Cherry valley duck hepatocarcinoma cell line DMH and its application

Abstract

This invention discloses a Cherry Valley duck hepatoma cell line (DMH) and its applications. It relates to the field of biotechnology. It provides a method for establishing immortalized duck hepatoma cells and related applications. This invention has good market demand and application value. It provides stable cell material for scientific research on duck viral hepatitis virus and the development and production of vaccines. Based on a duck hepatoma model induced by diethylnitrosamine (DEN), this invention obtains a highly active and pure duck hepatoma cell line through standardized primary tumor cell isolation, culture, and identification procedures. This cell line can efficiently support the proliferation of waterfowl hepatotropic viruses, and is particularly suitable for the culture of duck viral hepatitis virus, providing an ideal cell carrier for waterfowl viral pathogen research and vaccine development.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a Cherry Valley duck liver cancer cell line DMH and its applications. Background Technology

[0002] Duck hepatoma cell lines are immortalized passaged cell lines derived from duck liver tumor tissue. As a cell model derived from hepatocytes, they combine the biological characteristics of hepatocytes with the core advantages of passaged cell lines. These cell lines retain the target cell properties of duck hepatocytes, exhibit high compatibility with the natural host cells of viruses, and possess strong passage stability, uniform biological characteristics, high scalability for in vitro culture, and are not limited by the supply of duck embryos or live ducks, enabling standardized and reproducible in vitro culture operations. Compared to primary hepatocytes, duck hepatoma cell lines do not require frequent isolation and preparation from living tissue, effectively avoiding batch-to-batch variations in primary cells and providing a stable cell carrier for in vitro virus culture.

[0003] Waterfowl viruses typically grow well on primary duck liver cells. Duck viral hepatitis is an acute, highly lethal infectious disease primarily caused by duck hepatitis A virus types I and III, belonging to the genus *AvianhepatitisVirus* of the family Picornaviridae. The virus mainly attacks the liver, with typical symptoms including opisthotonus and hepatic hemorrhage, leading to mass acute mortality in duck flocks. Currently, the virus is commonly isolated and cultured using avian embryos. It can grow on some conventional cells, but at low titers.

[0004] Currently, duck embryonic fibroblasts (DEF) are the primary vector for in vitro culture of duck viral hepatitis virus, with chicken embryonic fibroblasts (CEF) also used as an adjunct. However, factors such as blurred avian embryo background, exogenous pathogen contamination, and interference from maternal antibodies lead to inaccurate results between batches. Other primary duck cells, such as duck embryonic hepatocytes and duck kidney cells, are also explored. However, these cells, like duck embryonic fibroblasts, suffer from inherent drawbacks such as dependence on duck embryos / live organisms, weak passage capacity, large batch-to-batch variability, and susceptibility to pathogen contamination. Primary cell isolation is cumbersome and costly, further reducing research efficiency and failing to meet the demands for efficient and stable large-scale culture. Currently, there are no duck hepatoma cancer cell lines.

[0005] Developing a duck liver cancer cell line adapted to this virus culture can provide an ideal cell model, improve research efficiency and accuracy, and improve the disease prevention and control system. This is of great significance to promoting the healthy development of the waterfowl farming industry and is a key issue that urgently needs to be solved in this field. Summary of the Invention

[0006] In view of this, the present invention provides a Cherry Valley duck hepatoma cell line, DMH, and its applications. The established duck hepatoma cell line can currently be used to isolate and culture waterfowl viruses such as duck hepatitis A virus type I and III, and some of its derivative products (receptor overexpression cell lines, etc.) have good market demand and application value. It provides stable cell material for scientific research on duck viral hepatitis viruses and the development and production of vaccines.

[0007] This invention is based on a duck liver cancer model induced by diethylnitrosamine (DEN). Through standardized procedures for the isolation, culture, and identification of primary tumor cells, a highly active and pure duck liver cancer cell line is obtained. This cell line can efficiently support the proliferation of waterfowl hepatotropic viruses, and is particularly suitable for the culture of duck viral hepatitis viruses, providing an ideal cell vector for waterfowl viral pathogen research and vaccine development.

