This document discusses various methods for scaling up suspension and monolayer cell cultures, including increasing culture volume, agitation, gas exchange, continuous culture, perfused systems, and bioreactors. For suspension cultures, scaling involves maintaining adequate mixing and gas exchange at larger volumes. For monolayer cultures, it requires increasing the growth surface area proportionally. Various devices like cell factories, macrocarriers, hollow fiber reactors, and microencapsulation aim to achieve this. Process control includes monitoring pH, oxygen, nutrients and waste products. Cryptic contamination can be detected using specialized assays.

1. Scale-up of suspension cultures
• Involves, primarily, an increase in the volume of the culture medium.
• Agitation of the medium is necessary when the depth exceeds 5 mm.
• Above 5–10 cm, sparging with CO2 and air is required to maintain
adequate gas exchange.
• Stirring of such cultures is best done slowly with a magnet encased in a
glass pendulum or with a large-surface area paddle (Bellco).
• The stirring speed should be between 30 and 100 rpm
– sufficient to prevent cell sedimentation
– not so fast as to create shear forces that would damage the cells.
• Antifoam or Pluronic F68 (0.01–0.1%)- serum concentration is above 2%.

3. Continuous Culture
• Cells can be maintained at set concentrations in two ways: Chemostat or Biostat.
• Chemostat: the cells are grown to the mid-log phase; a measured volume of cells is
removed each day and replaced with an equal volume of medium.
• Biostat: the cells may be run off continuously, at a constant rate, at mid-log phase,
and medium added at the same rate.
• require a stirrer vessel with four ports, two for CO2 inlet and outlet, one for medium
inlet, and one for spent medium outlet, collecting into a reservoir

4. Steady State v/s Batch
• A steady state: for monitoring metabolic changes
related to cell density.
• Is more expensive in medium.
• Is more likely to lead to contamination.
• If the operation is in the 50- to 1000-L range:
• Then more investment and time are spent in generating the
culture.
• Batch method becomes more costly in time, materials, and down
time.

5. Scale and Complexity
• Standard bench-top stirrer cultures operate satisfactorily up to
around 10 L for the bulk production of cells or medium.
• For larger volumes: a controlled fermenter or bioreactor should
be used.
• These culture vessels have regulated input of medium and gas
and provide the capability for data collection from oxygen,CO2,
and glucose electrodes in the culture vessel.
• They are regulated by a programmable control unit that records
and outputs data and regulates accordingly.

6. Mixing and Aeration
• Problems of increased scale in suspension cultures revolve
around mixing and gas exchange.
• Basic requirement: Achieve maximum movement of liquid
with minimum shear stress for the cells.
• Successful designs usually employ a slowly rotating large-
bladed paddle with a relatively high surface area
• Some additive are designed to minimize the harmful effects of
shear—e.g., Pluronic F68, carboxymethylcellulose (CMC), or
polyvinylpyrrolidone (PVP)

7. FERMENTERS
• Culture bags: Plastic bags can be used to culture suspension cells.
• They are gas permeable andcan be agitated by rocking on trays on a flat
rocking platform.

8. Air lift fermenters.
• Large-scale fermenters:
• frequently use the air lift principle
• 5% CO2 in air is pumped into a porous
steel ring at the base of the central
cylinder, and bubbles stream up the
centre, carrying a flow of liquid with
them, and are released at the top.
• The medium is recycled to the bottom
of the cylinder.
• extensively in the biotechnology
industry, up to capacities of 20,000 L.

9. BelloCell aerator culture
• Device has a bellows medium compartment that alternately
forces medium over cells anchored in porous matrices and
withdraws it again, in a ‘‘breathing’’ motion of the
bellows.

10. Perfused suspension culture
• The cells are retained in a low-volume compartment at a very high
concentration, and medium is perfused through hollow fibres within the
cell compartment, or through an adjacent membrane compartment.
• Regulation of gas exchange in the medium is external to the culture
chamber.

11. Fluidized bed reactors for suspension cultures.
• Microcarriers were originally conceived for monolayer cultures.
• Porous microcarriers can accommodate suspension cells within the
interstices of the bead matrix.
• Because of the higher density of the microcarriers they can be perfused
slowly from below, at such a rate that their sedimentation rate matches the
flow rate.
• The beads therefore remain in stationary suspension.
• Constantly replenishing nutrients and collecting the product into a
downstream reservoir.
• No mechanical mixing is required.

12. SCALE-UP IN MONOLAYER
• For anchorage-dependent monolayer cultures, it is necessary to
increase the surface area of the substrate in proportion to the
number of cells and the volume of medium.
• This requirement has prompted a variety of different strategies,
some simple and others complex.

13. Multisurface Propagators
• Nunc Cell Factory.:
• This system is made up of rectangular Petri dish-like units, with a total surface area
of 600–24,000 cm2, interconnected at two adjacent corners by vertical tubes.
• Because of the positions of the apertures in the vertical tubes, medium can flow
between compartments only when the unit is placed on end.
• The cell factory has the advantage that it is not different in the geometry or the
nature of its substrate from a conventional flask or Petridish.

