CHAPTER 1

Cell Culture
INTRODUCTION

C ell biology traces its roots to the introduction of the concept of cells by Robert
Hooke in the second half of the 17th century. However, not until nearly halfway
through the 20th century were techniques for the culture of cells developed. In fact, 1998
marked the golden anniversary of the first continuous mammalian cell line. Cell culture
has become such an integral part of cell biology that it is somewhat difficult to imagine
the field in the B.C. (Before Culture) era. Cell culture also represents the primary way
in which cell biology reaches into related disciplines, since the maintenance and propa-
gation of cells has become an important component of biochemistry, biophysics, genetics,
immunology, physiology, molecular biology, and neuroscience. Accordingly, it is alto-
gether fitting that the first chapter of Current Protocols in Cell Biology should present
methods related to the culture of cells.
The immediate aim of cell culture is to maintain or expand a population of cells, and the
single most important consideration is cell viability. Determining the number of cells and
their viability is important in standardizing culture and experimental conditions. As viable
cells replicate in culture, passaging of the cells allows their number to be expanded to
meet experimental needs. The ability to freeze, store, and recover cells provides an
essential safeguard against losing a cell line to contamination, incubator malfunction, or
an error on the part of the investigator. In addition to preserving the cells, maintenance of
a frozen stock is desirable to avoid cellular senescence and genetic drift. Chapter 1
therefore begins with protocols for passaging cells, freezing and thawing cells, and
determining cell number and viability (UNIT 1.1).

Success in cell culture is highly dependent on the choice of a medium. At minimum, a

medium must provide the nutritional requirements of the cells as well as any required
growth factors, and maintain pH and osmolarity compatible with survival. The historical
development of a wide variety of culture media has influenced significantly the types of
cells that can be studied experimentally, since cell lines that proliferate in a particular
environment are always selected at the expense of those that do not. The second unit of
Chapter 1 therefore focuses on media used in culturing cells and provides descriptions of
standard, serum-free, and selective media, as well as the use of soft agar for anchorage-
independent growth (UNIT 1.2).
The next three units of this chapter deal with microbial contamination of cell cultures.
UNIT 1.3 describes basic aseptic techniques and the laminar flow hoods that are the main
weapons in the constant battle against contamination. UNIT 1.4 provides protocols related
to sterilization, namely filtration and heat sterilization (e.g., autoclaving), as well as the
use of disinfectants. UNIT 1.5 describes methods for detecting microbial contaminants
(bacteria, fungi, and mycoplasmas). While the best way to deal with such contamination
may well be to review the previous unit on autoclaving and faithfully apply its precepts,
situations do arise where an attempt to salvage a contaminated culture is warranted. UNIT
1.5 details the use of antibiotics for this purpose.

Of course there are cell biologists who do not see the growth of fungi as an annoying
contamination of their mammalian cell cultures but as a desirable goal. For scientists who
Cell Culture

Contributed by Joe B. Harford 1.0.1

Current Protocols in Cell Biology (2003) 1.0.1-1.0.2
Copyright 2003 by John Wiley & Sons, Inc. Supplement 19
 wish to propagate yeast, UNIT 1.6 provides recipes for media and descriptions of some basic
culture methodologies.

UNIT 1.7 represents the first unit of Chapter 1 dealing with culture of plants cells, specifically
the culture and transformation of BY-2 cells derived from tobacco. BY-2 cells have been
described as the HeLa cell of higher plants.

Future units in Chapter 1 will cover specialized systems for cell culture (e.g., cell cloning,
polarized cells, and three-dimensional cultures), as well as additional units on the
propagation of plant cells, cells from other so-called simpler organisms, and viruses.

For additional information on mammalian cell culture, readers are directed to Freshney
(1993).

LITERATURE CITED
Freshney, R.I. 1993. Culture of Animal Cells. A Manual of Basic Techniques, 3rd ed. Wiley-Liss, New York.

Joe B. Harford

Introduction

1.0.2
Supplement 19 Current Protocols in Cell Biology