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

⁎ 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 1993), but requires large culture volumes (i.e. 10–20 m3)
to compensate for the relatively low cell densities that are
Mammalian cells are widely used to produce recom- attained. Typically, the cell density in suspension culture
binant glycoproteins such as hormones, enzymes, cyto- is between 106 and 107 cells·ml− 1. Compared to batch
kines and antibodies for human therapy. Mammalian cells culture in stirred tanks, nearly 10-fold higher cell densities
are the preferred expression system for making recombi- (i.e. 107–108 cells·ml− 1) can be attained in perfusion
nant proteins for human use because of their ability to cultures in which the medium is perfused at an appropriate
express a wide variety of proteins with a glycosylation rate in a constant volume culture and the cells are retained
profile that resembles that of the natural human protein in the bioreactor by various means (Hu and Peshwa, 1991;
(Goochee et al., 1991; Jenkins and Curling, 1994; Jenkins Ozturk, 1996; Voisard et al., 2003).
et al., 1996). In view of their advantages, tremendous Because of a high cell density, the productivity of
effort has been invested in developing animal cells as perfusion systems can be as much as 10-fold greater than
commercial production vehicles. A variety of cell culture the productivity of a comparable fed-batch bioreactor. In
systems are now available, as summarized in Fig. 1. other words, a 2 m3 perfusion culture would be roughly
The demand for therapeutic proteins derived from equivalent to a 20 m3 fed-batch culture. Disadvantages of
mammalian cell culture continues to grow (Morrow, perfusion culture include their complexity and possible
2001; Gavrilescu and Chisti, 2005), as newer products are difficulty in scale-up. For example, large-scale cell reten-
approved. Some of the newer products such as antibodies tion devices for suspension cells are not yet entirely
and receptor binding proteins need to be administered in satisfactory (Voisard et al., 2003).
higher doses and this necessitates production of larger A bioreactor system that can provide extremely high
quantities than was the case with earlier products. productivity within a compact size is the packed-bed
Consequently, there is a continuing need to increase the bioreactor (PBR). Packed-beds have been used widely for
productivity of mammalian cell culture bioreactors with perfusion culture of immobilized mammalian cells. This
minimal investment in additional equipment (Kadouri, review focuses on the prospects of PBRs as a potential
1994; Gòdia and Sola, 1995; Brotherton and Chau, 1996). future preferred production tool for making cell-culture
Most cell culture derived biopharmaceutical proteins derived products. In addition, the use of PBRs as
are produced in stirred tank bioreactors operated in batch “artificial organs” (Allen et al., 2001) in biomedical
or fed-batch mode (Hu and Aunins, 1997; Varley and applications is discussed. A relatively well-known exam-
Birch, 1999; Chisti, 2001; Kretzmer, 2002). Production in ple of such application is the bioartificial liver device
stirred bioreactors is relatively simple to scale-up (Chisti, (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
Fig. 1. Bioreactor systems for mammalian cell culture. least of 108 cells·ml− 1 (Stapakis et al., 1995). Therefore,
generating a satisfactory BAL requires the ability to could lead to oxygen diffusion limitations within the
support viable and fully functional hepatocytes at a high depth of the carriers.
density. This is a significant challenge as hepatocytes Higher internal porosities ranging from 0.80 to 0.95
generally are poorly able to proliferate in vitro. were reached with the next generation of packing
Recent reviews (Allen et al., 2001; Allen and Bhatia, materials such as disks made of non-woven polyester
2002) highlight the various bioreactor systems that are and polypropylene screen, ceramic spheres and other
being evaluated as BAL devices. These include hollow shapes, glass fibers (Perry and Wang, 1989; Chiou et al.,
fiber reactors, flat plate monolayer culture, perfused PBRs/ 1991), polyurethane and polyvinyl foams or resins. (The
scaffolds and encapsulated cell suspension cultures. PBRs latter are discussed in the section on biomedical appli-
are a good alternative to other types of BAL bioreactors, as cations.) Among these carriers, the Fibra-Cel® proved
they can support high cell densities in a compact volume. quite popular. This disk carrier was developed mainly for
Here we review the packed-bed bioreactors used for PBR applications that involved a high rate of medium
mammalian cell culture and discuss their performance and perfusion (Bohak and Kadouri, 1987; Kadouri, 1994).
main applications. Common features of both bioprocess Since it became commercially available, Fibra-Cel® has
applications and biomedical applications of PBRs are been used widely for mammalian cell culture at labo-
reviewed with a view to identifying the challenges that ratory-scale and pilot industrial-scale. Fibra-Cel® is
must be overcome in developing the next generation of manufactured in conformance with the current Good
improved PBRs. Manufacturing Practice (cGMP) guidelines. The high
porosity of its polyester non-woven fibers and polypro-
2. Applications of PBRs in bioprocessing pylene mesh provides for efficient entrapment of cells and
reduces intra-carrier diffusion limitations. This provides
2.1. Packing materials conditions for attaining a high cell density.
Ceramic pieces of 0.85 to 0.90 void-fraction have also
The early attempts at culturing cells in PBRs focused been used to construct a lab-scale PBR of 3 l packed-bed
on identifying support materials that were compatible volume (Mitsuda et al., 1991). The high porosity of this
with mammalian cells and had the other necessary attri- carrier was shown to improve intra-particle convection
butes identified in Table 1. Solid glass beads for growing and, consequently, minimize oxygen limitations (Park and
cells as monolayers were identified as a suitable material Stephanopoulos, 1993). The physical characteristics of
as early as 1953 (Earle et al., 1953). However, surface this and other carriers are summarized in Table 2.
growth on beads limited the maximal cell density in the In summary, the two matrices that are currently the
bed to ∼ 106 cells·ml− 1 because solid spheres have a very most frequently used for bioprocess PBRs are the
low specific surface-to-volume ratio available for cell SIRAN® and Fibra-Cel® porous carriers. They have
proliferation. This limitation was overcome in the late gained widespread acceptance as they are versatile and
1980s with the introduction of porous glass spheres (e.g. can be used “generically” to entrap both anchorage-de-
SIRAN®) that provided a higher specific surface. The cell pendant cells and cells that would normally grow in
density was increased by about 10-fold and reached up to suspension. These carriers have proved successful with
∼ 107 cells·ml− 1 of packed-bed (Looby and Griffiths, both serum-containing and serum-free media.
