The fluidized bed technology was introduced for animal cell culture in the 1980s by the company Verax Corporation (274,275). The cells were grown on porous microspheres, made of collagen and weighted with a noncytotoxic steel to achieve a specific gravity of > 1.6, so that the carriers remained suspended in the high-velocity (~70 cm/min) upward-fluid flow of culture medium. The microspheres (500 mm diameter) had a sponge-like structure of interconnected pores and channels with a diameter in the order of 20-40 mm allowing the cells to enter easily and populate the interior of the carrier (276). The fluidized bed was contained in a column type bior-eactor that was connected to an external recirculation loop. A gas exchanger (hollow fiber cartridge), pO2, pH, and temperature sensors, heating elements, and a circulation pump that controlled the expansion of the fluidized bed were located in the loop. Dissolved oxygen levels were monitored at the inlet and outlet of the gas exchanger in order to measure the oxygen transfer rate of the reactor and to control the oxygen flow (277). Typically, oxygen transfer rates approached 10mmol/L/hr. Carbon dioxide could also be supplied to the gas exchanger for the purpose of pH control. Should the cell culture medium become to acidic addition of base kept the pH at a given set point. This type of bioreactor, now no longer on the market, was available from 0.4 L (research scale) to 24 L expanded bed volume size for production of proteins for human use. The design principles of the bioreactor as well as examples for cultivation of hybridoma and recombinant CHO cells at different scales are comprehensively described in Refs.(274,278,279). The general concept of the Verax-system inspired many research groups to investigate and optimize the fluidized bed technology for animal cell culture (Fig. 8).
One focus of research was the microcarrier itself. The growth of cells on different matrix materials, such as glass and polyethylene, was investigated. Porous SiranTM carriers made of borosilicate glass (Schottwerke, Mainz, Germany) were shown to be a cost-effective alternative to the weighted Verax microspheres (280282). Chemical modifications of borosilicate SiranTM glass resulted in similar cell densities per milliliter packed bed volume as reported for the Verax microspheres (283,284). In contrast to stirred tank systems, a significantly higher specific gravity of >1.5 of the carriers is typically chosen to achieve homogenous fluidization in a fluidized bed bioreactor.
A major disadvantage of the fluidized beds was the progressive depletion of oxygen from the bottom to the top of the expanded bed. This problem of an oxygen gradient along the axis of the tubular reactor could be overcome by integration of a membrane oxygenation module directly into the fluidized bed (285,286). Alternatively, the integration of an in-line gasification tube module developed at the
Research Center Jülich was suggested (287). This latter fluidized bed bioreactor with improved oxygenation is commercially available form B. Braun Biotech International (Melsungen, Germany). A schematic drawing of this bioreactor is presented in Fig. 9. This bioreactor type is available with settled carrier volumes ranging from 0.02 to 0.5 L. Typically, it is operated with gelatinized porous Siran™ microcarriers that are available in different diameters. This system is used in the biotech industry for large-scale production of proteins for research and medical applications (288).
An alternative fluidized bed system based on internal recirculation of the culture medium was developed at the Institute of Applied Microbiology (289,290) in cooperation with Vogelbusch GmbH, Vienna, Austria (CytopilotTM). This system is available from laboratory scale to production scale (Vogelbusch GmbH). The fundamental differences between a conventional fluidized bed system and the Cytopi-lotTM are illustrated in Fig. 9.
The CytopilotTM bioreactor comprises lower and upper cylindrical chambers. A draft tube in the fermentor replaces the external recycle loop. The lower chamber houses an axial flow impeller that provides a fluid flow to expand the bed. Oxygen is homogeneously microsparged into the downcomer close to the impeller and uniformly distributed in the upper chamber by a designed liquid flow and a gas distribution plate at the bottom entrance of the fluidized bed. The bed expands or contracts
as a function of the stirrer speed that creates the necessary hydrodynamic pressure to lift the settled microcarriers. The lower chamber is additionally equipped with a heating circuit (double water jacket), sampling and harvest ports, pH and pO2 sensors.
Bluml and colleagues developed a microcarrier type that consists of polyethylene weighted with chalk and silicates (291). The buoyant density of this carrier type is dependent on the ratio of these three compounds providing various densities suitable for use in stirred tank, fluidized bed or packed bed systems (291,292). This former "IAM-carrier" is now commercialized as Cytoline™ (Amersham Biosciences) and is very often used in combination with the Cytopilot™.
The application of different types of microcarriers is described elsewhere in this book and has been reviewed previously (3,241). Verax microspheres, SiranTM (Schottwerke) and Cytoline™ (Amersham Biosciences) carriers have been successfully evaluated for protein production with anchorage dependent cell lines and suspension cells such as hybridomas. Very often "laboratory home-made'' bioreactor designs were used for all kinds of comparative studies as summarized in Table 4.
Reliable determination of the cell density in immobilized cultures used to be a draw-back when using SiranTM or CytolineTM carriers, since both carrier types cannot be solubilized enzymatically as is possible with proteinaceous materials such as collagen. This problem of growth monitoring has been overcome recently by the use of dielectric spectroscopy (284,293).
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