A duck corneal epithelial cell suspension cell line, its construction method and application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a duck corneal epithelial cell suspension cell line, its construction method, and application. The present invention is the first to isolate and obtain adult corneal epithelial cells from ducks and successfully suspend and domesticate them. The cell line is named Duck Corneal Epithelial Suspension Cells (DCE-S) and was deposited in the China Center for Type Culture Collection at Wuhan University, Wuhan, China on June 28, 2024, with the deposit number CCTCC NO: C2024189. The cell line is easy to culture, grows rapidly, can be stably passaged, and has a high cell density of 6 to 8×10 6 cells / mL, exhibiting individually dispersed growth, plump morphology, uniform size, and high cell viability, making it suitable for serum-free suspension culture in bioreactors. This cell line can be used to culture, isolate, and detect avian viruses, prepare avian vaccines, and screen for drugs used to prevent or treat diseases caused by avian viral infections.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a duck corneal epithelial cell suspension cell line, a construction method thereof, and an application thereof. Background Art

[0002] China is a major country in meat duck production and consumption. More than 3 billion meat ducks are produced and consumed annually, accounting for more than 70% of the global meat duck sales, with an annual output value of more than 100 billion yuan. Along with the increase in the breeding volume, poultry diseases occur frequently. Novel viral infectious diseases such as avian influenza (subtypes H5, H7, and H9, etc.), duck plague, duck viral hepatitis, gosling plague of young muscovy ducks, muscovy duck parvovirus disease, duck parvovirus disease, duck coronavirus enteritis, duck pox, muscovy duck reovirus disease, novel duck reovirus disease, duck paramyxovirus disease, duck circovirus, duck tembusu virus disease, etc. are all the most serious diseases endangering the waterfowl breeding industry, seriously restricting the development of the poultry industry.

[0003] Vaccine immunization is the most effective means to prevent animal diseases. The production of domestic poultry vaccines mainly relies on chicken embryo culture technology, avian primary cell culture technology, and continuous cell culture technology. Among them, the chicken embryo culture technology has a long culture cycle, cumbersome operation steps, and high production costs. There are problems of unstable chicken embryo quality and potential exogenous virus contamination during large-scale production. After harvesting, it is difficult to harmlessly treat the waste embryos, and there are risks of virus spread and environmental pollution. For the avian primary cell culture technology, fresh primary cells need to be prepared each time, and virus vaccines can only be produced through adherent culture technology. On the one hand, serum is required to maintain the culture, resulting in high costs; on the other hand, the scale-up is limited, and the virus yield decreases due to the limitation of the matrix surface area, and it is easy to be contaminated and the quality between batches is inconsistent. For the continuous cell culture technology, the existing cell-derived vaccines mainly use heterologous cells (such as using canine kidney cells (MDCK suspension cells) to culture avian influenza virus) as the culture matrix for virus production, and the immune effect of the vaccine is slightly worse than that of chicken embryo culture.

[0004] The research and development of domestic poultry cells and the achievements are few, and there is no commercial duck-derived cell line. In order to better and more effectively isolate, proliferate, and produce poultry viruses, develop waterfowl-specific vaccines, and prevent disease outbreaks, it is of great significance to develop a duck-derived cell suspension cell line suitable for industrial large-scale production.

[0005] Application No. 202111338851.3, "Goose Retinal Epithelial Cell Line and Its Construction Method and Application", mainly obtains 1 low-serum adherent culture type goose retinal epithelial cell line, which is not a duck-derived cell line and cannot be cultured in serum-free suspension.

[0006] Application No. 202210113778.8, "Porcine Retinal Epithelial Cell Line and Its Construction Method and Application", mainly aims to obtain a porcine retinal epithelial cell line, not a duck-derived cell line, and it cannot be cultured in serum-free suspension. Summary of the Invention

[0007] The purpose of the first aspect of the present invention is to provide a cell line.

[0008] The purpose of the second aspect of the present invention is to provide a construction method of the cell line of the first aspect of the present invention.

[0009] The purpose of the third aspect of the present invention is to provide an application.

[0010] The purpose of the fourth aspect of the present invention is to provide a method for culturing a virus.

[0011] The purpose of the fifth aspect of the present invention is to provide a method for a virus vaccine.

[0012] The purpose of the sixth aspect of the present invention is to provide a virus vaccine.

