Cell suspension cultures

Definition of cell culture A cell suspension culture can be defined in a practical way has

an homogenous suspension of dividing cells easily to take up

some aliquot only by a glass-pipette

A cell suspension culture consists of cell aggregates dispersed and growing in moving liquid media

Cell suspension culture uses

Understanding of biosynthetic pathway

Mutant selection

Secondary metabolite production

Use of suspension cultures in plant

propagation.

Establishment steps for cell suspension culture

1. Choice of the explants and induction to cell

division

2. Inoculum in a liquid culture medium.

3. Subculture of cell suspension culture

Obtaining friable callus.

1) Choice of explant

2) Identification of a suitable growth medium

Cell suspension culture from friable callus

Cell suspension culture is normally initiated by transferring

pieces of undifferentiated and friable calli to a liquid medium.

Callus as inoculum in liquid culture medium

Callus:

is separated from the parent explant and transferred to a

fresh medium to build up reasonable amount of callus tissue.

is transferred to fresh medium every 4-6 weeks.

is an essential step to avoid cell aging that is visible as

reduction of growing and dark spot.

Compact callus

Callus compact can be an alternative to

friable callus.

Transfer it with part of explant in the

liquid culture medium and after a period

variable from 7-10 days collect cells and

small clumps by a glass pipette. Transfer

it in fresh medium.

Media composition

• A wide variety of explant and media composition has been

used : Heller, B5 Gamborg and MS

• To these media are added, vitamins, inositol, sucrose, and

auxin (2,4D) at 1-5 M concentrations for cell to divide

Culture vessels • Wide-mounthed Erlemeyer flasks

are widely used as culture vessels.

• The flasks are normally sealed with

aluminium foil.

• Cotton wool plugs may be used for sealing flasks during

autoclaving but not for culturing cells. They are a common source of contamination on flasks that are sitting on a shaker for several weeks.

• Flasks closure must maintain sterility, allow gas exchange and reduce evaporation.

Orbital shaker • Platform shaker are widely used for the initiation and serial

propagation of plant cell suspension culture.

• They should have a variable speed control (30-150 rpm) and

the stroke should be in the range of 4-8 com orbital motion.

• The shaker should be kept in the air-conditioned room wit

good temperature control

Agitation of medium serves two purposes.

1. It exerts a mild pressure on cell aggregates, breaking them

into smaller clumps and single cells.

2. It maintains uniform distribution of cell and cell clumps in

the medium. Movement of the medium also provides good

gaseous exchange between the culture medium and air.

Fine cell suspension culture

In an ideal cell suspension culture there are single isodiametric cells and few clumps of 20-100 cells.

Mostly of cell suspension culture is made up by an heterogeneous cell population either by size than by specific density.

Methods for obtaining a well-dispersed suspension culture

De-Long flasks Sieving

Siringe Adding in the

medium cellulase and pectinase

Cell size

Mesh 150 x 150 m

Use of cell density for obtaining a fine cell suspension culture.

Cell can contain: vacuole, starch and other

Therefore cell can be separated on own density by a

centrifugation.

Discontinuous gradient in an appropriate solution

Ficoll is largely used as solute due to this features :

can be sterilized by autoclave

has got low osmomolarity

at high concentration (10-20%) has low viscosity

Establishment steps for cell suspension culture

1. Choice of the explant and induction to cell division

2. Inocolum in a liquid culture medium.

3. Subculturing of cell suspension culture

Subculturing

Cell suspension culture must be frequently and on regular bases

transfer in a fresh medium.

The lag period is usually between one or two weeks.

The ratio of dilution (cell vs medium) is experimentally

determined-

As general rule

1: 4 after one week

1:10 after two weeks

Is a cell suspension culture not sterile ?

• The sterility of plant cell suspension can be monitored by

several parameters:

– change in colour of solution

– change in pH

– smell

– interface analysis

– use of microscope

Establishment of cell culture

The accurate, fast, and reliable

determination of cell growth is of

critical importance in plant cell and

tissue culture

However, the measurement of growth parameters in the different types of cultures, and concomitantly the use of

various containers along with the heterogenity in cell morphology, introduce diverse problems that must be

addressed by using a specific methodology for each case callus and cell suspension cultures represent two of the most

common in vitro systems

Monitoring cell suspension culture.

