Packed-bed bioreactors for mammalian cell culture: Bioprocess and biomedical applications

F. Meuwly a , P.-A. Ruffieux b , A. Kadouri a , U. von Stockar c,

c

Serono Biotech Center, Laboratoires Serono S.A., Zone Industrielle B, CH-1809 Fenil-sur-Corsier, Switzerland b Biotechnology Development, Novartis Pharma A.G., CH-4002 Basel, Switzerland Institute of Chemical Engineering, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland

Abstract This article describes the development history of packed-bed bioreactors (PBRs) used for the culture of mammalian cells. It further reviews the current applications of PBRs and discusses the steps forward in the development of these systems for bioprocess and biomedical applications. The latest generation of PBRs used in bioprocess applications achieve very high cell densities (N 108 cells ml 1) leading to outstandingly high volumetric productivity. However, a major bottleneck of such PBRs is their relatively small volume. The current maximal volume appears to be in the range of 10 to 30 l. A scale-up of more than 10-fold would be necessary for these PBRs to be used in production processes. In biomedical applications, PBRs have proved themselves as compact bioartificial organs, but their metabolic activity declines frequently within 1 to 2 weeks of operation. A main challenge in this field is to develop cell lines that grow consistently to high cell density in vitro and maintain a stable phenotype for a minimum of 1 to 2 months. Achieving this will greatly enhance the usefulness of PBR technology in clinical practice. 2006 Published by Elsevier Inc.
Keywords: Bioreactors; Mammalian cells; Packed-beds; Bioartificial organs

Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications of PBRs in bioprocessing . . . . . . . . . . . . . . . . . . . . . . 2.1. Packing materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Packed-bed bioreactor configurations . . . . . . . . . . . . . . . . . . . . 2.3. PBR development for bioprocess applications . . . . . . . . . . . . . . . 2.4. Limitations and prospects for improved PBRs for bioprocess applications . PBRs as bioartificial organs and tissues . . . . . . . . . . . . . . . . . . . . . . 3.1. Bioartificial liver (BAL) . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Artificial organs for drugs toxicology testing . . . . . . . . . . . . . . . . 3.3. Limitations and development prospects for improved biomedical PBRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 47 47 47 48 50 51 52 52 53

3.

Corresponding author. Tel.: +41 21 6933185. E-mail address: urs.vonstockar@epfl.ch (U. von Stockar).

4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

1. Introduction Mammalian cells are widely used to produce recombinant glycoproteins such as hormones, enzymes, cytokines and antibodies for human therapy. Mammalian cells are the preferred expression system for making recombinant proteins for human use because of their ability to express a wide variety of proteins with a glycosylation profile that resembles that of the natural human protein (Goochee et al., 1991; Jenkins and Curling, 1994; Jenkins et al., 1996). In view of their advantages, tremendous effort has been invested in developing animal cells as commercial production vehicles. A variety of cell culture systems are now available, as summarized in Fig. 1. The demand for therapeutic proteins derived from mammalian cell culture continues to grow (Morrow, 2001; Gavrilescu and Chisti, 2005), as newer products are approved. Some of the newer products such as antibodies and receptor binding proteins need to be administered in higher doses and this necessitates production of larger quantities than was the case with earlier products. Consequently, there is a continuing need to increase the productivity of mammalian cell culture bioreactors with minimal investment in additional equipment (Kadouri, 1994; Gdia and Sola, 1995; Brotherton and Chau, 1996). Most cell culture derived biopharmaceutical proteins are produced in stirred tank bioreactors operated in batch or fed-batch mode (Hu and Aunins, 1997; Varley and Birch, 1999; Chisti, 2001; Kretzmer, 2002). Production in stirred bioreactors is relatively simple to scale-up (Chisti,

Fig. 1. Bioreactor systems for mammalian cell culture.

1993), but requires large culture volumes (i.e. 1020 m3) to compensate for the relatively low cell densities that are attained. Typically, the cell density in suspension culture is between 106 and 107 cellsml 1. Compared to batch culture in stirred tanks, nearly 10-fold higher cell densities (i.e. 107108 cellsml 1) can be attained in perfusion cultures in which the medium is perfused at an appropriate rate in a constant volume culture and the cells are retained in the bioreactor by various means (Hu and Peshwa, 1991; Ozturk, 1996; Voisard et al., 2003). Because of a high cell density, the productivity of perfusion systems can be as much as 10-fold greater than the productivity of a comparable fed-batch bioreactor. In other words, a 2 m3 perfusion culture would be roughly equivalent to a 20 m3 fed-batch culture. Disadvantages of perfusion culture include their complexity and possible difficulty in scale-up. For example, large-scale cell retention devices for suspension cells are not yet entirely satisfactory (Voisard et al., 2003). A bioreactor system that can provide extremely high productivity within a compact size is the packed-bed bioreactor (PBR). Packed-beds have been used widely for perfusion culture of immobilized mammalian cells. This review focuses on the prospects of PBRs as a potential future preferred production tool for making cell-culture derived products. In addition, the use of PBRs as artificial organs (Allen et al., 2001) in biomedical applications is discussed. A relatively well-known example of such application is the bioartificial liver device (BAL) (Allen and Bhatia, 2002). BAL is intended to assist patients experiencing liver failure (acute failure), or entirely replace a liver until a compatible organ becomes available for transplant (Ambrosino and D'Amico, 2003). A BAL is expected to perform all the multiple functions of a liver that are essential to maintaining life. These functions include carbohydrate metabolism, synthesis of proteins, amino acid metabolism, urea synthesis, lipid metabolism, drug biotransformation and waste removal. A BAL device capable of these varied functions is best produced by culturing intact liver cells, or hepatocytes, that can function in vitro. The human liver is a massive organ that typically contains at least 1011 cells in an average volume of 1.3 l. This is equivalent to a cell density of at least of 108 cellsml 1 (Stapakis et al., 1995). Therefore,