1988). However, SIRAN® glass spheres were still limited
by a relatively low internal porosity (εmatrix = 0.56) that 2.2. Packed-bed bioreactor configurations

The PBRs typically consist of a packed-bed that

Table 1 supports the cells on or within carriers and a reservoir that
Requirements of carrier materials for packed-bed bioreactors (Bliem is used to recirculate the oxygenated nutrient medium
et al., 1990) through the bed. Two major configurations are possible
Simple physical configuration and made of non-toxic materials (Fig. 2), with the packed-bed compartment located either
High surface to volume ratio external to, or within, the reservoir of the medium (Wang
Optimal diffusion from the bulk phase to the center of the carrier
et al., 1992a,b). Furthermore, the flow of the medium
Chemical and mechanical stability
Autoclavable through the bed may be arranged to parallel the long-
Suitable for adherent and non-adherent cells itudinal axis of the bed, or the medium may flow radially.
Chemically and biologically inert, no reaction with the product A frequent approach in developing PBRs is to first use
Low cost and reusable if possible a small-scale model bed to identify the optimal packing
Of nonanimal origin
matrix for the cell line of interest. An optimal matrix is one
Table 2
Physical characteristics of various packed-bed carriers
Carrier type and material Size a (mm) εmatrix (-) S/V a, b (× 103 m-1) Reference
Glass Solid glass spheres 3 0 2 (Bliem et al., 1990)
Glass Hollow glass cylinders 9/25 0 0.9 (Moro et al., 1994)
Glass Commercial fiberglass mat 0.02 0.91 15 (Chiou et al., 1991)
Glass Commercial fiberglass mat 0.08/5 0.90 5 (Perry and Wang, 1989)
SIRAN® Macroporous glass bead 1–6 0.56 74 (Fassnacht et al., 2001)
Ceramic pieces 3–6 0.9 – (Mitsuda et al., 1991)
Ceramic cylinders Uniform square channels 0.5/300 – 3.2 (Lyderson et al., 1985)
Cytodex-3 Cross-linked dextran matrix 0.2 – 34 (Ghanem and Shuler, 2000)
coated with denatured collagen
Cellsnow® Cellulose porous cubes charged 3–5 0.95 – (Ong et al., 1994)
with polyethyleneimine (PEI)
Fibra-Cel® Polyester non-woven fiber and polypropylene disks 6/0.5 0.90 119 (Bohak and Kadouri, 1987)
NWPF Polyester non-woven fiber and polypropylene disks 15/0.2 0.90 119 (Petti et al., 1994)
Polyester Polyester non-woven fiber material 0.55 0.94 119 (Kaufmann et al., 2001)
Polyurethane Membrane discs 30/6 0.9 11 (Kurosawa et al., 2000)
Polyurethane Macroporous sponge cubes 0.5 0.9 11 (Lazar et al., 1993)
Polyvinyl fluoride Resin cubes 2 0.8 – (Miyoshi et al., 1996)
BioNOC II™ Polyester strips 5/10 0.94 15 (Hu et al., 2003)
Ceramic Ceramic pieces 3–6 0.90 – (Mitsuda et al., 1991)
Ceramic Cylinder with uniform square channels 0.5/300 0 3.2 (Lyderson et al., 1985)
a
Size (diameter/length), internal porosity (εmatrix) and specific surface-to-volume ratio (S/V).
b
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, achieved with different cell lines are summarized in
proliferation and productivity. This matrix is then used to Table 3.
optimize the operational parameters (e.g. packed-bed
height and volume, medium perfusion rate, linear velocity 2.3. PBR development for bioprocess applications
of the medium across the packed-bed) of the PBR through
perfusion experiments that are generally performed at The first application of PBRs in bioprocessing was
laboratory-scale. the production of foot-and-mouth disease virus using
A great deal of research effort has been invested in BHK cells grown on the surface of solid glass beads for
developing PBRs with enhanced productivity and stab- 4–5 days in batch culture. The support beads were 3 mm
ility. The matrices, operation variables and cell densities 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-depen-
dent and anchorage-independent cells. Cells were suc-
cessfully grown in these reactors in media with and
without serum (Table 3).
Pörtner and coworkers promoted the use of SIRAN®
Fig. 2. Packed-beds with (a) external and (b) internal recirculation of
nutrient medium. The main design variables for the packed-bed are its
spheres for cultivation of hybridomas (Bohmann et al.,
volume (VPBR), height (hPBR) and the linear velocity of the medium at 1995). Fassnacht et al. (2001) scaled-up the SIRAN®
the entrance of the bed (U0). DO = dissolved oxygen sensor. packed-beds to 5.6 l packed-bed volume and also
Table 3
A summary of packed-bed bioreactors used with animal cells
Matrix PBR VPBR hPBR UPBR DPBR Xmax,PBR Run time Cell line Reference
type (l) (cm) (mm·s− 1) (day− 1) (cells·ml−FB1) (days) (product)
Glass External 0.03–3 3.5 0.33 – 1.5 × 106 4–5 BHK (Spier and
Whiteside, 1976)
Beads External 0.03–30 – 0.33 – 1.4 × 106 4–5 BHK (Whiteside and
Spier, 1981)
Beads External 15 15 – 0.4–4 1–5 × 108 40–200 Hybridoma (IgG1) (Bliem et al., 1990)
Hollow Internal 3 – – – – 21–36 Hybridoma (IgG2a) (Moro et al., 1994)
cylinders
Fibres External 0.02 0.6 10 – 0.5–3.2 × 106 17–21 CHO (γ-interferon) (Perry and Wang, 1989)
Fibres Internal 1.2 10 3.7 4 6.8 × 107 66 CHO (γ-interferon) (Chiou et al., 1991)
SIRAN® External 1 – 0.3–0.8 – 1.4×106– 5 GPK, Vero (Looby and
1.4 × 107 Griffiths, 1988)
Internal 0.01–5.6 b30 0.2–1.0 2–4 107–108 b21 Hybridoma (Fassnacht et al., 2001)
Internal 0.04 10.5 0.2–1.0 6.3 8.5 × 107 75 Immortalized mouse (Fassnacht et al., 2001)
hepatocyte (mHep-R1)
