-
Protocol
Fetal Thymus
Organ Culture
INTRODUCTION
The generation of
functionally competent T-cells from
their
progenitors involves a
series of developmental events
including
proliferation,
differentiation, and survival.
T-cell
development
is a non-cell-
autonomous event, and requires interactions
with
thymic stromal cells.
Fetal thymus organ cultures provide an
in vitro system in which
isolated embryonic thymus lobes
can
be maintained in
culture, allowing the study of
T-cell
development
as well as
thymic stromal cell function. This system
remains
the
only
in vitro system that supports a complete program
of
T-cell development,
including positive and negative
selection
of the developing
T-cell receptor repertoire.
Modifications
of
the basic fetal thymus organ culture system, such
as hanging
drop
cultures and reaggregate thymus organ
cultures, provide
useful
systems to analyze
thymus colonization
and thymic stromal
cell
function, respectively.
MATERIALS
Reagents
All media should be
prewarmed to 37°
C before
use.
10%
CO
2
in air,
contained
in gas
cylinder
2'-deoxyguanosine (dGuo) (Sigma-
Aldrich, D0901)
Prepare a 9 mM stock in 1X PBS. It
takes ~1 h at 37?
C for
the dGuo to dissolve.
Mix the solution well during this
period.
Dilute the dGuo
stock to a final
concentration of 1.35
mM in
1X PBS (see Step
3).
Dulbecco
’s
modified
Eagle’s medium
(DMEM) for thymus organ culture
70% ethanol
Mice, pregnant female
(gestational age E14-E16)
1X PBS (Ca
++
/Mg
++
-free)
RF10-H medium
10X trypsin (Sigma-
Aldrich,
T4674)
1
Prepare a 1X trypsin solution by
diluting the 10X stock in Ca
++
/Mg
++
-free
PBS
containing 0.02%
EDTA.
Equipment
Artiwrap sponges, 1
cm
2
(Medipost
Ltd.)
Aspirator
tube assembly (Sigma-Aldrich, A5177)
Boxes (rectangular,
plastic) with fitted lids (Watkins and
Doncaster,
E6052)
Bunsen burner, fish-
tail
Filters
(isopore membrane) with 0.8-?
m pore
size (Millipore,
ATTP01300)
Forceps, watchmaker, Dumont #5
(TAAB)
Incubator, preset to
37°
C
Microcentrifuge
Microcentrifuge tubes, 1.5
mL
Micropipette,
1 mL
Microscope
(stereo-dissecting) with magnification range
0.8X-5X
(e.g., Zeiss, Stemi
SV)
Petri dishes, 90-mm diameter, sterile
bacteriological grade
(Sterilin)
Plate inserts for multiwell plates
(e.g., for a six-well plate,
use Millicell 0.4-?
m plate
inserts; Millipore,
PICM03050)
(optional; see
Step 2.i)
Plates, Terasaki (Sterilin)
Scissors,
surgical
Tape
Tubing, glass, to make capillary
pipettes (Fisher Scientific
UK, FB51460)
Vortex mixer
METHOD
2
The standard fetal thymus organ culture
(FTOC) method described
in
Steps 1 and 2
can be
modified to study thymus colonization
using hanging drop cultures
(
Steps
3-5
;
Jenkinson et al. 1982).
In
addition, reaggregate thymus organ cultures (RTOC)
can be
used, in
which three-dimensional organ cultures are
generated
from defined
mixtures of thymic stromal cells and
thymocytes
(
Steps
6-16
). This latter method is
particularly useful in
studying
thymic stromal cell
function and the development of
an
individual
cohort of
thymocytes at a defined developmental
stage.
Fetal
Thymus Organ Culture (FTOC)
1. To dissect fetal thymus
lobes:
i. Swab
the abdominal region
of a
sacrificed female mouse using
70% ethanol.
Reflect
the
skin using scissors and
forceps, and
cut through the
abdominal
wall to reveal the
uterus. Separate
the uterus
from its attachments
at the
two
uterine horns laterally,
and from the bladder
anteriorly.
ii.
Transfer the uterus to
a
90-mm Petri dish. Using scissors
and forceps, cut
open
the
uterus
lengthwise
to remove the
embryos.
iii.
