Animal cell culture and their use in virology

Mr. Parag Chauhan

Introduction

  1. Animal cell culture is the process of growing animal cells in vitro under controlled laboratory conditions.
  2. In virology, cell culture systems are indispensable for isolating, propagating, and studying viruses.
  3. Since viruses are obligate intracellular parasites, they cannot replicate without a host cell.
  4. Animal cell culture provides a controlled environment where viruses can grow, facilitating research in viral replication, pathogenicity, diagnostics, vaccine development, and drug screening.

Key Features of Animal Cell Culture in Virology:

  • Controlled environment for viral replication.
  • Replacement for live animals in research.
  • Widely used for vaccine production and diagnostic purposes.

 

Types of Cell Cultures in Virology

Primary Cell Cultures

  • Definition: Cells freshly isolated from animal tissues that retain many of the characteristics of the parent tissue.
  • Characteristics:
    • Finite lifespan: Cells divide for a limited number of passages before senescence.
    • Maintain in vivo-like physiological characteristics, making them suitable for virus-host interaction studies.
    • Require constant replenishment, as they cannot be subcultured indefinitely.
  • Examples:
    • Chick embryo cells: Used for influenza virus growth.
    • Human amnion or kidney cells: Common for isolating adenoviruses or polioviruses.
  • Advantages:
    • Closely mimic in vivo conditions.
    • Useful for studying viruses that are difficult to grow in continuous cell lines.
  • Disadvantages:
    • High variability between preparations.
    • Time- and labor-intensive.

Continuous Cell Lines

  • Definition: Immortalized cells that can grow indefinitely due to transformation or being derived from cancer cells.
  • Characteristics:
    • Homogeneous population of cells.
    • It can be passed multiple times without losing growth potential.
    • Easier to handle and grow compared to primary cells.
  • Examples:
    • Vero cells (African green monkey kidney cells): Widely used for SARS-CoV-2, measles, and rabies viruses.
    • HeLa cells: First immortalized human cell line; used for various viruses.
    • MDCK cells (Madin-Darby Canine Kidney cells): Preferred for influenza virus research.
  • Advantages:
    • Easily maintained and reproducible.
    • Cost-effective for large-scale virus production (e.g., vaccines).
  • Disadvantages:
    • Lack of in vivo-like characteristics.
    • It may not support the replication of all viruses.

Organ Cultures

  • Definition: Small fragments of specific organs (e.g., trachea, intestine) grown in vitro.
  • Characteristics:
    • Preserve tissue architecture and specialized functions.
    • Suitable for studying viruses with organ-specific tropism.
  • Examples:
    • Respiratory tract organ culture for influenza and respiratory syncytial virus.
  • Advantages:
    • Better mimicry of natural infection compared to monolayer cultures.
  • Disadvantages:
    • Technically challenging and less commonly used.

Explant Cultures

  • Definition: Tissue fragments maintained on solid or semi-solid media.
  • Applications in Virology:
    • Useful for studying latent virus infections (e.g., herpes simplex virus).

Suspension Cultures

  • Definition: Cells grown in liquid media without attaching to a surface.
  • Applications in Virology:
    • Used for large-scale production of viruses for vaccines (e.g., poliovirus in suspension HeLa cells).

 

Animal Cell Culture Techniques in Virology

 Cell Culture Systems

  1. Monolayer Culture:
    • Cells grow as a single layer on a flat surface (e.g., Petri dishes, flasks).
    • Applications: Most commonly used for virus isolation and propagation.
  2. Suspension Culture:
    • Cells grow freely in liquid media, ideal for non-adherent cell types.
    • Applications: Industrial-scale production of viruses.
  3. 3D Culture Systems:
    • Cells grow in three dimensions (e.g., on scaffolds or in hydrogels).
    • Better mimic natural tissue architecture for studying complex virus-host interactions.

Virus Infection Techniques

  1. Inoculation:
    • Viral suspension is added to cultured cells, allowing the virus to attach and enter cells.
    • Adsorption is carried out for a defined period before washing to remove unbound viruses.
  2. Plaque Assay:
    • A quantitative method to measure virus infectivity.
    • Infected cells are covered with an overlay medium (e.g., agar), restricting virus spread to adjacent cells.
    • Result: Clear zones (plaques) indicate sites of virus-induced cell lysis.
  3. TCID50 (Tissue Culture Infectious Dose):
    • Determines the dose of virus required to infect 50% of cultured cells.
  4. Cytopathic Effect (CPE) Observation:
    • Morphological changes in infected cells (e.g., rounding, fusion, detachment).
    • Used to identify and quantify viruses.

