Dual pretreatment conditioned medium for non-cytopathic virus sf9 cell screening method

Abstract

The application discloses a rhabdovirus-free Sf9 cell screening method of double pretreated conditioned medium, relates to the technical field of biology, and obtains the rhabdovirus-free Sf9 cell by double pretreatment of the conditioned medium through cross-flow filtration and beta-propiolactone inactivation and hydrolysis, and combination of single cell cloning screening. The method realizes efficient virus interception and high-activity retention of nutritional factors, the single cell cloning formation rate is greater than 40%, and is suitable for virus-free cell bank construction and biological product production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for screening Sf9 cells free of rhabdoviruses using a dual-pretreatment culture medium. Background Technology

[0002] Sf9 cells, as the core host of the insect cell-baculovirus expression system (BICS), are widely used in the production of recombinant proteins, viral vectors (such as AAV), and vaccines. However, in 2014, three different research teams discovered that Sf9 cells in different insect cell lines were generally contaminated with a novel rhabdovirus (Sf-RV), posing a potential risk to the safety of biopharmaceuticals. Existing methods for screening Sf9 cells free of rhabdovirus mainly rely on single-cell cloning and drug treatment (such as 6-azauridine), which suffer from low screening efficiency, impaired cell viability, and loss of nutrient factors, making it difficult to efficiently remove the virus while preserving the activity of key growth factors and metabolites in the culture medium.

[0003] Conditioned culture media contain cell-secreted growth factors, extracellular matrix components, and metabolites, which can significantly promote cell proliferation and colony formation. However, traditional conditioned culture media preparation methods are not optimized for viral contamination and toxicity residues. Direct use may introduce exogenous viruses or chemical reagent residues, limiting their application in virus-free cell screening. In particular, existing technologies have significant shortcomings in achieving the dual goals of "efficient retention of rhabdoviruses and low loss of nutrient factors with high activity retention": on the one hand, a single filtration method is insufficient to achieve efficient virus retention and low loss of nutrient factors; on the other hand, while chemical inactivation methods such as β-propiolactone can effectively inactivate viruses, their residual toxicity and impact on culture medium activity have not been effectively controlled.

[0004] Therefore, developing a conditioned medium pretreatment technology that can efficiently remove viruses, retain nutritional activity, and leave no toxic residues is of great significance for improving the screening efficiency of Sf9 cells without rhabdovirus. Summary of the Invention

[0005] To address the problems of low cell viability and poor clone formation rate in existing screening methods for Sf9 cells without rhabdomyovirus, this invention provides a screening method based on a dual pretreatment conditioned medium. Through a combination of nanoscale filtration and targeted inactivation-hydrolysis, the method achieves efficient removal of viruses, retention of nutritional factors, and degradation of chemical toxicity in the conditioned medium, significantly improving the activity and formation rate of Sf9 single-cell clones.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for screening Sf9 cells free of rhabdoviruses using a dual-preconditioned culture medium includes the following steps: S1: Preparation of original conditioned medium: Take Sf9 cells and suspend them in serum-free insect cell culture medium until the viability is >95%. Collect the supernatant, filter it coarsely, and obtain the original conditioned medium. S2: Dual pretreatment process: The original conditioned medium is subjected to cross-flow filtration, and the filtrate is collected; β-propiolactone is added to the filtrate, inactivated, and hydrolyzed to obtain the dual pretreated conditioned medium; S3: Single-cell clone screening: Sf9 single-cell suspension was seeded into 96-well plates, and 300 μL of double preconditioning medium was added to each well. The medium was replaced with 50% double preconditioning medium every 3 days. The cells were cultured statically at 27-28℃ for 14-21 days. Sf-RV in the cloned cells was detected by RT-PCR. Negative clones were screened and expanded to obtain rhabdovirus-free Sf9 cells. Furthermore, the concentration of the Sf9 single-cell suspension is 1-10 cells / mL.

[0007] Furthermore, the Sf9 cells have the accession number ATCC CRL-1711; the serum-free insect cell culture medium is SF-900 III SFM.

[0008] Furthermore, the cross-flow filtration parameters are as follows: cross-flow filtration operating pressure is 0.1-0.3 MPa, temperature is 25-30℃, and cross-flow rate is 1.5-2 L / min / m. 2 The filtration time is 30-60 minutes.

