Page 154 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

P1: GRB/GUB P2: FYD Final Pages
Encyclopedia of Physical Science and Technology EN009G-399 July 6, 2001 20:4

Mammalian Cell Culture 39

laboratory. The largest stationary flask routinely used is 1. Cell Factory (A/S NUNC; Roskilde, Denmark): The
2
the Roux bottle (or disposable plastic T-flask), which has trays are 335 × 205 mm (600 cm ) and can be obtained
2
a surface area of 175 to 200 cm (depending upon make in multiples of 1, 2, 10, and 40. The largest has a surface
2
and type), requires 100 to 150 mL medium, and utilizes area of 24,000 cm and requires 8 L medium (equivalent
3
750 to 1000 cm of storage space. Such a vessel will yield to 14 large roller bottles but requiring half the incubation
8
7
2 × 10 diploid, or 10 heteroploid cells; thus, to produce a space and no ancillary roller equipment).
modest 10 10 cells, over 100 replicate cultures are needed 2. CellCube (Costar; Cambridge, MA, U.S.): Although
(i.e., manipulations have to be repeated 100 times) and this system also has parallel polystyrene trays, it is a mod-
100 L of culture space are required. Although a few pro- ular closed-looped perfusion system that includes an oxy-
cesses (mainly for classical viral vaccines) still use this genator, pumps, and system controller (pH, O 2 , level con-
methodology, the need to move to larger units is obvi- trol). The unit is very compact, with the trays being only
2
ous and is approached by increasing the ratio of surface 1 mm apart, thus the smallest unit of 21,250 cm is un-
area to volume. The first step in scale-up usually involves der 5 L total volume (1.25 L medium). Additional units of
2
2
a change from stationary flasks to roller bottles (i.e., a 42,500 cm (2.5 L medium) and 85,000 cm (5 L medium)
dynamic system) and is currently in widespread use for are available, and four units can be run in parallel in the
2
2
many products. Roller bottles can be up to 1750 cm , use system, giving 340,000 cm growth area.
350 mL medium, and have a volume of 2.5 L (i.e., a nine-
fold increase in surface area, but only a threefold increase
3. High-Volume Units
in medium and total volume). This is possible because
the cells use the total internal surface area for growth. In These are true unit process systems, analogous in volume
addition, more efficient aeration occurs because the cells and performance to suspension cell fermenter vessel pro-
move in and out of the culture fluid. This method has been cesses:
used industrially to produce viral vaccines, veterinary vac-
cines in multiples of 28,000, interferon, and EPO. It has 1. Glass bead culture: Packed beds of 3- to 5-mm glass
a place in modern processes largely because it has been spheres through which medium is continuously perfused
automated using robotic systems such as Cellmate (The have a demonstrated scale-up to 100 L. Spheres of 3-mm
Automation Partnership; Royston, Herts, U.K.). All the diameter pack sufficiently tightly to prevent the bed from
routine manipulations of cell seeding, media changing, shifting but allow sufficient medium flow up the column
bottle gassing, cell sheet rinsing, trypsinization, and cell so that fast flow rates, which would cause shear damage,
collection by scraping can be carried out automatically, are not required. Medium can be circulated by pump or by
and reproducibly, with very precise volumes. Examples airlift (for better oxygenation). Glass-sphere packed beds
®
of products produced by this method are Varivax (Merck constitute a simple system that minimizes moving parts
varicella vaccine) and Saizen (Serono recombinant human and the risk of mechanical failure and has an inexpensive
growth hormone). and reusable substrate capable of considerable radial and
Modifications to increase the ratio of surface area to reasonable vertical scale-up to operate beds of over 200-L
volume for the roller bottle culture include the Spira- volume using 5-mm spheres. The disadvantages are that,
Cell Multi-Surface Roller Bottle (Bibby Sterlin, Ltd.), ex- as spheres have the minimum surface area per unit volume,
tended surface area roller bottles (ESRB) (Bibby Sterlin, the culture will always be bulky, with most of the volume
Ltd.), and TexturSil, a silicone rubber matrix that coats being dead space. Also, the system offers limited secreted
roller bottles to increase the surface area (Ashby Scien- products, as it is difficult to harvest cells from the bed,
tific, Ltd.; Coalville, Leics., U.K.) so it is ideal for long-term continuous cultures rather than
The roller bottle is a well-established technique and is batch cultures.
still widely used, in both the research laboratory and the 2. Microcarrier culture: Microcarriers are small parti-
industrial plant. Some cell lines (particularly epithelial) cles, usually spheres 100 to 300 µm in diameter that are
may not be as successfully grown in roller bottles due to suspended in stirred culture medium. The technique was
streaking, clumping, or inadequate spreading over the total initiated in 1967 but required considerable developmental
surface (i.e., nonlocomotory cell lines) as in stationary work to produce a range of suitable microcarriers (e.g.,
bottles. An alternative scale-up route is to use multisurface the Cytodex series by Pharmacia). The first industrial pro-
plate stationary systems. cess based on microcarriers was for FMDV. Subsequently,
a wide range of microcarriers based on gelatin, collagen,
polystyrene, glass, cellulose, polyacrylamide, and silica
2. Multisurface Plate Units
have been manufactured to meet all situations. The key
Two commercially available examples of multiple-layered criteria in the design of effective microcarriers were to
polystyrene plates stacked within a polystyrene box are make the surface chemically and electrostatically correct