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Introduction to Animal Cell Culture

Technical Bulletin


John A. Ryan, Ph.D. Introduction

Corning Incorporated
Cell culture has become one of the major tools
Life Sciences
used in the life sciences today. This guide is
45 Nagog Park
designed to serve as a basic introduction to animal
Acton, MA 01720
cell culture. It is appropriate for laboratory workers
who are using it for the first time, as well as for
Table of Contents those who interact with cell culture researchers
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 and who want a better understanding of the key
concepts and terminology in this interesting and
What is Cell and Tissue Culture? . . . . . . . . . . . 1 rapidly growing field.

How are Cell Cultures Obtained? . . . . . . . . . . . 2 What is Cell and Tissue Culture?
What Are Cultured Cells Like? . . . . . . . . . . . . . 3 Tissue Culture is the general term for the
removal of cells, tissues, or organs from an animal
What Are Some of the Problems Faced or plant and their subsequent placement into an
by Cultured Cells? . . . . . . . . . . . . . . . . . . . . . . . 4 artificial environment conducive to growth. This
How to Decide if Cultured Cells environment usually consists of a suitable glass or
Are “Happy”? . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 plastic culture vessel containing a liquid or semi-
solid medium that supplies the nutrients essential
What is Cell Culture Used For? . . . . . . . . . . . . 6 for survival and growth. The culture of whole
organs or intact organ fragments with the intent
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 of studying their continued function or develop-
ment is called Organ Culture. When the cells
are removed from the organ fragments prior to,
or during cultivation, thus disrupting their normal
relationships with neighboring cells, it is called
Cell Culture.
Although animal cell culture was first
successfully undertaken by Ross Harrison
in 1907, it was not until the late 1940’s tissue
to early 1950’s that several developments
occurred that made cell culture widely
Additional cell culture available as a tool for scientists. First, there Mince or
terminology and usage chop
was the development of antibiotics that
information can be
made it easier to avoid many of the con-
found on the Society
for In Vitro Biology tamination problems that plagued earlier
web site at cell culture attempts. Second was the development of the techniques, such as Digest with
terminology.asp. the use of trypsin to remove cells from enzymes
culture vessels, necessary to obtain con-
tinuously growing cell lines (such as
HeLa cells). Third, using these cell lines,
scientists were able to develop standard- Place in
ized, chemically defined culture media culture
that made it far easier to grow cells.
These three areas combined to allow
many more scientists to use cell, tissue Enzymatic Dissociation
and organ culture in their research.
During the 1960’s and 1970’s, commer-
cialization of this technology had further creates a suspension of single cells that
impact on cell culture that continues to are then placed into culture vessels con-
this day. Companies, such as Corning, taining culture medium and allowed to
Fixed and stained human began to develop and sell disposable plastic grow and divide. This method is called
foreskin explants on the sur- and glass cell culture products, improved Enzymatic Dissociation.
face of a 150 mm culture dish. filtration products and materials, liquid
The explants were cultured for
approximately two weeks. Two
and powdered tissue culture media, and Subculturing
of the nine explants (bottom laminar flow hoods. The overall result of When the cells in the primary culture
left and right corners) failed to these and other continuing technological vessel have grown and filled up all of the
grow. The remaining explants developments has been a widespread available culture substrate, they must be
show good growth. Each square increase in the number of laboratories
is approximately 2 cm across.
Subcultured to give them room for con-
and industries using cell culture today. tinued growth. This is usually done by
removing them as gently as possible from
How Are Cell Cultures Obtained? the substrate with enzymes. These are
Primary Culture similar to the enzymes used in obtaining
the primary culture and are used to break
When cells are surgically removed from
the protein bonds attaching the cells to the
an organism and placed into a suitable
substrate. Some cell lines can be harvested
culture environment, they will attach,
by gently scraping the cells off the bot-
divide and grow. This is called a Primary
tom of the culture vessel. Once released,
Culture. There are two basic methods for
the cell suspension can then be subdivid-
doing this. First, for Explant Cultures,
ed and placed into new culture vessels.
small pieces of tissue are attached to a
Primary culture from the fish glass or treated plastic culture vessel and Once a surplus of cells is available, they
Poeciliopsis lucida. Embryos bathed in culture medium. After a few can be treated with suitable cryoprotec-
were minced and dissociated days, individual cells will move from the tive agents, such as dimethylsulfoxide
with a trypsin solution. These tissue explant out onto the culture vessel (DMSO) or glycerol, carefully frozen and
cells were in culture for about then stored at cryogenic temperatures
surface or substrate where they will begin
1 week and have formed a
confluent monolayer. to divide and grow. The second, more (below -130°C) until they are needed.
widely used method, speeds up this pro- The theory and techniques for cryopre-
cess by adding digesting (proteolytic) serving cells are covered in the Corning
enzymes, such as trypsin or collagenase, Technical Bulletin: General Guide for
to the tissue fragments to dissolve the Cryogenically Storing Animal Cell
cement holding the cells together. This Cultures (Ref. 9).