[0009] Furthermore, the β-propiolactone is added at a final concentration of 0.1-0.5 v / v; the inactivation temperature is 24-26℃ for 22-26 h; and the hydrolysis temperature is 36-38℃ for 1-3 h.

[0010] Furthermore, in the coarse filtration process, a 0.22 μm pore size polyethersulfone filter membrane is used; in the cross-flow filtration process, a 35 nm pore size polyethersulfone filter membrane is used.

[0011] Furthermore, in the cross-flow filtration process, a composite filter composed of a 35nm pore size polyethersulfone filter membrane and a modified nanofiber cellulose base membrane is used. Furthermore, the preparation method of the modified nanocellulose-based membrane includes the following steps: Crystalline cellulose was added to sulfuric acid solution and heated to 45-50℃ for 60-90 min to hydrolyze the solution. After dialyzing to neutral, the solution was centrifuged and freeze-dried to obtain nanocellulose powder. The nanocellulose powder was added to deionized water, ultrasonically dispersed, vacuum filtered, and then dried under pressure at 80-85℃ for 2-3 h to obtain a nanocellulose-based membrane. Dopamine hydrochloride was added to Tris-HCl buffer and stirred until homogeneous to obtain a PDA solution. Tannic acid and 3-aminopropyltrimethoxysilane were added to deionized water, mixed, and then sodium periodate solution was added. The mixture was stirred thoroughly to obtain a composite solution. The nanocellulose-based membrane was immersed in the PDA solution and shaken at 25-30℃ for 1-2 hours. It was then removed and rinsed with deionized water until the filtrate was colorless to obtain a PDA-modified nanocellulose-based membrane. The PDA-modified nanocellulose-based membrane was immersed in the composite solution and reacted at 60-65℃ for 1-1.5 hours. It was then removed and vacuum dried at 50-55℃ to obtain a modified nanocellulose-based membrane.

[0012] Furthermore, in the preparation process of the nanocellulose base membrane, the mass ratio of nanocellulose powder to deionized water is 1:(40-60); the thickness of the nanocellulose base membrane is 30-50μm, and the pore size is 0.18-0.3μm.

[0013] Furthermore, in the preparation of the PDA solution, the concentration of the PDA solution is 2.5-3 mg / mL, and the concentration of Tris-HCl is 45-50 mM; in the preparation of the composite solution, the mass ratio of tannic acid to 3-aminopropyltrimethoxysilane is 1:(1.2-1.5); and the concentration of the sodium periodate solution is 4-5 mg / mL.

[0014] Furthermore, the surface of the PDA-modified nanocellulose base film contains a polydopamine coating with a thickness of 50-80 nm; a tannic acid-silicon coating with a thickness of 30-50 nm is also deposited on the polydopamine coating on the surface of the modified nanocellulose base film.

[0015] Furthermore, the modified nanocellulose-based membrane has a pore size of 20-30 nm.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention forms a dual virus removal system through the physical interception of a composite filter and the subsequent chemical inactivation of β-propiolactone. The composite filter first intercepts more than 99% of rhabdoviruses, significantly reducing the inactivation load of β-propiolactone. This allows β-propiolactone to achieve complete inactivation of residual viruses at a low concentration of only 0.1-0.5 v / v%, avoiding the toxic residues caused by incomplete hydrolysis of high-concentration β-propiolactone. The hydrophilic modified coating of the composite filter membrane does not chemically react with or adsorb β-propiolactone, ensuring inactivation efficiency and thoroughness of subsequent hydrolysis, while also avoiding chemical contamination of the culture medium by the filter membrane coating.

[0017] 2. This invention employs a composite structure of polyethersulfone filter membrane and modified nanocellulose base membrane, utilizing the hierarchical retention effect of the double membrane; the polyethersulfone filter membrane first coarsely retains large-particle-size bullet-shaped viruses in the original conditioned culture medium, reducing the viral load on the subsequent modified nanocellulose base membrane; the modified nanocellulose base membrane finely retains small-particle-size viruses that were missed, achieving a virus retention rate of nearly 100%.

