422
Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
of the single-cell type, possibly because
of
their
relatively
higher
degree
of
differentiation.
In our experience with cell lines of
T. cuspidata
, the stabilization of cell lines
is a relatively lengthy process, normally
requiring 6 to 12 months for completion.
This is not only the case for yew species,
but also for many woody species, as a
result of their relatively slow growth and
often recalcitrant behavior
in vitro
.
3
Analyses of Taxol
and Taxane Production
3.1
Extraction Procedure
At least 0.5 g of fresh callus tissue or 1 g of
fresh cells from suspensions is collected
in glass test tubes (previously weighed and
identiFed), frozen (–20
C), and homog-
enized with a glass rod in 1 to 2 mL of
hexane, followed by 1 h of sonication at
room temperature. After 12 h in hexane
at 25
C, the samples are centrifuged at
2500
×
g
for 20 min to pellet the cells,
and the supernatants are discarded. The
pellets are then extracted in 1 to 2 mL
of methanol/dichloromethane (1 : 1) with
glass rod rehomogenization, followed by
sonication for 2 h at room temperature. Af-
ter centrifugation at 2500
×
g
for 30 min,
the supernatants containing the taxanes
are collected; the pellets are used for deter-
mining the extracted dry weights at 60
C
until constant weight.
The crude extracts are dried under vac-
uum or nitrogen at 25
C, redissolved
in 1 mL of dichloromethane, partitioned
with 1 mL of water, and centrifuged at
2500
×
g
for 20 min. The dichloromethane
fraction is collected, dried under vac-
uum or nitrogen at 25
C, redissolved
in
an
exact
volume
of
high
perfor-
mance
liquid
chromatography
(HPLC)
grade methanol (100–500
µ
L), and Fltered
through 0.45
µ
m Flters for HPLC analysis
(±ig. 2).
Alternatively, the tissue can be extracted
with 100% methanol. The hexane step
can be avoided for cleaner extracts (e.g.
cell suspension extracts) or if the column
for liquid chromatography to be used in
extract analysis is equipped with a precol-
umn, which prevents irreversible binding
of impurities to the column. The Taxol
ex-
traction step (methanol/dichloromethane,
1 : 1) can be repeated two or three times
with the pellet of the Frst Taxol
extraction
to achieve more complete Taxol
recov-
eries. In experiments involving several
treatment comparisons and large number
of samples with respective replicates, how-
ever, a single methanol/dichloromethane
extraction step is more practical; this pro-
cedure allows for Taxol
recovery of 80 to
85%. Media from cell cultures can be ex-
tracted for Taxol
with dichloromethane
(1 : 1 v/v ratio of medium to solvent). ±or
small volumes, the mixture contained in
glass tubes is vigorously agitated on a tube
mixer for 2 to 5 min, followed by soni-
cation for an hour at room temperature.
The tubes are briefly mixed again and cen-
trifuged for 30 min at 2000
×
g
at 20
C.
±or large volumes, the mixture is agitated
overnight in flask shakers and partitioned
f
o
ranh
ou
rins
ep
a
r
a
t
i
onfunn
e
l
s
.Th
e
upperaqueouslayerisd
iscardedandthe
organic fraction is dried under vacuum at
25
C. The preparation for HPLC analy-
sis is similar to the one just described,
although the Fltering of media extracts
tends to be more difFcult than in the case
of plant material extracts. This difFculty
in Fltering media extracts prior to HPLC
analysis is probably a result of the pres-
ence of relatively large amounts of soluble
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