Lecture 5 cell growth phases

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Industrial Microbiology Dr. Butler 2011

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Lecture 5 cell growth phases

  1. 1. Lecture 5 - Animal Cell Biotechnology Cell growth
  2. 2. Lecture 5 Animal Cell Biotechnology The Phases of Cell GrowthButler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P50.
  3. 3. Lecture 5 Animal Cell Biotechnology The Lag Phase no apparent increase in growth phase is associated with the synthesis of growth factors that must reach a critical concentration before growth startsLength of lag phase is dependent on:a) health of cells (metabolic status) → lag phase will be shorter if inoculum is taken from a dense culture of highly active cells
  4. 4. Lecture 5 Animal Cell Biotechnology The Lag PhaseEnergy Charge energy charge (EC) gives an indication of viability of a cell population for healthy cells, EC = 0.8, 0.9 [ ATP] 1/2 [ ADP]energy charge [ ATP] [ ADP] [ AMP]
  5. 5. Lecture 5 Animal Cell Biotechnology The Lag Phaseb) need for metabolic adaptation → may need to adapt to different medium, temperature, synthesize different enzymes, growth factorsc) cell density of inoculum → should inoculate at 104-105 cells/mL → a high density of inoculum increases the ability of cells to reach the initial critical concentration of growth factors and enzymes more quickly
  6. 6. Lecture 5 Animal Cell Biotechnology The Lag Phase not all inoculum cells are viable → use trypan blue dye test → viable cells exclude trypan blue non stainedcells % viable cells x 100 total numberof cells clones may require a feeder layer of cells –irradiated cells that can’t grow but release growthfactors into the medium
  7. 7. Lecture 5 Animal Cell Biotechnology Growth/Exponential Phase cells undergo mitosis and divide mammalian cells double 18-24 hours follows exponential growth
  8. 8. Lecture 5 Animal Cell Biotechnology Growth/Exponential Phase xN No.2log10 N log10 No x.log10 2 N = final cell concentration No = initial cell concentration X = number of generations of cell growthEquation only works for exponential growth
  9. 9. Lecture 5 Animal Cell Biotechnology Growth/Exponential Phasedoublingtime(t D ) T (h) X T = total elapsed time (h) X = number of generations
  10. 10. Lecture 5 Animal Cell Biotechnology Growth/Exponential PhaseSpecific growth rate (μ) = measure of the rate of dN 1 (h 1 ) increase of cell number or biomass dT N or ln N ln No T ln2 0.6931 μ tD tD
  11. 11. Lecture 5 Animal Cell Biotechnology Growth/Exponential Phase – Cell Cycle G1 – gap1 – uncharacterized phase after mitosis S – synthesis – period of DNA synthesis G2 – gap2 - uncharacterized phase after synthesis M – mitosis – cell division
  12. 12. Lecture 5 Animal Cell Biotechnology Growth/Exponential Phase – Cell Cycle Analysis  Stained cells forced through a nozzle  Stream of cells exposed to a laser ► ► ►  Fluorescence emission detected by photomultiplier  Fluorescence intensity directly proportional to the DNA content  Extrapolate distribution of cells by DNA contentButler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P80.
  13. 13. Fig. 5.12 Flow cytometer and cell sorter
  14. 14. Lecture 5 Animal Cell Biotechnology Growth/Exponential Phase – Cell Cycle Analysis G1 – normal diploid content (1x) S – 1-2x diploid content G2 – 2x diploid content M – 2x diploid contentButler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P79.
  15. 15. Lecture 5 Animal Cell Biotechnology Stationary Phase stationary phase occurs when there is no further increase in cell concentration → death rate = growth rateCell growth is limited by a number of conditions:1) nutrients may have been depleted to a level that cannot support cell growth2) the accumulation of metabolic by-products to a level that inhibits growth → build up of ammonia, lactic acid, etc
