Monoclonal antibody production has always been a foreboding task. To maximize production, traditional methods often involve investing resources in animal use for ascites production, or roller systems to culture hybridomas, resources many small labs cannot afford. Static tissue culture is an alternative, but the antibody concentration generated by this type of culture is low and the large volume of medium required to generate research quantities complicates purification. The CELLine™ cell cultivation system by BD Biosciences eliminates many of these problems and makes monoclonal antibody production feasible for any lab.
The CELLine™ device is a two-chambered tissue culture flask in which the two chambers are separated by a semi-permeable membrane. The flask is very similar in design to a regular T175 tissue culture flask and is approximately the same size and, therefore, can fit into any conventional tissue culture incubator. Cells are seeded at high density in 15 ml in the bottom chamber (cultivation chamber) and the top chamber is filled with 1000 ml tissue culture medium. Each chamber can be easily accessed with a serological pipette through different ports. Nutrients and waste products can diffuse across the membrane whereas the cells and larger molecules are trapped in the cultivation chamber. With this design, the monoclonal antibodies are trapped in the 15 ml cultivation chamber, while the cells continue to receive nutrients from the large volume of medium in the nutrient chamber, permitting continued cell growth and antibody production resulting in a high concentration of antibody in a relative small volume of culture medium. Depending on the specific hybridoma, the flask can generate several mg per milliliter of antibody within 7 days. One advantage of the CELLine™ device is the cells and medium in the cultivation chamber can be harvested, and the chamber reseeded for another round of antibody production, every 7 days for 21 days without changing the medium in the nutrient chamber. This protocol can generate tens of milligrams of antibody with little more than one liter of culture medium.
The instruction leaflet with the device is well written and generally easy to follow. However, at first, the design of the cultivation chamber port may be a little confusing. Instead of an open, free-flowing port like the nutrient chamber into which you can freely pipette medium similar to any tissue culture flask, the cultivation chamber port is connected to the chamber by a rubber gasket. The pipette must be pushed slightly into the gasket to form a slight seal around the pipette before you can pipette into and out of the chamber. The pipette does not extend into the cultivation chamber. BD Falcon brand serological pipettes are recommended, but other brands work equally well. I have the best results with loading and removing medium from the chamber with a 5 ml plastic pipette and have used several different brands. It is also recommended that any bubbles that get introduced into the cultivation chamber be removed. This can be a little tricky at first but by tilting the flasks so that the bubbles float to the corner where the port is located, it becomes a very simple task. My experience has been to remove as much air from the chamber as possible before inoculating the chamber with medium. This can be done by following the directions up to the point of inoculating the cultivation chamber, then use a 5 ml pipette and slowly “aspirate” out the air from the chamber as if you were transferring a liquid. Because the gasket seals the chamber from the outside, when you remove the pipette air will not re-enter the chamber. The few bubbles that may remain after loading the chamber with medium can be remove by following the instructions.
The manufacturer recommends the use of their growth medium, BD Cell MAb medium, for maximum antibody production. The estimates of antibody production in the instruction leaflet are based on cell growth in the BD Cell MAb Medium. There are several formulations available, a basal medium that requires serum supplementation, and a serum-free formula. Hybridomas must be slowly adapted to growth in this medium, and depending on the hybridoma, can take several weeks to months before the cells are fully adapted and producing antibody. However, the device works with other tissue culture medium and the use of Cell MAb medium is a recommendation, not an absolute requirement. I have obtained milligram quantities of antibody using DMEM supplemented with 10% serum. Regardless of choice of culture medium, if serum is used, only a few milliliters are required since only the 15 ml cultivation chamber requires serum, further reducing the cost of antibody production and eliminating some of the problems of purifying antibodies from large volumes of medium containing serum.
In summary, the CELLine™ device is a great advancement in monoclonal antibody production. It is simple to use and makes it possible for any lab with basic tissue culture facilities to produce their own monoclonal antibodies in large quantities without having to invest large amounts of money in equipment and animals.