generating a satisfactory BAL requires the ability to support viable and fully functional hepatocytes at a high density. This is a significant challenge as hepatocytes generally are poorly able to proliferate in vitro. Recent reviews (Allen et al., 2001; Allen and Bhatia, 2002) highlight the various bioreactor systems that are being evaluated as BAL devices. These include hollow fiber reactors, flat plate monolayer culture, perfused PBRs/ scaffolds and encapsulated cell suspension cultures. PBRs are a good alternative to other types of BAL bioreactors, as they can support high cell densities in a compact volume. Here we review the packed-bed bioreactors used for mammalian cell culture and discuss their performance and main applications. Common features of both bioprocess applications and biomedical applications of PBRs are reviewed with a view to identifying the challenges that must be overcome in developing the next generation of improved PBRs. 2. Applications of PBRs in bioprocessing 2.1. Packing materials The early attempts at culturing cells in PBRs focused on identifying support materials that were compatible with mammalian cells and had the other necessary attributes identified in Table 1. Solid glass beads for growing cells as monolayers were identified as a suitable material as early as 1953 (Earle et al., 1953). However, surface growth on beads limited the maximal cell density in the bed to 106 cellsml 1 because solid spheres have a very low specific surface-to-volume ratio available for cell proliferation. This limitation was overcome in the late 1980s with the introduction of porous glass spheres (e.g. SIRAN) that provided a higher specific surface. The cell density was increased by about 10-fold and reached up to 107 cellsml 1 of packed-bed (Looby and Griffiths, 1988). However, SIRAN glass spheres were still limited by a relatively low internal porosity (matrix = 0.56) that

could lead to oxygen diffusion limitations within the depth of the carriers. Higher internal porosities ranging from 0.80 to 0.95 were reached with the next generation of packing materials such as disks made of non-woven polyester and polypropylene screen, ceramic spheres and other shapes, glass fibers (Perry and Wang, 1989; Chiou et al., 1991), polyurethane and polyvinyl foams or resins. (The latter are discussed in the section on biomedical applications.) Among these carriers, the Fibra-Cel proved quite popular. This disk carrier was developed mainly for PBR applications that involved a high rate of medium perfusion (Bohak and Kadouri, 1987; Kadouri, 1994). Since it became commercially available, Fibra-Cel has been used widely for mammalian cell culture at laboratory-scale and pilot industrial-scale. Fibra-Cel is manufactured in conformance with the current Good Manufacturing Practice (cGMP) guidelines. The high porosity of its polyester non-woven fibers and polypropylene mesh provides for efficient entrapment of cells and reduces intra-carrier diffusion limitations. This provides conditions for attaining a high cell density. Ceramic pieces of 0.85 to 0.90 void-fraction have also been used to construct a lab-scale PBR of 3 l packed-bed volume (Mitsuda et al., 1991). The high porosity of this carrier was shown to improve intra-particle convection and, consequently, minimize oxygen limitations (Park and Stephanopoulos, 1993). The physical characteristics of this and other carriers are summarized in Table 2. In summary, the two matrices that are currently the most frequently used for bioprocess PBRs are the SIRAN and Fibra-Cel porous carriers. They have gained widespread acceptance as they are versatile and can be used generically to entrap both anchorage-dependant cells and cells that would normally grow in suspension. These carriers have proved successful with both serum-containing and serum-free media. 2.2. Packed-bed bioreactor configurations The PBRs typically consist of a packed-bed that supports the cells on or within carriers and a reservoir that is used to recirculate the oxygenated nutrient medium through the bed. Two major configurations are possible (Fig. 2), with the packed-bed compartment located either external to, or within, the reservoir of the medium (Wang et al., 1992a,b). Furthermore, the flow of the medium through the bed may be arranged to parallel the longitudinal axis of the bed, or the medium may flow radially. A frequent approach in developing PBRs is to first use a small-scale model bed to identify the optimal packing matrix for the cell line of interest. An optimal matrix is one

Table 1 Requirements of carrier materials for packed-bed bioreactors (Bliem et al., 1990) Simple physical configuration and made of non-toxic materials High surface to volume ratio Optimal diffusion from the bulk phase to the center of the carrier Chemical and mechanical stability Autoclavable Suitable for adherent and non-adherent cells Chemically and biologically inert, no reaction with the product Low cost and reusable if possible Of nonanimal origin

Table 2 Physical characteristics of various packed-bed carriers Carrier type and material Glass Glass Glass Glass SIRAN Ceramic pieces Ceramic cylinders Cytodex-3 Solid glass spheres Hollow glass cylinders Commercial fiberglass mat Commercial fiberglass mat Macroporous glass bead Size a (mm) matrix (-) S/V a, b ( 103 m-1) Reference 3 9/25 0.02 0.08/5 16 36 0.5/300 0.2 35 6/0.5 15/0.2 0.55 30/6 0.5 2 5/10 36 0.5/300 0 0 0.91 0.90 0.56 0.9 0.95 0.90 0.90 0.94 0.9 0.9 0.8 0.94 0.90 0 2 0.9 15 5 74 3.2 34 119 119 119 11 11 15 3.2 (Bliem et al., 1990) (Moro et al., 1994) (Chiou et al., 1991) (Perry and Wang, 1989) (Fassnacht et al., 2001) (Mitsuda et al., 1991) (Lyderson et al., 1985) (Ghanem and Shuler, 2000) (Ong et al., 1994) (Bohak and Kadouri, 1987) (Petti et al., 1994) (Kaufmann et al., 2001) (Kurosawa et al., 2000) (Lazar et al., 1993) (Miyoshi et al., 1996) (Hu et al., 2003) (Mitsuda et al., 1991) (Lyderson et al., 1985)

Uniform square channels Cross-linked dextran matrix coated with denatured collagen Cellsnow Cellulose porous cubes charged with polyethyleneimine (PEI) Fibra-Cel Polyester non-woven fiber and polypropylene disks NWPF Polyester non-woven fiber and polypropylene disks Polyester Polyester non-woven fiber material Polyurethane Membrane discs Polyurethane Macroporous sponge cubes Polyvinyl fluoride Resin cubes BioNOC II Polyester strips Ceramic Ceramic pieces Ceramic Cylinder with uniform square channels
a b

Size (diameter/length), internal porosity (matrix) and specific surface-to-volume ratio (S/V). Data from supplier; when available the internal surface is considered for the calculation of S/V ratio.

