Tissue culture: a vital tool for studying viruses

Introduction

 

What is tissue culture and why is it important for virology?

 

Tissue culture is a method of growing animal cells in a laboratory setting using various media and conditions. This technique allows scientists to study the behavior, growth, and interactions of cells, as well as to isolate and identify viruses that infect them.

 

Virology is the branch of science that deals with viruses, which are microscopic agents that can only replicate inside living cells. Viruses can cause various diseases in humans, animals, and plants, such as influenza, AIDS, COVID-19, rabies, and polio. To understand how viruses work and how to prevent or treat viral infections, virologists need to isolate and characterize them using tissue culture methods.

 

One of the advantages of tissue culture for virology is that it enables the detection of viruses that are difficult or impossible to grow in other systems, such as eggs or animals. Tissue culture also allows the observation of viral replication and propagation in different types of cells, as well as the measurement of viral infectivity, titers, and antigenicity. Furthermore, tissue culture can be used to create reporter cell lines that express specific proteins or genes in response to viral infection, which can facilitate the diagnosis and identification of viruses.

 

The tissue culture of animal viruses involves two main steps: seeding and infection. 

Seeding refers to the process of preparing and maintaining a monolayer of cells in a flask or a plate using appropriate growth media and conditions. 

Infection refers to the process of adding a virus sample to the cell monolayer and incubating it for a certain period. 

The virus sample can be obtained from clinical specimens, such as blood, saliva, urine, or tissue biopsies, or from reference strains stored in laboratories.

The outcome of viral infection in tissue culture can be observed by different methods, such as microscopy, immunofluorescence, plaque assay, hemadsorption assay, cytopathic effect assay, or molecular techniques. Depending on the type and dose of virus and the susceptibility and permissivity of cells, the infection can result in visible changes in cell morphology or behavior (cytopathic effect), formation of clear zones of cell lysis (plaques), binding of red blood cells to infected cells (hemadsorption), expression of viral antigens or reporter genes on the cell surface or cytoplasm (immunofluorescence), or amplification and detection of viral nucleic acids (PCR).

 

Tissue culture is a valuable tool for virology research and diagnostics. It helps with the isolation and identification of new or emerging viruses, the characterization and comparison of viral strains, the development and evaluation of antiviral drugs and vaccines, and the investigation of viral pathogenesis and host response.

 

How tissue culture was developed and used to isolate and identify viruses

 

Tissue culture is a technique that involves growing cells or tissues in a laboratory environment under controlled conditions. Tissue culture has been widely used in various fields of biology, medicine, and biotechnology, but one of its most important applications is in virology, the study of viruses.

 

Viruses are microscopic agents that can infect living cells and cause diseases. However, unlike bacteria or fungi, viruses cannot grow or reproduce on their own. They need a host cell to provide them with the necessary materials and machinery to make more copies of themselves. This makes studying viruses challenging, as they cannot be easily observed or cultured in isolation.

 

Tissue culture was developed to overcome this challenge and provide a suitable environment for virus growth and identification. The history of tissue culture dates to the late 19th and early 20th centuries, when scientists began experimenting with different methods of keeping animal cells alive outside the body. One of the pioneers of tissue culture was Alexis Carrel, a French surgeon who received the Nobel Prize in 1912 for his work on vascular suturing and organ transplantation. Carrel developed a technique of maintaining chick embryo cells in a nutrient solution for several years, demonstrating that cells could survive and multiply indefinitely in vitro (in glass).

 

However, it was not until the 1930s that tissue culture was used to isolate and identify viruses. The first virus to be successfully cultured in tissue was the vaccinia virus, which causes cowpox and is related to the smallpox virus. In 1931, Ernest Goodpasture and Alice Woodruff at Vanderbilt University used chicken embryo membranes as a substrate for growing the vaccinia virus. They also showed that the virus could be transferred from one membrane to another, creating a serial passage method that allowed them to increase the virus yield and purity.