Buying And Borrowing 2. In stationary culture vessels such as
An alternative to establishing cultures by T-flasks and bottles where, although
primary culture is to buy established cell the cells are not kept agitated, they are
cultures from organizations such as the unable to attach firmly to the substrate.
American Type Culture Collection (ATCC; Many cell lines, especially those derived or the Coriell Institute for from normal tissues, are considered to be
Medical Research ( Anchorage-Dependent, that is, they can
These two nonprofit organizations pro- only grow when attached to a suitable
vide high quality cell lines that are care- substrate.
fully tested to ensure the authenticity of
the cells. Some cell lines that are no longer consid-
Corning® cryogenic vials are ered normal (frequently designated as
available in a variety of sizes More frequently, researchers will obtain Transformed Cells) are frequently able
for freezing cells for long term (borrow) cell lines from other laboratories. to grow either attached to a substrate or
While this practice is widespread, it has floating free in suspension; they are
one major drawback. There is a high prob- Anchorage-Independent. In addition,
ability that the cells obtained in this man- some normal cells, such as those found in
ner will not be healthy, useful cultures. the blood, do not normally attach to sub-
This is usually due to previous mix-ups strates and always grow in suspension.
or contamination with other cell lines, or
the result of contamination with micro- Types of Cells
organisms such as mycoplasmas, bacteria, Cultured cells are usually described based
fungi or yeast. These problems are cov- on their morphology (shape and appear-
ered in detail in a Corning Technical ance) or their functional characteristics.
Corning culture dishes are
Bulletin: Understanding and Managing There are three basic morphologies:
available in a variety of sizes Cell Culture Contamination (Ref. 7).
1. Epithelial-like: cells that are attached
and shapes for growing
to a substrate and appear flattened and
anchorage-dependent cells. What Are Cultured Cells Like?
polygonal in shape.
Once in culture, cells exhibit a wide range 2. Lymphoblast-like: cells that do not
of behaviors, characteristics and shapes. attach normally to a substrate but
Some of the more common ones are remain in suspension with a spherical
described below. John Paul discusses shape.
these issues in detail in Chapter 3 of
Cell and Tissue Culture (Ref. 3). 3. Fibroblast-like: cells that are attached
to a substrate and appear elongated and
Cell Culture Systems bipolar, frequently forming swirls in
Two basic culture systems are used for heavy cultures.
growing cells. These are based primarily It is important to remember that the
Corning culture flasks are used
for growing anchorage-
upon the ability of the cells to either culture conditions play an important role
dependent cells. grow attached to a glass or treated plastic in determining shape and that many cell
substrate (Monolayer Culture Sytems) cultures are capable of exhibiting multiple
or floating free in the culture medium morphologies.
(Suspension Culture Systems).
Using cell fusion techniques, it is also
Monolayer cultures are usually grown possible to obtain hybrid cells by fusing
in tissue culture treated dishes, T-flasks, cells from two different parents. These
roller bottles, or multiple well plates, the may exhibit characteristics of either par-
choice being based on the number of cells ent or both parents. This technique was
needed, the nature of the culture environ- used in 1975 to create cells capable of
ment, cost and personal preference. producing custom tailored monoclonal
Suspension cultures are usually grown antibodies. These hybrid cells (called
Corning spinner vessels are either: Hybridomas) are formed by fusing two
