Murray J. Clifford. Angiogenesis Protocols - Methods in Molecular Medicine, Vol. 46
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Pericyte Isolation and Culture 257
Fig. 5. Identification of pericytes by iso-actin expression. (A) Phase-contrast photo-
micrograph; (B) same field as in A. Stained for total F-actin (filamentous actin) with
phalloidin; (C) same field as in A and B stained with antibody to vascular smooth
muscle-actin; asterisk (*) identifies a cell which is not a pericyte (i.e., negative for
vascular smooth muscle actin expression).
258 Nayak and Herman
3.5.3. Fixation and Permeabilization (27)
1. Wash cells once with DMEM (no serum) to remove serum components.
2. Add 1.0 mL of fixative to each well and incubate 5 minutes at room temperature.
3. Wash three times with 1.5 mL of PBS (containing 0.02% sodium azide), 5 min
per wash; change the first wash immediately.
4. Add 1.0 mL of lysis buffer for cell permeabilization and incubate for 60–90 sec
at room temperature.
5. Wash three times with 1.5 mL of PBS (containing 0.02% sodium azide), 5 min
per wash; change the first wash immediately.
3.5.4. Immunostaining
1. Cover a small piece of perspex (plexiglass) with parafilm and drop 25 µL of
diluted primary antibody per cover slip on it.
2. Place cover slips, cell side down, on the droplet of antibody solution.
3. Incubate in a humidified box for 45 min at room temperature.
4. Return the cover slips to the 12-well plate.
5. Wash three times with 1.5 mL of PBS (containing 0.02% sodium azide), 5 min
per wash, change the first wash immediately.
6. Drop 25 µL of diluted secondary antibody per cover slip on a parafilm sheet,
place cover slips, cell side down, on the droplet of antibody solution, and incu-
bate in a humidified box for 45 min at room temperature.
7. Return the cover slips to the 12-well plate and wash three times with 1.5 mL of
PBS (containing 0.02% sodium azide), 5 min per wash; change the first wash
immediately.
Table 1
Markers for Identification of Pericytes and Potential Cell Contaminants
Pericytes EC SMC Fibroblasts RPEC Astrocytes
Smooth
muscle actin + – + – – –
Cortical
nonmuscle + + +? +++
actin
3G5 + – – – – +
GFAP – – – – – +
Cytokeratin 18 – – – – + –
FVIIIRA – + – – – –
Acetylated
LDL uptake – + – – – –
EC, endothelial cells; SMC, smooth muscle cells; RPEC, retinal pigment epithelial cells; ?,
quantitatively decreased.
Pericyte Isolation and Culture 259
3.5.5. Mounting
1. To labeled microscope slides add 1 drop of mounting fluid (9/1 glycerol–PBS/
azide) with a pasteur pipet.
2. Rinse back of cover slips with water to remove salts and dry with a lint free paper
tissue (e.g., Kimwipes) then mount cover slips cell side down on glycerol droplet.
3. Wick away excess glycerol with a Kimwipe and seal the cover slip edges onto the
slides with nail polish.
4. When the nail polish has hardened the cells can be viewed with a fluorescence
microscope.
3.5.6. DiI-acetylated LDL Staining (28)
DiI-acetylated LDL staining must be performed on viable cells, therefore
the cells on cover slips should be fixed after the DiI-acetylated LDL staining
procedure given below.
1. DiI-acetylated LDL at 10 µg/mL in serum supplemented culture medium is added
to cells on cover slips (1.0 mL).
2. Incubate for 4 h at 37°C in a tissue culture incubator, then wash with PBS, and fix
as described above.
3. Mount and view as described above.
3.6. Cryopreservation and Recovery of Pericytes
3.6.1. Cryopreservation of Pericytes
Pericytes can be cryopreserved in liquid nitrogen using the same methods
that are in general use for other cell types. Pericytes can be recovered from
liquid nitrogen at least 1–2 yr after freezing them.
1. Trypsinize and centrifuge pericyte cultures by the procedure in Subheading 3.4.
2. Resuspend pericytes in 10 mL of growth medium, remove 10 µL of the cell sus-
pension and add it to 10 µL of trypan blue solution . After 5 min count the num-
ber of trypan blue-excluding (viable) cells in a hemocytometer. Adjust the
pericyte suspension to 1 × 10
6
cells/mL and add 1mL/vial.
3. Add an equal volume of freezing medium and transfer the vials to a polystyrene
foam tube rack and cover with a second rack (taping them together). Place the
assembly in a –80°C freezer.
4. The next day transfer the frozen vials to a liquid nitrogen freezer.
3.6.2. Recovery of Cryopreserved Pericytes
1. Remove a vial of pericytes from the liquid nitrogen freezer and defrost rapidly in
a 37°C water bath.
2. In the tissue culture hood, transfer the defrosted cell suspension to a sterile 10
mL tube and dilute slowly by dropwise addition of 9 mL of growth medium,
shaking the tube gently after each drop.
