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Cell Culture BASICS

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  • 1. TYPES OF CELL CULTURE IN BIOREACTORS
  • 2. 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
  • 3. 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.
  • 4. 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.
  • 5. 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.
  • 6. 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.
  • 7. 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
  • 8. Characteristics of Animal Cell Culture
    • Nutritionally demanding
    • Sensitive to shear and extremes of osmolality
    • Doubling time 12 to 48 hrs
    • Cell Density
  • 9. Current Choices of Host Cells in Biotech Bacteria Cells Yeast Transgenic Animals Transgenic Plants Animal Cells
  • 10. 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
  • 11. The Majority of Biotech Products on the Market Are Made in Animal Cells
  • 12. 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
  • 13. 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.
  • 14. 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
  • 15. 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
  • 16. 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
  • 17. 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"
  • 18. Cell Culture Morphology
    • Morphologically cell cultures take one of two forms:
      • Anchorage independent cells (Suspension culture)
      • Anchorage dependent cells (Adherent Culture)
  • 19. 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
  • 20. Development of Cell Lines
  • 21. Bioreactor
    • A bioreactor may refer to any device or system that supports a biologically active environment.
  • 22. 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
  • 23. 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)
  • 24. 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
  • 25. Types on the basis of mode of operation
    • Batch
    • Fed Batch
    • Continuous
  • 26. 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
  • 27. 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)
  • 28. 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.
  • 29. 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.
  • 30. 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
  • 31. 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.
  • 32. 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
  • 33. 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
  • 34. Batch Curve
  • 35. 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
  • 36. 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.
  • 37. 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.
  • 38. 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
  • 39.  
  • 40. 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.
  • 41. 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
  • 42. 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.
  • 43. 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
  • 44. Perfusion Culture
  • 45. 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
  • 46. Disadvantages of Perfusion Technology
    • Contamination risk
    • Equipment failure
    • Increased analytical costs
    • Long validation time
    • Potential regulatory/licensing issues
  • 47.
    • Thank you
  • 48.
    • Stirred Tank Bioreactor
    • Bubble Column Bioreactor
    • Air lift Bioreactor
    • Fluidized bed Bioreactor
    • Packed Bed Bioreactor
    • Flocculated Cell reactors
    • Wave
    • Hollow fiber
    • Perfusion
    • Encapsulation
  • 49. McLimans' group developed the first "spinner flasks" in 1957. Present Model Original Model
  • 50. Advantages of Spinner Flasks
    • Easy
    • Visible
    • Cheap
    • Depyrogenation feasible
  • 51. Disadvantages of Spinner Flasks
    • Poor aeration
    • Impeller jams
    • Requires cleaning siliconizing & sterilization
    • High space requirements in incubator
  • 52. 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
  • 53. Bioreactor Design Airlift Reactors Stirred Tank Reactor
  • 54. Stirred Tank Bioreactor
  • 55. Advantages of Stirred Tank Bioreactor
    • Versatility
    • Multi-gas and pH control
    • Increased Capacity( 5 L to 500 L +)
  • 56. Disadvantages of Stirred Tank Bioreactor
    • Costly
    • Size (footprint)/ Weight
    • Preparation - siliconizing, cleaning,
    • Sterilization, depyrogenation
    • Maintenance -Chiller, parts, o-rings
  • 57. 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
  • 58. Wave Bioreactor
  • 59. Wave Bioreactor
  • 60. Wave-induced Agitation
  • 61. 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
  • 62. 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
  • 63. Wave Bioreactor in Perfusion Mode
  • 64. Packed-bed and fluidized-bed biofilm or immobilized-cell bioreactor
  • 65. Tissue culture flasks (T-flasks)
  • 66. Hollow Fiber Bioreactor
  • 67. 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
  • 68. 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

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