Suspended serum-free PK cell based on composite synergist and method for culturing porcine circovirus by using suspended serum-free PK cell

Abstract

The invention relates to the technical field of veterinary biological product production, and discloses a suspension serum-free PK cell based on a composite synergist and a method for culturing porcine circovirus by using the suspension serum-free PK cell, a high-quality suspension cell strain is obtained by constructing an adherent PK cell suspension technical system of gradient domestication-monoclonal screening-performance verification; according to the method, a composite synergist composed of IGF1R, L-selenomethionine, polyvinylpyrrolidone (PVP) and a Cu / Mn compound is innovatively introduced, and specific proliferation parameter optimization of PCV2 and PCV3 strains is combined, so that efficient and stable culture of PCV2 / PCV3 is realized, and the virus titer and production efficiency are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of laser processing technology, specifically to a method for culturing porcine circovirus using suspended serum-free PK cells based on a composite synergist. Background Technology

[0002] In the process of intensive and large-scale development of the pig farming industry, porcine circovirus (PCV)-related diseases (PCVAD) have become a key factor restricting the healthy development of the industry. PCV mainly includes PCV2 (including mainstream subtypes such as 2a, 2b, and 2d) and PCV3. Both are highly pathogenic and have a wide range of effects: piglets aged 2-10 weeks are prone to developing post-weaning multisystemic wasting syndrome (PMWS) after infection, which manifests as typical symptoms such as depression, progressive emaciation, and difficulty breathing, with a mortality rate as high as 10%-30%; adult sows infected with PCV experience reproductive disorders such as abortion, stillbirth, and weak piglets, resulting in a decrease in sow reproductive efficiency of more than 30%; more seriously, PCV can damage the pig's immune system, induce immunosuppression, and greatly increase the susceptibility of the pig herd to diseases such as classical swine fever and porcine reproductive and respiratory syndrome (PRRS), with a mixed infection rate of 40%-60%. According to industry statistics, large-scale pig farms suffer annual economic losses of 15%-20% of their total revenue due to PCVAD, causing over $10 billion in economic losses to the global pig farming industry each year. Currently, the production of PCV-related vaccines mainly relies on traditional adherent PK cell culture technology, but this technology has many inherent defects and is difficult to meet the needs of industrial production.

[0003] Serum dependence and safety risks are prominent: Traditional processes require the addition of 8%-10% fetal bovine serum, which not only has high procurement costs (average market price of 500-800 yuan / L), but may also carry exogenous factors such as mycoplasma (detection rate of about 5%-8%) and bovine viral diarrhea virus (BVDV, detection rate of about 2%-3%) (last page), which can easily lead to vaccine contamination; at the same time, serum residues can cause allergic reactions in piglets, with an incidence rate of about 5%, which seriously affects the safety and immunization effect of vaccine use.

[0004] Low production efficiency: Adherent PK cells are limited by growth space, with a maximum density of only 5×10⁻⁶. 6 cells / mL, PCV proliferation titer (TCID) 50 The viral load is ≤5.5 / mL; and due to its adherent growth characteristics, it can only achieve "single harvest". Taking a single batch of 10L roller bottles as an example, the actual harvested viral fluid is only 1L. The yield is far from meeting the vaccine demand of large-scale pig farms, resulting in a prominent contradiction between vaccine supply and demand.

[0005] The process is complicated and the stability is poor: the production process requires manual operations such as trypsin digestion and passage, and multiple liquid changes, and the production cycle is as long as 45-50 days. More importantly, the difference in serum composition between batches (such as the fluctuation of growth factor content ±15%) will lead to a large fluctuation in cell proliferation efficiency and viral titer, with a batch variation coefficient ≥20%, which significantly increases the difficulty of quality control and makes it difficult to ensure the uniformity of vaccine quality.

[0006] Poor strain adaptability: PCV genotypes are prone to recombination and mutation, especially PCV3, which is a new strain discovered in 2016. Traditional adherent cells require 6-8 months for cell adaptation and process optimization, often missing the best time for prevention and control, resulting in "mismatch between vaccine strain and epidemic strain", which greatly reduces the prevention and control effect and cannot effectively curb the spread of PCV3.

[0007] To address these issues, serum-free suspension cell culture technology has become a key area of ​​innovation in the industry. However, existing technologies still face two major bottlenecks: Firstly, there is a lack of mature acclimatization techniques for adhering PK cells to suspension cells. Most acclimatization protocols only achieve "semi-suspension growth," resulting in cells with weak serum-free tolerance and rapid viability decay after passage. After 20 passages, the viability drops below 85%, making it difficult to obtain stable suspension cell lines that combine high proliferative activity with viral sensitivity. Secondly, there is insufficient optimization of suspension culture parameters for PCV3 strains. PCV3 is sensitive to parameters such as culture temperature and MOI (multiple of infection), and under current processes, its proliferation titer can only reach 6.0-6.5 TCID. 50 / mL, which cannot meet the requirements for viral titer (≥6.8 TCID) in industrial vaccine production. 50 The requirement of ( / mL) and the lack of a dedicated synergistic system that can simultaneously enhance the potency of PCV2 and PCV3 further limit vaccine production efficiency.

[0008] Therefore, developing a suspension serum-free culture method that includes a mature adherent PK cell domestication process, adapts to PCV2 / PCV3 strains, and introduces a special synergist is of great significance for improving PCV vaccine production efficiency and ensuring the safety of the pig industry. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a suspension of serum-free PK cells based on a composite synergist and a method for culturing porcine circovirus, thereby solving the problems existing in the production of PK cells and PCV2 / PCV3 strains in the aforementioned background technology.

[0010] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a suspension of serum-free PK cells based on a compound synergist, wherein the PK cells are the PK-S2 strain, and it is suggested to classify and name them as porcine kidney epithelial cells (PK-S2). Pig kidney cellsIt was deposited at the China General Microbiological Culture Collection Center on November 18, 2025, with accession number CGMCC No. 46750.

[0011] This invention also provides a method for culturing porcine circovirus in serum-free PK cells based on a composite synergist, comprising: (1) In the cell pretreatment stage, the adherent PK cells frozen in liquid nitrogen were taken out, and PK cells with stable proliferation rate were obtained by thawing in a water bath, resuspending culture, and passage culture. (2) In the serum gradient reduction acclimatization stage, a four-dimensional synergistic acclimatization strategy of "gradually decreasing serum concentration + gradually increasing serum-free culture medium + gradually increasing shaking intensity + adjusting the concentration gradient of compound synergist" was adopted to acclimatize and culture the obtained stable proliferating PK cells, and screened to obtain PK cell lines that are fully adapted to serum-free full suspension culture. (3) Suspension adaptation enhancement stage: the selected PK cell lines were transferred to Erlenmeyer flasks for expansion culture, and then single-clone screening was performed using the limiting dilution method to obtain single-clone suspension PK-S2 cell lines. (4) Virus seed preparation: Porcine circovirus PCV2 and PCV3 were screened from lymph node samples of diseased pigs. The obtained PCV2 and PCV3 seed viruses were cultured and screened in PK-S2 cells on serum-free medium at an MOI of 0.01. 0.12% compound synergist was added to the culture medium of PCV2 and 0.15% compound synergist was added to the culture medium of PCV3. (5) Large-scale virus culture: PK-S2 cells were first expanded through primary amplification in 125ml shake flasks, intermediate amplification in 500ml shake flasks, and advanced amplification in 5L shake flasks. 0.12% compound synergist was added at each amplification stage. Then, the seed cells amplified in 5L shake flasks were cultured at a rate of 1×10⁻⁶. 6Cells / mL were inoculated into 50L bioreactors and cultured in serum-free medium. PCV2 and PCV3 virus strains were inoculated at an MOI of 0.01. PCV2 was cultured at 37℃ throughout the entire process, as this temperature resulted in the highest PCV2 DNA polymerase activity and optimal viral replication efficiency. For PCV3, a segmented temperature control strategy was employed. The first 24 hours were cultured at 37℃ to promote viral adsorption and invasion, during which the PCV3 capsid protein exhibited the highest binding efficiency to cell receptors. The subsequent 48 hours were cultured at 35℃ to induce PCV3 viral particle assembly, where the viral capsid protein folded more stably, increasing particle assembly efficiency by 20%–30%. The pH for both viruses was maintained at 7.1–7.3, and the dissolved oxygen concentration was maintained at 30%–50%. A paddle agitator was used, with the rotation speed gradually increased from 50 rpm to 80 rpm. The concentration of the compound synergist was maintained at 0.12% during PCV2 culture and adjusted to 0.15% during PCV3 culture. (6) Harvest the venom by adopting a strategy of "continuous harvesting-feeding culture" to harvest multiple generations of virus.

[0012] Preferably, the composite synergist mother liquor contains IGF1R, L-selenomethionine, polyvinylpyrrolidone, and Cu. 2 + / Mn 2+ The complex consisted of four substances dissolved in a serum-free culture medium at a mass ratio of 3:2:1:0.5 to prepare a 10% stock solution.

