by Anita M. Joshi, (Email: anitamjoshi@hotmail.com)
Biotechnology Consultant, Raj Udyog, India.

Biotechnology is a multi-disciplinary science, which harnesses different areas of life sciences for the betterment of mankind. Of the different areas, animal cell biotechnology has developed into an important field in Biotechnology. The study of animal cells has helped us gain an insight not only in the structure and function of cells and tissues but also in different physiological, biochemical and immunological processes. Animal cells are known to produce many chemicals of great value to humans. There is an ever-increasing list of biologicals available from animal cells. Some of the different products obtained from mammalian cells have been tabulated below (Griffiths 1985; Spier and Fowler 1985).

Ever since recombinant DNA technology came into being, it was easy to produce these products in bacteria or fungi on a large scale. Large-scale culture of bacteria and fungi is relatively easy. But the main drawbacks of prokaryotes is that they lack post-translational modifications such as phosphorylation, glycosylation, amidation, carboxylation etc. as a result of which the protein is different and may not be functional. Besides, bacteria and yeasts may not excrete their products. Very often, the wild type cells overgrow the recombinants. Animal cells soon came to be considered as alternatives to bacteria and yeasts for recombinant products. Mammalian cells possess post-translational systems and extraction of products from these cells is possible. As a result, cell lines are being preferred to bacteria and yeasts as cloning systems for developing recombinant products.

Different methods of mass cultivation of animal cells have been devised to meet the growing demand for these cells. The choice of the method mainly depends on the cells to be used. Basically, cell lines may be roughly divided into those that grow as a suspension and those that are anchorage dependent.

The following review article tries to take a look at different methods of mass cultivation with special reference to the microcarrier system.

Anchorage dependent culture

Anchorage dependent cell lines can be grown by the following methods.?

Conventional methods

These methods are followed routinely in our laboratories. They include MD bottles, T flasks, Roux bottles and Rollers. If production is to be increased, the number of units has to be increased making the process time consuming and laborious. The result is that the product may not be cost effective. On the other hand, newer trends in manufacture aim at increasing productivity of a single unit.

?

New trends?

i) Multiple surface tissue culture propagator This consists of stacks of glass plates maintained in a supporting rack within a glass or acrylic culture vessel. These serve as the substratum on which cells can grow. There is an air-lift pump which circulates the medium at a predetermined rate. A sparger is provided to pass air and CO₂ (Weiss and Schleicher; Schleicher and Weiss 1968).?ii) Capillaries Semi permeable membranes, in the shape of capillaries 200-300 u in diameter, are bundled together in a glass culture vessel and medium is circulated continuously. The cells grow along the walls of the capillary. The nutrients can diffuse in and products can diffuse out of the semi-permeable membrane (Knazek et al 1972). Thus it is possible to operate such a system continuously.?iii) As aggregates This is possible mainly with insect cell lines. Enzyme treatment can release cells growing as a monolayer and they form aggregates ranging from 20-500 u in diameter. These aggregates are then grown as a suspension culture. The cells in the aggregates multiply and release products in the surrounding medium (Tolbert and Feder 1978).?iv) EncapsulationCells can be trapped inside beads made up of agarose, karageenan, calcium alginate etc. These beads are then introduced into stirred reactors. The products released remain localized in the material which goes to make the beads thereby making downstream processing very easy (Nilsson and Mosbach 1980).?v) Microcarriers These are bead-like structures ranging from 100-180 u in diameter that can be held in homogeneous suspension in stirred reactors. They are capable of providing a large surface area to cells in small volumes of media. Commercially available microcarriers have been made from a variety of materials such as DEAE Sephadex, cellulose, glass, gelatin etc. Cells attach and spread on the microcarriers and gradually grow into confluent monolayers. The microcarriers are held in a medium that is constantly stirred. The features of both suspension and monolayer culture are brought together in this system. Porous microcarriers, which allow growth of cells inside them, are termed as macrocarriers (Butler 1988; Reuveny and Thoma 1986; Spier 1988; Spier 1982). Macrocarriers are also available commercially eg. Cultispher-G. These type of microcarriers will produce better yields, the cells growing in them are not exposed to shearing forces in fermentors. Such microcarriers have been used for cultivating different cells like Vero and CHO and also for viruses such as VSV (Nikolai and Hu 1992). vi) Packed bed reactors

Microcarriers, macrocarriers or encapsulated beads could be used in fixed-bed reactors. The cells are immobilised in a matrix and the culture fluid is circulated in a closed loop. There is no agitation system. If the bed of immobilised cells is disturbed by the circulating medium it is said to be a fluidised bed reactor. Such a process achieves a high degree of aeration and agitation (Karkare et al 1985).?

Suspension Culture

The following methods are available for growing cells as a suspension.?

