis of the existing method, thereby obtaining the liver cells with good survival rate, high adherence rate, small cell damage degree and normal metabolic function.

Reference to the literature

[1] Li Yuehong, Gishangrei, Wudongming, etc. Cyprinus carpio liver cell primary culture research [ J ] Anhui agricultural science, 2012,40(22): 11278-.

[2] Giri, Cao Li Ping, D Wei Dong, et al optimization of the isolation and primary culture methods of crucian hepatocytes [ J ] North China agro-Proc, 2011,26(s2): 206-.

[3] Study on the protective effect of Lycium barbarum polysaccharides on carbon tetrachloride-induced primary hepatocyte injury of Jian carp [ J ]. school of Shanghai ocean university, 2014,23(5):718-725.

Claims

1. a method for separating and cultivating primary hepatocytes of Dory auratus, is characterized in that comprising the following steps:

(1) Pretreatment of culture plate: before plating, coat the culture plate with fibronectin at a concentration of 1-2 μg/ ^cm2 ;

(2) Liver isolation and digestion: Select healthy juvenile dory with a weight of 30-50 g, soak and disinfect with 1% potassium permanganate for 10 min, and then use a sterile syringe to collect blood from the tail vein until the gills are pale. Then wipe the fish body with alcohol cotton balls; use sterilized ophthalmic scissors to cut from the anus of the dory, cut off the left body wall of the fish along the lower fin, lateral line and opercular edge of the lateral line, open the abdominal cavity and use sterilized ophthalmic forceps Extract the liver; wash the liver with DPBS, remove impurities, cut it into 1-3 ^mm3 size, collect the liver into a sterile centrifuge tube, add 5-6 times the volume of collagenase IV or collagenase II to digest at 28°C for 30-50 min, add an equal volume of complete medium to terminate the digestion, pass the digested solution through a 200-mesh cell sieve, and centrifuge at 1000 rpm for 10 min to remove the supernatant to obtain the cell pellet;

(3) Preparation and purification of cell suspension: add erythrocyte lysate to the cell pellet obtained in the step (2), pipette repeatedly to form a suspension, let stand for 5 minutes, and remove by centrifugation at 800 rmp for 10 minutes The supernatant was cell pellet, then pre-warmed complete medium was added, and the remaining erythrocyte lysate was diluted by repeated pipetting with a pipette to dilute the remaining erythrocyte lysate. Purified cells were obtained after centrifugation;

(4) Dory hepatocyte culture: before plating, suspend the cells in serum-free DMEM/F12 medium, take 20 μl of the suspension, stain it with an equal volume of trypan blue, and count it with a cell counter and count the cell viability. , the cells were seeded at a concentration of 10 ⁶ -10 ⁷ cells/ml, and the hepatocytes were cultured at 28°C, 5% CO ₂ , placed in the incubator for 30 minutes, and then added 15% fetal bovine to the culture plate. Serum and 5% Dory bream serum were used for cell culture in the formed complete medium, thereby increasing the adherence rate of hepatocytes, and finally obtaining primary hepatocytes from Dory bream in vitro; the complete medium formula is: DMEM /F12, 15% fetal bovine serum, 5% bream serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, 20 ng/ml hepatocyte growth factor, 10 μg/ml bovine insulin, and 2 mM glutamine , the cell plating density was 10 ⁶ -10 ⁷ cells/ml, the cell culture temperature was 28°C, and the CO ₂ content was 5%.

2 . The method for separating and culturing primary hepatocytes of the dory according to claim 1 , wherein in step (1), the fibronectin of 1 mg/ml is used without Ca ²⁺ , Mg ²⁺ . 3 . The ionic phosphate buffer was diluted 100 times, then added to the culture plate, incubated at 37°C for 1 h or 4°C overnight, and washed twice with sterile double-distilled water before use.

3. The method for separating and culturing primary hepatocytes of Dory auratus according to claim 1, wherein the liver obtained in step (2) is washed 4-5 times with DPBS, and the liver is cut into pieces of ^1-3 mm Small pieces, the concentration of type IV collagenase or type II collagenase is 0.1% by weight, the amount of type IV collagenase or type II collagenase added is 5 times the volume of the liver, the cell sieve used is 200 mesh, and the centrifugation speed is 1000 rmp , the time is 10 min.

4. A method for separating and culturing primary hepatocytes of Dory auratus according to claim 1, characterized in that in step (3), after adding red blood cell lysate, the centrifugal speed is 800 rmp, and the time is 10 min, and complete culture medium is added. The post-centrifugation speed was 500 rpm and the time was 10 min.