used for growing anchorage- different but related cells. The first is a
independent cells in 1. In magnetically rotated spinner flasks spleen-derived lymphocyte that is capable
suspension. or shaken Erlenmeyer flasks where the of producing the desired antibody. The
cells are kept actively suspended in the second is a rapidly dividing myeloma cell
(a type of cancer cell) that has the machin- endotoxins, plasticizers, metal ions or
ery for making antibodies but is not pro- traces of chemical disinfectants, that are
grammed to produce any antibody. The invisible. The cell culture effects associat-
resulting hybridomas can produce large ed with endotoxins are covered in detail
quantities of the desired antibody. These in the Technical Bulletin: Endotoxins and
antibodies, called Monoclonal Antibodies Cell Culture (Ref. 10). Biological conta-
due to their purity, have many important minants in the form of fast growing yeast,
clinical, diagnostic, and industrial applica- bacteria and fungi usually have visible
Fibroblast-like 3T3 cells derived tions with a yearly value of well over a effects on the culture (changes in medium
from mouse embryos billion dollars. turbidity or pH) and thus are easier to
detect (especially if antibiotics are omit-
Functional Characteristics ted from the culture medium). However,
The characteristics of cultured cells result two other forms of biological contamina-
from both their origin (liver, heart, etc.) tion, mycoplasmas and viruses, are not
and how well they adapt to the culture easy to detect visually and usually require
conditions. Biochemical markers can be special detection methods.
used to determine if cells are still carrying
There are two major requirements to
on specialized functions that they per-
avoiding contamination. First, proper
formed in vivo (e.g., liver cells secreting
training in and use of good aseptic tech-
Epithelial-like cell line (Cl-9) albumin). Morphological or ultrastructur-
nique on the part of the cell culturist.
derived from rat liver. The al markers can also be examined (e.g.,
mitotic cells indicates this Second, properly designed, maintained
beating heart cells). Frequently, these
culture is actively growing. and sterilized equipment, plasticware,
characteristics are either lost or changed
glassware, and media. The careful and
as a result of being placed in an artificial
selective (limited) use of antibiotics
environment. Some cell lines will even-
designed for use in tissue culture can also
tually stop dividing and show signs of
help avoid culture loss due to biological
aging. These lines are called Finite. Other
contamination. These concepts are cov-
lines are, or become immortal; these can
ered in detail in a Corning Technical
continue to divide indefinitely and are
Bulletin: Understanding and Managing
called Continuous cell lines. When a
Cell Culture Contamination (Ref. 7).
“normal” finite cell line becomes immor-
tal, it has undergone a fundamental irre- Finding A “Happy” Environment
versible change or “transformation”. This
CHO-K1 cells – a widely used To cell culturists, a “happy” environment
can occur spontaneously or be brought
continuous (transformed) cell is one that does more than just allow cells
about intentionally using drugs, radiation
line derived from adult to survive in culture. Usually, it means an
Chinese hamster ovary tissue or viruses. Transformed Cells are usual- environment that, at the very least, allows
in 1957. ly easier and faster growing, may often cells to increase in number by undergoing
have extra or abnormal chromosomes and cell division (mitosis). Even better, when
frequently can be grown in suspension. conditions are just right, some cultured
Cells that have the normal number of cells will express their “happiness” with
chromosomes are called Diploid cells; their environment by carrying out impor-

