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Cell Culture

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable
artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic
or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already
been already established.

Primary Culture
Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated
under the appropriate conditions until they occupy all of the available substrate. At this stage, the cells have
to be subcultured (i.e., passaged) by transferring them to a new vessel with fresh growth medium to provide
more room for continued growth.

Cell Line
After the first subculture, the primary culture becomes known as a cell line or subclone. Cell lines derived
from primary cultures have a limited life span and as they are passaged, cells with the highest growth
capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population.

Cell Strain
If a subpopulation of a cell line is positively selected from the culture by cloning or some other method,
this cell line becomes a cell strain. A cell strain often acquires additional genetic changes subsequent to the
initiation of the parent line.

If a surplus of cells are available from subculturing, they should be treated with the appropriate protective
agent (e.g., DMSO or glycerol) and stored at temperatures below 130C (cryopreservation) until they are
needed. For more information on subculturing and cryopreserving cells, refer to the Guidelines for
Maintaining Cultured Cells,

Mycoplasma are simple bacteria that lack a cell wall, and they are considered the smallest self-replicating
organism. Because of their extremely small size (typically less than one micrometer), mycoplasma are very
difficult to detect until they achieve extremely high densities and cause the cell culture to deteriorate; until
then, there are often no visible signs of infection. Some slow growing mycoplasma may persists in culture
without causing cell death, but they can alter the behavior and metabolism of the host cells in the culture.
Chronic mycoplasma infections might manifest themselves with decreased rate of cell proliferation,
reduced saturation density, and agglutination in suspension cultures; however, the only assured way of
detecting mycoplasma contamination is by testing the cultures periodically using fluorescent stainin ELISA,
PCR, immunostaining, autoradiography, or microbiological assays.

Morphology of Cells in Culture

Cells in culture can be divided in to three basic categories based on their shape and appearance (i.e.,

Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated shapes, and grow
attached to a substrate.
Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a
substrate in discrete patches.
Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to
a surface.

Applications of Cell Culture

Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model
systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the
effects of drugs and toxic compounds on the cells, and mutagenesis and carcinogenesis. It is also used in
drug screening and development, and large scale manufacturing of biological compounds (e.g., vaccines,
therapeutic proteins). The major advantage of using cell culture for any of the these applications is the
consistency and reproducibility of results that can be obtained from using a batch of clonal cells.

Cell Culture Hood

The cell culture hood provides an aseptic work area while allowing the containment of infectious splashes
or aerosols generated by many microbiological procedures. Three kinds of cell culture hoods, designated
as Class I, II and III, have been developed to meet varying research and clinical needs.
Classes of Cell Culture Hoods
Class I
Cell culture hoods offer significant levels of protection to laboratory personnel and to the environment
when used with good microbiological techniques, but they do not provide cultures protection from
contamination. They are similar in design and air flow characteristics to chemical fume hoods.
Class II
Cell culture hoods are designed for work involving BSL-1, 2, and 3 materials, and they also provide an
aseptic environment necessary for cell culture experiments. A Class II biosafety cabinet should be used for
handling potentially hazardous materials (e.g., primate-derived cultures, virally infected cultures,
radioisotopes, carcinogenic or toxic reagents).
Class III
Biosafety cabinets are gas-tight, and they provide the highest attainable level of protection to personnel and
the environment. A Class III biosafety cabinet is required for work involving known human pathogens and
other BSL-4 materials.

