Best practices for media selection for mammalian cells

Abstract

Cell culture medium is a complex mixture of nutrients and growth factors that, along with the physical environment, can either help or destroy your experiment or production run. Nutritional requirements differ with different cell types and functions, as do optimal pH and osmolality. As cell growth proceeds, different cells will utilize amino acids and other components at different rates. By controlling for ammonia, free radicals, heavy metal toxicity, pH shifts, fluctuations in osmolality, nutrient depletion, and chemical and biological contaminants, you will optimize the chances of success. The contribution of each component of the medium is essential for the maintenance of the cell type of interest. While some cell types, such as established human cancer cell lines, may be quite able to tolerate a range of media and supplements, many normal cells and stems cells are not. Optimization of each component may be required to successfully maintain the latter cell types. The procedures for selecting and optimizing cell culture media and supplements are presented.

Appendix

Glossary of Terms (Schaeffer, WI 1990)

Adventitious: Agents which can contaminate cell cultures such as viruses and bacteria

Anchorage-dependent cells or cultures: Cells, or cultures derived from them, which will grow, survive, or maintain function only when attached to a surface such as glass or plastic. The use of this term does not imply that the cells are normal or that they are or are not neoplastically transformed.

Anchorage independent: Cells able to proliferate in suspension

Aneuploid: The situation which exists when the nucleus of a cell does not contain an exact multiple of the haploid number of chromosomes; one or more chromosomes being present in greater or lesser number than the rest. The chromosomes may or may not show rearrangements.

Cell line: A cell line arises from a primary culture at the time of the first successful subculture. The term cell line implies that cultures from it consist of lineages of cells originally present in the primary culture. The terms finite or continuous are used as prefixes if the status of the culture is known. If not, the term line will suffice. The term “continuous line” replaces the term “established line.” In any published description of a culture, one must make every attempt to publish the characterization or history of the culture. If such has already been published, a reference to the original publication must be made. In obtaining a culture from another laboratory, the proper designation of the culture, as originally named and described, must be maintained and any deviations in cultivation from the original must be reported in any publication.

Cell strain: A cell strain is derived either from a primary culture or a cell line by the selection or cloning of cells having specific properties or markers. In describing a cell strain, its specific features must be defined. The terms finite or continuous are to be used as prefixes if the status of the culture is known. If not, the term strain will suffice. In any published description of a cell strain, one must make every attempt to publish the characterization or history of the strain. If such has already been published, a reference to the original publication must be made. In obtaining a culture from another laboratory, the proper designation of the culture, as originally named and described, must be maintained and any deviations in cultivation from the original must be reported in any publication.

Chemically defined medium: A nutritive solution for culturing cells in which each component is of known chemical structure.

Clone: In animal cell culture terminology, a population of cells derived from a single cell by mitoses. A clone is not necessarily homogeneous, and therefore, the terms clone and cloned do not indicate homogeneity in a cell population, genetic or otherwise.

Continuous cell culture: A culture which is apparently capable of an unlimited number of population doublings, often referred to as an immortal cell culture. Such cells may or may not express the characteristics of in vitro neoplastic or malignant transformation.

Cryopreservation: Ultra-low temperature storage of cells.

Differentiated: Cells that maintain, in culture, all or much of the specialized structure and function typical of the cell type in vivo.

Finite cell culture: A culture which is capable of only a limited number of population doublings after which the culture ceases proliferation.

Hybridoma: The cell which results from the fusion of an antibody producing tumor cell (myeloma) and an antigenically stimulated normal plasma cell. Such cells are constructed because they produce a single antibody directed against the antigen epitope which stimulated the plasma cell. This antibody is referred to as a monoclonal antibody.

In vitro transformation: A heritable change, occurring in cells in culture, either intrinsically or from treatment with chemical carcinogens, oncogenic viruses, irradiation, transfection with oncogenes, etc., and leading to the acquisition of altered morphological, antigenic, neoplastic, proliferative, or other properties. This expression is distinguished from “in vitro neoplastic transformation,” in that the alterations occurring in the cell population may not always include the ability of the cells to produce tumors in appropriate hosts. The type of transformation should always be specified in any description.

Mutant: A phenotypic variant resulting from a changed or new gene.

Passage: The transfer or transplantation of cells, with or without dilution, from one culture vessel to another. It is understood that any time cells are transferred from one vessel to another, a certain portion of the cells may be lost, and therefore, dilution of cells, whether deliberate or not, may occur. This term is synonymous with the term “subculture.”

Passage number: The number of times the cells in the culture have been subcultured or passaged. In descriptions of this process, the ratio or dilution of the cells should be stated so that the relative cultural age can be ascertained.

Plating efficiency: This is a term which originally encompassed the terms, “attachment (“seeding”) efficiency,” “cloning efficiency,” and “colony forming efficiency” and which is now better described by using one or more of them in its place as the term “plating” is not sufficiently descriptive of what is taking place.

Population density: The number of cells per unit area or volume of a culture vessel; also the number of cells per unit volume of medium in a suspension culture.

Population doubling level: The total number of population doublings of a cell line or strain since its initiation in vitro. A formula to use for the calculation of “population doublings” in a single passage is number of population doublings = log (N/N _o ) × 3.33, where N = number of cells in the growth vessel at the end of a period of growth and N _o  = number of cells plated in the growth vessel. It is best to use the number of viable cells or number of attached cells for this determination. Population doubling level is synonymous with “cumulative population doublings.”

Population doubling time: The interval, calculated during the logarithmic phase of growth in which, for example, 1.0 × 10⁶ cells increase to 2.0 × 10⁶ cells. This term is not synonymous with “cell generation time.”

Primary culture: A culture started from cells, tissues, or organs taken directly from organisms. A primary culture may be regarded as such until it is successfully subcultured for the first time. It then becomes a “cell line.”

Saturation density: The maximum cell number attainable, under specified culture conditions, in a culture vessel. This term is usually expressed as the number of cells per square centimeter in a monolayer culture or the number of cell per cubic centimeter in a suspension culture.

Suspension culture: A type of culture in which cells, or aggregates of cells, multiply while suspended in liquid medium.

Undifferentiated: With animal cells, this is the state wherein the cell in culture lacks the specialized structure and/or function of the cell type in vivo.