Chicken embryo fibroblast cell, its preparation method and application

Abstract

This invention discloses a chicken embryo fibroblast, its preparation method, and its application, relating to the field of biopharmaceutical technology. The preparation method of the chicken embryo fibroblast includes the following steps: S1, providing chicken embryos and obtaining the muscle tissue of the chicken embryos; S2, mixing the muscle tissue with a plant-derived compound digestion solution, and sequentially performing digestion, purification, and primary culture treatment using a serum-free special culture medium to obtain primary chicken embryo fibroblasts; wherein, the plant-derived compound digestion solution includes bromelain and papain. This invention, using a plant-derived compound digestion solution and a serum-free special culture medium, significantly improves the batch-to-batch stability, cell yield, and cell purity of the cells, reducing the risk of exogenous viral contamination. Therefore, the prepared chicken embryo fibroblasts exhibit high batch-to-batch stability and good reproducibility in viral culture.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a chicken embryo fibroblast, its preparation method, and its application. Background Technology

[0002] Chicken embryo fibroblasts (CEFs) are primary fibroblast-like cells isolated from the muscle tissue of chicken embryos. They exhibit strong adherent growth and proliferation capabilities. CEFs are widely used in the culture of viral vaccines such as those for Newcastle disease, Marek's disease, and fowlpox.

[0003] Currently, the suspension culture process for chicken embryo fibroblasts mainly uses a medium containing 8% fetal bovine serum and relies on animal-derived trypsin for digestion. This process has the following drawbacks: (1) Using culture medium containing fetal bovine serum affects cell stability, resulting in large batch differences and the risk of exogenous virus contamination. Existing serum-free special culture medium with specific feed formulation still has the problem of poor culture medium compatibility. (2) Relying on single animal-derived trypsin digestion results in a high cell damage rate, with a survival rate of only 80%-85%, and the risk of exogenous virus contamination. (3) The product contains a high proportion of impurity cells (such as red blood cells and epithelial cells), with a purity of less than 90%. (4) Large-scale culture relies on adherent square bottles, resulting in low production capacity and high cost, making it difficult to meet the needs of large-scale vaccine production. Summary of the Invention

[0004] The main objective of this invention is to propose a chicken embryo fibroblast, its preparation method, and its application, aiming to solve the problems of large batch-to-batch variations and easy introduction of exogenous viruses in the chicken embryo fibroblasts prepared in the prior art.

[0005] To achieve the above objectives, the present invention proposes a method for preparing chicken embryo fibroblasts, the method comprising the following steps: S1. Provide a chicken embryo and obtain the trunk muscle tissue of the chicken embryo; S2. The trunk muscle tissue is mixed with plant-derived compound digestion solution and then digested, purified, and cultured in a serum-free special culture medium to obtain primary chicken embryo fibroblasts, i.e., chicken embryo fibroblasts. The plant-derived complex digestive fluid includes bromelain and papain.

[0006] In one embodiment, an incubation process is included prior to the digestion process.

[0007] In one embodiment, the mass ratio of bromelain to papain in the plant-derived complex digestive solution is (1-2):1; and / or, The plant-derived complex digestive solution also includes EDTA and phosphate buffer: the mass percentage of bromelain is 0.1%-0.2%, the mass percentage of papain is 0.05%-0.15%, the mass percentage of EDTA is 0.01%-0.02%, and the remainder is phosphate buffer.

[0008] In one embodiment, step S2 includes: S21. Mix the trunk muscle tissue with the plant-derived compound digestive solution, incubate and digest to obtain the digestive solution. S22. Add an animal-free neutralizing solution to the digestion solution, centrifuge, wash, and obtain a precipitate; the animal-free neutralizing solution includes a phosphate buffer containing plant peptone, soybean trypsin inhibitor, and trehalose. S23. The precipitate is resuspended in a serum-free culture medium and purified by passing it through a cell sieve and an immunomagnetic bead method. The filtrate is collected to obtain a suspension of primary chicken embryo fibroblasts. S24. The primary chicken embryo fibroblast suspension is inoculated by pre-coating and primary culture is carried out in serum-free special culture medium to obtain primary chicken embryo fibroblasts, i.e., chicken embryo fibroblasts.

