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ANEUPLOIDY
IN
THE
DEGENERATIVE
PHASE
OF
SERIAL
CULTIVATION
OF
HU1iAN
CELL
STRAINS*
BY
EERO
SAKSELAt
AND
PAUL
S.
-MOORHEAD
THE
WISTAR
INSTITUTE
OF
ANATOMY
AND
BIOLOGY,
PHILADELPHIA
Communicated
by
Warren
H.
Lewis,
June
26,
1963
The
factors
affecting
chromosomal
stability
of
mammalian
cells
in
long-term
cul-
ture
are
not
well
understood,
aside
from
the
effects
of
experimentally
applied
agents
such
as
irradiation
and
viruses.
Species
differences'-'
and
perhaps
cell
types
ap-
pear
to
be
important
with
respect
to
the
occurrence
of
spontaneous
alteration
or
heteroploid
transformation.4'
Cells
cultured
from
mouse
tissues
seem
to
be
ex-
tremely
labile
with
regard
to
chromosome
changes
and
associated
morphologic
and
growth
alterations.
6,
On
the
other
hand,
in
metaphase
studies
to
date,
cul-
tured
human
cells
of
the
fibroblastic
type
remain
diploid
and
show
no
tendency
to
undergo
spontaneous
transformation.
Interest
in
spontaneous
occurrences
of
in
vitro
transformation
of
mammalian
cells
has
been
based
upon
hope
for
its
confirmation
as
a
model
of
the
in
vivo
process
of
malignant
change.
The
two
processes
have
many
features
in
common,5'
12
aid
early
proposals
that
viruses
present
in
the
original
tissue
or
serum
might
be
causative
agents
have
been
strengthened
by
the
numerous
studies
showing
that
certain
viruses
do
produce
transformation
in
vitro.
Experiments
with
SV40
virus
and
human
cells
have
provided
another
reproducible
cell-virus
system
for
the
study
of
transformation
throughout
its
course."'
14
Furthermore,
the
discovery
of
chromosome
lesions
in
human
cells
in
association
with
measles
infections
has
increased
our
interest
in
the
nature
and
extent
of
chromosomal
changes
which
occur
in
vitro
in
the
absence
of
any
known
agent.
This
report
is
concerned
with
a
study
of
metaphase
chromosomes
in
two
human
cell
strains
during
their
total
period
of
in
vitro
cultivation.
In
a
previous
less
ex-
tensive
study
no
aberrations
were
seen
in
various
human
diploid
cell
strains
in
material
from
the
9th
to
the
40th
subcultivation
in
vitro.
"'
In
the
present
study
spontaneous
chromosome
changes
were
encountered,
but
only
in
those
cultures
which
had
been
subcultivated
for
40
or
more
times,
i.e.,
during
the
degenerative
phase
of
their
characteristically
limited
in
vitro
life.
Although
this
aneuploidy
seems
to
be
associated
with
the
degenerative
period
in
long-term
cultivation,
on
no
occasion
did
transformation
in
terms
of
altered
morphology,
growth
rate,
or
capac-
ity
for
indefinite
cultivation
occur
among
many
parallel
cultures
of
these
two
strains.
In
long-term
cultures
of
human
fibroblasts
Sax
and
Passano
16
have
previously
shown
an
association
between
"age
in
vitro"
and
increase
in
spontaneous
rate
of
anaphase
aberrations.
Materials
and
Methods.-Cell
strains:
Cell
strains
WI-26
and
WI-38,
male
and
female,
re-
spectively,
were
derived
from
fetal
lung
tissue
by
L.
Hayflick
according
to
procedures
previously
described.'0
In
essence,
cultures
were
grown
as
monolayers
of
fibroblastic-like
cells
in
Eagle's
basal
medium
with
10%10
calf
serum
and
50
jug
aureomycin
per
ml
and
subcultivated
by
trypsinization
with
0.25%
trypsin
(Difco
250:1).
Subcultivation
from
one
to
two
milk-dilution
bottles
was
per-
formed
twice
weekly,
i.e.,
when
the
monolayer
had
become
confluent.
Confluency
was
achieved
every
3-4
days
during
the
major
period
(4-5
months)
of
total
in
vitro
cultivation.
After
approxi-
mately
35
total
passages
in
vitro,
cultures
required
longer
periods
(5-8
days)
to
achieve
confluency;
390
Voio.
50,
1963
GENETICS:
SAKSELA
AND
MOORHEAD
391
eventually
(50
±
10
passages),
all
cultures
of
these
diploid
cell
strains
failed
to
replicate
suffi-
ciently
to
permit
any
further
subcultivation.
