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, Switzerlandb 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. Itfurther reviews the current applications of PBRs and discusses the steps forward in the development of these systems for bioprocessand biomedical applications. The latest generation of PBRs used in bioprocess applications achieve very high cell densities(N108 cells ml−1) leading to outstandingly high volumetric productivity. However, a major bottleneck of such PBRs is theirrelatively small volume. The current maximal volume appears to be in the range of 10 to 30 l. A scale-up of more than 10-foldwould be necessary for these PBRs to be used in production processes. In biomedical applications, PBRs have proved themselvesas compact bioartificial organs, but their metabolic activity declines frequently within 1 to 2 weeks of operation. A main challengein this field is to develop cell lines that grow consistently to high cell density in vitro and maintain a stable phenotype for aminimum 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462. Applications of PBRs in bioprocessing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

2.1. Packing materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472.2. Packed-bed bioreactor configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472.3. PBR development for bioprocess applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482.4. Limitations and prospects for improved PBRs for bioprocess applications . . . . . . . . . . . . . . . . . . . 50

3. PBRs as bioartificial organs and tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513.1. Bioartificial liver (BAL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523.2. Artificial organs for drugs toxicology testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523.3. 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 cellsare the preferred expression system for making recombi-nant proteins for human use because of their ability toexpress a wide variety of proteins with a glycosylationprofile that resembles that of the natural human protein(Goochee et al., 1991; Jenkins and Curling, 1994; Jenkinset al., 1996). In view of their advantages, tremendouseffort has been invested in developing animal cells ascommercial production vehicles. A variety of cell culturesystems are now available, as summarized in Fig. 1.

The demand for therapeutic proteins derived frommammalian cell culture continues to grow (Morrow,2001; Gavrilescu and Chisti, 2005), as newer products areapproved. Some of the newer products such as antibodiesand receptor binding proteins need to be administered inhigher doses and this necessitates production of largerquantities than was the case with earlier products.Consequently, there is a continuing need to increase theproductivity of mammalian cell culture bioreactors withminimal investment in additional equipment (Kadouri,1994; Gòdia and Sola, 1995; Brotherton and Chau, 1996).

Most cell culture derived biopharmaceutical proteinsare produced in stirred tank bioreactors operated in batchor fed-batch mode (Hu and Aunins, 1997; Varley andBirch, 1999; Chisti, 2001; Kretzmer, 2002). Production instirred bioreactors is relatively simple to scale-up (Chisti,

1993), but requires large culture volumes (i.e. 10–20 m3)to compensate for the relatively low cell densities that areattained. Typically, the cell density in suspension cultureis between 106 and 107 cells·ml−1. Compared to batchculture in stirred tanks, nearly 10-fold higher cell densities(i.e. 107–108 cells·ml−1) can be attained in perfusioncultures inwhich themedium is perfused at an appropriaterate in a constant volume culture and the cells are retainedin the bioreactor by variousmeans (Hu and Peshwa, 1991;Ozturk, 1996; Voisard et al., 2003).

Because of a high cell density, the productivity ofperfusion systems can be as much as 10-fold greater thanthe productivity of a comparable fed-batch bioreactor. Inother words, a 2 m3 perfusion culture would be roughlyequivalent to a 20 m3 fed-batch culture. Disadvantages ofperfusion culture include their complexity and possibledifficulty in scale-up. For example, large-scale cell reten-tion devices for suspension cells are not yet entirelysatisfactory (Voisard et al., 2003).

A bioreactor system that can provide extremely highproductivity within a compact size is the packed-bedbioreactor (PBR). Packed-beds have been used widely forperfusion culture of immobilized mammalian cells. Thisreview focuses on the prospects of PBRs as a potentialfuture preferred production tool for making cell-culturederived products. In addition, the use of PBRs as“artificial organs” (Allen et al., 2001) in biomedicalapplications 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 liverfailure (acute failure), or entirely replace a liver until acompatible organ becomes available for transplant(Ambrosino and D'Amico, 2003). A BAL is expectedto perform all the multiple functions of a liver that areessential to maintaining life. These functions includecarbohydrate metabolism, synthesis of proteins, aminoacid metabolism, urea synthesis, lipid metabolism, drugbiotransformation and waste removal. A BAL devicecapable of these varied functions is best produced byculturing intact liver cells, or hepatocytes, that canfunction in vitro. The human liver is a massive organthat typically contains at least 1011 cells in an averagevolume of 1.3 l. This is equivalent to a cell density of atleast of 108 cells·ml−1 (Stapakis et al., 1995). Therefore,Fig. 1. Bioreactor systems for mammalian cell culture.

generating a satisfactory BAL requires the ability tosupport viable and fully functional hepatocytes at a highdensity. This is a significant challenge as hepatocytesgenerally are poorly able to proliferate in vitro.

