BIOREACTORS

We have dealt with laboratory based animal cell culture which is a small scale process.This is used for the following purposes:•To study the cell morphology•To study the effect of different drugs on a cell line•To study the different concentrations of same drug

Commercial products obtained

Product Examples

Enzymes Urokinase,

tissue plasminogen activator

Hormones growth hormone

Growth factors Viral vaccines Human-rabies, mumps, rubella etc. Veterinary-

FMD vaccine, New Castle's Disease etc

Monoclonal antibodies Diagnostic tools

Insect virus Baculovirus Bioinsecticides

Immunoregulators Interferons and interleukins

Whole cells Toxicological testing

Scale up of cultureSmall scale Large Industrial ScaleResearch purpose Application purpose (1 Litre) (10,000 lts)

Scale up of culture is done as

•Monolayer culture•Suspension culture•Immobilized cell systems.

Types of scale-up for Anchorage dependant or monolayer cultures is done using these culture vessels

Scale up I•Plates-Roux plates•Roller bottles

Scale up IIModifications of Roller bottles

Scale up III•Multi-tray units•Hollow fiber cartridge•Plastic bags•Plastic film propagators

The following make the monolayer culture system similar to suspension cultures.•Glass bead reactors•Stacked plate reactors•Microcarrier systems

Types of scale up for Suspension cultures

Scale up is much easier than the anchorage dependant cell cultures.

There are 3 main types of reactors •Stirred tank Mode of operation may be

Batch or continuous•Looped reactors•Continuous flow reactors

Roux flasks Roller bottles Modifications of Roller bottles

One side of bottle for the cell culture to grow

Onto the surface of the bottle

Spiral Glass tubes

Stationery flask Bottles rotated at 10-20 rph

Bottles filled with film of plastic , roller

Bottles packed with glass tubes separated from each other by silicone spacer rings. Bottles rolled clockwise and anti clockwise

SA of 175cm2 SA of 750-1500 cm2 SA increased 5-10 times SA increased

Medium 100-150ml 200- 500ml

2x107 diploid cells 2x108 diploid cells

No gas exchange They are dipped in the medium for 25% of the time and are aerated for 75% of the time. Medium aerated, cells alternately fed, Gas exchange permitted.

Oxygen needs to be provided.

Oxygen needs to be provided.

Multi-tray units:

•Usually 10 flat chambers stacked on each other, interconnecting at corners with each others. the tubes have apertures, medium can flow between compartments. This is a polystyrene unit •SA of each 600 cm2.

Harvesting of cells•Cell harvesting by placing the unit on its one end and draining off the medium •wash with buffer •trypsinize the culture, gets removed from the surface of the unit.•Can be reused after proper washing•Interferons production

Disadv: •large amount of medium utilized•Not being able to completely remove the cells attached

Hollow-fibre catridge

•Synthetic •Fibres enclosed in sealed cartridge provide a large SA for cell attachment to the outside of fibres•Fibres of acrylic polymers (suspension) and polysulphone (anchorage), 350 um in diameter, 75um in thick walls.•The SA to volume ratio is very high supports 108 cells/ ml.•Medium perfused and becomes available to all cells.•Hollow fibre enables the continuous removal of product from other side. •Increasingly used for production of anti bodies

Plastic bags•Made up of Teflon, which is biologically inert but very gas permeable.•Measures 5x30 cm and medium filled to the depth of 2-10 mm.•Cells attach to both the sides of the plastic bag•Bag can be placed in the incubatorCells harvested by trypsinization

•Alternatively, plastic films used which is in from of long plastic tube wrapped around a reel.•The medium is pumped through the tube and cells grow on the inside of the tube.•10 mts long and gives SA of 25,000cm2.

•Culture vesssel also called as Stericell.

All scale up systems described so far can be placed inside conventional incubators to maintain an even temperature.

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

Hence, scale up performed in a single unit by increasing the vessel volume. These need independent heating, filtration and gas exchange facilities.. This is done using Bioreactors

Though ideal for the suspension cultures, anchorage dependant cells grow by having substratum in suspension

The three types are: •Glass bead reactors•Staked bed reactors•Microcarrier systems

Bioreactor

May be referred to any device or system that supports biologically active environment.

It is a vessel in which a chemical process is carried out.

Bioreactor design is rather complex engineering task.It must provide the optimum conditions in a controlled manner so that the physiological of a cell is maintained and cells are able to grow and give the desired biological product.

The bioreactor's environmental conditions like gas (i.e., air, oxygen, nitrogen, carbon dioxide) flow rates, temperature, pH and dissolved oxygen levels, and agitation speed/circulation rate need to be closely monitored and controlled.Most industrial bioreactor manufacturers use vessels, sensors and a control system networked together.

Bead bed reactors:Reactors tightly packed with 3-5 mm glass beads which provide SA for the cell attachmentMedium pumped through this system either up or down the bead column.

Stacked plate reactors:

•Circular glass or steel plates are fitted 5-7 mm apart across onto a central shaft.

