Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
TYPES OF CELL CULTURE IN BIOREACTORS
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
What is Cell Culture?
In vitro culture (maintain and/or proliferate) of cells, tissues or organs
Types of tissue culture Organ culture Tissue culture Cell culture
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Organ Culture The entire embryos or organs are excised
from the body and culture Advantages
Normal physiological functions are maintained. Cells remain fully differentiated.
Disadvantages Scale-up is not recommended. Growth is slow. Fresh explantation is required for every
experiment.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Tissue Culture Fragments of excised tissue are
grown in culture media Advantages
Some normal functions may be maintained.
Better than organ culture for scale-up but not ideal.
Disadvantages Original organization of tissue is lost.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Cell Culture Tissue from an explant is dispersed, mostly
enzymatically, into a cell suspension which may then be cultured as a monolayer or suspension culture.
Advantages Development of a cell line over several
generations Scale-up is possible
Disadvantages Cells may lose some differentiated
characteristics.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Why do we need Cell culture? Research
To overcome problems in studying cellular behavior such as: confounding effects of the surrounding tissues variations that might arise in animals under
experimental stress Reduce animal use
Commercial or large-scale production Production of cell material: vaccine, MAbs,
hormone etc which are impossible to produce synthetically.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Advantages of Cell culture Advantages:
Absolute control of physical environment Homogeneity of sample Less compound needed than in animal models
Disadvantages: Hard to maintain Only grow small amount of tissue at high cost Dedifferentiation Instability, aneuploidy
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Characteristics of Animal Cell Culture
Nutritionally demanding Sensitive to shear and extremes of
osmolality Doubling time 12 to 48 hrs Cell Density
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Current Choices of Host Cells in Biotech
Bacteria Cells
Yeast Transgenic Animals
Transgenic Plants Animal Cells
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Comparison of Monoclonal Antibody Produced from CHO & Transgenic Goats
Batch Yield
(grams/L)
AnnualYield
(Kg/yr)Assumption
CHOBioreactor
3.4 4000
Grange Castle
6 X 12,500 LBioreactors
TransgenicGoats
5.0 4060 goat herd350 L/animal
year
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
The Majority of Biotech Products on the Market Are Made in Animal Cells
Animal60%
Yeast10%
Microbial30%
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Comparison of Animal and Microbial Culture
Features Microbes Animal Cells
Cell wall Generally present Generally absent
Cell membrane Present Present
Growth Rate 10-50% per hour 1-5% per hour
O2 Requirement High Low
Nutritional Rqmt Usually simple Complex
CO2 Requirement Sometimes Key for buffering
Environmental FX Less affected Very susceptible
Size 100-2000 nm 10000-100000 nm
Seeding density 1 cell 105 cells/mL
Growth density 109-1010 cells/mL 106 cells/mL
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Types of Animal Cell culture1. Primary Cultures
Derived directly from excised tissue and cultured either as
Outgrowth of excised tissue in culture Dissociation into single cells (by enzymatic digestion or
mechanical dispersion) Advantages:
usually retain many of the differentiated characteristics of the cell in vivo
Disadvantages: initially heterogeneous but later become dominated by
fibroblasts. the preparation of primary cultures is labor intensive can be maintained in vitro only for a limited period of
time.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Types of Cell culture2. Continuous Cultures
derived from subculture (or passage, or transfer) of primary culture Subculture = the process of dispersion and re-
culture the cells after they have increased to occupy all of the available substrate in the culture
usually comprised of a single cell type can be serially propagated in culture for several
passages There are two types of continuous cultures
Cell lines Continuous cell lines
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Types of continuous culture
1) Cell lines finite life, senesce after approximately
thirty cycles of division usually diploid and maintain some degree
of differentiation. it is essential to establish a system of
Master and Working banks in order to maintain such lines for long periods
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Types of continuous culture
2) Continuous cell lines can be propagated indefinitely generally have this ability because they
have been transformed tumor cells. viral oncogenes chemical treatments.
the disadvantage of having retained very little of the original in vivo characteristics
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Immortality of continuous culture
Telomeres lose about 100 base pairs from their telomeric DNA at each mitosis which impose a finite life span on cells after 125 mitotic divisions, the telomeres would be completely gone
Immortal cells maintain telomere length with the aid of an enzyme Telomerase adds telomere repeat sequences to the 3' end
of DNA strands help complete the synthesis of the
"incomplete ends"
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Cell Culture Morphology
Morphologically cell cultures take one of two forms: Anchorage independent cells
(Suspension culture) Anchorage dependent cells (Adherent
Culture)
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Cell Culture Morphology Morphologically cell cultures take one of two forms:
growing in suspension (as single cells or small free-floating clumps) are able to survive and proliferate without attachment to the
culture vessel cells from blood, spleen, bone marrow, etc advantage: large numbers, ease of harvesting
growing as a monolayer that is attached to any surface. grow in monolayer, attached to the surfaces of the culture
vessels from ectodermal or endodermal embryonic cells, e.g.
fibroblasts, epithelial cells various shapes but generally are flat (rounded in suspension) Advantage: spread on surfaces such as coverslips, easy for
microscopy or other functional assays
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Development of Cell Lines
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Bioreactor
A bioreactor may refer to any device or system that supports a biologically active environment.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Requirements for a bioreactor for animal cell culture
1) well-controlled environment (T, pH, DO, nutrients, and wastes)
2) supply of nutrients3) gentle mixing (avoid shear damage to
cells)4) gentle aeration (add oxygen slowly to
the culture medium, but avoid the formation of large bubbles which can damage cells on contact).
