October 1, 2008 (Vol. 28, No. 17)

System for Maintaining High Cell Growth Developed to Simplify Experimental Practices

Cell–based research today is an area of intense interest for the life science and pharmaceutical industries. It provides a useful and viable tool for many scientists, allowing in-depth study of cellular and molecular interactions using a model that is highly representative of in vivo conditions. The methodologies and associated technologies, however, are under constant review and development, in order to establish the best system possible for cell culture and analysis.

Cell culture itself is not a new phenomenon; it has been utilized by many scientists with varying degrees of success for the past 60 years. These days demands for the guaranteed quality and consistency of cell cultures top the agenda.

With numerous parameters that often vary depending on the cell type, these demands remain a daily challenge for the cell biologist. In this article, we look at the practical requirements of cell culture, and the benefits that ready-made optimized culture systems can bring to a number of applications.

Successful mammalian cell culture requires the careful control of variables. Cells may require different media formulations and additives depending on their individual physiology and growth requirements. In addition, correct gaseous concentrations of O2 and CO2 are required for normal cell metabolism and the efficient maintenance of media pH.

The preferred format for adherent and nonadherent cell growth and expansion will also differ; growth can occur on a solid surface, which in itself may be modified for optimal cell adhesion and growth, or in suspension in a liquid medium.

Lastly, any microbial contamination introduced during the culture process can have a significant impact, not only on cell viability and phenotype but also on the subsequent data collected from downstream experimentation. Such errors are costly; thus tight control of these parameters is essential.

Obviously, experimental best practice plays an important role since any deviation from set protocols can increase the likelihood of operator error and subsequent contamination of cultures. Media consumption can, of course, present a significant cost to a laboratory. When expanding cells for large-scale experimentation, media replacement—or feeding—can prove time consuming, and its extra step risks the introduction of contamination.

Furthermore, large amounts of cells necessitate an increasing number of flasks and incubation space for expansion. As the use of cell-based assays has steadily grown for screening applications, these demands have become commonplace, and the number of cells needed for any one experiment has greatly increased.

Thus, there has not only been a drive to develop techniques for large-scale expansion, but also for a solution that can be used in an automated set-up. Traditional culture vessels and flasks do not always lend themselves to use with automation.

Also, as they are not hermetically closed containers, they can allow the evaporation of media, which can greatly impact pH and cell viability. They also require the stringent control of gaseous O2 and CO2 and, with careless handling and splashing of the seals, allow contaminants to enter culture media.

Respiratory Active Membranes

One solution lies with the development of a unique cell culture device that deviates from traditional concepts of culture ware. Thermo Scientific Nunc OptiCell™ consists of two parallel, 75 µm polystyrene (PS) membranes 2 mm apart, and attached to a PS frame.

These respiratory active membranes (RAM) provide a cell-culture treated, interactive substrate for anchorage-dependent cell types and a barrier to separate the cell from the external environment, while still remaining breathable. The RAM permits optimal O2 and CO2 exchange between the atmosphere and the internal environment, maintaining aerobic metabolism of the cells and the balanced pH of the medium, which is necessary for optimal cell growth (Figure 1).

In culture vessels, the depth of the medium can influence the diffusion rate of O2; optimal levels range between 2 and 5 mm depth. The OptiCell, when filled with 10 mL has a media depth of 2 mm. This is measured as the distance between the two membranes. Thus, the furthest distance any one cell can be from the gaseous phase is 1 mm.

The internal growth surface of the RAM has been treated to maintain the optimal hydrophilicity for the growth of most adherent mammalian cell types. The gaseous permeability of the RAM allows for the incubation of cells within the system for several days in air, without the need for additional CO2 control. Moreover, because of its impermeability to water and low permeability to water vapor, the system reduces media loss through evaporation and can be maintained for long periods of time in a regular atmosphere without the need for humidity control.

These properties are invaluable when transporting cells from lab to lab or shipping cell cultures. The OptiCell chamber meets the 95 kPa pressure requirement in the temperature range of -40°C to 55°C for International Air Transportation Association approval. Lastly, it is also suitable for the effective and convenient short- (at -80°C) and long-term (-152°C) storage of cells.

Cell detachment via enzyme (trypsin/EDTA) or nonenzyme methods is simple; the use of trypsin can be eliminated if preferred by briefly using an ultrasonic water bath to shake the cells off the membrane, minimizing reagent cost, cell damage, and the shearing of surface molecules that can occur.

OptiCell also provides a fast and easy method for cell separation using magnetic beads. The RAM allows the magnet to sit more closely to the cell populations, minimizing cell manipulation and allowing the continued growth of the desired cell in the same device.

Individually packaged, sterile, and bar-coded, the OptiCell also provides a completely enclosed environment in a hermetically sealed chamber. As an additional contamination control measure there are two resealing access ports that allow for the penetration of dispensing cannulae (commonly used with automated systems) or micropipette tips.

The enclosed chamber allows researchers to perform cell culture without the need for a laminar hood or biosafety cabinet. For applications such as hybridoma antibody production, the easy-access ports allow a centrifuge-free supernatant collection.

As a completely self-sealed, sterile unit, the OptiCell MAX provides a higher antibody yield than a traditional T75 flask and will continuously produce antibodies for more than a month. Harvesting can take place three times a week.

Figure 1. O2 and CO2 content in OptiCell.

Saving Laboratory Space

When expanding cells for large yields, laboratory space is at a premium. Efficient optimization should minimize the size and volume of the vessel (i.e., to the amount of culture media required for proliferation) while maximizing the surface area for adhesion and growth. The OptiCell has a growth area of 100 cm2 (50 cm2 on both sides), holding 10 mL (OptiCell 1100) or 30 mL (OptiCell MAX) of media depending on the model.

For a standard T75 flask, the volume-to-surface area required for optimal growth is 4 cc per cm2, compared to the OptiCell ratio of 0.6 cc per cm2. To translate, 20 OptiCells occupy one-fifth the incubation space of 26 T75 flasks, yet they provide an equal surface area for cell growth.

This means nearly a sevenfold reduction in space requirement, and the ability to maximize available incubator space. Furthermore, following the cell attachment to the membrane, incubation can take place in any orientation and individual units can be placed in racks (Figure 2).

Figure 2. Twenty OptiCells occupy one-fifth the incubation space of 26 T75 flasks.

Additional Functionality

To facilitate easy monitoring of cell cultures, the slim and clear PS membranes allow microscope objectives (of standard, inverted, and confocal microscope systems) to be set at close proximity to the cells for high-clarity 3-D images. Furthermore, cultures can be fixed and stained in situ with no background fluorescence. The membrane can be sectioned for small-scale staining and sections stored for repeats.

Also relevant for transfection studies, the RAM makes it easy to observe a transfection result through fluorescent markers and microscopy. Furthermore, the closed environment of the OptiCell means there is no need to change the transfection reagents and protocols, and single cells/clones are easy to isolate/cut out of the membrane.

The cell-based scientist may utilize many techniques during the course of research. Sourcing a multifunctional tool that provides the optimal conditions for cell growth and expansion, within a small footprint, and functionality for specific culture applications can improve cost- and time-efficiency and simplify experimental practices.

Cindy Neeley, Ph.D. ([email protected]), is a technical field specialist at Thermo Fisher Scientific. Web: www.thermo.com.

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