Culture Media

  • Nutritional Requirements:
    • Basal media like DMEM or RPMI supply essential nutrients (e.g., glucose, amino acids).
    • Supplements:
      • Serum (e.g., Fetal Bovine Serum, FBS): Provides growth factors and hormones.
      • Antibiotics: Prevent bacterial contamination.
      • Buffers: Maintain pH stability.

 

Use of Animal Cell Culture in Virology

In virology, animal cell cultures are used for several critical applications:

Virus Isolation and Propagation

Animal cell cultures are essential for growing viruses that require replicating living cells. Many viruses do not grow in media or require specific host cells to replicate. For example:

  • Influenza virus: Grown in MDCK cells (canine kidney cells).
  • Poliovirus: Grown in Vero cells (African green monkey kidney cells).
  • Herpes simplex virus (HSV): Grown in human diploid fibroblast cells (e.g., WI-38).

Process of Virus Isolation in Cell Culture:

  1. Inoculation: A suspected virus sample is introduced into a monolayer of animal cells.
  2. Incubation: The cells are incubated, allowing the virus to infect and replicate within the cells.
  3. Observation: After a specific period, cytopathic effects (CPE), such as cell lysis or rounding, are observed, indicating viral replication.

Virus Identification

Animal cell cultures are crucial for identifying viruses based on the effects they have on the cells or by using specific molecular or serological assays:

  1. Cytopathic Effects (CPE): Different viruses produce characteristic CPEs in cell cultures, such as:
    • Viral plaques: Clear areas where the virus has killed cells.
    • Syncytia formation: Giant multinucleated cells, typical of herpesviruses.
    • Cell rounding or detachment: Common with rhinoviruses.
  2. Molecular Techniques: Viral genome can be detected by PCR, RT-PCR, or in situ hybridization within infected cells.
  3. Serological Assays: Immunofluorescence, Western blotting, or enzyme-linked immunosorbent assays (ELISA) can detect viral proteins or antibodies produced in response to viral infection.

Vaccine Production

Animal cell cultures produce vaccines, particularly those involving live attenuated viruses or inactivated virus particles. This method offers several advantages over using whole animals for vaccine production.

  1. Poliovirus Vaccine: In Vero cells or human diploid cells, the virus is grown, inactivated, and purified to make the inactivated polio vaccine (IPV).
  2. Rabies Vaccine: In Madin-Darby canine kidney (MDCK) cells or Vero cells, the rabies virus is cultured to produce the rabies vaccine.
  3. Influenza Vaccine: A virus grown in MDCK cells prepares seasonal flu vaccines.

Using animal cell culture systems for vaccine production eliminates the need for infecting animals directly, making the process more efficient and ethical.

Research on Viral Pathogenesis

Animal cell cultures provide a model for how viruses interact with host cells. This includes:

  • Viral entry and uncoating: Understanding how viruses attach to host cell receptors and enter the cell.
  • Viral replication: Studying how viruses replicate their genomes and assemble new virions within the host cell.
  • Host immune response: Investigating how host cells respond to viral infection, including the production of interferons and other cytokines.

Cell culture models allow researchers to dissect the molecular mechanisms of viral diseases and assess therapeutic strategies to prevent or treat infections.

Antiviral Drug screening

Cell culture is an essential tool for testing the efficacy of antiviral agents. Researchers can assess which drugs effectively inhibit viral replication by inoculating cultured cells with a virus and then treating them with different compounds. Common antiviral drugs tested in cell cultures include:

  • Acyclovir (for herpesviruses)
  • Oseltamivir (Tamiflu) (for influenza)
  • Remdesivir (for Ebola and COVID-19)

This in vitro screening is a critical step in the drug development pipeline.

 

Advantages of Animal Cell Culture in Virology

  1. Controlled Environment: Cell cultures allow for precise control over the experimental conditions (temperature, pH, nutrients), making it easier to study viral infections under reproducible conditions.
  2. Ethical Considerations: Using cultured cells reduces the need for live animal experimentation, offering a more ethical alternative.
  3. High Yield: Cell cultures can produce large quantities of virus for research, diagnostic, and vaccine production purposes.
  4. Wide Range of Applications: Cell cultures can be used for viral studies, including vaccine development, virus-host interactions, and antiviral drug testing.

 

Limitations of Animal Cell Culture in Virology

  1. Not All Viruses Can Be Cultured: Some viruses, especially those that infect specific animal tissues or organisms, may not grow well in culture.
  2. Cell Line Specificity: Some viruses only infect specific cell types, which may not be available in cultured cell lines.
  3. Labor Intensive: Maintaining cell cultures and detecting viral infections requires technical expertise, time, and resources.
  4. Ethical Concerns: Despite reducing the use of live animals, animal-derived cell lines raise some ethical questions related to animal welfare.

 

Leave a Reply

Your email address will not be published. Required fields are marked *