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Cell Cultures in Virology

For medical students3 min readUpdated 2026-10-10

Cell culture is a foundational modern tool in virology. It allows human or animal tissue cells to be propagated outside the body in specialized artificial media, providing virtually unlimited opportunities for studying viruses.

DiscoveryThe method was developed in the 1950s by J. Enders and colleagues, for which they were awarded a Nobel Prize.

MediaComplex formulations such as Medium 199 or Eagle's minimum essential medium (MEM) are used, stabilized with buffer solutions.

LimitationsContinuous (immortalized) cell lines are strictly prohibited for vaccine production.

GrowthCells can grow as a monolayer on glass, as a suspension in liquid, or as whole tissue fragments (organ cultures).

Basic Principles and Culture Conditions

Embryonic or tumor (malignantly transformed) tissues are most frequently chosen for cell cultures. Their primary advantage over normal adult cells is a high capacity for active growth and division.

For a culture to grow successfully outside the organism, five key parameters must be strictly maintained:

Asepsis. Absolute sterility must be maintained at all stages of work.
Laboratory glassware. Only chemically neutral glass (culture flasks, tubes, bottles) or specialized industrial bioreactors are used.
Growth media. Cells require complex multicomponent mixtures. Medium 199 or Eagle's medium are commonly used, containing amino acids, vitamins, mineral salts, glucose, and serum. Buffer solutions are always added to stabilize the pH level.
Antibacterial protection. Antibiotics are added to the growth medium to suppress accidental contaminating microflora that could destroy the culture.
Temperature control. Incubation must be maintained strictly within the range of 36–38.5 °C.

Types of Cell Cultures by Growth Characteristics

Depending on tissue organization and how cells interact with the medium, three main forms are distinguished:

Monolayer cultures. Cells attach firmly to the surface of chemically neutral glass or plastic and multiply to form a continuous sheet exactly one cell thick. This is the most popular and frequently used method in practical virology.
Suspension cultures. Cells do not attach to surfaces; instead, they float freely and multiply throughout the volume of the liquid growth medium. Continuous agitation (using magnetic stirrers or rotating bottles) is required to ensure nutrient access and adequate aeration. This type is ideal for industrial biomass production, such as viral vaccine manufacturing.
Organ cultures. In this approach, whole fragments of tissues and organs are used rather than isolated cells. The main feature of this method is the preservation of the original tissue architecture. However, due to the complexity of maintaining viability, the application of this type is extremely limited.

Classification by Number of Generations (Passages)

Cell viability in the laboratory is assessed by the number of subcultivations (passages) they can withstand.

1. Primary (primary trypsinized) cultures These are derived from tumor, normal, or embryonic tissues. The starting material is treated with proteolytic enzymes (most commonly trypsin). The enzyme breaks down intercellular connections to yield a suspension of fully isolated cells. The main drawback of such cultures is their extremely limited lifespan: they survive only 5 to 10 passages.

2. Continuous (immortalized or established) cell lines Derived from tissues with extremely high growth potential—such as embryonic or tumor tissues. They can survive in the laboratory indefinitely (for decades) and withstand countless passages.

Advantages: cost-effectiveness, low labor input, high replication rate, ability to be stored frozen for long periods, and standardization (uniform international lines are used worldwide).
Disadvantages: cells are of malignant origin and accumulate somatic mutations during multiple passages. Because of their marked oncogenic potential, they are strictly prohibited for vaccine manufacturing.

3. Semi-continuous (finite / diploid) cell lines Occupy an intermediate position, most commonly derived from human embryonic tissues. They have greater viability than primary cultures (surviving 40–50 passages) but are not immortal. Their key genetic feature is the preservation of a normal diploid chromosome set typical of somatic cells, without malignant transformation. Due to their safety, they are widely used for both viral diagnostics and vaccine production.

Significance of the Method for Virology

The introduction of cell cultures was a truly revolutionary milestone in the development of science:

Identification. The method allowed scientists to isolate and describe a vast number of previously unknown viruses.
Specificity. Researchers gained the ability to precisely select the most sensitive cell lines for cultivating specific viral species.
Research depth. It enabled detailed investigation of the molecular interactions between viruses and living host cells.

Mnemonic

To remember the classification by passages: Primary (just starting, 5–10 passages, needs trypsin), Pi-semi (halfway there, 40–50 passages, safe for vaccines), Permanent/Continuous (outlive everything, endless passages, but tumor-derived and mutated).

Frequently asked questions

Which specific continuous cell lines (strains) are most commonly used in virological practice?

Sources mention the following specific continuous cell lines used for virus propagation:

HeLa — a continuous line noted for cultivating paramyxoviruses.
Vero — a continuous line noted for cultivating paramyxoviruses.
BHK-21 — a continuous line noted for cultivating togaviruses.
BHK-21 — hamster kidney cell culture, which clearly shows cytopathic effect (CPE) for flaviviruses.
SPEV — pig kidney cell culture, which clearly shows CPE for flaviviruses.

What methods (phenomena) are used to indicate viral replication in cell culture?

Viral replication in cell culture is indicated using the following specific phenomena:

Cytopathic effect (CPE) — detection of morphological changes or destruction of infected cells.
Intracellular inclusions — observation of viral particle aggregates or components in the cytoplasm or nucleus.
Plaque formation — detection of negative viral colonies ("sterile" spots) in the cell monolayer.
Hemadsorption — attachment of erythrocytes to the membranes of infected cells.
Hemagglutination — interaction of released viruses with erythrocytes in the culture fluid.
"Color" test — pH indicator color change in the growth medium due to metabolic shifts.

What are the main types of cytopathic effect (CPE) caused by viruses on a cell monolayer?

Sources describe the following manifestations of CPE and related signs of viral reproduction in cell culture:

Morphological cellular changes — cytopathic effect (CPE).
Cell destruction — CPE can manifest as the destruction of cells.
Syncytium formation — giant multinucleated cells; characteristic of paramyxoviruses due to F-protein activity.
Intracellular inclusion bodies — specific inclusions visible by microscopy; they can be cytoplasmic or nuclear.

The severity of CPE can vary: it is pronounced in togaviruses and mild in flaviviruses.

Why are continuous cell lines prohibited for vaccine production?

Continuous lines originate from malignant tissues and accumulate somatic mutations during endless passages. Consequently, they possess oncogenic potential and cannot be considered safe for humans.

Why is the enzyme trypsin used when preparing primary cultures?

Trypsin is a proteolytic enzyme that cleaves proteins. It is necessary to break down strong intercellular bonds in the initial tissue fragment to obtain a suspension of separated, isolated cells.

How do suspension cultures differ from monolayer cultures?

In monolayer cultures, cells multiply only after attaching to a surface in a single layer. In suspension cultures, cells remain suspended throughout the liquid medium, requiring constant agitation to supply them with oxygen and nutrients.

Go deeper

Detailed composition and purpose of Medium 199 and Eagle's medium components
Molecular mechanisms of viral interaction with susceptible host cells
Technologies for industrial viral vaccine production in bioreactors
Cryopreservation: protocols for long-term storage of frozen continuous cell lines