Recent reviews (Allen et al., 2001; Allen and Bhatia,2002) highlight the various bioreactor systems that arebeing evaluated as BAL devices. These include hollowfiber reactors, flat platemonolayer culture, perfused PBRs/scaffolds and encapsulated cell suspension cultures. PBRsare a good alternative to other types of BAL bioreactors, asthey can support high cell densities in a compact volume.

Here we review the packed-bed bioreactors used formammalian cell culture and discuss their performance andmain applications. Common features of both bioprocessapplications and biomedical applications of PBRs arereviewed with a view to identifying the challenges thatmust be overcome in developing the next generation ofimproved PBRs.

2. Applications of PBRs in bioprocessing

2.1. Packing materials

The early attempts at culturing cells in PBRs focusedon identifying support materials that were compatiblewith mammalian cells and had the other necessary attri-butes identified in Table 1. Solid glass beads for growingcells as monolayers were identified as a suitable materialas early as 1953 (Earle et al., 1953). However, surfacegrowth on beads limited the maximal cell density in thebed to∼ 106 cells·ml−1 because solid spheres have a verylow specific surface-to-volume ratio available for cellproliferation. This limitation was overcome in the late1980s with the introduction of porous glass spheres (e.g.SIRAN®) that provided a higher specific surface. The celldensity 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 limitedby a relatively low internal porosity (εmatrix=0.56) that

could lead to oxygen diffusion limitations within thedepth of the carriers.

Higher internal porosities ranging from 0.80 to 0.95were reached with the next generation of packingmaterials such as disks made of non-woven polyesterand polypropylene screen, ceramic spheres and othershapes, glass fibers (Perry and Wang, 1989; Chiou et al.,1991), polyurethane and polyvinyl foams or resins. (Thelatter are discussed in the section on biomedical appli-cations.) Among these carriers, the Fibra-Cel® provedquite popular. This disk carrier was developed mainly forPBR applications that involved a high rate of mediumperfusion (Bohak and Kadouri, 1987; Kadouri, 1994).Since it became commercially available, Fibra-Cel® hasbeen used widely for mammalian cell culture at labo-ratory-scale and pilot industrial-scale. Fibra-Cel® ismanufactured in conformance with the current GoodManufacturing Practice (cGMP) guidelines. The highporosity of its polyester non-woven fibers and polypro-pylenemesh provides for efficient entrapment of cells andreduces intra-carrier diffusion limitations. This providesconditions for attaining a high cell density.

Ceramic pieces of 0.85 to 0.90 void-fraction have alsobeen used to construct a lab-scale PBR of 3 l packed-bedvolume (Mitsuda et al., 1991). The high porosity of thiscarrier was shown to improve intra-particle convectionand, consequently,minimize oxygen limitations (Park andStephanopoulos, 1993). The physical characteristics ofthis and other carriers are summarized in Table 2.

In summary, the two matrices that are currently themost frequently used for bioprocess PBRs are theSIRAN® and Fibra-Cel® porous carriers. They havegained widespread acceptance as they are versatile andcan be used “generically” to entrap both anchorage-de-pendant cells and cells that would normally grow insuspension. These carriers have proved successful withboth serum-containing and serum-free media.

2.2. Packed-bed bioreactor configurations

The PBRs typically consist of a packed-bed thatsupports the cells on or within carriers and a reservoir thatis used to recirculate the oxygenated nutrient mediumthrough the bed. Two major configurations are possible(Fig. 2), with the packed-bed compartment located eitherexternal to, or within, the reservoir of the medium (Wanget al., 1992a,b). Furthermore, the flow of the mediumthrough 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 usea small-scale model bed to identify the optimal packingmatrix for the cell line of interest. An optimalmatrix is one

Table 1Requirements of carrier materials for packed-bed bioreactors (Bliemet al., 1990)

Simple physical configuration and made of non-toxic materialsHigh surface to volume ratioOptimal diffusion from the bulk phase to the center of the carrierChemical and mechanical stabilityAutoclavableSuitable for adherent and non-adherent cellsChemically and biologically inert, no reaction with the productLow cost and reusable if possibleOf nonanimal origin

that provides the requisite combination of cell attachment,proliferation and productivity. This matrix is then used tooptimize the operational parameters (e.g. packed-bedheight and volume, medium perfusion rate, linear velocityof the medium across the packed-bed) of the PBR throughperfusion experiments that are generally performed atlaboratory-scale.

