Endothelial Cell Culture - Hollow fiber cell culture bioreactor

FiberCell^® Systems hollow fiber cell culture modules are used primarily to produce proteins or other things that are secreted by cells.

Other characteristics of hollow fiber bioreactors make them very suitable for culturing cells at high density and in a post confluent state. FiberCell hollow fiber bioreactors can be used to study the effects of cytokines and specific matrices on proliferation and development and the generation of 3 dimensional cultures.

What cell types have been cultured in the FiberCell^® Hollow Fiber bioreactor system.

Essentially if the cells can be grown in flask or other conventional systems then they will grow in a hollow fiber system. It is dependent upon your research and production goals as to whether the cells will behave in the desired fashion. The most common cells types successfully used are:

Hollow fiber bioreactors support cells at 10-100X higher density than regular cell culture methods. This means the cells are in a more in vivo like environment and require less serum, can be more easily adapted to a serum free medium or can be supported with a simplified serum replacement like CDM-HD.

Secreted products will be concentrated by the filter-like behavior of the hollow fibers, typically 100X higher concentration than that found with traditional bioreactors.

The effects of cytokines such as TGF-Beta or TNF Alpha can be controlled by the selection of the pore size of the fiber.

Hollow fiber bioreactors permit the culture and handling of large numbers of cells in a way that might not be practical using other methods in most laboratories.

Cells are bound to a porous support so they are free to grow in a post confluent fashion. Cells do not need to be split and can grow for extended periods of time. Hybridomas will typically produce antibody for 6 months or longer, CHO and 293 for 3-6 months of continuous production. The record is 2 years of continuous growth of a glioma cell line.

The FiberCell^® Systems Duet Pump uses a positive pressure displacement method that incorporates two one way check valves to drive the medium through the cartridge. This ensures reliable flow and long cartridge life.

There is a loop of silicone tubing wrapped around the core of the cartridge flow path stand. Silicone tubing is very gas permeable and the gas composition of the medium will be the same as the gas composition in the incubator.

The Duet pump is designed to fit inside a standard CO² incubator, the thin cord is designed to fit through the glass door.

The rate limiting factor in hollow fiber cell culture is the low partial pressure of oxygen due to lower solubility at 37 degrees. For this reason generally flow rate should be at the higher levels, between 26-30 on the Duet Control Box for the larger cartridges (C2003 and C2018) and between 22-26 for the medium sized cartridges. At the initiation of culture you will want to use a somewhat lower flow rate in order to allow cytokines to concentrate around the cells.

I want to produce a monoclonal antibody. What cartridge should I use and how much monoclonal antibody can I produce?

Cat #C2011 or C5011 should be used. This MWCO allows TGF Beta to diffuse away while retaining the produced antibody.

C2011 will support up to 1-2 X 10⁹ cells, this is equal to a one-liter culture or more.

Produce 5-50mg of antibody every 2 days, average is 20 mg per harvest
Continue to produce antibody for up to 6 months of continuous culture
Consumes about 1 liter of cell culture medium every two days. To reduce medium consumption harvest more cells out.
A single mouse used for ascites fluid production will produce 10-20 mg total antibody; each harvest is equal to a single mouse.
FiberCell^® Systems cartridge has about 4-5 times the production capacity of the CellLine flask per harvest.
Endotoxin burden is 1/10 that of ascites fluid production.
Cannot be re-used but can be stored and re-inoculated with the same cell line. product in the supernatant will be your antibody, simplifying your purification.
Hybridomas grown in the FiberCell^® Systems cartridge can be more easily adapted to serum free cell culture medium or adapted to as low as 2% FBS. When CDM-HD is used the only

C5011 Will support up to 2-4 X10 10 cells ; this is equal to a two liter culture.

Hollow fiber bioreactors can be used with any cell type that will grow in flask or spinner culture. Stable transfectants should be used to take full advantage of the long term production potential offered. CHO, 293, HEP G2 and many other cell types have been used. Insect cell culture is not ideal due to the transient nature of the culture but constituitive expression in S2 cells have had excellent results at lower induction agent concentrations

The molecular weight of the protein to be produced determines the fiber MWCO to use.

Production will be typically 100X that of flasks with harvested product concentrations between 100 µg and 300 µg/mL/day.

4300-C2011 Has 2200cm 2 of surface area, equal to 12.5 T175 flasks. Because of the way cells grow when attached to a fiber the total number of cells will be equal to 50-60 T175 flasks.