•Either the shaft can be rotated around its axis or the medium can be moved across the plates

•SA of 2x105 cm2

Microcarriers

•Developed by van Wezel in 1967. •As the name suggests, small particles which will harbour cells on them.•The growth of anchorage dependant culture as suspension culture became possible

Structure•These systems use 90-300um diammeter particles/spheres as substrate provide SA of 4.5-6.0x103 cm2 per gm of spheres•Mostly spherical in form but can be in cylindrical form too. •Made up of dextran, polystyrene,polyacrylamide,DEAE,collagen and gelatine

Types of microcarriersSurface macroporous, high density macroporous, microspheres

Advantages

•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

Disadv•Sterilization is a problem

Initiation of microcarrier cultures•3lts of log phase culture inoculated in the fresh medium to which 2-3 g/l of microcarrier added•cult stirred at 15-25 rpm for 3-8 hours•Cells attach to the microcarriers, grow as monolayer•Vol of culture increased, to 3lts and stirring enhanced to normal rates (20-100rpm)•Cells grow, microcarriers become heavy, settle down therefore needs to be agitated.•medium changed every 3 days.•samples checked intermitttently

Harvesting of cells: is simple•stirring stopped•medium drained off•beads washed in buffer•treated with trypsin•culture shaken at 75-125 rpm for 20-30 mins, cells detach•stopped shaking•allowed to settle, supernatent is poured and collected passed through funnel with appropriate size filter, so that cells pass thru and the microcarriers remain

Alternatively, gelatin beads dissolved by trypsin/collagen beads treated with collagenase/dextranase treated with dextran , set the cells free.

Applications:A wide range of cells have been cultured on microcarriers. •For instance, cells from invertebrates, from fish, birds and cells of mammalian origin have been cultivated on microcarriers.

•Transformed and normal cell lines, fibroblastic and epithelial cells and even genetically engineered cells for various biologicals such as for the production of immunologicals like interferons, interleukins, growth factors etc. •Cells cultured on microcarriers also serve as hosts for a variety of viruses that are used as vaccines like foot and mouth disease or rabies.

Some of the parameters to be considered for scale-up of culturesto maintain the physiological conditions for cell growth

•Proper medium and supplements need to be provided:Depletion of nutrients can be growth limitingImportant to regularly supplement the medium with glucose in small amounts.

•OxygenSupply of O2 necessary throughout the culture. However, it is poorly soluble.Therefore aeration devices neededSurface aeration, sparging, medium perfusion, increase partial pressure of O2 in headspace.Surface aeration through passing gas through nozzle into headspaceSparging is bubbling of gas into the medium.Medium continuously taken out and passed through an oxygenated chamber and then returned to the chamber (used for microcarrier systems).Increased partial pressure helps to dissolve O2 and diffusion rates in the medium

•StirringThe medium must be suitably stirred to keep cells in suspension to make culture homogenousDifferent stirrers used, magnetic/ flat blade impellers, air lift. Aim of stirring is to mix well and not to damage the cells. Hence, the speed of rotation has to be optimized. Also the impeller design has to be appropriate.

•Shear forcesSodium carboxymethyl cellulose,(Na-CMC) added to protect cells against mechanical damagePluronic F-68 added to reduce foaming and prevent cells from attaching to the reactor sides(a mixture of polyoxyethylene and polyoxypropylene) is a non-ionic surfactant.Strengthening of the cell membrane making the cell more resistant to shear. It gets absorbed onto the cell membrane, suggested that effect is due to the creation of an artificial wall around the cell.

•pH: Require 7.4.pH influenced by buffering components of medium, amount of head-space and concentration of glucose.Medium buffered with CO2-bicarbonate system.Checked by the pH inserted in the medium. This is attached to reservoirs of sodium bicarbonate or dilute sodium hydroxide which can be programmed to release enough alkali into medium to maintain pH.

•Temperature controlLarge vessels either supplied with External water jackets or internal heating/cooling coils

•Redox potentialAffected by the proportion of oxidizing and reducing agents in the medium.The redox values falls during the log phase of the culture and lowest when the culture reaches stationery phaseRegular monitoring of the redox is done with the probe.

•Material used to construct bioreactorGenerally,made up of stainless steel.Leaching of the metal ions, therefore must be the material must treated to prevent these toxic ions from leaching into medium

•Sterilization•Fouling can harm the overall sterility and efficiency of the bioreactor, especially the heat exchangers. To avoid it, the bioreactor must be easily cleaned and as smooth as possible •Vessel connected to the steam generator which injects super- heated steam into the vessel.•The feeder pipes, outlet pipes, probes, and taps valves must also be sterilized.

Bioreactor design: bioreactor is provided with the following

Vertical sight glass and ports for pH, temperature and DO sensors.

Connections for acid and alkali (for pH control), antifoam agents and inoculum are located above the liquid level in the reactor vessel.

O2 and other gases (CO2 or NH3 for pH control; N2 for O2 control) can be introduced through a sparger at the bottom.

Foam breakers are used when antifoam is ineffective or the antifoam interferes with downstream processing (antifoam tends to foul the membrane during filtration).

