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
Mammalian cells are widely used to produce recom-
binant glycoproteins such as hormones, enzymes, cyto-
kines and antibodies for human therapy. Mammalian cells
are the preferred expression system for making recombi-
nant 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; Gòdia 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. 10–20 m3)
to compensate for the relatively low cell densities that are
attained. Typically, the cell density in suspension culture
is between 106 and 107 cells·ml− 1. Compared to batch
culture in stirred tanks, nearly 10-fold higher cell densities
(i.e. 107–108 cells·ml− 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 reten-
tion 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 exam-
ple 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 cells·ml− 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 attri-
butes 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 cells·ml− 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 cells·ml− 1 of packed-bed (Looby and Griffiths,
1988). However, SIRAN® glass spheres were still limited
by a relatively low internal porosity (εmatrix = 0.56) that
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
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 appli-
cations.) 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 labo-
ratory-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 polypro-
pylene 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-de-
pendant 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 long-
itudinal 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 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,
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 stab-
ility. The matrices, operation variables and cell densities
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.
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
4–5 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-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®
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
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
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 combina-
tions of cells and culture media.
Bioreactor manufacturers have developed commer-
cial 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 packed-
bed volume appears to be in the range of 10–30 l.
2.4. Limitations and prospects for improved PBRs for
bioprocess applications
Assessing a maximal potentially attainable perfor-
mance 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 ¼ 0:74= prcell ð1Þ
3
where r is the radius of the cell. Because mammalian cells
have an average diameter in the range of 12–15 μm, the
Xmax for PBRs is in the range of 4–8 × 108 cells·ml− 1.
Maximal cell densities of up to 1–5 × 108 cells·ml− 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):
U0 COin2 −COout2
hPBR ¼ d ð2Þ
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 εPBR considers both the space
occupied by the matrix and by the cells; thus,
XPBR
ePBR ¼ ematrix −ecells ¼ ematrix −0:74d ð3Þ
Xmax
where εmatrix and εcell 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 satu-
ration and cOout
2
remains above 20%, the cells have a
constant specific oxygen consumption rate (qO2) of
2 × 10− 13 mol·cell− 1 h− 1 (Ruffieux et al., 1998), the
packing is highly porous (εmatrix = 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 cells·ml− 1) and
superficial velocity U0 (0.2 b U0 b 1.0 cm·s− 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 pub-
lished 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 chromatog-
raphy 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 cm·s− 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 (Leven-
spiel, 1999), PBRs are expected to behave similarly to
large-scale chromatography columns. Columns for indus-
trial 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 substan-
tial scope for increasing the diameter of PBRs compared
with the diameters that have been used in the past (Table 3).
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).
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 cells·ml− 1 would have a productivity roughly
equivalent to that of a 2–9 m3 fed-batch bioreactor
operating at ∼ 107 cells·ml− 1. Such scaled-up PBRs can
be quite competitive with conventional bioreactors, for
producing therapeutic proteins that are needed in rela-
tively 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 cul-
ture conditions, packing materials and entrapment pro-
cedures 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 entrap-
ment (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 bio-
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
(Kurosawa et al., 2000), porous microcarriers (Ghanem
and Shuler, 2000), polyester strips (Hu et al., 2003) and
polyurethane foams (PUF).
 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 immobi-
lized hepatocytes were increased from 106 cells·ml− 1 in
the early stages of BAL development to the current levels
of 107–108 cells·ml− 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 extra-
corporeal 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 (2–4 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 cells·ml− 1) during short-term cultures
(b 26 h). Transport of nutrients and oxygen to immobi-
lized 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 cells·ml− 1,
Fig. 4. Flow setup for a biomedical packed-bed bioreactor connected to
a patient as an extra corporeal bioartificial liver device (BAL).
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 cells·ml− 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 at-
tained a density of 4 × 107 cells·ml− 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 pre-
clinical trials in pigs with liver ischemia.
Naruse et al. (1996) developed a BAL device based
on packed-bed of polyester matrix. These modules at-
tained a density ranging from 2 × 107 cells·ml− 1 (50 ml
device) up to 5 × 107 cells·ml− 1 (200 ml device) and
could support hepatocytes with reasonably high meta-
bolic 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 × 10-
7
cells·ml− 1 was attained using immortalized human
hepatocytes grown on a Cellsnow™ matrix. The bio-
reactor 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 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
As a consequence of their ability to support mamma-
lian 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 compart-
ments 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 com-
plexity 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 im-
proved biomedical PBRs
The maximum cell densities that have been reported
(Table 3) for biomedical PBRs are generally lower (∼ 107
cells·ml− 1) than for bioprocess PBRs (∼ 108 cells·ml− 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 ob-
served in the native liver organ (i.e. 108 cells·ml− 1), a
BAL device of 10–20 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 vol-
ume of 1 l and attain a total of 1010 immobilized hepa-
tocytes, 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 recom-
mended 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 cells·ml− 1
was maintained for 14–40 days by Kawada et al.
(1998) and 8.5 × 107 cells·ml− 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 1–2 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 packed-
bed bioreactors (PBRs) have achieved cells densities in
the range of 1 × 108 to 5 × 108 cells·ml− 1. These values
are close to the maximum density of ∼ 8 × 108 cells·ml− 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 chal-
lenge therefore is to demonstrate the scalability of packed-
bed 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
0.2–0.9 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 quan-
tities. 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 10–20 m3 may be the only
realistic option.
In biomedical applications of PBRs, the maximal cell
densities have been generally relatively low (e.g. ∼ 107
cells·ml− 1) compared with the bioprocess PBRs. How-
ever, replicating the performance of a whole adult liver in
a compact volume of 1–2 l would require attaining a
hepatocyte cell density of at least 108 cells·ml− 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 activ-
ity of in vitro cultured cells typically declines within
1–2 weeks of initiation, to unacceptably low levels.
Once stable cell lines are available, scale-up of bio-
medical PBRs to desired size of 1–2 l should not be
a problem, as these devices have been already scaled-
up to much larger volumes in animal cell culture bio-
process applications.
Nomenclature
BAL Bioartificial liver
cOin
2
Oxygen concentration at PBR inlet
cOout Oxygen concentration at PBR outlet
2
DPBR Medium dilution rate (in medium volume per
packed-bed volume per day)
h Packing material height
hPBR Packed-bed depth
NWF Non-woven polyester fabric
PBR Packed-bed bioreactor
PUF Polyurethane foam
PUM Polyurethane membrane
PVF Polyvinyl fluoride resin
qO 2 Specific oxygen consumption rate of the cells
rcell Radius of cell
U0 Superficial velocity of circulating fluid before
the packed-bed
U Superficial velocity of circulating fluid in the
packed-bed
VPBR Packed-bed volume
Xmax Maximum cell density in the bed
XPBR Viable cell density (in cells per unit packed-bed
volume)
εint Packing material internal porosity
εPBR Packed-bed porosity
εmatrix Porosity of carrier matrix
εcells Volume fraction of cells in the bed
∅ Packing material diameter
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