External 0.05–0.2 4–9 0.2–1.1 5.7 – 21 Hybridoma (IgG1) (Pörtner et al., 1997)
Internal 0.1–0.2 5–9 0.7 10–20 1.8 × 108 52 Hybridoma (IgG) (Fassnacht and
Pörtner, 1999)
External 0.6 – – – N/D 77 Hybridoma (IgG) (Bohmann et al., 1995)
Internal 0.05 4.0 0.1–0.8 – N/D 96 Hybridoma (IgG) (Bohmann et al., 1992)
External 1 – 0.3–3.3 – 4 × 107 18 Hybridoma (IgG) (Racher et al.,
1990a,b, 1993)
External 0.1 – 1.2–4.6 3–10 1.8–2.3 × 107 100 BHK (IgG) (Griffiths and
Racher, 1994; Racher
and Griffiths, 1993)
External 0.4 – – – 1.1 × 108 14 Human hepatocarcinoma (Kawada et al., 1998)
cells (FLC-7)
Fibra-Cel® Internal 1.75 – 5.2 3 × 107 35 CHO (r-hEPO) (Jixian et al., 1998)
– – – – – 2.4 × 107 – CHO (Ducommun
3.6 × 107 et al., 2002a,b)
Internal 0.5–1.0 14.6 ∼ 10 3–4 1.0–1.2 × 108 30 Hybridoma (IgG1) (Wang et al., 1992a,b)
External
External 0.5 30.5 2.0 4 108 46 Hybridoma (IgG1) (Wang et al., 1992a,b)
– – 14.6 – – – 10 HeLa (Hu et al., 2000)
External 0.05 – – – 6 × 106 8 Insect (β-galactosidase) (Kompier et al., 1991)
– – – – – 1 × 108 26 MRC-5 human lung (Petti et al., 1994)
diploid fibroblasts
Ceramic 0.01 1–5 – – 5.1 × 108 40 Rat pituitary (Park and
Stephanopoulos, 1993)
External 2.6 15 0.02–0.08 0.5–1.6 3.3 × 105 40 Human embryonic (Mitsuda et al., 1991)
lung diploid fibroblast
IMR-90 cells (t-PA)
External 1–5 30 1–5 – 1.8 × 107 7–8 10 different cell lines (Lyderson et al., 1985)
Cellsnow® External 0.05 4 0.4 – 5 × 107 – Hybridoma (Ong et al., 1994)
Cytodex 3 External 0.05–0.20 – – – – – Rat hepatoma (H4IIE) (Ghanem and
Rat lung (L2) Shuler, 2000)
NWF External 0.05–0.35 – – – 2–5 × 107 b6 Porcine hepatocytes (Flendrig et al.,
1997; Naruse et al.,
1996, 1998, 2001)
PUM External 0.032 – – – 1–3 × 107 7 Rat hepatocytes (Kurosawa et al., 2000)
External 0.0032 – 0.47 – 1–5 × 107 7 Rat hepatocytes (Kurosawa et al., 2000)
Internal 0.6 – – 1 6 × 106 19 HEK 293 (Lazar et al., 1993)
PUF External 0.01–0.3 6–17 – – 1.0 × 107 1–3 Dog hepatocytes (Ijima et al., 2000a,b)
External 0.26 – – 1 6.8 × 107 25 HEK 293 (tPA) (Kawakubo et al., 1994)
External 0.03 – – Batch 2.5 × 106 7 Hybridoma (Murdin et al., 1989)
PVF External 0.02 0.55 – – 5 × 106 9 Rat hepatocytes (Miyoshi et al., 1996)
proposed a design for an industrial-scale reactor. The If we assume cells to be spherical and packed as a bed
proposed system consisted of an internal PBR of 84.5 l of cells, the maximum attainable volume fraction of cells
volume that was placed inside a 300 l bioreactor but the in the bed would be 0.74 and, therefore, the maximal cell
feasibility of this approach was not tested in culture density Xmax could be estimated using the following
conditions. equation:
At Baxter, Bliem et al. (1990) followed the same  
4 3
approach to design a 14 l external PBR of glass beads. Xmax ¼ 0:74= prcell ð1Þ
The system was used for producing immunoglobulins 3
over 40 days using perfused cultures of hybridomas.
where r is the radius of the cell. Because mammalian cells
The authors suggested that a further four-fold scale-up
have an average diameter in the range of 12–15 μm, the
was feasible, to bring the packed-bed volume to 56 l.
Xmax for PBRs is in the range of 4–8 × 108 cells·ml− 1.
The scale-up strategy was not actually tested.
Maximal cell densities of up to 1–5 × 108 cells·ml− 1 have
Use of polyester packing materials of various shapes
actually been already reported (Table 3) and, therefore,
(e.g. disks, strips, fibers) has been reported by many
further increases are unlikely.
sources in PBRs with packed-bed volumes that have
In establishing the maximum size that packed-beds can
ranged from 0.05 to 1.75 l (Table 3). Because of their
be scaled-up to, we need to understand that the main
mesh-like structure, polyester fibers are capable of
limitation on increasing the height of the bed originates
easily supporting both attached and entrapped cells and
from the unavoidable occurrence of axial gradients in
have proved successful with many different combina-
concentrations of nutrients. The nutrient that generally
tions of cells and culture media.
limits the depth is oxygen, as the maximum attainable
Bioreactor manufacturers have developed commer-
concentration of oxygen at the inlet of the bed depends on
cial PBRs that can accommodate many different types
the solubility of oxygen which is quite low. Of course the
of carriers. Most of these PBRs are of the internal
oxygen concentration anywhere in the bed must not fall
configuration. The CelliGen Plus® system (New
below the critical level that would jeopardize survival of
Brunswick Scientific; www.nbsc.com) was primarily
the cells and their ability to produce the desired protein.
developed for use in combination with Fibra-Cel®
Models have been developed to estimate the axial
polyester disk carriers and has been scaled-up from
gradients in dissolved oxygen in packed-beds (Chisti
0.7 l to 5 l packed-bed volume. The TideCell®
and Moo-Young, 1994; Fassnacht et al., 2001). The depth
bioreactors (CESCO Bioengineering Co. Ltd.; www.
of the bed (hPBR) depends on the superficial fluid velocity
cescobio.com.tw) are available in 5 and 25 l sizes and
(U0) through it, the cell density XPBR and the specific
have been designed to operate with an internal
oxygen consumption rate (qO2), as follows (Chisti and
packed-bed of BioNOC II® polyester strips carriers.