[0018] 3. This invention uses a polydopamine coating as a "coarse pore adjuster" to achieve initial shrinkage of the original membrane pore size; and a tannic acid-silicon coating as a "fine pore adjuster" to precisely control the pore size within 20-30 nm. The combination of the two avoids the problems of uneven pore size or excessive clogging caused by single-step pore adjustment, and precisely matches the particle size differences of viruses / nutrients. The catechol groups of dopamine and the hydroxyl and carboxyl groups of tannic acid-silicon form a synergistic hydration layer, which is denser and more stable than the hydration layer of a single dopamine or tannic acid-silicon coating, significantly reducing the non-specific adsorption of nutrients on the membrane surface, while avoiding secondary pollution caused by coating peeling.

[0019] 4. In this invention, the dense hydration layer on the membrane surface blocks the hydrophobic contact between the nutrient factors and the membrane surface, greatly reducing the non-specific adsorption loss of the nutrient factors; the treatment conditions without adsorption, shearing, and high temperature ensure that the spatial structure and biological activity of the nutrient factors are not destroyed, solving the problem of loss of nutrient factor activity caused by adsorption, high pressure, and high temperature in traditional filter membranes.

[0020] 5. Based on a composite filter membrane prepared from polyethersulfone filter membrane and modified nanocellulose base membrane, this invention precisely controls the coating thickness and membrane pore size, and combines the composite filter membrane with cross-flow filtration process to achieve the dual goals of efficient interception of rhabdovirus and low loss of nutrient factors with high activity retention, providing core technical support for the efficient screening of rhabdovirus-free Sf9 cells. Attached Figure Description

[0021] Figure 1 This is a flowchart of the dual pretreatment process in the method for screening Sf9 cells without rhabdomyovirus using dual pretreatment culture medium according to the present invention. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0023] In the following embodiments, the preparation method of the modified nanocellulose-based membrane includes the following steps: Crystalline cellulose was added to a 64wt% sulfuric acid solution, heated to 45℃ for 60 min for hydrolysis, dialyzed to neutral, centrifuged, and freeze-dried to obtain nanocellulose powder; 1 g of nanocellulose powder was added to 50 mL of deionized water, ultrasonically dispersed, vacuum filtered, and dried at 80℃ for 2 h to obtain a nanocellulose-based membrane. Dopamine hydrochloride was added to 50 mL Tris-HCl buffer and stirred until homogeneous to obtain a 2 mg / mL PDA solution. 1 g tannic acid and 1.2 g 3-aminopropyltrimethoxysilane were added to deionized water, mixed, and then 4 mg / mL sodium periodate solution was added. The mixture was stirred thoroughly to obtain a composite solution. The nanocellulose membrane was immersed in the PDA solution and shaken at 25-30 °C for 1 h. It was then removed and rinsed with deionized water until the filtrate was colorless to obtain a PDA-modified nanocellulose membrane. The PDA-modified nanocellulose membrane was immersed in the composite solution and reacted at 60 °C for 1 h. It was then removed and vacuum dried at 50 °C to obtain a modified nanocellulose membrane.

[0024] Example 1: Screening method for Sf9 cells free of rhabdovirus using dual-pretreated conditioned medium. The screening method includes the following steps: S1: Preparation of original conditioned medium: ATCC CRL-1711 Sf9 cells are taken and cultured in suspension using SF-900 III SFM serum-free insect cell medium until the density reaches 5 × 10⁻⁶ cells / year. 6 Cells / mL, centrifuged at 1000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the original conditioned medium; S2: Dual pretreatment process: The original conditioned medium was cross-flow filtered using a composite filter to trap rhabdoviruses and retain small molecule nutrients. The filtrate was collected. β-propiolactone was added to the filtrate to a final concentration of 0.3 v / v%, inactivated at 25°C for 24 h, and hydrolyzed at 37°C for 2 h. HPLC analysis showed that the β-propiolactone residue was <0.1 μg / mL, thus obtaining the dual pretreatment conditioned medium. The composite filter consists of a 35nm polyethersulfone filter membrane and a modified nanocellulose base membrane, with a pore size of 25nm; the modified nanocellulose base membrane has a 50nm polydopamine coating and a 30nm tannic acid-silicon coating. The cross-flow filtration parameters are as follows: cross-flow filtration operating pressure is 0.2 MPa, temperature is 25℃, and cross-flow rate is 1.5 L / min / m. 2 The filtration time is 30 minutes; S3: Single-cell clone screening: Sf9 cells contaminated with Sf-RV were taken, digested with trypsin, and then the density was adjusted to 10 cells / mL with double pretreatment medium to obtain Sf9 single-cell suspension; the Sf9 single-cell suspension was seeded into 96-well plates, and 300 μL of double pretreatment medium was added to each well. The 50% double pretreatment medium was replaced every 3 days, and the cells were cultured statically at 27.5℃ for 18 days. Sf-RV in the cloned cells was detected by RT-PCR, and negative clones were screened and expanded to obtain rhabdovirus-free Sf9 cells.