[0013] Preferably, the specific process of the cell pretreatment stage in step (1) is as follows: take the frozen adherent PK cells out of liquid nitrogen, quickly place them in a 37°C water bath for 1-2 minutes to thaw them rapidly, so as to avoid damage to the cells due to prolonged low temperature; then add 10 mL of DMEM medium containing 5% fetal bovine serum and 0.05% compound synergist, centrifuge at 1000 rpm for 5 minutes to remove the cryopreservation solution, reduce the toxic effect of cryopreservation agent on cells, resuspend the cells and seed them into T75 culture flasks, add 10 mL of DMEM medium containing 5% fetal bovine serum and 0.05% compound synergist to each flask, and place them in a 37°C, 5% CO2 incubator for static culture; Once the cell confluence reaches 80%–90%, discard the supernatant and gently wash twice with PBS buffer (pH 7.2–7.4) to remove residual culture medium and metabolic waste. Add 2 mL of 0.25% trypsin-EDTA digestion solution and incubate at 37°C for 1–2 minutes. After observing increased intercellular spaces and rounded cells under a microscope, immediately add 5 mL of DMEM medium containing 5% fetal bovine serum and 0.05% compound synergist to terminate digestion. Gently pipette the cells to form a single-cell suspension, avoiding excessive pipetting that could cause cell breakage. Cells were seeded in new T75 culture flasks at a passage ratio of 1:3 and passaged 5 times consecutively. After each passage, cell viability was assessed using trypan blue staining and was required to be stable at 92%–95%. Simultaneously, cell proliferation activity was assessed using the CCK-8 assay to ensure a stable cell proliferation rate.

[0014] Preferably, step (2), the serum gradient reduction acclimatization stage, specifically includes, Low serum adaptation period: This stage is completed during the 6th to 10th generation of cells. The culture medium formula is 2% fetal bovine serum + 30% serum-free special medium + 67% DMEM basal medium to remove antibiotics + 0.08% compound synergist. Add 10 mL of this mixed medium to each T75 culture flask. Adjust the cell seeding density to 2 × 10⁶ cells / year during passage. 5 The cells / mL culture is still statically cultured, but the culture flask is gently shaken manually 1-2 times a day for 5 seconds each time to promote full contact between cells and culture medium and avoid uneven cell growth due to local nutrient deficiency. After each passage, the cells are passaged after 48 hours of culture. The cell morphology is observed under a microscope. The cells should maintain a typical epithelial spindle shape without obvious shrinkage or increased granules. The viability is detected by trypan blue staining and should be ≥90%. The cell adhesion rate is counted by a cell counting chamber and should be ≥90%. If the adhesion rate is lower than 85%, the serum degradation should be paused, and the current serum concentration and compound synergist concentration should be maintained for passage 1-2 more times to ensure that the cells adapt before entering the next stage. Semi-serum-free shaking induction phase: This phase is completed between passages 11 and 15. The culture medium formulation is adjusted to 1% fetal bovine serum + 60% serum-free medium + 39% DMEM basal medium + 0.1% compound synergist, further reducing serum dependence and increasing the proportion of serum-free medium. Simultaneously, the concentration of the compound synergist is increased to enhance cell suspension adaptability. Cells are transferred to 125mL Erlenmeyer flasks, with 20mL of cell suspension inoculated into each flask, increasing the inoculation density to 3 × 10⁶ cells / mL. 5 Cells / mL were cultured in a shaker at 37°C with 5% CO2. The shaker speed was initially set at 30 rpm, increasing by 10 rpm every two generations until it stabilized at 50 rpm. The shaking intensity was maintained at a level where cells did not adhere to the flask and were slightly suspended, avoiding excessive speed which could cause shear damage. At this stage, under a microscope, some cells could be seen detaching from the flask wall and appearing as single cells or small clusters in suspension. After each passage, the suspension rate was assessed using a hemocytometer, gradually increasing from an initial 10% to over 80%. Viability was assessed using trypan blue staining, requiring a result of ≥88%. If the suspension rate increased slowly, the shaker speed was increased by 15 rpm per generation, while maintaining the concentration of the compound synergist unchanged. 203. Serum-Free Adaptation Phase: This phase is completed between passages 16 and 20, completely eliminating serum dependence and adhesion dependence. A 100% serum-free culture medium with 0.12% of a compound synergist is used. The compound synergist is supplemented with 0.1% Pluronic F-68 to further reduce shear stress damage to cells. Increasing the concentration of the compound synergist ensures cell viability and proliferation in a completely serum-free environment. The shaking speed starts at 60 rpm, increasing by 20 rpm per passage, eventually stabilizing at 140 rpm. The culture temperature is maintained at 37°C, with a CO2 concentration of 5%. Cells are passaged every 3 days. Trypsin digestion is not required during passage; the suspended cell suspension is directly centrifuged at 1000 rpm for 5 min, the supernatant is discarded, and the cells are resuspended in fresh serum-free culture medium containing 0.12% of the compound synergist. The seeding density is adjusted to 1 × 10⁶ cells / year. 6 At this stage, cell populations still relying on adherent growth must be strictly eliminated, retaining only cell populations with a suspension rate ≥95%, round or short spindle-shaped cells, and uniform size; trypan blue staining is used to detect cell viability, which must be ≥90%; CCK-8 assay is used to detect cell proliferation activity, ensuring doubling time ≤26h; a total of 3-5 candidate cell populations are screened to enter the next stage to ensure that high-quality suspension cell lines are obtained subsequently.

[0015] Preferably, the shake-flask expansion culture process in step (3) specifically involves transferring the selected candidate cell populations to 125mL Erlenmeyer shake flasks, adding 30mL of serum-free culture medium containing 0.12% compound synergist to each flask, and inoculating at a density of 1×10⁻⁶ cells / mL. 6 Cells / mL, shaken at 140 rpm, cultured at 37°C and 5% CO2, passaged every 3 days, with daily sampling and analysis. Cell density was counted using a cell counting chamber to ensure a cell density of 6 × 10⁶ cells / mL every 3 days. 6 ~7×10 6Cells / mL; viability was detected using trypan blue staining to ensure viability ≥90%; apoptosis rate was detected using flow cytometry to ensure apoptosis rate ≤5%; the above tests verified the stable proliferation ability and activity of cells in suspension, eliminating candidate cell populations with slow cell density growth or apoptosis rate >5%, retaining 2-3 high-performance candidate cell populations for the monoclonal screening stage; the monoclonal screening process was as follows: candidate suspension cells in logarithmic growth phase were taken and diluted to 1 cell / 100μL with serum-free medium containing 0.12% compound synergist, ensuring that each well contained only one cell on average, avoiding the impact of multiple cell clones on cell line homogeneity; 100μL of the diluted cell suspension was added to each well of a 96-well cell culture plate and incubated at 37℃, 5%... Static culture was performed in a CO2 incubator; after 72 hours, wells containing single cells were observed and labeled under a microscope, excluding multicellular and cellless wells; when monoclonal cells grew to cover 50% of the bottom of the well in a 96-well plate, a single-cell suspension was formed by gently pipetting and transferred to a 24-well plate, with 1 mL of serum-free culture medium containing 0.12% compound synergist added to each well for further culture; when the cell density in the 24-well plate reached 1×10⁶ cells / well... 6 Cells / well were transferred to T25 culture flasks and cultured in 5 mL of serum-free culture medium containing 0.12% compound synergist until the cell density in the T25 culture flasks reached 5 × 10⁶ cells / well. 6 The cells / mL were eventually expanded to 125mL shake flasks to obtain a monoclonal suspension PK-S2 cell line.

[0016] Preferably, the amplification culture process in step (5) specifically involves: Primary expansion: PK-S2 cell line was expanded at a rate of 1×10⁻⁶. 6 Cells / mL were seeded in new 125mL shake flasks and cultured in serum-free medium containing 0.12% compound synergist at 37℃ and 140rpm for 72h; target cell density 6×10⁶. 6 The cell count / mL should be ≥95%; the apoptosis rate should be ≤3% when detected by flow cytometry to avoid apoptotic cells affecting subsequent expansion. Intermediate expansion: Transfer the cells from the primary expansion to 500 mL shake flasks, seeding at a density of 1 × 10⁶ cells / mL. 6 Cells / mL, cultured in serum-free medium containing 0.12% compound synergist at 37°C and 140 rpm for 72 h; target cell density 6.5 × 10⁻⁶. 6 The cell count / mL should be ≥95%; the pH of the culture medium should be measured with a pH meter and maintained between 7.1 and 7.3. If the pH deviates, adjust it by adding 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution to ensure a stable cell growth environment. Advanced amplification: Transfer the cells from the intermediate amplification stage to 5L shake flasks, seeding at a density of 1×10⁶ cells / year. 6 Cells / mL were cultured in serum-free medium containing 0.12% compound synergist at 37°C and 140 rpm for 72 h; target cell density was 7 × 10⁶ cells / mL. 6 Cells / mL, viability ≥95%; dissolved oxygen concentration in the culture medium is measured using a dissolved oxygen meter and needs to be maintained at 30%~50%. Dissolved oxygen is adjusted by changing the shaking speed of the flask to ensure that the cell respiration needs are met; cell scale is gradually expanded through stepwise amplification.

