In recent years, advances in mammalian cell-based
research have enabled many scientific and medical
discoveries. The ability to study and develop interactions
at the cellular or molecular level has had a
significant impact on research practices in both academic
and industrial research facilities, as evidenced
by the multibillion-dollar cell culture market.
In vitro models that closely mimic the behavior of cells
in vivo have been used to gain deeper insights into
areas such as functional biology, disease states and pathways,
and testing cellular responses to drug candidates.
Cell culture is also used in the production of biopharmaceuticals
and in support of the emerging field of cell-based
therapies. As a result of the broad applicability of
cell-based research, a diverse range of cell culture techniques
has become widely incorporated into academic
and industrial research environments.
Across these diverse applications, the success and reproducibility
of a cell-based experiment relies on eliminating
interference from contaminants and preserving the
phenotypic response to external stimuli. To do this,
there are numerous variables that must be controlled
to minimize contamination risks. For example, proper
facility design and equipment selection can minimize
the risks of contamination from aerosols, dust, and
microbes. These risks can be reduced even further by
working within a controlled environment, such as the
correct level of biological safety cabinet (Figure 1).
Figure 1 - Performing experiments within a biological safety
cabinet and utilizing sterile, single-use disposable products can
reduce the risks of contamination.
Another variable is the selection of suitable vessels
and reagents in which to conduct the procedure, since
the conditions required to culture different cell lines
toward various ends can vary widely. Not only do
reagents, growth media, culture flasks, and other consumables
need to be sterile, they also need to be compatible
with the specific cell type and application at
hand. With the wide variety of culture vessels and
growth media available, finding the right combination
for a specific experiment can be a daunting process.
To help address the daily challenges associated with the
culture of mammalian cells, Thermo Fisher Scientific
(Milford, MA) has brought together an array of equipment,
instruments, consumables, reagents, and media
in its Cell Culture Excellence™ program (referred to
throughout as Thermo Scientific products). This program
seeks to provide researchers with products proven
to work in specific applications while addressing key
issues such as security, reliability, and reproducibility.
This article examines two aspects of the culture process:
the use and supply of reagents and consumables, and how
these can best be applied to improve culture practices.
Importance of clean consumables
Many sources of contamination can affect the success
of cell cultures due to the numerous external
stimuli that play a role in mediating the signaling
processes that control cell growth and proliferation,
as well as the many routes of cell death. For example,
contamination or inconsistency in the surface of
products used to culture cells, or to store and transfer
growth media, can pose a threat to the experiment or
process success and reproducibility. Thus, inefficient
cleaning and sterilization of reusable containers can
provide a source of contamination. While the use of
disposable, single-use products has increased greatly, reusable containers are still widely employed in the
production and storage of media and reagents.
There is a wide range of disposable products available—including single-use flexible containers, flasks, wellplates,
bottles, and pipet tips—that support a sterile
environment for cell incubation, reagent storage, and
culture manipulations. Disposable culture-ware and
storage vessels are supplied with certificates of analysis
that enable full traceability while removing the time
and cost required to validate the sterility of the vessels
used during the research and development process.
Pipets and pipet tips are among the most commonly
used items in the laboratory, and yet without careful
operation, cross-contamination can easily occur.
While good experimental technique can dramatically
reduce this potential source of user error, electronic pipets such as the Thermo Scientific Finnpipette
Novus Electronic™ have been developed to ensure
correct aspiration during every step. For some delicate
applications, even the use of an electronic pipet
may not be enough to eliminate carryover contamination
by aerosolized materials. This is where specialized
filter tips, such as the Thermo Scientific Finntip
Filter™ and MBP brand ART (Aerosol Resistant
Tips) can play a key role in minimizing contamination
and ensuring experiment reproducibility.
Qualification and batch consistency
The quality of the reagents and materials used for a
cell-based experiment play a key role in achieving
success, since the presence and concentration of nutrients,
oxygen, and carbon dioxide all affect cell growth
and response. Growth media are of paramount importance
to any culture experiment, and need to provide
the optimal balance of nutrients, growth factors, and
pH buffers. With different cell types having different
requirements and varying levels of sensitivity to their
culture environment, this is an area of active research
and development. A number of sophisticated refinements
have already been made that allow researchers
to choose media specifically designed for the identified application and cell type, providing enormous benefits
to experimental and process control.
Serum can be derived from various sources, and as a
result its composition can vary greatly. This means that
researchers and manufacturers alike must frequently
qualify new lots of serum by examining its performance
with their particular cell lines and processes. This variability
not only wastes time and resources, but also leads
to poor reproducibility across experiments, which can
hinder both cellular, physiology-dependent quantitative
analysis and/or maintenance of acceptable bioproduction
performance. By moving to a more defined
growth medium, such as the Thermo Scientific line of
HyClone serum-free products, experiments become
more reproducible and the transfer from small-scale to
large-scale production is markedly simplified.
