Systems for Either Suspension or Attached Cells not on microcarriers

These systems can be broadly classified on the basis of whether the medium is mixed within a single compartment, or is intentionally segregated into two compartments separated by a semipermeable membrane.

10.3.3.1 Single-compartment systems

10.3.3.1a CelliGen®

The CelliGen® is a small to medium-sized, round-bottomed, stirred-tank bioreactor from New Brunswick Scientific that is best characterized by the mixing principle of its cell lift impeller. The rotation of the ports of this specially designed impeller through the upper level of the culture medium creates a negative pressure inside the hollow impeller shaft. This pulls medium up from the bottom of the vessel, through the hollow shaft and out of the impeller ports, thus inducing circulation of medium within the vessel (Figure 10.8). The rate of rotation of the impeller required to induce this flow is relatively low, and thus shear at the impeller tips is kept within acceptable limits. As with other stirred-tanks, it can be run in batch, fed-batch, or perfusion mode.

This bioreactor has been available, in slightly different forms, for more than 20 years, and has attracted a relatively small but loyal user base. It can be used not only as a stirred tank with cells growing in suspension or on microcarrier beads, but also as a perfusion device for use with packed-bed cultures using FibraCel® (see below) or similar bed materials that can both trap or encourage the adherence of suspension cells and support the growth of attached cells. It seems to have found most favour either for the culture of hybridomas (Wang et al. 1992; Mercille et al. 2000; Kunkel et al. 2000) or, with microcarriers or packed beds, for the growth of attached cells for the production of virus particles (Mendonca et al. 1994; Gallegos et al. 1995; Wang et al. 1996; Zhang et al. 1997; Hu et al. 2000; Merten et al. 2001), most notably Gendicine (see below).

The CelliGen® is currently supplied in sizes ranging from 2.2 litres to 14 litres working volume. If required, it can be fitted with conventional impellers, spin filters, etc., so that it can also be used as a normal small stirred tank.

Cell Lift Impeller
Figure 10.8 Diagram of New Brunswick Scientific's patented low-shear cell lift impeller as used in the CelliGen.

10.3.3.1b Systems for FibraCel-type disks

Fibra-Cel® is a matrix sold by New Brunswick Scientific (NBS) and composed of polyester non-woven fibre and polypropylene (in a 1:1 mixture by volume), ultrasonically bonded together and cut into 6-mm diameter disks. These are then treated to produce a net surface charge. The manufacturer claims that this surface charge facilitates both the attachment of anchorage-dependent cells, and the adherence of suspension cell lines. CESCO Bio of Taiwan produces a similar matrix called BioNOC II®.

These disks are frequently used in packed-bed reactors, although they can also be used in normal stirred vessels such as spinner flasks. Their effective surface area in a packed bed is around 120 cm2/cm3, much higher than that normally obtained with microcarriers in suspension, and comparable to that in hollow fibre culture units. At small scale, the packed beds can be run

Small Scale Filter Bed Fibre

Figure 10.9 Diagram of the principle of action of the BelloCell® culture bottle. (a) As the bellows compress, medium flows through the bed of cell carriers to immerse all the cells. (b) As the bellows expand, medium flows back down the bed, exposing it to the atmosphere.

Figure 10.9 Diagram of the principle of action of the BelloCell® culture bottle. (a) As the bellows compress, medium flows through the bed of cell carriers to immerse all the cells. (b) As the bellows expand, medium flows back down the bed, exposing it to the atmosphere.

in systems such as the CelliGen® (see Section 10.3.3.1a) or in purpose-made 'bellows bottles' in the CESCO Bio BelloCell® system (also sold by NBS under the FibraStage™ name - NBS, 2005) In these 'bellows bottles' the packed bed is suspended across the middle of a plastic bottle with a bellows-shaped bottom. Medium is moved up through the bed as the bellows are compressed, and drains out as the bellows expand (Figure 10.9). This cycle repeats indefinitely, achieving mixing of the medium simply by the pumping action of the bellows. A larger-scale system using the same principle is available from CESCO Bio, called the TideCell®, in which the matrix is packed in the inner of two concentric chambers that are interconnected, and air pressure is used to move the medium first into the chamber containing the matrix, then out into the outer chamber (Figure 10.10). Vessels of 5 litres and 25 litres working volume are currently available.