those that have other than the normal

tant in vivo physiological or biochemical
number are Aneuploid. If the cells
functions, such as muscle contraction or
form tumors when they are injected
➞ the secretion of hormones and enzymes.
into animals, they are considered to To provide this environment, it is impor-
be Neoplastically Transformed.
Figure 10. Photomicrograph of tant to provide the cells with the appro-
a low level yeast infection in a priate temperature, a good substrate for
liver cell line (PLHC-1, ATCC # What Are Some of the Problems attachment, and the proper culture medi-
CRL-2406). Budding yeast cells Faced by Cultured Cells? um. Many of the issues and problems
can been seen in several areas
(arrows). At this low level of associated with keeping cells “happy”
Avoiding Contamination
contamination, no medium are covered in the Corning Technical
turbidity would be seen; Cell culture contamination is of two main Bulletin: General Guide for Identifying
however, in the absence of types: chemical and biological. Chemical and Correcting Common Cell Culture
antibiotics, the culture medium contamination is the most difficult to Growth and Attachment Problems
will probably become turbid detect since it is caused by agents, such as
within a day. (Ref. 8).

Temperature is usually set at the same carbohydrates. These allow the cells to
Basic environmental
point as the body temperature of the host build new proteins and other components
Requirements for
“Happy” Cells: from which the cells were obtained. With essential for growth and function as well
cold-blooded vertebrates, a temperature as providing the energy necessary for
◗ Controlled range of 18° to 25°C is suitable; most metabolism. For additional information
mammalian cells require 36° to 37°C. on this topic, see the article: Construction
◗ Good substrate for
This temperature range is usually main- of Tissue Culture Media by C. Waymouth
cell attachment
tained by use of carefully calibrated, and in Growth, Nutrition and Metabolism of
◗ Appropriate medium
and incubator that frequently checked, incubators. Cells in Culture, Volume 1, (1972; Ref. 5).
maintains the correct Anchorage-dependent cells also require a The growth factors and hormones help
pH and osmolality good substrate for attachment and growth. regulate and control the cells’ growth rate
Glass and specially treated plastics (to and functional characteristics. Instead of
make the normally hydrophobic plastic being added directly to the medium, they
surface hydrophilic or wettable) are the are often added in an undefined manner
most commonly used substrates. How- by adding 5 to 20% of various animal
ever, Attachment Factors, such as colla- sera to the medium. Unfortunately, the
gen, gelatin, fibronectin and laminin, can types and concentration of these factors
be used as substrate coatings to improve in serum vary considerably from batch to
growth and function of normal cells batch. This often results in problems
derived from brain, blood vessels, kidney, controlling growth and function. When
liver, skin, etc. Often normal anchorage- growing normal functional cells, sera are
dependent cells will also function better if often replaced by specific growth factors.
Corning® Transwell permeable they are grown on a permeable or porous The medium also controls the pH range
supports are used to study cell surface. This allows them to polarize (have of the culture and buffers the cells from
transport and migration. a top and bottom through which things abrupt changes in pH. Usually a CO2-
can enter and leave the cell) as they do in
bicarbonate based buffer or an organic
the body. Transwell® inserts are Corning
buffer, such as HEPES, is used to help
vessels with membrane-based permeable keep the medium pH in a range from 7.0
supports that allow these cells to develop to 7.4 depending on the type of cell being
polarity and acquire the ability to exhibit cultured. When using a CO2-bicarbonate
special functions such as transport. Many buffer, it is necessary to regulate the
specialized cells can only be truly “happy” amount of CO2 dissolved in the medium.
(function normally) when grown on a This is usually done using an incubator
porous substrate in serum-free medium with CO2 controls set to provide an
with the appropriate mixture of growth atmosphere with between 2% and 10%
and attachment factors. CO2 (for Earle’s salts-based buffers). How-
Cells can also be grown in suspension on ever, some media use a CO2-bicarbonate
beads made from glass, plastic, polyacry- buffer (for Hanks’ salts-based buffers)
Suspension and microcarrier lamide and cross-linked dextran mole- that requires no additional CO2, but it
cultures can be grown in glass cules. This technique has been used to must be used in a sealed vessel (not dishes
spinner vessels. enable anchorage-dependent cells to be or plates). For additional information
grown in suspension culture systems and on this topic, see the article: The Gaseous
is increasingly important for the manu- Environment of the Cell in Culture by
facture of cell-based biologicals. W.F. McLimans in Growth, Nutrition
and Metabolism of Cells in Culture (1972;
The culture medium is the most impor-
Ref. 5).
tant and complex factor to control in
making cells “happy”. Besides meeting Finally, the osmolality (osmotic pressure)
the basic nutritional requirement of the of the culture medium is important since
cells, the culture medium should also it helps regulate the flow of substances in
have any necessary growth factors, regu- and out of the cell. It is controlled by the
late the pH and osmolality, and provide addition or subtraction of salt in the cul-
CHO-K1 cells growing on a
microcarrier bead
essential gases (O2 and CO2). The ‘food’ ture medium. Evaporation of culture
portion of the culture medium consists media from open culture vessels (dishes,
of amino acids, vitamins, minerals, and etc.) will rapidly increase the osmolality