[0025] Example 2: Screening method for Sf9 cells free of rhabdovirus using dual-preconditioned culture medium, the screening method comprising the following steps: S1: Preparation of original conditioned culture medium: ATCC CRL-1711 Sf9 cells were taken and cultured in suspension using SF-900 III SFM serum-free insect cell culture medium until the density reached 5 × 10⁶ cells / year. 6 Cells / mL, centrifuged at 1000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the original conditioned medium; S2: Dual pretreatment process: The original conditioned medium was cross-flow filtered using a composite filter to trap rhabdoviruses and retain small molecule nutrients. The filtrate was collected. β-propiolactone was added to the filtrate to a final concentration of 0.3 v / v%, inactivated at 25°C for 24 h, and hydrolyzed at 37°C for 2 h. HPLC analysis showed that the β-propiolactone residue was <0.1 μg / mL, thus obtaining the dual pretreatment conditioned medium. The composite filter is composed of a 35nm polyethersulfone filter membrane and a modified nanocellulose base membrane, with a pore size of 22nm; the modified nanocellulose base membrane has a polydopamine coating of 70nm and a tannic acid-silica coating of 40nm. The cross-flow filtration parameters are as follows: cross-flow filtration operating pressure is 0.3 MPa, temperature is 30℃, and cross-flow rate is 2 L / min / m. 2 The filtration time is 40 minutes; S3: Single-cell clone screening: Sf9 cells contaminated with Sf-RV were taken, digested with trypsin, and then the density was adjusted to 10 cells / mL with double pretreatment medium to obtain Sf9 single-cell suspension; the Sf9 single-cell suspension was seeded into 96-well plates, and 300 μL of double pretreatment medium was added to each well. The 50% double pretreatment medium was replaced every 3 days, and the cells were cultured statically at 27.5℃ for 18 days. Sf-RV in the cloned cells was detected by RT-PCR, and negative clones were screened and expanded to obtain rhabdovirus-free Sf9 cells.

[0026] Example 3: Screening method for Sf9 cells free of rhabdovirus using dual-pretreated conditioned medium, the screening method comprising the following steps: S1: Preparation of original conditioned medium: ATCC CRL-1711 Sf9 cells were taken and cultured in suspension using SF-900 III SFM serum-free insect cell medium until the density reached 5 × 10⁶ cells / year. 6 Cells / mL, centrifuged at 1000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the original conditioned medium; S2: Dual pretreatment process: The original conditioned medium was cross-flow filtered using a 35nm polyethersulfone filter membrane to trap rhabdoviruses and retain small molecule nutrients. The filtrate was collected. β-propiolactone was added to the filtrate to a final concentration of 0.3 v / v%, inactivated at 25℃ for 24 h, and hydrolyzed at 37℃ for 2 h. HPLC analysis showed that the β-propiolactone residue was <0.1 μg / mL, thus obtaining the dual pretreated conditioned medium. The cross-flow filtration parameters are as follows: cross-flow filtration operating pressure is 0.2 MPa, temperature is 25℃, and cross-flow rate is 1.5 L / min / m. 2 The filtration time is 30 minutes; S3: Single-cell clone screening: Sf9 cells contaminated with Sf-RV were taken, digested with trypsin, and then the density was adjusted to 10 cells / mL with double pretreatment medium to obtain Sf9 single-cell suspension; the Sf9 single-cell suspension was seeded into 96-well plates, and 300 μL of double pretreatment medium was added to each well. The 50% double pretreatment medium was replaced every 3 days, and the cells were cultured statically at 27.5℃ for 18 days. Sf-RV in the cloned cells was detected by RT-PCR, and negative clones were screened and expanded to obtain rhabdovirus-free Sf9 cells.