BioreactorsThese are essentially the classical fermentors used for the cultivation of bacterial and fungal cultures. They consist of a glass or metal vessel covered by a head plate. There is an agitator shaft with impellers, which stir the culture. Different probes are introduced into the vessel and these monitor process parameters like pH, temperature, dissolved Oxygen and CO₂ etc. Fermentors can be scaled up to hundreds of liters. Largest reported fermentor is of 8000 liters (Hu and Dodge 1985; Mowat and Chapman 1962; Phillips et al 1985).?

Hollow fiber reactorThese consist of semi-permeable membranes packed together. In the lumen of these "fibers", cells can grow. The semi-permeable membrane has a definite cut-off and hence nutrients can diffuse into the lumen and products and by-products of metabolism can diffuse out. Since the medium is constantly circulated, products can be obtained continuously. Large surface area to volume is provided for cell growth (Berg 1985; Altshuler et al 1986). Such systems are now available as highly automised systems.?

Air-lift fermentorsThese have a cylinder inside the culture vessel through which air is passed for circulating the medium. It is a very well aerated system. This type of fermentor is used mainly for monoclonal antibody production (Birch et al 1985).?

ChemostatsThis is a continuously stirred tank reactor with an inlet and outlet so that medium can be passed continuously at a fixed rate keeping the volume constant. The theory for chemostats was established by 1) Monod and 2) Novick and Szillard independently in 1942. The culture is first grown in batch mode and when cells reach the log phase they are grown in continuous mode. The medium employed has one growth-limiting nutrient and the rest are in excess. Hence cell growth is proportional to the growth-limiting nutrient. Steady state is obtained by a continuous and constant feed and harvesting rate where the dilution rate is determined by growth rate (Karkare et al 1985).

Suspension culture technology: Different modes

The following modes of cultivation are available for cells grown as a suspension in bioreactors.?

Batch CultureUntil a few years ago, most biologicals were produced as a single batch. Growth is determined by the rate of nutrient depletion and toxic metabolite accumulation. Vaccines and interferons are still produced in this mode. The yields are low compared to other modes. In this type of culture, cells are inoculated into a fixed volume of medium. They consume nutrients as they grow and release toxic metabolites into the surrounding medium. The environment changes continuously. Eventually, cell multiplication ceases due to accumulation of toxic metabolites and depletion of nutrients (Griffiths 1992). ?

Fed-batch culture

The culture is fed intermittently either with the limiting nutrient or with the whole growth medium. Cell growth is limited not by nutrients but by accumulation of waste products. In this mode, a higher cell concentration is achieved and cell viability is maintained for larger periods compared to the batch mode (Griffiths 1992).?

Continuous culture This mode is based on the principle established by Monod (1942). Homoestatic culture conditions are maintained in chemostats with no fluctuations of nutrients, metabolites or cell numbers. Once established they can be maintained at high cell density and high product yield for a long period (Griffiths 1992).?

Perfusion culture

In contrast to continuous culture where steady state is maintained by constant dilution of the culture, in perfusion cultures, the cells are physically retained in the vessel. Spent medium is withdrawn continuously from the culture system and an equal volume of fresh medium is added. Cell concentration increases constantly until it becomes limited at high cell population densities (Mizrahi 1989).?

Fermentation technology for the growth of animal cells and their products:

Fermentors have been used for the cultivation of bacteria and yeasts for a long time. Initially, fermentation was synonymous with alcohol production. Later, bacteriologists learnt to use the same principles for the preparation of vitamins, organic acids, antibiotics etc. This led to the rapid development of a variety of fermentors and methodologies.

It is but natural that the same principles be applied for the mass cultivation of animal and plant cells. However, adaptation of these processes is required. Cultivation of plant and animal cells is difficult, mainly due to the slow metabolism of these cells which is reflected by slow cell growth. Animal cells have complex nutritional requirements as compared to bacteria and yeasts. Thus, the media are complex and costly and prone to contamination. Animal cells lack the cell wall present in bacterial cells making them fragile and shear sensitive. Therefore, the agitation and aeration systems have to be designed differently. Cell densities achieved are low resulting in low concentration of products. Downstream processing procedures required to concentrate and purify the product lead to increased costs (Mizrahi 1989). Inspite of these drawbacks, fermentors have been used for growing animal cells for the last few decades (Mc Liman et al 1957; Telling and Elsworth 1965; Klein et al 1971; Tovey and Brouty-Boye 1976). Different cell lines like BHK-21 (Berg 1985; Altshuler et al 1986; Telling and Elsworth 1965), LS, Namalwa cells etc have been grown in fermentors as submerged cultures (Tovey et al 1973) for the production of viral vaccines and other products. The shear sensitivity and fragility of animal cells are overcome by the introduction of novel impellers, which are paddle shaped. Direct gas sparging produces bubbles capable of rupturing cells and hence supply of gases needs to be done via diffusion through a silicone tubing. The medium contains plenty of serum proteins capable of generating froth. Therefore, agitation has to be slow and gentle. For high-density cultivation, oxygen supply becomes critical (Balin et al 1976). The silicone tubing method of aeration has many advantages. No bubbles are formed and the oxygen transfer rate has been found to be satisfactory. However, the winding of the fragile tubing is difficult and is limited to small scale reactors used in laboratories (Beyeler et al 1989).