[0013] In order to achieve the above purposes of the present invention, the technical solutions adopted by the present invention are as follows:

[0014] In the first aspect of the present invention, a duck corneal epithelial cell line named Shelduck Corneal Epithelial Suspension Cells DCE-S is provided, which was deposited on June 28, 2024, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C2024189.

[0015] In the second aspect of the present invention, a construction method of the duck corneal epithelial cell line of the first aspect of the present invention is provided, including the following steps:

[0016] 1) Separate the eyeballs of duck embryos, separate the corneas, and cut them into tissue blocks;

[0017] 2) Add the first culture medium, mix well, culture, and passage 5 - 15 generations;

[0018] 3) Culture with the second culture medium and passage 3 - 5 generations;

[0019] 4) Culture with the third culture medium and passage 3 - 5 generations;

[0020] 5) Culture with the fourth culture medium and passage 3 - 5 generations,

[0021] 6) Culture with serum-free suspension medium and passage 20 - 30 generations to obtain.

[0022] Preferably, the first culture medium includes 8 - 10 v / v% fetal bovine serum and a basal medium.

[0023] Preferably, the second culture medium comprises 6-8 v / v% fetal bovine serum and a basal medium.

[0024] Preferably, the third culture medium comprises 4-6 v / v% fetal bovine serum and a basal medium.

[0025] Preferably, the fourth culture medium comprises 2-3 v / v% fetal bovine serum and a basal medium.

[0026] Preferably, the basal medium comprises at least one of MEM medium, DMEM medium, and DMEM / F12 medium.

[0027] In some embodiments of the present invention, the construction method comprises: 1) Take 3 duck embryos at 7-11 days old, separate the eyeballs with ophthalmic scissors, place them in a clean sterile petri dish, wash them 3 times with pre-cooled D-Hank's solution, separate the corneas, cut them into pieces, add a small amount of MEM with 8-10 v / v% fetal bovine serum, blow and break the tissue blocks, transfer them into a culture flask for culture, and the culture conditions are 37°C in a 5% CO2 incubator; 2) 12-24 h after cell separation and culture, gently shake the cell flask, discard the supernatant, only leave the adherent cells, supplement with fresh MEM with 8-10 v / v% fetal bovine serum, and continue the culture; when the cells grow into a monolayer, digest them with 0.25 wt% EDTA-trypsin until single cells, then inoculate them into a 96-well plate at a density of 0.5 cells / well on average, mark the wells inoculated with single cells, and after the cells in these single-cell wells grow into clone clusters, a duck corneal epithelial single-cell strain can be obtained, and continuously passage it more than 10 generations; 3) Culture with MEM medium containing 6-8 v / v% fetal bovine serum, and the culture conditions are 37°C in a 5% CO2 incubator. After continuously culturing for 3-5 generations, culture with MEM medium containing 4-6 v / v% fetal bovine serum; after continuously culturing for 3-5 generations, culture with MEM medium containing 2-3 v / v% fetal bovine serum; after continuously culturing for 3-5 generations, obtain low-serum adherent culture-type duck corneal epithelial cells; 4) When the low-serum adherent culture-type duck corneal epithelial cells grow into a monolayer, digest them with 0.25 wt% EDTA-trypsin, collect the cell suspension, centrifuge at 800-1000 rpm for 6 min, discard the supernatant, leave the cell pellet, resuspend the cell pellet with a serum-free suspension medium, adjust the cell density to 1.0×10 6 cells / mL, and the culture conditions are 37°C, 5% CO2, and culture on a shaker at 100-130 r / min. Take samples every 24 hours to observe the cell morphology and count. After culturing for 2-3 days, centrifuge and change the medium for passage until the number of cells in each generation is ≥6.0×10 6cells / mL. The cells were stably and continuously cultured and passaged more than 30 generations to obtain serum-free fully suspended cultured duck corneal epithelial cells, namely the duck corneal epithelial cell suspension cell line.

[0028] Preferably, the size of the tissue fragments in step 1) is 1-2 cm 3 / piece.

[0029] Preferably, the culture conditions are 31-39 °C and 4-6% CO2; more preferably 33-37 °C and 5% CO2.

