Animal Cell Culture

Indus Biotherapeutics
Cell Culture Process Development
TYPES OF CELL CULTURE IN BIOREACTORS
Types Of Cell Culture In Bioreactors
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
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.
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.
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.
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.
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
Characteristics of Animal Cell Culture

Nutritionally demanding Sensitive to shear and extremes of osmolality Doubling time 12 to 48 hrs Cell Density
Current Choices of Host Cells in Biotech
Yeast
Transgenic Animals
Bacteria Cells
Transgenic Plants
Animal Cells
Comparison of Monoclonal Antibody Produced from CHO & Transgenic Goats

Batch Yield (grams/L ) CHO Bioreactor 3.4 Annual Yield (Kg/yr)
Assumption
Grange Castle 6 X 12,500 L Bioreactors 60 goat herd 350 L/animal year
4000
Transgenic Goats
5.0
40
The Majority of Biotech Products on the Market Are Made in Animal Cells
Animal 60%
Yeast 10%
Microbial 30%
Comparison of Animal and Microbial Culture

Features
Cell wall Cell membrane Growth Rate O2 Requirement Nutritional Rqmt CO2 Requirement Environmental FX Size Seeding density Growth density
Microbes
Generally present Present 10-50% per hour High Usually simple Sometimes Less affected 100-2000 nm 1 cell 109-1010 cells/mL
Animal Cells
Generally absent Present 1-5% per hour Low Complex Key for buffering Very susceptible 10000-100000 nm 105 cells/mL 106 cells/mL
Types of Animal Cell culture

1. 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) usually retain many of the differentiated characteristics of the cell in vivo 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.
Advantages:

Disadvantages:

Types of Cell culture

2. Continuous Cultures
derived from subculture (or passage, or transfer) of primary culture Subculture = the process of dispersion and reculture 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 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 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
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"
Cell Culture Morphology

Morphologically cell cultures take one of two forms:
Anchorage independent cells (Suspension culture) Anchorage dependent cells (Adherent Culture)
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 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
growing as a monolayer that is attached to any surface.

Development of Cell Lines
Bioreactor
A bioreactor may refer to any device or system that supports a biologically active environment.
Requirements for a bioreactor for animal cell culture

1) well-controlled environment (T, pH, DO, nutrients, and wastes) 2) supply of nutrients 3) 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
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)
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 on the basis of mode of operation

Batch
Fed Batch
Continuous
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
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)
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 = ( kd) X dt where X is the cell concentration, 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.
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.
Exponential Phase
Monod batch kinetics is represented mathematically in the following equation:
= max S Ks+ S
where is the specific growth rate, 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
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.
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
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
Batch Curve
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
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.
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.
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
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.

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
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.
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
Perfusion technologies
Product is unstable Product concentration is low

Enhanced sedimentation
Centrifugation Spin filters
Conical settlers Incline settlers Lamellar settlers External Internal
Perfusion Culture
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
Disadvantages of Perfusion Technology Contamination risk Equipment failure Increased analytical costs Long validation time Potential regulatory/licensing issues
Thank you
Stirred Tank Bioreactor Bubble Column Bioreactor Air lift Bioreactor Fluidized bed Bioreactor Packed Bed Bioreactor Flocculated Cell reactors Wave Hollow fiber Perfusion Encapsulation
McLimans' group developed the first "spinner flasks" in 1957.
Original Model
Present Model
Advantages of Spinner Flasks

Easy Visible Cheap Depyrogenation feasible
Disadvantages of Spinner Flasks

Poor aeration Impeller jams Requires cleaning siliconizing & sterilization High space requirements in incubator
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
Bioreactor Design
Airlift Reactors
Stirred Tank Reactor

Stirred Tank Bioreactor
Advantages of Stirred Tank Bioreactor

Versatility
Multi-gas and pH control

Increased Capacity( 5 L to 500 L +)
Disadvantages of Stirred Tank Bioreactor

Costly Size (footprint)/ Weight Preparation - siliconizing, cleaning, Sterilization, depyrogenation Maintenance -Chiller, parts, o-rings
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
Wave Bioreactor
Cellbag Disposable Chamber Exhaust Filter Inlet Air Filter Probe Ports
Harvest Lines
Sampling Port
Aeration Pump
Speed Control
Temp Control
Base 20/50 Shown
Wave Bioreactor
Wave-induced Agitation
Advantage of Wave Bioreactor

DISPOSABLE BIOREACTOR CHAMBER . No cross-contamination, cleaning, sterilization or other validation headaches. SEED PREPARATION Seed 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
Advantage of Wave Bioreactor

COMPLETELY CLOSED SYSTEM Ideal for cell culture, GMP operations. OPERATES WITH OR WITHOUT AN INCUBATOR PROVEN FOR GMP OPERATIONS Used in the GMP production of human therapeutics. Closed system is easy to validate. All contact materials are FDA approved. PERFUSION CULTURE OPTION Patented internal perfusion filters enable perfusion of media for high-density cell culture. EASY TO OPERATE No complex piping or sterilization sequences. Simply place a new presterile Cellbag on the rocker; fill with media, and add your cells
Wave Bioreactor in Perfusion Mode
Packed-bed and fluidized-bed biofilm or immobilized-cell bioreactor
Tissue culture flasks (T-flasks)
Hollow Fiber Bioreactor
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
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 reactors Disposable bioreactors Airlift bioreactors Spin filter stirred tank
Stirred tank bioreactors are most widely used

Animal Cell Culture

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Animal Cell Culture

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Indus Biotherapeutics

Cell Culture Process Development

TYPES OF CELL CULTURE IN BIOREACTORS

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

What is Cell Culture?

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Cell Culture Process Development

Cell Culture Process Development

Cell Culture Process Development

Why do we need Cell culture?

Commercial or large-scale production

Cell Culture Process Development

Advantages of Cell culture

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Characteristics of Animal Cell Culture

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Current Choices of Host Cells in Biotech

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Comparison of Monoclonal Antibody Produced from CHO & Transgenic Goats

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Comparison of Animal and Microbial Culture

Cell Culture Process Development

Types of Animal Cell culture

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Types of Cell culture

Cell Culture Process Development

Types of continuous culture

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Types of continuous culture

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Immortality of continuous culture

Cell Culture Process Development

Cell Culture Morphology

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Cell Culture Morphology

growing in suspension (as single cells or small free-floating clumps)

growing as a monolayer that is attached to any surface.

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Development of Cell Lines

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Types Of Cell Culture In Bioreactors

Cell Culture Process Development

Requirements for a bioreactor for animal cell culture

Cell Culture Process Development

Cell Culture Process Development

Cell Culture Process Development

Types on the basis of mode of operation

Types Of Cell Culture In Bioreactors

Cell Culture Process Development