CN109161515B - Isolation culture method of primary hepatocytes of megalobrama amblycephala - Google Patents

Isolation culture method of primary hepatocytes of megalobrama amblycephala Download PDF

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CN109161515B
CN109161515B CN201810886568.6A CN201810886568A CN109161515B CN 109161515 B CN109161515 B CN 109161515B CN 201810886568 A CN201810886568 A CN 201810886568A CN 109161515 B CN109161515 B CN 109161515B
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megalobrama amblycephala
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刘文斌
曹秀飞
蒋广震
戴永军
袁向阳
王聪聪
黄洋洋
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Nanjing Agricultural University
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract

The invention discloses a method for separating and culturing primary hepatocytes of megalobrama amblycephala. Selecting healthy young megalobrama amblycephala fish with the weight of 30-50g, sterilizing the whole body by using 1% potassium permanganate, taking blood from tail vein, and taking liver by aseptic dissection. Digesting with collagenase, filtering with a 200-mesh cell sieve to obtain a cell suspension, removing redundant red blood cells in liver cells by using a red blood cell lysate, and removing cell debris by gradient centrifugation. Adding appropriate amount of complete culture medium into the obtained cell precipitate to suspend cells, calculating and adjusting cell concentration with cell counter, plating, adding 5% CO at 28 deg.C2The cell culture box is used for culturing, and the adherence condition is observed after 48 hours. The invention innovatively combines the characteristics of research species to separate and culture the liver cells, the obtained cell viability is up to more than 90%, the cell viability meets the requirement of primary culture, and theoretical basis and technical support are provided for further carrying out primary liver thin related experiments of megalobrama amblycephala.

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 (4)

1. A method for separating and culturing primary hepatocytes of megalobrama amblycephala is characterized by comprising 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: selecting young fish of healthy megalobrama amblycephala with weight of 30-50g, soaking in 1% potassium permanganate for sterilization for 10min, collecting blood from tail vein with sterile injector until gill is whitish, wiping fish body with alcohol cotton ball, shearing with sterilized ophthalmic scissors from anus of megalobrama amblycephala, cutting left body wall of fish along fin, lateral line and gill cap edge under lateral line, opening abdominal cavity, picking liver with sterilized ophthalmic forceps, cleaning liver with DPBS, removing impurity, and cutting into 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: adding erythrocyte lysate into the cell sediment obtained in the step (2), repeatedly blowing and beating the cell sediment into suspension by using a pipette, standing for 5 minutes, centrifuging for 10 minutes at 800rmp to remove supernatant to obtain cell sediment, 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 for 10 minutes at 500rmp 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 106-107 Planting cells at cell/ml concentration to make megalobrama amblycephala liver cells at 28 deg.C and 5% CO2Culturing under the condition, placing the culture plate into an incubator for 30min, adding 15% fetal calf serum and 5% megalobrama amblycephala serum into the culture plate, and performing cell culture by using a formed complete culture medium to increase the adherence rate of the hepatocytes so as to finally obtain the primary hepatocytes of the in vitro megalobrama amblycephala; the complete culture medium formula is as follows: DMEM/F12, 15% fetal bovine serum, 5% megalobrama amblycephala serum, 100IU/ml penicillin, 100 mug/ml streptomycin, 20ng/ml hepatocyte growth factor, 10 mug/ml bovine insulin and 2mM glutamine, the cell plating density is 106-107 cell/ml, cell culture temperature 28 ℃, CO2The content is 5%.
2. The method for isolated culture of the primary hepatocytes of the megalobrama amblycephala in claim 1, wherein in the step (1), the fibronectin (1 mg/ml) is Ca-free2+,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.
3. The method for isolated culture of megalobrama amblycephala primary hepatocytes as claimed in claim 1, wherein the liver obtained in step (2) is washed with DPBS for 4-5 times, and cut into pieces of 1-3mm3Small pieces, the concentration of type IV collagenase or type II collagenase is 0.1% by mass volume, 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.
4. The method for separating and culturing the primary hepatocytes of the megalobrama amblycephala according to claim 1, wherein the centrifugation speed after the erythrocyte lysate is added in the step (3) is 800rmp for 10min, and the centrifugation speed after the complete culture medium is added is 500rmp for 10 min.
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