 

The breakthrough of tissue culture for virology came in 1949, when John Enders, Thomas Weller, and Frederick Robbins at Harvard University managed to grow poliovirus in human embryonic kidney cells. Poliovirus is the causative agent of poliomyelitis, a devastating disease that affects the nervous system and can cause paralysis or death. Before Enders and his colleagues, poliovirus could only be studied in live animals, such as monkeys or mice, which were expensive and difficult to handle. The discovery of tissue culture for poliovirus opened up new possibilities for research and vaccine development. Enders, Weller, and Robbins shared the Nobel Prize in 1954 for their achievement.

 

Since then, tissue culture has been used to isolate and identify many other viruses, such as measles, mumps, rubella, herpes, influenza, hepatitis, HIV, and SARS-CoV-2. Tissue culture has also enabled scientists to manipulate viruses genetically, study their structure and function, test their sensitivity to drugs and antibodies, and produce vaccines and antiviral agents. Tissue culture remains an indispensable tool for virology and public health today.

 

How tissue culture is performed and what types of cells and media are used

 

Tissue culture is a technique of growing cells in a laboratory under controlled conditions. It can be used for various purposes, such as studying cell biology, genetics, biochemistry, physiology, pathology, pharmacology, toxicology, and biotechnology. Tissue culture can also be used for producing vaccines, antibodies, hormones, enzymes, and other biologically active substances.

 

There are different types of cells that can be cultured in vitro, depending on the source and the purpose of the culture. Some examples are:

 

Primary cells: These are cells that are isolated directly from a living organism and have not been subcultured or passaged. Primary cells retain most of the characteristics of their original tissue and are useful for studying normal cell functions and responses. However, primary cells have a limited lifespan and may undergo senescence or transformation after a few passages.

Cell lines: These are cells that have been subcultured or passaged for many generations and have acquired the ability to grow indefinitely in culture. Cell lines may be derived from primary cells or from tumors. Cell lines are useful for studying specific cell functions and responses, as well as for producing large quantities of cells or products. However, cell lines may lose some of the characteristics of their original tissue and may undergo genetic and phenotypic changes over time.

Stem cells: These are cells that have the potential to differentiate into various cell types under certain conditions. Stem cells can be obtained from embryonic, fetal, or adult sources. Stem cells are useful for studying developmental biology, regenerative medicine, and gene therapy. However, stem cells may have ethical issues and technical challenges associated with their isolation and manipulation.

 

The choice of the culture medium is crucial for the growth and maintenance of the cells in vitro. The culture medium must provide all the necessary nutrients and environmental factors for the cells to survive and function properly. The culture medium may consist of:

 

• Biological components: These are substances that are derived from living sources, such as blood serum, tissue extract, or growth factors. Biological components provide various nutrients, hormones, cytokines, and other factors that support cell growth and differentiation. However, biological components may also introduce variability, contamination, or immunogenicity to the culture.
•  Synthetic components: These are substances that are chemically defined and synthesized in the laboratory, such as amino acids, glucose, vitamins, inorganic salts, and lipids. Synthetic components provide a more consistent and controllable composition of the culture medium. However, synthetic components may not provide all the factors that are required for some cell types or functions.

 

The culture medium may be either liquid or solid (agarose or gelatin), depending on the type of culture. Liquid media are used for suspension cultures or for submerging adherent cultures. Solid media are used for supporting adherent cultures or for inducing differentiation or organogenesis.

 

The culture medium must also have an appropriate pH and osmolarity for the cells to thrive. The pH is usually adjusted to 7.2–7.4 by adding buffers such as HEPES or bicarbonate. The osmolarity is usually adjusted to 280–320 mOsm by adding sodium chloride or other salts.

 

The culture medium must be sterilized before use to prevent microbial contamination. Sterilization methods include filtration, autoclaving, or irradiation.