[0017] Preferably, the specific process of harvesting the virus through multiple generations in step (6) is as follows: First-generation harvest: 72 hours after inoculation, when the viral titer reaches its peak, harvest 75% of the culture to quickly harvest a high concentration of virus; during the harvesting process, maintain a stirring speed of 50 rpm to avoid cell precipitation that would cause turbidity in the harvested solution; after harvesting, test the viral titer and antigen content of the harvested solution to ensure that it meets quality standards; Second-generation harvest: Add fresh serum-free culture medium containing the corresponding concentration of compound synergist to the remaining 25% of the culture to a volume of 50L, and continue culturing for 72h; at this time, the virus titer remains stable, and all cultures are harvested; when feeding, add the culture medium at a uniform rate through the reactor feeding system to avoid shocking the cells and causing a decrease in viability; during the culture process, maintain the process parameters unchanged to ensure continuous virus proliferation; Third-generation harvest: Add fresh serum-free culture medium containing the corresponding concentration of compound synergist to 50L and continue culturing for 72h. At this time, the virus titer will decrease slightly but still meet the production requirements. Harvest all the culture. After harvesting, test the cell viability. If the viability is <80%, stop harvesting to avoid the subsequent harvest liquid having too low a titer. If the viability is ≥80%, you can continue to add material and culture to harvest the fourth generation. However, it should be noted that the virus titer of the fourth generation may decrease further. Whether to continue harvesting should be decided according to production needs.

[0018] This invention provides a suspension of serum-free PK cells based on a composite synergist and a method for culturing porcine circovirus. It has the following beneficial effects: (1) This invention innovatively adopts a four-dimensional synergistic domestication strategy of "serum gradient reduction + serum-free culture medium increment + shaking intensity enhancement + compound synergist adjustment" to solve the problems of weak serum-free cell tolerance and rapid decline in cell activity during passage in existing domestication processes. The obtained PK-S2 strain achieved a maximum density ≥7.8×10⁻⁶ in serum-free culture medium. 6The cell / mL viability remains ≥95% after 50 consecutive passages, which is a significant improvement in stability compared to the existing acclimatization program (the viability drops to 85% after 20 passages). At the same time, the PK-S2 strain is highly sensitive to both PCV2 and PCV3 and can maintain good performance without relying on compound synergists, solving the dual problems of weak adaptability of traditional adherent cell strains and poor stability of suspension cells.

[0019] (2) In view of the low-temperature preference of PCV3, this invention innovatively proposes a segmented temperature control strategy of "37℃ for 24h + 35℃ for 48h", combined with an increase in the concentration of the compound synergist (0.15%), to promote the adsorption and particle assembly of PCV3 virus, so that the PCV3 titer is stable at ≥7.5 TCID. 50 / mL; meanwhile, PCV2, when cultured at 37℃ throughout and with a 0.12% compound synergist concentration, maintained a stable potency ≥8.0 TCID. 50 / mL, enabling simultaneous large-scale culture of PCV2 / PCV3, solving the problem that traditional processes cannot efficiently adapt to PCV3, and rapidly responding to the prevention and control needs of PCVAD mixed infection.

[0020] (3) The production cycle of the culture method provided by the present invention is shortened from 45-50 days in the traditional wall-attached process to 35-40 days, and the production efficiency is increased by 22%. The innovative "continuous harvest-feed culture" strategy enables the total output of a single batch of 50L reactor to reach 137.5L, which is 1.7 times that of the same scale wall-attached roller bottle. The output of 500L reactor can reach 350-450L, which fully meets the needs of large-scale vaccine production. The virus recovery rate is over 92%, which is 31% higher than the traditional process, further improving the virus utilization rate.

[0021] (4) The serum-free culture system used in this invention completely eliminates the risk of contamination by exogenous factors (mycoplasma, BVDV, etc.) in serum, and the allergy rate of piglets is reduced from 5% to less than 0.5%, which significantly improves the safety of vaccine use; the viral titer fluctuation between batches is ≤8%, which is 60% more stable than the traditional process (≥20%), ensuring uniform and reliable vaccine quality and reducing the difficulty of quality control.

[0022] (5) The average price of serum-free culture medium is RMB 300 / L, while that of serum-containing culture medium is about RMB 430 / L. The culture medium cost of the method of the present invention is reduced by 30% compared with the traditional process. The cost of adding compound synergist is only 1 / 5 of the cost of serum, and it can significantly improve the yield and potency, further reducing the unit cost of virus solution. At the same time, the serum-free culture medium, compound synergist and bioreactor used are all domestically produced, with stable supply and cost 20%-30% lower than imports, completely eliminating import dependence. The process steps are simplified (no need for pancreatic enzyme digestion, multi-step liquid change), manual operation is reduced by 80%, and the labor cost per batch is only 1 / 3 of that of the traditional process. Based on an annual production capacity of 100 million doses of vaccine, the annual cost of the method of the present invention is RMB 20-25 million lower than that of the traditional process and RMB 10-13 million lower than that of the suspension process without compound synergist. The economic benefits are outstanding and can effectively promote cost reduction and efficiency improvement in the vaccine industry.

[0023] (6) The equipment (bioreactor, shake flask) and raw materials (serum-free culture medium, compound synergist) used in this invention are all commonly used industrial products, which are easy to obtain and have a stable supply; the process parameters are highly standardized and can be directly replicated to reactors of different scales from 50L to 2000L without the need for extensive equipment modification and process adjustment; at the same time, the PK-S2 cell line has been preserved, the source of the seed virus is traceable, and the quality control methods meet the requirements of the "Pharmacopoeia of the People's Republic of China", which facilitates vaccine manufacturers to quickly set up production lines, accelerate technology transformation and industrial application, and provide strong technical support for PCVAD prevention and control. Attached Figure Description

[0024] Figure 1 A nucleic acid agarose gel electrophoresis image of the amplified products from suspected PCV2 pathogen samples; Figure 2 This is an agarose gel electrophoresis image of nucleic acid products after amplification of suspected PCV3 pathogen samples. Detailed Implementation

[0025] 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.

[0026] Example 1 This embodiment provides a composite synergist suitable for PK cell culture. The composite synergist in this embodiment includes substance A, insulin-like growth factor 1 receptor (IGF1R), substance B, L-selenomethionine, substance C, polyvinylpyrrolidone (PVP), and substance D, Cu. 2+ / Mn 2+The complex consists of four substances in a mass ratio of 3:2:1:0.5. Substance A regulates cellular glucose metabolism pathways and maintains carbon source homeostasis. Substance B reduces reactive oxygen species damage to cells during culture. Substance C enhances cell resistance to agitation and shear stress. Substance D simultaneously promotes the assembly and maturation of PCV virus particles, thereby increasing viral infectivity. The following experiments were designed to verify the effects of the complex synergist.

[0027] Experimental materials: Cells: ATCC CCL-33 standard adherent PK cell line; Culture media: DMEM high glucose medium (gibco), PK201 serum-free medium; Reagent: trypan blue staining solution; Instruments: CO2 incubator, microscope.

[0028] Experimental methods: Group settings: Set 5 groups, with 3 repeating holes in each group: Control group: DMEM medium containing 5% fetal bovine serum; Experimental group 1: DMEM medium containing 5% fetal bovine serum + 0.05% compound synergist; Experimental group 2: DMEM medium containing 5% fetal bovine serum + 0.1% compound synergist; Experimental group 3: DMEM medium containing 5% fetal bovine serum + 0.15% compound synergist; Experimental group 4: DMEM medium containing 5% fetal bovine serum + 0.2% compound synergist.

[0029] Cell seeding: Adjust PK cells to 1×10⁶ 5 cells / mL, seeded in 96-well plates, 100 μL per well; Culture conditions: 37℃, 5% CO2 incubator for 72 hours; Detection indicators: Cell viability: The viability was calculated by counting live and dead cells under a microscope using trypan blue staining (number of live cells / total number of cells × 100%). Cell density: Counted using a cell counting chamber; Experimental results: Group Cell viability (%) <![CDATA[Cell density (×10 6 cells / mL)]]> control group 91.2±1.5 0.85±0.05 Experimental group 1 93.5±1.2 0.98±0.04 Experimental group 2 95.8±0.8 1.12±0.03 Experimental group 3 94.6±1.0 1.05±0.04 Experimental group 4 90.3±1.3 0.92±0.05 Results analysis: The experimental results show that adding the compound synergist can significantly improve the viability, density, and proliferation activity of PK cells. Among them, experimental group 2 (0.1% compound synergist) showed the best results: cell viability increased by 4.6 percentage points compared with the control group, cell density increased by 31.8% compared with the control group, and OD value increased by 41.9% compared with the control group. When the concentration of the compound synergist exceeded 0.1%, cell viability, density, and proliferation activity decreased (experimental groups 3 and 4), possibly because the high concentration of synergist had a slight toxic effect on the cells. Therefore, the appropriate concentration of the compound synergist in cell culture was determined to be 0.1%-0.12%, providing a basis for subsequent domestication processes.

[0030] Example 2 This embodiment provides a method for constructing serum-free PK cells based on a compound synergist, the specific process of which is as follows: Step (1) Pretreatment stage (passages 1-5): Stabilize basic cell activity Cryopreserved adherent PK cells were removed from liquid nitrogen and rapidly thawed in a 37°C water bath for 1-2 minutes to prevent cell damage from prolonged low temperatures. Then, 10 mL of DMEM medium containing 5% fetal bovine serum and 0.05% combined synergist was added, and the cells were centrifuged at 1000 rpm for 5 minutes to remove the cryopreservation solution and reduce the toxic effects of the cryoprotectant on the cells. After resuspending the cells, they were seeded into T75 culture flasks, each containing 10 mL of DMEM medium containing 5% fetal bovine serum and 0.05% combined synergist, and incubated statically at 37°C in a 5% CO2 incubator.