It
is
recognized
that
each
subcultivation
or
"pas-
sage
generation"
is
only
an
approximation
to
cell
population
doubling,
and
the
total
number
of
passages
has
no
significance
except
as
a
crude
measure
of
the
in
vitro
stage
any
strain
has
reached.
The
terminal
portion
of
the
finite
period
of
serial
subcultivation
is
referred
to
as
Phase
11I10
or
as
the
degenerative
phase,
and
in
the
absence
of
heteroploid
transformation
this
limitation
on
cultivation
has
long
been
recognized.
Ampoules
containing
2
X
106
cells
were
frozen
and
stored
in
liquid
nitrogen
by
procedures
described
previously.10
Substrain
designations
in
Tables
1
and
2
refer
to
cultures
reconstituted
from
such
ampoules
and
carried
independently.
Chromosome
preparations:
Chromosome
studies
were
made
from
permanent
mounts
of
Giemsa
stained
air-dried
metaphase
preparations.'7
Ordinarily
cells
were
harvested
with
trypsin
on
the
second
day
after
subcultivation,
following
3-5
hr
of
treatment
with
Colcemid
(CIBA),
0.05
ug
per
ml
of
medium.
The
cell
suspension
was
concentrated
in
1/2
ml
of
trypsin
solution,
and
a
4-fold
volume
of
distilled
water
was
added
to
swell
the
metaphase
cells
hypotonically
for
8-10
min.
Fixation
was
made
with
3:
1
methanol:
acetic
acid.
Spreading
was
done
by
ignition
of
a
drop
of
fixative,
containing
cells
in
suspension,
immediately
after
its
application
to
the
surface
of
a
clean
wet
slide.
Suitable
metaphases
were
selected
under
low
power
(150X)
observation,
and
those
judged
to
be
free
from
excessive
spreading
were
then
studied
under
oil
immersion
optics.
In
all
cells
so
selected,
the
chromosomes
were
counted,
and
25-40%
of
the
metaphases
of
each
sample
were
subjected
to
detailed
karyotypic
analysis,
involving
the
identification
of
individual
chromosome
pairs
or
groups
of
the
human
karyotype
according
to
the
Denver
convention:
Nos.
1,
2,
3,
4-5,
6-X-12,
13-15,
16,
17-18,
19-20,
21-22-Y.
Metaphase
counts
considered
to
be
arti-
facts
because
of
scattering
of
some
chromosomes
or
because
of
accidental
contamination
of
one
metaphase
with
chromosomes
of
another
were
not
excluded
from
the
data
on
the
various
samples
studied.
For
each
determination
of
the
level
of
tetraploidy
existent
in
the
dividing
cell
population
250-300
unselected
metaphases
were
examined
and
roughly
estimated
as
being
diploid
or
tetra-
ploid.
Observations:
WI-26:
Chromosome
counts,
numbers
of
cells
analyzed,
and
other
karyologic
observations
from
seven
different
cultures
of
strain
WI-26
are
presented
in
Table
1.
Chromosome
preparations
from
diploid
cell
strain
WI-26
were
first
made
at
its
19th
subcultiva-
tion
passage.
In
a
sample
of
100
cells
the
exact
chromsome
number
was
determined,
and
36
cells
were
analyzed
in
detail.
It
could
be
seen
that
most
of
the
cells
had
a
normal
male
chromosome
complement,
and
in
the
hypodiploid
cells
no
pattern
concerning
the
missing
chromosomes
could
be
deduced.
Of
the
dividing
cell
population
3.1
%
were
tetraploid,
and
no
abnormal
chromosomes
were
found.
Between
the
28th
and
37th
in
vitro
passages,
106
cells
were
examined,
and
of
these
28
were
analyzed
in
detail.
A
normal
distribution
of
chromosome
numbers,
skewed
toward
hypo-
diploid
counts,
was
observed.
The
analysis
of
each
of
the
9
hypodiploid
cells
of
the
sample
did
not
reveal
any
consistency
as
to
the
chromosomes
which
were
missing,
and
these
counts
are
pre-
sumed
to
be
artifacts.
A
somewhat
higher
value
of
tetraploidy
was
recorded
at
the
28th
passage,
and
again
at
the
37th
passage,
4.5
and
4.4%,
respectively.
In
the
sample
from
the
32nd
passage,
1.6%
of
the
dividing
cells
were
tetraploid.
An
acentric
fragment
was
observed
in
one
of
45
cells
examined
at
the
37th
passage.
It
was
found
in
a
-cell
with
46
(excluding
the
fragment)
chromo-
somes,
which
was
lacking
a
No.