[0008] Preservation Information: Cherry Valley duck liver cancer cells DMH ( Anas platyrhynchos domesticus (), deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China; deposit date: December 2, 2025; accession number: CCTCC NO: C2025364.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] An immortalized Cherry Valley duck liver cancer cell line DMH (Anas platyrhynchos domesticus), with accession number CCTCC NO: C2025364.

[0011] The present invention also provides the application of the above-mentioned immortalized Cherry Valley duck liver cancer cells DMH in scientific research on waterfowl viruses, and in the development of vaccines, antibodies or drugs.

[0012] Preferred: Waterfowl virus: Duck viral hepatitis virus.

[0013] This invention also provides a method for establishing immortalized duck liver cancer cells, comprising the following steps: (1) Wash and chop the isolated duck liver tumor; (2) Add EDTA to the chopped tissue and incubate, centrifuge and discard the supernatant, add collagenase IV and incubate, then filter; (3) Centrifuge the cells, discard the supernatant, and then resuspend them in the culture medium; spread the obtained tumor cells evenly; (4) Cell suspensions were obtained using conventional digestion and passage methods, followed by serial dilution; (5) After mixing the diluted cell suspension, spread it onto a cell plate; (6) Observe the color of the culture medium. If the color turns yellow, it indicates cell proliferation wells. Change the medium. (7) After the cells in a single well have grown to full capacity, they are routinely digested and transferred, plated for observation, recorded, and cultured on a larger scale.

[0014] Preferred method: Step (1) includes a step of inducing a duck liver cancer model using DEN: The liver cancer is induced in stages by a combination of drinking water and intraperitoneal injection: 0-9 weeks: basic induction is performed by adding 100ppm DEN to the drinking water; 10-15 weeks: intraperitoneal injection of DEN once a week; 16-30 weeks: intraperitoneal injection of DEN once every two weeks at a dose of 100mg / kg; combined with an increase of more than 4 times in ALT, AST, AFP, and APT levels as biochemical indicators of tumor occurrence, to ensure that the induced liver cancer tissue meets the experimental requirements.

[0015] Preferred: Step (1) specifically involves: removing blood and capsule from isolated duck liver tumors, washing them three times with pre-cooled Hank's solution, and then mincing the tissue in PBS; Step (2) is as follows: Add 10 times the volume of 0.02% EDTA to the chopped tissue and incubate at 4℃ for 30 min. Centrifuge at 800 rpm for 5 min and discard the supernatant. Add 10 times the volume of collagenase IV and incubate at 37℃ for 2 h. Shake every 10 min and filter the separated cells through a 70µm sieve. Step (3) is as follows: Centrifuge the cells at 450 × g for 10 minutes, discard the supernatant, and then resuspend them in DMEM / F12+10%FBS medium; Spread the obtained tumor cells in a 6-well tissue culture plate or culture flask coated with 0.1 mg / ml poly-L-lysine, with a concentration of 2 million cells per well; Step (4) specifically involves: obtaining a cell suspension using conventional digestion and passage methods, and counting the cells, with a standard of 1-2 × 10⁷; then serially diluting the cell suspension to 10-20 cells / mL. Step (5) is as follows: After mixing the diluted cell suspension, add 100 μL / well to a 96-well plate, plate 3-4 cell plates, and randomly select 8-10 wells from each plate to observe the number of cells. Step (6) specifically involves observing the color of the culture medium after 6-7 days. If the color turns yellow, it indicates cell proliferation wells. After marking, the medium is changed. Step (7) is as follows: After the cells in a single well have grown to confluence in 3-4 days, they are routinely digested and transferred to a 48-well plate, 0.5 mL per well. After 3 hours of plating, the morphology of the cells in each well is observed. Wells with consistent morphology are the target wells, which are marked and recorded. After 3 days of culture, the cells are further cultured.

[0016] Preferred: Step (7) also includes impurity removal and identification: using cell cloning method to remove mixed fibroblasts to ensure the purity of duck liver cancer cells; performing karyotype analysis to identify and verify the genetic stability of cells.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a Cherry Valley duck liver cancer cell line DMH and its application, and the technical effects achieved are as follows: The duck liver cancer cell line prepared by this invention provides a culture system for scientific research on waterfowl viruses and the development of vaccines and antibody drugs.

