Analysis of Tumor Cell Invasion in
Organotypic Brain Slices Using
Confocal Laser-Scanning Microscopy
Organotypic cultures of nervous tissue, including
those of the hippocampal and cortical regions, have
been produced successfully with a simple method in
which brain slices are maintained in a culture at the
interface between air and culture medium (Yamamoto et al.
, 1989, 1992; Stoppini et al.
, 1991; Tanaka et al.
1994). In these organotypic brain slice cultures, not
only is the normal cytoarchitecture such as cortical
lamination and pyramidal cells preserved, but the
biochemical and electrophysiological properties of
neuronal cells are also maintained for 2 or 3 months.
By modifying this organotypic culture of nervous
tissues, we established a model for glial tumor cell
invasion with conditions analogous to those of normal
brains in situ (Ohnishi et al.
, 1998; Matsumura et al.
2000). This model enables not only to quantitatively
analyze the tumor cell invasion in brain tissues, but also
to investigate molecular events in vitro
occur between transplanted cells and brains in vivo
Hanks' balanced salt solution (HBSS) (Cat. No.
H9269), Eagle's minimum essential medium (MEM)
with HEPES (Cat. No. M7278), D-glucose (Cat. No.
G7021), penicillin-streptomycin solution (Cat. No.
P0781), amphotericin B (Cat. No. A2942), propidium
iodide (PI) (Cat. No. P4170), L
-glutamine (Cat. No. G5763), N-methyl-D-aspartate (NMDA) (Cat. No.
M3262), and agar (Cat. No. A5431) are from Sigma.
Dulbecco's phosphate-buffered saline, calciummagnesium
free [PBS(-), pH 7.4] (Cat. No. 14190-250),
horse serum (Cat. No. 16050-122), and fetal bovine
serum (FBS) (Cat. No. 16000-044) are from Invitrogen
Corp. Culture plate inserts with a 0.4-µm-pore membrane,
30mm (Millicell-CM) (Cat. No. PICM 030 50)
are from Millipore Corp. Six-well culture plates (Cat.
No. 3506), 60-mm culture dishes (Cat. No. 430166), and
100-mm culture dishes (Cat. No. 430167) are from
Corning. The PKH2 fluorescent cell staining kit is from
ZYNAXIS Cell Science. C6 rat glioma cells and T98G
human glioma cells are from American Type Culture
Collection. For these cell cultures, Ham's F10 powder
(Cat. No. N6635) and MEM (Cat. No. M4655) are from
A. Preparation of Brain Slice and
the Oraganotypic Culture
This procedure is modified from the method of
Stoppini et al.
Solutions and Instruments
Scissors (large one for decapitation and small one for
dissection of brains)
Microforceps with fine chips
10% povidone-iodine solution
Phoshate-buffered saline without calcium and
magnesium (pH 7.4)
Microslicer with a sliding cut mode (possible to cut
nonfrozen fresh brains with a range of 50 to 1000 µm
Culture plate inserts with 0.4µm-pore membranes
(30mm in diameter) (Millicell-CM)
Six-well culture plates (35 mm in diameter/well)
60-mm culture dishes
Culture medium: 50% Eagle's MEM (Earle salt with L
HEPES, and NaHCO3), 25%
HBSS, 25% heat-inactivated horse serum, 6.5 mg/ml
D-glucose, 100U/ml penicillin, 100µg/ml streptomycin,
and 2.5µg/ml amphotericin B. To make
200ml, add 96ml of a Eagle's MEM solution, 50ml
of HBSS, 50ml of horse serum, 1.3 g
2ml of a penicillin (10,000U/ml)-streptomycin
(10mg/ml) solution, and 2ml of an amphotericin
B solution (250µ/ml). Keep at 4°C.
- Anesthetize a 2-day-old neonatal rat with
diethyl ether and plunge into a 10% povidone-iodine
- Cut off the head with large scissors, remove the
skin and the skull with a small scissors, and take out
the whole brain quickly and place in a 60mm culture
dish with HBSS.
- Cut the brain vertically to the base, 1 mm inward
from both rostral and caudal ends of the cerebrum
with a blade, and mount on the stage of a microslicer,
which is sterilized with 70% ethyl alcohol.
