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Plant Cell, Tissue and Organ CultureHORT 515
Cell Suspension Cultures
1. Definition and Background
2. Initiation and Maintenance of Cell Suspension Cultures
3. Suspension Culture Types and Growth Patterns
1. Definition and Background
Cell Suspension - Cultures of single cells (minority) and small cell aggregates (majority) that proliferate and complete a growth cycle while suspended in liquid medium
For a batch culture (finite amount of medium), a growth cycle is referred to as a passage, example
Nickell - 1965 - First report of a continuously maintained cell suspension culture, Phaseolus vulgaris
1. Definition and Background
Cell Suspensions - Cultures of single cells (minority) and small cell aggregates (majority) that proliferate and complete a growth cycle while suspended in liquid medium.
For a batch culture (finite amount of medium), a growth cycle is referred to as a passage.
Nickell - 1965 - First report of a continuously maintained cell suspension culture, Phaseolus vulgaris
2. Initiation and Maintenance of Cell Suspension Cultures
I. Explant materialII. Nutrient mediumIII. Inoculum density/medium conditioning
I. Explant material - Cell suspensions typically are initiated by inoculating the friable callus into liquid medium
Individual cells and/or cell aggregates are maintained in suspension by agitation or aeration, which also minimizes hypoxia.
After the initial passage, culture is typically filtered to eliminate large tissue masses, example
II. Nutrient medium - Medium that results in friable callus proliferation, high auxin relative to cytokinin, w/o agar
Medium Effects on Tobacco Callus Morphology
0.1 mg/L kinetin3.0 mg/L 2,4-D
2.0 mg/L IAA3.0 mg/L 2-iP
friable callus compact callus
Sieve (300 to 500 m) to filter suspension
2nd Passage1st PassageFriable Callus
Procedure for Initiation of a Cell Suspension Culture from Callus
2. Initiation and Maintenance of Cell Suspension Cultures
I. Explant material - Cell suspensions typically are initiated by inoculating the friable callus into liquid medium
Individual cells and/or cell aggregates are maintained in suspension by agitation or aeration, which also minimizes hypoxia
After the initial passage, the culture is usually filtered (300 to 500 µm) to eliminate large tissue masses, example
II. Nutrient medium - Medium that results in friable callus proliferation, high auxin relative to cytokinin, w/o agar
III. Inoculum (minimum) density/medium conditioning - Critical initial cell density/minimum effective density (minimum density) - lowest inoculum density per volume of medium at which a cell culture will grow
Medium conditioning - Cells release metabolites into the medium that accumulate to sufficient levels for growth to initiate, i.e. cell cycle initiation
Medium condition occurs during the lag phase prior to initiation of cell division.
“Artificial” medium conditioning can reduce the minimum density, e.g. from 104 cells/ml to below 103 cells/ml
Conditioning factors are nonspecific, i.e. cells of one genotypecan condition for cells of another
Cells of inoculated into a medium above the minimum density can beremoved after 24 hr and then cells of a different genotype can beinoculated into the medium below the minimum density will besustained
Conditioning factors are low molecular weight compounds,e.g. mitogenic peptide phytosulfokine-, example
Some conditioning factors are volatile
Conditioning requirement can be replaced, to some extent, byconstituent additions to the medium
Phytosulfokine--sulfonated pentapeptide (Tyr(SO3H)-Ile- Tyr(SO3H)-Thr-Gln), 89 aa (precursor)
Medium containing sycamore cells at 1.0 x 103
cells/ml (below the minimum density of 1.0 x 104
cells/ml)
Glass tube connected at the bottom with dialysis tubing (<3500 daltons), inside sycamore cells at high
density
Low Molecular Weight Conditioning Factors
Conditioning factors are nonspecific, i.e. cells of one genotypecan condition for cells of another
Cells of inoculated into a medium above the minimum density can beremoved after 24 hr and then cells of a different genotype can beinoculated into the medium below the minimum density will besustained
Conditioning factors are low molecular weight compounds,e.g. mitogenic peptide phytosulfokine-
Some conditioning factors are volatile, example
Conditioning requirement can be replaced, to some extent, byconstituent additions to the medium
Volatile Conditioning Factors
Sycamore cells inoculated at 600 cells/ml:
1. Medium conditioned – no growth
2. Medium conditioned, second flask (top) contains cells inoculated at high density with a semi-permeable closure - growth
3. Medium conditioned, second flask (top) contains cells inoculated at high density with a semi-permeable closure and 40% KOH trap (captures CO2) – no growth