A great deal of research effort has been invested indeveloping PBRs with enhanced productivity and stab-ility. The matrices, operation variables and cell densities

achieved with different cell lines are summarized inTable 3.

2.3. PBR development for bioprocess applications

The first application of PBRs in bioprocessing wasthe production of foot-and-mouth disease virus usingBHK cells grown on the surface of solid glass beads for4–5 days in batch culture. The support beads were 3 mmin diameter. The bed of beads was connected to anexternal reservoir of the medium. Liquid flowed throughthe bed in an axial direction. In the 1970s, this type ofPBR was scaled-up 1000-fold for production purposesto a 100 l system that had an effective packed-bedvolume of 30 l (Spier and Whiteside, 1976; Whitesideand Spier, 1981).

During the 1990s, lab-scale PBRs of SIRAN®porous glass spheres with packed-bed volumes rangingfrom 0.01 to 5.6 l were used successfully for cultivatingmany types of cells, including both anchorage-depen-dent and anchorage-independent cells. Cells were suc-cessfully grown in these reactors in media with andwithout 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

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

Table 2Physical characteristics of various packed-bed carriers

Carrier type and material Size a (mm) εmatrix (-) S/Va, 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

coated with denatured collagen0.2 – 34 (Ghanem and Shuler, 2000)

Cellsnow® Cellulose porous cubes chargedwith polyethyleneimine (PEI)

3–5 0.95 – (Ong et al., 1994)

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.

Table 3A summary of packed-bed bioreactors used with animal cells

Matrix PBRtype

VPBR

(l)hPBR(cm)

UPBR

(mm·s−1)DPBR

(day− 1)Xmax,PBR

(cells·mlFB−1)

Run time(days)

Cell line(product)

Reference

Glass External 0.03–3 3.5 0.33 – 1.5×106 4–5 BHK (Spier andWhiteside, 1976)

Beads External 0.03–30 – 0.33 – 1.4×106 4–5 BHK (Whiteside andSpier, 1981)

Beads External 15 15 – 0.4–4 1–5×108 40–200 Hybridoma (IgG1) (Bliem et al., 1990)Hollow

cylindersInternal 3 – – – – 21–36 Hybridoma (IgG2a) (Moro et al., 1994)

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–

1.4×1075 GPK, Vero (Looby and

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

hepatocyte (mHep-R1)(Fassnacht et al., 2001)

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; Racherand Griffiths, 1993)

External 0.4 – – – 1.1×108 14 Human hepatocarcinomacells (FLC-7)

(Kawada et al., 1998)

Fibra-Cel® Internal 1.75 – 5.2 3×107 35 CHO (r-hEPO) (Jixian et al., 1998)– – – – – 2.4×107 – CHO (Ducommun

et al., 2002a,b)3.6×107

Internal 0.5–1.0 14.6 ∼10 3–4 1.0–1.2×108 30 Hybridoma (IgG1) (Wang et al., 1992a,b)ExternalExternal 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

diploid fibroblasts(Petti et al., 1994)

Ceramic 0.01 1–5 – – 5.1×108 40 Rat pituitary (Park andStephanopoulos, 1993)

External 2.6 15 0.02–0.08 0.5–1.6 3.3×105 40 Human embryoniclung diploid fibroblastIMR-90 cells (t-PA)

(Mitsuda et al., 1991)

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

Shuler, 2000)Rat lung (L2)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. Theproposed system consisted of an internal PBR of 84.5 lvolume that was placed inside a 300 l bioreactor but thefeasibility of this approach was not tested in cultureconditions.

