July 1, 2005 (Vol. 25, No. 13)
Using Disposables to Reduce Labor and Increase Yields
Single-use cell culture systems are the hot topic of the moment, and with good reason. Disposable systems, such as T-flasks, roller bottles, and bags, can offer a variety of advantages over fully instrumented bioreactors. These benefits include elimination of:
Cleaning, as they come presterilized, saving work in the lab, as well as eliminating the need to validate the cleaning process;
Autoclaving, as they are presterilized and ready to go;
Lengthy learning curves, as these systems tend to be plug-and-play simple;
Costly start-up, as there is no need for expensive designs, piped-in steam, water lines, or other utilities;
Cross-contamination issues between batches, as the system is disposed of when done.
Until now, disposables have had many disadvantages. They:
Produce low cell densities and product yields, especially when compared to stirred tank bioreactors;
Occupy a lot of lab space to produce equivalent yields to those of more capable systems;
Are labor-intensiveespecially when you are caring for dozens or hundreds of cultures.
New Brunswick Scientific (NBS; Edison, NJ) has introduced FibraStage, a new, high-yield, disposable cell culture system. The labor-saving system produces high yields of protein, virus, or cell mass from anchorage-dependent or suspension cultures, while offering all the benefits of disposable technology.
The complete system is comprised of up to four disposable FibraStage 500 bottles, a FibraStage console that controls bottle movement, and an NBS or user-supplied CO2 incubator to control temperature and gas exchange.
Disposable FibraStage 500
Disposable FibraStage 500 bottles are designed to hold up to 500 mL of liquid, and come presterilized and preloaded with 10 g of FibraCel disks. The multilayer FibraCel disks form a solid-support matrix for producing high-density cultures. Cells can grow in, on, and between the disk layers. The system therefore provides a large surface area for cell growth, equivalent to 100 cm3.
The high surface area enables high cell yields. Depending on the cell line used and desired end product, up to four FibraStage 500 bottles can produce cell yields of 2 x 1010 cells/mL, while a single bottle can produce cell densities of 6 x 109 cells/mL. To achieve an equivalent surface area of a single FibraStage 500 bottle, one would need to maintain cultures in approximately 2040 roller bottles (bottle size: 850 cm2) or 350400 T-flasks (flask size: 250 mL).
Furthermore, cells remain entrapped in the disk bed during the entire run, keeping the medium virtually cell free. This further reduces labor by simplifying media replacement as well as facilitating product recovery.
Gas exchange and media mixing are accomplished by placing up to four bottles in a FibraStage console, which slowly raises and lowers the media level in the bottles. (Figure 1). This up/down movement of the FibraStage device collapses or expands bellows at the base of each bottle.
In the “up” position, the media is forced through the disk bed, facilitating nutrient feeding and removal of waste from the cells. During the “down” cycle, cells are exposed to air and gasses. This process enables efficient oxygen and nutrient transfer to the cells while maintaining a low-foam, shear-free environment.
A controller allows setting variable speeds for the up and down motions, and variable “hold times” during which the cells are exposed to media or gas. A 0.22-m filter is supplied in the bottle cap for ventilation.
The FibraStage system can be operated with just one or up to four disposable bottles. Because each bottle works as an independent reactor, the system can be used as a screening device to test different media formulations or various cell lines.
The compact system measures 10.24″ W x 14.17″ L x 6.89″ H and fits into most standard CO2 incubators. The controller has a magnetized plate, allowing it to be stored on the outside wall of the incubator.
The FibraStage system has been proven effective when tested with a variety of anchorage-dependent and suspension cultures. A sample protocol for growing Hybridoma cells is outlined in this article.
Protocol for Hybridoma Growth
This study used one FibraStage 500 bottle with a 50-mL inoculum. A general protocol for using the system is provided. Optimum parameters will vary depending on the cell line used. Table 1 shows the materials and results of culturing Hybridoma cells.
Preparation. Inoculate a 250-mL flask with 2.5 x 105 cells/mL in 130 mL of media. Culture at 90 rpm, 28C for 3 days. When the cell density exceeds 9.7 x 108 cells/mL and viability remains above 95%, harvest, centrifuge and re-suspend the cells to provide an inoculum.
Place the FibraStage device in a 28C CO2 incubator. Warm the culture medium in a 28C water bath. Aseptically add 450 mL of fresh culture medium and introduce 50 mL of the inoculum. Swirl the bottle to evenly distribute the cells in the medium.
Place the FibraStage 500 bottle on the FibraStage platform and twist the bottle to lock in place. Using the FibraStage controller, set Inoculation Parameters as shown in Table 2, and press “Start”. Usually more than 90% of the cells will be immobilized in the disk bed within 30 minutes. After 2 to 3 hours, reset the controller to the Culture Parameter as indicated.
Monitor the pH, residual glucose concentration, and other metabolic activities by taking samples every day.
During the culture, CO2 was maintained at 5%, and glucose concentration (Table 3) was measured daily in order to determine the medium change. The glucose concentration was measured using a YSI Biochemistry Analyzer (Figure 2); protein was determined by the ELISA method and an NBS NucleoCounter was used to count viable cells.
Figure 3 shows protein concentration data from daily samples. For protein or virus production, the FibraStage 500 system is equally efficient (Table 4). The system can be operated in batch and fed-batch modes and will soon be available as a continuous perfusion system.
FibraCel disks are manufactured according to cGMP guidelines, and have been used in hundreds of labs worldwide. While primarily used in NBS packed-bed bioreactors for production of secreted products, they have also been found to be effective in increasing product yields when used in shake flasks and bags. FibraCel disks are currently being used in the production of the world’s first licensed gene therapy drug. Their specifications are shown in Table 5.
The disposable FibraStage 500 system is a versatile, labor-saving tool for culturing high yields of biomass, proteins, or virus from a variety of anchorage-dependent or suspension cultures. Its diverse range of applications includes:
Monoclonal antibody production
Recombinant protein production
Virus and vaccine production
Seed production for inoculating larger vessels
Gene therapy/drug discovery
Cell therapy and tissue engineering
The system is easy to learn, simple to operate, compact in size and low in cost, making it ideal for the R&D lab.