that provides the requisite combination of cell attachment, proliferation and productivity. This matrix is then used to optimize the operational parameters (e.g. packed-bed height and volume, medium perfusion rate, linear velocity of the medium across the packed-bed) of the PBR through perfusion experiments that are generally performed at laboratory-scale. A great deal of research effort has been invested in developing PBRs with enhanced productivity and stability. The matrices, operation variables and cell densities

achieved with different cell lines are summarized in Table 3. 2.3. PBR development for bioprocess applications The first application of PBRs in bioprocessing was the production of foot-and-mouth disease virus using BHK cells grown on the surface of solid glass beads for 45 days in batch culture. The support beads were 3 mm in diameter. The bed of beads was connected to an external reservoir of the medium. Liquid flowed through the bed in an axial direction. In the 1970s, this type of PBR was scaled-up 1000-fold for production purposes to a 100 l system that had an effective packed-bed volume of 30 l (Spier and Whiteside, 1976; Whiteside and Spier, 1981). During the 1990s, lab-scale PBRs of SIRAN porous glass spheres with packed-bed volumes ranging from 0.01 to 5.6 l were used successfully for cultivating many types of cells, including both anchorage-dependent and anchorage-independent cells. Cells were successfully grown in these reactors in media with and without serum (Table 3). Prtner and coworkers promoted the use of SIRAN spheres for cultivation of hybridomas (Bohmann et al., 1995). Fassnacht et al. (2001) scaled-up the SIRAN packed-beds to 5.6 l packed-bed volume and also

Fig. 2. Packed-beds with (a) external and (b) internal recirculation of nutrient medium. The main design variables for the packed-bed are its volume (VPBR), height (hPBR) and the linear velocity of the medium at the entrance of the bed (U0). DO = dissolved oxygen sensor.

Table 3 A summary of packed-bed bioreactors used with animal cells Matrix Glass Beads Beads Hollow cylinders Fibres Fibres SIRAN PBR type VPBR (l) hPBR (cm) 3.5 15 0.6 10 b30 10.5 49 59 4.0 14.6 30.5 14.6 15 15 UPBR (mms 1) 0.33 0.33 10 3.7 0.30.8 0.21.0 0.21.0 0.21.1 0.7 0.10.8 0.33.3 1.24.6 DPBR Xmax,PBR (day 1) (cellsml 1) FB 0.44 4 24 6.3 5.7 1020 310 1.5 106 1.4 106 15 108 0.53.2 106 6.8 107 1.4106 1.4 107 107108 8.5 107 1.8 108 N/D N/D 4 107 Run time Cell line (days) (product) 45 45 40200 2136 1721 66 5 b21 75 21 52 77 96 18 BHK BHK Hybridoma (IgG1) Hybridoma (IgG2a) CHO (-interferon) CHO (-interferon) GPK, Vero Hybridoma Immortalized mouse hepatocyte (mHep-R1) Hybridoma (IgG1) Hybridoma (IgG) Reference (Spier and Whiteside, 1976) (Whiteside and Spier, 1981) (Bliem et al., 1990) (Moro et al., 1994) (Perry and Wang, 1989) (Chiou et al., 1991) (Looby and Griffiths, 1988) (Fassnacht et al., 2001) (Fassnacht et al., 2001)

External 0.033 External 0.0330 External 15 Internal 3 External 0.02 Internal 1.2 External 1 Internal Internal 0.015.6 0.04

External 0.050.2 Internal 0.10.2 External 0.6 Internal 0.05 External 1 External 0.1

1.82.3 107 100

External 0.4 Fibra-Cel Internal Internal External External External Ceramic 1.75 0.51.0 0.5 0.05 0.01 External 2.6

10 2.0

5.2 34 4

1.1 108

14

35 3 107 2.4 107 3.6 107 1.01.2 108 30 108 6 106 1 108 5.1 108 3.3 105 46 10 8 26 40 40

(Prtner et al., 1997) (Fassnacht and Prtner, 1999) Hybridoma (IgG) (Bohmann et al., 1995) Hybridoma (IgG) (Bohmann et al., 1992) Hybridoma (IgG) (Racher et al., 1990a,b, 1993) BHK (IgG) (Griffiths and Racher, 1994; Racher and Griffiths, 1993) Human hepatocarcinoma (Kawada et al., 1998) cells (FLC-7) CHO (r-hEPO) (Jixian et al., 1998) CHO (Ducommun et al., 2002a,b) Hybridoma (IgG1) (Wang et al., 1992a,b) Hybridoma (IgG1) HeLa Insect (-galactosidase) MRC-5 human lung diploid fibroblasts Rat pituitary Human embryonic lung diploid fibroblast IMR-90 cells (t-PA) 10 different cell lines Hybridoma Rat hepatoma (H4IIE) Rat lung (L2) Porcine hepatocytes (Wang et al., 1992a,b) (Hu et al., 2000) (Kompier et al., 1991) (Petti et al., 1994) (Park and Stephanopoulos, 1993) (Mitsuda et al., 1991)

0.020.08 0.51.6

Cellsnow Cytodex 3 NWF

External 15 30 External 0.05 4 External 0.050.20 External 0.050.35 617 0.55

15 0.4 0.47

1 1 Batch

1.8 107 5 107 25 107

78 b6

PUM

PUF

PVF

External External Internal External External External External

0.032 0.0032 0.6 0.010.3 0.26 0.03 0.02

13 107 15 107 6 106 1.0 107 6.8 107 2.5 106 5 106

7 7 19 13 25 7 9

Rat hepatocytes Rat hepatocytes HEK 293 Dog hepatocytes HEK 293 (tPA) Hybridoma Rat hepatocytes

(Lyderson et al., 1985) (Ong et al., 1994) (Ghanem and Shuler, 2000) (Flendrig et al., 1997; Naruse et al., 1996, 1998, 2001) (Kurosawa et al., 2000) (Kurosawa et al., 2000) (Lazar et al., 1993) (Ijima et al., 2000a,b) (Kawakubo et al., 1994) (Murdin et al., 1989) (Miyoshi et al., 1996)

proposed a design for an industrial-scale reactor. The proposed system consisted of an internal PBR of 84.5 l volume that was placed inside a 300 l bioreactor but the feasibility of this approach was not tested in culture conditions. At Baxter, Bliem et al. (1990) followed the same approach to design a 14 l external PBR of glass beads. The system was used for producing immunoglobulins over 40 days using perfused cultures of hybridomas. The authors suggested that a further four-fold scale-up was feasible, to bring the packed-bed volume to 56 l. The scale-up strategy was not actually tested. Use of polyester packing materials of various shapes (e.g. disks, strips, fibers) has been reported by many sources in PBRs with packed-bed volumes that have ranged from 0.05 to 1.75 l (Table 3). Because of their mesh-like structure, polyester fibers are capable of easily supporting both attached and entrapped cells and have proved successful with many different combinations of cells and culture media. Bioreactor manufacturers have developed commercial PBRs that can accommodate many different types of carriers. Most of these PBRs are of the internal configuration. The CelliGen Plus system (New Brunswick Scientific; www.nbsc.com) was primarily developed for use in combination with Fibra-Cel polyester disk carriers and has been scaled-up from 0.7 l to 5 l packed-bed volume. The TideCell bioreactors (CESCO Bioengineering Co. Ltd.; www. cescobio.com.tw) are available in 5 and 25 l sizes and have been designed to operate with an internal packed-bed of BioNOC II polyester strips carriers. The Swiss company Bioengineering AG (www. bioengineering.ch), has also developed internal PBRs that can be placed within bioreactors and offer fixed bed volumes from 0.9 to 1.2 l. In summary, although a large variety of PBR systems have been assessed successfully in the laboratory (Table 3), few pilot-scale and industrial installations have been described. Indeed, most of published data deals with PBRs of less than 5 l of packed-bed volume. Only a few systems have been scaled-up to above 5 l. The current maximal packedbed volume appears to be in the range of 1030 l. 2.4. Limitations and prospects for improved PBRs for bioprocess applications Assessing a maximal potentially attainable performance for PBRs requires a knowledge of the maximum cell density that can be attained in the bed and the maximum size that a PBR can be scaled-up to.

