Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be subcultured (i.e., passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth.
Introduction to Cell Culture
What is Cell Culture?
Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established.
After the first subculture, the primary culture becomes known as a cell line or subclone. Cell lines derived from primary cultures have a limited life span (i.e., they are finite; see below), and as they are passaged, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population.
If a subpopulation of a cell line is positively selected from the culture by cloning or some other method, this cell line becomes a cell strain. A cell strain often acquires additional genetic changes subsequent to the initiation of the parent line.
Finite vs Continuous Cell Line
Normal cells usually divide only a limited number of times before losing their ability to proliferate, which is a genetically determined event known as senescence; these cell lines are known as finite. However, some cell lines become immortal through a process called transformation, which can occur spontaneously or can be chemically or virally induced. When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line.
Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured invariably consists of a suitable vessel containing the following:
- a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals)
- growth factors
- gases (O2, CO2)
- a regulated physico-chemical environment (pH, osmotic pressure, temperature)
Most cells are anchorage-dependent and must be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture), while others can be grown floating in the culture medium (suspension culture).
If a surplus of cells are available from subculturing, they should be treated with the appropriate protective agent (e.g., DMSO or glycerol) and stored at temperatures below –130°C (cryopreservation) until they are needed. For more information on subculturing and cryopreserving cells, refer to the Guidelines for Maintaining Cultured Cells.
Morphology of Cells in Culture
Cells in culture can be divided into three basic categories based on their shape and appearance (i.e., morphology).
Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate.
Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches.
Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface.
Applications of Cell Culture
Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis. It is also used in drug screening and development, and large scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins). The major advantage of using cell culture for any of these applications is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.