[0030] The third aspect of the present invention lies in providing the application of the duck corneal epithelial cell line of the first aspect of the present invention in any one of 1)-8):

[0031] 1) Culturing viruses;

[0032] 2) Preparing products for culturing viruses;

[0033] 3) Isolating viruses;

[0034] 4) Preparing products for isolating viruses;

[0035] 5) Detecting viruses for non-diagnostic and therapeutic purposes;

[0036] 6) Preparing products for detecting viruses;

[0037] 7) Preparing virus vaccines;

[0038] 8) Drug screening;

[0039] The drug is a drug for preventing or treating diseases caused by viral infections.

[0040] Preferably, the virus includes at least one of avian influenza virus, duck circovirus, and duck Tembusu virus.

[0041] Preferably, the avian influenza virus includes H9 subtype avian influenza virus.

[0042] The fourth aspect of the present invention provides a method for culturing viruses, inoculating the virus into the duck corneal epithelial cell line of the first aspect of the present invention and culturing.

[0043] Preferably, the virus includes at least one of avian influenza virus, duck circovirus, and duck Tembusu virus.

[0044] Preferably, the avian influenza virus includes H9 subtype avian influenza virus.

[0045] Preferably, the culture conditions are 31-39 °C and 4-6% CO2; more preferably 33-37 °C and 5% CO2.

[0046] In a fifth aspect of the present invention, there is provided a method for preparing a viral vaccine, comprising inoculating a virus into the duck corneal epithelial cell line of the first aspect of the present invention, culturing, and inactivating to obtain the viral vaccine.

[0047] Preferably, the virus includes at least one of avian influenza virus, duck circovirus, and duck Tembusu virus.

[0048] Preferably, the avian influenza virus includes H9 subtype avian influenza virus.

[0049] Preferably, the culturing conditions are 31 - 39°C and 4 - 6% CO2; more preferably 33 - 37°C and 5% CO2.

[0050] In a sixth aspect of the present invention, there is provided a viral vaccine prepared by the method of the fifth aspect of the present invention.

[0051] The beneficial effects of the present invention are as follows:

[0052] Through a large amount of creative work by the inventors, duck corneal epithelial cells were isolated from ducks for the first time, successfully suspended and domesticated, and a duck corneal epithelial cell line named Muscovy duck corneal epithelial suspension cells DCE - S was provided. It was deposited with the China Center for Type Culture Collection, Wuhan University, Wuhan, China on June 28, 2024, with the deposit number CCTCC NO: C2024189. This cell line is easy to culture, has a relatively fast growth rate, can be stably passaged, has a high cell density, up to 6 - 8×10 6 cells / mL, grows in a single - dispersed manner, has a plump morphology, uniform size, a high cell viability, and is suitable for serum - free suspension culture in a bioreactor. This cell line can be used for culturing, isolating, and detecting avian viruses, preparing avian vaccines, and screening drugs for preventing or treating diseases caused by avian virus infections. This cell line provides an important culture substrate for the large - scale suspension culture of avian vaccines and cost reduction and efficiency improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The present invention will be further described below in conjunction with the drawings and examples, where:

[0054] Figure 1 It is the duck corneal epithelial cells in low - serum adherent culture type in Example 1; the left side shows the result after 24 - hour culture, and the right side shows the result after 48 - hour culture.

[0055] Figure 2 It is the growth state result of the 25th passage of Muscovy duck corneal epithelial suspension cells DCE - S.

[0056] Figure 3It shows the stable subculture of different passages of corneal epithelial suspension cells DCE-S (F9 - F30) of mallard ducks.

[0057] Figure 4 It shows the cytopathic effect of corneal epithelial suspension cells DCE-S of mallard ducks inoculated with H9 subtype avian influenza virus for 72 hours.

[0058] Figure 5 It shows the cytopathic effect of corneal epithelial suspension cells DCE-S of mallard ducks inoculated with duck circovirus for 60 hours.

[0059] Figure 6 It shows the cytopathic effect of corneal epithelial suspension cells DCE-S of mallard ducks inoculated with duck Tembusu virus for 50 hours. Detailed implementation mode

[0060] The following will clearly and completely describe the concept and technical effects of the present invention in combination with embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work belong to the scope of protection of the present invention.

[0061] Example 1 Construction of duck corneal epithelial cell suspension cell line

[0062] 1. Rapid isolation and culture of primary duck corneal epithelial cells

[0063] (1) Take duck embryos at 10 days of incubation. After candling, draw the boundary of the air chamber and the fetal position.

