Question

Basic techniques for mammalian cell tissue culture proceducer typsin edta

Answer 1

Tissue culture technology has found wide application in the field of cell biology. Cell cultures are utilized in cytogenetic, biochemical, and molecular laboratories for diagnostic as well as research studies. In most cases, cells or tissues must be grown in culture for days or weeks to obtain sufficient numbers of cells for analysis. 

An important factor influencing the growth of cells in culture is the choice of tissue culture medium. Many different recipes for tissue culture media are available and each laboratory must determine which medium best suits their needs. Individual laboratories may elect to use commercially prepared medium or prepare their own. Commercially available medium can be obtained as a sterile and ready-to-use liquid, in a concentrated liquid form, or in a powdered form. Besides providing nutrients for growing cells, medium is generally supplemented with antibiotics, fungicides, or both to inhibit contamination.

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually cell death. The first step in subculturing monolayers is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth, fed periodically, and may be subsequently subcultured to produce tertiary cultures, etc. The time between passaging cells depends on the growth rate and varies with the cell line.

The Basic Protocol describes subculturing of a monolayer culture grown in petri plates or flasks; the Alternate Protocol 1 describes passaging of suspension cultures. Support Protocols describe freezing of monolayer cells, thawing and recovery of cells, counting cells using a hemacytometer, and preparing cells for transport. Alternate Protocol 2 describes freezing of suspension cells.
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