Cheryl D. Helgason
Cindy L. Miller
METHODS IN MOLECULAR BIOLOGYTMTM
Basic Cell Culture Techniques
From: Methods in Molecular Biology, vol. 290: Basic Cell Culture Protocols, Third Edition
Edited by: C. D. Helgason and C. L. Miller © Humana Press Inc., Totowa, NJ
Culture of Primary Adherent Cells
and a Continuously Growing Nonadherent Cell Line
Cheryl D. Helgason
Cell culture is an invaluable tool for investigators in numerous fields. It facilitates
analysis of biological properties and processes that are not readily accessible at the level
of the intact organism. Successful maintenance of cells in culture, whether primary or
immortalized, requires knowledge and practice of a few essential techniques. The pur-
pose of this chapter is to explain the basic principles of cell culture using the mainte-
nance of a nonadherent cell line, the P815 mouse mastocytoma cell line, and the isolation
and culture of adherent primary mouse embryonic fibroblasts (MEFs) as examples.
Procedures for thawing, culture, determination of cell numbers and viability, and
cryopreservation are described.
Key Words: Cell culture; nonadherent cell line; adherent cells; P815; primary mouse
embryonic fibroblasts; MEF; hemocytometer; viability; subculturing; cryopreservation.
There are four basic requirements for successful cell culture. Each of these
will be briefly reviewed in this introduction. However, a more detailed
description is beyond the scope of this chapter. Instead, the reader is referred to
one of a number of valuable resources that provide the information necessary
to establish a tissue culture laboratory, as well as describe the basic principles
of sterile technique (1–4).
The first necessity is a well-established and properly equipped cell culture
facility. The level of biocontainment required (Levels 1–4) is dependent on the
type of cells cultured and the risk that these cells might contain, and transmit,