Thus suitably modified fermentors can be used for the mass culture of animal cells growing as suspension cultures. If an anchorage-dependent cell line needs to be grown, it becomes necessary to use a carrier system such as microcarriers.

Development of microcarriers:

With the development of microcarriers in 1967 by van Wezel the production of biologicals from anchorage dependent cells is a dream come true. van Wezel made microcarriers composed of cross-linked dextran beads charged with tertiary amine groups (DEAE) having an exchange capacity of 3.5 milli equivalent / gm dry weight. He demonstrated the growth of primary cells and cells from a human diploid cell strain on them as well as the propagation of poliomyelitis virus in the cells grown on microcarriers.

These microcarriers faced the problem of cell attachment due to large charge densities on their surfaces (van Wezel 1973; 1977). It was observed that above a bead concentration of 1 gm/liter, there were increased innoculum losses, long lag periods and diminished capacity for cell growth. Complete cell death was seen at bead concentrations of more than 2 gm/liter. To overcome this problem, many approaches were tried like coating the beads with serum proteins, nitrocellulose or carboxymethyl cellulose.

It was Levine et al 1979 who developed microcarriers of reduced ion exchange capacity (1.5 meq./gm dry weight). These could be used at concentrations as high as 6 gms/liter with no deleterious effect on cell growth. These low charged beads could be used to cultivate primary, diploid as well as established cell lines. Subsequently a lot of work was done to improve the quality of the beads. Different types of microcarriers were developed and patented. Henderson T. M has obtained a patent (US patent no. 4448884) for glass microcarriers. These have an outer layer of glass below which a layer of magnetic material has been added so that the microcarriers may be separated from the culture medium under the influence of a magnetic field. Cross-linked dextran has been found to be the best material for making microcarriers. Also, microcarriers were derived such that the surface charge was limited to the surface layers only. In such microcarriers the absorption of proteins like IgG or albumin present in the serum was low. Collagen has also been used for producing microcarriers. These beads have the advantage that they can be digested by enzymes, thereby facilitating harvesting (Gebb et al 1984).

Advantages of microcarrier cultures:

This system has the following advantages over other methods of large-scale cultivation:

• High surface area to volume ratio can be achieved which, can be varied by changing the microcarrier concentration. This leads to high cell densities per unit volume with a potential for obtaining highly concentrated cell products.
• Cell propagation can be carried out in a single high productivity vessel instead of using many low productivity units, thus achieving a better utilization and a considerable saving of medium.
• Since the microcarrier culture is well mixed, it is easy to monitor and control different environmental conditions such as pH, pO₂, pCO₂ etc.
• Cell sampling is easy.
• Since the beads settle down easily, cell harvesting and downstream processing of products is easy.
• Microcarrier cultures can be relatively easily scaled up using conventional equipment like fermenters that have been suitably modified (Spier and Horaud 1985).

Because of the many advantages of the technique itself, it has gained great popularity. Thus, a large variety of microcarriers are available in the market.

Commercially available microcarriers:

Due to the growing need for large quantities of anchorage-dependent cells, a variety of microcarriers are now available in the market. A list of some of the commercially available microcarriers, together with the names of their manufacturers is given in Table 2.

The availability of a wide range of microcarrier products in the market makes it necessary to define the exact requirements of a good microcarrier.

Requirements for an optimum microcarrier:

• The surface properties of the beads should be such that cells can adhere and proliferate rapidly. In other words, the contour should be even.
• The density of the beads should be slightly more than that of the culture medium, so as to facilitate easy separation. Conventional culture media are aqueous in nature and have densities ranging from 1.03-1.09 g/cc. However the density should not exceed a certain limit the optimum range being 1.03-1.045 gms/ml. Gentle stirring, which will not harm the shear-sensitive cells, should be sufficient to keep them in suspension. If the beads settle down cell growth will be prevented.
• The size-distribution of the beads should be narrow so that an even suspension of all microcarriers is achieved and cells attain confluency at approximately the same time.
• The optical properties should enable easy microscopic observation.
• They should be non-toxic not only for the survival and good growth of the cells but also for cell culture products that are used for veterinary or clinical purposes.
• The matrix of the beads should be such that collisions, which occur during stirring of the culture, do not cause fragmentation of the beads.
• Ideally, the beads should be such that they can be reused (Levine et al 1979).