[0031] Once the cells reach 80%-90% confluence (approximately 48 hours), discard the supernatant and gently wash twice with PBS buffer (pH 7.2-7.4) to remove residual culture medium and metabolic waste. Add 2 mL of 0.25% trypsin-EDTA digestion solution and incubate at 37°C for 1-2 minutes. After observing increased intercellular spaces and rounded cells under a microscope, immediately add 5 mL of DMEM medium containing 5% fetal bovine serum and 0.05% compound synergist to terminate digestion. Gently pipette the cells to form a single-cell suspension, avoiding excessive pipetting that could cause cell breakage.

[0032] Cells were seeded at a 1:3 passage ratio in new T75 culture flasks and passaged five times consecutively. After each passage, cell viability was assessed using trypan blue staining, and the viability should be stabilized at 92%-95%. Simultaneously, cell proliferation activity was assessed using the CCK-8 assay to ensure a stable cell proliferation rate (doubling time ≤ 28 hours), laying a good foundation for subsequent acclimatization. Cell morphology: Under a microscope, cells exhibited a typical spindle shape, without abnormal shrinkage or increased granulation. Step (2) Serum gradient reduction and acclimatization stage (6th-20th generation): Inducing cells to break free from serum dependence and adhesion dependence.

[0033] This invention innovatively employs a four-dimensional synergistic acclimatization strategy: "gradually decreasing serum concentration + gradually increasing serum-free culture medium + progressively increasing shaking intensity + gradient adjustment of compound synergist concentration." This strategy is implemented in three orderly stages to prevent a sudden drop in cell activity due to environmental mutations. The specific process is as follows: 201. Generations 6-10 (Low Serum Adaptation Phase): The core objective of this stage is to gradually adapt the cells to the low serum environment. The culture medium formulation is 2% fetal bovine serum + 30% serum-free medium + 67% DMEM basal medium (with antibiotics removed) + 0.08% compound synergist. Add 10 mL of this mixed medium to each T75 culture flask. Adjust the cell seeding density to 2 × 10⁶ cells / year during passaging. 5 Cells / mL were cultured statically, but the culture flask was gently shaken manually 1-2 times daily (5 seconds each time) to promote full contact between cells and the culture medium and avoid uneven cell growth due to local nutrient deficiency. Cells were passaged after 48 hours of culture per generation. Cell morphology was observed under a microscope; they should maintain a typical epithelial-like spindle shape without obvious shrinkage or increased granules. Viability was assessed using trypan blue staining: 91.5% for passage 6, 92.3% for passage 7, 91.8% for passage 8, 92.5% for passage 9, and 93.1% for passage 10, all ≥90%. Cell adhesion was counted using a cell counting chamber: 92% for passage 6, 93% for passage 7, 91% for passage 8, 92% for passage 9, and 94% for passage 10, all ≥90%, indicating successful cell adaptation to the low serum environment. If adhesion fell below 85%, serum degradation was paused, and the current serum and compound synergist concentrations were maintained for another 1-2 passages to ensure cell adaptation before proceeding to the next stage.

[0034] 202. Passages 11-15 (Semi-serum-free shaking induction period): The focus of this stage is to induce the cells to transition from adherent growth to semi-suspension growth. The culture medium formula is adjusted to 1% fetal bovine serum + 60% serum-free medium + 39% DMEM basal medium + 0.1% compound synergist, further reducing serum dependence, increasing the proportion of serum-free medium, and increasing the concentration of compound synergist to enhance cell suspension adaptability. Cells are transferred to 125mL Erlenmeyer flasks (with 0.22μm breathable caps to ensure gas exchange), and each flask is inoculated with 20mL of cell suspension, increasing the inoculation density to 3×10⁻⁶ cells / mL. 5Cells / mL were cultured in a shaker at 37°C with 5% CO2. The shaker speed was started at 30 rpm and increased by 10 rpm every two generations, eventually stabilizing at 50 rpm. The shaking intensity was such that the cells did not adhere to the wall and were slightly suspended, avoiding excessive shaking speed that could cause cell shearing damage. During this stage, under the microscope, some cells can be seen detached from the flask wall and suspended in single or small clusters (3-5 cells). After each passage, the suspension rate (number of suspended cells / total number of cells × 100%) is detected using a hemocytometer. The rate is 12% for passage 11, 25% for passage 12, 48% for passage 13, 65% for passage 14, and 82% for passage 15, gradually increasing to over 80%. The viability is detected using trypan blue staining. The viability is ≥88% for passage 11, 88.5% for passage 12, 89.8% for passage 13, 88.9% for passage 14, and 89.5% for passage 15, indicating that the cells have successfully transitioned from adherent growth to semi-suspension growth. If the increase in suspension rate is slow (<10% increase per passage), the shaking speed should be increased appropriately (15 rpm per passage) while maintaining the concentration of the compound synergist to ensure that the cells gradually adapt to the suspension environment.

[0035] 203. Generations 16-20 (Serium-Free Complete Adaptation Phase): During this stage, cells are completely free from serum dependence and adhesion. Serum is completely removed, and 100% serum-free culture medium with 0.12% compound synergist (containing 0.1% Pluronic F-68 to further reduce cell shear damage) is used. Increasing the concentration of the compound synergist ensures cell viability and proliferation in a completely serum-free environment. The shaking speed starts at 60 rpm, increasing by 20 rpm per generation, eventually stabilizing at 140 rpm. The culture temperature is maintained at 37°C, with a CO2 concentration of 5%. Cells are passaged every 3 days. Trypsin digestion is not required during passage; the cell suspension is directly collected, centrifuged at 1000 rpm for 5 min, the supernatant is discarded, and the cells are resuspended in fresh serum-free culture medium (containing 0.12% compound synergist). The seeding density is adjusted to 1 × 10⁶ cells / year. 6 cells / mL. At this stage, cell populations still relying on adherence for growth must be strictly eliminated (shake flasks with adherent cells >10% should be discarded directly). The results of the remaining 3 cell populations showed... Cell suspension rate: all three strains were ≥95% (95.2%, 96.5%, and 95.8%, respectively). Cell viability: all three strains were ≥90% (91.5%, 92.8%, and 91.2%, respectively). Cell morphology: All cells were round or short spindle-shaped and of uniform size, and three candidate suspension cell populations were successfully obtained.

[0036] Step (3) Suspension Adaptability Enhancement Stage (Generations 21-30): 301. Shake-flask expansion culture: The three selected candidate cell populations were transferred to 125mL Erlenmeyer flasks, and 30mL of serum-free culture medium (containing 0.12% compound synergist) was added to each flask. The inoculation density was 1×10⁻⁶. 6 Cells / mL, shaken at 140 rpm, cultured at 37°C with 5% CO2, passaged every 3 days. Daily sampling and analysis: Cell density was counted using a cell counting chamber to ensure a cell density of 6 × 10⁶ cells / mL every 3 days. 6 -7×10 6 cells / mL (Candidate group 1: 6.8 × 10⁻⁶) 6 cells / mL, Candidate group 2: 7.2 × 10⁻⁶ 6 cells / mL, candidate population 3: 6.5 × 10 6 Cell viability was assessed using trypan blue staining, with all candidates showing viability ≥90% (candidate population 1: 91.5%, candidate population 2: 93.2%, candidate population 3: 90.8%). Apoptosis rates were assessed using flow cytometry, with all candidates showing apoptosis rate ≤5% (candidate population 1: 4.2%, candidate population 2: 3.5%, candidate population 3: 4.8%). All three candidate cell populations exhibited good suspension adaptability.

[0037] 302. Monoclonal screening (limiting dilution method): In order to obtain suspension cell lines with uniform genetic background and stable performance, the limiting dilution method was used to screen three candidate cell populations for monoclonal cells. The specific steps are as follows: Candidate suspension cells in the logarithmic growth phase are seeded into 96-well plates for static culture. The cells are diluted to 1 cell / 100 μL with serum-free culture medium (containing 0.12% compound enhancer) to ensure an average of only one cell per well, avoiding the influence of multiple cell clones on cell line homogeneity. 100 μL of the diluted cell suspension is added to each well of the 96-well cell culture plate, and the plates are placed in a 37℃, 5% CO2 incubator for static culture (with shaking stopped to prevent cell aggregation and polyclonal formation). After 72 hours, wells containing single cells are observed and labeled under a microscope, excluding wells with multiple cells and those without cells. Once the single-cell clones have grown to cover 50% of the bottom of the wells in the 96-well plate (approximately 10-14 days), the cells are gently pipetted to form a single-cell suspension, which is then transferred to 24-well plates. 1 mL of serum-free culture medium (containing 0.12% compound enhancer) is added to each well for continued culture. The culture continues until the cell density in the 24-well plate reaches 1 × 10⁶ cells / well. 6 Cells / well were transferred to a T25 culture flask and 5 mL of serum-free culture medium (containing 0.12% compound enhancer) was added for culture; the cells were cultured until the cell density in the T25 culture flask reached 5 × 10⁶ cells / well. 6 The cells / mL were eventually expanded to 125mL shake flasks. After 12 days of culture, monoclonal cells with a clonal formation rate of 80% were selected. One monoclonal cell line showed the best performance and was named PK-S2.

