Scale-up cell culture

20166014 - Naman
20166015 - Netra
20166016 - Reshma
20166017 - Sakshi
SCALE UP OF SUSPENSION CULTURE
A cell suspension or suspension culture is a type of cell culture in which single cells or small
aggregates of cells are allowed to function and multiply in an agitated growth medium, thus
forming a suspension.

Scale-up involves the development of culture systems

in stages from (small scale) laboratory to (large scale)
industry.
The methodology adopted to increase
the scale of a culture depends on the proliferation of cells
SUSPENSION CELL LINES
Scale-Up in Suspension: Scale-up in suspension is the preferred method as it is simpler. Scale-up of
suspension culture primarily involves an increase in the volume of the culture. Small scale generally means
the culture capacity less than 2 litres volume (or sometimes 5 litres).

● Stirred suspension cultures: It is usually necessary to maintain cell strains in stirred suspension
cultures, by agitation (or stirring) of the medium. The stirring of the culture medium is achieved by a
magnet encased in a glass pendulum or by a large surface area paddle. The stirring is usually done at
a speed of 30-100 rpm. This is sufficient to prevent sedimentation of cells without creating shear forces
that would damage cells.

● Static suspension cultures: Some cells can grow in suspension cultures, without stirring or agitation of
the medium, and form monolayer cells. However, static suspension cultures are unsuitable for scale-
up.
FACTORS:
For appropriate scale-up, the physical and chemical requirements of cells have to
be satisfied.

Physical parameters: Chemical parameters:

i. Configuration of the bioreactor. i. Medium and nutrients.

ii. Supply of power. ii. Oxygen.

iii. Stirring of the medium.
iii. pH and buffer systems.

iv. Removal of waste products.

Stirrer Culture:
The size of the stirrer flask is in the range of 2-10 litres.
It is fitted with a magnetized rotating pendulum, and
two side arms — one for the addition of cells and medium,
and the other for the supply of CO2.

The stirrer culture vessel is autoclaved

(at 15 lb/ in2 for 15 minutes) The flask is seeded with the culture.

Then medium along with an antifoam agent is added

The flask is connected to CO2 and stirred at a speed of 60 rpm.

The flask is incubated for about 2 hours.
The contents of the small stirrer flask are transferred to
a large flask and the entire set up is restarted.
Incubation at 37°C is carried out for 4-7 days.

The growth of the cells is monitored daily, and the cells

are counted.
There is a tendency of the cells to enter apoptosis,
if the concentration exceeds 1 × 106 cells/ml.
BATCH CULTURE
Batch culture is a closed culture system that contains
limited amounts of nutrients.
In batch culture cells grow in a
finite volume of liquid medium and are usually maintained in
conical flasks on orbital shakers at a speed of 80–120 rpm.

AIRLIFT FERMENTORS:

Pt-100: Monitors the temperature in the culture vessel.

Foam probe: It senses foam formation.
pH electrode: Monitors the pH in the culture vessel.
Oxygen sensor: Maintains the dissolved oxygen content level.
Heating pad: Provides heat to the medium.
Cold finger: It is a pipe that passes cold water inside a vessel
to cool the contents.
Rotameter: Provides variable airflow into the culture vessel.
Pressure valve: Maintains the pressure.
Air pump: Supplies air throughout the medium.
Peristaltic pump: It pumps acid, base and antifoam into the medium.
Continuous Culture
● Continuous culture is a continuous process where nutrients are continually added to the bioreactor
and the culture broth (containing cells and metabolites) is removed at the same time.
● The volume of the culture broth is constant due to a constant feed-in and feed-out rate (i.e
consumed nutrients are replaced and toxic metabolites are removed from the culture).
● The continuous culture method produces larger biomass leading to a higher yield of the desired
protein.
Principle of Continuous Batch
● A continuous flow system consists of a reactor into which reactants are pumped at a
steady rate and from which products are emitted.

The factors governing their operation are:

a. how material passes through the reactor (which depends upon its design);
b. the kinetics of the reaction taking place.
● In continuous culture, growth-limiting nutrients can be maintained at steady-state
concentrations, which permits microorganisms to grow at submaximal rates.
Process of Continuous culture
Continuous culture involves feeding feed
streams with necessary nutrients and removing effluent
streams with cells, products, and residuals.

A steady-state is established by maintaining equal flow rates

for feed and effluent streams, maintaining constant
culture volume and nutrient concentrations.
A) Turbidostat method
● Cell growth is controlled and remains constant in this system,
but the flow rate of fresh media varies.
● Cell density is controlled based on the set value for turbidity,
which is created by the cell population while fresh
media is continuously supplied.

A) Chemostat method
● In a chemostat, the nutrients are continuously supplied
at a constant flow rate, and the density of the cells is adjusted
according to the supplied essential nutrients for growth.
● In a chemostat, the growth rate is determined by adjusting
the concentration of substrates
like carbon, nitrogen, and phosphorus.
C) Plug-flow reactor
● In this type of continuous culture, the culture solution flows through a tubular reactor without
back mixing.
● In a plug flow reactor, nutrients (reactants) enter the reactor in the form of “plugs,” which
flow in an axial direction through the reactor.
● The culture medium flows steadily through a tube and the cells are recycled from the outlet
to the inlet.
Application of Continuous Culture
1. Continuous culture fermentation has been used for the production of single-cell protein, organic
solvents, starter cultures, etc.
2. It has been used in the production of beer, fodder yeast, vinegar, baker’s yeast, etc.
3. In the industrial production of secondary metabolites (such as antibiotics from a Penicillium or a
Streptomyces sp.)

