A variety of useful products like interferon, hormones, interleukins, enzymes, antibodies, virus vaccines such as polio, measles, rabies etc. are obtained from cell cultures. For these objectives, scale up of cell cultures are essential and large scale fermentors of up to 10,000 liters are used for this purpose. The method employed to increase the scale of a culture depends on whether the cell is proliferating in suspension or is anchored to the substrate.
The scaling up of cell cultures may be done as:
- Monolayer culture
- Suspension culture
- Immobilized cell cultures
Monolayer Culture This type of culture is essential for anchorage dependent cells. Scaling up of such cultures is based on increasing the surface area of the substrate in proportion to the number of cells and the volume of medium and therefore tends to be more complex than suspension cultures. The available surface area can be increased by using plates, spirals, ceramics and micro carriers.
The various culture vessels used are:`
- Roux bottle
- Plastic film
- Roller bottle
- Helicell vessels
- Multitray unit
- Bead bed reactor
- Synthetic hollow fibre cartridge
- Heterogenous reactors
- Opticell culture system
Immobilized cultures By immobilizing the cultures, there stability and specificity increases. Two basic mechanisms used for immobilization are; Immurement culture methods and Entrapment culture methods
- Immurement culture– In this type of culture, cells are encapsulated in a polymeric matrix by adsorption. Material used matrix is gelatin, polylysine, alginate and agarose.
- Entrapment culture– In this type of culture, the cells are held within an open matrix through which the medium flows freely. The cells may be entrapped within the porous ceramic walls of the unit or cells also can be enmeshed in cellulose fibres.
Roux Bottle It is commonly used in laboratory and is kept stationary so that only a portion of its internal surface is available for cell anchorage. Each bottle provides Ca. 175- 200 cm2 surface area for cell attachment and occupies 750-1000 cm3 space.
Roller Bottle This vessel permits a limited scale up as it is rocked or preferably rolled so that its entire internal surface is available for anchorage. Several modifications of roller bottle further enhance the available surface, e.g.,
(i) Spira-Cel (spiral polystyrene cartridge),
(ii) glass tube (roller bottle packed with a parallel cluster of small glass tubes separated by silicone spacer rings)
(iii) extended surface area roller bottle (the bottle surface is corrugated enhancing the surface by a factor of two), etc.
Multitray Unit A standard unit has 10 chambers stacked on each other, which have interconnecting channels; this enables the various operations to be carried out in one go for all the chambers. Each chamber has a surface area of 600 cm2. This polystyrene unit is disposable and gives good results similar to plastic flasks.
Synthetic Hollow Fibre Cartridge The fibres enclosed in a sealed cartridge provide a large surface area for cell attachment on the outside surface of fibres. The capillary fibres, made up of acrylic polymer, are 350 μm in diameter with 75 μm thick walls. The medium is pumped in through the fiber; it perfuses through the fiber walls and becomes available to the cells.
Opticell Culture System It consists of a cylindrical ceramic cartridge in which 1 mm2 channels run through the length of the unit and perfusion loop to a reservoir is provided for environmental (medium, gas, etc.) control. It gives about 40 cm2 surface area/ml of medium. It is suitable for virus, cell surface antigen and monoclonal antibody production and for both suspension and monolayer cell cultures.
Plastic Film Teflon (fluoroethylenepropylene copolymer) is biologically inert and highly permeable to gas. Teflon bags (5 x 30 cm) filled with cells and medium (2-10 mm deep) serve as good culture vessels; cells attach to the inside surface of bags. Alternatively, teflon tubes are wrapped round a reel with a spacer and the medium is pumped through the tube; cells grow on the inside surface of tube.
Heli-Cell Vessels These vessels are packed with polystyrene ribbons (3 mm x 5-10 mm x 100 μm) that are twisted in helical shape. The medium is pumped through the vessel, the helical shape of ribbons ensuring good circulation; the cells adhere to the ribbon surfaces. All the culture vessels, in addition to the increased surface area due to the vessel design, allow further scaling up by the use of multiple units of the vessels.
Bead Bed Reactors These reactors are packed with 3-5 mm glass beads (which provide the surface for cell attachment) and the medium is pumped either up or down the bead column. Use of 5 mm beads gives better cell yields than that of 3 mm beads.
Heterogeneous Reactors These reactors contain circular glass or stainless steel plates stacked 5-7 mm apart and fitted to a central shaft. Either an airlift pump is used for mixing or the shaft is rotated either vertically or horizontally. The chief disadvantage of the system is very low ratio of surface area to medium volume (1-2 cm2/ml).
Microcarrier Cultures These systems use 90-300 pm dia particles as substrate for cell attachment. Initially, Dextran beads (Sephadex A-50) were used by Van Wezel in 1967; these were not entirely satisfactory due to the unsuitable charge of beads and possibly due to toxic effects.
- Suspension culture These cultures are used for anchorage independent cells. Depending on optimal, physical and chemical factors results are obtained. To keep the cells in suspension and also to make the culture homogenous, the medium must be suitably stirred. The various reactors used for large scale suspension culture are of three types
- Stirred Bioreactors
- Continuous flow reactors
- Air lift fermentors
- Stirred Tank Bioreactors These are glass (smaller vessels) or stainless steel (larger volumes) vessels. These are closed systems with fixed volumes and are usually agitated with motor-driven stirrers with considerable variations in design details, e.g., water jacket in place of heater type temperature control, curved bottom for better mixing at low speeds, mirror internal finishes to reduce cell damage, etc. Many heteroploid cell lines can be grown in such vessels.
- Continuous-Flow Cultures These culture systems are either of chemostat or turbidostat type. In both the types, cultures begin as a batch culture. In a chemostat type, inoculated cells grow to the maximum density when some nutrient, e.g., a vitamin, becomes growth limiting. Fresh medium is added after 24-48 hours of growth, at a constant rate (usually lower than the maximum growth rate of culture) and at an equal rate the culture is withdrawn.
- Airlift Fermenters Cultures in such vessels are both aerated and agitated by air (5% CO2 in air) bubbles introduced at the bottom of vessels. The vessel has an inner draft tube through which the air bubbles and the aerated medium rise