[0038] Validation of cell performance after acclimatization: ensuring suitability for PCV culture requirements.

[0039] Five core performance tests were performed on the monoclonal cell lines to screen for the optimal cell line for production (named PK-S2, with a suggested classification as porcine kidney epithelial cells, submitted to the China General Microbiological Culture Collection Center on November 18, 2025, accession number CGMCC No. 46750). The specific testing standards are as follows: Growth performance: Cells were cultured using the shake-flask method with a 30 mL culture system (serum-free medium + 0.12% compound synergist) at 140 rpm at 37°C for 7 days. Cell density was counted daily using a cell counting chamber, viability was assessed using trypan blue staining, and doubling time was calculated using the CCK-8 assay. The selection criterion was a maximum cell density ≥8 × 10⁶ cells / year. 6 The cells / mL doubling time is ≤24h, and the viability on day 5 of culture is ≥95%, ensuring that the cells have high proliferative capacity in a serum-free suspension environment.

[0040] Passage stability: Cells were passaged 50 times consecutively, and growth performance (maximum cell density, viability, and doubling time) was assessed every 10 passages. Cell morphology (microscopic observation) and apoptosis rate (flow cytometry) were also measured. The selection criteria were: maximum cell density, viability, and doubling time at passage 50 differing from passage 10 by ≤5%; cell morphology remained uniform (round or short spindle-shaped); and the apoptosis rate was ≤5%, ensuring stable cell performance after long-term passage and meeting the long-term needs of industrial production.

[0041] Virus susceptibility: Inoculated with PCV2d and PCV3 strains, MOI=0.01, cultured for 72 h and then subjected to TCID. 50 The viral titer was detected using the PK-S2 cell monolayer culture method, with a control group not supplemented with the compound synergist. The screening criterion was a PCV2d titer ≥ 8.0 TCID in the group supplemented with the compound synergist. 50 / mL, PCV3 titer ≥7.5 TCID 50 / mL, and the potency was increased by ≥0.8 TCID compared to the control group. 50 / mL, ensuring that cells are highly sensitive to the target virus, and the compound synergist can effectively enhance the viral titer.

[0042] Example 3 This embodiment provides a method for culturing porcine circovirus in serum-free suspension PK cells based on a composite synergist. The specific process is as follows: Step (1) Preparation of seed viruses: Sources of PCV2 and PCV3 seed viruses: During the epidemiological investigation, two samples of lymph node tissue from pigs suspected of having PCV2 and PCV3 were ground and pulverized. After soaking in PBS for 1 hour, the samples were placed in a -70℃ freezer for freeze-thaw cycles, repeated 3 times. The supernatant was collected by centrifugation and filtered through a 0.22μm filter membrane for later use. Nucleic acid was extracted and then verified by PCR.

[0043] DNA extraction was performed according to the instructions of the kit from Tiangen Biotech (Beijing) Co., Ltd., and primer synthesis was completed by BGI Genomics.

[0044] PCV2 identification primers: PCV2 upstream: 5'-GACACCGCCCCCGCAGCCAT- 3' PCV2 downstream: 5'-GTAGTTTGTAGTCTCAGCCA-3' PCV3 identification primers PCV3 upstream: 5'-aaggagacgacgacgccacagg-3' PCV3 downstream: 5'-gctttgtcctgggtgagctc-3' Amplification system: DNA polymerase 1 μL, 5x DNA polymerase buffer 5 μL, dNTP 2 μL, DNA 1 μL, primers PCV2 upstream / downstream, and PCV3 upstream / downstream, add deionized water to a total volume of 25 μL.

[0045] Reaction conditions: 98℃ for 5 min, 90℃ for 25 s, 50℃ for 50 s, 70℃ for 55 s, 40 cycles, 72℃ for 7 min.

[0046] The amplified product from suspected PCV2 pathogen samples was subjected to nucleic acid agarose gel electrophoresis, which amplified the target band to 535 bp, consistent with the theoretical size. (See the instruction manual appendix.) Figure 1 , Appendix Figure 1 Lane 1 contains suspected PCV2 case material, lane 3 is a positive control, and lanes 2 and 4 are negative controls.

[0047] The amplified product from suspected PCV3 pathogen samples was subjected to nucleic acid agarose gel electrophoresis, which amplified the target band to 484 bp, consistent with the theoretical size. (See the instruction manual appendix.) Figure 2 , Appendix Figure 2 Lane 2 contains suspected PCV3 cases, lane 4 is a positive control, and lanes 1 and 3 are negative controls.

[0048] The virus solutions verified as PCV2 and PCV3 were simultaneously inoculated into T75 culture flasks containing cultured PK adherent cells at a volume ratio of 5% or 8%, respectively, and 1% of 300 mM D-Gluc was added. The flasks were then incubated at 37°C in a 5% CO2 incubator for 96 hours.

[0049] Virus collection: After repeated freeze-thaw cycles 3 times, the cultured T75 culture flasks are centrifuged at 5000-6000 rpm for 10 min at 4℃ and stored at -80℃.

[0050] And adopt TCID 50 For potency testing, PCV2 must be ≥7.0 TCID. 50 / mL, PCV3 must be ≥7.0 TCID 50 / mL.

[0051] PCV2 seed preparation: Resuscitate PK-S2 cells from the working seed bank and expand to a cell density of 6 × 10⁶ cells / mL in 125 mL shake flasks. 6 cells / mL, adjusted to 1×10⁻⁶ cells / mL using serum-free culture medium. 6 The virus was inoculated at a concentration of cells / mL with PCV2d seed at an MOI of 0.01, with 0.12% of a compound synergist added. The culture was carried out at 37°C and 140 rpm for 72 h using a shaker. After culture, the virus solution was harvested, centrifuged at 1000 rpm for 10 min to remove cell debris, and then filtered through a 0.22 μm filter for sterilization. TCID45 was then used. 50 The potency must be ≥8.2 TCID. 50 / mL; ELISA method was used to detect antigen content (≥1:20480); after aliquoting, the virus was stored at -80℃ to avoid repeated freeze-thaw cycles affecting viral activity. The entire preparation cycle took only 3 days, significantly shorter than the traditional adherence process (7 days), thus improving the efficiency of seed virus preparation.

[0052] PCV3 seed preparation: Innovative and optimized inoculation conditions were developed based on the characteristics of the PCV3 strain: PK-S2 cell density reached 6×10⁻⁶. 6 When the cell density is 1 × 10⁶ cells / mL, adjust the cell density to 1 × 10⁶ cells / mL using serum-free culture medium. 6 Cells / mL were inoculated with PCV3 seed at an MOI of 0.01, with 0.15% of a compound synergist added (to increase the concentration of the compound synergist to enhance PCV3 replication ability). The cells were first incubated at 37°C for 24 h (to promote virus adsorption and invasion), then transferred to 35°C for 48 h (low temperature induces PCV3 virus particle assembly). After incubation, the virus solution was harvested, centrifuged at 1000 rpm for 10 min to remove cell debris, and sterilized by filtration through a 0.45 μm filter membrane; TCID45 was used for further analysis. 50 For potency testing, the titer must be ≥7.8 TCID. 50 / mL; antigen content is detected by capture ELISA method, requiring ≥1:15360; store at -80℃. The preparation cycle is only 5 days, without relying on adherent cell propagation, which is more than 50% shorter than the traditional process (12 days), enabling rapid response to the needs of PCV3 epidemic prevention and control.

[0053] Step (2) Large-scale virus culture, PK-S2 cell expansion employed a stepwise approach of "shake-flask pilot-reactor inoculation," adding 0.12% of a compound synergist at each stage. Cell density and viability were rigorously monitored to ensure stable seed cell quality. The specific steps were as follows: Resuscitation Phase: PK-S2 cells were resuscitated from the working seed bank and seeded in 125 mL shake flasks using serum-free culture medium (containing 0.12% compound enhancer) at a seeding density of 5 × 10⁶ cells / mL. 5 Cells / mL, cultured at 37℃ and 140 rpm for 72 h; cell density was counted using a cell counting chamber, with a target density of 4 × 10⁶ cells / mL. 6 cells / mL; viability was detected using trypan blue staining and had to be ≥95%; if viability was <95%, cells needed to be revived to ensure good initial viability of the seed cells.

[0054] Primary expansion: Cells in the recovery phase were divided into groups of 1×10⁻⁶. 6 Cells / mL were seeded into new 125mL shake flasks and cultured in serum-free medium (containing 0.12% compound enhancer) at 37℃ and 140rpm for 72h; target cell density was 6×10⁶. 6 cells / mL, viability 98%;

[0055] Intermediate expansion: Transfer the cells from the primary expansion to 500 mL shake flasks, seeding at a density of 1 × 10⁶ cells / mL. 6 Cells / mL were cultured in serum-free medium (containing 0.12% compound synergist) at 37°C and 140 rpm for 72 h; target cell density was 6.5 × 10⁻⁶. 6 The cell count / mL should be ≥97%; the pH of the culture medium should be measured using a pH meter and maintained between 7.1 and 7.3. If the pH deviates from the specified range, adjust it by adding 1 mol / L hydrochloric acid or 1 mol / L sodium oxide solution to ensure a stable cell growth environment.

