Cell Culture Techniques
Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37°C ,5% CO2 for mammalian cells) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed.
Cells can be grown in suspension or adherent cultures. Some cells naturally live in suspension, without being attached to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified to be able to survive in suspension cultures so that they can be grown to a higher density than adherent conditions would allow. Adherent cells require a surface, such as tissue culture plastic, which may be coated with extracellular matrix components to increase adhesion properties and provide other signals needed for growth and differentiation. Most cells derived from solid tissues are adherent. Another type of adherent culture is organotypic culture which involves growing cells in a three-dimensional environment as opposed to two-dimensional culture dishes. This 3D culture system is biochemically and physiologically more similar to in vivo tissue, but is technically challenging to maintain because of many factors (e.g. diffusion).
Suspension Cell Culture
Circulating blood cells or cells easily obtained by lavage such as peritoneal and alveolarmacrophages can normally survive in suspension culture when provided with a suitablemedium. Cells from organized solid organs or tissues must be separated fromtissue and, if possible, separated into cell types, before being suspended in such a medium. Cell association within organs depends on protein complex formation, which in turn is Ca 2 + dependent. Consequently dissociation media generally contain a proteolytic enzyme and the Ca 2 + chelator EDTA. Cells in suspension may be maintained for a limited period of time in definedmedia or for longer periods in nutrient, but less well-defined, media. In either case these cultures are often used for studies of xenobiotic metabolism.
Monolayer Cell Culture
Proliferation of most cells in culture requires attachment to a substrate and occurs until limited by cell-to-cell contact, resulting in the formation of a cellular monolayer. The substrate provided for attachment is usually polystyrene modified to carry a charge.The medium for continued maintenance and growth contains salts and glucose, usuallywith a bicarbonate buffer. Because of the bicarbonate buffering system these cultures are maintained in a 5–10% CO 2 atmosphere in a temperature and humidity controlled incubator. Many cells require serum for optimal growth, inducing considerable variability into the experimental system.
Indicators of Toxicity in Cultured Cells
Observation of cultured is usually carried out by phase contrast microscopy,utilizing the inverted phase contrast microscope. More recently, more detailed observations have become possible utilizing fluorescent tags and inverted fluorescent microscopes.
Toxicity to cultured cells may be the result either of inadequacies in the culture orthe toxicity effects of the chemical being investigated. Short-term toxicity is usually evaluated by examination of end points that indicate effects on cellular organelles such as leakage of cell constituents into the medium, uptake of dyes into the cell.