In both the BelloCell® and TideCell® systems, oxygen transfer is achieved by the periodic exposure of the cells to the air, although one might speculate that there are significant differences in the supply of oxygen to cells at the top and bottom of the bed, as the proportion of the cycle time during which the cells are exposed to the air will decrease as one moves down the bed.

It is more difficult to recover cells from such matrices than from microcarriers, and thus they are not recommended for biomass production. However, the packed-bed system has proven useful for the production of secreted or released cell products, and a 14-litre CelliGen Plus® reactor (see Section 10.3.3.1a) with 200 g of FibraCel® disks is employed for the production of the adenoviral vector used in the world's first licensed gene therapy, Shenzhen SiBiono GeneTech's Gendicine (Peng 2004).

Suspension Therapy Bed
Figure 10.10 The TideCell®, shown with (a) the bed of cell carriers immersed in the culture medium, and (b) the bed of cell carriers exposed to the atmosphere.

10.3.3.2 Dual-compartment systems

10.3.3.2a Dialysis tubing culture systems

These are the simplest type of dual compartment system, being composed of a cell suspension retained within a length or lengths of dialysis tubing sealed at both ends. The tubing is then suspended in a much larger volume of medium within a roller bottle, fermenter or other stirred vessel (see, for example, Pannell & Milstein 1992). The medium and cells are incubated at culture temperature, and high molecular weight secreted products are generated by the cells and retained within the semipermeable dialysis tubing for later harvesting. Such systems are popular for generating quantities of monoclonal antibodies for research use, but are generally home-made, and are not suitable for the generation of material for use in humans.

10.3.3.2b Hollow-fibre systems

Hollow-fibre cell culture systems were first designed with the intention of mimicking the in vivo cell environment (Knazek et al. 1972). Within tissues, cells live immobilized at high density, and are perfused via capillaries having semipermeable walls. Fluid (blood) circulating through the capillaries delivers oxygen and nutrients to the cells whilst removing CO2 and other waste products. Hollow-fibre culture systems work in a similar way, with the cells being immobilized on the outside [i.e. in the extracapillary space, (ECS)] of artificial hollow fibres made from semipermeable membranes, while medium is circulated through the lumen of the fibres. This separation of the cells and other contents of the ECS from the majority of the nutrient medium gives the hollow-fibre system a number of advantages, particularly for the production of high molecular weight secreted products such as monoclonal antibodies. In such cases, the molecular weight cut-off (MWCO) of the fibres is chosen such that it is very much smaller than the molecular weight of the product of interest, so that the product is retained in the ECS with the cells whilst nutrients and waste products of low molecular weight equilibrate across the membrane. Thus for the production of an IgG antibody (typical molecular weight 160 kD) an MWCO of 10 kD might be chosen. Only small volumes of high molecular weight nutrients/growth factors then need to be introduced directly into the ECS, and the antibody product can be removed at the same time in a similarly small volume. Some of the advantages of hollow-fibre systems arise directly from the above properties:

• High secreted product concentrations can be attained, typically 20- to 100-times those obtained using the same cells and media in T-flasks (Hanak & Davis 1995). In part this is because the product is contained in only a small fraction (5-10 %) of the total nutrient medium required by the cells, not diluted in the entire volume as would be the case in a homogeneous system.

• Requirements for high molecular weight supplements, such as serum, are low. If needed at all, these need only be added to the small volume of medium in the ECS, and it may be possible to reduce or stop their use without loss of productivity (Tiebout 1990) once the culture is well established, as the cells frequently secrete factors that support their own viability, and these may be retained in the ECS.

• A high ratio of product to medium-derived contaminants is attained. This is a consequence of the first point above, but the effect may be increased further by the second.

• The cells are maintained in a low-shear environment. The cells are separated from the main medium flow in the lumen of the fibres, and oxygenation is performed in a remote gas exchange cartridge, minimizing the cells' exposure to potentially damaging gas bubbles.

Other advantages arise indirectly:

• For equivalent cell numbers, a hollow-fibre system is much smaller than a homogeneous system (e.g. a stirred tank). This is because viable cells are largely retained within the hollow fibre cartridge, meaning that cell densities reach levels of around 108/ml in the ECS. Consequently, a bench-top system such as the Biovest AcuSyst-Jr (Figure 10.11) or Maximizer can contain in the region of 1-4 X 1010 cells, whereas a production-scale system such as the Biovest Xcell (Figure 10.12) or Xcellerator, despite only being the size of a large fridge/freezer, can maintain in culture 2-4 X 1011 cells.