- 300-µm-thick cut brain slices and transfer each
slice onto a porous (0.4µm pore size) membrane of a
culture plate insert, which is placed in a well of a sixwell
culture plate filled with PBS.
- After aspiration of PBS from the outer well of the
six-well culture plate, add 1 ml of culture medium to
the outer well but without covering the brain slice
placed on the membrane.
- Incubate the brain slice at 37°C under standard
conditions of 100% humidity, 95% air, and 5% CO2.
- After 3 days of the culture, replace half of the
medium with fresh medium twice a week. Reduce the
volume of the medium after the second change to
0.8 ml so that the slices remain well exposed to the air.
(This is critical for long-term survival of the neuronal
cells.) (Fig. 1).
|FIGURE 1 Illustrative procedures of brain slice culture. A rat whole brain slice 300µm thick is placed on a
porous membrane affixed to the culture plate insert and cultured at the interface between air and culture
B. Assessment of Viability of Brain Slices
The viability of cultured brain slices can be assessed
by morphological observation, neuronal activity, electrophysiological
features, and production of bioactive
substances such as γ-aminobutyric acid and neuropeptides.
Normal cytoarchitecture such as cortical
lamination and hippocampal structure is clearly observed for about 2 months after the slice culture if
the culture condition is kept properly (Fig. 2). This
section describes the method used to assess the neuronal
viability of brain slices by NMDA insult that
can induce early and delayed neuronal cell death
(Sakaguchi et al.
Solutions and Instruments
100 µM N-methyl-D-aspartate solution
|FIGURE 2 Morphological pictures of a rat brain slice after 8 days of culture.
including cortical lamination and hippocampal structure,
(left: macroscopic picture, ×0.5;
right: phase contrast, ×40).
: Dissolve 1.47 mg
of NMDA in 100ml of artificial cerebrospinal fluid
(CSF). Prepare artificial CSF from three stock solutions,
A, B, and C, before use. Stock solution A consists
of NaCl, 0.488g
of KCl, and 1.232 g of MgSO4
O in 100ml
O. Stock solution B contains 2.22 g of CaCl2
100 ml of H2
O, and stock solution C contains 4.62 g
in 100ml of H2
O. Store at 4°C.To make
100 ml of artificial CSF, add 4 ml of stock solution A,
4 ml of stock solution C, and 91 ml of H2
O and then
place the mixed solution under the current of 95%
air and 5% CO2
to lower the pH of the solution.
Then, add 1 ml of solution B and 0.18g of D
to the mixed solution (the final pH is 7.4).
4.6µg/ml propidium iodide solution
. Dissolve PI in a
serum-free solution containing 75% MEM, 25%
HBSS, 2mM L
-glutamine, and 6.5 mg/ml D-glucose
to a final concentration of 4.6µg/ml.
Fluorescence microscope with a tetramethylrhodamine
isothiocyanate (TRITC) filter
|FIGURE 3 Neuronal viability of rat brain slices assessed
uptake of propidium iodide (PI) without
(upper) and with
(lower) treatment of
N-methyl-D-aspartate (NMDA). Normally functioned
neurons can exclude PI and show early or delayed
cell death by NMDA insult, thus permitting entry
of the PI into the
C. Preparation of Tumor Cell Spheroids
Tumor Cells, Solutions, and Instruments
- Incubate brain slices in 1 ml of the artificial CSF
solution containing 100 µM NMDA, which is placed in the bathing well of a six-well culture plate for 15 min.
Incubate the slices in PI solution for 1 h to measure
early neuronal death or for 24h to measure delayed
neuronal cell death.
- View PI signals under a fluorescence microscope
with a TRITC filter.
- As a control, incubate brain slices in PI solution
for 1 h or 24 h following incubation in the CSF solution
without NMDA for 15 min. (Fig. 3).
Tumor cells in culture and their culture medium
(Ham's F10 medium containing 10% FBS is used for
C6 rat glioma cells, and MEM supplemented with
1% nonessential amino acid, 1% sodium pyruvate,
and 10% FBS is used for T98G human glioma cells)
PKH2 fluorescent cell-staining kit
1.25% agar-coated culture dish (100mm in diameter)
Place 5 ml of 1.25% agar solution on a culture dish and
dry under air.
