A Long Term Culture Model for Primary Hepatocytes from Cynomolgus Monkeys
In
vitro
models
of
animal
liver
;ssues
are
of
great
interest
to
drug
developers
and
regulatory
agencies
because
of
their
poten;al
to
reduce
costs
and
live
animal
use
during
drug
development.
Furthermore,
drug
metabolism
and
toxicity
studies
in
vitro
across
human
and
animal
species
are
u;lized
for
selec;on
of
the
appropriate
animal
species
for
in
vivo
tes;ng
in
rodent
and
non-‐rodent
species.
Primary
monkey
hepatocytes
are
widely
considered
to
be
the
most
suitable
for
drug
metabolism
and
toxicity
studies;
however,
these
cells
display
a
precipitous
decline
in
phenotypic
func;on
when
kept
in
suspension
or
plated
in
a
sandwich
of
extracellular
matrix.
We
have
developed
a
microscale
model
of
the
Cynomolgus
monkey
liver
in
which
microfabrica;on
tools
adapted
from
the
semiconductor
industry
were
u;lized
to
organize
primary
hepatocytes
into
colonies
of
empirically
op;mized
dimensions
and
these
colonies
were
subsequently
surrounded
by
suppor;ve
murine
embryonic
3T3-‐J2
fibroblasts
(cynomolgus
monkey
micropaQerned
co-‐cultures
or
CM-‐MPCC).
We
have
determined
the
effects
of
hepatocyte
cell
seeding
densi;es,
donor
lots,
and
fresh
versus
cryopreserved
sources
on
the
magnitude
and
life;me
of
CM-‐MPCCs.
Several
liver-‐specific
func;ons
were
measured
over
four
weeks
including
urea
synthesis,
cytochrome
P450
ac;vi;es,
and
forma;on
of
func;onal
bile
canaliculi.
Our
results
indicate
that
CM-‐MPCCs
display
higher
levels
of
measured
func;ons
for
at
least
4
weeks
as
compared
to
tradi;onal
cell
culture
models.
For
instance,
bile
canalicular
transport
was
observed
with
5-‐(and-‐6)-‐carboxy-‐2´,7´-‐dichlorofluorescein
diacetate
for
at
least
24
days
in
culture,
while
urea
synthesis
was
nearly
45-‐fold
higher
in
CM-‐MPCCs
than
in
sandwich
cultures
on
Day
10
of
culture
(266.87
μg/day/million
cells
vs.
5.99
μg/day/million
cells).
In
addi;on,
cultures
showed
higher
func;onality
when
prolifera;ng
fibroblasts
were
used
as
compared
to
growth-‐arrested
ones.
The
u;lity
of
CM-‐MPCCs
for
drug
metabolism
and
toxicity
studies
is
now
currently
under
inves;ga;on.
In
the
future,
CM-‐MPCCs
may
be
used
as
an
in
vitro
predic;ve
tool
for
refining
and
reducing
tes;ng
in
live
monkeys
during
drug
development.
vitro
models
of
animal
liver
;ssues
are
of
great
interest
to
drug
developers
and
regulatory
agencies
because
of
their
poten;al
to
reduce
costs
and
live
animal
use
during
drug
development.
Furthermore,
drug
metabolism
and
toxicity
studies
in
vitro
across
human
and
animal
species
are
u;lized
for
selec;on
of
the
appropriate
animal
species
for
in
vivo
tes;ng
in
rodent
and
non-‐rodent
species.
Primary
monkey
hepatocytes
are
widely
considered
to
be
the
most
suitable
for
drug
metabolism
and
toxicity
studies;
however,
these
cells
display
a
precipitous
decline
in
phenotypic
func;on
when
kept
in
suspension
or
plated
in
a
sandwich
of
extracellular
matrix.
We
have
developed
a
microscale
model
of
the
Cynomolgus
monkey
liver
in
which
microfabrica;on
tools
adapted
from
the
semiconductor
industry
were
u;lized
to
organize
primary
hepatocytes
into
colonies
of
empirically
op;mized
dimensions
and
these
colonies
were
subsequently
surrounded
by
suppor;ve
murine
embryonic
3T3-‐J2
fibroblasts
(cynomolgus
monkey
micropaQerned
co-‐cultures
or
CM-‐MPCC).
We
have
determined
the
effects
of
hepatocyte
cell
seeding
densi;es,
donor
lots,
and
fresh
versus
cryopreserved
sources
on
the
magnitude
and
life;me
of
CM-‐MPCCs.
Several
liver-‐specific
func;ons
were
measured
over
four
weeks
including
urea
synthesis,
cytochrome
P450
ac;vi;es,
and
forma;on
of
func;onal
bile
canaliculi.
Our
results
indicate
that
CM-‐MPCCs
display
higher
levels
of
measured
func;ons
for
at
least
4
weeks
as
compared
to
tradi;onal
cell
culture
models.
For
instance,
bile
canalicular
transport
was
observed
with
5-‐(and-‐6)-‐carboxy-‐2´,7´-‐dichlorofluorescein
diacetate
for
at
least
24
days
in
culture,
while
urea
synthesis
was
nearly
45-‐fold
higher
in
CM-‐MPCCs
than
in
sandwich
cultures
on
Day
10
of
culture
(266.87
μg/day/million
cells
vs.
5.99
μg/day/million
cells).
In
addi;on,
cultures
showed
higher
func;onality
when
prolifera;ng
fibroblasts
were
used
as
compared
to
growth-‐arrested
ones.
The
u;lity
of
CM-‐MPCCs
for
drug
metabolism
and
toxicity
studies
is
now
currently
under
inves;ga;on.
In
the
future,
CM-‐MPCCs
may
be
used
as
an
in
vitro
predic;ve
tool
for
refining
and
reducing
tes;ng
in
live
monkeys
during
drug
development.