[0009] In one embodiment, in step S21, the incubation temperature is 0-4°C; and / or, In step S21, the incubation time is 35-45 min; and / or, In step S21, the digestion treatment temperature is 30-40℃; and / or, In step S21, the digestion process takes 15-25 minutes; and / or, In step S22, the volume ratio of the digestive fluid to the animal-free neutralizing fluid is 1:(1.5-2.5). In step S22, the centrifugation speed is 800-900 r / min; and / or, In step S22, the centrifugation time is 8-12 min; and / or, In step S24, the inoculation density is 4.0 × 10⁻⁶. 5 -5.0×10 5 cells / cm 2 .

[0010] In one embodiment, the following steps are included after step S2: S3. Pass the primary chicken embryo fibroblasts to the P2-P3 generation and screen to obtain seed cells; S4. The seed cells are inoculated into microcarriers and cultured in a segmented fed-batch manner. The cells are then recovered and purified to obtain passaged chicken embryo fibroblasts, also known as chicken embryo fibroblasts.

[0011] In one embodiment, in step S3: the survival rate of the seed cells is ≥96%, and the purity of the seed cells is ≥99%.

[0012] In one embodiment, in step S4: The seed cells were inoculated into the microcarrier at a density of 4.0 × 10⁶. 4 -5.0×10 4 cells / cm 2 ; and / or, The microcarriers include dextran microcarriers, wherein the particle size of the dextran microcarriers is 120-180 μm and the pore size of the dextran microcarriers is 20-30 μm; and / or, The parameters for the segmented fed-batch culture are: temperature 36-37℃, pH 7.1-7.3, dissolved oxygen 35%-45%, and stirring speed 50-55 r / min; after culturing for 24-48h, 0.6% glucose solution is added, and after culturing for 48-72h, 10% serum-free culture medium at twice the concentration is added.

[0013] The present invention also provides a chicken embryo fibroblast, which is prepared according to the aforementioned method for preparing chicken embryo fibroblasts.

[0014] The present invention also provides an application of chicken embryo fibroblasts in virus culture, wherein the chicken embryo fibroblasts include the aforementioned chicken embryo fibroblasts or chicken embryo fibroblasts prepared by the aforementioned chicken embryo fibroblast preparation method; The virus includes at least one of Newcastle disease virus, Marek's disease virus, and fowlpox virus.

[0015] In this invention, the use of a plant-derived compound digestive solution to digest the trunk muscle tissue of chicken embryos reduces damage to trunk muscle cells and improves the survival rate and yield of primary chicken embryo fibroblasts, thereby enhancing batch-to-batch stability. Primary culture is performed using a serum-free culture medium, which avoids the introduction of blood cell impurities and exogenous animal viruses, thus improving the purity of the primary chicken embryo fibroblasts. This invention, by using a plant-derived compound digestive solution and a serum-free culture medium, significantly improves batch-to-batch stability, cell yield, and cell purity. Therefore, the prepared chicken embryo fibroblasts cultured with viruses exhibit high batch-to-batch stability and good reproducibility. Attached Figure Description

[0016] 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 some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0017] Figure 1 This is an optical microscope image of the primary chicken embryo fibroblast suspension after double purification in Example 1 of the present invention; Figure 2 This is an optical microscope image of primary chicken embryo fibroblasts in Example 1 of the present invention.

[0018] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. 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.

[0020] Chicken embryo fibroblasts (CEFs) are primary fibroblast-like cells isolated from the muscle tissue of chicken embryos. They exhibit strong adherent growth and proliferation capabilities. CEFs are widely used in the culture of viral vaccines such as those for Newcastle disease, Marek's disease, and fowlpox.

[0021] Currently, the suspension culture process for chicken embryo fibroblasts mainly uses a medium containing 8% fetal bovine serum and relies on animal-derived trypsin for digestion. This process has the following drawbacks: (1) Using culture medium containing fetal bovine serum affects cell stability, resulting in large batch differences and the risk of exogenous virus contamination. Existing serum-free special culture medium with specific feed formulation still has the problem of poor culture medium compatibility. (2) Relying on single animal-derived pancreatic enzyme digestion results in a high cell damage rate, with a survival rate of only 80%-85%, and the risk of exogenous virus contamination. (3) The product contains a high proportion of miscellaneous cells (such as red blood cells and epithelial cells), with a purity of less than 90%. (4) Large-scale culture relies on adherent square bottles, resulting in low production capacity and high cost, making it difficult to meet the needs of large-scale vaccine production.