4-5
chromosome
and
contained
an
extra
chromosome
in
the
6-12
group.
It
is
possible
that
the
fragment
resulted
from
breakage
in
the
long
arm
of
a
4-5
chromo-
some;
this
deficient
chromosome
would
then
be
indistinguishable
from
members
of
the
6-12
group.
Thus,
from
a
total
of
over
200
cells
counted
in
cultures
of
the
19th,
28th,
32nd,
and
37th
passages,
of
which
64
were
studied
in
detail,
only
one
cell
was
found
which
had
an
abnormality.
Between
the
41st
and
54th
in
vitro
passage,
58
cells
of
WI-26
were
karyologically
examined.
Six
of
25
metaphases
at
the
41st
passage
contained
abnormal
dicentric
chromosomes,
while
3
out
of
20
did
so
at
the
54th
passage.
A
small
sample
of
13
cells
from
43rd
passage
material
re-
vealed
no
abnormalities,
and
the
extremely
low
frequency
of
mitosis
prevented
extension
of
this
sample.
At
the
41st
passage,
3.6%
of
the
cells
were
tetraploid,
and
the
very
high
value
of
16%
tetraploidy
occurred
in
the
54th
passage
material.
Substrains
of
WI-26
(X,
XI,
XIII,
XIX,
and
XXIII)
could
not
be
subcultivated
more
than
40-49
total
passages;
XXVI
survived
for
56
passages
in
vitro.
392
GENETICS:
SAKSELA
AND
MOORHEAD
PROC.
N.
A.
S.
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*
z
VOL.
50,
1963
GENETICS:
SAKSELA
AND
MOORHEAD
393
WVI-38:
At
the
fourth
subcultivation
passage
200
WI-38
cells
were
examined
and,
of
these,
84
were
subjected
to
detailed
karyotypic
analysis.
The
distribution
of
the
exact
chromosome
counts
obtained
is
given
in
Table
2.
Among
14
hypodiploid
cells
no
consistent
pattern
as
to
the
identity
of
the
missing
chromosomes
could
be
determined.
Three
cells
with 47
chromosomes
were
ob-
served,
but
in
two
of
these
contamination
from
another
metaphase
was
definitely
indicated
by
a
differential
state
of
condensation
in
the
extra
chromosome.
In
all
other
cells
examined,
no
devia-
tion
from
the
normal
female
karyotype
was
observed.
At
this
early
stage
in
vitro,
the
frequency
of
tetraploid
cells
in
the
dividing
population
was
1.0%.
Essentially
similar
findings
were
obtained
concerning
the
chromosomal
constitution
of
various
WI-38
substrains
from
the
14th
to
the
37th
passage;
223
cells
were
examined
and
98
of
these
karyotypically
analyzed.
A
single
pseudodiploid
cell
was
observed
in
the
33rd
passage
sample.
The
level
of
tetraploidy
remained
low,
except
for
one
slightly
higher
value
at
the
37th
passage,
3.8%.
From
WI-38
cultures
between
the
41st
and
46th
in
vitro
passages,
the
chromosomes
of
165
cells
were
counted,
and
79
of
these
were
karyotypically
analyzed.
Aneuploid
changes
and
a
marked
increase
in
frequency
of
hypodiploid
cells
are
evident
in
all
of
these
late
passage
samples.
Although
no
obvious
pattern
as to
the
missing
chromosomes
can
be
observed
(Table
2),
the
fact
that
2%,o
(41st
passage)
to
30%
(46th
passage)
of
the
cells
exhibited
obviously
abnormal
chromosomes
would
imply
that
perhaps
many
of
the
hypodiploids
were
not
artifacts
of
technique.
The
ab-
normalities
observed
were
mainly
dicentrics
formed
by
translocations
between
two
chromosomes
of
the
complement,
in
some
cases
identifiable
chromosomes.
Less
frequently
(Table
2),
fragments,
minute
chromosomes,
and
abnormal
monocentric
chromosomes
were
observed.
In
--this
late
pas-
sage
WI-38
material,
the
frequency
of
tetraploid
cells
(2.7-5.1%)
was
elevated
above
values
seen
in
lower
passage
levels
(Table
2).
The
original
or
parent
strain
of
WI-38
was
cultivated
for
a
total
of
48
passages,
and
none
of
the
three
substrains
(II,
IV,
V)
could
be
subcultivated
more
than
43
to
48
passages
in
vitro.
Discussion.-Aneuploid
changes
appeared
in
different
substrains
of
each
strain
studied
following
a
long
period
of
apparent
chromosome
stability.