[0018] Currently, there are no immortalized cell lines available domestically or internationally for use in the production of waterfowl vaccines.

[0019] This invention discloses a method for preparing and applying an immortalized duck hepatoma cell line. The method uses Cherry Valley ducks as experimental animals. After standardized feeding and immunization, a duck hepatoma model is established through staged induction with gradient doses of DEN. Primary hepatoma cells are isolated through precise material collection, mechanical shearing combined with enzymatic digestion, and purified using an optimized culture system to maximize cell viability and purity, resulting in an immortalized cell line. This cell line exhibits stable genetic traits, is free from pathogen contamination, and remains genetically stable even after more than 30 passages. It does not rely on duck embryos / live organisms, completely eliminating source limitations. Cloning, purification, and aseptic handling ensure high purity and eliminate pathogen contamination, avoiding interfering factors. Derived from duck hepatoma tissue, it closely matches the growth requirements of waterfowl hepatotropic viruses, significantly increasing viral titers. It can grow under conventional culture conditions, is simple to operate, cost-effective, and easy to promote. It can efficiently support the proliferation of waterfowl hepatotropic viruses such as duck viral hepatitis virus, and is suitable for vaccine development and pathogen research.

[0020] The duck hepatoma cell line constructed in this invention can be used for the isolation, culture, and scientific research of waterfowl pathogens, such as the preparation of high-titer vaccines, and subsequently, the production of high-level antibodies. Furthermore, the obtained duck hepatoma cell line has a wide range of applications, suitable for viruses that cannot be cultured or proliferate at low titers in various heterologous cells. The cell line obtained in this invention exhibits high viral proliferation titers, effectively reducing production costs. The preparation and culture processes adhere to aseptic standards, use no harmful reagents, and meet the safety requirements of the biotechnology industry. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 The attached figure shows the morphology of duck liver cancer cell line passaged for 20 generations provided by the present invention.

[0023] Figure 2 The attached figure shows the morphology of duck liver cancer cell line passaged for 30 generations provided by the present invention.

[0024] Figure 3 The attached figure is a karyotype observation diagram of the cell line provided by the present invention.

[0025] Figure 4 The attached figure shows a CPE pattern in duck viral hepatitis virus-infected cells provided by this invention.

[0026] Figure 5 The attached figure shows the change in viral titer in cells infected with duck viral hepatitis virus provided by the present invention. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] This invention discloses a Cherry Valley duck liver cancer cell line DMH and its applications.

[0029] All raw materials and reagents not mentioned in the examples are commercially available; all experimental methods not mentioned are conventional experimental methods.

[0030] Example 1 Establishment of a diethylnitrosamine (DEN)-induced duck liver cancer model 1. Duck farming Two hundred Cherry Valley ducks were raised and divided into groups at 20 days of age. The control group consisted of 40 ducks, the low-dose group (50 mg / kg DEN) of 80 ducks, and the high-dose group (100 mg / kg DEN) of 80 ducks. Daily feed requirement: days × number of ducks × 0.016 catties. Immunization program: 1 day old, intramuscular injection of antibodies (duck hepatitis + parvovirus 0.3 ml / bird); 7 days old, subcutaneous injection of H5 bivalent / trivalent oil-based vaccine (0.5 ml / bird) in the neck; 15 days old, intramuscular injection (or drinking water) of duck plague live vaccine at 4 times the normal dose in the leg; 40 days old, subcutaneous injection of H5 bivalent / trivalent oil-based vaccine (0.5 ml / bird) in the neck; 90 days old, duck plague live vaccine at 4 times the normal dose in drinking water; 110 days old, subcutaneous injection of H5 bivalent / trivalent oil-based vaccine (0.5 ml / bird) in the neck. Nutritional requirements for medication: For infants aged 1-3 days, administer enrofloxacin solution in drinking water (1g / kg). For infants aged 1-5 days, administer 0.5% electrolyte compound multivitamin in drinking water. For infants aged 1 day, add 3% glucose. If symptoms appear at other ages, choose appropriate medication for treatment.

