Basic principles and cultivation conditions
Embryonic or tumor (malignantly transformed) tissues are most often chosen for growing cultures. Their main advantage over normal adult cells is a high capacity for active growth and division.
For a culture to grow successfully outside the organism, strict adherence to five key parameters is required:
- Asepsis. Absolute maintenance of sterility at all stages of work.
- Laboratory glassware. Exclusively chemically neutral glass (flat-bottomed flasks, test tubes, culture bottles) or specialized industrial bioreactors are used.
- Nutrient media. Cells require complex multicomponent mixtures. Medium 199 or Eagle's medium is commonly used. Their composition includes amino acids, vitamins, mineral salts, glucose, and blood serum. Buffer solutions are mandatory to stabilize the pH level.
- Antibacterial protection. Antibiotics are added to the nutrient medium. This is necessary to suppress the growth of accidental extraneous microflora that could ruin the culture.
- Temperature regime. Incubation in a thermostat is carried out strictly within the range of 36–38.5 °C.
Types of cell cultures by growth pattern
Depending on tissue organization and the nature of cell-medium interaction, three main forms are distinguished:
- Monolayer cultures. Cells tightly attach to the surface of chemically neutral glass and proliferate, forming a continuous layer exactly one cell thick. This is the most demanded and frequently used method in practical virology.
- Suspension cultures. Cells do not attach to the walls, but freely float and multiply throughout the entire volume of the liquid nutrient medium. Constant agitation of the liquid (using magnetic stirrers or rotating drums) is required to ensure access to nutrition and adequate aeration. This type is ideal for industrial biomass production, for example, in the manufacture of viral vaccines.
- Organ cultures. In this case, whole fragments of tissues and organs are used instead of individual cells. The main feature of the 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 of cultures by number of generations (passages)
Cell viability in the laboratory is assessed by the number of possible subcultures they are able to withstand.
1. Primary (primary-trypsinized) cultures They are derived from tumor, normal, or embryonic tissues. To do this, the starting material is treated with proteolytic enzymes (most often trypsin). The enzyme destroys intercellular connections and makes it possible to obtain a suspension of completely isolated cells. The main disadvantage of such cultures is their extremely limited viability: they withstand only 5 to 10 passages (generations).
2. Continuous (established) cultures Originate from tissues with an extremely high growth potential — embryonic or tumor tissues. They are capable of living in the laboratory indefinitely (decades) and withstanding countless subcultures.
- Advantages: cost-effectiveness, low labor intensity, high proliferation rate, ability to be stored frozen for long periods, and standardization of processes (uniform international lines are used worldwide).
- Disadvantages: cells have a malignant character and accumulate somatic mutations during multiple passages. Due to their pronounced oncogenic potential, they are strictly prohibited for use in vaccine production.
3. Semi-continuous cultures Occupy an intermediate position. Most often, they are obtained from human embryonic tissues. They have greater viability than primary ones (withstanding 40–50 passages), but are not immortal. Their main genetic feature is the preservation of the diploid set of chromosomes typical of normal somatic cells, and the absence of malignant transformation. Due to their safety, they are widely used for both diagnostics and vaccine production.
Significance of the method for virology
The introduction of cell cultures became a truly revolutionary stage in the development of science:
- Identification. The method allowed scientists to isolate and describe a huge number of viruses previously unknown to science.
- Specificity. Researchers gained the ability to precisely select the most sensitive cell lines for cultivating specific types of viruses.
- Depth of research. It became possible to study in detail the processes of interaction between the virus and the living cell at a fine molecular level.
Briefly: answers to topical questions
Which specific continuous cell lines (strains) are most commonly used in virological practice?
The sources mention the following specific continuous cell lines used in virus cultivation:
- HeLa — a continuous line indicated for the cultivation of paramyxoviruses.
- Vero — a continuous line indicated for the cultivation of paramyxoviruses.
- BHK-21 — a continuous line indicated for the cultivation of togaviruses.
- BHK-21 — a hamster kidney cell culture in which the cytopathic effect (CPE) of flaviviruses is clearly manifested.
- SPEV — a pig kidney cell culture in which the CPE of flaviviruses is clearly manifested.
What methods (phenomena) are used to indicate virus propagation in cell culture?
Indication of virus multiplication in cell culture is carried out using the following specific phenomena:
- Cytopathic effect — detection of morphological changes or destruction of infected cells.
- Formation of intracellular inclusions — detection of accumulations of viral particles or components in the cytoplasm and nucleus.
- Plaque formation — detection of negative virus colonies ("sterile" spots) in the cell layer.
- Hemadsorption — attachment of erythrocytes to the membranes of infected cells.
- Hemagglutination — interaction of viruses released from cells with erythrocytes in the culture fluid.
- "Color" reaction — change in the color of the nutrient medium indicator due to a pH shift.
What are the main types of cytopathic effect (CPE) of viruses on a cell monolayer?
The sources do not provide a complete classification of the types of viral CPE on a cell monolayer. The following manifestations of CPE and related signs of virus reproduction in cell culture are described:
- Morphological changes in cells — cytopathic effect (CPE).
- Cell destruction — CPE may manifest as cell destruction.
- Formation of symplasts — giant multinucleated cells; characteristic of paramyxoviruses due to the activity of the F protein.
- Formation of intracellular inclusions — specific inclusions visible by microscopy; they can be cytoplasmic or intranuclear.
The severity of CPE can vary: it is pronounced in togaviruses and mildly pronounced in flaviviruses.
To memorize the classification by passages (number of subcultures): Primary (just started, 5–10 passages, trypsin needed), Semi-continuous (halfway, 40–50 passages, safe for vaccines), Continuous (will outlive everyone, infinite passages, but tumor-derived and mutated).
Class and exam questions
Why is the use of continuous cell lines prohibited for vaccine production?
Continuous lines originate from malignant tissues and accumulate somatic mutations during endless passages. Because of this, they possess oncogenic potential and cannot be considered safe for humans.
Why is the enzyme trypsin used when obtaining primary cell cultures?
Trypsin is a proteolytic enzyme that breaks down proteins. It is necessary for the destruction of strong intercellular connections in the initial tissue fragment to obtain a suspension of separate, isolated cells.
How do suspension cultures differ from monolayer cultures?
In monolayer cultures, cells multiply only after attaching to the glass surface in a single layer. In suspension cultures, cells are in a suspended state throughout the entire volume of the liquid medium, which requires constant agitation to provide them with oxygen and nutrition.
What else to study on this topic
- Detailed composition and purpose of the components of Medium 199 and Eagle's medium
- Molecular mechanisms of virus interaction with a susceptible cell
- Technologies for industrial production of viral vaccines in bioreactors
- Cryopreservation: rules for long-term storage of continuous cell lines in a frozen state
Study the full topic in the «Microbiology» course
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