Introduction (Successful cell culture depends on the aseptic handling)

Successful cell culture depends heavily on keeping the cells free from contamination by microorganisms
such as bacterial, fungi, and viruses. Non-sterile supplies, media, and reagents, airborne particles laden with
microorganisms, unclean incubators, and dirty work surfaces are all sources of biological contamination.
Aseptic technique, designed to provide a barrier between the microorganisms in the environment and the
sterile cell culture, depends upon a set of procedures to reduce the probability of contamination from these

sources. The elements of aseptic technique are a sterile work area, good personal hygiene, sterile reagents
and media, and sterile handling.
Sterile Work Area
The simplest and most economical way to reduce contamination from airborne particles and aerosols (e.g.,
dust, spores, shed skin, sneezing) is to use a cell culture hood.
The cell culture hood should be properly set up and be located in an area that is restricted to cell
culture that is free from drafts from doors, windows, and other equipment, and with no through
The work surface should be uncluttered and contain only items required for a particular procedure;
it should not be used as a storage area.
Before and after use, the work surface should be disinfected thoroughly, and the surrounding areas
and equipment should be cleaned routinely.
For routine cleaning, wipe the work surface with 70% ethanol before and during work, especially
after any spillage.
You may use ultraviolet light to sterilize the air and exposed work surfaces in the cell culture hood
between uses.
Using a Bunsen burner for flaming is not necessary nor recommended in a cell culture hood.
Leave the cell culture hood running at all times, turning them off only when they will not be used
for extended periods of time.
Good Personal Hygiene
Wash your hands before and after working with cell cultures. In addition to protecting you from hazardous
materials, wearing personal protective equipment also reduces the probability of contamination from shed
skin as well as dirt and dust from your clothes.
Sterile Reagents and Media
Commercial reagents and media undergo strict quality control to ensure their sterility, but they can become
contaminated while handling. Follow the guidelines below for sterile handling to avoid contaminating them.
Always sterilize any reagents, media, or solutions prepared in the laboratory using the appropriate
sterilization procedure (e.g., autoclave, sterile filter).
Sterile Handling
Always wipe your hands and your work area with 70% ethanol.
Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before placing them
in the cell culture hood.
Avoid pouring media and reagents directly from bottles or flasks.
Use sterile glass or disposable plastic pipettes and a pipette or to work with liquids, and use each
pipette only once to avoid cross contamination. Do not unwrap sterile pipettes until they are to be
used. Keep your pipettes at your work area.
Always cap the bottles and flasks after use and seal multi-well plates with tape or place them in
resealable bags to prevent microorganisms and air born contaminants from gaining entry.
Never uncover a sterile flask, bottle, petri dish, etc. until the instant you are ready to use it and
never leave it open to the environment. Return the cover as soon as you are finished.
If you remove a cap or cover, and have to put it down on the work surface, place the cap with
opening facing down.

Use only sterile glassware and other equipment.

Be careful not to talk, sing, or whistle when you are performing sterile procedures.
Perform your experiments as rapidly as possible to minimize contamination.

Most laboratories will have, as an integral part of the research program, procedures in place for the
characterization of new cultures. Species identification (in case the cells are misidentified or crosscontaminated) will be unnecessary if DNA profiling is being used to confirm cell line identity; otherwise,
chromosome analysis or isoenzyme electrophoresis can be used. The lineage or tissue or origin can be
determined by using antibodies to intermediate filament proteins, for example, cytokeratins for epithelial
cells, vimentin for mesodermal cells such as fibroblasts, endothelium, and myoblasts, desmin for myocytes,
neurofilament protein for neuronal and some neuroendocrine cells, and glial fibrillary acidic protein for
astrocytes. Some cell surface markers are also lineage specific, for example, EMA in epithelial cells, A2B5
in glial cells, PECAM-1 in endothelial cells, and N-CAM in neural cells, and have the additional advantage
that they can be used in cell sorting by magnetic sorting or flow cytometry. Morphology can also be used,
but can be ambivalent as similarities can exist between cells of very different origins. Spontaneous
transformation is unlikely in normal cells of human origin, but indicators are a more refractile appearance
under phase contrast with a lower cytoplasmic/nuclear ratio, piling up of the cells and loss of contact
inhibition and density limitation of growth, increased clonogenicity in agar, and the ability to form tumors
in immune-deprived hosts, such as the Nude or SCID mouse. Where transformation is detected, it is more
likely to be due to cross-contamination, although it is possible that the tissue sample may have contained
some preneoplastic cells that have then progressed in culture.

