Animal Cell Culture

Interest in the in vitro cultivation of animal cells has developed because of the need for large scale produc tion of monoclonal antibodies, hormones, vaccines and other products which are difficult or impossible to produce synthetically or by using other culture techniques.

Animal cells are usually more nutritionally demanding than microbial cells. They lack cell walls which makes them sensitive to shear and extremes of os-molarity. The doubling times are normally 12 to 48 hours and cell densities in suspension cultures rarely exceed 106 to 107 cells cm™3. Two distinct modes of growth can be recognized:

1. Anchorage dependent cells. These cells require a solid support for their replication. They produce cellular protrusions (pseudopodia) which allow them to adhere to positively charged surfaces and often grow as monolayers.

2. Anchorage independent cells. These cells do not require a support and can grow as a suspension in submerged culture. Established and transformed cell lines are normally in this category.

of vessel was due to sparging and the break up of bubbles on the medium surface. This type of damage causing cell death might be reduced by increasing the height to diameter ratio in the vessel, increasing the bubble size, decreasing the gas flow rate or by adding protective agents. Other damage mechanisms in stirred fermenters are caused by cell-microcarrier eddies and microcarrier-microcarrier interactions. Damage of this type may be reduced by reducing the impeller speed, impeller diameter, microcarrier size and concentration or by increasing the viscosity of the medium.

The maximum cell densities obtainable in stirred and air lift fermenters are often only 106 cells cm™3. This is not ideal if secreted proteins are present only in very low concentrations and mixed with other proteins present in the original medium. A number of modified fermenters and reactors have been developed to grow cells at higher concentrations using microcarriers, encapsulation, perfusion, glass beads or hollow fibres to obtain the required product at higher concentrations.

There are also intermediate categories of cells which may be grown as anchorage dependent or suspension cells. It is also possible to grow some anchorage dependent cells in suspension, provided the cells can be grown on suitable microcarriers.

A range of free and immobilized culture systems have therefore been developed for the culture of different lines of animal cells and their products. The range of available equipment and techniques may be confusing to scientists not familiar with animal cells who may have to decide on the most appropriate culture systems for laboratory, pilot or production scale. Because of the relatively small quantities of product required, the volume at production scale may only be 100 to 10,000 dm3. Some useful introductions include Griffiths (1986,1988), Propst etal. (1989), Lavery (1990) and Bliem et al. (1991).

Shear is a phenomenon recognized as being critical to the scale up of animal cell culture processes, irrespective of the cell line or reactor configuration (Bliem and Katinger, 1988). Shear may influence the cell culture causing damage which may result in cell death or metabolic changes. The shear sensitivity of animal cells may vary between cell lines, the phase of growth or with a change to fresh medium

Mijnbeek (1991) has reviewed research on shear stress of free and immobilized animal cells in stirred fermenters and air-lift fermenters. It was concluded that the predominant damage mechanism in both types

Stirred fermenters

Unmodified stirred fermenters have been used for the batch production of some virus vaccines (Propst et al., 1989), but modified vessels are used for most cultures (Propst et al., 1989; Lavery, 1990). The modifications made to fermenters are to reduce the possibility of cell damage due to shear, heat or contamination. Marine propellers revolving at a slow speed (10 to 100 rpm) will normally provide adequate mixing. Hemispherical bottoms on the vessels will ensure better mixing of the broth at slow stirrer speeds. Water jacket heating is often preferred since heating probes may give rise to localized zones of high temperature which might damage some of the cells. Magnetic driven stirrers may be used to reduce the risk of contamination. A novel sparger-impeller design which improves aeration at slow speeds is incorporated into the Celligen system manufactured by New Brunswick, U.S.A. (Fig. 7.54; Beck et al., 1987). When the impeller rotates the swept-back ports produce a negative pressure inside the hollow impeller complex. This creates a suction lift that produces highly efficient circulation and gas transfer at low rpm without damaging the cells. Gases are introduced through a ring sparger into the medium as it circulates through a fine stainless steel mesh jacket which excludes cells and microcarriers. Because the gas sparging is restricted to a relatively small zone, foaming

Exhaust-gas out

Hollow agitator shaft Foam-eliminator chamber

Exhaust-gas out

Hollow agitator shaft Foam-eliminator chamber

Impeller port

Cell-free medium out

Ring sparger

Hollow Agitator Shaft

Medium in

Fig. 7.54. Sparger-impeller in the Celligen cell-culture fermenter (New Brunswick Ltd, Hatfield, England).

Impeller port

Cell-free medium out

Ring sparger

Medium in

Fig. 7.54. Sparger-impeller in the Celligen cell-culture fermenter (New Brunswick Ltd, Hatfield, England).

is reduced, and any foam formed is broken up by the mesh in the foam eliminator chamber.

Air-lift fermenters

Air-lift fermenters have proved ideal for growth of some cell lines because of the gentle mixing action and reduced shear forces when compared with those in stirred vessels. The absence of a stirrer and associated seals excludes a potential source of contamination (Griffiths, 1988; Propst etal, 1989; Lavery, 1990).