Cell vitality

Cell Growth

There are several methods for evaluating growth kinetics in plant cell cultures

Selected examples include:

• fresh cell weight,

• dry cell weight,

• settled cell volume,

• packed cell volume,

• cell counting,

• culture optical density,

• residual electrical conductivity,

• pH measurements

Growth of suspension cultures is commonly evaluated as the

settled cell volume (SCV),

packed cell volume (PCV),

fresh cell weight (FCW),

dry cell weight (DCW).

Indirect evaluations include pH

measurements and medium

residual conductivity

Finally, parameters describing growth efficiency, such as specific growth rate (μ), doubling time (dt), and growth index, can be determined

Settled Cell Volume (SCV) and Packed Cell Volume (PCV)

Both parameters allow the quick estimation of culture growth,

while maintaining sterile conditions.

These measurements are useful for monitoring growth in the same

flasks along a culture cycle, because suspensions may be returned

to prior culture conditions.

Care must be taken to maintain sterile conditions.

Settled Cell Volume (SCV) and Packed Cell Volume (PCV)

Volume estimation may not be an accurate way of monitoring

growth, given its dependence on cell morphology (cell and clump

size, cell density, and other).

SCV is determined by allowing a cell suspension to sediment in

graduated tubes. It is reported as the percentage of the total

volume of suspension occupied by the cell mass.

The PCV is determined in a similar way, after it has been

compacted by centrifugation.

Settled cell volume (SCV)

1. Pour the cell suspension in a graduated cylinder of adequate

volume.

2. Allow the suspension to settle for 30 min and record the cell

volume.

3. Take a second reading 30 min later. If the variation between

readings is higher than 5%, record a third measurement after

another 30-min wait period.

4. The volume fraction of the suspension occupied by the cells is

determined as the SCV.

PCV can be determined by centrifuging 10 mL of the

culture in a 15-mL graduated conical centrifuge tube at

200g for 5 min

Settled cell volume (SCV)

Fresh Cell Weight and Dry Cell Weight

• Fresh and dry cell weight represent more precise measurements

of cell growth than the sole culture volume.

• However, both require the manipulation of samples in non

sterile conditions.

• Fresh weight estimation involves less time than that required for

dry weight, but it may not reflect a real measurement of biomass

gain, particularly at the end of the culture period, when most of

the culture growth is because of water uptake.

Protocol for FW and DW

• Collect the cell mass by filtration, using a Büchner funnel

under vacuum.

• Wash the cell package with about 3 mL distilled water and

retain under vacuum for a fixed time period (e.g., 30 s).

• Weigh immediately to reduce variations caused by water

evaporation.

• Fresh and dry weights are determined as described earlier for

callus tissue.

Culture Cell Density In order to obtain a reliable value of the number of cells in a

suspension culture, clusters should be first disaggregated

This can be accomplished by incubating the suspension with an

8% chromium trioxide solution, or with hydrolytic enzymes, such

as cellulase and pectinase.

The chromium trioxide method is quicker and less complicated

than the use of enzymes; however, it hinders the estimation of

cell viability in the same sample.

Because a careful use of enzymes maintains cells viable, the

assessment of the number of living cells by the exclusion of vital

stains can be performed in the same sample.

Cell cluster disaggregation by

chromium and hydrolytic enzymes

1) Take 1 mL of the cell suspension and add 2 mL of 8%

chromium trioxide (CrO3).

2) Incubate the mixture for 15 min at 70°C.

3) Vortex the mixture vigorously for 15 min

A. Take 1 mL of the cell suspension and mix it with 0.5 mL of

10% cellulase and 0.5 mL 5% pectinase.

B. Incubate 30 min at 25°C with rotatory agitation (100 rpm)

Cell counting

Although complicated and time consuming, cell counting

represents the best way to assess culture growth in suspension

cultures.

Nevertheless, it often shows a good correlation with other

parameters, such as electric conductivity.

