Reducing Cell Culture Costs By Reducing Serum Levels

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 media

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 media

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 problems

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:

  1. 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%.
  2. 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.
  3. Use the CellBIND surface to obtain better cell attachment at lower serum concentrations.
  4. 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.

  5. Pre-equilibrate or pregas culture vessels, especially for larger flasks, roller bottles, and CellSTACK® culture chambers (Corning) (Figure 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.
  6. Seed cultures with at least 10,000–20,000 cells/cm2 as a minimum.
  7. 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.
  8. 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.
  9. 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 serum levels.
  10. Be patient! It may take several passages in the reduced serum medium for the cells to fully adapt.

For happier cells:

  1. Maintain better culture pH levels by using a medium that is supplemented with 5–10 mM HEPES organic buffer.
  2. 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.
  3. 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.
  4. Pretest several lots of serum to find the one that is best for the cell lines.
  5. 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.
  6. 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 the cells.
  7. 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.
  8. Use enough medium in the culture vessels. The author recommends using at least 0.2–0.3 mL of medium/cm2 of growth surface.
  9. 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.
  10. 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.

Conclusion

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.

References

  1. 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.
  2. 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.
  3. Waymouth, C. “Feeding the baby”—designing the culture milieu to enhance cell stability. J. Natl. Cancer Inst. 1974, 53(5), 1443–8.
  4. 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 Sciences, 45 Nagog Park, Acton, MA 01720, U.S.A.; tel.: 978-645-2281; e-mail: ryanj@corning.com.

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