Normal cells can divide a limited number of times; hence, cell lines derived from normal tissue will die out
after a fixed number of population doublings. This is a genetically determined event involving several
different genes and is known as senescence. It is thought to be determined, in part, by the inability of
terminal sequences of the DNA in the telomeres to replicate at each cell division. The result is a progressive
shortening of the telomeres until, finally, the cell is unable to divide further. Exceptions to this rule are
germ cells, stem cells, and transformed cells, which often express the enzyme telomerase, which is capable
of replicating the terminal sequences of DNA in the telomere and extending the life span of the cells,
infinitely in the case of germ cells and some tumor cells.

Cell Signaling

Cell proliferation, migration, differentiation, and apoptosis in vivo are regulated by cellcell interaction,
cellmatrix interaction, and nutritional and hormonal signals, as discussed above. factors. Signals that reach
the cell from another tissue via the systemic vasculature Some signaling is contact-mediated via cell
adhesion molecules, but signaling can also result from soluble, diffusible are called endocrine, and those
that diffuse from adjacent cells without entering the bloodstream are called paracrine. It is

Figure: Cell Interaction and Signaling. Routes of interaction among cells. (a) Factors influencing the behavior of a cell
include endocrine hormones from the vasculature, paracrine factors from the stroma, homocrine factors from adjacent
similar cells, and autocrine factors from the cell itself. Matrix, soluble, and cell-associated heparan sulfate (HS) may help
the activation, stabilization, and/or translocation of paracrine factors. (b) Uniformity of response intarget tissue is improved
by gap junctional communication, by calcium signaling, and, possibly, by homocrine factors from the stimulated cell.

useful to recognize that some soluble signals arise in, and interact with, the same type of cell. I will call this
homotypic paracrine, or homocrine, signaling . Signals that arise in a cell type different from the responding
cells are heterotypic paracrine and will be referred to simply as paracrine in any subsequent discussion. A
cell can also generate its own signaling factors that bind to its own receptors, and this is called autocrine
signaling. Although all of these forms of signaling occur in vivo, under normal conditions with basal media
in vitro, only autocrine and homocrine signaling will occur. The failure of many cultures to plate with a
high efficiency at low cell densities may be due, in part, to the dilution of one or more autocrine and
homocrine factors, and this is part of the rationale in using conditioned medium or feeder layers to enhance
plating efficiency. As the maintenance and proliferation of specialized cells, and the induction of their
differentiation, may depend on paracrine and endocrine factors, these must be identified and added to
differentiation medium. However, their action may be quite complex as not only may two or more factors
be required to act in synergy, but, in trying to simulate cellcell interaction by supplying exogenous
paracrine factors, it is necessary to take into account that the phenotype of interacting cells, and hence the
factors that they produce and the time frame in which they are produced, will change as a result of the
interaction. Heterotypic combinations of cells may be, initially at least, a simpler way of providing the
correct factors in the correct matrix microenvironment, and analysis of this interaction may then be possible
with blocking antibodies or antisense RNA

Evolution of Cell Lines

After the first subculture, or passage, the primary culture becomes known as a cell line and may be
propagated and subcultured several times. With each successive subculture, the component of the
population with the ability to proliferate most rapidly will gradually predominate, and nonproliferating or
slowly proliferating cells will be diluted out. This is most strikingly apparent after the first subculture, in
which differences in proliferative capacity are compounded with varying abilities to withstand the trauma
of trypsinization and transfer. Although some selection and phenotypic drift will continue, by the third
passage the culture becomes more stable and is typified by a rather hardy, rapidly proliferating cell. In the
presence of serum and without specific selection conditions, mesenchymal cells derived from connective
tissue fibroblasts or vascular elements frequently overgrow the culture. Although this has given rise to some
very useful cell lines (e.g., WI-38 human embryonic lung fibroblasts, BHK21 baby hamster kidney
fibroblasts, COS cells, CHO cells, and perhaps the most famous of all, the L-cell, a mouse subcutaneous
fibroblast treated with methylcholanthrene , this overgrowth represents one of the major challenges of tissue
culture since its inception namely, how to prevent the overgrowth of