Can be sterilized in-place using saturated steam at a minimum absolute pressure of 212 kPa. Over–pressure protection is provided by a rupture disc on the top of the reactor, which cracks to relieve the pressure to avoid explosion.

The vessel should have as few internals as possible and should be free of stagnant areas where pockets of solids or liquids may accumulate.

Maximum allowable working pressure of 377-412 kPa (absolute), allowable temperature is usually 150-180C (>121C for sterilization). The vessel should withstand full vacuum or it could collapse while cooling after sterilization.

Suspension cultures

Cultures which can be grown in suspension, haemaopoietic tissues, hybridoma lines

Stirred tank bioreactors

•Glass vessels(small) or steel (larger)•These are used for volumes >5 lts till 8000 lts

•Closed systemswit hfixed volumes, agitated with motor driven stirrers•STR enable homogenization, suspension of solids, dispersion of gas-liquid mixtures, aeration of liquid and heat exchange.•Stirred by means of properly designed impellers, so that homogenous culture conditions maintained. •The effectiveness of agitation depends upon the de-sign of the impeller blades, speed of agitation and the depth of liquid. •Usually batch type mode of operation.

•Many heteroploid cells grown, hybridoma cells

Looped bioreactors

They have features similar to stirred tank reactors. Called as pneumatic reactors

They have inner draft tube and the air is introduced in form of bubbles at the bottom of the vessel.

Cultures aerated and agitated by the turbulence caused by the air bubbles introduced at bottom of the vessel.

Air lifts to the top and passes out through the outlet. The medium and cells move out of the draft tube and are recirculated. Mixing is by the cyclic flow of culture around the vessel.

Gas type most gentle preferred for animal cell culture esp for monoclonal Antibody production

Gas type Impeller type Jet type

Continuous flow bioreactorsUsed for increasing cell densities by

Allow the cells to grow, with intermittent removal of the product and the addition of fresh medium to the culture.

•Cells experience a constant, and steady supply of limiting substrate and nutrients. Consequently, they can (over time) adjust their enzyme levels, pH and osmotic gradients, macromolecular composition etc. to achieve an “optimal growth”. This situation is generally referred to as “steady state” and may take up to some generations to achieve.

•The biomass concentration and growth rates are maintained at steady state and the physical/chemical environment of cells also remains in steady state.

These systems are either of chemostat or turbidostat

In chemostat, the inoculated cells grow to maximum density when some nutrient becomes growth limiting. Fresh medium added at constant rate and the culture withdrawn. Thus a steady state maintained where both cell density and medium composition remain constant to achieve max growth.

In turbidostat, a predecided cell density has to be maintained. A fixed volume of culture withdrawn and same volume of fresh medium added, lowers cell density , cells keep growing maximum density achieved and the process repeated.

Continuous production of cells for interferons and viruses.

Often used as two stage system first promotes cell growth and the next allows the product generation

Immobilized cultures:

Higher cell densities, stability and longetivity, protection from shear forces

Immurement and entrapment cultures

Immurement cells confined within a medium permeable barrier

Hollow fibres: Fibres in cartridge. Medium circulated through fibres, cells in suspension outside fibre. Very efficient for high cell densities.

Encapsulated:in a polymeric matrix by adsorption, covalent bonding, to matix material ,made up of gelatin, agarose.

The cells grow on surface and inside the pores, medium diffuses freely into the matrix and into cells while the cell products move out into the medium

Allows production of Antibodies, hormones, immunochemicals, enzymes

Entrapment culturesIn this approach cells are held within an open matrix through which the medium flows freely. An example is the Opticell in which the cells are entrapped within the porous ceramic walls of the unit. Opticell units of upto 210 m2 surface are available, which can yield upto 50 g monoclonal antibodies per day. The cells can also be enmeshed in cellulose fibres, e.g., DEAE, TLC, QAE, TEAE. These fibers are autoclaved and washed as prescribed, and added in a spinner/stirred bioreactor at a concentration of 3 g/1. .

Porous microcarriers are small beads of gelatin, collagen, glass or cellulose which have a network of interconnecting pores. These provide a tremendous enhancement in surface area/volume ratio, permit efficient diffusion of medium and product, are suitable for scaling up, and are equally useful for suspension and monolayer cultures. These can be arranged as fixed bed or fluidized bed reactors or used in stirred tanks

Advantages of Cell Immobilization1) Larger size, cells in or on a support material provide physically larger particles than free cells. Are more resistant to mechanical damage and easier for processors to handle.

2) IM cells are particulate so easily separated from the soluble product simplifying downstream process operation.

3) IM enables greater cell density to be achieved i.e. more cells per volume, and reduces reactor size but increases product compared to free cells.

4) IM provides a better local microenvironment sort of artificial tissue seems to stabilize cells and stimulate growth compared to free cells.

5) IM enables re-use of cells as product easily removed more substrate added. Also enables prolonged use for slow reactions by perfusion of substrate over the IM cells.

6) Cells inside support material are protected from antibodies and other components of the immune system compared to free cells.

7) Cost is low compared to the advantages gained over using free cell suspensions.

It is expected that future developments will make the immobilized cell systems the most dominant production systems.