Reciprocating shaker (usable in a CO2
D. Migration Assay of Tumor Cells on Brain
Slices (Fig. 4)
Instuments and Molecules
- Grow tumor cells as a monolayer culture under
- Harvest the tumor cells by trypsinization, wash
twice, and resuspend in labeling diluent "A" (provided
with the PKH2 staining kit) at a concentration
of 2 × 107 cells/ml (cell/diluent suspension).
- Add PKH2 dye to an equal volume of diluent
"A" to make a 4 µM solution. Add this solution to the
cell/diluent suspension and mix by gentle agitation.
- After incubating the cells at room temperature
for 5 min, stop the labeling reaction by adding a double
volume of the culture medium containing 10% FBS
and four times the volume of FBS into the sample
- Wash the cells and resuspend in the culture
medium with 10% FBS.
- Seed the labeled tumor cells (5 × 106) into a 1.25%
agar-coated culture dish and incubate under continuous
agitation at a speed of 40rpm on a reciprocating
shaker at 37°C in a humidified atmosphere of 5% CO2 and 95% air for 2 to 3 days.
- For the experiments, select cell aggregates with a
size of 150 to 200 µm.
Barin slices after 7 days culture
Tumor cell spheroids
Micropipette with a volume of 10µm
Molecules or agents affecting cell migration
Fluorescence microscope with a FITC filter
Color-chilled 3-CCD camera
|FIGURE 4 Illustrative procedures of tumor cell migration and invasion assay in cocultured brain slices. A
tumor (glioma) spheroid is placed on the brain slice and is cocultured at the interface between air and culture
medium. For tumor cell migration, the extent of the spread of fluorescent dye-stained tumor cells on the
surface of the slice is measured. For tumor cell invasion, the spatial extent of the tumor cell infiltration in the
slice is analyzed by confocal laser-scanning microscopy (CF-LSM). d, distance; S, area.
- Using a micropipette, take one spheroid of tumor
cells place on the surface of brain slice, and coculture
at 37°C under standard conditions.
- Four hours later, apply 2 µl of molecules in investigation
directly to the tumor spheroid. Carry out the
application of the molecule once a day for 3 to 6 days.
- To estimate the extent of cell migration, calculate
the distance between the margin of the initially placed
spheroids and the population of the migrating cells
showing half of the density (area) of the maximum
density of migrating cells from the tumor spheroid by
using computer images for which the original fluorescent
pictures of the slices are taken with a color-chilled
- For this calculation, draw concentric circles
10µm apart around the margin of the spheroid and
measure cell density (area of fluorescence-stained
cells) within each ring by an NIH image. Then, do the
summation of area of the cells contained in each ring
and plot as a function of the distance from the margin
of the tumor spheroid. Thus the distribution curve of
migrating cells outside the spheroid is constructed for
each brain slice (Fig. 5). The migratory strength of the
cells on the slice is defined as the distance (µm) that
shows half of the value of the maximum density (area)
of migrating cells on the distribution curve.
|FIGURE 5 Construction of distribution curves of migrating cells
for a quantitative analysis of tumor cell migration in brain slices. The
total area of the labeled cells contained in each ring is plotted as a
function of the distance from the margin of the tumor spheroid.
(Inset) Concentric circles 10µm apart are drawn around the margin
of the tumor spheroid, and cell density (area of fluorescent-stained
cells) within each ring is measured by an NIH image. Distribution
curves represent Ll-stimulated C6 glioma migration at day 2, day 4,
and day 6 after the coculture with brain slices. The migratory
strength of the cells is determined as the distance (µm) that shows
half of the value of the maximum density (area) of migrating cells
on the distribution curve (see the distribution curve on day 6).
E. Invasion Assay in Brain Slices (Fig. 4)
Barin slices after 7 days culture
Tumor cell spheroids
Micropipette with a volume of 10µm
Inverted confocal laser-scanning microscope with
FITC filter optics
- With a micropipette, take one spheroid of tumor
cells tagged with the PKH2-fluoresent dye, place on the surface of brain slice, and coculture at 37°C under
- To detect PKH2-stained tumor cells in brain
slices, use an inverted confocal laser-scanning microscope
with FITC (520nm) filter optics.