260 Nayak and Herman
3. Pellet the cells by centrifugation and aspirate off the supernatant. Resuspend the
cells in growth medium and estimate the viable cell number by trypan blue exclu-
sion as described in step 2 of Subheading 3.6.1. Approx 250,000 viable cells
are pipeted onto each sterile 10-cm Petri dish and the medium topped up to 8 mL
total volume/plate.
4. The following day exchange with fresh medium.
4. Notes
1. The vitreous may have to be gently teased to squeeze it out of the eye-cup. Avoid
disturbing the pigment epithelium beneath!
2. When removing the retina, start at an edge and gently stroke the retina in toward
the optic nerve head.
3. The Swinnex filter holders can be attached to each other allowing sequential fil-
tration over both meshes at once.
4. Do not leave out the “weeding” procedure, it is essential! The cultures will be
overgrown with nonpericytes if they are not weeded.
5. The number of times the cells can be passaged depends on the structure/function that
is to be studied and the conditions under which the pericytes are maintained (17).
This must be determined empirically for the specific function that you wish to study.
6. The single best definable criterion for the identification of pericytes is their ana-
tomical location within the microvascular basement membrane in vivo (18).
Clearly such a definition is of no value in culture systems as the anatomical rela-
tionships are not preserved. We are therefore constrained in using biochemical
and immunological means of identifying pericytes in vitro.
7. Iso-actin distribution (Fig. 5) is often used to distinguish pericytes from SMCs
(29). Both pericytes and SMCs express smooth muscle and cortical (sub-
plasmalemmal) nonmuscle actin. In SMCs, there is quantitatively less cortical
nonmuscle actin than in pericytes. This may often be observed by fluorescent
staining as a lack of cortical nonmuscle actin in smooth muscle cells (10). When
staining total F-actin with phalloidin, extreme caution is necessary (phalloidin is
very toxic). Phalloidin staining can be performed using the antibody staining
procedure detailed above. Add 0.45 U of phalloidin stock (0.3 units/µL in metha-
nol), i.e, 1.5 µL per cover slip; added to 25 µL of DMEM or PBS. The Sigma-
Aldrich Chemical Company also sells monoclonal antibodies to _-vascular smooth
muscle- and `-actin that are of different IgG isotypes and would be amenable to
double staining using the appropriate fluorescently labeled secondary antibodies.
8. The 3G5 antibody stains pericytes (Fig. 4) well at a protein concentration of
5–10 µg/mL. It does react with retinal neurons, but this is not problematic because
they do not survive in culture. Monoclonal antibody 3G5 also reacts with a sub-
population of GFAP positive neonatal rat brain astrocytes as well as a subpopula-
tion of GFAP positive human astrocytoma cells (RCN, unpublished
observations). The 3G5 monoclonal antibody will also react with a subpopula-
tion of SMCs in subconfluent cultures but does not react with smooth muscle
cultures that have been maintained at confluence for several days (RCN, unpub-
Pericyte Isolation and Culture 261
lished observations).
9. GFAP (glial fibrillary acidic protein) is the classic astrocyte marker although not
expressed by all astrocytes. In vivo, nonastrocytic cells have been shown to
express GFAP after tissue injury (19).
10. Cytokeratin 18 is a marker of simple epithelia (gastrointestinal, respiratory, uri-
nary, liver, and glandular) but does not react with most stratified squamous epi-
thelia (oesophageal, epidermal) (20). However, recent studies indicate that
microvascular endothelium from the synovium of rheumatoid arthritis patients
express cytokeratin 18 (21) while endothelia from other vascular beds do not (22).
11. Antifactor VIII-related antigen (von Willebrand’s factor) immunohistochemistry
and Di-I-acetylated LDL uptake are both extensively used as markers for vascu-
lar endothelial cells (23). Fluorescent staining with both reagents should show
punctate fluorescence associated with intracellular vesicles, although there are
organ and species specific exceptions (23). Care must be taken not to add too
much reagent when performing acetylated LDL uptake studies as nonspecific
uptake by pinocytosis will occur in both endothelial and nonendothelial cells.
12. Secondary antibodies are available from many commercial sources. When per-
forming double indirect immunofluorescence studies it is crucial that the second-
ary reagents are absolutely specific for their respective primary antibodies. Any
cross-reactions will invalidate the results. When the primary antibodies are from
different species, the secondary antibodies should have been absorbed with serum
from the other species. For example, if the primary antibodies are produced in
rabbits and mice, then the antimouse secondary reagent should have been
absorbed with rabbit serum and the antirabbit secondary reagent with mouse serum.
13. Shepro and colleagues have recently described a procedure for eliminating
endothelial and epithelial cells from retinal pericyte cultures by selective killing
with
L-leucine-methyl ester (24). We have tried this technique twice and it appears
to work as described. It is a much less tedious way of enriching pericytes than
weeding, and may replace weeding in the standard protocol.
Acknowledgments
We thank Alice Lin, Michael Papetti, Albert Perdon and Gregory
Sieczkiewicz for critical reading of the manuscript and insightful comments.
We would also like to thank Patricia Griffiths for assistance with the prepara-
tion of the manuscript. Dr. Nayak is supported by NIH grants EY12607 and
EY13054. Dr. Herman is supported by NIH grants GM55110 and EY09033.
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