[0064] (2) Disinfect the eggshell of the embryo with 75% alcohol. In a sterile operating table, gently break the eggshell at the air chamber part with sterile forceps and remove it. Use another sterile forceps to tear off the shell membrane at the air chamber part, and then use another sterile forceps to take out the embryo body and put it into a sterile petri dish.

[0065] (3) Separate the eyeball with ophthalmic scissors, put it into a clean sterile petri dish, wash it 3 times with pre-cooled D-Hank's solution, separate the cornea, cut it into pieces, add a small amount of MEM with 10 v / v% fetal bovine serum, blow and break the tissue blocks, and transfer them into a culture flask for culture. The culture conditions are 37°C and 5% CO2 incubator.

[0066] (4) 24 hours after cell separation and culture, gently shake the cell flask, discard the supernatant, and only leave the adherent cells. Supplement with fresh MEM with 10% fetal bovine serum and continue the culture.

[0067] (5) When the cells grow into a monolayer, digest them with 0.25 wt% EDTA-trypsin until single cells are obtained. Then, inoculate them into a 96-well plate at a density of 0.5 cells / well on average, mark the wells inoculated with single cells, and a duck corneal epithelial single-cell strain can be obtained after the cells in these single-cell wells grow into clone clusters.

[0068] (6) Continuously passage the duck corneal epithelial single-cell strain to the 10th generation to obtain an adherent culture type of duck corneal epithelial cells with a uniform morphology.

[0069] 2. Construction of duck corneal epithelial cell suspension cell strain

[0070] (1) Obtaining low-serum adherent culture type of duck corneal epithelial cells: When the duck corneal epithelial cells in step 1(6) grow into a monolayer, digest them with 0.25 wt% EDTA-trypsin, and culture them with MEM medium containing 8 v / v% fetal bovine serum. The culture conditions are 37 °C in a 5% CO2 incubator. After continuous culture for 5 generations, culture them with MEM medium containing 6 v / v% fetal bovine serum; after continuous culture for 5 generations, culture them with MEM medium containing 3 v / v% fetal bovine serum; after continuous culture for 5 generations, low-serum adherent culture type of duck corneal epithelial cells are obtained. The results are as Figure 1 shown.

[0071] (2) Obtaining duck corneal epithelial cell suspension cell strain: When the low-serum adherent culture type of duck corneal epithelial cells grow into a monolayer, digest them with 0.25 wt% EDTA-trypsin, collect the cell suspension, centrifuge at 800 rpm for 6 min, discard the supernatant, leave the cell pellet, resuspend the cell pellet with serum-free suspension medium (BSL-01 medium, Guangzhou Biosai Biopharmaceutical Technology Co., Ltd.), adjust the cell density to 1.0×10 6 cells / mL, and culture them at 37 °C, 5% CO2, and 120 r / min on a shaker. Sample and observe the cell morphology and count every 24 hours. After 3 days of culture, centrifuge and change the medium for passage until the cell number of each generation of cells is ≥6.0×10 6 cells / mL, the cells are stable and continuously cultured and passaged to the 30th generation to obtain serum-free fully suspended culture type of duck corneal epithelial cells, that is, duck corneal epithelial cell suspension cell strain.

[0072] During the construction process of the above duck corneal epithelial cell suspension cell strain, freeze the cells and establish a cell bank every 5 passages.

[0073] The above serum-free fully suspended cultured duck corneal epithelial cells are named Muscovy duck corneal epithelial suspended cells DCE-S. When subcultured to the 25th passage, they were deposited on June 28, 2024 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C2024189. Figure 2 It is the growth status result of the 25th passage of Muscovy duck corneal epithelial suspended cells DCE-S. Figure 3 It is the stable subculture situation of Muscovy duck corneal epithelial suspended cells DCE-S (F9 - F30) at different passages. The passage starting from suspension culture is recorded as F1. The cell density of Muscovy duck corneal epithelial suspended cells DCE-SD is stable above 6.0×106 cells / mL from F9 to F30 passages.

