Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
421
Stem explant of
Taxus
species
Callus develops on solid media
Two months
Transfer callus to
liquid media and development
of cell suspension after two to
three weeks
Released cells
transferred
Cells immobilized
to glass fiber mats
Free cells transferred
to liquid media
Wash, surface-sterilize,
and trim explant for culture
Fig. 1
Establishment of immobilized cell cultures and cell suspension cultures of
T. cuspidata
.
Within the frst days oF contact with
the substrate, the cells are spontaneously
immobilized. The cells grow on top oF and
within the mats, Forming several layers
oF cells; aFter the cells have saturated
the immobilization substrate, they begin
to be released into the liquid medium.
These cells can be used to establish new
suspension cultures or new immobilized
cultures. Alternatively, cell cultures can be
propagated by careFully removing external
cell layers with a sterile spatula, or by
cutting the mats in two or Four portions
and placing these on Fresh medium flasks
containing a new glass fber mat (±ig. 1).
Two-year-old cell suspensions oF
T. cuspi-
data
have various degrees oF cell aggre-
gation. Moreover, the shape oF the cells
is variable, From round to considerably
long, cylinder-like cells. In general, most
cell suspension cultures display a rela-
tively high number oF cell aggregates oF
various sizes, with relatively Fewer cells
occurring
individually.
Cell
aggregates
may constitute a problem in large-scale
cell cultures, which can be more sus-
ceptible to shear stress and may create
diFfculties in the mixing oF cells, gases,
and medium components. It is possi-
ble to select For smaller aggregate sizes
by allowing larger aggregates to settle
For a short period oF
time and then
transFerring the smaller aggregates and
single cells From the upper portion oF
the flask into a new culture flask. The
selection
For
the
single-cell
or
small-
a
g
g
r
e
g
a
t
et
y
p
eo
Fsu
s
p
en
s
i
on
,h
ow
e
v
e
r
,
may have implications For Taxol
produc-
tivity. Aggregated cell suspensions tend
to be more productive than suspensions
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