Page 155 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

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Encyclopedia of Physical Science and Technology EN009G-399 July 6, 2001 20:4

40 Mammalian Cell Culture

for cell attachment, spreading, and growth. The power of size of a single fermenter vessel than the specialized units
the method is exemplified by the following data: used for ADC. Although some cell lines will not grow in
2
a. 1 g Cytodex = 6000 cm which at 2 g/L = 12,000 suspension [e.g., human diploid cells (HDC) lines WI-38
2
cm /L (equivalent to eight large roller bottles) and MRC-5], many lines can be adapted to grow in free
b. Scale-up to 4000 L has been achieved (=3200 roller suspension by recognized procedures.
bottles) in an environmentally controlled and optimized Several physical factors must be satisfied for successful
process. scale-up. Good mixing is essential for homogeneity and
efficient mass transfer, but during scale-up the power re-
This method opened up both industrial production op- quired to produce these conditions can cause problems.
portunities and allowed research laboratories to easily Edy effects such as the energy generated at the tip of the
produce substantial quantities of developmental prod- stirrer blade, and particularly gas bubble rupture at the
ucts. Microcarrier culture is the most versatile, reliable, media surface, are limiting factors giving rise to damag-
and characterized procedure for unit volume scale-up of ing shear forces (created by fluctuating liquid velocities in
anchorage-dependent cells. It has had widespread use for turbulent areas). As mixing efficiency increases with tur-
industrial processes [vaccines, interferon, tPA, and human bulence, a compromise has to be reached to minimize cell
growth hormone (hGH)] as well as many developmental damage. Thus, cell cultures use large impellers running at
uses, has been scaled-up to 4000 L, and has the potential relatively low speeds. Magnetic bars used in spinner flasks
for process intensification by perfusion with spin filters or give only radical mixing, with no lift or turbulence, and
by the use of microporous microcarriers. scale-up means a move to marine (not turbine) impellers.
Alternative systems to avoid stirring have been developed:
B. Bioreactors for Suspension Cells
1. Air-lift fermenter: This fermenter uses the bubble
1. Laboratory Scale column principle to both agitate and aerate a culture. Air
The basic culture unit for suspension cells is the spinner bubbles are introduced into the bottom of a culture ves-
flask, so called because it has a magnetic bar operated sel (aspect ratio 8–12 : 1) and rise up an inner draft tube.
by standing the unit on a magnetic stirrer. Side arms are Aerated medium has a lower density than nonaerated so
usually fitted to allow gassing with CO 2 /O 2 through a fil- the medium rises through the draft tube and circulates
ter and for sampling. The spinner flask is usually glass down the outside of the vessel (upflow and downflow
with a silicone- or Teflon-coated magnet and is available of approximately equal volumes). The amount of energy
in sizes from 50 mL to 20 L, although 10 L should be required is very low and shear forces are absent; thus,
considered the maximum practical size. It is advisable to it is an ideal method for fragile cells. Air-flow rates of
siliconize the culture vessel and add medium supplements, about 300 mL/min are used. Vessels are available from
such as Pluronic F-68 (polyglycol) (BASF, Wyandot) at 2 L upwards, with 2000-L reactors being used in indus-
0.1% to protect against mechanical damage, especially if trial manufacturing processes. However, scale-up is more
low serum concentrations are used. Stirring speeds for sus- or less linear; a 90-L vessel requires 4 m headroom, so
pension cells are usually within the range of 75 to 250 rpm special manufacturing suites are needed for large-scale
(depending upon cell type and vessel geometry), and a cul- operations.
5
6
ture will expand from 1–2 × 10 /mL to 1–3 × 10 /mL in 2. Celligen fermenter: This fermenter is available from
4 to 5 days before requiring either harvesting or a medium New Brunswick Scientific and is designed so that the im-
change. Conventional spinner flasks are available from peller acts as a fluid pump and aerator. Macrocirculation
a wide range of laboratory suppliers. Modified spinner mixing is generated by the hollow central shaft filled with
cultures include a radial stirring action (Techne), a float- threerotatinghorizontaljettubesinalowshearbulkmove-
ing impeller (Techne BR-06 Bioreactor), the Superspin- ment of cells and medium (cell-lift effect).
ner (Braun Melsungen AG), CellSpin (Tecnomara AG,
Switzerland) and a dual overhead drive system to allow The other principal physical factor to be accommodated
perfusion (Bellco). For small-scale culture (5 to 10 mL) is aeration. Sparging can cause cellular damage unless
polypropylene tubes (50 mL) placed on a horizontal cir- carried out at very low rates. General guidelines are to
cular motion shaker at 100 to 200 rpm are a useful means use large bubbles (1 to 3 mm in diameter) and low flow
3
of carrying out process optimization studies. rates (5–10 cm /min) which means relatively inefficient
mass transfer, especially at the low stirring rates used.
Even though cell bioreactors have an aspect ratio of less
2. Scale-Up
than 2:1 in order to maximize surface diffusion, supply-
Suspension culture is the preferred method for scaling-up ing sufficient oxygen during scale-up is a limiting factor.
cell cultures as it is easier to volumetrically increase the Strategies to address this have included a surface aerator,