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Cell Culture  BASICS

Cell Culture BASICS






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    Cell Culture  BASICS Cell Culture BASICS Presentation Transcript

    • 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 Assumption Annual Yield (Kg/yr) Batch Yield (grams/L) 60 goat herd 350 L/animal year 40 5.0 Transgenic Goats Grange Castle 6 X 12,500 L Bioreactors 4000 3.4 CHO Bioreactor
    • The Majority of Biotech Products on the Market Are Made in Animal Cells
    • Comparison of Animal and Microbial Culture 10 6 cells/mL 10 9 -10 10 cells/mL Growth density 10 5 cells/mL 1 cell Seeding density 10000-100000 nm 100-2000 nm Size Very susceptible Less affected Environmental FX Key for buffering Sometimes CO 2 Requirement Complex Usually simple Nutritional Rqmt Low High O 2 Requirement 1-5% per hour 10-50% per hour Growth Rate Present Present Cell membrane Generally absent Generally present Cell wall Animal Cells Microbes Features
    • Types of Animal Cell culture
      • 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
      • 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
        • 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
        • 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 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 = (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 = μ max 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 = -k d 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 settlers
      • Incline settlers
      • Lamellar settlers
          • Centrifugation
          • Spin filters
      • 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. Present Model Original 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
    • Wave Bioreactor
    • Wave-induced Agitation
    • Advantage of Wave Bioreactor
      • DISPOSABLE BIOREACTOR CHAMBER . No cross-contamination, cleaning, sterilization or other validation headaches.
      • Seed culture can be prepared in the final system itself, i.e. batch can be started with 100ml and can go to 2000ml.
      • Lack of bubbles and mechanical devices
    • Advantage of Wave Bioreactor
      • COMPLETELY CLOSED SYSTEM Ideal for cell culture, GMP operations.
      • 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