Cell density is obtained by direct counting of cells under the

microscope, using a cell counting chamber, such as the Sedgewick

rafter cell (Graticulates Limited, Tonbridge England) or the

Newbauer chamber (Sigma-Aldrich, St. Louis,MO). Such devices

hold a fixed volume of the suspension over a defined area.

The base of the chamber is divided in squares, frequently

containing a 1 mm3 (1 μL) volume.

By observing the suspension with a low magnification objective,

cells contained in such a volume are identified and counted.

Cell counting

•Fill the counting cell chamber with the mixture, position

carefully the cover glass on top of the chamber, to avoid the

formation of bubbles.

•Observe under the microscope with the ×10 objective to locate

the squared field.

•Count all the cells contained in 10 squares. Add the values of

the 10 squares (do not obtain the average).

•This number represents the number of cells in 10 μL, so multiply

by 100 to determine the cell number per milliliter.

•Depending on the culture’s cell density, further dilution may be

required, which should be considered in the calculation.

Cell counting

Electric conductivity of culture medium decreases inversely to biomass gain

This is a consequence of ion uptake by cells.

The monitoring of this decrease to assess cell growth offers several

advantages over other methods, such as:

1) continuous and in situ or on-line monitoring of cell growth;

2) no sampling or wet chemical analysis is required;

3) it is economical and efficient;

4) it provides an accurate, reliable, and reproducible

measurement of plant cell growth rate; and

5) it is independent of cell aggregation, growth morphology,

and apparent viscosity

Parameter of growth efficiency

Fresh and dry weight are measurements of tissue’s absolute

biomass at a given sampling time.

Growth index (GI) is a relative estimation of such capacity as it

correlates the biomass data at the sampling time to that of the

initial condition.

It is calculated as the ratio of the accumulated and the initial

biomass. The accumulated biomass corresponds to the difference

between the final and the initial masses.

GI= (WF-WO)/WO

Where GI represents growth index, and WF and W0, represent the

final and initial masses, respectively (either as fresh or dry weight).

The specific growth rate (μ) refers to the steepness of such a curve, and

it is defined as the rate of increase of biomass of a cell population per

unit of biomass concentration.

It can be calculated in batch cultures, since during a defined period of

time, the rate of increase in biomass per unit of biomass concentration is

constant and measurable.

This period of time occurs between the lag and stationary phases. During

this period, the increase in the cell population fits a straight-line

equation

Ln X=t+ ln x0

=(lnx-lnx0)/t

Where xo is the initial biomass (or cell density), x is the

biomass (or cell density) at time t, and μ is the specific growth

rate.

Specific Growth Rate

Measurement of Cell Viability in In Vitro Cultures

• The accurate assessment of the number of viable cells in a

population is very important to prevent the inclusion of low

viable or dead cells in the calculations of results per cell or on a

fresh weight basis or to indicate the maximal attainable cell

density in production processes.

Viable Cell A cell is considered viable if it has the ability to grow and develop

Viability assays are based on either the physical properties of viable

cells, such as membrane integrity or cytoplasmic streaming, or on

their metabolic activity, such as reduction of tetrazolium salts or

hydrolysis of fluorogenic susbtrates.

• To assess cell membrane integrity, dyes such as Evans blue,

methylene blue, Trypan blue, neutral red and phenosaphranin

have been used.

• These compounds leak through the ruptured membranes and stain

the contents of dead cells and then, are accounted for via

microscopic observation or spectrometric estimation.

Assesment of cell viability

• To assess cell membrane integrity, dyes such as Evans blue,

methylene blue, Trypan blue, neutral red and phenosaphranin

have been used.

• These compounds leak through the ruptured membranes and

stain the contents of dead cells and then, are accounted for via

microscopic observation or spectrometric estimation.

Other methods rely on the measurement of the activity of some

enzymes REDUCTASE

MTT(3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl

tetrazolium bromide) and

TTC (2,3,5- triphenyl tetrazolium chloride),

accept electrons from the electron transport

chain of the mitochondria;

as a result, these molecules are converted to insoluble formazan

within viable cells with fully active mitochondria.

Esterase

intracellular esterases hydrolyze a fluorogenic

substrate (fluorescein diacetate), that can pass

through the cell membrane, whereupon they

cleave off the diacetate group producing the

highly fluorescent product fluorescein.