 

The culture medium must be changed regularly to replenish nutrients and remove waste products. The frequency of medium change depends on the type and density of the cells, as well as on the volume and composition of the medium.

 

Tissue culture is a powerful tool for studying various aspects of cell biology and biotechnology. However, tissue culture also requires careful planning, preparation, execution, and analysis to ensure optimal results.

  

How tissue culture is used to study virus replication, pathogenesis, evolution, and vaccines

 

Tissue culture is a technique that involves growing cells in an artificial environment outside of their natural host. Tissue culture can be used to study various aspects of virus biology, such as replication, pathogenesis, evolution, and vaccine development.

 

Virus replication is the process by which viruses produce new copies of themselves inside host cells. Tissue culture can be used to measure the rate and efficiency of virus replication, as well as the factors that affect it, such as temperature, pH, and antiviral drugs. Tissue culture can also be used to isolate and purify virus particles for further analysis.

 

Pathogenesis is the mechanism by which viruses cause disease in their hosts. Tissue culture can be used to study how viruses interact with host cells and tissues, and how they induce cellular damage, inflammation, and immune responses. Tissue culture can also be used to model different types of infections, such as acute, chronic, latent, or persistent.

 

Evolution is the change in genetic characteristics of viruses over time. Tissue culture can be used to study how viruses adapt to different environmental conditions, such as host species, cell types, or immune pressures. Tissue culture can also be used to monitor the emergence and spread of new virus variants, such as mutations or recombinants.

 

Vaccine development is the process of creating safe and effective immunizations against viral diseases. Tissue culture can be used to test the efficacy and safety of potential vaccines, such as live attenuated, inactivated, subunit, or vector-based vaccines. Tissue culture can also be used to produce large quantities of vaccine antigens or vectors for mass immunization.

 

What are the limitations and difficulties of tissue culture for virology?

 

We have explained above that tissue culture is a widely used technique for cultivating and studying viruses in the laboratory that involves growing animal cells in flasks using various broth media and then infecting these cells with virus samples. Tissue culture can help with the detection, identification, and characterization of viruses, as well as the development of vaccines and antivirals.

However, tissue culture also has some limitations and difficulties that need to be considered. Some of these are:

1. Not all viruses can infect or replicate in cell cultures. Some viruses have specific host or tissue tropism, meaning they can only infect certain types of cells or organs. For example, the hepatitis B virus can only infect liver cells, while the rabies virus can only infect nerve cells. Therefore, cell cultures need to be carefully selected and matched with the virus of interest.
2. Cell cultures can lose their permissivity or susceptibility to viruses over time. Permissivity refers to the ability of a cell to support viral replication, while susceptibility refers to the ability of a cell to be infected by a virus. Cell cultures can lose these properties due to genetic or phenotypic changes, such as mutations, senescence, or differentiation. Therefore, cell cultures need to be regularly monitored and maintained to ensure their quality and functionality.
3. Cell cultures can be contaminated by other microorganisms or agents. Cell cultures can be accidentally exposed to bacteria, fungi, mycoplasma, endotoxins, or other viruses during handling or storage. These contaminants can interfere with viral growth, detection, or analysis, as well as cause harm to the cells or the researcher. Therefore, cell cultures need to be handled under sterile conditions and tested for contamination regularly.
4. Cell cultures can have ethical or safety issues. Cell cultures are derived from animal tissues, which may raise ethical concerns about animal welfare or rights. Some cell cultures are also derived from human tissues, which may raise ethical concerns about informed consent or privacy. Moreover, some viruses are highly pathogenic or infectious, which may pose safety risks to the researcher or the environment. Therefore, cell cultures need to be obtained from reputable sources and handled under appropriate biosafety levels.

 

These are some of the main limitations and difficulties of tissue culture for virology. However, tissue culture is still a valuable and indispensable tool for virology research and diagnostics. With proper care and precautions, tissue culture can provide reliable and informative results for studying viruses and their interactions with host cells.