The
earliest
chromosome
aberrations
were
coincident,
with
the
beginning
of
the
decline
of
each
strain
during
its
serial
subcultivation.
Setting
aside
the
question
of
criteria
for
chromosomal
or
karyotypic
"normality,"
we
must
consider
whether
these
observa-
tions
are
generally
applicable
to
human
fibroblast
strains
in
long-term
culture.
Since
strains
WVI-26
and
WI-38
were
selected
for
extended
investigation
for
the
sole
reason
that
one
is
male
and
the
other
female,
it
seems
warranted
to
regard
them
as
representative
of
such
serially
propagated
strains,
at
least
for
those
of
embryonic
origin.
Further,
two
other
human
fibroblast
strains
cursorily
examined
also
showed
some
aneuploid
cells
at
high
passage
levels
of
cultivation.
That
our
previous
study'0
failed
to
reveal
aneuploid
changes
with
increased
"culture
age"
is
attributed
to
the
fact
that
only
two
of
the
16
samples
studied
from
13
strains
were
from
cultures
approaching
or
in
the
period
of
decline,
i.e.,
above
the
level
of
the
35th
passage.
At
its
40th
passage,
WI-12
revealed
no
abnormalities
in
a
sample
of
33
metaphases,
and
only
17
cells
were
available
from
the
39th
passage
of
WI-1
because
of
the
low
growth
rate
in
its
declining
phase.
No
aneuploidy
was
observed
in
earlier
studies
on
long-term
cultivated
human
fibroblasts.
Among
cultures
from
individuals
1-41
years
of
age,
Tjio
and
Puck8
reported
"no
variation
in
chromosome
number
and
morphology."
Their
cultures
were
studied
over
periods
comparable
to
those
reported
in
the
present
paper.
In
a
later
paper9
these
authors
remarked
upon
the
constancy
of
the
human
karyotype,
finding
no
change
in
number
or
morphology
over
"20
successive
harvests
(trans-
fers)...
involving
more
than
40
generations."
MAakino
and
co-workers"
reported
the
maintenance
of
a
"normal
complement
of
46
chromosomes"
in
cells
obtained
394
GENETICS:
SAKSELA
AND
1OORHEAD
PROC.
N.
A.
S.
from
the
2nd
to
the
44th
subculture;
however,
they
also
state,
"In
comparison
with
the
results
of
chromosome
counts
in
the
primary
cultures,
cells
with
hypo-
or
hyper-
diploid
chromosome
numbers
occurred
at
a
higher
incidence
in
the
subcultured
speci-
mens."
These
authors
did
not
present
details
concerning
the
proportion
of
their
large
total
sample
which
was
derived
from
the
late-passage
cultures
mentioned.
On
the
other
hand,
in
this
study
we
deliberately
sought
information
during
the
period
when
the
cultures'
growth
rates
had
begun
to
diminish.
This
fact
may
account
for
our
ob-
serving
aneuploidy,
whereas
they
reported
none.
Our
results,
in
respect
to
samples
taken
prior
to
about
the
40th
subculture,
are
in
accord
with
the
other
studies.
The
constancy
of
the
human
karyotype
in
such
material
is
impressive
when
compared
to
continuously
cultivatable
cell
lines
from
mammalian
tissue
which
are
near-
diploid,
i.e.,
containing
a
large
number
of
apparently
euploid
cells
but
with
some
pseudo-,
hypo-,
and
hyper-diploid
cells
also
detectable.2
For
a
priori
reasons,
criteria
for
the
"normal"
human
karyotype
should
be
based
upon
in
vivo
dividing
populations
(such
as
direct
preparations
from
bone
marrow)
or
upon
primary
or
very
early
tissue
cultures.
This
must
be
determined
against
a
background
of
a
certain
amount
of
hypodiploid
counts
which,
for
technical
reasons,
are
spurious.
Within
these
limitations
of
technique
and
of
the
experi-
ence
of
the
investigators,
prior
to
the
40th
passage
level,
both
strains
WI-26
and
WI-38
may
be
regarded
as
normal
or
classic
diploid.
A
minor
reservation
may
be
made
with
respect
to
strain
WI-26,
however,
as
the
tetraploid
percentages
ob-
served
at
the
28th
and
37th
passages
were
slightly
above
values
usually
ob-
served.
A
number
of
studies8'
0
11
have
shown
that
the
tetraploidy
level
in
pre-
sumably
normal
cultures
of
human
fibroblast
cells
is
seldom
greater
than
3
per
cent.
