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CN-121975719-A - Method for preparing HSV virus vector by amplification of domesticated low-serum Vero cell line and suspension culture cell

CN121975719ACN 121975719 ACN121975719 ACN 121975719ACN-121975719-A

Abstract

The invention relates to neurobiology, molecular virology, biomedical engineering and the like, and discloses a domesticated low-serum Vero cell line and a method for preparing HSV virus vectors by suspension culture cell expansion. Aiming at the bottleneck problem that the process of amplifying HSV by using the wall-attached culture Vero cells is difficult to realize large-scale and engineering amplification production, the invention researches and develops a method for preparing HSV based on suspension culture cell amplification. The introduction of Vero cells from ATCC was acclimatized, and the serum content of the medium was gradually reduced, making Vero cells suitable for suspension culture in microcarrier or pellet reactors using low serum or serum-free medium. Suspension culture of Vero cells and infection expansion of HSV viral vectors were performed using a dill bioengineering (Shanghai) limited T & J CloudReady TM bioreactor. The high-activity suspension culture and HSV amplification of Vero cells are realized by optimizing production conditions, cell density, biochemical detection and the like, and a stable and efficient method is provided for preparing the HSV virus vector with high titer and high purity by combining with purification process upgrading.

Inventors

  • WANG HUADONG
  • XU FUQIANG
  • Liu Tongtan

Assignees

  • 中国科学院深圳先进技术研究院

Dates

Publication Date
20260505
Application Date
20251222

Claims (10)

  1. 1. A low serum-acclimated Vero cell line, characterized in that the original strain is a standardized Vero cell line introduced from ATCC, said low serum-acclimated Vero cell line being acclimated to: (1) Culturing original Vero cells in MEM medium containing 10% fetal bovine serum, and recording as generation P1; (2) After 2 generations of continuous culture, the volume concentration of serum in the culture medium is reduced to 8%, and the volume concentration of serum is gradually reduced to 4%, so that P7 generation cells are obtained; (3) After 4 generations of cells of the P7 generation are cultured, the volume concentration of serum is reduced to 2 percent, and after 4 generations of cells are cultured, the volume concentration of serum is reduced to 1 percent, so that the Vero cells domesticated by the low serum of the P18 generation are obtained; The P18 generation cells can be cultured in suspension in a microcarrier or sheet-type carrier reactor in a low-serum or serum-free medium.
  2. 2. The application of the low-serum domesticated Vero cell line according to claim 1, wherein the P18 generation low-serum domesticated Vero cell is applied to the fields of virus vector amplification preparation, eukaryotic protein expression and vaccine production and preparation in a bioreactor suspension culture mode.
  3. 3. The low-serum domesticated Vero cell line according to claim 2, wherein the P18 generation low-serum domesticated Vero cells are well adapted to a suspension culture system based on a microcarrier bioreactor, and a corresponding Vero cell suspension culture method and parameters, and an HSV amplification preparation process method and parameters can be established by condition optimization; The P18 generation low serum domesticated Vero cells can be well adapted to a suspension culture system based on a basket bioreactor, and a corresponding Vero cell suspension culture method and parameters, and a HSV amplification preparation process method and parameters can be established through condition optimization.
  4. 4. The low serum domesticated Vero cell line of claim 3, wherein the P18 generation low serum domesticated Vero cells can efficiently amplify and produce herpes simplex virus vectors, or other viruses that can infect Vero cells and replicate.
  5. 5. A method for preparing HSV viral vectors using the Vero cell line of any one of claims 1-4, comprising the steps of: s1, inoculating P18 generation low-serum domesticated Vero cells into a bioreactor containing microcarriers or sheet carriers, and performing suspension culture in a low-serum or serum-free culture medium; S2, inoculating HSV virus for infection amplification when the cells grow to a target density on a carrier; S3, maintaining culture after infection, and collecting virus suspension; S4, purifying the virus suspension to obtain the HSV virus vector with high titer and high purity.
  6. 6. The method for preparing HSV virus vectors by using Vero cell line according to claim 5, wherein in step S1, the suspension culture is performed by perfusion culture.
  7. 7. The method for preparing HSV virus vectors by using Vero cell line according to claim 5, wherein in step S2, HSV virus is inoculated when cells are grown on the vectors to a density of 90% or more.
  8. 8. The method for preparing HSV virus vectors by using Vero cell line according to claim 5, wherein in step S1, the bioreactor is a microcarrier bioreactor, the culture parameters of the microcarrier bioreactor are that the initial working volume is 200 mL, the cell inoculation density is 5×10 5 cells/mL, the microcarrier concentration is 5 g/L, the stirring speed is 95-130 rpm, the temperature is 37 ℃, the DO value is 60%, the pH value is 7.2, and the air inlet mode is bottom aeration. Or in the step S1, the bioreactor is a basket bioreactor, the culture parameters of the basket bioreactor are that the working volume is 300 mL, the cell inoculation density is 6 multiplied by 10 5 cells/mL, the feeding amount of the flaky carrier is 6g, the surface area is 9000 cm 2 , the stirring speed is 250-300 rpm, the temperature is 37 ℃, the DO value is 60%, the pH value is 7.2, and the air inlet mode is bottom ventilation.
  9. 9. The method for preparing HSV virus vectors by using the Vero cell line according to claim 5, wherein in step S2, the multiplicity of infection of HSV virus is MOI=0.1, and the liquid change operation is performed before the infection.
  10. 10. The method for preparing HSV virus vector by Vero cell line according to claim 5, wherein in step S4, the purification procedure is that after thawing the virus suspension at 4 ℃, the virus suspension is centrifuged at 6500 Xg for 10min to remove cell debris, filtered by a 0.22 μm filter membrane, and the precipitate is collected by centrifugation for 3 hours at 50000 Xg, and resuspended in a buffer solution, and then centrifuged for 3 hours at 50000 Xg in a 20% sucrose solution to remove the supernatant, and the virus is resuspended in a sterile solvent and packaged for frozen storage at-80 ℃.