resulting in stressed, damaged or dead Plating Efficiency is a testing method
cells. For open (not sealed) culture sys- where small numbers of cells (20 to 200)
tems, incubators with high humidity are placed in a culture vessel and the num-
levels to reduce evaporation are essential. ber of colonies they form is measured.
For additional information, see article by The percentage of cells forming colonies
C. Waymouth: Osmolality of Mammalian is a measure of survival, while the colony
Blood and of Media for Culture of size is a measure of growth rate. This
Mammalian Cells (1970; Ref. 6). testing method is similar in application to
growth rate analysis but is more sensitive
How to Decide if Cultured Cells to small variations in culture conditions.
Are “Happy” The final characteristic, the Expression
Evaluating the general health or “happi- of Specialized Functions, is usually the
ness” of a culture is usually based on four most difficult to observe and measure.
important cell characteristics: morphology, Usually biochemical or immunological
Examining the morphology of growth rate, plating efficiency and expres- assays and tests are used. While cultured
these stained roller bottles sion of special functions. These same cells may grow very well in subobtimal
(containing MRC-5 human conditions, highly specialized functions
characteristics are also widely used in
fibroblasts) is a good way to
evaluating experimental results. usually require near perfect culture con-
check if the cells are “happy”.
The bottle on the left was ditions and are often quickly lost when
The Morphology or cell shape is the
rotated at too high a speed cells are placed in culture.
resulting in poor attachment
easiest to determine but is often the least
useful. While changes in morphology are
and growth and very What is Cell Culture Used For?
“unhappy” cells. frequently observed in cultures, it is often
difficult to relate these observations to Cell culture has become one of the major
the condition that caused them. It is also tools used in cell and molecular biology.
a very difficult characteristic to quantify Some of the important areas where cell
or to measure precisely. culture is currently playing a major role
are briefly described below:
Often, the first sign that something is
wrong with a culture occurs when the Model Systems
cells are microscopically examined and Cell cultures provide a good model sys-
poor or unusual patterns of cell attach- tem for studying 1) basic cell biology and
ment or growth are observed. When biochemistry, 2) the interactions between
problems are suspected, staining the cul- disease-causing agents and cells, 3) the
A colony of fixed and stained ture vessels with crystal violet or other
human fibroblast cells effects of drugs on cells, 4) the process
simple histological stains may show growth and triggers for aging, and 5) nutritional
patterns indicating a problem. These studies.
growth problems are discussed in detail
in the Corning Technical Bulletin: General Toxicity Testing
Guide for Identifying and Correcting Cultured cells are widely used alone or in
Common Cell Culture Growth and conjunction with animal tests to study the
Attachment Problems (Ref. 8). effects of new drugs, cosmetics and chem-
Cell counting and other methods for esti- icals on survival and growth in a wide
mating cell number, on the other hand, variety of cell types. Especially important
allow the determination of the Growth are liver- and kidney-derived cell cultures.
Rate, which is sensitive to major changes
HTS Transwell®-24 plates are
in the culture environment. This allows Cancer Research
used for toxicity testing and
the design of experiments to determine Since both normal cells and cancer cells
drug transport studies
which set of conditions (culture media, can be grown in culture, the basic differ-
substrate, serum, plasticware) is better ences between them can be closely studied.
for the cells, i.e., the conditions produc- In addition, it is possible, by the use of
ing the best growth rate. These same or chemicals, viruses and radiation, to con-
similar techniques can also be used to vert normal cultured cells to cancer causing
measure cell survival or death and are cells. Thus, the mechanisms that cause
often used for in vitro cytotoxicity assays. the change can be studied. Cultured