[0027] Comparative Example 1: A method for screening Sf9 cells free of rhabdovirus in a dual-preconditioned culture medium, the screening method comprising the following steps: S1: Preparation of original conditioning medium: ATCC CRL-1711 Sf9 cells were taken and cultured in suspension in SF-900 III SFM serum-free insect cell culture medium until the density reached 5 × 10⁶ cells / year. 6 Cells / mL, centrifuged at 1000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the original conditioned medium; S2: Single-cell clone screening: Sf9 cells contaminated with Sf-RV were taken, digested with trypsin, and then the density was adjusted to 10 cells / mL with the original conditioned medium to obtain Sf9 single-cell suspension; the Sf9 single-cell suspension was seeded into 96-well plates, and 300 μL of double preconditioning medium was added to each well. The 50% double preconditioning medium was replaced every 3 days, and the cells were cultured statically at 27.5℃ for 18 days. Sf-RV in the cloned cells was detected by RT-PCR, and negative clones were screened and expanded to obtain rhabdovirus-free Sf9 cells.

[0028] Comparative Example 2: Screening method for Sf9 cells free of rhabdovirus using dual-pretreated conditioned medium, the screening method comprising the following steps: S1: Preparation of original conditioned medium: ATCC CRL-1711 Sf9 cells were taken and cultured in suspension using SF-900 III SFM serum-free insect cell medium until the density reached 5 × 10⁶ cells / year. 6 Cells / mL, centrifuged at 1000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the original conditioned medium; S2: Dual pretreatment process: The original conditioned medium is cross-flow filtered using a composite filter to intercept the rhomboid virus, retain small molecule nutrients, and collect the filtrate to obtain the dual pretreated conditioned medium. The composite filter consists of a 35nm polyethersulfone filter membrane and a modified nanocellulose base membrane, with a pore size of 25nm; the modified nanocellulose base membrane has a 50nm polydopamine coating and a 30nm tannic acid-silicon coating. The cross-flow filtration parameters are as follows: cross-flow filtration operating pressure is 0.2 MPa, temperature is 25℃, and cross-flow rate is 1.5 L / min / m. 2 The filtration time is 30 minutes; S3: Single-cell clone screening: Sf9 cells contaminated with Sf-RV were taken, digested with trypsin, and then the density was adjusted to 10 cells / mL with double pretreatment medium to obtain Sf9 single-cell suspension; the Sf9 single-cell suspension was seeded into 96-well plates, and 300 μL of double pretreatment medium was added to each well. The 50% double pretreatment medium was replaced every 3 days, and the cells were cultured statically at 27.5℃ for 18 days. Sf-RV in the cloned cells was detected by RT-PCR, and negative clones were screened and expanded to obtain rhabdovirus-free Sf9 cells.

[0029] Comparative Example 3: Screening method for Sf9 cells free of rhabdovirus using dual-pretreated conditioned medium, the screening method comprising the following steps: S1: Preparation of original conditioned medium: ATCC CRL-1711 Sf9 cells were taken and cultured in suspension using SF-900 III SFM serum-free insect cell medium until the density reached 5 × 10⁶ cells / year. 6Cells / mL, centrifuged at 1000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the original conditioned medium; S2: Dual pretreatment process: β-propiolactone was added to the original conditioned medium to a final concentration of 0.3 v / v%, inactivated at 25℃ for 24 h, hydrolyzed at 37℃ for 2 h, and the residual β-propiolactone was detected by HPLC to be <0.1 μg / mL, thus obtaining the dual pretreatment conditioned medium; S3: Single-cell clone screening: Sf9 cells contaminated with Sf-RV were taken, digested with trypsin, and then the density was adjusted to 10 cells / mL with double pretreatment medium to obtain Sf9 single-cell suspension; the Sf9 single-cell suspension was seeded into 96-well plates, and 300 μL of double pretreatment medium was added to each well. The 50% double pretreatment medium was replaced every 3 days, and the cells were cultured statically at 27.5℃ for 18 days. Sf-RV in the cloned cells was detected by RT-PCR, and negative clones were screened and expanded to obtain rhabdovirus-free Sf9 cells.