Description

Method for preparing HSV virus vector by amplification of domesticated low-serum Vero cell line and suspension culture cell Technical Field The invention belongs to the technical field of biology, relates to neurobiology, molecular virology, biomedical engineering and the like, and in particular relates to a method for preparing an HSV virus vector by amplifying a domesticated low-serum Vero cell line and suspension culture cells. Background In the neuroscience field, resolving complex and highly interconnected neural network structures is the basis for understanding brain function and disease mechanisms. One of the key technologies to achieve this goal is the cross-synaptic virus tracing technology that exploits the property of certain neurotropic viruses to enable cross-synaptic transmission between neurons, labeling and revealing direct connection pathways between neurons in a high-brightness, highly specific manner. Herpes simplex virus (Herpes Simplex Virus, HSV) has become one of the most important viral vector tools in life science and biomedical fields because of its remarkable advantages of wide host range, large exogenous gene carrying capacity (up to 30-150 kb), high infection efficiency (especially natural tropism to neuronal cells), and capability of forming long-term stable episomes within the nucleus of host cells without integration into genome. The virus particles of HSV-1 are spherical and have a diameter of about 150-200 nm a. From the outside to the inside, HSV-1 has the structure of an envelope (Envelop), a matrix (Tegument), a nucleocapsid (CAPSID SHELL), and a viral Core (Core) in that order. The outermost envelope is embedded with about 12 glycoproteins that play an important role in the viral recognition of the receptor, entry into the cell and budding. The matrix contains 26 proteins that play a critical role in the viral life cycle. The nucleocapsid is a nonstandard regular icosahedral structure composed of a variety of capsid proteins, which is internally enveloped by the genome of HSV-1 up to 152 kbp. The genome of HSV-1 is a linear double-stranded DNA structure, consisting of covalently linked Unique Long (UL) and Unique Short (US) fragments. The application value of HSV virus vectors mainly appears in three key directions: 1) HSV is one of the preferred carriers for nerve loop retrograde tracing, synapse-crossing tracing and whole brain map drawing, and provides an irreplaceable technical means for analyzing the structure and function of a complex nerve network. 2) Gene therapy HSV-based vector systems offer great potential in delivering therapeutic genes to treat genetic disorders, neurodegenerative disorders, and the like. 3) Oncolytic viral therapy-native or engineered HSV-1 (e.g., T-VEC) has been approved for clinical cancer treatment by selectively lysing tumor cells and eliciting an anti-tumor immune response. Despite the broad application prospect, the large-scale and standardized production of HSV vectors still restricts the bottleneck of basic research transformation and clinical application. Currently, laboratory scale HSV vector production typically relies on adherent cultured cell lines such as Vero cells (African green monkey kidney cells) or BHK-21 cells. The traditional process has obvious limitations that 1) the scale-up is difficult, the adherence culture mode depends on a cell factory or a roller bottle, the operation is complex, the occupied area is large, the labor force is intensive, and the consistency among batches is difficult to ensure. 2) The cost and risk are high, the traditional process usually needs high concentration serum, the cost is high, animal-derived components are introduced, and batch-to-batch differences and potential exogenous factor pollution risks are increased. 3) The process control is difficult, and parameters (such as pH, dissolved oxygen and nutrient substances) in the adherent culture system are not monitored and controlled as precisely as those of the suspension culture system, so that the process optimization and standardization are not facilitated. In order to solve the bottleneck, the large-scale, engineering and scalable GMP-level production of the HSV vector is realized, and the production process is changed from the traditional adherence culture to the suspension culture, so that the technology development direction of the industry is clear. Suspension culture is well established in the biopharmaceutical industry (e.g., vaccine, antibody production), and has the advantages of easy amplification, controllable process parameters, high production efficiency, more cost-effectiveness compliance, and the like. However, the process of amplifying HSV vectors by culturing Vero cells by adherence is difficult to scale and is difficult to engineer and amplify the bottleneck problem of production. Therefore, development of a Vero cell line which can adapt to low-serum suspension culture and a corresponding efficient culture process t