At Baxter, Bliem et al. (1990) followed the sameapproach to design a 14 l external PBR of glass beads.The system was used for producing immunoglobulinsover 40 days using perfused cultures of hybridomas.The authors suggested that a further four-fold scale-upwas 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 manysources in PBRs with packed-bed volumes that haveranged from 0.05 to 1.75 l (Table 3). Because of theirmesh-like structure, polyester fibers are capable ofeasily supporting both attached and entrapped cells andhave proved successful with many different combina-tions of cells and culture media.

Bioreactor manufacturers have developed commer-cial PBRs that can accommodate many different typesof carriers. Most of these PBRs are of the internalconfiguration. The CelliGen Plus® system (NewBrunswick Scientific; www.nbsc.com) was primarilydeveloped for use in combination with Fibra-Cel®polyester disk carriers and has been scaled-up from0.7 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 andhave been designed to operate with an internalpacked-bed of BioNOC II® polyester strips carriers.The Swiss company Bioengineering AG (www.bioengineering.ch), has also developed internal PBRsthat can be placed within bioreactors and offer fixedbed volumes from 0.9 to 1.2 l.

In summary, although a large variety of PBRsystems have been assessed successfully in thelaboratory (Table 3), few pilot-scale and industrialinstallations have been described. Indeed, most ofpublished data deals with PBRs of less than 5 l ofpacked-bed volume. Only a few systems have beenscaled-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 forbioprocess applications

Assessing a maximal potentially attainable perfor-mance for PBRs requires a knowledge of the maximumcell density that can be attained in the bed and themaximum size that a PBR can be scaled-up to.

If we assume cells to be spherical and packed as a bedof cells, the maximum attainable volume fraction of cellsin the bed would be 0.74 and, therefore, the maximal celldensity Xmax could be estimated using the followingequation:

Xmax ¼ 0:74=43pr3cell

� �ð1Þ

where r is the radius of the cell. Because mammalian cellshave an average diameter in the range of 12–15 μm, theXmax 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 haveactually been already reported (Table 3) and, therefore,further increases are unlikely.

In establishing themaximum size that packed-beds canbe scaled-up to, we need to understand that the mainlimitation on increasing the height of the bed originatesfrom the unavoidable occurrence of axial gradients inconcentrations of nutrients. The nutrient that generallylimits the depth is oxygen, as the maximum attainableconcentration of oxygen at the inlet of the bed depends onthe solubility of oxygen which is quite low. Of course theoxygen concentration anywhere in the bed must not fallbelow the critical level that would jeopardize survival ofthe cells and their ability to produce the desired protein.Models have been developed to estimate the axialgradients in dissolved oxygen in packed-beds (ChistiandMoo-Young, 1994; Fassnacht et al., 2001). The depthof the bed (hPBR) depends on the superficial fluid velocity(U0) through it, the cell density XPBR and the specificoxygen consumption rate (qO2

), as follows (Chisti andMoo-Young, 1994; Fassnacht et al., 2001):

hPBR ¼ U0

ePBRdCinO2−Cout

O2

qO2 dXPBRð2Þ

In Eq. (2), cO2

in and cO2

out are the oxygen concentrationvalues at the inlet and outlet of the bed, respectively. InEq. (2) the bed porosity εPBR considers both the spaceoccupied by the matrix and by the cells; thus,

ePBR ¼ ematrix−ecells ¼ ematrix−0:74dXPBR

Xmaxð3Þ

where εmatrix and εcell are porosity of the matrix andvolume fraction of cells in bed, respectively.

If we assume that the PBR operates within a range ofoxygen concentrations such that b80% of oxygen satu-ration and cO2

out remains above 20%, the cells have aconstant specific oxygen consumption rate (qO2

) of2×10−13 mol·cell−1 h−1 (Ruffieux et al., 1998), thepacking is highly porous (εmatrix=0.90), and the cells are

evenly distributed in the bed at an average cell density ofXPBR, 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 immobilizedcell density XPBR (1×10

8bXPBRb5×108 cells·ml−1) and

superficial velocity U0 (0.2bU0b1.0 cm·s−1) of themedium. The U0 and XPBR values in Fig. 3 spanned themaximum and minimum values of these variables aspreviously published in the literature (Table 3). FromFig. 3, we can deduce that the maximal PBR depth thatcan be achieved under the specified conditions is in therange of 5bhPBRb30 cm. Indeed, values of hPBR pub-lished in Table 3 correspond well to these values.