[0056] Advanced amplification: Transfer the cells from the intermediate amplification stage to 5L shake flasks, seeding at a density of 1×10⁶ cells / year. 6 Cells / mL were cultured in serum-free medium (containing 0.12% compound synergist) at 37°C and 140 rpm for 72 h; target cell density was 7 × 10⁻⁶ cells / mL. 6The cell density was 98%; the density met the amplification target, and the seed cell quality met the standards. The dissolved oxygen concentration in the culture medium was monitored using a dissolved oxygen meter and maintained between 30% and 50%. Dissolved oxygen was adjusted by changing the shaking speed (±10 rpm) to ensure that the cells' respiration needs were met. A stepwise amplification method was used to gradually increase the cell scale, avoiding uneven cell growth and decreased activity caused by direct large-scale culture.

[0057] Reactor culture and parameter optimization: Seed cells expanded in 5L shake flasks were cultured at a rate of 1×10⁻⁶. 6 Cells / mL were introduced into a 50L bioreactor using serum-free culture medium. Based on the differences in growth characteristics between PCV2 and PCV3, and by adjusting the concentration of the compound synergist, process parameters were precisely optimized to ensure efficient proliferation of both viruses. Temperature control: PCV2 was cultured at 37℃ throughout the process, meeting its optimal proliferation temperature requirement, where PCV2 DNA polymerase activity was highest and viral replication efficiency was optimal. PCV3 employed a segmented temperature control strategy: the first 24 hours were at 37℃ (promoting viral adsorption and invasion; during this stage, the PCV3 capsid protein bound to cell receptors most efficiently), followed by 48 hours at 35℃ (low temperature induces PCV3 viral particle assembly; at this temperature, viral capsid protein folding is more stable, increasing particle assembly efficiency by 20%-30%). The reactor achieved precise temperature control with an accuracy of ±0.1℃ through its built-in temperature control system (equipped with heating rods and cooling water pipes), preventing temperature fluctuations from affecting viral proliferation. Temperature alarm thresholds were also set (37℃±0.5℃, 35℃±0.5℃); when the temperature exceeded the threshold, an automatic alarm was triggered and temperature regulation was activated to ensure temperature stability.

[0058] pH control: The pH for both virus cultures was controlled between 7.1 and 7.3, within which PK-S2 cells exhibited the highest activity and the optimal viral infection rate. The pH was adjusted using an automatic replenishment system (equipped with a 1 mol / L sodium bicarbonate solution tank and a peristaltic pump). When the pH fell below 7.1, the peristaltic pump automatically added sodium bicarbonate solution to raise the pH; when the pH rose above 7.3, the addition automatically stopped, and the pH was lowered by introducing CO2 gas (5% concentration). pH was monitored every 10 minutes to ensure real-time control and adjustment.

[0059] Dissolved oxygen control: The dissolved oxygen concentration is maintained between 30% and 50%. This concentration range meets the respiration requirements of PK-S2 cells (avoiding hypoxia and apoptosis caused by excessively low dissolved oxygen) while preventing oxidative damage caused by excessively high dissolved oxygen (reducing the destruction of viral particles by reactive oxygen species). Precise control is achieved by adjusting the air-to-oxygen ratio (air:oxygen = 10:1 to 5:1). When dissolved oxygen is below 30%, the oxygen ratio is increased (air:oxygen = 8:1 to 5:1); when dissolved oxygen is above 50%, the oxygen ratio is decreased (air:oxygen = 10:1 to 9:1). Dissolved oxygen detection uses an online dissolved oxygen electrode with a detection accuracy of ±2%, ensuring stable dissolved oxygen levels.

[0060] Stirring speed: A paddle-type stirrer (four-bladed, 8cm diameter) was used, with the speed gradually increased from 50 rpm to 80 rpm (gradual increase: 50 rpm for 0-24h after inoculation, 60 rpm for 24-48h, and 80 rpm for 48-72h) to avoid cell shearing damage caused by the initial high speed. The stirring speed for both virus cultures was controlled at 50-80 rpm. This speed range ensured thorough mixing of cells and culture medium (avoiding local nutrient deficiency and accumulation of metabolic waste) while maintaining stable cell morphology (apoptosis rate ≤5%). The stirring speed was adjusted by the reactor's variable frequency motor with an accuracy of ±1 rpm to ensure stable speed.

[0061] Concentration control of the compound synergist: During PCV2 culture, the concentration of the compound synergist was maintained at 0.12%, at which the PCV2 titer reached its maximum (≥8.0 TCID). 50 The concentration of the compound enhancer was adjusted to 0.15% ( / mL), and it did not cause cytotoxicity due to excessive concentration (viability ≥95%). During PCV3 culture, the concentration of the compound enhancer was adjusted to 0.15%. Because PCV3 is more sensitive to enhancers, higher concentrations can further improve viral particle assembly efficiency, resulting in a PCV3 titer ≥7.5 TCID. 50 / mL. The compound synergist is added through the reactor feeding system (equipped with a compound synergist stock solution storage tank and a peristaltic pump). 50% is added at the time of inoculation, and the remaining 50% is added after 24 hours of culture to avoid stimulation of cells by the initial high concentration, while ensuring the stability of the synergist concentration throughout the process.

[0062] Inoculation density and MOI: The inoculation density for both viruses was 1×10⁻⁶. 6 At this density, the cells are in the logarithmic growth phase, exhibiting strong proliferative capacity and efficiently supporting viral replication (viral infection rate ≥90%). However, if the inoculation density is too high (>1.2×10⁻⁶ cells / mL), the cells will be in the viral growth phase, exhibiting strong proliferative capacity and efficiently supporting viral replication. 6 If the inoculation density is too low (<0.8×10⁶ cells / mL), it will lead to competition for nutrients among cells and a decrease in cell viability; if the inoculation density is too low (<0.8×10⁶ cells / mL), it will cause cells to compete for nutrients and decrease their viability. 6A low MOI (number of cells / mL) can lead to insufficient target cell infection and reduced viral titer. The MOI for inoculation was 0.01. The inoculation amount was precisely calculated based on viral titer and cell density (inoculation amount = total cells × MOI / viral titer) to avoid premature cell death due to excessively high MOI (viability <80% at 48 hours post-infection) or decreased viral proliferation efficiency due to excessively low MOI (titer <7.0 TCID). 50 / mL).

[0063] Step (3) Harvesting the virulence using a multi-generation harvesting strategy: Taking advantage of the fact that suspended cells do not require adhesion, an innovative "continuous harvesting-feed culture" strategy is adopted to achieve multi-generation virus harvesting, significantly increasing single-batch yield and reducing virus damage. The specific process is as follows: First-generation results: 72 hours after infection, the viral titer reaches its peak (PCV2 ≥ 8.0 TCID). 50 / mL, PCV3 ≥ 7.5 TCID 50 The culture was harvested at a concentration of 75% (approximately 37.5 L) using a reactor harvesting system (equipped with a 0.45 μm filter membrane and harvesting tank), rapidly harvesting a high concentration of virus. During harvesting, the stirring speed was maintained at 50 rpm to prevent cell precipitation and turbidity of the harvested solution. After harvesting, the viral titer and antigen content of the harvested solution were tested to ensure compliance with quality standards (PCV2 titer ≥ 8.0 TCID). 50 / mL, antigen content ≥1:20480; PCV3 titer ≥7.5 TCID 50 / mL, antigen content ≥1:15360).

[0064] Second-generation harvest: Add fresh serum-free culture medium (containing the corresponding concentration of compound enhancer) to the remaining 25% of the culture (approximately 12.5L) to a final volume of 50L, and continue culturing for 72 hours; at this point, the viral titer remains stable (PCV2 ≥ 7.8 TCID). 50 / mL, PCV3 ≥ 7.3 TCID 50 Harvest all culture (approximately 50 L) at a rate of 1 L / min using the reactor feeding system; during feeding, add culture medium at a uniform rate (1 L / min) to avoid impacting cells and causing a decrease in viability; during the culture process, maintain constant process parameters (temperature, pH, dissolved oxygen, rotation speed) to ensure continuous virus proliferation.

[0065] Third-generation harvest: Fresh serum-free culture medium (containing the corresponding concentration of compound enhancer) was added again to 50L, and cultured for another 72 hours; at this time, the viral titer decreased slightly but still met the production requirements (PCV2 ≥ 7.5 TCID). 50 / mL, PCV3 ≥ 7.0 TCID 50Harvest all culture (approximately 50 L) at / mL. After harvesting, check cell viability. If viability is <80%, stop harvesting to avoid excessively low titers in subsequent harvest media; if viability is ≥80%, continue culture with added substrate and harvest the fourth generation, but note that the virus titer in the fourth generation may further decrease (PCV2 ≥ 7.2 TCID). 50 / mL, PCV3 ≥ 6.8 TCID 50 ( / mL), whether to continue harvesting needs to be determined based on production requirements.

[0066] This step yielded a total of 137.5 L of virus fluid per batch, representing a 72% increase in yield compared to traditional roller bottles of the same scale (≤80 L). Furthermore, the virus recovery rate reached 93%, significantly superior to the traditional process (70%). Simultaneously, the PCV3 titer was higher than the control group without the added synergist (6.5-6.8 TCID). 50 Increase by 0.7-1.0 TCID / mL 50 / mL, which fully verifies the high efficiency of the method of the present invention and the synergistic effect of the compound synergist.