If we assume cells to be spherical and packed as a bed of cells, the maximum attainable volume fraction of cells in the bed would be 0.74 and, therefore, the maximal cell density Xmax could be estimated using the following equation:   4 3 Xmax 0:74= prcell 1 3 where r is the radius of the cell. Because mammalian cells have an average diameter in the range of 1215 m, the Xmax for PBRs is in the range of 48 108 cellsml 1. Maximal cell densities of up to 15 108 cellsml 1 have actually been already reported (Table 3) and, therefore, further increases are unlikely. In establishing the maximum size that packed-beds can be scaled-up to, we need to understand that the main limitation on increasing the height of the bed originates from the unavoidable occurrence of axial gradients in concentrations of nutrients. The nutrient that generally limits the depth is oxygen, as the maximum attainable concentration of oxygen at the inlet of the bed depends on the solubility of oxygen which is quite low. Of course the oxygen concentration anywhere in the bed must not fall below the critical level that would jeopardize survival of the cells and their ability to produce the desired protein. Models have been developed to estimate the axial gradients in dissolved oxygen in packed-beds (Chisti and Moo-Young, 1994; Fassnacht et al., 2001). The depth of the bed (hPBR) depends on the superficial fluid velocity (U0) through it, the cell density XPBR and the specific oxygen consumption rate (qO2), as follows (Chisti and Moo-Young, 1994; Fassnacht et al., 2001): hPBR
in out U0 CO2 CO2 d ePBR qO2 dXPBR

In Eq. (2), cOin and cOout are the oxygen concentration 2 2 values at the inlet and outlet of the bed, respectively. In Eq. (2) the bed porosity PBR considers both the space occupied by the matrix and by the cells; thus, ePBR ematrix ecells ematrix 0:74d XPBR Xmax 3

where matrix and cell are porosity of the matrix and volume fraction of cells in bed, respectively. If we assume that the PBR operates within a range of oxygen concentrations such that b 80% of oxygen saturation and cOout remains above 20%, the cells have a 2 constant specific oxygen consumption rate (qO2) of 2 10 13 molcell 1 h 1 (Ruffieux et al., 1998), the packing is highly porous (matrix = 0.90), and the cells are

evenly distributed in the bed at an average cell density of XPBR, the depth of bed can be estimated using Eq. (2). Fig. 3 shows plots of the calculated maximal depth (hPBR) of the PBRs for various values of the immobilized cell density XPBR (1 108 b XPBR b 5 108 cellsml 1) and superficial velocity U0 (0.2 b U0 b 1.0 cms 1) of the medium. The U0 and XPBR values in Fig. 3 spanned the maximum and minimum values of these variables as previously published in the literature (Table 3). From Fig. 3, we can deduce that the maximal PBR depth that can be achieved under the specified conditions is in the range of 5 b hPBR b 30 cm. Indeed, values of hPBR published in Table 3 correspond well to these values. The maximum diameter of a packed-bed is limited by the ability to uniformly distribute the flow over the entire cross-section to prevent nonhomogeneities and channeling. The problem of achieving uniformity of flow over the cross-sectional area occurs also in packed-bed chromatography and has been investigated in some detail in relation to chromatography. Chromatography columns used for protein capture commonly have the same range of packing sizes (i.e. particles of 0.2 to 2 mm in diameter) and linear flow velocities (i.e. 0.1 bU0 b 0.3 cms 1) (Amersham Biosciences, 2003), as encountered in packed-beds of immobilized animal cells. Furthermore, because the dispersion coefficient in PBRs is relatively constant for a wide range of particle sizes and linear velocities (Levenspiel, 1999), PBRs are expected to behave similarly to large-scale chromatography columns. Columns for industrial chromatography are successfully operated with diameters as large as 2 m. Such large columns are commercially available from companies such as Millipore (www.millipore.com). Clearly, therefore, there is substantial scope for increasing the diameter of PBRs compared with the diameters that have been used in the past (Table 3).

Assuming that the depth of bed can vary between 5 and 30 cm and the maximum diameter cannot exceed 2 m, the range of reasonable volumes for the bed works out to be from 0.2 to 0.9 m3. PBRs of this size range operating with 108 cellsml 1 would have a productivity roughly equivalent to that of a 29 m3 fed-batch bioreactor operating at 107 cellsml 1. Such scaled-up PBRs can be quite competitive with conventional bioreactors, for producing therapeutic proteins that are needed in relatively small quantities. If the required bioreactor volume exceeds the limits established here, a PBR would not be technically feasible for the specific application. 3. PBRs as bioartificial organs and tissues The use of PBRs in biomedical applications began in the 1990s. Mainly, the focus has been in using PBRs to culture immobilized hepatocytes as a bioartificial liver device (BAL). Hepatocytes proliferate poorly in vitro (Ohshima et al., 1997); therefore, optimization of culture conditions, packing materials and entrapment procedures is important for supporting a large total number of immobilized hepatocytes in PBRs. As for bioprocess applications, the initial attempts to culture hepatocytes in PBRs were made using non-porous glass beads as the packing material. The first such PBR consisted of a 30 ml packed-bed BAL with glass beads of 1.5 mm in diameter. This system was good at cell entrapment (N80% immobilization efficiency) and allowed culture of metabolically active hepatocytes for more than 14 days (Li et al., 1993). In later designs, the solid glass beads were replaced with porous packing materials that supported even higher cell densities. Various materials with high porosities have been used to construct biomedical PBRs, as listed in Table 3. Immobilization matrices such as polyurethane membranes (PUM) have heterogeneous pores of about 100 m average diameter that facilitate cell immobilization with up to 99% efficiency and support cell densities ranging from 1 107 to 5 107 cellsml- 1 (Lazar et al., 1993). However, at least in some cases, clogging of packed-beds has been observed with PUM carriers for cell densities exceeding 5 107 cellsml 1 (Kurosawa et al., 2000). This phenomenon has been attributed to accumulation of cell debris in the dead-ended pores of the matrix. Clogging could be avoided by using other matrices having open-ended pores, such as non-woven polyester fabrics (Naruse et al., 1996, 2001; Flendrig et al., 1997), reticulated polyvinyl fluoride (PVF) resin scaffolds (Kurosawa et al., 2000), porous microcarriers (Ghanem and Shuler, 2000), polyester strips (Hu et al., 2003) and polyurethane foams (PUF).