The maximum diameter of a packed-bed is limited bythe ability to uniformly distribute the flow over the entirecross-section to prevent nonhomogeneities and channeling.The problem of achieving uniformity of flow over thecross-sectional area occurs also in packed-bed chromatog-raphy and has been investigated in some detail in relation tochromatography. Chromatography columns used forprotein capture commonly have the same range of packingsizes (i.e. particles of 0.2 to 2 mm in diameter) and linearflow velocities (i.e. 0.1bU0b0.3 cm·s−1) (AmershamBiosciences, 2003), as encountered in packed-beds ofimmobilized animal cells. Furthermore, because thedispersion coefficient in PBRs is relatively constant for awide range of particle sizes and linear velocities (Leven-spiel, 1999), PBRs are expected to behave similarly tolarge-scale chromatography columns. Columns for indus-trial chromatography are successfully operated withdiameters as large as 2 m. Such large columns arecommercially available from companies such as Millipore(www.millipore.com). Clearly, therefore, there is substan-tial scope for increasing the diameter of PBRs comparedwith the diameters that have been used in the past (Table 3).

Assuming that the depth of bed can vary between 5 and30 cm and the maximum diameter cannot exceed 2 m, therange of reasonable volumes for the bed works out to befrom 0.2 to 0.9 m3. PBRs of this size range operating with∼ 108 cells·ml−1 would have a productivity roughlyequivalent to that of a 2–9 m3 fed-batch bioreactoroperating at ∼ 107 cells·ml−1. Such scaled-up PBRs canbe quite competitive with conventional bioreactors, forproducing therapeutic proteins that are needed in rela-tively small quantities. If the required bioreactor volumeexceeds the limits established here, a PBR would not betechnically feasible for the specific application.

3. PBRs as bioartificial organs and tissues

The use of PBRs in biomedical applications began inthe 1990s. Mainly, the focus has been in using PBRs toculture immobilized hepatocytes as a bioartificial liverdevice (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 numberof immobilized hepatocytes in PBRs.

As for bioprocess applications, the initial attempts toculture hepatocytes in PBRs were made using non-porousglass beads as the packing material. The first such PBRconsisted of a 30 ml packed-bed BAL with glass beads of1.5 mm in diameter. This system was good at cell entrap-ment (N80% immobilization efficiency) and allowedculture of metabolically active hepatocytes for more than14 days (Li et al., 1993). In later designs, the solid glassbeads were replaced with porous packing materials thatsupported even higher cell densities. Various materialswith 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 μmaverage diameter that facilitate cell immobilization withup to 99% efficiency and support cell densities rangingfrom 1×107 to 5×107 cells·ml-1 (Lazar et al., 1993).However, at least in some cases, clogging of packed-bedshas been observed with PUM carriers for cell densitiesexceeding 5×107 cells·ml−1 (Kurosawa et al., 2000).This phenomenon has been attributed to accumulation ofcell debris in the dead-ended pores of the matrix.Clogging could be avoided by using other matriceshaving open-ended pores, such as non-woven polyesterfabrics (Naruse et al., 1996, 2001; Flendrig et al., 1997),reticulated polyvinyl fluoride (PVF) resin scaffolds(Kurosawa et al., 2000), porous microcarriers (Ghanemand Shuler, 2000), polyester strips (Hu et al., 2003) andpolyurethane foams (PUF).

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

Concurrently with advances in matrix design, methodswere optimized for immobilizing hepatocytes on andwithin them (Li et al., 1993; Miyoshi et al., 1996;Kurosawa et al., 2000). These improvements enhancedthe 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 inthe early stages of BAL development to the current levelsof 107–108 cells·ml−1 of PBR matrix volume.

3.1. Bioartificial liver (BAL)

Both the internal and external packed-bed bioreactorconfigurations have been used for PBRs in biomedicalapplications such as bioartificial liver device (BAL). ABAL is generally connected to the patient as an extra-corporeal BAL as shown in Fig. 4. A number of BALPBRs have been reported (Table 3).

Use of polyvinyl fluoride (PVF) cubes for primaryculture of hepatocytes in a small PBR (2–4ml packed-bedvolume) has been reported (Yanagi et al., 1992). Thesebeds were shown to reach quite high cell densities (from4×106 to 1.2×107 cells·ml−1) during short-term cultures(b26 h). Transport of nutrients and oxygen to immobi-lized cells in the beds were identified as being critical tomaintaining themetabolic activity of cells in vitro. Furthertrials demonstrated that hepatocytes cultured in serumcontaining medium for up to 9 days retained metabolicfunctionality 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 avolume that would allow its use in pre-clinical and clinicaltrials were not demonstrated.