Choice of microcarriers:

The choice of the microcarrier depends primarily on the objective of the culture. For instance, in the case of extracellular viruses and cell products obtained from established cell lines, dextran microcarriers prove to be the best. If the cells need to be trypsinised or detached then glass or gelatin beads are preferred. When a particular type of cell tends to have a low plating efficiency, it is important to use that microcarrier which enables attachment of the maximum number of cells. The choice of the microcarrier also depends on the morphology of the cells. For instance, when culturing cells with fibroblast-like morphology, microcarriers with a layer of surface charge on the periphery of the bead tends to show better attachment of cells as compared with others. For cells with an epithelial-like morphology, the attachment is better on beads coated with a protein like gelatin ( Levine et al 1979, Spier and Horaud 1985). Macrocarriers can be used when cells are extremely shear sensitive.

Adhesion of cells to microcarrier surface:

The surface on which the cells are grown as well as the cells at physiological pH have either a net negative or a positive charge. The charge density on these surfaces rather than their polarity is responsible for attachment and spreading of cells. It is also known that cell adhesion is mediated by specific cell surface receptors.

Maroudas’s theory of attachment states that cell surface contact is bridged by amphoteric proteins (Figure 1). According to him, the prerequisite for attachment of cells onto any surface is the adsorption of protein factors to culture surface (Mukhopadhay et al 1993) after which the contact between cells and surface can take place. The cells attach onto the substratum before which they produce their own glycoproteins and matrix proteins and then spread out (Figure 2). It is this matrix that adheres to the charged surface and the cells then bind to the matrix via specific receptors. Therefore many a times it has been observed that surfaces that have been conditioned by previous cell growth often provide a better surface for attachment.

Three major classes of transmembrane proteins have been shown to be involved in cell-cell and cell-substrate adhesion viz.

1. Cell-cell adhesion molecules (CAMS) which are calcium independent and Cadherins which are calcium dependent
2. Integrins which are receptors for matrix molecules such as fibronectin, laminin and collagen which bind to them with the help of a arginine-glycine-aspartic acid sequence known as the RGD motif
3. Transmembrane proteoglycans which interact with matrix molecules like collagen but not with the help of the RGD motif (Freshney 1994).

Cell adhesion also depends on a functional contractile system. It involves multiple contacts with the surface. Next numerous filopodia are formed. They fit into a lattice structure formed by the glycoproteins on the substratum. This is followed by cytoplasmic webbing and flattening of the cell mass. Thus the cells get attached onto the substratum (Hirtenstein et al 1980).

Once the cells have attached onto the microcarriers, they grow using the nutrients provided in their culture medium. The complexities of this culture system are mainly due to the large number of parameters affecting cell yield. Medium composition now assumes importance since it contains the carbon and nitrogen source, the energy source, growth factors and dissolved oxygen and other gases. Besides nutrient limitation, growth of cells is also affected by the accumulation of toxic metabolites. Other important considerations are environmental factors like pH and temperature and shear sensitivity of the cells, especially in case of microcarrier cultures employing spinner bottles and fermentors. (See figures 3 and 4).

Applications of microcarrier cultures:

Microcarrier cultures have found a wide number of applications other than mass cultivation as well. Cells growing on microcarriers serve as an excellent tool for studying different aspects of cell biology such as cell-to-cell or cell-to-substratum interactions. Cell differentiation and maturation, metabolic studies have also been carried using microcarriers (Tang et al 1994). Such cells can also be used for electron microscopic examinations or for the isolation of cell organelles such as the cell membrane. Also, this system is essentially a three-dimensional system and serves as a good 3-D model (Jessup et al 1997). Similarly, co-cultivation of cells can be done using this system (Johns et al 1995).

Microcarriers have also been used for the depletion of macrophages from a population of spleen cells. DEAE-dextran microcarriers can potentiate stimulation of lymphocytes by concanavalin A (con A). Microcarrier beads confluent with allogenic tumour cells can be injected in mice to increase humoral and cell-mediated immunity. Plant protoplasts can be immobilised on DEAE-dextran microcarriers (Maroudas 1977).

Due to the large surface area to volume ratio provided by microcarriers, they are now successfully being used for a variety of biologicals on a laboratory as well as an industrial scale of even 4000 liters (Meigner et al 1980; Griffiths 1992; Montagnon et al 1984; van Wezel et al 1978; 1984; Montagnon 1989).

In conclusion, it can be said that microcarrier technology is a wonderful tool for the industry since the technology can be scaled up easily to any extent and can be easily optimised.

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