Fluorescein will accumulate in cells, which

possess intact membranes, so the green

fluorescence can be used as a marker of cell

viability

MTT/TTC 1. Wash aseptically cell suspension samples (1 mL) with 50 mM

phosphate buffer, pH 7.5. Repeat twice

2. Resuspend the cells in 1 mL of the same buffer.

3. Add MTT or TTC to a final concentration of 1.25 or 2.5 mM,

respectively.

4. Incubate samples for 8 h in the dark at 25°C.

5. Solubilize formazan salts with 1.5 mL 50% methanol,

containing 1% SDS, at 60°C for a period of 30 min.

6. Centrifuge at 1875g for 5 min and recover the supernatant.

7. Repeat steps 5 and 6. Pool the supernatants.

8. Quantify absorbance at 570 nm for MTT and 485 nm for TTC

MTT assay in cells and protoplasts.

(a) Pink coloured cells (viables).

(b) Cell of purple colour

(viable).

(c) Colourless cell (non-viable).

(d Pink and red coloured

protoplasts (viable).

(e) Protoplast with red

cytoplasm (viable).

(f) Protoplast with purple

cytoplasm (viable).

(g) Control of non-viable cells

dead with FAA.

TTC EXAMPLE

Evans Blue Assay

1. Add Evans Blue (EB) stock solution to cell suspension

samples (1 mL) to a final concentration of 0.025%

(v/v).

2. Incubate for 15 min at room temperature.

3. Wash extensively with distilled water to remove

excess and unbound dye.

4. Solubilize dye bound to dead cells in 50% (v/v)

methanol with 1% (w/v) SDS at 60°C for 30 min.

Repeat twice and pool the supernatants.

5. Centrifuge at 1875g for 15 min.

6. Dilute the supernatant to a final volume of 7 ml

7. Quantify absorbance at 600 nm

Cells and protoplasts stained with

Evans blue.

(a) Non-stained cells (viable).

(b) Viable protoplast surrounded

by blue cellular aggregates

(c) Cell with blue cytoplasm.

(d) Control of Non-viable cells

dead with FAA.

FDA Assay

1. Mix cell samples (1 mL) with 10 μL of FDA stock solution.

2. Incubate for 15 min at room temperature in the dark.

3. Adjust the volume to 4 mL with distilled water.

4. Centrifuge at 1875g for 5 min. Resuspend the pellet in 1 mL 50 mM

phosphate buffer, pH 7.5.

5. Freeze quickly in liquid nitrogen. Thaw and dilute samples to 3 mL

with phosphate buffer.

6. Homogenize with a Brinkman polytron at high speed for 10 s.

7. Centrifuge at 1875g for 20 min.

8. Dilute a 100 μL sample of the supernatant to a final volume of 2 mL.

9. Determine fluorescence at 516 nm, using a 492 nm excitation beam

Microscopic Assay

1. Stain cell samples with FDA by mixing the samples (1 mL) with 10

μL of the stock solution.

2. Incubate for 15 min at room temperature in the dark.

3. Wash with 50 mM phosphate buffer, pH 7.5.

4. Centrifuge at 1875g for 5 min.

5. Resuspend in phosphate buffer (1 mL).

6. Counterstain with EB, following steps 1–5 but using 10 μL of the

EB stocksolution.

7. Determine the number of blue dead cells under a bright field and

yellow-green fluorescent viable cells under ultraviolet light

(excitation: BP 450-490 nm and emission: LP 520 nm) in an

Axioplan microscope in 10 randomized fields in Sedgewick

chamber.

The cells in culture exhibit the following five phases of a growth cycle

i. Lag phase, where cells prepare to

divide

ii. Exponential phase, where the rate

of cell division is highest

iii. Linear phase, where the cells

division slows but the rate of cells

expansion increases

iv. Deceleration phase, where the

rates of cell division and

elongation decreases

v. Stationary phase, where the

number and size of cells remain

constant

The Five phases

Some examples of metabolic variation in the batch culture (1)

Some examples of metabolic variation

in the batch culture (2)