An
increase
in
proportion
of
tetraploids
in
the
metaphase
population
is
a
common
feature
of
SV40
transformation
of
human
fibroblasts.18'
19
We
have
observed
a
similar
association
between
tetraploidy
increase
and
subsequent
spontaneous
heteroploid
transformation
in
serially
cultivated
cells
of
the
rhesus
monkey.
Considerations
as
to
the
tissue's
species
of
origin
and
the
possible
presence
of
in-
apparent
viruses
may
explain
the
fact
that
adult
rhesus
monkey
kidney
tissue
cultures
ordinarily
degenerate
within
a
few
weeks,
surviving
no
more
than
3
or
4
subcultiva-
tions.
These
monkey
kidney
subcultures
may
show
an
extremely
high
frequency
of
mitotic
aberrations
(16-40
per
cent)
even
before
the
second
week
of
in
vitro
cultiva-
tion.20
That
human
cell
types
other
than
fibroblasts
may
show
entirely
different
patterns
with
respect
to
their
fate
in
vitro
is
well
known.
For
example,
extra-em-
bryonic
amnion
can
usually
be
cultivated
for
only
a
few
subdivisions
before
it
degen-
erates
or,
on
occasion,
transforms.
It
is
possible
that
the
inherently
limited
in
vivo
growth
potential
of
amnion,
a
kind
of
"tissue
age,"
may
be
expressed
even
in
vitro.
The
three
WI-26
substrains
(XIII,
XXIII,
XXVI)
which
revealed
aneuploidy
survived
as
serial
cultures
no
longer
than
various
parallel
subcultures
from
this
strain
which
were
not
examined
for
chromosome
changes.
The
same
applies
to
the
particular
substrains
(Parental,
II
and
IV)
comprising
the
post-40th
passage
mate-
rial
in
which
aneuploid
changes
were
observed
in
the
WI-38
strain.
Among
the
re-
arrangements
and
chromosome
breaks
observed,
there
was
no
consistency
as
to
the
particular
chromosomes
affected.
We
cannot
exclude
the
possibility
that
the
two
particular
strains,
WI-26
and
VOL.
50,
1963
GENETICS:
SAKSELA
AND
MOORHEAD
395
WI-38,
were
in
some
way
predisposed
to
undergo
aneuploid
changes
or
that
some
un-
known
effect
of
the
medium
used
has
induced
an
instability.
However,
aneuploid
changes
were
noted
in
different
substrains,
even
though
they
were
carried
on
me-
dium
from
independent
commercial
sources.
It
therefore
appears
more
likely
that
our
findings
may
apply
generally
to
such
cells
and
confirm
the
study
by
Sax
and
Passano'6
in
which
anaphase
bridges,
lagging
chromosomes,
rod
and
dot
deletions
were
found
to
increase
approximately
3-fold
during
six
months
of
serial
cultivation
of
human
fibroblast
strains.
Interphase
nuclei
with
abnormal
sizes
and
shapes
are
seen
in
daughter
cells
in
later
passage
material'0
and
are
presumably
the
products
of
abnormal
division.
The
conditions
in
these
human
fibroblast
cultures,
after
loss
of
their
proliferative
capacity,
are
superficially
identical
to
those
reported
in
cultures
of
mouse
cells
just
prior
to
the
usual
reversal
of
the
latter's
declining
growth
rate."
I
This
increase
in
growth
rate
then
leads
to
establishment
of
the
mouse
cell
culture
as
a
continuously
propagated
and
usually
heteroploid
cell
line.
In
spite
of
the
degenerate
state
of
late
passage
cultures
of
strains
WI-26
and
WI-38,
with
chromosome
aberrations
and
reduced
mitotic
activity,
none
has
undergone
a
spontaneous
transformation,
al-
though
numerous
cultures
have
been
observed
1-3
months
after
cessation
of
growth.
Note
added
in
proof:
It
has
come
to
our
attention
that
M.
C.
Yoshida
and
S.
Makino
have
re-
ported
quite
similar
findings
in
the
Japan.
J.
Human
Genetics,
5,
39
(1963).
These
independent
observations
strengthen
our
conclusion
that
the
presence
of
aberrations
associated
with
in
vitro
decline
of
such
cell
strains
is
a
general
phenomenon.
*
Supported
in
part
by
USPHS
research
grant
CA-04534,
contract
PH-43-62-157,
and
career
development
award
5K3-CA-18,372
from
the
National
Cancer
Institute.
The
excellent
technical
assistance
of
Miss
P.
Mancinelli
and
Mrs.
M.
Lebowitz
is
gratefully
acknowledged.
t
Permanent
address:
University
Department
of
Pathology,
Maria
Hospital,
Helsinki.
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J.,
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