Cell Culture BASICS

Types Of Cell Culture In Bioreactors

Cell Culture Process Development Indus Biotherapeutics

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

Bacteria Cells

Yeast Transgenic Animals

Transgenic Plants Animal Cells



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



The Majority of Biotech Products on the Market Are Made in Animal Cells

Animal60%

Yeast10%

Microbial30%



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 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 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 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

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



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 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



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 = (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.



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:

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



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 Product is unstable Product concentration is low

Perfusion technologies Enhanced sedimentation

Conical settlersIncline settlersLamellar settlers

Centrifugation Spin filters

ExternalInternal



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.

Present ModelOriginal 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

Base 20/50 Shown

Inlet Air FilterExhaust FilterCellbag® Disposable Chamber

Sampling Port

Speed Control

Aeration Pump

Harvest Lines

Probe Ports

Temp Control



Wave Bioreactor



Wave-induced Agitation



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



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



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 reactorsDisposable bioreactorsAirlift bioreactorsSpin filter stirred tank

Stirred tank bioreactors are most widely used