Limitation of Continuous Culture:

4. In the long-term cultivation process, sterility maintenance can be tricky, and downstream processing can
prove challenging.
5. Controlling the production of some non-growth-related products is not easy Because of this, continuous
culture often requires fed-batch culturing as well as continuous nutrient supply.
6. As a result of the viscosity and heterogeneous nature of the mixture, it may be challenging to maintain
filamentous organisms.
MICRO-CARRIER CULTURE:
Monolayers can be grown on small spherical carriers or micro-beads (80-300 pm diameter) referred to as
micro-carriers.
Materials used ( Plastic, glass, Gelatin, collagen, cellulose)
The micro-beads provide maximum surface area for monolayer cultures.
The cell counting techniques are difficult to be used for micro-carrier cultures.
The growth rate can be detected by analyzing DNA or protein.
Cells are growing on a smooth surface at the solid–liquid interface

Factors affecting micro-carrier culture:

i. Composition and coating of beads (gelatin and collagen beads are preferred as they can be solubilized by
proteases).
ii. Higher stirring speed is usually required.
iii. Glass beads are used when the micro-carriers need to be recycled.
Roller Culture:

A round bottle or tube is rolled around its axis (by rollers) as the medium along with the cells runs
around inside of the bottle
As the cells are adhesive, they attach to inner surface of the bottle and grow forming a monolayer.
Adherent cells will attach to the bottle surface, and through the rotating motion, the cells will be
alternately bathed in the culture medium and exposed to gases in the bottle headspace.
No gradients are formed as the bottles are in constant rotating movement.

Advantages:
Increase in utilizable surface area for a given size of bottle;
The medium is gently and constantly agitated.

Perfused Monolayer Culture:

The growth surface areas of the monolayer cultures can be perfused to facilitate medium replacement
and improved product formation and recovery.
The perfusion can be carried out with pumps, oxygenators and other controllers.
Fixed-bed reactors:
The fixed-bed reactor has a bed of glass beads
The medium is perfused upwards through the bed. The cells are grown on the surfaces of the beads.
The products can be collected from the top along with the spent medium.
Instead of glass beads, porous ceramic matrix with micro-channels can also be used in fixed-bed
reactors.

Fluidized-bed reactors:
In a fluidized-bed reactor, the beads are suspended in a stream of medium
These beads are porous in nature, and are made up of ceramics or a mixture of ceramics mixed with
natural products such as collagen.
They are of low density and float in the medium.
The flow rate of the perfused medium is equal to
the sedimentation rate of the beads.
The cells can grow as monolayers on the outer surfaces and
inside of the porous beads
MICROENCAPSULATION
Microencapsulation may be defined as the packaging technology of solids, liquid or gaseous
material with thin polymeric coatings, forming small particles called microcapsules

The polymer acts as a protective film, isolating the core and avoiding the effect of its inadequate
exposure.
Sodium alginate is a natural polysaccharide with a linear structure, is biodegradable,
biocompatible and safe for the body, provides strength and flexibility to the tissue

It can be used industrially because it has gelling, viscous and stabilizing properties and the ability
to retain water

Alginate solutions can be mixed with a cell suspension, then the mixtures rapidly crosslinked by
divalent cations
PROCESS CONTROL
To maintain proper conditions for scale up cell cultures we need to take care of many
things like:-

a)monitor pH, oxygen, CO2 and Glucose

b)To check for any special probes electrons

c) to check for utilization of nutrients, various assays,if there is any build of

metabolites(lactate, ammonia)

d)Samples to be drawn regularly to check ATP, DNA, protein levels,and total biomass.

Temperature: preheating input medium and by heating surrounding it with water jacket:
temperature probe

Also to take care of flow rate of medium, its Stirring speed and its Viscosity
CHALLENGES

Monolayer cell cultures: cell observation difficult along with cell counting and
biomass determination

NMR: assay cell culture contents Enables identification and quantitation of specific
metabolites.

Used as imaging devices to check distribution of cells Distinguish between

proliferating and non proliferating zones of the culture
Culture of cells for production of various biologicals

Cell culture has various applications and on of the main benefits of cell cultures is
of that we can study molecular and cellular biology interactions

Create model systems for studies regarding physiology and biochemistry of cells

Check the effects of drugs and also toxicology studies can be conducted

Large scale manufacturing of biological compounds like vaccines and proteins for
therapeutic purpose
Manufacturing of Biological Systems
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Production
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Biologics
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REFERENCES:
Adherent cell scale up culture : Sakshi
https://www.mlsu.ac.in/econtents/1468_Unit%203-%20Scaling%20up%20ACC.pdf

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9603258/\

Continuous Cell Culture: Reshma

https://microbenotes.com/continuous-culture/

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/continuous-cell-culture

Suspension cell culture, batch and stirrer culture: Netra

https://www.sciencedirect.com/science/article/pii/S006521640762005X#:~:text=Laboratory%E2%80%90scale%20fermentations
%20were%20started,the%20substrate%20concentration%20in%20the4

https://pubmed.ncbi.nlm.nih.gov/17869604/

https://www.nottingham.ac.uk/ncmh/documents/bger/volume-8/bger8-12.pdf

Process control and Culture of Cells for production of various biologicals: Naman

https://www.ema.europa.eu/en/documents/presentation/presentation-manufacturing-process-biologics-kowid-ho-afssaps_en.pdf

https://www.researchgate.net/figure/Biologic-manufacturing-includes-multiple-steps-that-may-vary-between-manufacturers_fig1_262197373
Contribution:
20166017 - Sakshi
Micro-carrier culture, perfused monolayer culture, Roller culture, microencapsulation

20166016 - Reshma

Continuous cell culture, principle, types, advantages and limitation.

20166015 - Netra

Scale up of suspension cell culture, factors, stirrer culture, Batch Culture

20166014 - Naman

Process control, cell culture for production of biologicals, Manufacturing of biologicals