Hollow fiber cell culture is based on a cartridge system containing tube shaped filters with variable pore sizes (5kD to 1uM), enabling the system to be adaptable for different applications. Cell culture media is pumped into the cartridge and flows through the fibers, while cells are introduced into the extra-fiber space. Cells attach to the porous hollow fibers and can be grown for months at very high cell numbers (due to the large surface area of the fibers, cell concentrations can reach 1x108
/ml). Proteins secreted from cells remain in the extra-fiber space (based on pore size) and can reach concentrations of up to two orders of magnitude higher than in conventional flask culture.
Hollow fiber cell culture cartridges are frequently used in the production of mg to g quantities of monoclonal antibodies. However, this system is also gaining popularity for the production of secreted recombinant proteins, viruses and a number of other specialized applications. Indeed, Fibercell systems, Inc. can provide validated protocols for the culture of HEK-293, CHO and endothelial cells to name a few examples.
The system is relatively simple and the expense is minimal given an already established cell culture facility. The major purchase is the pump, which comes in two formats (one or two cartridges). The other required purchase is the cartridge which is available in a range of sizes (based on your application). The technical support for this system is excellent, particularly at assisting you in identifying the appropriate cartridge for your needs (www.fibercellsystems.com). A video CD is also available that details all procedures from initial set up to routine maintenance.
We have been using the hollow fiber cell system to express a recombinant, secreted enzyme that is stably transfected in HEK-293 cells. We have used the fibercell duet pump (can take two cartridges) with a low molecular weight cut off cartridge (5kD) to trap the secreted enzyme in the extra-fiber space. The procedure begins by culturing the HEK-293 cells in flasks that are expanded to 5x T-150cm2. Cells are harvested at this point and introduced into the extra-fiber space of the cartridge. While the expansion of the HEK-293 in flasks is occurring, the cartridge is primed with PBS and cell culture media. Once the cells are introduced into the cartridge, cell density increases require incremental increases in the volume of cell culture media in the reservoir (125ml, 250ml, 500ml, 1L) until 1L of media is reached. The time to increase the cell culture media reservoir is determined by monitoring the glucose concentration in the media. Once 50% of the glucose concentration in the media is used in a 24hr period, it is time to increase the reservoir volume. Glucose concentration is monitored with a glucometer with disposable test strips (we use the Roche Accu-chek monitor). Once the reservoir is up to 1L, the glucose concentration is monitored daily and the media is changed every 1-2 days. At this stage, we adapted the cells incrementally to a serum-free media that aids purification of our protein.
In summary, we have been able to maintain cultures of HEK-293 cells for over 6months in the cartridge using the Fibercell system. During that time, the cells continued to produce the recombinant protein with no observable decrease. The system has the advantages of reducing time in the lab (prior to using this system we performed scale–ups of up to 100 x T-150cm2 flasks which was a very time consuming process), as well as reducing the use of reagents and plastic ware. Once the system is running, daily maintenance requires no more than 15 minutes per day. As with any long term culture, sterile technique is essential and the hollow fiber system is no exception. In our lab however, after using the system for over 1 year we have not experienced any contamination.
Senior Research Scientist