Vessels of 1000 to 2000 dm3 are commercially available. Celltech Ltd, U.K., have used such vessels to produce monoclonal antibodies from hybridoma cells (Wilkinson, 1987).

Mitroearricrs

Microcarriers may provide a solution to the probl of growing anchorage-dependent cultures in suspension culture in fermenters, by providing the necessary su" face for attachment. Animal cells normally have a net negative surface charge and will attach to a positively' charged surface by electrostatic forces. Van Wezel (1967) made use of this property and attached anchorage dependent cells to chromatographic grade DEAE Sephadex A-50 resin beads and suspended the coated beads in a slowly stirred liquid medium. The density of the electrostatic charges on the microcarrier surface is critical if cell growth at high bead concentrations is to be achieved. If the net charge density on the bead surface is too low, cell attachment will be restricted. When the charge density is too high, apparent toxic effects will limit cell growth (Fleischaker, 1987). A number of microcarrier beads, manufactured from dex-tran, cellulose, gelatin, plastic or glass, are now commercially available (Fleischaker, 1987; Butler, 1988). Dextran microcarriers have been used for large scale production of viral vaccines and interferon. Unfortunately some of the microcarriers cost £1200 to £1500 kg"1.

Encapsulation

At least three methods of encapsulation have been developed (Griffiths, 1988; Lavery, 1990). Encapsel, a technique developed by Damon Biotechnology, U.S.A., traps the animal cells in sodium alginate spheres which are then coated with polylysine to form a semi-permeable membrane. The enclosed alginate gel is solubilized with sodium citrate to release the cells into free suspension within the capsules which are usually 50 to 500-/xm diameter. After a few weeks growth it is possible to obtain cell concentrations of 5 X 10 s cm ~3 and product levels 100 times higher than with free cells can be achieved. The high molecular weight products are retained within the capsules. This technique has been used commercially for monoclonal antibody production.

In a second method the cells are entrapped in calcium alginate which will allow high molecular weight products to diffuse into the medium. Unfortunately, the spheres tend to be 0.5 to 1.0 mm diameter and slow diffusion into the spheres may cause nutrient limitations. Alternatively the cells can be entrapped in agarose beads in which the cells are contained in a honeycomb matrix within the gel. These capsules have a wide size distribution and a low mechanical strength compared with alginate ones.

achieved by increasing the number of small vessels. This technique is available only as a contract production service from Bioresponse Inc. of California, U.S.A.

Hollow fibre chambers

Anchorage dependent cells can be cultured at densities of 108 cells cm~3 using bundles of hollow fibres held together in cartridge chambers (Hirschel and Gru-enberg, 1987; Griffiths, 1988). The cells are grown in the extra capillary spaces (ECS) within the cartridge. Medium and gases diffuse through from the capillary lumea to the ECS. The molecular weight cut-off of the fibre walls may be selected so that the product is retained in the ECS or released into the perfusing medium. Many chambers will be needed for scale-up because of diffusion limitations in larger chambers. These have been used to study production of monoclonal antibodies, viruses, gonadotropin, insulin and antigens.

Packed glass bead reactors

Packed glass bead reactors have proved to be useful for long term culture of attached dependent cell lines. It is possible to obtain cell densities of 1010 viable cells in a 1-dm3 vessel with moderate medium flow rates through the vessel (Fig. 7.55; Propst et al., 1989). Increasing the size of vessels causes problems with mass transfer of oxygen and nutrients and scale up can be

Perfusion cultures

Perfusion culture is a technique where modified fermenters of up to 100 dm3 are gently stirred and broth is withdrawn continuously from the vessel and passed through a stainless steel or ceramic filter. This type of culture is sometimes referred to as spin culture, since the filter is spun to prevent blocking with cells. The filtered medium is pumped to a product reservoir and fresh medium is pumped into the culture vessel. The rate of addition and removal can be regulated depending on the cell concentration in the vessel. With this method it is possible to obtain cell densities 10 to 30 times higher than the maximum cell density in an unmodified fermenter (Lydersen, 1987; Tolbert et al., 1988; Lavery, 1990). This procedure has been used commercially by Invitron Corp. U.S.A. This company has patented a gentle 'sail' agitator to prevent cell damage in a 100-dm3 vessel. Attachment dependent cells have been grown on microcarriers using sail agitators rotating at 8 to 12 rpm.

At Hybridtech Inc. USA, animal cells have been immobilized on a ceramic matrix and medium is perfused through this matrix. In this way high cell densities can be maintained. The apparatus is marketed as the 'Opticell' (Lydersen, 1987).

Sample port

Sample port

Glass Bead Reactor

Direction of flow

Flo. 7.55. Schematic diagram of a glass bead reactor (Browne et al., 1988).

Direction of flow

Flo. 7.55. Schematic diagram of a glass bead reactor (Browne et al., 1988).

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