[0022] In view of this, the present invention provides a method for preparing chicken embryo fibroblasts, the method comprising the following steps: Step S1: Provide a chicken embryo and obtain the trunk muscle tissue of the chicken embryo; Step S2: Mix the trunk muscle tissue with plant-derived compound digestion solution, and perform digestion treatment, purification treatment and primary culture treatment using serum-free special culture medium to obtain primary chicken embryo fibroblasts, i.e. chicken embryo fibroblasts. The plant-derived complex digestive fluid includes bromelain and papain.

[0023] In this invention, the use of a plant-derived compound digestive solution to digest the trunk muscle tissue of chicken embryos reduces damage to trunk muscle cells and improves the survival rate and yield of primary chicken embryo fibroblasts, thereby enhancing batch-to-batch stability. Primary culture is performed using a serum-free culture medium, which avoids the introduction of blood cell impurities and exogenous animal viruses, thus improving the purity of the primary chicken embryo fibroblasts. This invention, by using a plant-derived compound digestive solution and a serum-free culture medium, significantly improves batch-to-batch stability, cell yield, and cell purity. Therefore, the prepared chicken embryo fibroblasts cultured with viruses exhibit high batch-to-batch stability and good reproducibility.

[0024] In some embodiments, an incubation treatment is included prior to the digestion treatment. The incubation treatment is performed at a temperature lower than that of the digestion treatment. During the incubation treatment, the plant-derived compound digestive solution does not digest the cells, preventing premature cell damage. Furthermore, the incubation treatment primarily allows the plant-derived compound digestive solution sufficient time to penetrate the trunk muscle tissue, thereby improving the efficiency of subsequent digestion treatments.

[0025] In some embodiments, the mass ratio of bromelain to papain in the plant-derived compound digestive solution is (1-2):1. That is, the mass ratio of bromelain to papain can be 1:1, 1.5:1, or 2:1. A mass ratio within the above range can ensure a good digestive effect on trunk muscle tissue without excessively damaging cells.

[0026] In some embodiments, the plant-derived compound digestive solution further includes EDTA and phosphate buffer: the mass percentage of bromelain is 0.1%-0.2%, the mass percentage of papain is 0.05%-0.15%, the mass percentage of EDTA is 0.01%-0.02%, and the balance is phosphate buffer. That is, the mass percentage of bromelain can be 0.1%, 0.15%, or 0.2%, the mass percentage of papain can be 0.05%, 0.1%, or 0.15%, and the mass percentage of EDTA can be 0.01%, 0.015%, or 0.02%. Maintaining the mass percentages of these substances within appropriate ranges ensures good digestion of trunk muscle cells with minimal cell damage, ultimately improving the survival rate of primary chicken embryo fibroblasts.

[0027] In some embodiments, step S2 includes: Step S21: Mix the trunk muscle tissue with the plant-derived compound digestive solution, incubate and digest to obtain the digestive solution; Step S22: Add animal-free neutralizing solution to the digestion solution, centrifuge, wash, and obtain a precipitate; the animal-free neutralizing solution includes phosphate buffer containing plant peptone, soybean trypsin inhibitor and trehalose. Step S23: Resuspend the precipitate in serum-free culture medium, and purify it by passing it through a cell sieve and immunomagnetic beads in sequence. Collect the filtrate to obtain a suspension of primary chicken embryo fibroblasts. Step S24: The primary chicken embryo fibroblast suspension is inoculated by pre-coating and primary culture is carried out by adding serum-free special culture medium to obtain primary chicken embryo fibroblasts, i.e., chicken embryo fibroblasts.

[0028] In the technical solution of this invention, the incubation followed by digestion in step S21 improves the uniform distribution of the plant-derived compound digestive solution in the trunk muscle tissue; in step S22, the animal-free neutralizing solution can terminate the digestion reaction initiated by the plant-derived compound digestive solution. Soybean trypsin inhibitors are used to inhibit the activity of bromelain and papain, while plant peptone and trehalose protect the cells. The entire process does not introduce any animal-derived substances (animal serum), maintaining the animal-free characteristics of the preparation process; in step S23, the precipitate is re-selected and pre-purified using a cell sieve to remove undigested tissue blocks and cell clumps. Then, a secondary purification is performed using immunomagnetic beads to remove non-fibroblasts (such as epithelial cells and immune cells), obtaining a high-purity primary chicken embryo fibroblast suspension; in step S24, the pre-coating technology can significantly enhance the adhesion efficiency of primary chicken embryo fibroblasts, thereby reducing experimental errors caused by cell detachment, ensuring the uniformity of cell morphology, and providing a more reliable cell model for subsequent virus inoculation.