Conditioning factors are nonspecific, i.e. cells of one genotypecan condition for cells of another
Cells of inoculated into a medium above the minimum density can beremoved after 24 hr and then cells of a different genotype can beinoculated into the medium below the minimum density will besustained
Conditioning factors are low molecular weight compounds,e.g. mitogenic peptide phytosulfokine-
Some conditioning factors are volatile
Conditioning requirement can be replaced, to some extent, byconstituent additions to the medium, example
A. Conditioning factors are nonspecific, i.e. cells of one genotype can condition for cells of another; 24 hr growth period of cells at above the minimum density, remove the cells by filtering and reinoculate new cells at below the minimum density
B. Conditioning factors are low molecular weight compounds, e.g. phytosulfokine-
C. Some conditioning factors are volatile
D. Conditioning requirement can be replaced, to some extent, by additions to the medium, example
Hormones Are Conditioning Factors
Medium Minimum density (cells/ml) Basal 1.0 x 104
Basal + GA + Amino acids + Cytokinin 2.0 x 103
Sycamore cells
Plant Cell, Tissue and Organ Culture HORT 515
Cell Suspension Cultures
1. Definition and Background
2. Initiation and Maintenance of Cell Suspension Cultures
3. Suspension Culture Types and Growth Patterns
3. Cell Suspension Culture Types and Growth Patterns
Culture types
Batch culture – medium volume is finite throughout the culture passage,i.e. growth continues until a nutrient becomes limiting (passage), usuallycarbon
Continuous culture - medium is replenished during culture, i.e. sustainedgrowth
Liquid suspensions of plant cells are grown using a variety of systems that keep the medium aerated and facilitate cell separation
Gyratory or reciprocating shakers or chemostatic airlift fermentation systems
Lag phase - cells activate metabolism for cell proliferation,conditioning is occurring
Exponential phase - cell mass gain is exponential due primarily tocell division
Linear/progressive deceleration phase - linear mass gain dueprimarily to cell expansion
Stationary phase - growth ceases, example
I. Batch culture - finite amount of medium that is not replenished and final cell mass is dependent on the quantity of the limiting nutrient
Growth cycle phases - typical growth cycle is 3 to 6 cell doublings,12 to 14 days in duration
Batch Culture Growth Cycle (Passage)
Linear
Log
Time (days)
Lag
Exponential
Linear/progressive deceleration
Stationary
Cell volume changes - during exponential phase cell size decreases (cell divisionfresh weight gain) and during linear phase cell size increases (cell divisionfresh weight gain)
Cell aggregate size - cells are smaller but more aggregated during the division phase and are larger but less aggregated during the expansion phase
Inoculum density effects - higher inoculum density reduces the length of the lag phase and number of cell doublings per culture interval, example
Growth( )
Cell Volume(----)
Cell Units<10 cells (%)
RelativeGrowth
(log)
Cell Volume
Cell Aggregation
2.0 x 105 cells/ml
6.2 x 105 cells/ml
Inoculum Density
15
5
Time (days)
Continuous culture - culture which is replenished with medium
Closed continuous culture - fresh medium is supplied concomitant with medium harvest, however, cells are not harvested
Culture has a very extended exponential, linear and stationary growth phases
Cell viability is maintained in stationary phase; may be useful for active synthesis of secondary products, example
Open continuous cultures - medium input is balanced with culture (cells + medium) harvest
Growth may be maintained at any growth phase
Closed Continuous Culture
Batch Culture
Time (days)
Growth
0 5 10 15 20 25
Growth of a Closed Continuous Culture
Continuous culture - growth rate and cell density are held constant by a continuous input of nutrient and harvest of culture (medium and cells)
Steady-state growth rate can be established at any growth stage
Semi-continuous culture - medium is supplied periodically at the same time that an equivalent volume of culture is harvested
Cultures are allowed to reach a specific cell density over periodic interval and growth rate is averaged over the interval examples
Open continuous cultures - medium input is balanced with culture (cells + medium) harvest
Figure 3. Establishment of steady-state kinetics for dry weight accumulation by tobacco cells grown in semicontinuous culture (see Materials and Methods section). Dilution rate, D, 0.1 day-1 and steady state cell density, ss = 12 g DW L-1. Each point represents two replicate samples from a single flask (DW, dry weight).
Days After Inoculation
Dry
Wei
gh
t (g
L-1)
Xss
0 10 20 30 40 50 60 700
5
10
15
20
Establishment of a Semi-continuous Culture