[0030] Comparative Example 4: Screening method for Sf9 cells free of rhabdovirus using dual-pretreated conditioned medium, the screening method comprising the following steps: S1: Preparation of original conditioned medium: ATCC CRL-1711 Sf9 cells were taken and cultured in suspension using SF-900 III SFM serum-free insect cell medium until the density reached 5 × 10⁶ cells / year. 6 Cells / mL, centrifuged at 1000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the original conditioned medium; S2: Dual pretreatment process: The original conditioned medium was cross-flow filtered using a 0.1 μm cellulose acetate monolayer filter membrane to trap rhabdoviruses and retain small molecule nutrients. The filtrate was collected. β-propiolactone was added to the filtrate to a final concentration of 0.3 v / v%, inactivated at 25 °C for 24 h, and hydrolyzed at 37 °C for 2 h. HPLC analysis showed that the β-propiolactone residue was <0.1 μg / mL, thus obtaining the dual pretreated conditioned medium. The cross-flow filtration parameters are as follows: cross-flow filtration operating pressure is 0.2 MPa, temperature is 25℃, and cross-flow rate is 1.5 L / min / m. 2 The filtration time is 30 minutes; S3: Single-cell clone screening: Sf9 cells contaminated with Sf-RV were taken, digested with trypsin, and then the density was adjusted to 10 cells / mL with double pretreatment medium to obtain Sf9 single-cell suspension; the Sf9 single-cell suspension was seeded into 96-well plates, and 300 μL of double pretreatment medium was added to each well. The 50% double pretreatment medium was replaced every 3 days, and the cells were cultured statically at 27.5℃ for 18 days. Sf-RV in the cloned cells was detected by RT-PCR, and negative clones were screened and expanded to obtain rhabdovirus-free Sf9 cells.

[0031] Comparative Example 5: Screening method for Sf9 cells free of rhabdovirus using dual-pretreated conditioned medium, the screening method comprising the following steps: S1: Preparation of original conditioned medium: ATCC CRL-1711 Sf9 cells were taken and cultured in suspension using SF-900 III SFM serum-free insect cell medium until the density reached 5 × 10⁶ cells / year. 6 Cells / mL, centrifuged at 1000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the original conditioned medium; S2: Dual pretreatment process: The original conditioned medium was cross-flow filtered using a composite filter to trap rhabdoviruses and retain small molecule nutrients. The filtrate was collected. β-propiolactone was added to the filtrate to a final concentration of 0.3 v / v%, inactivated at 25°C for 24 h, and hydrolyzed at 37°C for 2 h. HPLC analysis showed that the β-propiolactone residue was <0.1 μg / mL, thus obtaining the dual pretreatment conditioned medium. The composite filter consists of a 35nm polyethersulfone filter membrane and a modified nanocellulose base membrane, with a pore size of 35nm; the modified nanocellulose base membrane contains only a polydopamine coating of 80nm. The cross-flow filtration parameters are as follows: cross-flow filtration operating pressure is 0.2 MPa, temperature is 25℃, and cross-flow rate is 1.5 L / min / m. 2 The filtration time is 30 minutes; S3: Single-cell clone screening: Sf9 cells contaminated with Sf-RV were taken, digested with trypsin, and then the density was adjusted to 10 cells / mL with double pretreatment medium to obtain Sf9 single-cell suspension; the Sf9 single-cell suspension was seeded into 96-well plates, and 300 μL of double pretreatment medium was added to each well. The 50% double pretreatment medium was replaced every 3 days, and the cells were cultured statically at 27.5℃ for 18 days. Sf-RV in the cloned cells was detected by RT-PCR, and negative clones were screened and expanded to obtain rhabdovirus-free Sf9 cells.

