Cell culture is a very important research tool today,
but one that is also very expensive to use. A major
component of that expense is fetal bovine serum
(FBS), an often essential part of cell culture. Many
researchers routinely supplement their culture media
with 10–20% FBS for growing their cells; however,
these levels may be higher than the cells require.
By reducing FBS levels by 50% or more, researchers
may be able to reduce costs considerably (Table 1). It
should be noted that although many cell lines will do
well at lower FBS levels, not all will.
This short guide will help readers better understand
the steps they can take to reduce culture costs by
reducing the cultures’ FBS levels. It will also offer
practical suggestions and steps to keep cultures happy
under lower serum conditions.
Disadvantages of using basic
Sometimes high serum levels are necessary because
the medium does not supply all of the necessary
nutrients required by the cells. This is common
when a basic “bare bones” medium, such as Eagle’s
Minimum Essential Medium (EMEM), is used. This
widely used medium was developed to determine the
minimum requirements (as they were known in the
1950s) for highly transformed mouse L-cells to grow
in the presence of small amounts of dialyzed serum.
It was not designed to promote optimum growth of
these cells. Other very basic minimal media include
Eagle’s Basal Medium (BME) and Dulbecco’s Modification
of Eagle’s Medium (DMEM), which is often
confused with EMEM. These media were all designed
for growing transformed cell lines (HeLa, L-cells,
etc.), and often do not contain any lipids, selenium,
zinc and other trace elements, nonessential amino
acids, and other important micronutrients.1–3
In order to obtain good growth for many widely used
cell lines utilizing these basic media, higher levels of
serum must be used to supply the missing micronutrients.
The widely used Chinese hamster ovary (CHO)
cell line illustrates a good example of this problem.
Due to a mutation, these cells have a requirement for
proline (normally a nonessential amino acid), which
is not a component of standard EMEM. As a result,
to support good growth of these cells, EMEM requires
either supplementation with proline or higher levels
of serum from which the cells can obtain their proline.
Switching to a richer, more complex medium
containing proline will lower the need for high levels
of serum while reducing culturing costs.4
Figure 1 - The Corning CellBIND surface can improve cell
attachment without the use of expensive coatings.
Figure 2 - The Corning CellBIND surface increases HEK-293 cell yields in 1% FBS. Cells were grown in 10% serum
prior to seeding into Iscove’s Modified Dulbecco’s Medium
(IMDM) containing 1% serum at an initial seeding density of
1.8 × 106 cells/T-75 flask (Corning). Cultures grown on the
Corning CellBIND surface had better cell attachment with corresponding
49.5% higher cell yields. Data represent the average
count ± SE (standard error) from six flasks from two separate
experiments for each condition tested.
Advantages of using enriched
To help researchers reduce their serum usage, some
media manufacturers have taken the traditional
basic media formulations and enriched them by adding
lipids, insulin, trace metals, and other ingredients
to develop new proprietary media that are
specifically designed to be used with lower serum
levels. Although they are sometimes slightly more
expensive than basic media formulations, by allowing
researchers to reduce FBS levels to only 2–4%,
these enriched media formulations can reduce culturing
costs even further. The media suppliers can be
contacted for recommendations.
Reducing cell attachment
Besides providing nutrients, growth factors, and hormones
for the cells, serum also contains fibronectin
and vitronectin, two key proteins cells use to
attach to the culture vessel. Thus, when serum levels
are reduced, the corresponding reduction in these
attachment proteins sometimes leads to a problem
with cell attachment. Traditional solutions to this
problem have been to either add these attachment
proteins to the reduced-serum medium or to coat the
culture vessels with collagen or other extracellular
matrix proteins. However, both of these solutions
are very expensive and would eliminate any savings
obtained from reducing the serum levels (Figure 1).
There is, however, a third alternative for better cell
attachment: the Corning CellBIND® surface (Corning®
Incorporated, Acton, MA). This surface is created
using a patented (U.S. Patent No. 6,617,512) plasma
treatment process to create a surface that incorporates
significantly more oxygen (50–60%) than traditional
tissue culture surface treatments, while creating a more
stable surface. These higher oxygen levels increase
surface wettability, giving superior cell attachment
even in very low serum conditions (Figures 1 and 2).
By combining the Corning CellBIND surface with
commercially available enriched reduced-serum media,
researchers can save money and have happier cells.
Some helpful hints
Lastly, high serum levels also help cells survive or
recover from harsh treatment by researchers. Poor
techniques such as over-trypsinization, centrifuging
cells too long or too hard, and leaving harvested
cell suspensions at room temperature all take a toll
on cell viability. While using high levels of serum
can sometimes reduce or prevent these losses, there
is no substitute for good technique and practice. If
a researcher is currently using high levels of serum
(10% or more), he or she may be able to reduce this
amount by 50% or more while reducing overall costs
by following these simple suggestions.