PBRs based on polyurethane membrane (PUM) havebeen used to culture hepatocytes (Kurosawa et al.,2000). At the optimum density of 2.5×107 cells·ml−1,

the hepatocytes expression level could be maintained forup to 1 week in serum-free medium, but declined duringthe second week of culture. Another BAL system basedon polyurethane materials was developed by Ijima et al.(2000a,b). Hydrophilic polyurethane foam was used tomake packed-beds of 14.5 ml and 300 ml for in vitro andin vivo studies, respectively. The in vivo work wascarried out in dogs (Ijima et al., 2000b). The 300 mlBAL device containing 30 g of hepatocytes (i.e. celldensity ∼ 107 cells·ml−1) was shown to extend thesurvival rate of dogs afflicted with liver failure. TheBAL module had to be freshly prepared (not more than1 day old) to be effective.

Another BAL module used non-woven polyesterfibers for cell immobilization and had internal tubing fordirect oxygenation of the hepatocytes. This module at-tained a density of 4×107 cells·ml−1. This BAL devicewas initially tested in mice (Flendrig et al., 1997) andlater 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 basedon packed-bed of polyester matrix. These modules at-tained a density ranging from 2×107 cells·ml−1 (50 mldevice) up to 5×107 cells·ml−1 (200 ml device) andcould support hepatocytes with reasonably high meta-bolic activity over a period of 6 days. These BALdevices were further scaled-up and successfully tested inanimal models (Naruse et al., 1998, 2001).

BAL devices with porous microcarriers have beentested successfully. For example, a cell density of 8.5×10-7 cells·ml−1 was attained using immortalized humanhepatocytes grown on a Cellsnow™ matrix. The bio-reactor used in this workwas an internal PBRwith a 40mlpacked-bed volume (Fassnacht et al., 2001). The cellsstably retained metabolic activity over the 40-day culture(Fassnacht et al., 2001).

The highest hepatocytes cell density reported in PBRshas been 1.1×108 cells·ml−1 and was attained withporous glass microcarriers in a packed-bed of 400 mlvolume. The cells remained viable and metabolicallyactive during the 14-day experiment (Kawada et al.,1998). Another study was successful in extending the runtime to 40 days (Nagamori et al., 2000).

In summary, although several PBR systems haveproved 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

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

for drugs toxicology testing, where the cells' response to atest-compound is evaluated under conditions comparableto those in vivo. For example, Ghanem and Shuler (2000)developed a model system that mimicked the lung andliver functions of an adult rat. This system was made ofthree PBR compartments that represented the lung, theliver and the other tissues, respectively. The compart-ments were configured as a series of packed-bedswith cellculture medium exchanged between compartments atphysiological flow rates, enabling interactions of the“organs” and to mimic the whole animal. This system hasbeen used to test the toxicity of chemicals on lung andliver functions.

Another system, based on a radial-flow PBR wasused to cultivate human hepatocyte-derived cells(HepG2) for possible use in in vitro evaluation oftoxicity of drugs (Nagamori et al., 2000). Developingbioreactor systems that mimic with fidelity the com-plexity of animal metabolism is challenging. Individualbioreactor compartments generally support a single typeof cell and do not reproduce the complexity of any singleorgan, the interactions of organs and regulation ofmetabolism. This problem notwithstanding suitablyconfigured PBRs do offer an alternative to the use ofwhole animals and can potentially provide usefulinformation about the effect of chemicals on in vivometabolism.

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 highdensities. Because the cell densities obtained in vitro arecurrently generally 10-fold lower than the densities ob-served in the native liver organ (i.e. 108 cells·ml−1), aBAL device of 10–20 l would be needed to provide thefunctionality of an adult liver.

The BAL devices under clinical evaluation (mainlyhollow-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 adulthuman liver (Allen and Bhatia, 2002). Although it isgenerally accepted that 10% of the total cell numberexisting in a real liver would suffice in replacing liverfunction in many urgent clinical applications (Yanget al., 2001; Ijima et al., 2000a), this situation is notideal and there is a need to develop compact BALdevices that provide the full functionality of an adultliver.

