Achieving Consistency and Reproducibility in Cell-Based Research

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 cell types.3

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, or polycarbonate. 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 system.

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.

References

  1. Broedel, S.E.; Papiciak, S.M. BioProcess Int. 2003, 1(2), 56.
  2. Keselowsky, B.G.; Collard, D.M.; Garcia, A.J. Biomaterials
    2004, 25, 5947.
  3. 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.
  4. Staggert, J. Gen. Eng. News July 1, 2008, 28(13).
  5. 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: jeffrey.goldman@thermofisher.com.

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