Moo-Young, 1994; Fassnacht et al., 2001):
The Swiss company Bioengineering AG (www.
bioengineering.ch), has also developed internal PBRs
U0 COin2 −COout2
that can be placed within bioreactors and offer fixed hPBR ¼ d ð2Þ
ePBR qO2 dXPBR
bed volumes from 0.9 to 1.2 l.
In summary, although a large variety of PBR In Eq. (2), cOin and cOout are the oxygen concentration
2 2
systems have been assessed successfully in the values at the inlet and outlet of the bed, respectively. In
laboratory (Table 3), few pilot-scale and industrial Eq. (2) the bed porosity εPBR considers both the space
installations have been described. Indeed, most of occupied by the matrix and by the cells; thus,
published data deals with PBRs of less than 5 l of
packed-bed volume. Only a few systems have been XPBR
scaled-up to above 5 l. The current maximal packed- ePBR ¼ ematrix −ecells ¼ ematrix −0:74d ð3Þ
Xmax
bed volume appears to be in the range of 10–30 l.
where εmatrix and εcell are porosity of the matrix and
2.4. Limitations and prospects for improved PBRs for volume fraction of cells in bed, respectively.
bioprocess applications If we assume that the PBR operates within a range of
oxygen concentrations such that b 80% of oxygen satu-
Assessing a maximal potentially attainable perfor- ration and cOout
2
remains above 20%, the cells have a
mance for PBRs requires a knowledge of the maximum constant specific oxygen consumption rate (qO2) of
cell density that can be attained in the bed and the 2 × 10− 13 mol·cell− 1 h− 1 (Ruffieux et al., 1998), the
maximum size that a PBR can be scaled-up to. packing is highly porous (εmatrix = 0.90), and the cells are
evenly distributed in the bed at an average cell density of Assuming that the depth of bed can vary between 5 and
XPBR, the depth of bed can be estimated using Eq. (2). 30 cm and the maximum diameter cannot exceed 2 m, the
Fig. 3 shows plots of the calculated maximal depth range of reasonable volumes for the bed works out to be
(hPBR) of the PBRs for various values of the immobilized from 0.2 to 0.9 m3. PBRs of this size range operating with
cell density XPBR (1 × 108 b XPBR b 5 × 108 cells·ml− 1) and ∼ 108 cells·ml− 1 would have a productivity roughly
superficial velocity U0 (0.2 b U0 b 1.0 cm·s− 1) of the equivalent to that of a 2–9 m3 fed-batch bioreactor
medium. The U0 and XPBR values in Fig. 3 spanned the operating at ∼ 107 cells·ml− 1. Such scaled-up PBRs can
maximum and minimum values of these variables as be quite competitive with conventional bioreactors, for
previously published in the literature (Table 3). From producing therapeutic proteins that are needed in rela-
Fig. 3, we can deduce that the maximal PBR depth that tively small quantities. If the required bioreactor volume
can be achieved under the specified conditions is in the exceeds the limits established here, a PBR would not be
range of 5 b hPBR b 30 cm. Indeed, values of hPBR pub- technically feasible for the specific application.
lished in Table 3 correspond well to these values.
The maximum diameter of a packed-bed is limited by 3. PBRs as bioartificial organs and tissues
the ability to uniformly distribute the flow over the entire
cross-section to prevent nonhomogeneities and channeling. The use of PBRs in biomedical applications began in
The problem of achieving uniformity of flow over the the 1990s. Mainly, the focus has been in using PBRs to
cross-sectional area occurs also in packed-bed chromatog- culture immobilized hepatocytes as a bioartificial liver
raphy and has been investigated in some detail in relation to device (BAL). Hepatocytes proliferate poorly in vitro
chromatography. Chromatography columns used for (Ohshima et al., 1997); therefore, optimization of cul-
protein capture commonly have the same range of packing ture conditions, packing materials and entrapment pro-
sizes (i.e. particles of 0.2 to 2 mm in diameter) and linear cedures is important for supporting a large total number
flow velocities (i.e. 0.1 bU0 b 0.3 cm·s− 1) (Amersham of immobilized hepatocytes in PBRs.
Biosciences, 2003), as encountered in packed-beds of As for bioprocess applications, the initial attempts to
immobilized animal cells. Furthermore, because the culture hepatocytes in PBRs were made using non-porous
dispersion coefficient in PBRs is relatively constant for a glass beads as the packing material. The first such PBR
wide range of particle sizes and linear velocities (Leven- consisted of a 30 ml packed-bed BAL with glass beads of
spiel, 1999), PBRs are expected to behave similarly to 1.5 mm in diameter. This system was good at cell entrap-
large-scale chromatography columns. Columns for indus- ment (N80% immobilization efficiency) and allowed
trial chromatography are successfully operated with culture of metabolically active hepatocytes for more than
diameters as large as 2 m. Such large columns are 14 days (Li et al., 1993). In later designs, the solid glass
commercially available from companies such as Millipore beads were replaced with porous packing materials that
(www.millipore.com). Clearly, therefore, there is substan- supported even higher cell densities. Various materials
tial scope for increasing the diameter of PBRs compared with high porosities have been used to construct bio-
with the diameters that have been used in the past (Table 3). medical PBRs, as listed in Table 3.
Immobilization matrices such as polyurethane mem-
branes (PUM) have heterogeneous pores of about 100 μm
average diameter that facilitate cell immobilization with
up to 99% efficiency and support cell densities ranging
from 1 × 107 to 5 × 107 cells·ml- 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 cells·ml− 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
Fig. 3. Estimated values of the maximal packed-bed depth (hPBR) in
(Kurosawa et al., 2000), porous microcarriers (Ghanem
packed-bed bioreactors, as a function of the cell density (XPBR), for and Shuler, 2000), polyester strips (Hu et al., 2003) and
different values of superficial fluid velocity (U0). polyurethane foams (PUF).