Technologies have been developed to consistentlyattain a density of immobilized cells in PBRs that iscomparable to the density of hepatocytes in the liver(Table 3). Use of porous packing materials is recom-mended for attaining the requisite cell density withoutclogging the bed. A number of other objectives must beattained for developing the next generation of improvedbiomedical devices based on PBR technology. Theseinclude the following:

1. A cell line with stable expression and good ability togrow in vitro needs to be identified. Ongoing workon this aspect was recently reviewed by Allen andBhatia (2002). With most of the cell lines that areavailable currently, metabolic activity is lost within2 weeks. This has been attributed to unstable cellphenotype and the inability to supply the cells withoxygen and other nutrients. To be viable in clinicalapplications, a BAL device should ideally be capableof functioning for a typical 30-day treatment period(Ambrosino and D'Amico, 2003).

2. Optimization of nutrients/oxygen supply in the PBRs tokeep the cells viable and productive for at least 30 daysthat are desired for clinical application. This appears tobe possible, as cells have been maintained at highdensities in PBRs for extended periods, at least in a fewcases. 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 for40 days by Fassnacht et al. (2001). These reports arehowever 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, butnot further in order to provide a compact BAL devicefor convenient clinical use. The largest biomedicalPBR device that has been reported had a volume of400 ml (Table 3). This objective is still out of reach;however, the technical feasibility of such a scale-uphas been proved with PBRs in various bioprocessapplications with animal cells.

4. Concluding remarks

The latest generation of animal cell culture packed-bed bioreactors (PBRs) have achieved cells densities inthe range of 1×108 to 5×108 cells·ml−1. These valuesare close to the maximum density of∼ 8×108 cells·ml−1

that can be attained theoretically if the cells are packedin a bed as spheres.

In bioprocess applications, the largest PBRs reportedhad a maximal volume of 30 l. This is a small fraction ofthe bed sizes that are commonly used in nonanimal cell

culture bioprocesses such as in wastewater treatment andbiotransformations 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 theexistence of substantial axial gradients in concentrationsof nutrients such as oxygen. This limits packed-bed depthto ∼ 30 cm. The bed diameter is generally limitedto∼ 2 m, as uniform distribution of nutrient fluid over thebed cross-section becomes difficult with further increasein diameter. In view of these limitations, the maximumestimated volume for PBRs appears to be in the range of0.2–0.9 m3. Notwithstanding their limited potential forscale-up, PBRs can be quite competitive with otherbioreactor systems in producing proteins (e.g. cytokinesor hormones) that are needed in relatively small quan-tities. This is because of the exceptionally high volumetricproductivity of the PBRs.When large quantity of a proteinmust be produced, suspension culture in large fed-batch orperfusion bioreactors of 10–20 m3 may be the onlyrealistic option.

In biomedical applications of PBRs, the maximal celldensities 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 ina compact volume of 1–2 l would require attaining ahepatocyte cell density of at least 108 cells·ml−1.Developing a hepatocyte cell line that consistently attainsthese densities in vitro remains a challenge. Furthermore,any cell line for use in bioartificial organs must stablyretain functionality for at least 30 days to be usefulin clinical applications. At present, metabolic activ-ity of in vitro cultured cells typically declines within1–2 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 bea problem, as these devices have been already scaled-up to much larger volumes in animal cell culture bio-process applications.

NomenclatureBAL Bioartificial livercO2

in Oxygen concentration at PBR inletcO2

out Oxygen concentration at PBR outletDPBR Medium dilution rate (in medium volume per

packed-bed volume per day)h Packing material heighthPBR Packed-bed depthNWF Non-woven polyester fabricPBR Packed-bed bioreactorPUF Polyurethane foamPUM Polyurethane membrane

PVF Polyvinyl fluoride resinqO2

Specific oxygen consumption rate of the cellsrcell Radius of cellU0 Superficial velocity of circulating fluid before

the packed-bedU Superficial velocity of circulating fluid in the

packed-bedVPBR Packed-bed volumeXmax Maximum cell density in the bedXPBR Viable cell density (in cells per unit packed-bed

volume)εint Packing material internal porosityεPBR Packed-bed porosityεmatrix Porosity of carrier matrixεcells Volume fraction of cells in the bed∅ Packing material diameter

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