Part II: Safety and handling considerations for animal cell culture
Welcome to the second of a new series of articles aimed at providing useful
hints for culturing animal cells. This article describes considerations
for working with animal cell cultures as well as information on cell culture
contamination. The series will continue in future issues of QIAGEN News
with information on cell culture growth conditions, followed by cell culture
Legislation and regulatory guidelines
Before undertaking any work with human or animal tissue (e.g., to establish
a primary cell culture), it is necessary to ensure that the nature of the
work conforms to the appropriate medical-ethical and animal-experiment legislation
and guidelines. It may be necessary to seek approval from the relevant regulatory
authorities and/or individuals.
Safety considerations and biohazards
When working with potentially hazardous material, it is important to be
aware of the possible risks associated with both the material and the experimental
protocol. All cell cultures are considered a biohazard because of their
potential to harbor an infectious agent (e.g., a virus). The degree of hazard
depends on the cells being used and the experimental protocol. Primary cell
cultures in particular should be handled carefully as these cultures have
a high risk of containing undetected viruses. Although commonly used cell
lines are generally assumed to be free of infectious agents, care should
still be exercised when working with these cell lines as it is possible
that they contain infectious agents, such as latent viruses. Cell cultures
used to stud
y specific viruses should be assumed to have the same degree
of hazard as the virus under study. We recommend handling all material as
potentially infectious to ensure the safest possible working environment.
Work should be performed in an approved laminar flow hood using aseptic
technique, and the creation of aerosols should be avoided (see below). After
the work is complete, all waste media and equipment (i.e., used flasks, pipets, etc.)
should be disinfected by autoclaving or immersion in a suitable disinfectant
according to institutional and regional guidelines.
Handling cell cultures
Adherence to good laboratory practice when working with cell cultures is
essential for two reasons: first, to reduce the risk of exposure of the
worker to any potentially infectious agent(s) in the cell culture, and second,
to prevent contamination of the cell culture with microbial or other animal
cells (see below).
Aseptic technique and minimization of aerosols
Aseptic technique and the proper use of laboratory equipment are essential
when working with cell cultures. Always use sterile equipment and reagents,
and wash hands, reagent bottles, and work surfaces with a biocide or 70%
ethanol before beginning work.
Creation of aerosols should be avoided aerosols represent an inhalation
hazard, and can potentially lead to cross-contamination between cultures.
To avoid aerosols, use TD (to deliver) pipets, and not TC (to contain) pipets;
use pipets plugged with cotton; do not mix liquids by rapidly pipetting
up and down; do not use excessive force to expel material from pipets; and
do not bubble air through liquids with a pipet. Avoid releasing the contents
of a pipet
from a height into the receiving vessel. Expel liquids as close
as possible to the level of liquid of the receiving vessel, or allow the
liquid to run down the sides of the vessel.
Proper use of equipment can also help minimize the risk of aerosols. For
example, when using a centrifuge, ensure the vessel to be centrifuged is
properly sealed, avoid drops of liquid near the top of the vessel, and use
centrifuge buckets with caps and sealed centrifuge heads to prevent contamination
Laminar flow hoods
For the most efficient operation, laminar flow hoods should be located in
an area of the laboratory where there is minimal disturbance to air currents.
Avoid placing laminar flow hoods near doorways, air vents, or locations
where there is high activity. Hoods are often placed in dedicated cell culture
Tip - Keep laminar flow hoods clean, and avoid storing equipment inside
Tip - Before starting work, disinfect the work surface of the hood as well
as the outside of any bottles (e.g., by wiping with 70% ethanol), and then
place everything needed for the cell culture procedure in the hood.
Tip - Arrange equipment, pipets, waste containers, and reagent bottles so
that used items are not placed near clean items, and avoid passing used
items over clean items.
Tip - Place used items (e.g., pipets) in a container inside the hood, and
disinfect or seal before removing from the hood.
The presence of microorganisms can inhibit cell growth, kill cells, and
lead to inconsistent results. Contamination of cell cultures can occur with
both cell cultu
re novices and experts. Potential contamination routes are
numerous. For example, cultures can be infected through poor handling, from
contaminated media, reagents, and equipment (e.g., pipets), and from microorganisms
present in incubators, refrigerators, and laminar flow hoods, as well as
on the skin of the worker and in cultures coming from other laboratories.