[0032] Comparative Example 6: Screening method for Sf9 cells free of rhabdovirus using dual-pretreated conditioned medium, the screening method comprising the following steps: S1: Preparation of original conditioned medium: ATCC CRL-1711 Sf9 cells were taken and cultured in suspension using SF-900 III SFM serum-free insect cell medium until the density reached 5 × 10⁶ cells / year. 6 Cells / mL, centrifuged at 1000 rpm for 5 min, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the original conditioned medium; S2: Dual pretreatment process: The original conditioned medium was cross-flow filtered using a composite filter to trap rhabdoviruses and retain small molecule nutrients. The filtrate was collected. β-propiolactone was added to the filtrate to a final concentration of 1.0 v / v%, inactivated at 25°C for 24 h, and hydrolyzed at 37°C for 2 h. HPLC analysis showed that the β-propiolactone residue was <0.1 μg / mL, thus obtaining the dual pretreatment conditioned medium. The composite filter consists of a 35nm polyethersulfone filter membrane and a modified nanocellulose base membrane, with a pore size of 25nm; the modified nanocellulose base membrane has a 50nm polydopamine coating and a 30nm tannic acid-silicon coating. The cross-flow filtration parameters are as follows: cross-flow filtration operating pressure is 0.2 MPa, temperature is 25℃, and cross-flow rate is 1.5 L / min / m. 2 The filtration time is 30 minutes; S3: Single-cell clone screening: Sf9 cells contaminated with Sf-RV were taken, digested with trypsin, and then the density was adjusted to 10 cells / mL with double pretreatment medium to obtain Sf9 single-cell suspension; the Sf9 single-cell suspension was seeded into 96-well plates, and 300 μL of double pretreatment medium was added to each well. The 50% double pretreatment medium was replaced every 3 days, and the cells were cultured statically at 27.5℃ for 18 days. Sf-RV in the cloned cells was detected by RT-PCR, and negative clones were screened and expanded to obtain rhabdovirus-free Sf9 cells.

[0033] Cell line validation: The selected cell lines were passaged continuously (≥50 passages) to detect cell growth rate, viability, baculovirus infectivity and recombinant protein expression level, and to verify their stability and production performance.

[0034] Performance testing: Single-cell colony formation rate test: Colony formation rate (%) = (Number of wells with colonies / Total number of inoculated wells) × 100%.

[0035] The test results are shown in Table 1 below.

[0036] Table 1. Comparison of Sf9 cell single-cell colony formation rate under different pretreatment conditions.

[0037] Conclusion: The dual pretreatment culture medium used in this invention can promote the growth of single-cell clones. After 50 passages, the growth rate (doubling time 24±2 h) and baculovirus titer (TCID50 10) of this cell bank were significantly improved. 8.5 The expression levels of recombinant GFP (200 mg / L) and Sf9 were not significantly different from those of wild-type Sf9, and no Sf-RV was detected. It can be used in conjunction with existing single-cell cloning techniques such as flow cytometry and limiting dilution, and is suitable for the construction of large-scale virus-free cell banks.

[0038] Comparative Example 1 was prepared directly from the original culture medium after coarse filtration at 0.22 μm without any pretreatment. Without filtration and β-propiolactone treatment, the virus was not removed, resulting in a high risk of cell contamination. At the same time, the virus and inhibitors produced by cell metabolism were not removed, and the nutrients were not enriched or protected for activity, resulting in extremely low colony formation ability.

[0039] Comparative Example 2 uses a single cross-flow filtration system without β-propiolactone inactivation hydrolysis. Physical filtration can remove most viruses, but it cannot inactivate residual microviruses, resulting in viral contamination during the cloning process and affecting cell viability.

[0040] Comparative Example 3 was inactivated by β-propiolactone alone without cross-flow filtration. Although β-propiolactone can inactivate the virus, it cannot remove the virus particles themselves or cellular metabolic inhibitors. Furthermore, high concentrations of chemical reagents may damage the activity of nutrient factors.

[0041] Comparative Example 4 is a traditional filter membrane, a 0.1μm cellulose acetate monolayer filter membrane. The pore size is too large, resulting in incomplete virus interception. At the same time, the monolayer membrane suffers from severe non-specific adsorption, leading to significant loss of nutrient factors.

[0042] Comparative Example 5 had a single polydopamine coating, which resulted in uneven pore size control and virus leakage; moreover, the single coating lacked sufficient hydrophilicity, leading to a high loss of nutrient adsorption.

[0043] Comparative Example 6 contained a high concentration of β-propiolactone, which still left trace amounts of toxic residues even after hydrolysis. Furthermore, the high concentration may have damaged the structure of nutrient factors and affected cell growth.

