Bioreactors

Presented by- Bhawna Kushawaha P.hd Biotech DUVASU (Mathura)

Contents

Bioreactor designing

Classification of Bioreactor process

Components/ Parameter of bioreactor

Types of bioreactor

Recent advance in bioreactor design

Application

Bioreactor

A bioreactor may refer to a device or system meant to grow animal cells or tissues in the context of cell culture. These devices are being developed for use in tissue engineering or biochemical engineering.

Fermenter

Fermenters are well established for the cultivation of microbes ,proteins ,industrial product(acetic acid, alcohol etc.) under monitored ,controlled environmental and operational conditions up to an industrial scale.

Consideration for bioreactor designing• cell size (10-20µm)• more fragile .• grow more slowly than most

bacteria and fungi• toxic metabolites e.g.

ammonium & lactate produced during growthProperties of

animal cell that set constrains on design of animal cell bioreactor :

In order to ensure adequate mixing at low stirring speeds, the culture vessels are designed with a round bottom, which distinguishes them from the flat-bottom bacterial bioreactors .

Impeller blades which are fitted at the end of mechanical drive shafts are designed to allow vertical as well as horizontal liquid flow.

Vessel should be made up of glass or stainless steel .

Classification of Bioreactor process for suspension culture

1. In terms of process requirements they are of following types-

(i) Aerobic

(ii) Anaerobic

(iv)immobilized cell

bioreactors

2. On the basis of mode of operation, it may be

classified as-

Batch e.g -stirred tank bioreactor

Fed batch. e.g- fluidized bed bioreactor

Continuous -An example of a continuous bioreactor

is the chemostat.

Characteristics principles of fed-batch and batch

Fed-batch batch

Continuous medium addition.

Addition of selected components

energy source (e.g. glucose, glutamine), amino acids, vitamins, salts, metal trace, growth factors.

Dilution of the by-products, etc.

e.g. toxic lactate, ammonia.

Changing environment for the cells.

lower stability of the product of interest.

Alkali addition

Continuous addition and removal of medium can not be done.

• Addition of complete medium at once .

Removal / Dilution of the by-products can not done .

Constant environment for the cells

higher stability of the product of interest.

Less alkali addition for pH control

Fed-batch batch

Continuous dilution of the product of

interest lower concentration

Multiple harvests variation

In total larger volume of harvest increase work load of down-stream

Smaller bioreactor (up to 500 or 1000 L)

less available, less ‘convenient’

Technically more complex higher risk for failure higher risk for contamination

accumulation of the product of

interest

Single cell harvest

Smaller/limited harvest

Larger bioreactor (up to 20000 L)

more available, even reusable

Technically less complex

Cell culture dynamics

Components/ Parameter of

bioreactor1.Agitation Agitation is required

for homogeneous distribution of cells

and nutrient media in the cells.

it can be done by magnetic

stirred ,turbine impeller, marine

impeller. Maximum stirring

rates for suspension – 100-150 rpmMicrocarrier -40

rpm (suspension and anchorage dependent

cells)

2. Aeration

Through bubbling air

Silicon tubing-highly gas permeable

( inconvenient to use)

By medium perfusion-medium is continuously taken from culture vessel , passed through oxygenation chamber

(risk of o2 toxicity).

3. Baffles-used to prevent vortex

formation.

4. Sparger –used to pass air into

vessel.

e.g.- porous sparger

orifice sparger

nozzle sparger

5. Foam control-produced either by

agitation or by component used in

medium like protein. foaming cause

adhesion of cell to inner surface of vessel. to avoid

foaming antifoams are used . like

pluronic f68,liquid paraffin , oil in some

culture.

6.Temperature

Usually set at the same point as the body temp of the host from

which the cell obtained

Cold-blooded vertebrates – 18-

25°C

Mammalian cells – 36-37°C

Temp maintained by use of carefully calibrated and

frequently checked incubators

7.pHMost cells in culture grow best at pH 7.4

Common used buffer bicarbonate-

CO2 or HEPESKeep the pH

medium in a range 7-7.4

When using bicarbonate-CO2

buffer, need to regulate the amount of CO2 dissolved in

the medium

Done by using an incubator with CO2

control set to provide an

atmosphere with between 2% and

10% CO2.

8.Viscosity

In absence of serum in media ,it is necessary to

increase viscosity of media with the help of

carboxymethyle cellulose.

The viscosity can be determined by using commercial available viscometers, for example, cone and plate viscometers, coaxial

cylinders viscometers, and impeller viscometers.

9.Sterilization

Heating –dry heat -7o c for 1 hr

moist heat -121 c for 30 min

Radiation-kill bacteria as well as virus.

X-ray ,UV ray

Chemicals- formaldehyde ,H2O2

, ethylene oxide .

Filtration-syringe filter , depth filter

screen filter.

10.Scale up

Scale up means-to increase volume of culture.

Firstly ,it should be done as pilot experiments.