5) removal of wastes
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Scale-up Start with small volume reactors
T flasks, shaker flasks (5-25 mL)
Intermediate scale Small, highly controlled bioreactors (1-5 L)
Production scale Large reactors (20-1,000 L)
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Reactor types Tissue flasks
Easy to use for small scale Cell factories
Production of large numbers of cells Labor intensive
Roller bottles Good control of gas phase Labor intensive
Hollow fiber systems High cell densities, good oxygenation Difficult to remove cells
Spinner flasks Mimic a traditional stirred tank reactor
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Types on the basis of mode of operation
Batch
Fed Batch
Continuous
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Batch Culture A closed culture system
which contains an initial, limited amount of nutrient. The inoculated culture will pass through a number of phases following a growth curve. The growth curve contains four distinct regions as Lag Phase Exponential Phase Stationary Phase Death Phase
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Lag Phase
The first major phase of growth in a batch bioreactor
A period of adaptation of the cells to their new environment
Minimal increase in cell density May be absent in some Bioreactors
(depends on seed culture)
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Exponential Phase
Also known as the logarithmic growth phase Cells have adjusted to their new environment The cells are dividing
at a constant rate resulting in an exponential increase in the number of cells present. This is known as the specific growth rate and is represented mathematically by first order growth rate
dX = (μ – kd) X dt
where X is the cell concentration, μ is the cell growth rate
kd is the cell death rate. The cell death rate is sometimes neglected if it is considerably
smaller than the cell growth rate.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Exponential Phase
Cell growth rate is often substrate limited, as depicted in the figure to limited the right.
The growth curve is well represented by Monod batch kinetics, which is mathematically depicted on the following slide.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Exponential Phase
Monod batch kinetics is represented mathematically in the following equation:
μ = μmax S Ks+ S
where μ is the specific growth rate, μ max is the maximum specific growth rate, S is the growth limiting substrate concentration and Ks is the saturation constant which is equal to the substrate concentration that produces a specific growth rate equal to half the max specific growth rate
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Exponential Phase
For Primary Metabolite production conditions to extend the exponential phase accompanied by product excretion
For Secondary Metabolite production, conditions giving a short exponential phase and an extended production phase, or conditions giving a decreased growth rate in the log phase resulting in earlier secondary metabolitwe formation.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Stationary Phase The third major phase of microbial growth in a batch
process occur when the number of cells dividing and dying is in equilibrium and can be the result of the following Depletion of one or more essential growth nutrients
Primary metabolite, or growth associated, production stops
Secondary metabolite or non-growth associated, production may continue
Accumulation of toxic growth associated by-products Stress associated with the induction of a recombinant
gene
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Death Phase
The rate of cells dying is greater than the rate of cells dividing
represented mathematically by first order kinetics as following
dx = -kd X
dt
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Batch Curve
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Fed Batch Culture
Types of Fed Batch Culture
Intermittent Harvest Grow up the culture, harvest and refill with
fresh medium Fed Batch Culture
Extended Fed Batch Culture
Fed Batch Culture with metabolic shift
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Intermittent Harvest
In general, fed batch processes do not deviate significantly from batch cultures.
Cells are inoculated at a lower viable cell density in a medium that is usually very similar in composition to a typical batch medium.
Cells are allowed to grow exponentially with essentially no external manipulation until nutrients are somewhat depleted and cells are approaching the stationary growth phase.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Intermittent Harvest
At this point, a portion of the cells and product are harvested, and the removed culture fluid is replenished with fresh medium
This process is repeated several times, as it allows for an extended production period.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Fed Batch Culture
While cells are still growing exponentially, but nutrients are becoming depleted, concentrated feed medium (usually a 10-15 times concentrated basal medium) is added either continuously (as shown) or intermittently to supply additional nutrients, allowing for a further increase in cell concentration and the length of the production phase.
In contrast to an intermittent-harvest strategy, fresh medium is added proportionally to cell concentration without any removal of culture broth.
To accommodate the addition of medium, a fedbatch culture is started in a volume much lower than the full capacity of the bioreactor
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Extended Fed Batch Culture
Grow up the cells, then begin to feed concentrate of medium components, viability continues to decrease but cell and product concentrations continue to increase.
Can reach very high product and cell concentration.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Fed Batch Culture with Metabolic Shift
In batch cultures and most fedbatch processes, lactate, ammonium, and other metabolites eventually accumulate in the culture broth over time, affecting cell growth, glycoform of the product and productivity.