[0074] Example 2 Testing of inoculating Muscovy duck corneal epithelial suspended cells DCE-S with H9 subtype avian influenza virus

[0075] Take the 25th passage cells of Muscovy duck corneal epithelial suspended cells DCE-S, place them in a shaker at 37°C, 5% CO2, and 120 r / min for 72 h. Wait until the cell density reaches above 6×10 6 cells / mL, then dilute the cells to 4×10 6 cells / mL with fresh medium. At the same time, inoculate with H9 subtype avian influenza virus at 1‰ v / v (virus titer HA = 9log2, EID 50 = 10 -8.0 / 0.1 mL), and add TPCK trypsin with a final concentration of 5 μg / mL. Adjust the temperature to 35°C and culture for 72 h to harvest the virus solution and detect HA. The results show that Muscovy duck corneal epithelial suspended cells DCE-S are sensitive to H9 subtype avian influenza virus, and HA ≥ 9log2. Figure 4 It is the cytopathic effect map of Muscovy duck corneal epithelial suspended cells DCE-S inoculated with H9 subtype avian influenza virus for 72 h. A large number of cells died and lysed, there were more cell debris, and the number of live cells decreased significantly.

[0076] The above strain has been disclosed in the literature of Qi, Xuefeng et al. “Down-regulation of cellular protein heme oxygenase-1 inhibits proliferation of avian influenza virus H9N2 in chicken oviduct epithelial cells.” The Journal of general virology vol.99, 1 (2018): 36 - 43. doi:10.1099 / jgv.0.000986.

[0077] Example 3: Test on duck circovirus (DuCV) inoculation of corneal epithelial suspension cells DCE-S of duck eyes

[0078] The 25th generation of corneal epithelial suspension cells of duck eyes, DCE-S, were cultured in a shaking incubator at 37°C, 5% CO2, and 120 rpm for 72 h. 6 cells / mL, add fresh culture medium to dilute the cells to 4×10 6 cells / mL, and duck circovirus was inoculated at 5‰ v / v (virus titer TCID 50 =10 -6.0 / 0.1mL), adjust the temperature to 37°C, and harvest the virus solution after 60h of incubation. Detect TCID 50 The results showed that the corneal epithelial suspension cells DCE-S of ducks were sensitive to duck circovirus, and the TCID 50 ≥10 -7.0 / 0.1mL. Figure 5 This is a cytopathic effect image of duck corneal epithelial suspension cells DCE-S inoculated with duck circovirus for 60 hours. A large number of cells died and lysed, there were many cell fragments, and the number of live cells was significantly reduced.

[0079] The above strains have been published in the literature of Xinyou et al. Establishment of a dual fluorescence quantitative PCR detection method for duck hepatitis A virus type 3 and duck circovirus [J]. China Poultry.

[0080] Example 4: Test of duck corneal epithelial suspension cells DCE-S inoculated with duck Tembusu virus (DTMUV)

[0081] The 25th generation of corneal epithelial suspension cells of DCE-S were taken from duck eyes and cultured in a shaking incubator at 37°C, 5% CO2, and 120 rpm for 72 h. 6 cells / mL, add fresh culture medium to dilute the cells to 4×10 6 cells / mL, and duck Tembusu virus was inoculated at 0.5‰ v / v (virus titer TCID 50 =10 -7.0 / 0.1mL), adjust the temperature to 37°C, and harvest the virus solution after 50h of incubation. Detect TCID 50 The results showed that the duck corneal epithelial cell suspension strain was sensitive to duck Tembusu virus, and the TCID 50 ≥10 -7.5 / 0.1mL. Figure 6 This is a cytopathic effect image of duck corneal epithelial suspension cells DCE-S inoculated with duck Tembusu virus 50 hours later. A large number of cells died and lysed, there were many cell fragments, and the number of live cells was significantly reduced.

[0082] The above-mentioned virus strains have been disclosed in the literature "Establishment and Preliminary Application of a One-Step RT-PCR Detection Method for Duck Tembusu Virus" by Zu Lichuang et al., published in Guangdong Journal of Animal Husbandry and Veterinary Science, 2015 (Vol. 40), No. 5, pp. 37-40.

Claims

1. A duck corneal epithelial cell line named Muscovy duck corneal epithelial suspension cell DCE-S was deposited on June 28, 2024 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: C2024189.

2. Use of the duck corneal epithelial cell line according to claim 1 in any one of 1) to 4): 1) Culturing a virus; 2) Preparing a product for culturing a virus; 3) Isolating a virus; 4) Preparing a product for isolating a virus; The virus includes at least one of avian influenza virus, duck circovirus, and duck Tembusu virus.

3. A method for culturing a virus, comprising inoculating the virus into the duck corneal epithelial cell line according to claim 1 and culturing; The virus includes at least one of avian influenza virus, duck circovirus, and duck Tembusu virus.

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