Acusyst Maximizer
Figure 10.11 Biovest AcuSyst-Jr. bench-scale hollow fibre culture system.
Figure 10.12 Biovest Xcell production-scale hollow fibre culture system.

• Because the units are relatively small, they can be housed within rooms of normal height. This is in contrast to fermenters, where those much larger than 100 litres require additional height to permit removal of the head-plate for cleaning and servicing, and large vessels may take up several storeys of a building specially built or adapted for the purpose. This could have major implications in the case where hollow-fibre systems could be housed in an existing facility but fermenters would require a new building.

• These systems, even at full production scale, are relatively easy and rapid to install and set up, as the only services they require are CO2 and single-phase electricity, in contrast to a fermenter which may require up to ten different services (see Section 10.3.2.4).

There are, of course, disadvantages to hollow-fibre systems:

• The nature of the supporting material of the fibre walls entraps suspension cells effectively, but is less well suited to the culture of attachment-dependent cells. While such cells can be grown in hollow-fibre systems, they often require larger inocula for the initiation of a successful culture.

• Because of the entrapment of the cells, it is not possible during the course of a culture (which can last many months) to remove a sample of cells for the enumeration of viable cell concentrations and similar purposes. Thus surrogate markers such as lactate production rate must be used, and the relationship of such parameters to cell number will vary not only with the cell line in use, but also with culture conditions such as pH, temperature, and the phase of the culture.

• Hollow-fibre culture is suited to the production of secreted products, but because it is extremely difficult to remove cells from the ECS effectively, it is not suited to the production of biomass.

Scale-up beyond a certain point is limited. Scale-up is achieved not by increasing the size of the hollow-fibre cartridges (as this would merely increase the gradients of oxygen, nutrients and waste products, leading to inefficient colonization of the cartridge), but by the use of multiple cartridges. Whilst up to 20 can be accommodated within a single production-scale machine, further scale-up is achieved through the use of multiple machines. At this point no further advantages of scale-up are available, and more machines mean an essentially linear increase in staffing levels, with batch sizes remaining unaltered. This has limited the application of hollow-fibre systems to markets requiring no more than a few kilograms of secreted protein per year, as a good cell line in a single production-scale machine will probably not produce more than 1-2 kg of product per year. Thus Cytogen's ProstaScint, a radiolabelled monoclonal antibody used for the in vivo diagnosis of prostate cancer. is produced in hollow-fibre culture, and was licensed by the FDA in 1997 (and by Health Canada in 2002). However, its unit dose size is only 0.5 mg (see http://www.cytogen. com), very much smaller than most therapeutic monoclonal antibodies where the dose sizes are tens or, more commonly, hundreds of milligrams. To satisfy the world-wide markets for these, tens to hundreds of kilograms of material are required per year, and culture is performed almost exclusively in fermenters. Hollow-fibre systems have, however, proved very popular in the production of monoclonal antibodies for in vitro diagnostics, where rather smaller quantities of material are required.

When considering scale up/scale down in hollow-fibre systems, care should be taken (see Section 10.4.1) that the smaller system represents the larger accurately. For example, the AcuSyst-Jr or Maximizer is, in the author's experience, a good scaled-down model for the AcuSyst Xcell. However, attempts to scale-down further would necessitate the use of equipment like the Unisyn C100 that has no in-line pH control, no constant medium replenishment, no constant harvesting from the ECS, and different hollow-fibre membranes. Data obtained from such systems may at best be of limited use, and may even be positively misleading.

10.3.3.2c CELLine flasks

These flasks from Integra Biosciences look similar at first sight to T-flasks, and have a similar footprint, but are actually two-compartment bioreactors. The cells are separated from the bulk of the medium by a semipermeable membrane, as is also the case in hollow-fibre culture systems and the MiniPERM bioreactor (see below). Some of the advantages of a hollow-fibre system, i.e. the higher secreted product concentrations, lower serum requirements, and higher ratio of product-to-medium derived impurities, can be realized at a smaller scale and without the expense of a hollow-fibre system, with the added advantage that cells can be easily removed from the vessel. However, flasks must be kept within a CO2 incubator, there is not the degree of control available that there is in a hollow-fibre system, and scale-up can only be achieved by the use of multiple units. Thus realistically these flasks can only deliver a limited degree of scale-up from T-flasks.