  16. 16. Lecture 5 Animal Cell Biotechnology Stationary Phase3) limitation of growth surface → cells have reached confluence (single monolayer of cells covering the available substratum)Cells may still be metabolically active in the absence of growth → high cell density → many may still be viable → secrete product into media
  17. 17. Lecture 5 Animal Cell Biotechnology The Decline Phase - NecrosisTwo different death mechanisms:1) Necrosis passive process that normally occurs when cells are subjected to sudden severe cellular stress leads to breakdown of the plasma membrane, leading to cell swelling and eventual cell rupture “extended” stationary phase
  18. 18. Lecture 5 Animal Cell Biotechnology The Decline Phase - Apoptosis2) Apoptosis (programmed cell death) cell suicide mechanism that occurs in culture or in vivo under normal physiological conditions genetically programmed pattern of cellular events abnormalities in process also lead to transformation endogenous endonucleases are activated, cleave DNA into fragments, forming a DNA ladder
  19. 19. Apoptosis Definition: Cell death process which occurs during the development and aging of animals Also induced By: Cytotoxic lymphocytes, drugs, UV irradiation, deprivation of survival factors and cytokines called death factors.
  20. 20. Apoptosis• -Cells Shrink• -Microviolli disappear• -Nucleus condensed and fragmented• -Cells themselves fragmented with cellular content inside.• -Biochemical hallmark of apoptosis is the fragmentation of chromosomal DNA into nucleosomal size units (180bp)
  21. 21. Lecture 5 Animal Cell Biotechnology The Decline Phase - ApoptosisLane 1: Mr DNA markersLanes 2-4: from mouse thymocytesshowing DNA laddering Smith and Wood, Eds. 1996. Cell Biology 2nd Ed. London:Chapman and Hall. P 507.
  22. 22. Lecture 5 Animal Cell Biotechnology The Decline Phase - ApoptosisButler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P51.
  23. 23. Lecture 5 Animal Cell Biotechnology The Decline Phase - Apoptosis cell shrinks, the nucleus condenses, and the cell fragments into apoptotic bodies, phagocytosed by adjacent cells have identified anti-apoptosis genes (gene products inhibit apoptosis proteins) → inserted into cells to reduce/delay apoptosis → extends stationary phase, production period
  24. 24. Lecture 5 Animal Cell Biotechnology Necrosis vs. Apoptosis – a comparisonhttp://www.niaaa.nih.gov/publications/arh25-31/images0.1.gif - accessed Jan 12/05
  25. 25. Fig. 11.2 Measurement of specific productivity
  26. 26. The final yield of the product will depend on :- • the specific productivity of each viable cell - expressed as μg of product formed per 106 cell-day. • the viable cell density of the culture (x106 cells/ml).
  27. 27. Cell specific productivity Q s= P. t 1 N dt= 0P.
  28. 28. 14 80 Mab from TB/C3.bcl2 Mab from TB/C3.pEFCell density (x106 cells/ml) 12 Growth of TB/C3.bcl2 IgG concentration (ug/ml) Growth of TB/C3.pEF 60 10 8 40 6 4 20 2 0 0 0 20 40 60 80 100 120 140 Time (hour)
  29. 29. Determination of specific rate of productivity 80 28.5 pg/cell per day TBC3.bcl-2 60 TBC3.pEF 40 18.3 pg/cell per dayIgG (mg/L) 20 0 -20 0 20 40 60 80 100 Viability index t 0 X.dt (105 cell-hours/ml)
  30. 30. Wurm,F (2004) Nature Biotech 22: 1393
  31. 31. Problem demonstration 1A bioreactor containing 20 liters of medium was inoculated with a 1.5 L inoculum (3 x 106 cells/ml). A lag phase was observed for the first 26 hours after which cells grew exponentially until they reached a maximum density of 2 x 106 cells/ml after 4 days from the initial inoculation.i) Determine the number of generations of cell growth.ii) Determine the doubling time during cell growth.iii) Determine the specific growth rate.

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