 Concurrently with advances in matrix design, methods the hepatocytes expression level could be maintained for
were optimized for immobilizing hepatocytes on and up to 1 week in serum-free medium, but declined during
within them (Li et al., 1993; Miyoshi et al., 1996; the second week of culture. Another BAL system based
Kurosawa et al., 2000). These improvements enhanced on polyurethane materials was developed by Ijima et al.
the cell attachment yield from 10% in early trials to 40% (2000a,b). Hydrophilic polyurethane foam was used to
and, in some cases, as much as 99% (Yang et al., 2001). make packed-beds of 14.5 ml and 300 ml for in vitro and
Consequently, the maximum cell densities of immobi- in vivo studies, respectively. The in vivo work was
lized hepatocytes were increased from 106 cells·ml− 1 in carried out in dogs (Ijima et al., 2000b). The 300 ml
the early stages of BAL development to the current levels BAL device containing 30 g of hepatocytes (i.e. cell
of 107–108 cells·ml− 1 of PBR matrix volume. density ∼ 107 cells·ml− 1) was shown to extend the
survival rate of dogs afflicted with liver failure. The
3.1. Bioartificial liver (BAL) BAL module had to be freshly prepared (not more than
1 day old) to be effective.
Both the internal and external packed-bed bioreactor Another BAL module used non-woven polyester
configurations have been used for PBRs in biomedical fibers for cell immobilization and had internal tubing for
applications such as bioartificial liver device (BAL). A direct oxygenation of the hepatocytes. This module at-
BAL is generally connected to the patient as an extra- tained a density of 4 × 107 cells·ml− 1. This BAL device
corporeal BAL as shown in Fig. 4. A number of BAL was initially tested in mice (Flendrig et al., 1997) and
PBRs have been reported (Table 3). later scaled-up to a 400 ml packed-bed BAL for a pre-
Use of polyvinyl fluoride (PVF) cubes for primary clinical trials in pigs with liver ischemia.
culture of hepatocytes in a small PBR (2–4 ml packed-bed Naruse et al. (1996) developed a BAL device based
volume) has been reported (Yanagi et al., 1992). These on packed-bed of polyester matrix. These modules at-
beds were shown to reach quite high cell densities (from tained a density ranging from 2 × 107 cells·ml− 1 (50 ml
4 × 106 to 1.2 × 107 cells·ml− 1) during short-term cultures device) up to 5 × 107 cells·ml− 1 (200 ml device) and
(b 26 h). Transport of nutrients and oxygen to immobi- could support hepatocytes with reasonably high meta-
lized cells in the beds were identified as being critical to bolic activity over a period of 6 days. These BAL
maintaining the metabolic activity of cells in vitro. Further devices were further scaled-up and successfully tested in
trials demonstrated that hepatocytes cultured in serum animal models (Naruse et al., 1998, 2001).
containing medium for up to 9 days retained metabolic BAL devices with porous microcarriers have been
functionality that was comparable to the cells in vivo tested successfully. For example, a cell density of 8.5 × 10-
(Miyoshi et al., 1996; Ohshima et al., 1997). However, 7
cells·ml− 1 was attained using immortalized human
stable operation in serum-free medium and scale-up to a hepatocytes grown on a Cellsnow™ matrix. The bio-
volume that would allow its use in pre-clinical and clinical reactor used in this work was an internal PBR with a 40 ml
trials were not demonstrated. packed-bed volume (Fassnacht et al., 2001). The cells
PBRs based on polyurethane membrane (PUM) have stably retained metabolic activity over the 40-day culture
been used to culture hepatocytes (Kurosawa et al., (Fassnacht et al., 2001).
2000). At the optimum density of 2.5 × 107 cells·ml− 1, The highest hepatocytes cell density reported in PBRs
has been 1.1 × 108 cells·ml− 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 standard-
ized process has been developed for culturing hepato-
cytes 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 As a consequence of their ability to support mamma-
a patient as an extra corporeal bioartificial liver device (BAL). lian cells under tissue-like conditions, PBRs can be used
for drugs toxicology testing, where the cells' response to a Technologies have been developed to consistently
test-compound is evaluated under conditions comparable attain a density of immobilized cells in PBRs that is
to those in vivo. For example, Ghanem and Shuler (2000) comparable to the density of hepatocytes in the liver
developed a model system that mimicked the lung and (Table 3). Use of porous packing materials is recom-
liver functions of an adult rat. This system was made of mended for attaining the requisite cell density without
three PBR compartments that represented the lung, the clogging the bed. A number of other objectives must be
liver and the other tissues, respectively. The compart- attained for developing the next generation of improved
ments were configured as a series of packed-beds with cell biomedical devices based on PBR technology. These
culture medium exchanged between compartments at include the following:
physiological flow rates, enabling interactions of the
“organs” and to mimic the whole animal. This system has 1. A cell line with stable expression and good ability to
been used to test the toxicity of chemicals on lung and grow in vitro needs to be identified. Ongoing work
liver functions. on this aspect was recently reviewed by Allen and
Another system, based on a radial-flow PBR was Bhatia (2002). With most of the cell lines that are
used to cultivate human hepatocyte-derived cells available currently, metabolic activity is lost within
(HepG2) for possible use in in vitro evaluation of 2 weeks. This has been attributed to unstable cell
toxicity of drugs (Nagamori et al., 2000). Developing phenotype and the inability to supply the cells with
bioreactor systems that mimic with fidelity the com- oxygen and other nutrients. To be viable in clinical
plexity of animal metabolism is challenging. Individual applications, a BAL device should ideally be capable
bioreactor compartments generally support a single type of functioning for a typical 30-day treatment period
of cell and do not reproduce the complexity of any single (Ambrosino and D'Amico, 2003).
organ, the interactions of organs and regulation of 2. Optimization of nutrients/oxygen supply in the PBRs to
metabolism. This problem notwithstanding suitably keep the cells viable and productive for at least 30 days
configured PBRs do offer an alternative to the use of that are desired for clinical application. This appears to
whole animals and can potentially provide useful be possible, as cells have been maintained at high
information about the effect of chemicals on in vivo densities in PBRs for extended periods, at least in a few
metabolism. cases. For example, a density of 1.1 × 108 cells·ml− 1
was maintained for 14–40 days by Kawada et al.
3.3. Limitations and development prospects for im- (1998) and 8.5 × 107 cells·ml− 1 were maintained for
proved biomedical PBRs 40 days by Fassnacht et al. (2001). These reports are
however an exception to the norm (e.g. Yang et al.,
The maximum cell densities that have been reported 2001; Ducommun et al., 2002a,b).
