420
Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
and B5Phe8/0GA (B5bPhe salts and or-
ganic supplements with 8.0 mg L
1
of
2,4-D and 0.5 mg L
1
of gibberellic acid).
After autoclaving, gibberellic acid (GA3)
is added to the culture medium by Fltra-
tion through 0.22-
µ
m sterile Flters. All
other
medium components
are
added
prior to autoclaving. The overall analy-
sis of the results showed a trend toward
an inverse relationship between Taxol
production and growth in the developing
callus cultures.
Cell suspension cultures can be initi-
ated from approximately two-month-old
callus cultures developed from stem seg-
ments. Depending on the availability of
callus biomass for inoculum and on in-
cubator space, cell cultures can be grown
in 125-, 250-, 500-, and 1000-mL Erlen-
meyer flasks containing one-Ffth of their
total volume of liquid medium. Basic in-
cubation conditions are 22–25
Cindark
on gyratory shakers at 110 rpm. Experi-
ments done in callus and some additional
optimization for cell suspension culture
medium yielded the following basic for-
mulations: B5 medium salts, threefold
the regular amount of B5 organic supple-
ments, 3.0% sucrose (w/v), 1.5% soluble
PVP (MW
40
×
10
3
), and 1 mg L
1
of
2,4-D (B5C1) or the same composition of
B5C1, but with a modiFed balance of plant
growth regulators (i.e. 4 mg L
1
of 2,4-D
and 1 mg L
1
of kinetin (B5C2)).
Medium preparation, pH adjustment,
and sterilization procedure are the same
as in callus culture medium. Insoluble
PVP is replaced with soluble PVP in
liquid
cell
cultures
to
facilitate
visual
and microscopic observation of the cells
and cell harvesting. Inoculum weights
are preferably high (e.g. one-third of the
volume of the liquid medium) to initially
establish cell suspension cultures. After 1
to 2 weeks in culture on a gyratory shaker
at 110 rpm, the cells released from the calli
are collected and transferred into fresh
liquid medium (1 : 1 or 2 : 1 proportion
of old to fresh medium). The calli with
attendant stem debris can be discarded
or replated in solid medium to regenerate
more callus tissue. During the Frst months
of culture, the media can be replaced
at 2-week intervals following 10 min of
centrifugation at 600
×
g
at 20
Cunde
r
sterile conditions to pellet the cells; in this
way, the maximum amount of cell biomass
is saved until the density is great enough
to start suspension culture propagation
in new flasks. Once suspensions have
been stabilized (generally after several
cycles of propagation), inoculum amounts
can be reduced to one-sixth (w/v) of the
liquid medium for propagation of new
cell suspension flasks. Transfers to fresh
medium in stock cultures can be done
every 20 days. Viability checks of newly
developing cell lines can be performed
via dye exclusion of cells in the presence
of Evans Blue (cells staining blue under
the microscope are considered not viable)
or other cytochemical method, and/or by
analyzing the occurrence of growth in
microcalli rescued from the liquid culture
and plated in solid B5bPVP. Microscopic
examination of cell viability and culture
sterility should be performed regularly,
particularly during the Frst few months
of culture in liquid medium.
Cell cultures can also be immobilized
after the Frst months of culture to facilitate
medium replacement and to aid in the
establishment of recalcitrant cell lines;
this approach has been particularly useful
in our efforts to establish
T. cuspidata
cell
lines (±ig. 1). Immobilization is performed
by spontaneous adhesion to 5 cm
×
5cm,
10- to 15-mm thick glass Fber mats with
a uniform diameter of 10
µ
mandFb
e
r
density
of
approximately
61 mg cm
2
.
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