Other Reactor Types

Hollow Fiber Bioreactor

In contrast to the homogeneous bioreactor systems described so far, which support in the range of 107 cells/mL under perfusion conditions, the heterogeneous hollow fiber bioreactor (HFBR) is a high-intensity system where tissue-like cell densities (>108mL_1) and structures are obtained. This bioreactor type was originally developed by Knazek in 1972 (178) starting from commercially available ultrafiltration modules containing cellulose acetate capillaries and further developed by adding gas permeable silicone polycarbonate capillaries for improved oxygen supply and CO2 removal. The units are composed of a large number of semipermeable capillaries potted into a cylindrical housing. In many reports especially from research laboratories simple hemodialysis cartridges (8,58,179,180) have been applied. However, during the last 30 years a number of companies have commercially developed and automated systems for mammalian cell culture [some examples are: Technomou-seTM (Integra Biosciences) (181), Cell-PharmTM (Unisyn), Acusyst™ (Biovest International), Cellstasis™ (Genespan)].

The membranes are either of an ultrafiltration type (10-100 kDa) (58,178,180,182) or microporous (0.1-0.2 mm). Different membrane materials, for example, cellulose acetate (178), polypropylene (183), and polysulfone (0.2 mm) (184,185), have been used. In most applications the cells are grown in the extracapillary space (EC) while oxygen enriched medium is recirculated through the fibers (intracapillary space, IC) (179,186,187). A general setup is shown in Fig. 6. In many cases, 10 kDa ultrafiltration membranes are applied to enable the use of inexpensive basal medium where expensive large molecular weight growth factors are supplied only in the EC space (182). Cell secreted proteins are also retained in the EC space leading to a significant accumulation of the product in the EC space which substantially reduces downstream processing efforts. However, it was reported that inhibitory components might accumulate in the EC space, which negatively affect cell growth and productivity (188). Furthermore, proteases secreted by the cells or released from dead cells may cause product degradation during long-term operation. Due to product accumulation this bioreactor type is not suitable for toxic or feedback inhibited products. To improve nutrient supply and waste removal, protocols have been developed where fresh medium is supplied to a medium reservoir connected to the IC space after removal of spent medium (180). In other cases the medium reservoir is regularly replaced with fresh medium (185).

The basic concept of an axial flow hollow fiber bioreactor has been developed further over the years and a number of alternative designs have been described. An overview of cell culture systems and related operation modes and transport phenomena was given by Tharakan et al. (189), Piret and Cooney (190), and Brotherton and Chau (191). Uludag et al. (192) reviewed the technology in relation to its application in cell therapy and tissue transplantation.

In principle, three operation modes of hollow fiber systems can be distinguished: (a) open shell ultrafiltration, (b) closed shell ultrafiltration, and (c) crossflow

Figure 6 Schematic drawing of a hollow fiber cell culture bioreactor. The cells are kept in the extracapillary space while fresh oxygen enriched medium is recycled through the intracapillary space. [Reprinted from Ref. (182), with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc., Copyright ©1999 John Wiley & Sons.]

Figure 6 Schematic drawing of a hollow fiber cell culture bioreactor. The cells are kept in the extracapillary space while fresh oxygen enriched medium is recycled through the intracapillary space. [Reprinted from Ref. (182), with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc., Copyright ©1999 John Wiley & Sons.]

ultrafiltration (see Fig. 7). For a detailed description see Refs.(189,193). In the open shell ultrafiltration mode the transmembrane pressure is decreasing along the length of the fibers. In the closed shell ultrafiltration mode the transmembrane pressure is positive in the entrance half of the module and a back-flow of medium from the EC is obtained in the distal part. These pressure gradients along the axial flow bioreactor types (a, b) cause nutrient and oxygen gradients, which may result in uneven cell growth. These gradient phenomena are the reason for the limited scale-up

Figure 7 Three operation modes of hollow fiber systems (top) and their corresponding transmembrane pressure profile P (bottom). (Left) open shell ultrafiltration, (center) closed shell ultrafiltration, (right) crossflow ultrafiltration. [Reprinted from Ref. (193), with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc., Copyright ©1986 John Wiley & Sons.]

Figure 7 Three operation modes of hollow fiber systems (top) and their corresponding transmembrane pressure profile P (bottom). (Left) open shell ultrafiltration, (center) closed shell ultrafiltration, (right) crossflow ultrafiltration. [Reprinted from Ref. (193), with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc., Copyright ©1986 John Wiley & Sons.]

potential of these modules and a solution is to use multiple parallel units. A detailed review to model axial flow hollow fiber cell culture bioreactors is provided in Ref. (191).