(Table 3) for biomedical PBRs are generally lower (∼ 107 3. Scale-up the packed-bed volume to about 1–2 l, but
cells·ml− 1) than for bioprocess PBRs (∼ 108 cells·ml− 1). not further in order to provide a compact BAL device
This reflects the difficulty in growing hepatocytes to high for convenient clinical use. The largest biomedical
densities. Because the cell densities obtained in vitro are PBR device that has been reported had a volume of
currently generally 10-fold lower than the densities ob- 400 ml (Table 3). This objective is still out of reach;
served in the native liver organ (i.e. 108 cells·ml− 1), a however, the technical feasibility of such a scale-up
BAL device of 10–20 l would be needed to provide the has been proved with PBRs in various bioprocess
functionality of an adult liver. applications with animal cells.
The BAL devices under clinical evaluation (mainly
hollow-fiber technology) generally have a maximum vol- 4. Concluding remarks
ume of 1 l and attain a total of 1010 immobilized hepa-
tocytes, or merely 10% of the total cell number in an adult The latest generation of animal cell culture packed-
human liver (Allen and Bhatia, 2002). Although it is bed bioreactors (PBRs) have achieved cells densities in
generally accepted that 10% of the total cell number the range of 1 × 108 to 5 × 108 cells·ml− 1. These values
existing in a real liver would suffice in replacing liver are close to the maximum density of ∼ 8 × 108 cells·ml− 1
function in many urgent clinical applications (Yang that can be attained theoretically if the cells are packed
et al., 2001; Ijima et al., 2000a), this situation is not in a bed as spheres.
ideal and there is a need to develop compact BAL In bioprocess applications, the largest PBRs reported
devices that provide the full functionality of an adult had a maximal volume of 30 l. This is a small fraction of
liver. the bed sizes that are commonly used in nonanimal cell
culture bioprocesses such as in wastewater treatment and PVF Polyvinyl fluoride resin
biotransformations with immobilized enzymes. A chal- qO 2 Specific oxygen consumption rate of the cells
lenge therefore is to demonstrate the scalability of packed- rcell Radius of cell
bed technology in animal cell culture applications. U0 Superficial velocity of circulating fluid before
A major limitation to further scale-up originates in the the packed-bed
existence of substantial axial gradients in concentrations U Superficial velocity of circulating fluid in the
of nutrients such as oxygen. This limits packed-bed depth packed-bed
to ∼ 30 cm. The bed diameter is generally limited VPBR Packed-bed volume
to ∼ 2 m, as uniform distribution of nutrient fluid over the Xmax Maximum cell density in the bed
bed cross-section becomes difficult with further increase XPBR Viable cell density (in cells per unit packed-bed
in diameter. In view of these limitations, the maximum volume)
estimated volume for PBRs appears to be in the range of εint Packing material internal porosity
0.2–0.9 m3. Notwithstanding their limited potential for εPBR Packed-bed porosity
scale-up, PBRs can be quite competitive with other εmatrix Porosity of carrier matrix
bioreactor systems in producing proteins (e.g. cytokines εcells Volume fraction of cells in the bed
or hormones) that are needed in relatively small quan- ∅ Packing material diameter
tities. This is because of the exceptionally high volumetric
productivity of the PBRs. When large quantity of a protein
References
must be produced, suspension culture in large fed-batch or
perfusion bioreactors of 10–20 m3 may be the only Allen JW, Hassanein T, Bhatia SN. Advances in bioartificial liver
realistic option. devices. Hepatology 2001;34:447–55.
In biomedical applications of PBRs, the maximal cell Allen JW, Bhatia SN. Improving the next generation of bioartificial
densities have been generally relatively low (e.g. ∼ 107 liver devices. Cell & Dev Biol 2002;13:447–54.
cells·ml− 1) compared with the bioprocess PBRs. How- Ambrosino G, D'Amico DF. Bioartificial liver support. Minerva Chir
2003;58:649–56.
ever, replicating the performance of a whole adult liver in Amersham Biosciences. Protein Purification Handbook; 2003. 27–34 pp.
a compact volume of 1–2 l would require attaining a Bliem R, Oakley R, Matsuoka K, Varecka R, Taiariol V. Antibody
hepatocyte cell density of at least 108 cells·ml− 1. production in packed bed reactors using serum-free and protein-
Developing a hepatocyte cell line that consistently attains free medium. Cytotechnology 1990;4:279–83.
these densities in vitro remains a challenge. Furthermore, Bohak Z, Kadouri A. Novel anchorage matrices for suspension culture
of mammalian cells. Biopolymers 1987;26:S205–13.
any cell line for use in bioartificial organs must stably Bohmann A, Pörtner R, Schmieding J, Kasche V, Märkl H. The
retain functionality for at least 30 days to be useful membrane dialysis bioreactor with integrated radial-flow fixed bed—
in clinical applications. At present, metabolic activ- a new approach for continuous cultivation of animal cells. Cyto-
ity of in vitro cultured cells typically declines within technology 1992;9:51–7.
1–2 weeks of initiation, to unacceptably low levels. Bohmann A, Pörtner R, Märkl H. Performance of a membrane-dialysis
bioreactor with a radial-flow fixed bed for the cultivation of a
Once stable cell lines are available, scale-up of bio- hybridoma cell line. Appl Microbiol Biotechnol 1995;43:772–80.
medical PBRs to desired size of 1–2 l should not be Brotherton JD, Chau PC. Modeling of axial-flow hollow fiber cell
a problem, as these devices have been already scaled- culture bioreactors. Biotechnol Prog 1996;12:575–90.
up to much larger volumes in animal cell culture bio- Chiou T-W, Murakami S, Wang DIC. A fiber-bed bioreactor for
anchorage-dependent animal cell cultures: Part I. Bioreactor design
process applications.
and operations. Biotechnol Bioeng 1991;37:755–61.
Chisti Y. Animal cell culture in stirred bioreactors: observations on
Nomenclature scale-up. BioProcess Eng 1993;9:191–6.
BAL Bioartificial liver Chisti Y. Hydrodynamic damage to animal cells. Crit Rev Biotechnol
cOin
2
Oxygen concentration at PBR inlet 2001;21:67-110.
cOout Oxygen concentration at PBR outlet Chisti Y, Moo-Young M. Anchorage dependent animal cell culture in
2
packed beds with airlift driven liquid circulation: a theoretical
DPBR Medium dilution rate (in medium volume per analysis of oxygen transfer and comparison with stirred tank
packed-bed volume per day) microcarrier culture system. Trans Inst Chem Eng 1994;72C:92–4.
h Packing material height Ducommun P, Kadouri A, von Stockar U, Marison IW. On-line
hPBR Packed-bed depth determination of animal cell concentration in two industrial high-
NWF Non-woven polyester fabric density culture processes by dielectric spectroscopy. Biotechnol
Bioeng 2002a;77:316–23.