[0067] The advantages of this harvesting strategy are: it eliminates the need for trypsin digestion, avoiding viral particle damage caused by trypsin in traditional adherent cells (trypsin destroys viral capsid proteins, leading to a 30%-40% decrease in viral infectivity), and achieving a virus recovery rate of over 92% (compared to only 70% in traditional adherent cells); at the same time, each generation of harvested fluid is filtered through a 0.45μm filter membrane to remove cell debris (fragment content <0.1%), laying a good foundation for subsequent purification processes, reducing impurities during purification, and lowering purification costs.

[0068] Virus fluid quality testing (after each generation harvest): Virus titer: using TCID 50 The acceptable standard for PCV2 assay (PK-S2 cell monolayer culture) is ≥7.5 TCID2. 50 / mL, PCV3 ≥ 7.0 TCID 50 / mL, to ensure that the virus concentration meets the requirements for vaccine production; Antigen content: The ELISA method was used for detection. The qualified standards were PCV2 ≥ 1:15360 and PCV3 ≥ 1:10240 to ensure sufficient viral antigen content and guarantee vaccine immunogenicity.

[0069] To visually demonstrate the advantages of the method of this invention, it was compared and validated in multiple dimensions with traditional adherent cell culture methods and suspension culture methods without compound synergists. The results are as follows: Comparison Dimensions Traditional adherent cell culture Suspension culture without compound synergists The method of this invention (including compound synergists) Cell serum dependence 8%-10% fetal bovine serum none none <![CDATA[Maximum cell density (×10 6 cells / mL)]]> 5.0±0.3 7.2±0.4 7.8±0.3 <![CDATA[PCV2 titer (TCID 50 / mL)]]> ≤5.5 7.0±0.2 8.2±0.3 <![CDATA[PCV3 titer (TCID 50 / mL)]]> ≤5.0 6.8±0.2 7.7±0.2 Production cycle (days) 45-50 38-42 35-40 Single batch production (50L reactor, L) ≤80 120-130 135-140 Virus recovery rate (%) 70±5 85±3 92±2 Piglet allergy rate (%) Approximately 5 ≤1.0 ≤0.5 Inter-batch fluctuation (%) ≥20 12±2 8±1 Cost per unit of annual production capacity of 100 million units (ten thousand yuan) Approximately 8000 6500-6800 5500-5800 The comparative results show that the method of the present invention is significantly superior to the traditional adherence process and the suspension process without composite synergists in terms of cell performance, virus proliferation efficiency, production efficiency, safety, stability and cost control. The maximum cell density was increased by 56% compared to the traditional process and by 8.3% compared to the suspension process without synergists; PCV2 potency was improved by 49.1% compared to the traditional process and by 17.1% compared to the suspension process without synergists; PCV3 potency was improved by 54% compared to the traditional process and by 13.2% compared to the suspension process without synergists. The production cycle is shortened by 22.2% compared to the traditional process and by 7.7% compared to the suspension process without synergists; The single-batch yield is increased by 75% compared to the traditional process and by 7.7% compared to the suspension process without synergists; The virus recovery rate is 31.4% higher than that of traditional processes and 8.2% higher than that of suspension processes without synergists. The allergy rate in piglets is reduced by 90% compared to the traditional process and by 50% compared to the suspension process without synergists. Batch-to-batch variation is reduced by 60% compared to traditional processes and by 33.3% compared to suspension processes without synergists; The annual cost is reduced by 31.25% compared to the traditional process and by 15.4% compared to the suspension process without synergists.

[0070] The above comparison results fully demonstrate that the present invention significantly improves the efficiency and quality of PCV culture by introducing a compound synergist and optimizing the domestication process, and has extremely high industrial application value.

[0071] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A suspension of serum-free PK cells based on a compound synergist, characterized in that, The PK cells were strain PK-S2, and it is recommended to classify them as porcine kidney epithelial cells. They were deposited at the China General Microbiological Culture Collection Center on November 18, 2025, with accession number CGMCC No. 46750.

2. A method for constructing serum-free PK cells based on a composite synergist as described in claim 1, characterized in that, The specific steps include: (1) In the cell pretreatment stage, the adherent PK cells frozen in liquid nitrogen were taken out, and PK cells with stable proliferation rate were obtained by thawing in a water bath, resuspending culture, and passage culture. (2) In the serum gradient reduction acclimatization stage, a four-dimensional synergistic acclimatization strategy of "gradually decreasing serum concentration + gradually increasing serum-free culture medium + gradually increasing shaking intensity + adjusting the concentration gradient of compound synergist" was adopted to acclimatize and culture the obtained stable proliferating PK cells, and screened to obtain PK cell lines that are fully adapted to serum-free full suspension culture. (3) In the suspension adaptation enhancement stage, the selected PK cell lines were transferred to Erlenmeyer flasks for expansion culture, and then the limiting dilution method was used for single-clone screening to obtain single-clone suspension PK-S2 cell lines.

3. The method for constructing serum-free PK cells based on a composite synergist according to claim 2, characterized in that: The composite synergist mother liquor contains IGF1R, L-selenomethionine, polyvinylpyrrolidone, and Cu²⁺. + The Mn²⁺ complex was prepared by dissolving four substances in a mass ratio of 3:2:1:0.5 in a serum-free culture medium to form a 10% stock solution.

4. The method for constructing serum-free PK cells based on a composite synergist according to claim 3, characterized in that: The specific process of the cell pretreatment stage in step (1) is as follows: take the frozen adherent PK cells out of liquid nitrogen, quickly place them in a 37℃ water bath for 1-2 minutes to thaw them rapidly, so as to avoid damage to the cells due to prolonged low temperature; then add 10 mL of DMEM medium containing 5% fetal bovine serum and 0.05% compound synergist, centrifuge at 1000 rpm for 5 minutes to remove the cryopreservation solution, reduce the toxic effect of cryopreservation agent on cells, resuspend the cells and seed them into T75 culture flasks, add 10 mL of DMEM medium containing 5% fetal bovine serum and 0.05% compound synergist to each flask, and place them in a 37℃, 5% CO2 incubator for static culture; Once the cell confluence reaches 80%–90%, discard the supernatant and gently wash twice with PBS buffer (pH 7.2–7.4) to remove residual culture medium and metabolic waste. Add 2 mL of 0.25% trypsin-EDTA digestion solution and incubate at 37°C for 1–2 minutes. After observing increased intercellular spaces and rounded cells under a microscope, immediately add 5 mL of DMEM medium containing 5% fetal bovine serum and 0.05% compound synergist to terminate digestion. Gently pipette the cells to form a single-cell suspension, avoiding excessive pipetting that could cause cell breakage. Cells were seeded in new T75 culture flasks at a passage ratio of 1:3 and passaged 5 times consecutively. After each passage, cell viability was assessed using trypan blue staining and was required to be stable at 92%–95%. Simultaneously, cell proliferation activity was assessed using the CCK-8 assay to ensure a stable cell proliferation rate.

5. The method for constructing serum-free PK cells based on a composite synergist according to claim 3, characterized in that: The specific steps of step (2), the serum gradient reduction acclimatization phase, include: Low serum adaptation period: This stage is completed during the 6th to 10th generation of cells. The culture medium formula is 2% fetal bovine serum + 30% serum-free special medium + 67% DMEM basal medium to remove antibiotics + 0.08% compound synergist. Add 10 mL of this mixed medium to each T75 culture flask. Adjust the cell seeding density to 2 × 10⁶ cells / year during passage. 5 The cells / mL culture is still statically cultured, but the culture flask is gently shaken manually 1-2 times a day for 5 seconds each time to promote full contact between cells and culture medium and avoid uneven cell growth due to local nutrient deficiency. After each passage, the cells are passaged after 48 hours of culture. The cell morphology is observed under a microscope. The cells should maintain a typical epithelial spindle shape without obvious shrinkage or increased granules. The viability is detected by trypan blue staining and should be ≥90%. The cell adhesion rate is counted by a cell counting chamber and should be ≥90%. If the adhesion rate is lower than 85%, the serum degradation should be paused, and the current serum concentration and compound synergist concentration should be maintained for passage 1-2 more times to ensure that the cells adapt before entering the next stage. Semi-serum-free shaking induction phase: This phase is completed between passages 11 and 15. The culture medium formulation is adjusted to 1% fetal bovine serum + 60% serum-free medium + 39% DMEM basal medium + 0.1% compound synergist, further reducing serum dependence and increasing the proportion of serum-free medium. Simultaneously, the concentration of the compound synergist is increased to enhance cell suspension adaptability. Cells are transferred to 125mL Erlenmeyer flasks, with 20mL of cell suspension inoculated into each flask, increasing the inoculation density to 3 × 10⁶ cells / mL. 5 Cells / mL were cultured in a shaker at 37°C with 5% CO2. The shaker speed was started at 30 rpm and increased by 10 rpm every two generations, eventually stabilizing at 50 rpm. The shaking intensity was such that the cells did not adhere to the flask and were slightly suspended, avoiding excessive shaking that could cause shear damage to the cells. At this stage, under a microscope, some cells could be seen detaching from the flask wall and appearing as single cells or small clusters in suspension. After each passage, the suspension rate was checked using a hemocytometer, and it needed to be gradually increased from the initial 10% to over 80%. The viability should be ≥88% when tested using trypan blue staining. If the suspension rate increases slowly, the shaking speed should be increased by 15 rpm per generation, while maintaining the concentration of the compound synergist unchanged.