Fig. 3. Estimated values of the maximal packed-bed depth (hPBR) in packed-bed bioreactors, as a function of the cell density (XPBR), for different values of superficial fluid velocity (U0).

Concurrently with advances in matrix design, methods were optimized for immobilizing hepatocytes on and within them (Li et al., 1993; Miyoshi et al., 1996; Kurosawa et al., 2000). These improvements enhanced the cell attachment yield from 10% in early trials to 40% and, in some cases, as much as 99% (Yang et al., 2001). Consequently, the maximum cell densities of immobilized hepatocytes were increased from 106 cellsml 1 in the early stages of BAL development to the current levels of 107108 cellsml 1 of PBR matrix volume. 3.1. Bioartificial liver (BAL) Both the internal and external packed-bed bioreactor configurations have been used for PBRs in biomedical applications such as bioartificial liver device (BAL). A BAL is generally connected to the patient as an extracorporeal BAL as shown in Fig. 4. A number of BAL PBRs have been reported (Table 3). Use of polyvinyl fluoride (PVF) cubes for primary culture of hepatocytes in a small PBR (24 ml packed-bed volume) has been reported (Yanagi et al., 1992). These beds were shown to reach quite high cell densities (from 4 106 to 1.2 107 cellsml 1) during short-term cultures (b 26 h). Transport of nutrients and oxygen to immobilized cells in the beds were identified as being critical to maintaining the metabolic activity of cells in vitro. Further trials demonstrated that hepatocytes cultured in serum containing medium for up to 9 days retained metabolic functionality that was comparable to the cells in vivo (Miyoshi et al., 1996; Ohshima et al., 1997). However, stable operation in serum-free medium and scale-up to a volume that would allow its use in pre-clinical and clinical trials were not demonstrated. PBRs based on polyurethane membrane (PUM) have been used to culture hepatocytes (Kurosawa et al., 2000). At the optimum density of 2.5 107 cellsml 1,

the hepatocytes expression level could be maintained for up to 1 week in serum-free medium, but declined during the second week of culture. Another BAL system based on polyurethane materials was developed by Ijima et al. (2000a,b). Hydrophilic polyurethane foam was used to make packed-beds of 14.5 ml and 300 ml for in vitro and in vivo studies, respectively. The in vivo work was carried out in dogs (Ijima et al., 2000b). The 300 ml BAL device containing 30 g of hepatocytes (i.e. cell density 107 cellsml 1) was shown to extend the survival rate of dogs afflicted with liver failure. The BAL module had to be freshly prepared (not more than 1 day old) to be effective. Another BAL module used non-woven polyester fibers for cell immobilization and had internal tubing for direct oxygenation of the hepatocytes. This module attained a density of 4 107 cellsml 1. This BAL device was initially tested in mice (Flendrig et al., 1997) and later scaled-up to a 400 ml packed-bed BAL for a preclinical trials in pigs with liver ischemia. Naruse et al. (1996) developed a BAL device based on packed-bed of polyester matrix. These modules attained a density ranging from 2 107 cellsml 1 (50 ml device) up to 5 107 cellsml 1 (200 ml device) and could support hepatocytes with reasonably high metabolic activity over a period of 6 days. These BAL devices were further scaled-up and successfully tested in animal models (Naruse et al., 1998, 2001). BAL devices with porous microcarriers have been tested successfully. For example, a cell density of 8.5 107 cellsml 1 was attained using immortalized human hepatocytes grown on a Cellsnow matrix. The bioreactor used in this work was an internal PBR with a 40 ml packed-bed volume (Fassnacht et al., 2001). The cells stably retained metabolic activity over the 40-day culture (Fassnacht et al., 2001). The highest hepatocytes cell density reported in PBRs has been 1.1 108 cellsml 1 and was attained with porous glass microcarriers in a packed-bed of 400 ml volume. The cells remained viable and metabolically active during the 14-day experiment (Kawada et al., 1998). Another study was successful in extending the run time to 40 days (Nagamori et al., 2000). In summary, although several PBR systems have proved successful as BAL devices, no single standardized process has been developed for culturing hepatocytes for use as a BAL device. 3.2. Artificial organs for drugs toxicology testing

Fig. 4. Flow setup for a biomedical packed-bed bioreactor connected to a patient as an extra corporeal bioartificial liver device (BAL).

As a consequence of their ability to support mammalian cells under tissue-like conditions, PBRs can be used

for drugs toxicology testing, where the cells' response to a test-compound is evaluated under conditions comparable to those in vivo. For example, Ghanem and Shuler (2000) developed a model system that mimicked the lung and liver functions of an adult rat. This system was made of three PBR compartments that represented the lung, the liver and the other tissues, respectively. The compartments were configured as a series of packed-beds with cell culture medium exchanged between compartments at physiological flow rates, enabling interactions of the organs and to mimic the whole animal. This system has been used to test the toxicity of chemicals on lung and liver functions. Another system, based on a radial-flow PBR was used to cultivate human hepatocyte-derived cells (HepG2) for possible use in in vitro evaluation of toxicity of drugs (Nagamori et al., 2000). Developing bioreactor systems that mimic with fidelity the complexity of animal metabolism is challenging. Individual bioreactor compartments generally support a single type of cell and do not reproduce the complexity of any single organ, the interactions of organs and regulation of metabolism. This problem notwithstanding suitably configured PBRs do offer an alternative to the use of whole animals and can potentially provide useful information about the effect of chemicals on in vivo metabolism. 3.3. Limitations and development prospects for improved biomedical PBRs The maximum cell densities that have been reported (Table 3) for biomedical PBRs are generally lower ( 107 cellsml 1) than for bioprocess PBRs ( 108 cellsml 1). This reflects the difficulty in growing hepatocytes to high densities. Because the cell densities obtained in vitro are currently generally 10-fold lower than the densities observed in the native liver organ (i.e. 108 cellsml 1), a BAL device of 1020 l would be needed to provide the functionality of an adult liver. The BAL devices under clinical evaluation (mainly hollow-fiber technology) generally have a maximum volume of 1 l and attain a total of 1010 immobilized hepatocytes, or merely 10% of the total cell number in an adult human liver (Allen and Bhatia, 2002). Although it is generally accepted that 10% of the total cell number existing in a real liver would suffice in replacing liver function in many urgent clinical applications (Yang et al., 2001; Ijima et al., 2000a), this situation is not ideal and there is a need to develop compact BAL devices that provide the full functionality of an adult liver.