PBR Packed-bed bioreactor Ducommun P, Ruffieux P-A, von Stockar U, Marison IW. Monitoring
PUF Polyurethane foam of temperature effects on animal cell metabolism in a packed bed
PUM Polyurethane membrane process. Biotechnol Bioeng 2002b;77:838–42.
Earle WR, Bryant JC, Schilling EL. Certain factors limiting the size of enhanced hepatocarcinoma cell lines. In Vitro Cell Dev Biol, Anim
the tissue culture and the development of massive cultures. Ann 1998;34:109–15.
NY Acad Sci 1953;58:1000–11. Kawakubo Y, Matsushita T, Funatsu K. Tissue plasminogen activator
Fassnacht D, Pörtner R. Experimental and theoretical considerations (t-PA) production by a high density culture of weakly adherent
on oxygen supply for animal cell growth in fixed-bed reactors. human embryonic kidney cells using a polyurethane-foam packed-
J Biotechnol 1999;72:169–84. bed culture system. Appl Microbiol Biotechnol 1994;41:413–8.
Fassnacht D, Reimann I, Pörtner R, Markl H. Scale-up of fixed bed Kompier R, Kislev N, Segal I, Kadouri A. Use of a stationary bed
reactors for cultivating animal cells. Chemie Ingenieur Technik, reactor and serum-free medium for the production of recombinant
vol. 73; 2001. p. 1075–9. proteins in insect cells. Enzyme Microb Technol 1991;13:822–7.
Flendrig LM, Velde AA, Chamuleau RAFM. Semipermeable hollow Kretzmer G. Industrial processes with animal cells. Appl Biochem
fiber membranes in hepatocyte bioreactors: a prerequisite for a Biotechnol 2002;59:135–42.
successful bioartificial liver? Artif Organs 1997;21:1177–81. Kurosawa H, Yasumoto K, Kimura T, Amano Y. Polyurethane
Gavrilescu M, Chisti Y. Biotechnology—a sustainable alternative for membrane as an efficient immobilization carrier for high-density
chemical industry. Biotechnol Adv 2005;23:471–99. culture of rat hepatocytes in the fixed-bed reactor. Biotechnol
Ghanem A, Shuler ML. Characterization of a perfusion reactor Bioeng 2000;70:160–6.
utilizing mammalian cells on microcarrier beads. Biotechnol Prog Lazar A, Reuveny S, Kronman C, Velan B, Shafferman A. Evaluation of
2000;16:471–9. anchorage-dependent cell propagation systems for production of
Gòdia F, Sola C. Fluidized-bed bioreactors. Biotechnol Prog human acetylcholinesterase by recombinant 293 cells. Cytotechnol-
1995;11:479–97. ogy 1993;13:115–23.
Goochee CF, Gramer MJ, Andersen DC, Bahr JB, Rasmussen JR. The Levenspiel O. Chemical Reaction Engineering. 3rd ed. New York:
oligosaccharides of glycoproteins: bioprocess factors affecting Wiley; 1999.
oligosaccharide structure and their effect on glycoprotein proper- Li AP, Barker G, Beck D, Colburn S, Monsell R, Pellegrin C.
ties. Biotechnology (NY) 1991;9:1347–55. Culturing of primary hepatocytes as entrapped aggregates in a
Griffiths JB, Racher AJ. Cultural and physiological factors affecting packed bed bioreactor: a potential bioartificial liver. In Vitro Cell
expression of recombinant proteins. Cytotechnology 1994;15:3–9. Dev Biol, Anim 1993;29A:249–54.
Hu W-S, Aunins JG. Large-scale mammalian cell culture. Curr Opin Looby D, Griffiths JB. Fixed bed porous glass sphere (porosphere)
Biotechnol 1997;8:148–53. bioreactors for animal cells. Cytotechnology 1988;1:339–46.
Hu W-S, Peshwa MV. Animal cell bioreactors—recent advances and Lyderson BK, Pugh GC, Paris MS, Avender P, Sherma BH, Noll LA.
challenges to scale-up. Can J Chem Eng 1991;69:409–20. Ceramic matrix for large scale animal cell culture. Bio/technology
Hu Y-C, Kaufman J, Cho MW, Golding H, Shiloach J. Production of 1985;3:62–6.
HIV-1 gp120 in packed-bed bioreactor using the vaccinia virus/T7 Mitsuda S, Matsuda Y, Kobayashi N, Suzuki A, Itagaki Y, Kumazawa E,
expression system. Biotechnol Prog 2000;16:744–50. et al. Continuous production of tissue plasminogen activator (t-PA)
Hu Y-C, Lu J-T, Chung Y-C. High-density cultivation of insect cells by human embryonic lung diploid fibroblast, IMR-90 cells, using
and production of recombinant baculovirus using a novel ceramic bed reactor. Cytotechnology 1991;6:23–31.
oscillating bioreactor. Cytotechnology 2003;42:145–53. Miyoshi H, Yanagi K, Fukuda H, Ohshima N. Long-term performance
Ijima H, Nakazawa K, Koyama S, Kaneko M, Matsushita T, Gion T, et al. of albumin secretion of hepatocytes cultures in a packed-bed
Development of a hybrid artificial liver using a polyurethane foam/ reactor utilizing porous resin. Artif Organs 1996;20:803–7.
hepatocyte-spheroid packed-bed module. Int J Artif Organs Moro AM, Alves Rodrigues MT, Gouvea MN, Zucheran Silvestri
2000I;23:389–97. ML, Kalil JE, Raw I. Multiparametric analysis of hybridoma
Ijima H, Nakazawa K, Koyama S, Kaneko M, Matsushita T, Gion T, et growth on glass cylinders in a packed-bed bioreactor system with
al. Conditions required for a hybrid artificial liver support system internal aeration. Serum-supplemented and serum-free media
using a PUF/hepatocyte-spheroid packed-bed module and it's use comparison for MAb production. J Immunol Methods 1994;176:
in dogs with liver failure. Int J Artif Organs 2000b;23:446–53. 67–77.
