32.2
Cell Culture
Most vertebrate cells grow in vitro attached to a substrate as a monolayer, called adherent cultures. The flasks and plates used to grow cells are che…
Cell culturing is a technique of growing eukaryotic cells in a laboratory setting.
It provides a model system to study the physiology of cells outside their natural environment.
When cells from a tissue sample are grown directly in culture medium, the resultant cell cultures are called primary cultures.
Such cells need artificially controlled conditions, to help them grow, such as correct cultureware, depending on if they are adherent or suspension cultures; an optimal temperature; a suitable growth medium that can provide essential nutrients, like amino acids, vitamins, salts, and growth factors; and a pH buffering system.
As the primary cell adherent culture multiplies, it gradually occupies the entire surface area of the culture dish.
At this stage, the cells must be treated with a solution of protease and chelating agent, such as Trypsin-EDTA, to loosen the cell-substrate and cell-cell interactions to obtain single cells.
A small number of these cells can then be subcultured into a new dish containing a fresh growth medium to facilitate continued growth. Such subcultured cells form secondary cultures.
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Q1: What is the difference between primary and secondary cell cultures?
Primary cultures are eukaryotic cells grown directly from tissue samples in culture medium. As primary adherent cultures multiply and occupy the entire dish surface, cells must be treated with protease and chelating agents like Trypsin-EDTA to obtain single cells. A small number of these cells are then subcultured into fresh medium, forming secondary cultures that continue growing.
Q2: What are the key components of a growth medium for mammalian cell culture?
Growth media contain amino acids, vitamins, inorganic salts, and glucose as a carbon source. Fetal bovine serum is added as a source of growth factors, hormones, lipids, and minerals. Antibiotics prevent microbial contamination, while buffering systems like sodium bicarbonate maintain pH at 7.4. A pH indicator such as phenol red shows pH changes in the culture.
Q3: How do adherent and suspension cultures differ in their growth requirements?
Adherent cultures grow as monolayers attached to chemically treated flasks and plates. Suspension cultures, such as hematopoietic cells, grow in non-treated cultureware and require magnetic stirrers or spinner flasks to agitate the media. Both culture types need optimal temperature, suitable growth medium, and pH buffering systems for proper development.
Q4: What role do plant growth hormones play in plant cell culture?
Plant growth hormones, specifically auxins and cytokinins, control tissue development in plant cell cultures. A balanced ratio of these hormones produces undifferentiated cells called callus. By altering the hormone ratio, the callus can be differentiated into roots or shoots, generating a complete plant from the explant tissue.
Q5: What conditions must be maintained in an incubator for optimal mammalian cell growth?
Mammalian cells require an optimal temperature of 37°C and a CO2 concentration of 5% in the incubator. The growth medium pH is maintained at 7.4 using buffering systems with sodium bicarbonate and exogenous CO2. These controlled conditions, combined with aseptic culturing protocols, ensure proper cell growth and prevent microbial contamination.
Q6: How are plant cell cultures used in agricultural and conservation applications?
Plant cell cultures produce improved hybrid plants and conserve endangered plant species. Explant cultures are also used in large-scale production of plant-derived products. Plant tissue, known as explant, is cultured in nutrient medium containing plant growth hormones, micronutrients, and a carbon source under controlled temperature, light intensity, and photoperiod conditions.
Q7: Why is Trypsin-EDTA treatment necessary when subculturing adherent cells?
Trypsin-EDTA is a protease and chelating agent solution that loosens cell-substrate and cell-cell interactions in adherent cultures. As primary cultures multiply and occupy the entire dish surface, this treatment is essential to obtain single cells for subculturation. The dissociated cells can then be transferred to fresh growth medium to establish secondary cultures.