Figure: Evolution of a Cell Line. The vertical (Y) axis represents total cell growth (assuming no reduction at passage) for a
hypothetical cell culture. Total cell number (cell yield) is represented on this axis on a log scale, and the time in culture is
shown on the X-axis on a linear scale. Although a continuous cell line is depicted as arising at 14 weeks, with different cells
it could arise at any time. Likewise, senescence may occur at any time, but for human diploid fibroblasts it is most likely to
occur between 30 and 60 cell doublings, or 10 to20 weeks, depending on the doubling time. Terms and definitions used are
as in the glossary.

the more fragile or slower-growing specialized cells such as hepatic parenchyma or epidermal
keratinocytes. Inadequacy of the culture conditions is largely to blame for this problem, and considerable
progress has now been made in the use of selective media and substrates for the maintenance of many
specialized cell lines.

Buffering Systems
Maintaining the pH of the cell culture medium is critical to cell viability. Most cell lines grow well at pH
7.4 and are inhibited by growth at pH 6.8. Glucose is usually the sugar included in media and is metabolized
by the cells very rapidly, at a rate faster than it is needed. Byproducts of this metabolism include pyruvic,
lactic acids, and CO2. To reduce the amount of lactic acid, glucose can be replaced with other sugars such

as galactose or fructose. These sugars are utilized at a slower rate, but also result in a slower rate of cell
growth. This replacement can slow down the onset of a pH shift. The bicarbonate and carbon dioxide
buffering system is most commonly used to maintain physiological pH 7.2-7.4 of a culture. Bicarbonate is
a weak buffer with a pKa of 6.1 making a pH range of 7.2-7.6 more difficult to prevent rapid pH changes.
Bicarbonate, however, is non-toxic, and has nutritional value.
Bicarbonate/CO2 Systems
The amount of dissolved CO2 in water is dependent on the amount of atmospheric CO2 and the temperature.
Increasing CO2 tension in the absence of sodium bicarbonate leaves the medium acidic. The addition of
sodium bicarbonate in the presence of CO2 will drive the equation above to the left to allow for an
equilibrium to be achieved and the pH maintained at 7.2-7.4.

Although CO2 is produced by growing cells, it is not produced in a high enough level when growing at a
low cell density or during lag phase to maintain an optimal pH. Use of a CO2incubator helps to control the
CO2 tension and the temperature.

Balanced Salt Solution

Sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium acetate and sodium
citrate solution
Balanced Salt Solution is a sterile physiological solution, each mL containing sodium chloride (NaCl)
0.64%, potassium chloride (KCl) 0.075%, calcium chloride dihydrate (CaCl22H2O) 0.048%, magnesium
chloride hexahydrate (MgCl26H2O) 0.03%, sodium acetate trihydrate (C2H3NaO23H2O) 0.39%,
sodium citrate dihydrate (C6H5Na3O72H2O) 0.17%, sodium hydroxide and/or hydrochloric acid (to
adjust pH), and water for injection. The pH is approximately 7.0. The osmolality is approximately 300
mOsm/ Kg.
Balanced salt solutions can provide an environment that maintains the structural and physiological integrity
of cells in vitro. All GIBCO balanced salt solutions are manufactured in state-of-the-art cGMP, ISO
certified facilities to ensure the highest quality and consistency for reproducible results every day. Tests
include osmolality, pH, stability, the absence of bacterial and fungal contamination and endotoxin tests. All
powdered balanced salt solutions are produced without sodium bicarbonate to increase stability.