Plant Cell, Tissue and Organ Culture HORT 515 Cell Suspension Cultures 1.Definition and Background 2.Initiation and Maintenance of Cell Suspension Cultures

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


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


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


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


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 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 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



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

36 37 38 390

2

4

6

8

10

12

Days After Inoculation

Su

gar

or

Dry

Wei

gh

t (g

L-1)

Cell density

Specific growth rate

Periodic fluctuations in specific growth rate (—) and cell density (---) during steady state growth in a semi-continuous culture.

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Plant Cell, Tissue and Organ Culture HORT 515 Cell Suspension Cultures 1.Definition and Background 2.Initiation and Maintenance of Cell Suspension Cultures