Technologies have been developed to consistently attain a density of immobilized cells in PBRs that is comparable to the density of hepatocytes in the liver (Table 3). Use of porous packing materials is recommended for attaining the requisite cell density without clogging the bed. A number of other objectives must be attained for developing the next generation of improved biomedical devices based on PBR technology. These include the following: 1. A cell line with stable expression and good ability to grow in vitro needs to be identified. Ongoing work on this aspect was recently reviewed by Allen and Bhatia (2002). With most of the cell lines that are available currently, metabolic activity is lost within 2 weeks. This has been attributed to unstable cell phenotype and the inability to supply the cells with oxygen and other nutrients. To be viable in clinical applications, a BAL device should ideally be capable of functioning for a typical 30-day treatment period (Ambrosino and D'Amico, 2003). 2. Optimization of nutrients/oxygen supply in the PBRs to keep the cells viable and productive for at least 30 days that are desired for clinical application. This appears to be possible, as cells have been maintained at high densities in PBRs for extended periods, at least in a few cases. For example, a density of 1.1 108 cellsml 1 was maintained for 1440 days by Kawada et al. (1998) and 8.5 107 cellsml 1 were maintained for 40 days by Fassnacht et al. (2001). These reports are however an exception to the norm (e.g. Yang et al., 2001; Ducommun et al., 2002a,b). 3. Scale-up the packed-bed volume to about 12 l, but not further in order to provide a compact BAL device for convenient clinical use. The largest biomedical PBR device that has been reported had a volume of 400 ml (Table 3). This objective is still out of reach; however, the technical feasibility of such a scale-up has been proved with PBRs in various bioprocess applications with animal cells. 4. Concluding remarks The latest generation of animal cell culture packedbed bioreactors (PBRs) have achieved cells densities in the range of 1 108 to 5 108 cellsml 1. These values are close to the maximum density of 8 108 cellsml 1 that can be attained theoretically if the cells are packed in a bed as spheres. In bioprocess applications, the largest PBRs reported had a maximal volume of 30 l. This is a small fraction of the bed sizes that are commonly used in nonanimal cell

culture bioprocesses such as in wastewater treatment and biotransformations with immobilized enzymes. A challenge therefore is to demonstrate the scalability of packedbed technology in animal cell culture applications. A major limitation to further scale-up originates in the existence of substantial axial gradients in concentrations of nutrients such as oxygen. This limits packed-bed depth to 30 cm. The bed diameter is generally limited to 2 m, as uniform distribution of nutrient fluid over the bed cross-section becomes difficult with further increase in diameter. In view of these limitations, the maximum estimated volume for PBRs appears to be in the range of 0.20.9 m3. Notwithstanding their limited potential for scale-up, PBRs can be quite competitive with other bioreactor systems in producing proteins (e.g. cytokines or hormones) that are needed in relatively small quantities. This is because of the exceptionally high volumetric productivity of the PBRs. When large quantity of a protein must be produced, suspension culture in large fed-batch or perfusion bioreactors of 1020 m3 may be the only realistic option. In biomedical applications of PBRs, the maximal cell densities have been generally relatively low (e.g. 107 cellsml 1) compared with the bioprocess PBRs. However, replicating the performance of a whole adult liver in a compact volume of 12 l would require attaining a hepatocyte cell density of at least 108 cellsml 1. Developing a hepatocyte cell line that consistently attains these densities in vitro remains a challenge. Furthermore, any cell line for use in bioartificial organs must stably retain functionality for at least 30 days to be useful in clinical applications. At present, metabolic activity of in vitro cultured cells typically declines within 12 weeks of initiation, to unacceptably low levels. Once stable cell lines are available, scale-up of biomedical PBRs to desired size of 12 l should not be a problem, as these devices have been already scaledup to much larger volumes in animal cell culture bioprocess applications. Nomenclature BAL Bioartificial liver cOin Oxygen concentration at PBR inlet 2 Oxygen concentration at PBR outlet cOout 2 DPBR Medium dilution rate (in medium volume per packed-bed volume per day) h Packing material height Packed-bed depth hPBR NWF Non-woven polyester fabric PBR Packed-bed bioreactor PUF Polyurethane foam PUM Polyurethane membrane

PVF qO 2 rcell U0 U VPBR Xmax XPBR int PBR matrix cells

Polyvinyl fluoride resin Specific oxygen consumption rate of the cells Radius of cell Superficial velocity of circulating fluid before the packed-bed Superficial velocity of circulating fluid in the packed-bed Packed-bed volume Maximum cell density in the bed Viable cell density (in cells per unit packed-bed volume) Packing material internal porosity Packed-bed porosity Porosity of carrier matrix Volume fraction of cells in the bed Packing material diameter

