1. Plant Cell, Tissue and Organ Culture Cell Suspension Cultures
HORT 515
Cell Suspension Cultures
Definition and Background
Initiation and Maintenance of Cell Suspension Cultures
3. Suspension Culture Types and Growth Patterns

2. 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 First report of a continuously maintained cell suspension culture, Phaseolus vulgaris

3. Potato Cell Suspension Culture

4. 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 First report of a continuously maintained cell suspension culture, Phaseolus vulgaris

5. Initiation and Maintenance of Cell Suspension Cultures
I. Explant material
II. Nutrient medium
Inoculum density/medium conditioning
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

6. Medium Effects on Tobacco Callus Morphology
2.0 mg/L IAA
3.0 mg/L 2-iP
0.1 mg/L kinetin
3.0 mg/L 2,4-D
friable callus
compact callus

7. Procedure for Initiation of a Cell Suspension Culture from Callus
Sieve (300 to 500 m) to filter suspension  
Friable Callus
1st Passage
2nd Passage

8. Initiation and Maintenance of Cell Suspension Cultures
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

9. 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

10. Conditioning factors are nonspecific, i.e. cells of one genotype
can condition for cells of another
Cells of inoculated into a medium above the minimum density can be
removed after 24 hr and then cells of a different genotype can be
inoculated into the medium below the minimum density will be
sustained
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, by
constituent additions to the medium

11. Low Molecular Weight Conditioning Factors
Glass tube connected at the bottom with dialysis tubing (<3500 daltons), inside sycamore cells at high density 
Medium containing sycamore cells at 1.0 x 103
cells/ml (below the minimum density of 1.0 x 104
cells/ml)
Phytosulfokine--sulfonated pentapeptide (Tyr(SO3H)-Ile- Tyr(SO3H)-Thr-Gln), 89 aa (precursor)

12. Conditioning factors are nonspecific, i.e. cells of one genotype
can condition for cells of another
Cells of inoculated into a medium above the minimum density can be
removed after 24 hr and then cells of a different genotype can be
inoculated into the medium below the minimum density will be
sustained
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, by
constituent additions to the medium

13. Volatile Conditioning Factors
Sycamore cells inoculated at 600 cells/ml:
1. Medium conditioned – no 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
2. Medium conditioned, second flask (top) contains cells inoculated at high density with a semi-permeable closure - growth  

14. Conditioning factors are nonspecific, i.e. cells of one genotype
can condition for cells of another
Cells of inoculated into a medium above the minimum density can be
removed after 24 hr and then cells of a different genotype can be
inoculated into the medium below the minimum density will be
sustained
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, by
constituent additions to the medium, example

15. Conditioning factors are nonspecific, i. e
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
Conditioning factors are low molecular weight compounds, e.g. phytosulfokine-
Some conditioning factors are volatile
D. Conditioning requirement can be replaced, to some extent, by additions to the medium, example

16. Hormones Are Conditioning Factors
Medium Minimum density (cells/ml)
Basal x 104
Basal + GA +
Amino acids + Cytokinin 2.0 x 103
Sycamore cells

17. Plant Cell, Tissue and Organ Culture Cell Suspension Cultures
HORT 515
Cell Suspension Cultures
Definition and Background
Initiation and Maintenance of Cell Suspension Cultures
3. Suspension Culture Types and Growth Patterns

18. 3. Cell Suspension Culture Types and Growth Patterns
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
Culture types
Batch culture – medium volume is finite throughout the culture passage,
i.e. growth continues until a nutrient becomes limiting (passage), usually
carbon
Continuous culture - medium is replenished during culture, i.e. sustained
growth

19. 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
Lag phase - cells activate metabolism for cell proliferation,
conditioning is occurring
Exponential phase - cell mass gain is exponential due primarily to
cell division
Linear/progressive deceleration phase - linear mass gain due
primarily to cell expansion
Stationary phase - growth ceases, example

20. Batch Culture Growth Cycle (Passage)
Linear
Stationary
Linear/progressive deceleration
Log
Exponential
Lag
Time (days)

21. Cell volume changes - during exponential phase cell size decreases (cell divisionfresh weight gain) and during linear phase cell size increases (cell divisionfresh 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

22. ( ) (----) Time (days) Growth Cell Volume Cell Volume 15 Cell Units
( )
Cell Volume
(----)
Cell Volume 15
Cell Units
<10 cells (%)
Cell Aggregation 5
2.0 x 105 cells/ml
Relative
Growth
(log)
6.2 x 105 cells/ml
Inoculum Density
Time (days)

23. 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

24. Growth of a Closed Continuous Culture
Closed Continuous Culture
Growth
Batch Culture 5 10 15 20 25
Time (days)

25. Open continuous cultures - medium input is balanced with culture (cells + medium) harvest
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

26. Days After Inoculation
Establishment of a Semi-continuous Culture 20
Dry Weight (gL-1) 15
Xss 10 5 10 20 30 40 50 60 70
Days After Inoculation
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).

27. Days After Inoculation12
Cell density 10 8
Sugar or Dry Weight (g L-1) 6 4
Specific
growth rate 2 36 37 38 39
Days After Inoculation
Periodic fluctuations in specific growth rate (—) and cell density (---) during steady state growth in a semi-continuous culture.