[0044] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for screening Sf9 cells free of rhabdoviruses using a dual-pretreatment conditioned medium, characterized in that: Includes the following steps: S1: Preparation of original conditioned medium: Take Sf9 cells and suspend them in serum-free insect cell culture medium until the viability is >95%. Collect the supernatant, filter it coarsely, and obtain the original conditioned medium. S2: Dual pretreatment process: The original conditioned medium is subjected to cross-flow filtration, and the filtrate is collected; β-propiolactone is added to the filtrate, inactivated, and hydrolyzed to obtain the dual pretreated conditioned medium; The amount of β-propiolactone added was 0.1-0.5 v / v% at a final concentration. S3: Single-cell clone screening: Sf9 single-cell suspension was inoculated into culture plates containing double-pretreated medium, and Sf-RV negative clones were screened to obtain rhabdovirus-free Sf9 cells.

2. The method for screening Sf9 cells without rhabdovirus in a dual-pretreatment culture medium according to claim 1, characterized in that: The inactivation temperature is 24-26℃ and the time is 22-26h; the hydrolysis temperature is 36-38℃ and the time is 1-3h.

3. The method for screening Sf9 cells without rhabdovirus in a dual-pretreatment culture medium according to claim 1, characterized in that: In the coarse filtration process, a 0.22 μm pore size polyethersulfone filter membrane is used; in the cross-flow filtration process, a 35 nm pore size polyethersulfone filter membrane is used.

4. The method for screening Sf9 cells without rhabdovirus in a dual-pretreatment culture medium according to claim 3, characterized in that: In the cross-flow filtration process, a composite filter composed of a 35nm pore size polyethersulfone filter membrane and a modified nanocellulose base membrane is used. The method for preparing the modified nanocellulose-based membrane includes the following steps: Crystalline cellulose was added to sulfuric acid solution and heated to 45-50℃ for 60-90 min to hydrolyze the solution. After dialyzing to neutral, the solution was centrifuged and freeze-dried to obtain nanocellulose powder. The nanocellulose powder was added to deionized water, ultrasonically dispersed, vacuum filtered, and then dried under pressure at 80-85℃ for 2-3 h to obtain a nanocellulose-based membrane. Dopamine hydrochloride was added to Tris-HCl buffer and stirred until homogeneous to obtain a PDA solution. Tannic acid and 3-aminopropyltrimethoxysilane were added to deionized water, mixed, and then sodium periodate solution was added. The mixture was stirred thoroughly to obtain a composite solution. The nanocellulose-based membrane was immersed in the PDA solution and shaken at 25-30℃ for 1-2 hours. It was then removed and rinsed with deionized water until the filtrate was colorless to obtain a PDA-modified nanocellulose-based membrane. The PDA-modified nanocellulose-based membrane was immersed in the composite solution and reacted at 60-65℃ for 1-1.5 hours. It was then removed and vacuum dried at 50-55℃ to obtain a modified nanocellulose-based membrane.

5. The method for screening Sf9 cells without rhabdovirus in a dual-pretreatment culture medium according to claim 4, characterized in that: In the preparation of the nanocellulose-based membrane, the mass ratio of nanocellulose powder to deionized water is 1:(40-60); the thickness of the nanocellulose-based membrane is 30-50μm, and the pore size is 0.18-0.3μm.

6. The method for screening Sf9 cells without rhabdovirus in a dual-pretreatment culture medium according to claim 4, characterized in that: In the preparation of the PDA solution, the concentration of the PDA solution is 2.5-3 mg / mL, and the concentration of Tris-HCl is 45-50 mM; in the preparation of the composite solution, the mass ratio of tannic acid to 3-aminopropyltrimethoxysilane is 1:(1.2-1.5); and the concentration of the sodium periodate solution is 4-5 mg / mL.

7. The method for screening Sf9 cells without rhabdovirus in a dual-pretreatment culture medium according to claim 4, characterized in that: The surface of the PDA-modified nanocellulose base film contains a polydopamine coating with a thickness of 50-80 nm; a tannic acid-silicon coating with a thickness of 30-50 nm is also deposited on the polydopamine coating on the surface of the modified nanocellulose base film.

8. The method for screening Sf9 cells without rhabdovirus in a dual-pretreatment culture medium according to claim 4, characterized in that: The modified nanocellulose-based membrane has a pore size of 20-30 nm.

9. The method for screening Sf9 cells without rhabdovirus using a dual-pretreatment culture medium according to claim 1, characterized in that: The cross-flow filtration parameters are as follows: cross-flow filtration operating pressure is 0.1-0.3 MPa, temperature is 25-30℃, and cross-flow rate is 1.5-2 L / min / m. 2 The filtration time is 30-60 minutes.

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