References
Allen JW, Hassanein T, Bhatia SN. Advances in bioartificial liver devices. Hepatology 2001;34:44755. Allen JW, Bhatia SN. Improving the next generation of bioartificial liver devices. Cell & Dev Biol 2002;13:44754. Ambrosino G, D'Amico DF. Bioartificial liver support. Minerva Chir 2003;58:64956. Amersham Biosciences. Protein Purification Handbook; 2003. 2734 pp. Bliem R, Oakley R, Matsuoka K, Varecka R, Taiariol V. Antibody production in packed bed reactors using serum-free and proteinfree medium. Cytotechnology 1990;4:27983. Bohak Z, Kadouri A. Novel anchorage matrices for suspension culture of mammalian cells. Biopolymers 1987;26:S20513. Bohmann A, Prtner R, Schmieding J, Kasche V, Mrkl H. The membrane dialysis bioreactor with integrated radial-flow fixed bed a new approach for continuous cultivation of animal cells. Cytotechnology 1992;9:517. Bohmann A, Prtner R, Mrkl H. Performance of a membrane-dialysis bioreactor with a radial-flow fixed bed for the cultivation of a hybridoma cell line. Appl Microbiol Biotechnol 1995;43:77280. Brotherton JD, Chau PC. Modeling of axial-flow hollow fiber cell culture bioreactors. Biotechnol Prog 1996;12:57590. Chiou T-W, Murakami S, Wang DIC. A fiber-bed bioreactor for anchorage-dependent animal cell cultures: Part I. Bioreactor design and operations. Biotechnol Bioeng 1991;37:75561. Chisti Y. Animal cell culture in stirred bioreactors: observations on scale-up. BioProcess Eng 1993;9:1916. Chisti Y. Hydrodynamic damage to animal cells. Crit Rev Biotechnol 2001;21:67-110. Chisti Y, Moo-Young M. Anchorage dependent animal cell culture in packed beds with airlift driven liquid circulation: a theoretical analysis of oxygen transfer and comparison with stirred tank microcarrier culture system. Trans Inst Chem Eng 1994;72C:924. Ducommun P, Kadouri A, von Stockar U, Marison IW. On-line determination of animal cell concentration in two industrial highdensity culture processes by dielectric spectroscopy. Biotechnol Bioeng 2002a;77:31623. Ducommun P, Ruffieux P-A, von Stockar U, Marison IW. Monitoring of temperature effects on animal cell metabolism in a packed bed process. Biotechnol Bioeng 2002b;77:83842.

Earle WR, Bryant JC, Schilling EL. Certain factors limiting the size of the tissue culture and the development of massive cultures. Ann NY Acad Sci 1953;58:100011. Fassnacht D, Prtner R. Experimental and theoretical considerations on oxygen supply for animal cell growth in fixed-bed reactors. J Biotechnol 1999;72:16984. Fassnacht D, Reimann I, Prtner R, Markl H. Scale-up of fixed bed reactors for cultivating animal cells. Chemie Ingenieur Technik, vol. 73; 2001. p. 10759. Flendrig LM, Velde AA, Chamuleau RAFM. Semipermeable hollow fiber membranes in hepatocyte bioreactors: a prerequisite for a successful bioartificial liver? Artif Organs 1997;21:117781. Gavrilescu M, Chisti Y. Biotechnologya sustainable alternative for chemical industry. Biotechnol Adv 2005;23:47199. Ghanem A, Shuler ML. Characterization of a perfusion reactor utilizing mammalian cells on microcarrier beads. Biotechnol Prog 2000;16:4719. Gdia F, Sola C. Fluidized-bed bioreactors. Biotechnol Prog 1995;11:47997. Goochee CF, Gramer MJ, Andersen DC, Bahr JB, Rasmussen JR. The oligosaccharides of glycoproteins: bioprocess factors affecting oligosaccharide structure and their effect on glycoprotein properties. Biotechnology (NY) 1991;9:134755. Griffiths JB, Racher AJ. Cultural and physiological factors affecting expression of recombinant proteins. Cytotechnology 1994;15:39. Hu W-S, Aunins JG. Large-scale mammalian cell culture. Curr Opin Biotechnol 1997;8:14853. Hu W-S, Peshwa MV. Animal cell bioreactorsrecent advances and challenges to scale-up. Can J Chem Eng 1991;69:40920. Hu Y-C, Kaufman J, Cho MW, Golding H, Shiloach J. Production of HIV-1 gp120 in packed-bed bioreactor using the vaccinia virus/T7 expression system. Biotechnol Prog 2000;16:74450. Hu Y-C, Lu J-T, Chung Y-C. High-density cultivation of insect cells and production of recombinant baculovirus using a novel oscillating bioreactor. Cytotechnology 2003;42:14553. Ijima H, Nakazawa K, Koyama S, Kaneko M, Matsushita T, Gion T, et al. Development of a hybrid artificial liver using a polyurethane foam/ hepatocyte-spheroid packed-bed module. Int J Artif Organs 2000I;23:38997. Ijima H, Nakazawa K, Koyama S, Kaneko M, Matsushita T, Gion T, et al. Conditions required for a hybrid artificial liver support system using a PUF/hepatocyte-spheroid packed-bed module and it's use in dogs with liver failure. Int J Artif Organs 2000b;23:44653. Jenkins N, Curling EMA. Glycosylation of recombinant proteins: problems and prospects. Enzyme Microb Technol 1994;16: 35464. Jenkins N, Parekh RB, James DC. Getting the glycosylation right: implications for the biotechnology industry. Nat Biotechnol 1996;14:97581. Jixian D, Qin Y, Xuan C, Jiang Z. Production of rhEPO with a serumfree medium in the packed bed bioreactor. Chin J Biotechnol 1998;13:24752. Kadouri A. Cultivation of anchorage-dependent mammalian cells and production of various metabolites. Colloids Surf, B Biointerfaces 1994;2:26572. Kaufmann H, Mazur X, Marone R, Bailey JE, Fussenegger M. Comparative analysis of two controlled proliferation strategies regarding product quality, influence on tetracycline-regulated gene expression, and productivity. Biotechnol Bioeng 2001;72:592602. Kawada M, Nagamori S, Aizaki H, Fukaya K, Niiya M, Matsuura T, et al. Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: ultrafine structure of functionally