Furthermore, certain animal-derived or serum-based
media and components have been identified as potential
sources of contamination. For example, the REO
and Cache Valley viruses can pose a significant safety
risk1 if used for the production of biopharmaceuticals.
Even some types of serum-free media that are
composed of materials obtained from animal sources
may contain infectious agents if manufacturers do not
adhere to stringent quality control measures.
This has led many biopharmaceutical manufacturers to
use serum- and animal-product-free media, which offer
a lower risk of contamination compared with serum-containing
media. Chemically defined serum-free media
have a simplified composition and are better defined,
leading to greater batch-to-batch consistency. However,
some serum-free media can still have a protein concentration
between 50 mg/L and 1000 mg/L, which has the
potential to cause downstream purification problems for
researchers or manufacturers of recombinant proteins
that are often produced in a similar concentration range.
These contamination issues can be solved using
animal-product and protein-free media such as the
Thermo Scientific HyClone CDM4Mab™ media
for producing antibodies and recombinant proteins
(Figure 2). Developed using the Metabolic Pathway
Design™ approach, these targeted media formulations
contain all the nutrients and growth factors for
specific cell lines, and are less complex and better
defined than earlier-generation serum-free media.
Figure 2 - HyClone serum-free media.
Advanced surface technologies for cell culture
The surface of a culture vessel can play a critical role
in controlling the success of a cell culture experiment by influencing cell clustering and
cell signaling processes that regulate cell
migration, survival, proliferation, and differentiation.2 A key component of this
surface dependence is how well proteins—such as fibronectin, immunoglobulins, vitronectin,
and fibrinogen—can adsorb onto
the surface for integrin receptor binding
and subsequent cellular adhesion events.
The hydrophobicity of the surface has also
been observed to have an effect on the
gene expression profiles exhibited by several
Protein–plastic binding is a complicated
phenomenon affected by numerous variables,
including temperature, pH, and the
presence of surfactants, salts, and other
proteins, and the plastic itself. Plastics
made from polyolefins and fluoropolymers
typically bind fewer proteins than
those made of polystyrene, polyethylene
terephthalate G copolymer,
To enable researchers to control
the amount of cell binding, a range
of products has been developed to suit
the requirements of different cell types
and applications. For example, Thermo
Scientific Nunc™ Low Cell Binding™ dishes deter cell binding, while Nunc
EasyFlasks™, coated with either poly-D-lysine or collagen I, promote it. Because
surface materials are so important in controlling the density and quality of cell
growth, maintaining the same surface when transferring from small-scale development
to midscale production is desirable. Using vessels made from the same
material, such as the Thermo Scientific Nunclon surface, can dramatically simplify
the scale-up process.4
Use of a sealed growth environment to reduce contamination risks
Figure 3 - Nunc OptiCell cell culture
While researchers make every effort to ensure that aerosols, dust, and microbes do
not contaminate cell cultures, it is not always possible to eliminate human error,
accidents, and spillages. The Thermo Scientific Nunc OptiCell™ cell culture
system provides a sterile, sealed growth environment between two optically clear,
gas-permeable, polystyrene membranes. This design reduces the risks associated
with accidents or contamination. Two self-sealing ports allow easy cell seeding and
media replacement without the introduction of contaminants. Not only does the
Nunc OptiCell system provide a large surface area for cell growth within a small
footprint, but its membranes maintain the aerobic metabolism of the cells by permitting
optimal O2 and CO2 exchange with the atmosphere (Figure 3).
The Nunc OptiCell system also enables researchers to view cells under a microscope
without having to transfer them to a microscope slide, thereby reducing the
risk of damaging cells or introducing contaminants. Additionally, it makes transporting
and handling cultures much easier since the cells are constantly covered
in media in any orientation. This protected environment minimizes the risks of
dehydration or other cell trauma,5 and maintains viability without the need for
expensive temperature and gaseous control units.
Optimal tools plus best practices
Cell culture has become an indispensable part of the life science researcher’s toolkit.
For experiments to be successful, many conditions mimicking those in vivo
need to be reproduced while minimizing the risks of contamination. This can be
achieved not only through best practices, but also through the careful selection of
equipment, instruments, consumables, reagents, and media.
- Broedel, S.E.; Papiciak, S.M. BioProcess Int. 2003, 1(2), 56.
- Keselowsky, B.G.; Collard, D.M.; Garcia, A.J. Biomaterials
2004, 25, 5947.
- Allen, L.T.; Fox, E.J.; Blute, I.; Kelly, Z.D.; Rochev, Y.; Keenan, A.K.; Dawson, K.A.; Gallagher, W.M. Proc. Nat. Acad. Sci. USA 2003, 100, 6331.
- Staggert, J. Gen. Eng. News July 1, 2008, 28(13).
- Barbera-Guillem, E. Am. Biotechnol. Lab. 2001, 19(6), 18.
Mr. Goldman is in Strategic Marketing, Thermo Fisher Scientific,
450 Fortune Blvd., Milford,
MA 01757, U.S.A.; tel.: 774-452-6977; e-mail: email@example.com.