Attempts to overcome the gradient-related problems have been made by modifying the operation of conventional HFBR. Manipulation of the bioreactor orientation (194) or the osmotic environment of the EC space (195) were suggested in addition to periodically reversing the flow direction in the IC space (194) or change the pressure in the EC space to generate a backflow of medium into the IC space (196). In order to improve oxygen supply to the EC space oxygen carriers were added to the medium that was recirculated through the IC space, which led to improved antibody production (197). Another approach is the development of alternative designs. In bioreactors with extra capillaries for oxygen supply the IC flow rate can be reduced and axial oxygen gradients are eliminated. By additional rotation of the culture chamber further improvement of the culture performance was claimed (Cellstasis™ product information, Genespan). Cima et al. (183) developed a radial flow hollow fiber reactor based on concentric microporous polypropylene fibers with a 200 mm annular space for cell growth. They investigated different operation modes resulting in a more uniform distribution of nutrients by forcing medium through the EC space. The modeling of an intercalated dead end hollow fiber bioreactor with arterial (inlet) and venous (outlet) fibers is presented in Ref. (198). An example for the crossflow operation mode is the flat-bed bioreactor developed by Ku et al. (199) where gradients are substantially reduced compared to the axial configurations. The fibers are used for oxygen supply and pH control. The cells were retained in the unit using microporous stainless-steel filters. However, in most commercially available systems modified axial flow configurations are used (Cell-PharmTM, Unisyn; TechnomouseTM, Integra Biosciences; CellstatTM, Genespan).

Hollow fiber bioreactors have mainly been used for the growth of suspension cells. However, using other membrane materials such as polypropylene (183) tissue-like cell densities of adherent cells were obtained and could be maintained over several weeks to months. Also coating of the fibers with polycationic materials such as polylysine (200) can be used to enable adherent cell growth. Many different types of mammalian cells have been cultured in hollow fiber bioreactors such as primary cells, tumor cells, and stable cell lines [BHK 21 and Vero cells as reviewed in Refs.(189,201)]. The most important application is the production of monoclonal antibodies (58,180,184,186,202) mainly for diagnostic and research purposes. Considerable attempts have been made to use the technology for the production of therapeutic antibodies, which resulted in the first registration of a hollow fiber produced drug, ProstaScint [Cytogen (203)] in 1996 (204). Other, more recent applications are the production of cells for cell and tissue therapy (205) and the use as artificial organs [liver (8,9), kidney (13), pancreas (10)].

Miscellaneous Dialysis Bioreactors

Several other bioreactors based on the dialysis principle were developed in the past.

Generally, dialysis culture offers the advantage of continuous supply of nutrients and removal of low molecular weight metabolic waste products while accumulating the product in the cell culture compartment. The stirred tank dialysis bioreactor setup eliminates the formation of gradients, the major disadvantage of the hollow fiber bioreactor. Compared to stirred-tank perfusion systems mass transfer is still diffusion controlled. Culturing hybridoma cells in simple dialysis bags

(206) placed in a spinner was further developed to setups where dialysis membranes (207,208) or dialysis modules (209) were submerged in STRs. In the latter systems the cells are cultured in suspension in the stirred vessels and medium from a reservoir is recycled through the dialysis membranes. Cell densities of more than 1 x 107mL_1 and significantly increased product concentrations have been reported in an industrial application (209). Another modification of this principle is a two-compartment stirred tank vessel with cells on one side of the dialysis membrane and dialysis medium on the other side (210). Dialysis bioreactor technology was reviewed by Portner (211). Some historical examples of complex systems mainly for cell maintenance are the InVitron Static Maintenance Reactor (212) or the Membroferm bioreactor (137,213).

Microencapsulation Technique

A completely different technology for suspension cell culture based on immobilization and compartmentalization is microencapsulation. It was originally introduced by Lim and Sun (214) for the immobilization of mammalian cells used in bioartificial organ applications . This technique has been further developed and applied to monoclonal antibody production for commercial use (215,216). The cells are suspended in a solution of a naturally gelling polymer and the microspheres are generated subsequently. The microcapsules can be "cultured" in suspension in different bioreactor types. Various materials have been described for suspension cell entrapment in microcapsules; Ca-alginate (217), Na-alginate (215,216), agarose (218), cellulose sulphate (219). Additionally, composite gels combining the properties of alginate and agarose (220) or PEG and alginate (221) were developed. Collagen (222) and fibrin (67) were suggested for the cultivation of anchorage dependent cells. The diameters of the capsules range from 0.5 to 1mm. Agarose beads have a lower mechanical strength compared to alginate (223,224). Alginate beads have been coated with poly-lysine to generate a semipermeable membrane with controllable molecular weight cut-off (225). Improvements of the mechanical strength were obtained using photosensitive polymers (226). The cell leakage of conventional alginate could be reduced using PEG-alginate composite beads (221). Even more materials and modifications are used for cell and gene therapy and tissue transplantation as reviewed by Uludag (192).