14. NUNC FACTORY

15. Multiarray Disks, Spirals, and Tubes
• Roller Culture:
• If cells are seeded into a round bottle or tube that is then
rolled around its long axis on a roller rack, the medium carrying the cells
runs around the inside of the bottle.
• Adhesive cells will gradually attach to the inner surface
of the bottle and grow to form a monolayer.
• Three major advantages over static monolayer culture:
• Increase in utilizable surface area for a given size of bottle
• Constant, but gentle, agitation of the medium
• Allows gas exchange

16. Macrocarriers
• Porous microcarriers:
• Which allow cells to grow within the interstices of the bead
• That are larger with a macroscopic structure made up of a number
of different materials, such as polylactic acid (PLA),
polyglycolic acid (PGA), collagen, or gelatin (Gelfoam) in a
variety of different geometries.
• These can be loaded with cells and stirred in a bioreactor or
perfused in a fixed-bed or fluidized bed reactor

17. Perfused Monolayer Culture
• Perfusion is frequently used to facilitate medium replacement
and product recovery.
• Membrane perfusion. Many systems depend on filter
membrane technology in which the culture bed is a
flat, permeable sheet.
• Membroferm is compartmentalized in such a way that the cells,
medium supply, and product occupy different membrane
compartments.

18. • Hollow fiber perfusion. There are a number of hollow
fiber perfusion systems in which adherent cells grow on the
outer surface of the perfused microcapillary bundles.
• Highmolecular-weight products concentrate in the outer space with the
cells, while nutrients are supplied and metabolites
removed via the inner space.

19. • Fixed-bed reactors. Systems have also been
developed with glass or matrix beads with the
medium being perfused upward through the bed or
percolating downward by gravity.
• The product is collected with the
spent medium in a reservoir.

20. Microencapsulation
• Sodium alginate behaves as a gel, depending on the
concentration.
• It will gel as a hollow sphere around cells in suspension in a
• Because the alginate acts as a barrier to high-molecular-weight
molecules, macromolecules secreted by the cells are trapped
within the vesicle.
• Nutrients, metabolites, and gas freely permeate the gel.

21. PROCESS CONTROL
• The progress of suspension cultures is monitored:
1. via pH, oxygen, CO2, and glucose electrodes that read from the
culture in situ
2. by assaying the utilization of nutrients, such as glucose and
amino acids, or the buildup of metabolites, such as lactate and
ammonia, and products, such as immunoglobulin from
hybridomas.
• The number of cells are determined in samples drawn from the
culture and are used to calculate the total biomass.
• The temperature of the medium is regulated by preheating the
input medium and by heating the surrounding water jacket
regulated by feedback from the temperature probe.

22. • There is a recurrent problem when monolayer cell cultures are scaled up,
particularly in a fixed-bed or hollow fiber bioreactor:
• It is no longer possible to observe the cells directly.
• Monitoring the progress of a culture by cell counting and determining the
biomass become difficult.

23. TYPES OF MICROBIAL
CONTAMINATION
• Bacteria, yeasts, fungi, molds, mycoplasmas, and occasionally protozoa,
can all appear as contaminants in tissue culture.
• In general, rapidly growing organisms are less problematic as they are often
overt and readily detected, whereupon the culture can be discarded.
• Difficulties arise when the contaminant is cryptic, either because it is too
small to be seen on the microscope, e.g., mycoplasma, or slow growing
such that the level is so low that it escapes detection.
• Use of antibiotics can be a common cause of cryptic contaminations
remaining undetected

24. Visible Microbial Contamination
• Characteristic features of microbial contamination :
1. A sudden change in pH, usually a decrease with most
bacterial infections, very little change with yeast until the contamination is
heavy, and sometimes an increase in pH with fungal contamination.
2. Cloudiness in the medium sometimes with a slight film or scum on the
surface or spots on the growth surface.
3. Under a low-power microscope spaces between cells will appear granular
and may shimmer with bacterial contamination.
4. With a slide preparation, the morphology of the bacteria can be resolved at
×1000, but this is not usually necessary.
5. Microbial infection may be confused with precipitates of media
constituents (particularly protein) or with cell debris, but can be
distinguished by their regular morphology.

25. • Mycoplasma
• Detection of mycoplasmal infections is not obvious by routine microscopy,
other than through signs of deterioration in the culture.
• Requires fluorescent staining, PCR, ELISA assay or microbiological assay.
• Fluorescent staining of DNA by Hoechst 33258 is the easiest and most reliable
method.
• Reveals mycoplasmal infections as a fine particulate or filamentous staining
over the cytoplasm at ×500 magnification .
• The nuclei of the cultured cells are also brightly stained by this method and
thereby act as a positive control for the staining procedure.

26. • Many mycoplasma contaminants, particularly in continuous cell lines, grow
slowly and do not destroy host cells. However, they can alter the
metabolism of the culture in many different ways.
• Because mycoplasmas take up thymidine from the medium, infected
cultures show abnormal labeling with [3H]thymidine .
• Mycoplasmas can alter cell behavior and metabolism in many other ways,
so there is an absolute requirement for routine, periodic assays to detect
possible covert contamination of all cell cultures, particularly continuous
cell lines.