[0029] It should be noted that, through the synergistic effect of stepwise digestion in step S21 and immunomagnetic bead purification in step S23, the present invention achieves a survival rate of ≥96% and a purity of ≥99% for primary chicken embryo fibroblasts, which is significantly superior to traditional methods.

[0030] It should be noted that the pre-coating technique involves pre-coating the culture vessel with polylysine and laminin, followed by inoculating a suspension of primary chicken embryo fibroblasts into the pre-coated vessel for primary culture. Polylysine carries a positive charge, increasing the hydrophilicity of the culture vessel surface and attracting negatively charged cell membranes via electrostatic attraction, promoting initial adhesion of primary chicken embryo fibroblasts to the vessel surface. The integrin receptors present on the surface of primary chicken embryo fibroblasts specifically recognize and bind to laminin, thereby signaling and promoting cell spreading, proliferation, and differentiation. The combined use of polylysine and laminin significantly improves the adhesion rate and survival rate of primary chicken embryo fibroblasts.

[0031] In some embodiments, in step S21, the incubation temperature is 0-4℃; and / or, in step S21, the incubation time is 35-45 min; and / or, in step S21, the digestion treatment temperature is 30-40℃; and / or, in step S21, the digestion treatment time is 15-25 min. That is, the incubation temperature can be 0℃, 2℃, or 4℃, and the incubation time can be 35 min, 40 min, or 45 min. Low-temperature incubation allows plant-derived digestive enzymes to fully penetrate into the interstitial spaces, while low temperature inhibits enzyme activity, preventing premature digestion and cell damage. The digestion treatment temperature can be 35℃, 40℃, or 45℃, and the digestion treatment time can be 15 min, 20 min, or 25 min. Controlling the incubation temperature and time, as well as the digestion temperature and time, within appropriate ranges ensures good cell digestion and a high cell integrity rate.

[0032] In some embodiments, in step S22, the volume ratio of the digestive fluid to the animal-free neutralizing solution is 1:(1.5-2.5); in step S22, the centrifugation speed is 800-900 r / min; and / or, in step S22, the centrifugation time is 8-12 min. That is, the volume ratio of the digestive fluid to the animal-free neutralizing solution can be 1:1.5, 1:2, or 1:2.5, and a suitable volume ratio can ensure a good termination effect on the digestion reaction.

[0033] In some embodiments, in step S24, the inoculation density is 4.0 × 10⁻⁶. 5 -5.0×10 5 cells / cm 2 This seeding density helps ensure sufficient close contact between cells, thus forming an effective cell communication network. Cells can secrete and share essential signaling molecules such as growth factors and cytokines; this "paracrine support" is crucial for the survival and initiation of proliferation of primary cells.

[0034] In some embodiments, step S2 is followed by the following steps: Step S3: Passage the primary chicken embryo fibroblasts to P2-P3 generation and screen to obtain seed cells; Step S4: Inoculate the seed cells into a microcarrier and perform segmented fed-batch culture, recover and purify to obtain passaged chicken embryo fibroblasts, i.e., chicken embryo fibroblasts.

[0035] While primary chicken embryo fibroblasts (P0) most closely resemble the in vivo state of chicken embryos, they still contain a small number of impurity cells, which can easily lead to significant batch-to-batch stability differences. In the technical solution of this invention, primary chicken embryo fibroblasts are first passaged to P2-P3 generations. Utilizing principles such as differential adhesion, fibroblasts with faster growth rates and stronger adhesion abilities become the dominant population, thus achieving preliminary purification and screening to obtain seed cells. Subsequently, the seed cells are inoculated onto microcarriers and subjected to segmented fed-batch culture. The microcarriers can be tiny particles with a diameter of 50-350 micrometers. The microcarriers provide a large three-dimensional growth surface area for the adhesion-dependent chicken embryo fibroblasts in the suspended bioreactor, thereby significantly increasing the cell yield per unit volume and meeting the needs of large-scale cell or vaccine production.

[0036] In some embodiments, in step S3: the survival rate of the seed cells is ≥96%, and the purity of the seed cells is ≥99%. Purity refers to the proportion of chicken embryo fibroblasts in the total cell population. Higher purity is used to ensure the uniformity of the chicken embryo fibroblast product, and higher survival rate is used to ensure the vigorous growth of the seed cells.