[0031] 2. Establishment of a liver cancer model The control group was fed normally, while the experimental group was temporarily fed a combined diet with 100 ppm DEN added to their drinking water until week 9. The experimental groups were then separated into a low-dose group and a high-dose group, each with 80 birds. From weeks 10 to 15, the experimental group received intraperitoneal injections of DEN once a week. The control group received an equal dose of physiological saline. From weeks 16 to 30, the experimental group received intraperitoneal injections of DEN every two weeks at a dose of 100 mg / kg. The mental state of the experimental group ducks was observed daily, especially the characteristics of abdominal distension, which was palpable as a fluctuation; these were the key monitoring targets. When the ascites volume decreased and the ducks' weight significantly decreased, they were euthanized and the tumor tissue was removed.

[0032] 3. Indicator Monitoring (1) From 20 days old to 30 weeks old, the weight of each group of ducks (3 ducks / group) was measured every week, and the weight change curve was plotted. (2) Record the survival rate and mental state of each group of ducks (take photos); (3) Blood samples were collected from each group of ducks (3 ducks / group) at 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 weeks. Serum was prepared by centrifugation. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (hepatitis markers) were measured using an automated biochemical analyzer. Alpha-fetoprotein (AFP) and abnormal prothrombin (APT) (liver tumor markers) were detected using an ELISA kit.

[0033] Once the duck flock develops ascites symptoms and tumor monitoring indicators are more than 4 times higher than normal (18-20 weeks), 3 ducks from each group are selected each week for necropsy to observe and record liver lesions.

[0034] Preparation of primary duck hepatoma cells 1. Perform gross excision of the duck liver tumor, remove blood and capsule, wash three times with pre-cooled Hank's solution, and mince the tissue in a small amount of PBS using ophthalmic scissors.

[0035] 2. Add 10 volumes of 0.02% EDTA to the chopped tissue and incubate at 4°C for 30 min. Centrifuge at 800 rpm for 5 min and discard the supernatant. Add 10 volumes of collagenase IV and incubate at 37°C for 2 h, gently shaking every 10 min. Filter the separated cells through a 70µm sieve.

[0036] 3. Centrifuge the cells at 450 × g for 10 minutes, discard the supernatant, and then resuspend them in culture medium (DMEM / F12 + 10% FBS). Spread the tumor cells onto 6-well tissue culture plates or culture flasks coated with poly-L-lysine (0.1 mg / ml) at a concentration of 2 million cells per well.

[0037] 4. Obtain cell suspensions using standard digestion and passage methods, and count the cells (typically 5 × 10⁶ cells per flask). 7 The cell has a relatively large surface area, approximately 1-2 × 10⁻⁶.7 The cell suspension was serially diluted to approximately 10-20 cells / mL.

[0038] 5. After mixing the diluted cell suspension, add 100 μL / well to a 96-well plate, generally for 3-4 wells. Randomly select 8-10 wells from each plate to observe the cell count (preferably no more than 3).

[0039] 6. Generally, observe the color of the culture medium after 6-7 days. Wells that turn yellow indicate cell proliferation, while wells that remain red indicate dead cells or no cells. Observe the cell morphology of each well and record it. Change the medium after labeling.

[0040] 7. After 3-4 days, once the cells in each well have reached confluence, they are routinely digested and transferred to 48-well plates, 0.5 mL per well. Observe the cell morphology of each well 3 hours after plating; wells with consistent morphology are designated as target wells, marked, and recorded. Continue culture for 3 days to expand the scale-up.

[0041] To further optimize the technical solution, conventional cell cloning methods can also be used to remove any mixed fibroblasts, ensuring the purity of duck liver cancer cells.

[0042] Technical effectiveness verification: 1. Morphological identification ( Figure 1 , 2 ) When continuously passaged duck hepatoma cell lines are observed under an inverted microscope, the duck hepatoma cells appear as long, prismatic shapes, forming a single layer, tightly packed, exhibiting typical epithelial cell characteristics, and still retaining the growth characteristics of primary cells.

