Methods for Implantation of BD Matrigel™ Matrix into Mice and Tissue Fixation
1Kazuo Ohashi, M.D., Ph.D., 1Takashi Yokoyama, M.D., 1Yoshiyuki Nakajima, M.D., Ph.D., and 2Marshall Kosovsky, Ph.D.
1Nara Medical University, Nara City, Nara JAPAN; 2BD Biosciences – Discovery Labware, Billerica, MA
BD Matrigel™ Matrix is a solubilized Subcutaneous injection of
basement membrane preparation BD Matrigel Matrix into a mouse
extracted from Engelbreth-Holm-
Swarm (EHS) mouse sarcoma, a
tumor rich in ECM proteins. Its major
component is laminin, followed by
collagen IV, heparan sulfate proteo-
glycan, and entactin. BD Matrigel
Matrix is effective for the attachment
and differentiation of both normal
and transformed anchorage-dependent
epithelial and other cell types.
BD Matrigel Matrix is highly useful in
various studies including 3D cell culture,
cell invasion and migration assays,
drug metabolism/toxicology, in vitro
and in vivo angiogenesis assays. This
report describes the use of BD Matrigel
Matrix for in vivo applications such
as angiogenesis and human tumor cell
implantation in mice.1-8
1. BD Matrigel Matrix (Cat. Nos.
354234 and 356234) is suitable as a
scaffold for supporting the implan-
tation of various tumor cells. Growth
Factor Reduced (GFR) BD Matrigel
Matrix (Cat. Nos. 354230 and
356230) is also available for studies
in which a reduced growth factor
composition is required.
2. BD Matrigel Matrix phenol red-
free (Cat. No. 356237) and GFR
BD Matrigel Matrix, phenol red-free
(Cat. No. 356231) are typically used
for the cyan-metrohemoglobin method
that measures hemoglobin content
(measurement of reddish-brown
absorption) in angiogenesis studies.
BD Matrigel Matrix has been shown
to enhance the process of angiogenesis
3. BD Matrigel Matrix High
Concentration (HC) (Cat No. 354248)
is suited for in vivo applications where
a high protein concentration augments
growth of tumors. The high protein
concentration (18-22 mg/ml) also
allows the BD Matrigel Matrix plug
to maintain its integrity after subcu-
taneous injection into mice. This
keeps the injected tumor cells and/or
angiogenic compounds localized for
in situ analysis and/or future excision.
Subcutaneous injection of BD Matrigel Matrix into a mouse
1. Since BD Matrigel Matrix forms a gel
above 10°C, BD Matrigel Matrix
solution should be kept at low
temperatures, and thus all equip-
ment and reagents (syringes, needles,
BD Matrigel Matrix solution, etc.)
should be chilled on ice prior to
2. After mixing BD Matrigel Matrix
with a cell suspension [Note 1], the
BD Matrigel mixture is injected into
a mouse subcutaneously [Note 2]
(Figure 1). An appropriate needle
size (21-25G) should be selected
to prevent the destruction of cells.
To increase the contact area of the
injected BD Matrigel mixture into
subcutaneous tissues, a wide subcu-
taneous pocket should be formed by
swaying the needlepoint right and left
after a routine subcutaneous insertion.
The BD Matrigel mixture is then
injected into the pocket. When the
BD Matrigel mixture is injected into
a particular area without swaying the
needlepoint, the mixture will form
a large cell clump and a subsequent
growth defect may result due to
inefficient perfusion of nutrients to
the cells within the core of the clump.
Note 1: In this experiment, undiluted
BD Matrigel Matrix alone was injected into
the mouse. For tumor implantation appli-
cations, approximately 2x107 cells/ml of
cell suspension should be mixed with
BD Matrigel Matrix, resulting in a final cell
concentration of ~106 cells/ml. To prevent
incomplete gel formation in mice, do not
dilute BD Matrigel Matrix to a final concen-
tration below 4 mg/ml.
Note 2: In this experiment, 0.7 ml of the
BD Matrigel was injected. The injection
volume of BD Matrigel takes into account
the absorption of BD Matrigel into the
tissue and allows for easy removal of the
resultant tissue ‘plug’. The optimal injection
volume should be determined according to
the requirements of your experiment.
While the injection of ~0.1 ml of a
BD Matrigel mixture into mice may be
sufficient for the augmentation of tumor
growth, the injection of at least 0.5 ml is
recommended for in vivo angiogenesis studies.
Removal of the BD Matrigel plug
from the mouse
3. After an appropriate incubation
period [Note 3], the mouse is
anaesthetized and a square segment
of tissue is excised with scissors. To
ensure complete excision of the plug,
cut ~5 mm wider than the implantion
site on all sides.To maintain the shape
of the BD Matrigel plug, excise the
subcutaneous tissue, peritoneum, as
well as skin. These tissues are then
fixed with formalin. Figure 3 shows
the implanted BD Matrigel viewed
from the peritoneal side following
excision. The volume of the implanted
BD Matrigel is reduced from the
injected volume due to absorption and
partial degradation of BD Matrigel
in vivo. The excised BD Matrigel
plug is usually clear yellowish in
color. If blood vessels are formed
within the BD Matrigel plug, the
color of the BD Matrigel will
appear red (Figure 4).
Note 3: In this experiment, the BD Matrigel
plug was removed after one week. When
the quantity of hemoglobin is used to assess
angiogenesis, BD Matrigel containing VEGF
and heparin should be injected to promote
angiogenesis. After about three days, the
BD Matrigel plug containing newly formed
blood vessels can be easily removed.
Fixation of tissues including
BD Matrigel Matrix
4. The excised tissue should be stretched
and put on a sheet of thick paper
(e.g., poster board) to avoid the
formation of wrinkles. The tissue
is then placed in a nylon bag for
protection. Fix the tissue in 10%
neutralized formalin solution for at
least one day at room temperature
[Note 4]. This treatment will harden
the tissue in preparation for slicing
the sample (Figure 6). Care should
be taken to ensure that the thickness
of the slice is adequate to retain the
implanted BD Matrigel plug.
Note 4: Fixation of BD Matrigel under 8°C
may cause depolymerization of BD Matrigel.
Therefore, BD Matrigel should be fixed at
5. The fixed BD Matrigel plug can
be embedded in paraffin to prepare
sections for histochemical staining.
Figure 7 shows a section of the
BD Matrigel plug stained with
hematoxylin-eosin (HE). BD Matrigel
appears pink to light reddish in color
with HE staining.
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Data provided by Dr. Kazuo Ohashi and
Dr. Takashi Yokoyama at Nara Medical