cell suspension cultures · fine cell suspension culture in an ideal cell suspension culture there...
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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)