It provide the closer approximation /prediction for the large scale

production about various factor like pH , temp , aeration ,media.

This may save cost ,labour and time.

Types of bioreactor

Stirred types of bioreactor

Air lift bioreactor

Fluidized bed bioreactor

Tower bioreactor

Gaseous phase bioreactor

Continuous bioreactor

It is batch type of bioreactor.

These are closed system with fix volume.

Used for suspension culture.

It is easy to use.

Good temp control and less expensive.

Easy to maintain sterile condition.

Air Lift bioreactor

The bioreactor consists of two concentric

cylinders.The inner cylinder being shorter at both ends than the outer, thereby creating an outer and an inner

chamber.

The bottom of the inner chamber carries a sintered steel ring through which 5%

CO2/O2-8% in air is bubbled.

The bubbles rise, carrying the cell

suspension with them. O2/CO2 is vented from

the top, and displacement ensures the return of the cell suspension down the

outer chamber.

AdvantageSuited for aerobic

culture.

Low energy consumption.

No agitator shaft is needed.

Greater heat removal vs stirred tank.

DisadvantageGreater air throughput and

higher pressure needed.

No bubble breaker.

Continuous bioreactor

Chemostate-(chemical

environment is constant) cell grow at max density when some

nutrient like vitamin , is

growth limiting.

Turbidostate -cells

grow to achieve higher density.

AdvantageGenerally maintain cells in log phase for longer

period.

Process maintain at steady state .

Large amount of production can be done.

Disadvantage

Difficult to maintain pH and temp.

Tower bioreact

or

Elongated non - mechanically

stirred Fermenters .

Aspect ratio 6:1 (height : diameter )

Unidirectional flow of

gases .

Used for continues

production .

E.g used for singled cell

protien production.

Fluidized bed reactors (FBB)Cells are used as biocatalyst in three phase system (solid ,liquid and gas).

Basically particles used in fbb can be of three types-

Inert core in which cell can attached.

Porous particles in which biocatalyst is entrapped.

Cell aggregates or flocs.

Because of the higher density of the

microcarriers they can be perfused slowly

from below, at such a rate that their

sedimentation rate matches the flow rate.

The beads therefore remain in stationary suspension, perfused

by the medium, constantly

replenishing nutrients and collecting the

product into a downstream reservoir.

Gas exchange is external to the reactor, and no

mechanical mixing is required.

Advantage

Universal particles mixing.

Uniform temp

gradient.

Ability to operate reactor in continuous

state.

Downstream processing of

bioreactorFiltration –surface filtration , depth filtration, cross flow

filtration ,membrane filtration etc.

Centrifugation- difficulty arise due to small

differences in the density of particle and medium .

Ion exchange resins - dextrone , cellulose,

polyamines.

Chromatography- affinity chromatography

,adsorption chromatography.

Recent advance in bioreactor designThe cell seeding of

scaffolds is an important step in establishing a 3D

culture in a macroporous

scaffold.Not only seeding at high cell densities,

but also a homogeneous

distribution of cells within the scaffold

is essential.

As most of the scaffolds have

large, interconnected pores, during

seeding, cells are distributed quite uniformly. During

cultivation, medium flow through a

construct enhances the mass transfer of

substrates, particularly oxygen to immobilized cells

when interconnected cell

free pores are available.

The carriers are arranged in a column either packed (fixed

bed) or fluidized (floating bed). The column is permanently

perfused with a conditioned

medium from a medium

reservoir, mostly in a circulation

loop.

In recent studies, small

well-mixed bioreactors (e.g., shake

flasks, stirred vessels, and

super spinner) have been

suggested for cell

proliferation, in which the cells are grown on

microcarriers .

Offline measurement of dissolved oxygen and

dissolved CO2 were done by sampling the

chamber using a syringe and analyzing it

using a blood gas analyzer (Radiometer

ABL5).

Cell counts were done by

haemocytometer , and product

assays were done by

appropriate methods such

as ELISA.

A perfused flow-chamber

bioreactor with a new concept for

aeration has been introduced

recently ,in which tissue-specific

inserts for various types of tissue (e.g., cartilage, skin, and bone) can be applied.

NASA tissue cloning

bioreactor

In bioreactors in which cells or tissues grow for experimental or therapeutic purposes, the design

is significantly different from industrial bioreactors.

Many cells and tissues, especially mammalian ones, must have a surface or

other structural support in order to grow, and agitated environments are

often destructive to these cell types and tissues.

NASA has developed a new type of bioreactor that

artificially grows tissue in cell cultures.

NASA's tissue bioreactor can grow heart tissue, skeletal

tissue, ligaments, cancer tissue for study, and other types of

tissue.

ApplicationGenetic Engineering

Cell Therapy

Model SystemViral vaccines

Monoclonal antibodies

Recombinant proteins (glycoprotein)

Cancer Research

Toxicity Testing

Drug Screening and Development

Questions....????

Thank you