Place the embryos
in a Petri
dish containing 1X PBS to
wash off any blood
.
Use
forceps to
remove the placenta and
membranes
from each
embryo.
iv.
Place the embryos in a Petri dish
containing
RF10-H
medium.
Decapitate
the embryos by pinching them
with
forceps just
below
the chin.
Place each
embryo on its
back
,
and
open the
anterior
surface of the
chest wall by placing
the
tips
of
a
closed
pair
of
forceps
into
the
chest
cavity
and
allowing
the
forceps to
open.
v.
Under the dissecting
microscope,
remove
the
entire
thoracic
tree--heart,
lungs, trachea,
thymus lobes--by
grasping
below
the
heart.
Place the thoracic
trees in a Petri
dish containing
fresh RF10-H medium.
Problem 1:
When
removing the thoracic tree from the embryos,
thymus
lobes are left in the
chest cavity.
[Step
1.v]
Solution:
It is
important to
grasp the heart (a red,
apple-shaped
organ) firmly.
Sharp straight forceps
also help; if
forceps
are blunt or bent,
fine dissection is difficult.
vi. Check
each
tree for the presence
of the two thymus lobes.
Remove the
thymus
lobes from the thoracic tree using
forceps.
Thymus
lobes
appear
as
oval
encapsulated
organs
on
either
side
of
the
trachea
(
Fig. 1
).
Problem2:
Instruments
become
sticky
with
congealed
blood
and
tissue
during the preparation of large numbers
of embryos, making
it
difficult to
manipulate dissected
lobes.
[Step
]
Solution:
Wash
the instruments
regularly with 70%
alcohol and
allow them to
air-dry before use.
3
Figure 1.
Position of the fetal thymus lobes in
the thorax. (
a
)
When the embryo is placed on its
back
and the anterior chest wall is opened, the
two thymus lobes can be seen lying
above the
heart (H, heart;
L, liver). (
b
) These lobes
can be
View larger version
(39K):
individually dissected as
encapsulated organs.
[in this window]
[in a new window]
2.
To prepare
fetal thymus organ cultures:
i.
Place 4 mL of
prewarmed
DMEM in a 90-mm
Petri dish
, and
use
forceps to
place two 1-cm
2
Artiwrap sponges and two
0.8-?
m
filters
into the dish.
Make
sure that the medium wets the
filters
from
below.
See
Troubleshooting.
As
an
alternative
to
the
sponge-and-
filter
method,
multiwell plate inserts with
membranes
of the
appropriate
pore size can be
used.
Problem3:
Large areas of the
bottom of the Petri dish are not
covered by
medium.
[Step
2.i]
Solution:
Make sure the volume of medium is accurate;
insufficient
medium in the dishes can adversely
affect organ cultures. This
may also be due to sponge supports that
are too large and consequently
soak
up the
medium.
Problem4:
Filters slip off
the sponge supports and sink.
[Step 2.i]
Solution:
Adding
too much medium to the Petri dish
increases
the depth of the
medium,
which
can
cause
filters
to
float
off
their
support
and
sink.
Make
sure
the
volume of medium is
accurate.
Also, use sponge
supports that are larger in
area than
the filters.
ii.
Prepare
a
finely
drawn
glass
pipette
by
heating
the
glass
tubing
in
a
Bunsen
flame, and
allow the
pipette to cool
before
attaching to
the
aspirator
tube
.
iii.
Using a
mouth-controlled glass pipette
from Step ,
pick
up the
thymus
lobes individually and
transfer
them to
the
surface
of the filters.
See Troubleshooting. As
an alternative approach
to the glass
pipette method,
fine
forceps
can be
used.
Problem5:
A large volume of
medium is transferred to the filter
during the
placement
of
lobes
on
the
filter
.
[Step
]
Solution:
Use
a
fine
mouth-
controlled
glass
pipette
to
transfer
the
lobes.
The
diameter
of
the
glass pipette should be
approximately
half the size
of the thymus lobe.
If
forceps
are
used,
medium
is
easily
transferred
to
the
filter
,
which
can
submerge the
lobes.
4