- At the first observation, determine the level of
the basal plane (0µm) in accordance with the upper
surface of the brain slice.
- Obtain serial sections every 20µm downward
from the basal plane to the bottom of the slice (Fig. 6).
|FIGURE 6 Confocal laser-scanning microscopic pictures of invading
glioma cells in rat brain slices. At 24h after coculture of T98G
glioma spheroid and the brain slice, most glioma cells remain at the
top of the slice (0µm), while the glioma cells migrate extensively
within the brain slice (show the maximum spread at -40 µm from
the top of the slice) at 72h after coculture.
F. Quantitative Analysis of Tumor Invasion
The total area of PKH2-stained cells in each section
is calculated with NIH image software. The area is
plotted as a function of the distance from the basal
plane of the brain slice and the distribution curve is
constructed for each experiment. The extent of tumor
cell invasion in the slice is defined as the depth (µm)
that shows half of the maximum density (area) of invasive
cells on the distribution curve.
- As a source of brain slices, brain tissues from
mice and humans (obtained from epilepsy surgery) are
also applicable. In the case of rats, 2- to 5-day-old neonatal brains are best. As brains from much younger
rats are smaller and more soft, it is difficult to manuplate
the whole brain as intact slices. Brains prepared
from rats of an older age have a tendency to be resistant
to tumor invasion into the slices.
- For about 4-5 days after initiating the brain slice
culture, several neuronal death and glial cell migration
to the bottom of the slices occur. After 5 days in culture,
the exposure of slices to PI alone without NMDA insult
does not elicit a detectable fluorescence signal. Therefore,
for tumor invasion experiments, brain slices after
5 days in culture should be used. Usually, brain slices
after 7 days in culture are used for any kind of studies.
At this time, the thickness of the brain slices is reduced
to about 200 µm from the original thickness of 300 µm. Brain slices maintain their normal structures, such as
cortical lamination, and are functionally viable for
about 2 months after the culture, but it seems that the
best time for experiments is from 7 to 30 days after
- To detect migrating tumor cells in the brain slice,
a tumor-labeling method using green fluorescent
protein (GFP) is also applicable. Once tumor cell clones
with persistent expression of GFP are established, their
use is of great advantage in analyzing the behavior
of the tumor cells because the GFP is transmitted to
the tumor cells after cell division. An EGFP vector
(pEGFP-C1, obtained from Clonetics Corp., San Diego,
CA) can be used to transfect and label malignant
Matsumura, H., Ohnishi, T., Kanemura, Y., Maruno, M., and
Yoshimine, T. (2000). Quantitative analysis of glioma cell invasion
by confocal laser scanning microscopy in a novel brain slice
model. Biochem. Biophys. Res. Commun
Ohnishi, T., Matsumura, H., Izumoto, S., Hiraga, S., and Hayakawa,
T. (1998). A novel model of glioma cell invasion using organotypic
brain slice culture. Cancer Res
Sakaguchi, T., Okada, M., Kuno M., and Kawasaki, K. (1996).
Dual mode of N-methyl-D-aspartate-induced neuronal death in
hippocampal slice cultures in relation to N-methyl-D-aspartate
receptor properties. Neuroscience 76
Stoppini, L., Buchs, P.-A., and Muller, D. (1991). A simple method
for organotypic cultures of nervous tissue. J. Neurosci. Methods 37
Tanaka, M., Tomita, A., Yoshida, S., Yano, M., and Shimuzu, H.
(1994). Observation of the highly organized development of
granule cells in rat cerebellar organotypic cultures. Brain Res
Yamamoto, N., Kurotani, T., and Toyama, K. (1989). Neural connections
between the lateral geniculate nucleus and visual cortex in
. Science 245
Yamamoto, N., Yamada, K., Kurotani, T., and Toyama, K. (1992).
Laminar specificity of extrinsic cortical connections studied in
coculture preparations. Neuron 9