[0037] In some embodiments, in step S4: the seed cells are inoculated onto the microcarrier at an inoculation density of 4.0 × 10⁻⁶. 4 -5.0×10 4 cells / cm 2; and / or, the microcarriers include dextran microcarriers, the particle size of the dextran microcarriers being 120-180 μm and the pore size of the dextran microcarriers being 20-30 μm; and / or, the parameters of the segmented fed-batch culture are: temperature 36-37℃, pH 7.1-7.3, dissolved oxygen 35%-45%, stirring speed 50-55 r / min; after culturing for 24-48h, 0.6% volume of glucose solution is added, and after culturing for 48-72h, 10% volume of serum-free special culture medium at twice the concentration is added. That is, the particle size of the dextran microcarrier can be 120 μm, 150 μm or 180 μm, and the pore size of the dextran microcarrier can be 20 μm, 25 μm or 30 μm. In the segmented fed-batch culture conditions: the temperature can be 36℃, 36.5℃ or 37℃, the pH can be 7.1, 7.2 or 7.3, the dissolved oxygen can be 35%, 40% or 45%, and the stirring speed can be 50 r / min, 52 r / min or 55 r / min; glucose solution can be added after 24h, 30h or 40h of culture, and the volume of glucose solution is 0.6% of the culture system volume, and the concentration of glucose solution is 20%; serum-free special medium with a concentration of 2 times can be added after 48h, 60h or 72h of culture. The serum-free special medium with a concentration of 2 times means that the concentration of all nutrients (such as amino acids, vitamins, inorganic salts, glucose, etc.) in the serum-free special medium is twice the final concentration required for cell growth. Simultaneously controlling the seed cell inoculation density, microcarrier size, parameters of segmented fed-batch culture, and the order of adding supplemental medium throughout the segmented fed-batch culture process can ensure the maximum amount of cells harvested at the end.

[0038] The present invention also provides a chicken embryo fibroblast, which is prepared according to the aforementioned method for preparing chicken embryo fibroblasts. Therefore, this chicken embryo fibroblast possesses all the beneficial effects of the aforementioned method for preparing chicken embryo fibroblasts, which will not be elaborated further here.

[0039] This invention also provides the application of chicken embryo fibroblasts in virus culture, wherein the chicken embryo fibroblasts include the aforementioned chicken embryo fibroblasts or chicken embryo fibroblasts prepared by the aforementioned method for preparing chicken embryo fibroblasts; the virus includes at least one of Newcastle disease virus, Marek's disease virus, and fowlpox virus. The chicken embryo fibroblasts exhibit good batch-to-batch stability, resulting in good batch-to-batch stability for virus amplification.

[0040] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0041] Experimental materials 9-10 day old SPF (Specific Pathogen Free) chicken embryos; PBS (phosphate buffered saline); Plant-derived compound digestion solution: The solvent was PBS, and the solutes were 0.15% bromelain (purchased from Shanghai Sangon Biotech, catalog number A429790), 0.1% papain (purchased from Shanghai Sangon Biotech, catalog number A501612), and 0.015% EDTA (purchased from Solarbio, catalog number E803). Animal-free neutralizing solution: The solvent is PBS, and the solutes are 3% plant peptone (purchased from Angel Yeast, catalog number FP410), 0.8% soybean trypsin inhibitor (purchased from Shanghai Sangon Biotech, catalog number A666011), and 2% trehalose (purchased from Shanghai Sangon Biotech, catalog number A631024). CD45 antibody-modified immunomagnetic beads: purchased from Thermo Fisher Scientific for custom manufacturing; Serum-free culture medium: purchased from Benogi; Dextran microcarriers: purchased from HOLVES, catalog number HCD-001; Glucose: Purchased from Shanghai Sangon Biotech, product number A430191; Animal trypsin digestion solution: 0.25% trypsin: purchased from GIBCO, catalog number 2537224; Suspension culture medium containing 2% chicken serum: The suspension culture medium was KMSCSC suspension culture medium, purchased from Womei (custom-made); the chicken serum was purchased from Aupsay Biotechnology, catalog number: 20240730; Newcastle disease virus (La Sota strain): purchased from the China Institute of Virology, product number CVCCAV1615; Marek's disease virus (GA strain): purchased from the China Institute of Virology, product number CVCCAV1590; Chickenpox virus: purchased from the China Institute of Animal Disease Control and Prevention, product number CVCCAV1003.