Bacteria, yeasts, fungi, molds, mycoplasmas, and other cell cultures are
common contaminants in animal cell culture. To safeguard against accidental
cell culture loss by contamination, we recommend freezing aliquots of cultured
cells to re-establish the culture if necessary. A protocol for freezing
cell cultures will be provided in a future issue of QIAGEN News.
The characteristic features of microbial contamination are presented in
Table 1. The presence of an infectious agent sometimes can be detected by
turbidity and a sharp change in the pH of the medium (usually indicated
by a change in the color of the medium), and/or cell culture death. However,
for some infections, no turbidity is observed and adverse affects on the
cells are not easily observed.
Table 1. Characteristic features of microbial contamination
Cell cultures should be routinely evaluated for contamination.
Mycoplasmal infections are one of the more common and difficult-to-detect
infections; their detection and eradication are described in further detail
Mycoplasmal infection detection
Mycoplasmas are small, slow-growing prokaryotes that lack a cell wall and
commonly infect cell cultures. They are generally unaff
ected by the antibiotics
commonly used against bacteria and fungi. Furthermore, as mycoplasma do
not overgrow cell cultures and typically do not cause turbidity, they can
go undetected for long periods of time and can easily spread to other cell
cultures. The negative effects of mycoplasmal contamination include inhibition
of metabolism and growth, as well as interference with nucleic acid synthesis
and cell antigenicity. Acute infection causes total deterioration of the
cell culture, sometimes with a few apparently resistant colonies that may,
in fact, also be chronically infected. There are two main approaches to
detect mycoplasma Hoechst 33258 staining (1, 2) and mycoplasma-specific
DNA probes (Fisher Scientific). Alternatively, a PCR-based, mycoplasma-testing
service is offered by the ATCC (www.atcc.org
or BioReliance (www.biomeva.com
a fee-for-service basis.
Mycoplasmal infection - eradication
The best action to take with a culture containing chronic mycoplasmal infection
is to discard it by either autoclaving or incineration. Only if the cell
culture is absolutely irreplaceable should eradication be attempted. This
process should be performed by experienced personnel in an isolated hood
that is not used for cell culture, preferably in a separate room. Elimination
of mycoplasma is commonly achieved by treatment with various commercially
available antibiotics such as a quinolone derivative (Mycoplasma Removal
Agent), ciprofolxacin (Ciprobay), enrofloxacin (Baytril), and
a combination of tiamulin and minocycline (BM-Cyclin). Treatment procedures
and appropriate antibiotic concentrations can be found in the suppliers'
ns and in references 1 and 3.
Cross-contamination of cell lines
Cross-contamination of one cell culture with fast-growing cells from another
culture (such as HeLa) presents a serious risk. To avoid cross-contamination,
only use cell lines from a reputable cell bank; only work with one cell
line at a time in the hood; use different pipets, bottles of reagents, and
bottles of media for different cell lines; and check cells regularly for
the correct morphological and growth characteristics. The QIAGEN Guide to
Animal Cell Culture will continue in future issues of QIAGEN News with information
on cell culture growth conditions and cell culture protocols. If there is
any other information you would like to see on these pages of QIAGEN News,
please let us know by calling QIAGEN Technical Services or your local distributor.
1. Freshney, R.I. (1993)
Culture of Animal Cells,
a Manual of Basic
Technique. 3rd ed. New
2. Spector, D., Goldman,
R.R., and Leinwand, L.A.,
eds. (1998) Cells:
a Laboratory Manual.
Cold Spring Harbor, NY:
Cold Spring Harbor
3. Drexler, H.G. et al., eds.
(1997) DSMZ Catalog of
Human and Animal Cell
Lines. 6th ed.
Source:Page: All 1 2 3 4 5 6 Related biology technology :1
. The QIAGEN Guide to Animal Cell Culture2
. The QIAGEN Guide to Animal Cell Culture3
. The QIAGEN Guide to Animal Cell Culture4
. The QIAGEN Guide to Animal Cell Culture5
. The QIAGEN Guide to Animal Cell Culture6
. QIAGEN Instrument Service insist on the best in service and support7
. QIAGEN Multiplex PCR Handbook8
. QIAGEN Multiplex PCR Kit9
. QIAGEN Plasmid Kits10
. Laser microdissection and nucleic acid purification - a Leica - QIAGEN
. QIAGEN PCR CloningPlus Kit