[0043] 2. Chromosome karyotype identification ( Figure 3 ) On the day of the experiment, ensure the cells are in the logarithmic growth phase. Generally, one 6cm culture dish with 80% density is sufficient for one experiment. Remove the cells from the 37℃, 5% CO2 incubator, add colchicine (final concentration 0.2ug / ml), mix well, and incubate at 37℃ for 1-2 hours. While waiting, prepare a hypotonic solution of 0.075M KCl (prepared by adding 44.7mg KCl to 8ml of sterile water) and preheat in a 37℃ water bath. After incubation, digest the cells with trypsin, centrifuge at 1200 rpm for 5 minutes, collect in a centrifuge tube, remove the supernatant, resuspend in 8ml of hypotonic solution, mix well, and incubate in a 37℃ water bath for 40 minutes. Add 2 ml of freshly prepared fixative (methanol / glacial acetic acid = 2 / 1~3 / 1) and mix well. Incubate at room temperature for 10 min, then centrifuge at 1000 rpm for 10 min. Discard the supernatant (retain 500 μL; first, resuspend the cells at the bottom). Add 8 ml of fresh fixative, centrifuge at 1200 rpm for 10 min, and repeat three times. Drop the slide and age it in an 80℃ oven for 3 hours. Then, digest the dried slide in freshly prepared trypsin (0.25%) for 1 min (strictly control the time), and then stain it in Giemsa stain for 5-10 minutes (stain one slide for 5 minutes initially, adjusting the staining time as needed). Remove the slide, rinse off the stain with running water, and observe the results under a microscope.

[0044] The results showed that the cell line nuclei were diploid.

[0045] Example 2 Assay for the ability of duck hepatoma cell lines to culture duck viral hepatitis virus 1. Cell preparation: The self-prepared duck hepatoma cell line was revived and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 until the logarithmic growth phase. Cells were digested and collected, and the cell concentration was adjusted to 5 × 10⁶ cells / mL. 5 Cells / mL were seeded into 96-well cell culture plates, 100 μL per well, and cultured for 12-24 h until a dense monolayer of cells was formed.

[0046] 2. Virus inoculation and sampling: Duck viral hepatitis virus (DHAV) was inoculated into monolayer cells at a multiplicity of infection (MOI) of 0.1. After adsorption at 37°C for 1–2 h, the virus solution was discarded, and DMEM maintenance medium containing 2% fetal bovine serum was added. The cells were then incubated at 37°C in a 5% CO2 incubator. Sampling was performed at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h post-infection, with four replicates at each time point. A control group of normal cells without virus inoculation was also included.

[0047] 3. CPE Observation and Positive Well Count: Cell cultures from each sampling time point were collected, and the virus solution was diluted to 10^10 using a 10-fold serial dilution method. -1 ~10 -6 Virus solutions of various dilutions were inoculated into pre-cultured duck hepatoma cell monolayers in 96-well plates, 100 μL per well, with 8 replicates per dilution. After adsorption at 37°C for 1 h, 100 μL of maintenance medium was added. Culture was continued for 3–5 days, and cytopathic effect (CPE) was observed daily using an inverted microscope. The CPE positivity rate of each dilution well was recorded. Figure 4 As shown.

[0048] 4. Titer Calculation and Growth Curve Plotting: Based on CPE observation results, the Reed-Muench method was used to calculate the viral TCID at each sampling time point. 50 / mL value. Plot time post-infection on the x-axis, -lgTCID 50 A one-step growth curve of DHAV was plotted with 0.1 mL as the ordinate to analyze the proliferation dynamics of DHAV in duck hepatoma cell lines. The results are as follows: Figure 5 As shown, the proliferation kinetics of duck viral hepatitis virus in self-made duck hepatocellular carcinoma cell lines revealed that: the viral titer was at a low level 12 h post-infection, representing the viral latency period; the viral titer rapidly increased from 24 to 48 h, entering the logarithmic proliferation phase; and the proliferation peak (-lgTCID) was reached at 60 h. 50 (Approximately 5.3 per 0.1 mL), the titer decreased slightly after 72 h but remained at a high level, indicating that this duck hepatocellular carcinoma cell line can efficiently support the replication and proliferation of DHAV.