[0042] Example 1 1. Preparation method of primary chicken embryo fibroblasts (1) Chicken embryo pretreatment: Select 10 9-day-old SPF chicken embryos, wipe them with iodine for disinfection, remove the head, limbs and internal organs of the chicken embryos under sterile conditions, peel off the trunk muscle tissue, wash with PBS 3-5 times to obtain muscle tissue.

[0043] (2) Stepwise digestion: The above muscle tissue was cut into pieces and added to 50 mL of pre-cooled plant-derived compound digestion solution (PBS containing 0.15% bromelain, 0.1% papain and 0.015% EDTA). After pre-incubation at 4°C for 40 min, it was then shaken and digested at 35°C for 20 min. During the shaking digestion, the tissue was gently blown once every 5 min to obtain the digestion solution.

[0044] (3) Mild neutralization and washing: Add 2 times the volume of animal-free neutralization solution (PBS containing 3% plant peptone, 0.8% soybean trypsin inhibitor and 2% trehalose) to the above digestion solution to terminate digestion. Centrifuge at 800 r / min for 10 min, take the precipitate and wash the precipitate twice with serum-free special culture medium. Resuspend the washed precipitate with serum-free special culture medium to obtain the resuspension.

[0045] (4) Double purification: The above resuspension was filtered through a 200-mesh cell sieve to obtain the filtrate. Then, the epithelial cells and other cells in the filtrate were removed by immunomagnetic bead method (CD45 antibody-modified immunomagnetic beads). The filtrate was collected, which is the primary chicken embryo fibroblast suspension (its cell diagram is shown in Figure 1). Figure 1 As shown, the measurements were taken using an optical microscope.

[0046] (5) Cell culture: The above-mentioned primary chicken embryo fibroblast suspension was cultured at 5.0 × 10⁻⁶ cm⁻¹. 5 cells / cm 2 The cells were densely seeded into pre-coated (polylysine + laminin) culture containers, added to serum-free culture medium, and incubated at 37°C in a 5% CO2 incubator. After 24 hours, the culture medium was replaced with fresh medium to obtain primary chicken embryo fibroblasts (cell diagram shown). Figure 2 As shown in the figure, measurements were taken using an optical microscope. The preparation steps for the pre-coated culture vessel are as follows: First, add 2 mL of 0.01% polylysine (PLL) working solution to a 25 cm² bottle, ensuring that the liquid completely covers the bottom growth surface. Incubate overnight at room temperature. Gently wash the culture surface 2-3 times with sterile PBS to remove unbound PLL. Add 0°C pre-cooled laminin working solution (concentration 0.001%) to a 25 cm² flask that has been treated and cleaned by PLL. The volume is the same as in the PLL procedure. Incubate overnight at 4°C. After incubation, the laminin solution can be aspirated to obtain a pre-coated 25 cm² flask.

[0047] 2. Preparation method of passaged chicken embryo fibroblasts (1) Obtaining seed cells: Passage primary chicken embryo fibroblasts to P2-P3 generation (i.e., cells that have undergone 2-3 passages) and select cells with a survival rate ≥96% and a purity ≥99% as seed cells.

[0048] (2) Microcarrier adaptation and inoculation: Select dextran microcarriers (particle size 180 μm, pore size 20 μm), and inoculate at 4 × 10 4 cells / cm 2 Microcarriers were inoculated using surface area density. The initial stirring speed was 25 r / min. After 6 hours of adhesion to the wall, the stirring speed was gradually increased by 10 r / min every 30 minutes until the speed reached 55 r / min, thus obtaining the inoculated microcarriers.

[0049] (3) Segmented fed-batch culture: The inoculated microcarriers were transferred to a 10L stirred bioreactor, and serum-free culture medium was added. The parameters were set as follows: temperature 36.8℃, pH 7.1, dissolved oxygen 35%, and stirring speed stabilized at 55 r / min. After culturing for 36 h, 0.6% glucose solution was added, and after culturing for 60 h, 10% serum-free culture medium at twice the concentration was added. After 72 h of segmented fed-batch culture, the cell density reached 1.8 × 10⁻⁶ cells / year. 6 A suspension of passaged chicken embryo fibroblasts at cells / mL.

[0050] Example 2 The difference between Example 2 and Example 1 is that: In the stepwise digestion process, instead of incubating at 4°C for 40 minutes, the digestion is carried out directly at 35°C with shaking for 20 minutes.