enhanced hepatocarcinoma cell lines. In Vitro Cell Dev Biol, Anim 1998;34:10915. Kawakubo Y, Matsushita T, Funatsu K. Tissue plasminogen activator (t-PA) production by a high density culture of weakly adherent human embryonic kidney cells using a polyurethane-foam packedbed culture system. Appl Microbiol Biotechnol 1994;41:4138. Kompier R, Kislev N, Segal I, Kadouri A. Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells. Enzyme Microb Technol 1991;13:8227. Kretzmer G. Industrial processes with animal cells. Appl Biochem Biotechnol 2002;59:13542. Kurosawa H, Yasumoto K, Kimura T, Amano Y. Polyurethane membrane as an efficient immobilization carrier for high-density culture of rat hepatocytes in the fixed-bed reactor. Biotechnol Bioeng 2000;70:1606. Lazar A, Reuveny S, Kronman C, Velan B, Shafferman A. Evaluation of anchorage-dependent cell propagation systems for production of human acetylcholinesterase by recombinant 293 cells. Cytotechnology 1993;13:11523. Levenspiel O. Chemical Reaction Engineering. 3rd ed. New York: Wiley; 1999. Li AP, Barker G, Beck D, Colburn S, Monsell R, Pellegrin C. Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: a potential bioartificial liver. In Vitro Cell Dev Biol, Anim 1993;29A:24954. Looby D, Griffiths JB. Fixed bed porous glass sphere (porosphere) bioreactors for animal cells. Cytotechnology 1988;1:33946. Lyderson BK, Pugh GC, Paris MS, Avender P, Sherma BH, Noll LA. Ceramic matrix for large scale animal cell culture. Bio/technology 1985;3:626. Mitsuda S, Matsuda Y, Kobayashi N, Suzuki A, Itagaki Y, Kumazawa E, et al. Continuous production of tissue plasminogen activator (t-PA) by human embryonic lung diploid fibroblast, IMR-90 cells, using ceramic bed reactor. Cytotechnology 1991;6:2331. Miyoshi H, Yanagi K, Fukuda H, Ohshima N. Long-term performance of albumin secretion of hepatocytes cultures in a packed-bed reactor utilizing porous resin. Artif Organs 1996;20:8037. Moro AM, Alves Rodrigues MT, Gouvea MN, Zucheran Silvestri ML, Kalil JE, Raw I. Multiparametric analysis of hybridoma growth on glass cylinders in a packed-bed bioreactor system with internal aeration. Serum-supplemented and serum-free media comparison for MAb production. J Immunol Methods 1994;176: 6777. Morrow KJ. Antibody production. Genet Eng News 2001;21:1-77. Murdin AD, Spier RE, Groves DJ. Growth and metabolism of hybridomas immobilized in packed beds: comparison with static and suspension cultures. Enzyme Microb Technol 1989;11: 3416. Nagamori S, Hasumura S, Matsuura T, Aizaki H, Kawada M. Developments in bioartificial liver research: concepts, performance, and applications. J Gastroenterol 2000;35:493503. Naruse K, Sakai Y, Nagashima I, Jiang GX, Suzuki M, Muto T. Development of a novel bioartificial liver module filled with porcine hepatocytes immobilized on a non-woven fabric. Int J Artif Organs 1996;19:34752. Naruse K, Nagashima I, Sakai Y, Harihara Y, Jiang GX, Suzuki M, et al. Efficacy of a bioreactor filled with porcine hepatocytes immobilized on a nonwoven fabric for ex vivo direct hemoperfusion treatment of liver failure in pigs. Artif Organs 1998;22:10317. Naruse K, Sakai T, Lei G, Sakamoto Y, Kobayashi T, Puliatti C, et al. Efficacy of nonwoven fabric bioreactor immobilized with porcine hepatocytes for ex vivo xenogeneic perfusion treatment of liver failure in dogs. Artif Organs 2001;25:27380.

Ohshima N, Yanagi K, Miyoshi H. Packed-bed type reactor to attain high density culture of hepatocytes for use as a bioartificial liver. Artif Organs 1997;21:116976. Ong CP, Prtner R, Mrkl H, Yamazaki Y, Yasuda K, Matsumura M. High density cultivation of hybridoma in charged porous carriers. J Biotechnol 1994;34:25968. Ozturk SS. Engineering challenges in high density cell culture systems. Cytotechnology 1996;22:3-16. Petti SA, Lages AC, Sussman MV. Three-dimensional mammalian cell growth on nonwoven polyester fabric disks. Biotechnol Prog 1994;10:54850. Park S, Stephanopoulos G. Packed bed bioreactor with porous ceramic beads for animal cell culture. Biotechnol Bioeng 1993;41:2534. Perry ST, Wang DIC. Fiber bed reactor design for animal cell culture. Biotechnol Bioeng 1989;34:19. Prtner R, Rssing S, Koop M, Ldemann I. Kinetic studies on hybridoma cells immobilized in fixed bed reactors. Biotechnol Bioeng 1997;55:53541. Racher AJ, Looby D, Griffiths JB. Studies on monoclonal antibody production by a hybridoma cell line (C1E3) immobilised in a fixedbed, porosphere culture system. J Biotechnol 1990a;15:12946. Racher AJ, Looby D, Griffiths JB. Use of lactate dehydrogenase release to assess changes in culture viability. Cytotechnology 1990b;3: 3017. Racher AJ, Looby D, Griffiths JB. Influence of ammonium ion and glucose on MAb production in suspension and fixed bed hybridoma cultures. J Biotechnol 1993;29:14556. Racher AJ, Griffiths JB. Investigation of parameters affecting a fixed bed bioreactor process for recombinant cell lines. Cytotechnology 1993;13:12531. Ruffieux P-A, von Stockar U, Marison IW. Measurement of volumetric (OUR) and determination of specific (qO2) oxygen uptake rate in animal cell cultures. J Biotechnol 1998;63:8595.

Spier RE, Whiteside JP. The production of foot and mouth disease virus from BHK 21 C13 cells grown on the surface of glass spheres. Biotechnol Bioeng 1976;18:64967. Stapakis J, Stamm E, Townsend R, Thickman D. Liver volume assessment by conventional vs. helical CT. Abdom Imaging 1995;20:20910. Varley J, Birch J. Reactor design for large scale suspension animal cell culture. Cytotechnology 1999;29:177205. Voisard D, Meuwly F, Baer G, Kadouri A. Potential of cell retention techniques for large-scale high-density perfusion culture of suspended mammalian cells. Biotechnol Bioeng 2003;82: 75165. Wang G, Zhang W, Jacklin C, Freedman D, Eppstein L, Kadouri A. Modified CelliGen-packed bed bioreactors for hybridoma cell cultures. Cytotechnology 1992a;9:419. Wang G, Zhang W, Freedman D, Eppstein L, Kadouri A. Animal cell technology: developments: process and products. In: Spier RE, Griffiths G, MacDonald D, editors. Continuous production of monoclonal antibodies in CelliGen packed bed reactor using Fibracel carrier. Oxford: Butterworth-Heinemann; 1992b. p. 4604. Whiteside JP, Spier RE. The scale-up from 0.1 to 100 liter of a unit process system based on a 3 mm-diameter glass spheres for the production of four strains of FMDV from BHK mono layer cells. Biotechnol Bioeng 1981;23:55165. Yanagi K, Miyoshi H, Fukuda H, Ohshima N. A packed-bed reactor utilizing porous resin enables high density culture of hepatocytes. Appl Microbiol Biotechnol 1992;37:31620. Yang TH, Miyoshi H, Ohshima N. Novel cell immobilization method utilizing centrifugal force to achieve high-density hepatocyte culture in porous scaffold. J Biomed Mater Res 2001;55:37986.