For better cell attachment and subsequent growth:
Grow cells in a richer, more complex medium.
Media manufacturers have developed a variety
of enriched media specifically designed to be
used at serum levels as low as 2–3%.
- Adapt cells by reducing serum levels in stages,
allowing one or two passages at each stage, to
give the cells time to adapt; for instance, going
from 10 to 5% serum, waiting for two passages,
and then going to 2–3% serum.
- Use the CellBIND surface to obtain better cell
attachment at lower serum concentrations.
- Prewarm medium when initiating cultures to
speed up cell attachment.
Figure 3 - Larger culture vessels such as the CellSTACK-10 chamber should be pregassed to minimize medium pH shifts
for faster and more even cell attachment.
- Pre-equilibrate or pregas culture vessels, especially
for larger flasks, roller bottles, and
CellSTACK® culture chambers (Corning)
3) to minimize pH increases while cells
are initially attaching. The harder it is for cells
to attach initially, the more likely there will be
uneven attachment and reduced growth.
- Seed cultures with at least 10,000–20,000 cells/cm2 as a minimum.
- If cell attachment or slower growth is a problem,
try seeding cells at twice their normal density
the first few passages until they fully adapt to the
reduced serum medium.
- Harvest cells gently and quickly to avoid damage
to the cell surface so that cells can attach faster.
Keep exposure to proteolytic enzymes, such as
trypsin, as short as possible.
- Make sure the dissociating agent has been inactivated
or removed by centrifugation. Trypsin
is inactivated by proteins in serum, but some
activity may remain in cultures with very low
- Be patient! It may take several passages in the
reduced serum medium for the cells to fully adapt.
For happier cells:
Maintain better culture pH levels by using a
medium that is supplemented with 5–10 mM
HEPES organic buffer.
- Avoid storing medium or cultures where they
can be exposed to fluorescent light to prevent
photo-activated formation of hydrogen peroxide
and other toxic by-products.
- Buy glutamine-free media when possible and add
fresh glutamine solution immediately before use
to ensure its stability and freshness. Glutamine
has a relatively short half-life in medium but is
very important to cell growth.
- Pretest several lots of serum to find the one that
is best for the cell lines.
- Subculture cells before they are confluent, especially
epithelial-like cells, so that they have not formed as
many tight junctions with other cells and are thus
easier to dissociate without lowering their viability.
- Try centrifuging cells more gently. Spin at 100 ×
g for only 5 min or just long enough to get a soft
pellet that is easy to resuspend without damaging
- Keep cell suspensions chilled after harvesting,
while counting, etc. This will increase viability
and reduce clumping. Always store frozen cells
below –130 °C to prevent decreases in culture
viability during long-term storage.
- Use enough medium in the culture vessels. The
author recommends using at least 0.2–0.3 mL of
medium/cm2 of growth surface.
- Keep cultures well fed. Feed rapidly growing cultures
at least twice a week. Better yet, optimize
the feeding schedule by measuring glucose depletion
(using test strips or meters for monitoring
blood glucose) in the medium and then feeding
when it gets too low. By not overfeeding, one
can save time and reduce costs even further.
- Make sure the cultures are not contaminated
with mycoplasma. These tiny organisms cannot
be seen under the microscope even at concentrations
as high as 108 mycoplasma/mL, but will
have a big impact on the health of the cell cultures
and the experimental results.
Growing healthy, happy cell cultures will always
require the use of good-quality vessels, culture media,
and reagents. However, by following the above steps,
it is possible to significantly reduce culture costs
without sacrificing quality.
Mather, J.P. Making Informed Choices: Medium,
Serum, and Serum-Free Medium. In Methods in Cell
Biology: Animal Cell Culture Methods; Mather, J.P.,
Barnes, D., Eds.; Academic Press: New York, 1998;
Vol. 57, pp 20–9.
- Freshney, R.I. Media. In Culture of Animal Cells: A
Manual of Basic Technique, 4th Ed.; Alan R. Liss, New
York, 2000; Chapter 8, pp 89–104.
- Waymouth, C. “Feeding the baby”—designing the
culture milieu to enhance cell stability. J. Natl. Cancer
Inst. 1974, 53(5), 1443–8.
- Ham, R.G.; McKeehan, W.L. Media and Growth
Requirements. In Methods in Enzymology: Cell Culture;
Jacoby, W.B., Pasten, I.H., Eds.; Academic Press: New
York, 1979; Vol. 58, pp 110–16.
Dr. Ryan is Technical Marketing Manager, Corning Life
45 Nagog Park, Acton, MA 01720, U.S.A.; tel.:
978-645-2281; e-mail: firstname.lastname@example.org.