[0051] Comparative Example 1 The difference between Comparative Example 1 and Example 2 is as follows: In the preparation method of primary chicken embryo fibroblasts: the plant-derived compound digestion solution was replaced with an equal volume of animal trypsin digestion solution (concentration of 0.25%), the animal-free neutralization solution was replaced with an equal volume of animal-derived neutralization solution (M199 medium containing 8% fetal bovine serum), and the serum-free special medium was replaced with M199 medium containing 8% fetal bovine serum; uncoated 25 cubic flasks were used in the cell culture step; In the preparation method of passaged chicken embryo fibroblasts: in the segmented fed-batch culture, the serum-free special medium is replaced with a suspension medium containing 2% chicken serum.

[0052] Example 3: Passaged chicken embryo fibroblasts used in the field of virus propagation Ten batches of passaged chicken embryo fibroblasts were prepared consecutively using the steps in Example 1, and ten batches of passaged chicken embryo fibroblasts were also prepared consecutively using the steps in Comparative Example 1. Each batch of passaged chicken embryo fibroblasts was inoculated with Newcastle disease virus (La Sota strain) (MOI 0.05), Marek's disease virus (GA strain) (MOI 0.1), and fowlpox virus (MOI 0.02), respectively, and cultured for 96 h, 144 h, and 120 h, respectively. The virus fluid was harvested and its EID was measured.50 The PFU (Package Validation Unit) is used to verify batch consistency, and the test method is as follows: (1) EID 50 (Half-infection dose in chicken embryos), reflecting the viral titer in cell culture medium: refer to Chinese Veterinary Pharmacopoeia, Part III (2020 edition); (2) PFU (plaque forming unit), reflecting the number of virus particles with infectious activity: refer to Chinese Veterinary Pharmacopoeia, Part III (2020 edition); The test results are shown in Table 1.

[0053] Table 1 Batch Consistency Verification Results

[0054] As shown in Table 1, the average titer of Newcastle disease virus over 10 consecutive batches was 10. 9.26 EID 50 / 0.1mL, the average titer of Marek's disease virus in 10 consecutive batches was 10. 8.1 The average titer of fowlpox virus over 10 consecutive batches was 10 PFU / 0.1mL. 6.33 EID 50 / 0.1mL, the average titer of the three viruses was higher than the industry average (Newcastle disease virus 10). 8.63 EID 50 / 0.1mL, Marek's disease virus 10 7.83 PFU / 0.1mL, fowlpox virus 10 6.0 EID 50 (0.1 mL). Compared with Comparative Example 1, the method of the present invention has extremely high repeatability and stability, which can meet the requirements of large-scale virus culture.

[0055] The cell viability and purity of the primary chicken embryo fibroblasts prepared in Examples 1-2 and Comparative Example 1 were determined using the following methods: (1) Survival rate: trypan blue staining method; (2) Purity: Flow cytometry.

[0056] The test results are shown in Table 2.

[0057] Table 2. Cell viability and purity of primary chicken embryo fibroblasts in Examples 1-2 and Comparative Example 1.

[0058] As shown in Table 2, compared with Example 1, Example 2 did not involve incubation at 4°C. 4°C can partially inhibit enzyme activity, slowing down the digestion process and making it more gentle. At the same time, 4°C can stabilize the cell membrane structure, reduce membrane damage and apoptosis, thus resulting in a slightly lower cell viability. Compared with Comparative Example 1, Example 2 used animal-derived digestive enzymes, and trypsin easily destroyed cell surface adhesion proteins, affecting cell adhesion and proliferation, thus resulting in a lower cell viability.

[0059] In summary, this invention utilizes a plant-based compound digestion solution to digest the muscle tissue of 9-10 day old SPF chicken embryos. Stepwise digestion and immunomagnetic bead purification are then employed to obtain high-density chicken embryo fibroblasts cultured in a serum-free, specialized culture medium. Furthermore, a segmented fed-batch culture of chicken embryo fibroblasts using microcarriers and a bioreactor is employed for large-scale culture. After 72 hours, the cell count reaches 1.8 × 10⁻⁶ cells / year. 6 With a virus droplet concentration of over 100 cells / mL, subsequent inoculations with Newcastle disease, Marek's disease, and fowlpox viruses resulted in virus droplets exceeding the industry average for 10 consecutive batches, laying a solid foundation for the large-scale production of poultry disease vaccines.

