CH 7. Cell Suspension Culture

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CH 7. Cell Suspension Culture
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7- 1. Introduction
Tissue or Organ Culture Callus Culture
Cell Culture Protoplast Culture
Suspension Cell Culture Single Cell Clones
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Suspension Cell Culture

• Rapidly dividing
• Homogenous cells or cell aggregates
• Suspended in a liquid medium
• Cultured to produce a cell line?

Single Cell Clones
Cell lines established from single cell origin.
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Suspension Cell Culture
Cell culture
Embryogenic cells
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7- 2. Techniques of cell culture
7 - 2 - 1. Initiation of cell suspension culture
 Fragments of undifferentiated callus 2 - 3 g
/ 100 mL   ? Liquid
medium ? ?
? ?
Subculture ? ?
?   Suspension cell cultures
aeration --------
agitation --------
screen small size clumps
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7- 2- 2. Small batch culture
  Culture volume in a small fixed
volume
lt 100mL
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7- 2- 2. Small batch culture
Gaseous exchange   Moving  
The Steward apparatus
tumble tube
nipple flask
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7- 2- 2. Small batch culture
Plateform shaker   speed   stroke range
Orbital shaker
30-150 rpm
3/4 - 3/2 in.
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7- 2- 2. Small batch culture
  Measuring of cell growth
FW
DW
Cell No.
Packed Cell Volume (PCV)
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mL cells / mL medium
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7- 2- 3. Single cell clones
Bergmann (1960), Nicotiana tabacum, Phaseolus
vulgaris, Single cell isolated from plant
tissue materials ?

suspension cell cultures
sieve cells to obtain true single cell ?
plating culture
?   single cell clones
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plating culture
single cells suspended in agar medium
layer, ca. 1mm thick   observe the cell
growth with inverted microscope,
count the plating efficiency   No. of
colony formed / No. of cells plated
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plating culture
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plating culture
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7- 3. Growth patterns in suspension cell culture
Sigmoidal (S) growth Lag phase --
Logarithmic (log, exponential) -- Stationary
phase  
Stationary
Progressive deceleration
FW, DW,
Linear
Log
Lag
Exponential
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Factors affect the growth cycle
1. Interval of subculture   Subculture at
stationary phase --gt
Long lag phase
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  1. Interval of subculture Subculture
at log phase --gt
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Factors
2. Initial cell density High initial cell
density --gt
Low initial cell density --gt
short lag phase few cell
division
long lag phase long exponential growth
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Factors
2. Initial cell density
  Critical initial density --gt
In general, 0.5 2.5 X 105 cells / mL
4 6 division 1 4 X 106 cells / mL
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3. Cell growth rate
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7 - 4. Synchronous culture

• Cold treatment temperature shocks at 4? for few
  days.
• Starvation method
• Deprivation of an essential growth compound from
  suspension culture leads to a stationary growth
  phase.
• Resupplying the missing compound or subculturing
  to a fresh complete medium will allow growth to
  resume and may result in synchronization of cell
  growth.

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• 3. Use of inhibitors
• Cell synchrony at G1/S stage used below
• 5-fluorodeoxyuridine (FudR)
• Excess thymidine
• Hydroxyurea (HU)
• 4. Colchicine method
• One of most effective spindle fiber inhibitors.
• Suspension cultures in exponential growth are
  supplemented with 0.02 (w/v) colchicine.

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7 - 5. Characteristics of plant cell lines
1. A high degree of cell separation,
small size of cell clumps 2. Homogeneous cell
morphology 3. Distinct nuclei and dense
cytoplasm 4. Formation of starch granules 5.
Relatively few tracheal elements
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7 - 5. Characteristics of plant cell lines
6. Doubling time of 24-72 hr., superior
growth rate than that of callus 7. Loss of
totipotency 8. Hormone habituation 9. Increased
ploidy levels 10. Low resistance to shear
stress 11. Contamination during long term culture
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5 - 6. Applications of plant cell culture
Suspension culture