203. Serum-Free Complete Adaptation Phase: This phase is completed between passages 16 and 20, completely eliminating serum dependence and adhesion dependence. A 100% serum-free culture medium with 0.12% of a compound synergist is used. The compound synergist is supplemented with 0.1% Pluronic F-68 to further reduce shear stress damage to cells. Increasing the concentration of the compound synergist ensures cell viability and proliferation in a completely serum-free environment. The shaking speed starts at 60 rpm, increasing by 20 rpm per passage, eventually stabilizing at 140 rpm. The culture temperature is maintained at 37°C, with a carbon dioxide concentration of 5%. Cells are passaged every 3 days. During passage, trypsin digestion is not required; the suspended cell suspension is directly centrifuged at 1000 rpm for 5 min, the supernatant is discarded, and the cells are resuspended in fresh serum-free culture medium containing 0.12% of the compound synergist. The seeding density is adjusted to 1 × 10⁶ cells / year. 6 At this stage, cell populations still relying on adherent growth must be strictly eliminated, retaining only cell populations with a suspension rate ≥95%, round or short spindle-shaped cells, and uniform size; trypan blue staining is used to detect cell viability, which must be ≥90%; CCK-8 assay is used to detect cell proliferation activity, ensuring doubling time ≤26h; a total of 3-5 candidate cell populations are screened to enter the next stage to ensure that high-quality suspension cell lines are obtained subsequently.

6. The method for constructing serum-free PK cells based on a composite synergist according to claim 5, characterized in that: The specific process of expanding the culture in the shake flasks in step (3) involves transferring the selected candidate cell populations to 125mL Erlenmeyer shake flasks, adding 30mL of serum-free culture medium containing 0.12% compound synergist to each flask, and inoculating at a density of 1×10⁻⁶ cells / mL. 6 Cells / mL, shaken at 140 rpm, cultured at 37°C and 5% CO2, passaged every 3 days, with daily sampling and analysis. Cell density was counted using a cell counting chamber to ensure a cell density of 6 × 10⁶ cells / mL every 3 days. 6 ~7×10 6 cells / mL; Viability was assessed using trypan blue staining to ensure it was ≥90%; apoptosis rate was assessed using flow cytometry to ensure it was ≤5%. These tests validated the stable proliferation and activity of cells in suspension. Candidate cell populations with slow cell density growth or apoptosis rates >5% were eliminated, retaining 2-3 high-performing candidate cell populations for monoclonal screening. The monoclonal screening process involved taking candidate suspension cells in logarithmic growth phase and diluting them to 1 cell / 100 μL with serum-free medium containing 0.12% compound synergist, ensuring an average of only one cell per well to avoid multiple cell clones affecting cell line homogeneity. 100 μL of the diluted cell suspension was added to each well of a 96-well cell culture plate and incubated at 37°C with 5% [presumably referring to a specific temperature range]. Static culture was performed in a CO2 incubator; after 72 hours, wells containing single cells were observed and labeled under a microscope, excluding multicellular and cellless wells; when monoclonal cells grew to cover 50% of the bottom of the well in a 96-well plate, a single-cell suspension was formed by gently pipetting and transferred to a 24-well plate, with 1 mL of serum-free culture medium containing 0.12% compound synergist added to each well for further culture; when the cell density in the 24-well plate reached 1×10⁶ cells / well... 6 Cells / well were transferred to T25 culture flasks and cultured in 5 mL of serum-free culture medium containing 0.12% compound synergist until the cell density in the T25 culture flasks reached 5 × 10⁶ cells / well. 6 The cells / mL were eventually expanded to 125mL shake flasks to obtain a monoclonal suspension PK-S2 cell line.

7. A method for culturing porcine circovirus in serum-free PK cells based on a composite synergist, characterized in that: (1) Virus seed preparation: Porcine circovirus PCV2 and PCV3 were screened from lymph node samples of diseased pigs, and the obtained PCV2 and PCV3 seed viruses were cultured and screened in serum-free PK-S2 cells as described in claim 1 at an MOI of 0.

01. 0.12% compound synergist was added to the culture medium for PCV2, and 0.15% compound synergist was added to the culture medium for PCV3. (2) Large-scale virus culture: First, the PK-S2 cells as described in claim 1 were amplified and cultured. The amplification of PK-S2 cells was completed step by step through primary amplification in 125ml shake flasks, intermediate amplification in 500ml shake flasks, and advanced amplification in 5L shake flasks. 0.12% compound synergist was added at each amplification stage. Then, the seed cells amplified in 5L shake flasks were cultured at a ratio of 1×10⁻⁶. 6 Cells / mL were inoculated into 50L bioreactors and cultured in serum-free medium. PCV2 and PCV3 virus strains were inoculated at an MOI of 0.

01. PCV2 was cultured at 37℃ throughout the entire process, as this temperature resulted in the highest PCV2 DNA polymerase activity and optimal viral replication efficiency. For PCV3, a segmented temperature control strategy was employed. The first 24 hours were cultured at 37℃ to promote viral adsorption and invasion, during which the PCV3 capsid protein exhibited the highest binding efficiency to cell receptors. The subsequent 48 hours were cultured at 35℃ to induce PCV3 viral particle assembly, where the viral capsid protein folded more stably, increasing particle assembly efficiency by 20%–30%. The pH for both viruses was maintained at 7.1–7.3, and the dissolved oxygen concentration was maintained at 30%–50%. A paddle agitator was used, with the rotation speed gradually increased from 50 rpm to 80 rpm. The concentration of the compound synergist was maintained at 0.12% during PCV2 culture and adjusted to 0.15% during PCV3 culture. (3) Harvest the venom by adopting the strategy of "continuous harvesting-feeding culture" to harvest multiple generations of virus.

8. The method for culturing porcine circovirus in suspension serum-free PK cells based on a composite synergist according to claim 7, characterized in that: The amplification and culture process in step (2) is specifically as follows: Primary expansion: PK-S2 cell line was expanded at a rate of 1×10⁻⁶. 6 Cells / mL were seeded in new 125mL shake flasks and cultured in serum-free medium containing 0.12% compound synergist at 37℃ and 140rpm for 72h; target cell density 6×10⁶. 6 The cell count / mL should be ≥95%; the apoptosis rate should be ≤3% when detected by flow cytometry to avoid apoptotic cells affecting subsequent expansion. Intermediate expansion: Transfer the cells from the primary expansion to 500 mL shake flasks, seeding at a density of 1 × 10⁶ cells / mL. 6 Cells / mL, cultured in serum-free medium containing 0.12% compound synergist at 37°C and 140 rpm for 72 h; target cell density 6.5 × 10⁻⁶. 6 The cell count / mL should be ≥95%; the pH of the culture medium should be measured with a pH meter and maintained between 7.1 and 7.

3. If the pH deviates, adjust it by adding 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution to ensure a stable cell growth environment. Advanced amplification: Transfer the cells from the intermediate amplification stage to 5L shake flasks, seeding at a density of 1×10⁶ cells / year. 6 Cells / mL were cultured in serum-free medium containing 0.12% compound synergist at 37°C and 140 rpm for 72 h; target cell density was 7 × 10⁶ cells / mL. 6 Cells / mL, viability ≥95%; dissolved oxygen concentration in the culture medium is measured using a dissolved oxygen meter and needs to be maintained at 30%~50%. Dissolved oxygen is adjusted by changing the shaking speed of the flask to ensure that the cell respiration needs are met; cell scale is gradually expanded through stepwise amplification.

9. The method for culturing porcine circovirus in serum-free PK cells based on a composite synergist according to claim 8, characterized in that: The specific process of harvesting the virus through multiple generations in step (3) is as follows: First-generation harvest: 72 hours after inoculation, when the viral titer reaches its peak, harvest 75% of the culture to quickly harvest a high concentration of virus; during the harvesting process, maintain a stirring speed of 50 rpm to avoid cell precipitation that would cause turbidity in the harvested solution; after harvesting, test the viral titer and antigen content of the harvested solution to ensure that it meets quality standards; Second-generation harvest: Add fresh serum-free culture medium containing the corresponding concentration of compound synergist to the remaining 25% of the culture to a volume of 50L, and continue culturing for 72h; at this time, the virus titer remains stable, and all cultures are harvested; when feeding, add the culture medium at a uniform rate through the reactor feeding system to avoid shocking the cells and causing a decrease in viability; during the culture process, maintain the process parameters unchanged to ensure continuous virus proliferation; Third-generation harvest: Add fresh serum-free culture medium containing the corresponding concentration of compound synergist to 50L and continue culturing for 72h. At this time, the virus titer will decrease slightly but still meet the production requirements. Harvest all the culture. After harvesting, test the cell viability. If the viability is <80%, stop harvesting to avoid the subsequent harvest liquid having too low a titer. If the viability is ≥80%, you can continue to add material and culture to harvest the fourth generation. However, it should be noted that the virus titer of the fourth generation may decrease further. Whether to continue harvesting should be decided according to production needs.

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