Other factors, such as high osmolarity and accumulation of reactive oxygen species, are also growth inhibitory
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Fed Batch Culture with Metabolic Shift
After extended exposure to low glucose concentrations, cell metabolism is directed to a more efficient state, characterized by a dramatic reduction in the amount of lactate produced. Such a change in cell metabolism from the normally observed high lactate producing state to a much reduced lactate production state is often referred to as metabolic shift.
Very high cell concentrations and product titers were achieved in hybridoma cells.
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Cell retention and perfusion Characterized by the continuous addition of fresh nutrient
medium and the withdrawal of an equal volume of used medium.
Need of perfusion Product is unstable Product concentration is low
Perfusion technologies Enhanced sedimentation
Conical settlersIncline settlersLamellar settlers
Centrifugation Spin filters
ExternalInternal
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Perfusion Culture
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Advantages of Perfusion Technology
Better economics High cell density High productivity Longer operation duration Small fermenter size flexibility Fast start up in process development Constant nutrient supply Better controlled culture environment Steady state operation Ease of control Better product quality
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Disadvantages of Perfusion Technology
Contamination risk Equipment failure Increased analytical costs Long validation time Potential regulatory/licensing issues
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Thank you
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Stirred Tank Bioreactor Bubble Column Bioreactor Air lift Bioreactor Fluidized bed Bioreactor Packed Bed Bioreactor Flocculated Cell reactors Wave Hollow fiber Perfusion Encapsulation
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
McLimans' group developed the first "spinner flasks" in 1957.
Present ModelOriginal Model
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Advantages of Spinner Flasks
Easy Visible Cheap Depyrogenation feasible
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Disadvantages of Spinner Flasks
Poor aeration Impeller jams Requires cleaning siliconizing &
sterilization High space requirements in incubator
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Four Basic Bioreactor Designs
Stirred tank reactors (mechanical agitation for aeration)
Bubble column reactors (bubbling air into media for aeration)
Internal loop airlift reactors (air and media circulate together)
External loop airlift reactors
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Bioreactor Design
Airlift Reactors Stirred Tank Reactor
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Stirred Tank Bioreactor
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Advantages of Stirred Tank Bioreactor
Versatility
Multi-gas and pH control
Increased Capacity( 5 L to 500 L +)
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Disadvantages of Stirred Tank Bioreactor
Costly Size (footprint)/ Weight Preparation - siliconizing, cleaning, Sterilization, depyrogenation Maintenance -Chiller, parts, o-rings
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Disposable Bioreactor Can be scaled to at least 500 liters A non-invasive agitation mechanism Easy to use Disposable, presterile, and biocompatible Well instrumented, and can be sampled Useful for suspension and adherent culture Suitable for GMP operation
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Wave Bioreactor
Base 20/50 Shown
Inlet Air FilterExhaust FilterCellbag® Disposable Chamber
Sampling Port
Speed Control
Aeration Pump
Harvest Lines
Probe Ports
Temp Control
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Wave Bioreactor
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Wave-induced Agitation
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Advantage of Wave Bioreactor DISPOSABLE BIOREACTOR CHAMBER
. No cross-contamination, cleaning, sterilization or other validation headaches.
SEED PREPARATIONSeed culture can be prepared in the final
system itself, i.e. batch can be started with 100ml and can go to 2000ml.
MAINTAIN QUALITY OF CELLS Lack of bubbles and mechanical devices
SCALABLE TO 500 LITERS
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Advantage of Wave Bioreactor COMPLETELY CLOSED SYSTEM
Ideal for cell culture, GMP operations.
OPERATES WITH OR WITHOUT AN INCUBATOR
PROVEN FOR GMP OPERATIONSUsed in the GMP production of human therapeutics. Closed
system is easy to validate. All contact materials are FDA approved.
PERFUSION CULTURE OPTIONPatented internal perfusion filters enable perfusion of
media for high-density cell culture.
EASY TO OPERATENo complex piping or sterilization sequences. Simply place
a new presterile Cellbag on the rocker; fill with media, and add your cells
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Wave Bioreactor in Perfusion Mode
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Packed-bed and fluidized-bed biofilm or immobilized-cell bioreactor
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Tissue culture flasks (T-flasks)
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Hollow Fiber Bioreactor
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Hollow Fiber Bioreactor
Intraluminal (Cells inside fibers )
Extraluminal (Cells outside fibers)
Fibers are made of a porous material (PTFE and others).
Permits movement of small molecules (O2, glucose), but not cells
Types Of Cell Culture In Bioreactors
Cell Culture Process Development Indus Biotherapeutics
Cell Culture Systems Various cell culture systems were developed over a period of time
Small scale culture systems T-Flask Spinners
Large/production scale culture systems Roller bottle Multiple plate culture systems Bioreactors
Stirred tank reactorsDisposable bioreactorsAirlift bioreactorsSpin filter stirred tank
Stirred tank bioreactors are most widely used