If the protein of interest can be trapped by the molecular weight cut-off of the fiber it will reach a concentration 100 times that of the same cell line grown in flask culture when collected from the ECS (extra-capillary space)
Average productivity is around 100 µg/mL (of ECS volume) or up to 1mg per day.
Proteins greater than 100kd in molecular weight will be trapped by this fiber.
If the protein of interest is too small to be trapped (like cytokines and cell growth factors) it will reach a concentration of 10 times that of the same cell line grown in flask culture when collected from the reservoir bottle.
Harvest every day instead of every two days
Medium consumption same as for hybridomas.
Reduction in serum concentration or easier adaptation to serum-free medium makes purification of proteins or identification of low concentration growth factors easier.

4300-C2018 Has 1.2m 2 of surface area. This is equal to 68 T175 flasks. Because of the way that cells grow on hollow fibers this will be equal to over 400 T175 flasks.

Any cell culture medium that is used in flasks can be used with a hollow fiber bioreactor. However, there are some special considerations to keep in mind.

The high cell density allows the reduction of the amount of serum to 2% and can facilitate the adaptation to serum free mediums. The also means that cells can be supported using a simplified replacement for serum such as CDM HD offered by FiberCell^® Systems.
Protein free mediums, such as CDM-HD, can be used but keep in mind that protein free mediums generally do not contain any attachment factors. Generally we want the cells to attach to the fiber so it is preferred to seed the cells in the presence of serum and then adapt to serum free medium or CDM-HD once the cells have reached high density (i.e. consuming 1 gram or more of glucose per day)
Generally you want to avoid a medium such as RPMI due to the low (2.5 grams per liter) concentration of glucose. This simply means that you would need to change the medium more often, an inconvenience.
Cells are growing in a stable, shear free environment. The use of surfactants such as pluronic F60 is not required. CDM-HD does not contain any surfactants or other membrane protectants.

Unless you have a compelling reason to not use antibiotics in your culture medium FiberCell^® Systems recommends the use of standard concentrations of antibiotics. We can fix anything, as long as you don't contaminate the cartridge. Antibiotics can help prevent the occasional lapse in technique from spoiling a culture. If your concern is endotoxin, and you wish for the absolute lowest levels of endotoxin then it is recommended that you work without antibiotics. Antibiotics can shield an infection but permit endotoxin to accumulate.

The only other piece of equipment that may not be part of a standard cell culture laboratory would be a way to measure glucose. There are some fancy and expensive machines to measure glucose out on the market but a simple glucometer like the ones used by diabetics and available at just about any drugstore or pharmacy will do the trick. Keep in mind that their readings won't be that accurate above 3.5 grams per liter so it is important to know the starting glucose concentration of your medium.

For endothelial cell culture under chronic shear use C2025. This cartridge allows extra-cellular matrix proteins to be attached to the fiber. One of the few ways to grow endothelial cells (cells that line the interior of blood vessels) under conditions of medium flowing over them (like they experience in the body). When grown under these conditions they behave in a much more in vivo like manner. They lay down flat and form a monolayer, form tight junctions, and certain genes are turned on in response to this shear stress that are not expressed in static culture. Other cell types such as vascular smooth muscle and brain glial cells can be co-cultivated with these endothelial cells.

What are the advantages of using the hollow fiber system for in vitro toxicology?

Hollow fiber bioreactor cartridges from FiberCell^® Systems offer a simple way to set up a two-compartment model for in vitro toxicology with higher levels of reproducible control to complex growth, infection, treatment, and sampling regimens. This system permits more realistic simulation of in vivo drug effects in a dynamically controlled system providing data that more accurately reflects biological responses. The design is fully disposable and will take into account the potential use of weaponized pathogens and genetically modified organisms

Any type of therapeutic compound can be used. FiberCell^® Systems offers two fiber types, polysulfone and cellulosic. The polysulfone is preferred because the flux rate and therefore equilibration of drug across the fiber is quite rapid and the geometry of the fibers results in even distribution of the fiber bundle inside the housing. Cellulosic fibers are generally used when the compound to be tested is highly non-polar which can result in significant non-specific binding of the compound to the fiber. Cellulosic fibers will have much lower non-specific binding but also lower flux rates so somewhat slower equilibration times across the fiber.

Closed bio-safe system
Organism load can be high enough to match human infections. A high starting number is required to uncover the emergence of drug resistance.
Drug pharmacokinetics can be exactly modeled on human profile
Many experiments can be run simultaneously
Complex systems such as two drug or two cell type cultures can be easily set up.