10.3.3.2d miniPERM vessels miniPERM vessels can be viewed as a two-compartment roller bottles modified to improve gas exchange. The main, reusable compartment (volume ca 550 ml) contains the bulk of the medium and is separated from the disposable cell-containing compartment (volume 5-35 ml) by a semipermeable membrane. The opposite face of the cell compartment is a silicone membrane through which oxygen and carbon dioxide can be exchanged with the atmosphere within an incubator. Mixing within the two compartments should be better than in CELLine flasks, as the vessel is rotated in the same way as a roller bottle (although normally at higher speeds). It is claimed that cell densities beyond 1 X 107/ml (for hybridoma cells) can be attained in the cell compartment (Heraeus 1995). However, as with CELLine flasks, these vessels must be kept within a CO2 incubator, but also require equipment to rotate them. Scale-up can only be achieved by the use of multiple units, and there is neither the degree of control available that there is with hollow-fibre systems, nor the simplicity inherent in normal roller bottles that facilitates automation. Thus these vessels too are not likely to be used to produce large amounts of material but, like CELLine flasks, have proven popular for producing small quantities of monoclonal antibodies, in place of ascitic fluid production in vivo (Bruce et al. 2002).

10.3.3.2e Perfused rotary cell culture technology vessels

The Synthecon Rotary Cell Culture Technology systems are based on the rotating wall vessel bioreac-tor designed by NASA (NASA, 1997). Originally intended for the three-dimensional growth of cells under pseudo-microgravity, certain of their perfused systems combine features of the miniPERM and hollow-fibre systems. Like the miniPERM, the cells are cultured in one compartment of a rotating vessel, and like all dual-compartment systems, the cells are separated from the bulk of the medium by a semipermeable membrane. However like a hollow-fibre system, fresh medium is continuously fed into (and spent medium removed from) the compartment containing the bulk of the medium, and oxygen is supplied to the culture by passing the medium in this compartment through an external oxygenator. These systems are currently less sophisticated than most medium- and larger-scale hollow-fibre systems, and are not yet intended for continuous harvesting of cell-secreted products, thus again placing them somewhere between the miniPERM and hollow-fibre systems. For further details, see the Synthecon website - http://www.synthecon.com/index.htm.

10.3.3.2f Encapsulation

This technology, in a number of different formats including hollow spheres of polylysine (Duff 1985; Rupp 1985) or cellulose (Kloth et al. 1995), alginate spheres (Griffiths 1988), and agarose beads (Nilsson et al. 1983, 1987), was taken up enthusiastically in the 1980s and early 1990s. The perceived advantages were various. All the methods could potentially protect fragile cells from the shear forces encountered in some culture systems. Hollow spheres had to be semipermeable in order to allow nutrients and oxygen to reach the cells, and to allow waste products out, but the permeability could be easily controlled such that these requirements could be met whilst retaining high molecular weight secreted products such as monoclonal antibodies within the spheres. After harvesting the spheres, these products could then be released at high concentration. (Other encapsulation methods were simpler and allowed the cells access to high molecular weight growth stimulators in the medium, but the particles were not part of a dual-compartment system and did not concentrate secreted products, and the beads produced are perhaps more accurately viewed as macroporous microcarriers.)

A number of problems were encountered with encapsulation technology, most notably difficulties in getting an adequate flux of nutrients and oxygen into, and waste products out of, the beads (and thus of maintaining cell viability), as well as the logistical and technical complexities of applying the technology at large scale (Griffiths 1988). Consequently, the use of this approach became increasingly limited.

Interest has revived in recent years, however, as the technology's potential for use in cell and gene therapy has become apparent. In this case, cells are encapsulated before being introduced into the body of the recipient. The encapsulation process is designed to protect the cells from attack by, or dissemination in, the body into which they have been introduced. As the cells used are usually not those of the host, this prevents immune rejection, or the need to use immunosuppres-sive drugs. However, the encapsulating material still permits the cells to take up nutrients from the body and secrete the therapeutically active substance that they produce. This approach is proving promising for the treatment of a variety of disorders, such as epilepsy (Guttinger 2005), liver failure (Mai et al. 2005), diabetes (Black et al. 2006), cancer (Li et al. 2006), and haemophilia (Wen et al. 2006), and is already in the process of being commercialized (see, for example, http://www. lctglobal.com/).

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  • jennifer baier
    How microcarriers attach to cells?
    6 months ago

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