Introduction

Animal cell cultures

Depending on their origin, animal cells grow either as an adherent monolayer or in suspension.

Adherent cells are anchorage-dependent and propagate as a monolayer attached to the cell culture vessel. This attachment is essential for proliferation — many adherent cell cultures will cease proliferating once they become confluent (i.e., when they completely cover the surface of cell culture vessel), and some will die if they are left in this confluent state for too long. Most cells derived from tissues are anchorage-dependent.

Suspension cells can survive and proliferate without being attached to a substratum. Hematopoietic cells (derived from blood, spleen, or bone marrow) as well as some transformed cell lines and cells derived from malignant tumors can be grown in suspension.

Primary cells, finite cultures, and continuous cell lines differ in their proliferative potential (see below). Different cell types vary greatly with respect to their growth behavior and nutritional requirements. Optimization of cell culture conditions is necessary to ensure that cells are healthy and in optimal condition for downstream applications.

Extensive information on culturing cells can be found in reference 1.

Primary cell cultures come from the outgrowth of migrating cells from a piece of tissue or from tissue that is disaggregated by enzymatic, chemical, or mechanical methods. Primary cultures are formed from cells that survive the disaggregation process, attach to the cell culture vessel (or survive in suspension), and proliferate.

Primary cells are morphologically similar to the parent tissue. These cultures are capable of only a limited number of cell divisions, after which they enter a non-proliferative state called senescence and eventually die out. Adherent primary cells are particularly susceptible to contact inhibition, that is, they will stop growing when they have reached confluency. At lower cell densities, however, the normal phenotype can be maintained. Primary cell culture is generally more difficult than culture of continuous cell lines.

Primary cell cultures are sometimes preferred over continuous cell lines in experimental systems. Primary cells are considered by many researchers to be more physiologically similar to in vivo cells. In addition, cell lines cultured for extended periods of time can undergo phenotypic and genotypic changes that can lead to discrepancies when comparing results from different laboratories using the same cell line. Furthermore, many cell types are not available as continuous cell lines.

Finite cell cultures are formed after the first subculturing (passaging) of a primary cell culture. These cultures will proliferate for a limited number of cell divisions, after which they will senesce. The proliferative potential of some human finite cell cultures can be extended by introduction of viral transforming genes (e.g., the SV40 transforming-antigen genes). The phenotype of these cultures is intermediate between finite cultures and continuous cultures. The cells will proliferate for an extended time, but usually the culture will eventually cease dividing, similar to senescent primary cells. Use of such cells is sometimes easier than use of primary cell cultures, especially for generation of stably transfected clones.

Finite cell cultures will eventually either die out or acquire a stable, heritable mutation that gives rise to a continuous cell line that is capable of unlimited proliferative potential. This alteration is commonly known as in vitro transformation or immortalization and frequently correlates with tumorigenicity.

Rodent primary cell cultures form continuous cell lines relatively easily, either spontaneously or following exposure to a mutagenic agent. In contrast, human primary cell cultures rarely, if ever, become immortal in this way and require additional genetic manipulation to form a continuous cell line. However, cell cultures derived from human tumors are often immortal.

Continuous cell lines are generally easier to work with than primary or finite cell cultures. However, it should be remembered that these cells have undergone genetic alterations and their behavior in vitro may not represent the in vivo situation.

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