cancer cells also serve as a test system Genetic Engineering
to determine suitable drugs and methods The ability to transfect or reprogram
for selectively destroying types of cancer. cultured cells with new genetic material
(DNA and genes) has provided a major
tool to molecular biologists wishing to
One of the earliest and major uses of study the cellular effects of the expression
cell culture is the replication of viruses in of theses genes (new proteins). These tech-
cell cultures (in place of animals) for use niques can also be used to produce these
in vaccine production. Cell cultures are new proteins in large quantity in cultured
also widely used in the clinical detection cells for further study. Insect cells are
and isolation of viruses, as well as basic widely used as miniature cells factories to
Corning® roller bottles are research into how they grow and infect
widely used for producing express substantial quantities of proteins
viral vaccines.
organisms. that they manufacture after being infected
with genetically engineered baculoviruses.
Cell-Based Manufacturing
While cultured cells can be used to pro- Gene Therapy
duce many important products, three The ability to genetically engineer cells
areas are generating the most interest. has also led to their use for gene therapy.
The first is the large-scale production Cells can be removed from a patient lack-
of viruses for use in vaccine production. ing a functional gene and the missing or
These include vaccines for polio, rabies, damaged gene can then be replaced. The
chicken pox, hepatitis B and measles. cells can be grown for a while in culture
The Corning CellCube®
bioreactor system is ideal Second, is the large-scale production and then replaced into the patient. An
for mass production of of cells that have been genetically engi- alternative approach is to place the miss-
anchorage-dependent cells. neered to produce proteins that have ing gene into a viral vector and then
medicinal or commercial value. These “infect’’ the patient with the virus in the
include monoclonal antibodies, insulin, hope that the missing gene will then be
hormones, etc. Third, is the use of cells expressed in the patient’s cells.
as replacement tissues and organs. Arti-
ficial skin for use in treating burns and Drug Screening and Development
ulcers is the first commercially available Cell-based assays have become increas-
product. However, testing is underway ingly important for the pharmaceutical
on artificial organs such as pancreas, liver industry, not just for cytotoxicity testing
and kidney. A potential supply of replace- but also for high throughput screening
ment cells and tissues may come out of of compounds that may have potential
work currently being done with both use as drugs. Originally, these cell culture
Corning® Erlenmeyer flasks are
embryonic and adult stem cells. These tests were done in 96 well plates, but
often used for growing insect
cells in suspension. are cells that have the potential to differ- increasing use is now being made of
entiate into a variety of different cell 384 and 1536 well plates.
types. It is hoped that learning how to
control the development of these cells For additional product or technical
may offer new treatment approaches information, please visit our web site
for a wide variety of medical conditions. or call
1.800.492.1110. Outside the United
Genetic Counseling States, call 978.635.2200.
Amniocentesis, a diagnostic technique
that enables doctors to remove and cul-
1. Culture of Animal Cells, A Manual of
ture fetal cells from pregnant women, Basic Technique (1994) R. Ian Freshney,
Corning microplates are widely has given doctors an important tool for 3rd edition, Alan R. Liss, Inc., New York.
used for drug screening. the early diagnosis of fetal disorders. 2. Methods in Enzymology: Cell Culture,
These cells can then be examined for Vol. 58, (1979) W. B. Jacoby and I. H.
abnormalities in their chromosomes and Pasten, eds. Academic Press, New York.
genes using karyotyping, chromosome 3. Cell and Tissue Culture (1975) John Paul,
painting and other molecular techniques. 5th edition, Churchill Livingstone,

4. Animal Cell Culture Methods, Volume 57, 8. General Guide for Identifying and
(1998) J. Mather and D. Barnes, eds. Correcting Common Cell Culture
Methods in Cell Biology, Academic Growth and Attachment Problems
Press, San Diego, 1998. Corning Life Sciences Technical Bulletin.
5. Growth, Nutrition and Metabolism of This is available on the Corning Life
Cells in Culture (1972) G. H. Rothblat Sciences web site at
and V. J. Cristofalo eds. Volumes 1-3 by lifesciences.
Academic Press, New York. 9. General Guide for Cryogenically Storing
6. Osmolality of Mammalian Blood and of Animal Cell Cultures Corning Life
Media for Culture of Mammalian Cells, Sciences Technical Bulletin. This is avail-
(1970) C. Waymouth, In Vitro, Volume 6: able on the Corning Life Sciences web
109-127. site at
7. Understanding and Managing Cell 10. Endotoxins and Cell Culture Corning Life
Culture Contamination Corning Life Sciences Technical Bulletin. This is avail-
Sciences Technical Bulletin. This is avail- able on the Corning Life Sciences web
able on the Corning Life Sciences web site at
site at

For additional product or technical information, please visit

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