Microencapsulation of cells is a well-established technology in bacterial culture and especially in wastewater treatment. Much of the literature on the preparation of the microcapsules (227), their rheological and mechanical properties (224), as well as transport and mass transfer phenomena (228) is found there.

Typical advantages of microencapsulation are the shear protection of cells, the product concentration, and compartmentalization obtained with some microencap-sulation methods [e.g., polylysin coated Na-alginate beads (216)] and the high cell densities in the particles in the range of 107mL_1 (220) to 108mL_1 (67). Another advantage of the microencapsulation technique often cited is the increased specific antibody production rate (220). However, a critical review of the literature shows that an increase in specific antibody productivity is cell line specific (229) and dependent on the cultivation conditions and applied parameters (230). Furthermore, serum-free (217) and even protein-free cultivation (220) could be established facilitated by the compartmentalization of cells and the bulk of the medium. However, a drawback of the technology is the diffusion-controlled transport of nutrients, waste products and—most critically—oxygen, which may lead to necrosis in the center of larger microcapsules. In case of product retention in the capsule this system is not suitable for proteolytically sensitive and feedback inhibited products.

Microcapsules have been used in different bioreactor types such as stirred tanks (215,216), airlifts (231), and fluidized beds (220) mainly in small-scale batch operation mode. Although, from a historical perspective, the development of this technology was stimulated by the need for efficient and economic production methods for monoclonal antibodies it cannot be considered as suitable for industrial-scale mammalian cell culture (67,157). The major application area of this technology today is cell and gene therapy and tissue transplantation (232-234).


Adherent cell culture has a tradition going back to the beginning of the last century when tissue culture was first devised as a method to study the behavior of animal cells free of systemic variations. Basic techniques are described by Freshney (235) and an interesting historical review on the advances in tissue culture during the last century is given by Jensen (236). In anchorage-dependent monolayer culture, the achievable cell number is directly proportional to the available growth surface. Cell yields in the order of 105 cells/cm2 are obtained for HeLa cells (235), CHO and HEK 293 cell lines (own data). Typical small-scale culture systems include Petri dishes and multiwell pates that are kept in a humidified CO2 incubator. From a sterility point of view T-flasks available with and without vented caps are preferable (available surface area 25-225 cm2, 500 cm2 triple layer (some suppliers are NalgeNunc, Corning Costar, Integra Biosciences, Falcon). Culture flasks used to be made of glass. However, nowadays, disposable plastic ware is standard. Coating the plastic surface with l-lysine or other substances (235) may improve attachment of less anchorage dependent cells such as HEK 293 cells. Especially in roller bottle culture this helps preventing the formation of suspended aggregates. One of the simplest systems for scaling up monolayer cultures is the Cell FactoryTM (NalgeNunc) providing culture surface areas of 600-24 000 cm2 and larger (100 000 cm2) via multiple interconnected plastic layers (235). Gas transfer is obtained via diffusion and a critical liquid depth of a few millimeters should not be exceeded. Spier (21) reported oxygen transfer rates of 0.53 mM/cm2/hr for monolayer aeration.

Especially for commercial vaccine production roller bottles have been applied for over 40 years (37,236) (surface area 850 cm2, 1500 cm2, and other sizes; available from Falcon, Corning, etc.). Roller bottles require a specific apparatus for constant rotation. Major advantages of this traditional "large-scale" culture method are (a) the increased surface area, (b) the constant mixing preventing gradients, (c) very high oxygen transfer rates [200 h^1 (21)], and (d) the flexibility in number of units applied for a certain task. Handling large numbers of multiple culture units for industrial production of vaccines or recombinant protein is very tedious and prone to contamination. Therefore, successful attempts have been made toward automation using laboratory robots such as the CellmateTM (The Automation Partnership), which is able to handle roller bottles and T-flasks (237).

The CellCube™ system (Corning Costar, available surface area 8500— 85,000 cm2) combines the advantage of a large surface area typical for multilayer systems with good oxygen transfer and mixing capabilities. Medium is continuously pumped through the system which can be oxygen and nutrient enriched in an external loop. This system has been used at Merck for the development of Hepatitis A vaccine (238) and by other authors for virus propagation (239).

Although the described methods are feasible for large-scale production there are considerable drawbacks such as (a) high costs for labor, equipment, and consumables, (b) considerable risk of contamination, and (c) relatively poor opportunity to control culture parameters at optimum set-points. This led to the development of scaleable bioreactor systems for anchorage-dependent cell culture. The most prominent examples are microcarrier culture in stirred tanks (240), fluidized (241), or packed (242) beds or cell culture in solid bed bioreactors (243). These bioreactors are described in more detail Chapter 11. The initial impetus for the development of such techniques was provided by the mass vaccination campaigns of the 1950s against viral diseases and later by the advent of recombinant DNA technology used for the production of drugs in animal cells.

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