[0049] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0050] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An immortalized Cherry Valley duck liver cancer cell line, DMH ( Anas platyrhynchos domesticus ), characterized in that, The accession number is CCTCC NO: C2025364.

2. The application of the immortalized Cherry Valley duck liver cancer cells DMH as described in claim 1 in scientific research on waterfowl viruses, and in the development of vaccines, antibodies, or drugs.

3. The use according to claim 2, wherein Waterfowl virus: Duck viral hepatitis virus.

4. A method for establishing immortalized duck liver cancer cells, characterized in that, Includes the following steps: (1) Wash and chop the isolated duck liver tumor; (2) Add EDTA to the chopped tissue and incubate, centrifuge and discard the supernatant, add collagenase IV and incubate, then filter; (3) Centrifuge the cells, discard the supernatant, and then resuspend them in the culture medium; spread the obtained tumor cells evenly; (4) Cell suspensions were obtained using conventional digestion and passage methods, followed by serial dilution; (5) After mixing the diluted cell suspension, spread it onto a cell plate; (6) Observe the color of the culture medium. If the color turns yellow, it indicates cell proliferation wells. Change the medium. (7) After the cells in a single well have grown to full capacity, they are routinely digested and transferred, plated for observation, recorded, and cultured on a larger scale.

5. The method as described in claim 4, characterized in that, Before step (1), the following steps are included: inducing a duck liver cancer model using DEN: liver cancer is induced in stages by a combination of drinking water and intraperitoneal injection: 0-9 weeks are basal induction by adding 100ppm DEN to the drinking water; 10-15 weeks are intraperitoneal injection of DEN once a week; 16-30 weeks are intraperitoneal injection of DEN once every two weeks at a dose of 100mg / kg; combined with an increase of more than 4 times in ALT, AST, AFP, and APT levels as biochemical indicators of tumor development, to ensure that the induced liver cancer tissue meets the experimental requirements.

6. The method as described in claim 5, characterized in that, Step (1) specifically involves removing blood and capsule from isolated duck liver tumors, washing them three times with pre-cooled Hank's solution, and then mincing the tissue in PBS. Step (2) is as follows: Add 10 times the volume of 0.02% EDTA to the chopped tissue and incubate at 4℃ for 30 min. Centrifuge at 800 rpm for 5 min and discard the supernatant. Add 10 times the volume of collagenase IV and incubate at 37℃ for 2 h. Shake every 10 min and filter the separated cells through a 70µm sieve. Step (3) specifically involves centrifuging the cells at 450 × g for 10 minutes, discarding the supernatant, and then resuspending them in DMEM / F12+10%FBS medium. The obtained tumor cells were spread evenly in 6-well tissue culture plates or culture flasks coated with 0.1 mg / ml poly-L-lysine, with a concentration of 2 million cells per well; Step (4) is specifically: using conventional digestion passage to obtain cell suspension, and counting, standard is 1-2x10 7 ; gradient dilution is carried out to the cell suspension, to 10-20 / mL; Step (5) is as follows: After mixing the diluted cell suspension, add 100 μL / well to a 96-well plate, plate 3-4 cell plates, and randomly select 8-10 wells from each plate to observe the number of cells. Step (6) specifically involves observing the color of the culture medium after 6-7 days. If the color turns yellow, it indicates cell proliferation wells. After marking, the medium is changed. Step (7) is as follows: After the cells in a single well have grown to confluence in 3-4 days, they are routinely digested and transferred to a 48-well plate, 0.5 mL per well. After 3 hours of plating, the morphology of the cells in each well is observed. Wells with consistent morphology are the target wells, which are marked and recorded. After 3 days of culture, the cells are further cultured.

7. The method as described in claim 6, characterized in that, Step (7) also includes impurity removal and identification: cell cloning method is used to remove mixed fibroblasts to ensure the purity of duck liver cancer cells; karyotype analysis is performed to identify and verify the genetic stability of the cells.

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