[0060] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A method for preparing chicken embryo fibroblasts, characterized in that, The method for preparing the chicken embryo fibroblasts includes the following steps: S1. Provide a chicken embryo and obtain the trunk muscle tissue of the chicken embryo; S2. The trunk muscle tissue is mixed with plant-derived compound digestion solution and then digested, purified, and cultured in a serum-free special culture medium to obtain primary chicken embryo fibroblasts, i.e., chicken embryo fibroblasts. The plant-derived complex digestive fluid includes bromelain and papain.

2. The method for preparing chicken embryo fibroblasts as described in claim 1, characterized in that, The digestion process is preceded by an incubation process.

3. The method for preparing chicken embryo fibroblasts as described in claim 2, characterized in that, In the plant-derived complex digestive solution, the mass ratio of bromelain to papain is (1-2):1; and / or, The plant-derived complex digestive solution also includes EDTA and phosphate buffer: the mass percentage of bromelain is 0.1%-0.2%, the mass percentage of papain is 0.05%-0.15%, the mass percentage of EDTA is 0.01%-0.02%, and the remainder is phosphate buffer.

4. The method for preparing chicken embryo fibroblasts as described in claim 2, characterized in that, Step S2 includes: S21. Mix the trunk muscle tissue with the plant-derived compound digestive solution, incubate and digest to obtain the digestive solution. S22. Add an animal-free neutralizing solution to the digestion solution, centrifuge, wash, and obtain a precipitate; the animal-free neutralizing solution includes a phosphate buffer containing plant peptone, soybean trypsin inhibitor, and trehalose. S23. The precipitate is resuspended in a serum-free culture medium and purified by passing it through a cell sieve and an immunomagnetic bead method. The filtrate is collected to obtain a suspension of primary chicken embryo fibroblasts. S24. The primary chicken embryo fibroblast suspension is inoculated by pre-coating and primary culture is carried out in serum-free special culture medium to obtain primary chicken embryo fibroblasts, i.e., chicken embryo fibroblasts.

5. The method for preparing chicken embryo fibroblasts as described in claim 4, characterized in that, In step S21, the incubation temperature is 0-4℃; and / or, In step S21, the incubation time is 35-45 min; and / or, In step S21, the digestion treatment temperature is 30-40℃; and / or, In step S21, the digestion process takes 15-25 minutes; and / or, In step S22, the volume ratio of the digestive fluid to the animal-free neutralizing fluid is 1:(1.5-2.5). In step S22, the centrifugation speed is 800-900 r / min; and / or, In step S22, the centrifugation time is 8-12 min; and / or, In step S24, the inoculation density is 4.0 × 10⁻⁶. 5 -5.0×10 5 cells / cm 2 .

6. The method for preparing chicken embryo fibroblasts as described in claim 1, characterized in that, Step S2 is followed by the following steps: S3. Pass the primary chicken embryo fibroblasts to the P2-P3 generation and screen to obtain seed cells; S4. The seed cells are inoculated into microcarriers and cultured in a segmented fed-batch manner. The cells are then recovered and purified to obtain passaged chicken embryo fibroblasts, also known as chicken embryo fibroblasts.

7. The method for preparing chicken embryo fibroblasts as described in claim 6, characterized in that, In step S3: the survival rate of the seed cells is ≥96%, and the purity of the seed cells is ≥99%.

8. The method for preparing chicken embryo fibroblasts as described in claim 6, characterized in that, In step S4: The seed cells were inoculated into the microcarrier at a density of 4.0 × 10⁶. 4 -5.0×10 4 cells / cm 2 ; and / or, The microcarriers include dextran microcarriers, wherein the particle size of the dextran microcarriers is 120-180 μm and the pore size of the dextran microcarriers is 20-30 μm; and / or, The parameters for the segmented fed-batch culture are: temperature 36-37℃, pH 7.1-7.3, dissolved oxygen 35%-45%, and stirring speed 50-55 r / min; after culturing for 24-48h, 0.6% glucose solution is added, and after culturing for 48-72h, 10% serum-free culture medium at twice the concentration is added.

9. A type of chicken embryo fibroblast, characterized in that, The chicken embryo fibroblasts were prepared according to the method for preparing chicken embryo fibroblasts as described in any one of claims 1 to 8.

10. An application of chicken embryo fibroblasts in virus culture, characterized in that, The chicken embryo fibroblasts include the chicken embryo fibroblasts of claim 9 or chicken embryo fibroblasts prepared by the method for preparing chicken embryo fibroblasts according to any one of claims 1 to 8; The virus includes at least one of Newcastle disease virus, Marek's disease virus, and fowlpox virus.

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