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

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

Potato Cell Suspension Culture

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

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

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

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

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

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 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

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)

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

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  

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

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

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 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

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

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

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

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

( ) (----) 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)

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

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

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

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).

Days After Inoculation 12 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.