Jenkins N, Curling EMA. Glycosylation of recombinant proteins: Morrow KJ. Antibody production. Genet Eng News 2001;21:1-77.
problems and prospects. Enzyme Microb Technol 1994;16: Murdin AD, Spier RE, Groves DJ. Growth and metabolism of
354–64. hybridomas immobilized in packed beds: comparison with static
Jenkins N, Parekh RB, James DC. Getting the glycosylation right: and suspension cultures. Enzyme Microb Technol 1989;11: 341–6.
implications for the biotechnology industry. Nat Biotechnol Nagamori S, Hasumura S, Matsuura T, Aizaki H, Kawada M.
1996;14:975–81. Developments in bioartificial liver research: concepts, perfor-
Jixian D, Qin Y, Xuan C, Jiang Z. Production of rhEPO with a serum- mance, and applications. J Gastroenterol 2000;35:493–503.
free medium in the packed bed bioreactor. Chin J Biotechnol Naruse K, Sakai Y, Nagashima I, Jiang GX, Suzuki M, Muto T.
1998;13:247–52. Development of a novel bioartificial liver module filled with
Kadouri A. Cultivation of anchorage-dependent mammalian cells and porcine hepatocytes immobilized on a non-woven fabric. Int J Artif
production of various metabolites. Colloids Surf, B Biointerfaces Organs 1996;19:347–52.
1994;2:265–72. Naruse K, Nagashima I, Sakai Y, Harihara Y, Jiang GX, Suzuki M, et al.
Kaufmann H, Mazur X, Marone R, Bailey JE, Fussenegger M. Efficacy of a bioreactor filled with porcine hepatocytes immobilized
Comparative analysis of two controlled proliferation strategies on a nonwoven fabric for ex vivo direct hemoperfusion treatment of
regarding product quality, influence on tetracycline-regulated gene liver failure in pigs. Artif Organs 1998;22:1031–7.
expression, and productivity. Biotechnol Bioeng 2001;72:592–602. Naruse K, Sakai T, Lei G, Sakamoto Y, Kobayashi T, Puliatti C, et al.
Kawada M, Nagamori S, Aizaki H, Fukaya K, Niiya M, Matsuura T, et al. Efficacy of nonwoven fabric bioreactor immobilized with porcine
Massive culture of human liver cancer cells in a newly developed hepatocytes for ex vivo xenogeneic perfusion treatment of liver
radial flow bioreactor system: ultrafine structure of functionally failure in dogs. Artif Organs 2001;25:273–80.
Ohshima N, Yanagi K, Miyoshi H. Packed-bed type reactor to attain Spier RE, Whiteside JP. The production of foot and mouth disease
high density culture of hepatocytes for use as a bioartificial liver. virus from BHK 21 C13 cells grown on the surface of glass
Artif Organs 1997;21:1169–76. spheres. Biotechnol Bioeng 1976;18:649–67.
Ong CP, Pörtner R, Märkl H, Yamazaki Y, Yasuda K, Matsumura M. Stapakis J, Stamm E, Townsend R, Thickman D. Liver volume
High density cultivation of hybridoma in charged porous carriers. assessment by conventional vs. helical CT. Abdom Imaging
J Biotechnol 1994;34:259–68. 1995;20:209–10.
Ozturk SS. Engineering challenges in high density cell culture Varley J, Birch J. Reactor design for large scale suspension animal cell
systems. Cytotechnology 1996;22:3-16. culture. Cytotechnology 1999;29:177–205.
Petti SA, Lages AC, Sussman MV. Three-dimensional mammalian cell Voisard D, Meuwly F, Baer G, Kadouri A. Potential of cell retention
growth on nonwoven polyester fabric disks. Biotechnol Prog techniques for large-scale high-density perfusion culture of
1994;10:548–50. suspended mammalian cells. Biotechnol Bioeng 2003;82: 751–65.
Park S, Stephanopoulos G. Packed bed bioreactor with porous ceramic Wang G, Zhang W, Jacklin C, Freedman D, Eppstein L, Kadouri A.
beads for animal cell culture. Biotechnol Bioeng 1993;41:25–34. Modified CelliGen-packed bed bioreactors for hybridoma cell
Perry ST, Wang DIC. Fiber bed reactor design for animal cell culture. cultures. Cytotechnology 1992a;9:41–9.
Biotechnol Bioeng 1989;34:1–9. Wang G, Zhang W, Freedman D, Eppstein L, Kadouri A. Animal cell
Pörtner R, Rössing S, Koop M, Lüdemann I. Kinetic studies on technology: developments: process and products. In: Spier RE,
hybridoma cells immobilized in fixed bed reactors. Biotechnol Griffiths G, MacDonald D, editors. Continuous production of
Bioeng 1997;55:535–41. monoclonal antibodies in CelliGen packed bed reactor using Fibra-
Racher AJ, Looby D, Griffiths JB. Studies on monoclonal antibody cel carrier. Oxford: Butterworth-Heinemann; 1992b. p. 460–4.
production by a hybridoma cell line (C1E3) immobilised in a fixed- Whiteside JP, Spier RE. The scale-up from 0.1 to 100 liter of a unit
bed, porosphere culture system. J Biotechnol 1990a;15:129–46. process system based on a 3 mm-diameter glass spheres for the
Racher AJ, Looby D, Griffiths JB. Use of lactate dehydrogenase release production of four strains of FMDV from BHK mono layer cells.
to assess changes in culture viability. Cytotechnology 1990b;3: Biotechnol Bioeng 1981;23:551–65.
301–7. Yanagi K, Miyoshi H, Fukuda H, Ohshima N. A packed-bed reactor
Racher AJ, Looby D, Griffiths JB. Influence of ammonium ion and utilizing porous resin enables high density culture of hepatocytes.
glucose on MAb production in suspension and fixed bed Appl Microbiol Biotechnol 1992;37:316–20.
hybridoma cultures. J Biotechnol 1993;29:145–56. Yang TH, Miyoshi H, Ohshima N. Novel cell immobilization
Racher AJ, Griffiths JB. Investigation of parameters affecting a fixed method utilizing centrifugal force to achieve high-density
bed bioreactor process for recombinant cell lines. Cytotechnology hepatocyte culture in porous scaffold. J Biomed Mater Res
1993;13:125–31. 2001;55:379–86.
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;63:85–95.