Please click the main subject to get the list of sub-categories
| Section: Cell Biology Methods » Cell and Tissue Culture: Associated Techniques » Differentiation & Reprogramming of Somatic Cells
|Please share with your friends:
Cultured for Neuronal PC12 Ce lls: A Model Function, Differentiation, and Survival
|Cultured PC12 Ce lls: A Model
Since its initial description and characterization in
1976 (Greene and Tischler, 1976), the rat pheochromocytoma
PC12 cell line has become a commonly
employed model system for studies of neuronal development
and function. In particular, PC12 cells have
been a convenient alternative to cultured neurons for
studying the trophic and differentiative actions of
nerve growth factor (NGF; reviewed by Levi-Montalcini
and Angeletti, 1968; Levi and Alem& 1991). When
cultured in serum-containing medium, PC12 cells
adopt a round and phase-bright morphology and
proliferate to high density. Under these conditions,
PC12 cells display many of the properties associated
with immature adrenal chromaffin cells and sympathicoblasts.
When challenged with physiological levels
of NGF, these cells cease division, become electrically
excitable, extend long branching neurites, and gradually
acquire many characteristics of mature sympathetic
neurons. Under serum-free conditions, NGF
promotes not only neuronal differentiation of PC12
cells, but also their survival (Greene, 1978; Rukenstein et al.
Several attributes of PC12 cells have led to their
widespread popularity in neurobiological research.
These include their relatively high degree of differentiation
before and after NGF treatment, homogeneous
response to stimuli, availability in large numbers for
biochemical studies, and suitability for genetic manipulations.
This article details experience gained with
this cell line in terms of tissue culture requirements and treatment with NGF, as well as quantitative
assessment of NGF actions. In addition, we describe
some of the potential difficulties that one may
encounter when culturing PC12 cells and suggest possible
means to avoid or ameliorate these problems. The
reader is referred to several prior articles (Greene and
Tischler, 1982; Greene et al.
, 1987, 1991) for a more indepth
discussion of the properties and experimental
exploitation of the PC12 cell line.
II. MATERIALS AND
Rosewell Park Memorial Institute 1640 (RPMI 1640)
medium is purchased from Invitrogen (Carlsbad,
CA; Cat. No. 23400062) in powder form. Donor horse
serum (Cat. No. 12-44977P), fetal bovine serum (Cat.
No. 12-10377P), and penicillin/streptomycin (Cat. No.
59-60277P) are obtained from JRH Biosciences (Lenexa,
KA). It is recommended that sera be prescreened for
their capacities to promote PC12 cell growth and maintenance
The horse serum should be heat inactivated in
a 56°C water bath for 30min before use.
Tissue culture plasticwares are obtained from
Falcon, Becton Dickson and Company (Lincoln Park,
NJ). Freezing vials are purchased from Nunc,
Denmark (Cat. No. 377267). Millipak-60 filters
(0.22 µm, Cat. No. MPGL06SH2) are from Millipore
(Bedford, MA). Filter units (0.45 µm, Cat. No. 245-0045)
are obtained from Nalgene Company (Rochester, NY).
Ethylhexadecyldimethylammonium bromide (Cat. No. 117 9712) is purchased from Eastman Kodak
Company (Rochester, NY).
NGF is prepared from adult male mouse submaxillary
glands as described by Mobley et al.
glands can be purchased from Harlan Bioproducts for
Science (Indianapolis, IN; Cat. No. 516) and stored at -80°C until use. NGF stocks (>100µg/ml; in pH 5.0
acetate buffer, 0.4 M NaCl) are stored at -80°C and, once
thawed, can be kept at 4°C for at least I month without
significant loss of activity. Recombinant or purified NGF
may also be purchased from a variety of commercial
suppliers, including Harlan Bioproducts for Science,
Roche Molecular, and Upstate Biotechnology, Inc.
Rat tail collagen is prepared in 0.1% acetic acid as
described previously (Greene et al.
, 1991) from the
tendons of rat tails [see Fig. 14.2 of Kleitman et al.
(1991) for a photographic illustration of the procedure
for exposing and removing rat tail tendons]. Each large
tail furnishes approximately 200ml of stock collagen.
Aliquots of collagen stock are stored at-20°C. Once
thawed, the stock can be stored for up to several
months at 4°C. Sterile technique should be employed
throughout the preparation.
A. Routine Tissue Culture Techniques
- Complete growth medium: Prepare RPMI 1640
medium according to the supplier's protocol in reverse
osmosis/Milli-Q or double-distilled water. After the
addition of sodium bicarbonate (2g/liter), penicillin
(final concentration 25 U/ml), and streptomycin (final
concentration 25 µg/ml), sterilize the medium by pressure
filtration (driven by 90% air, 10% CO2 mixture)
through a Millipak-60 filter unit, dispense into 500-ml
autoclaved bottles, and store in the dark at 4°C. The
bottles should be dedicated to tissue culture only and
should be cleaned by thorough rinsing without soap
or detergent. To make up complete growth medium,
add 50 ml of heat-inactivated horse serum and 25 ml of
fetal bovine serum to 500ml of RPMI 1640 medium.
Store complete growth medium at 4°C.
- Medium for freezing of cells: Mix 1 volume of
dimethyl sulfoxide (DMSO) with 9 volumes of complete
growth medium. This medium should be freshly
prepared for immediate use only.
B. Promotion and Assessment of
NGF-Dependent Neurite Outgrowth
Low serum medium
- PC12 cells show optimal adherence to collagencoated
culture dishes. Before applying to dishes,
freshly dilute the stock collagen solution with autoclaved
reverse osmosis/Milli-Q water as noted later.
The optimal final dilution of the collagen should be
determined empirically by testing various concentrations
for their capacities to foster cell attachment and
NGF-promoted neurite outgrowth (Greene et al., 1991).
At too low a dilution, adhesion to substrate is poor,
whereas at too high a concentration, neurite outgrowth
is impeded and cells are difficult to dislodge for subculture.
For application to plates without the necessity
of spreading a thin layer, add the diluted collagen solution
(typically a 1:50 dilution of the stock is optimal)
to plastic tissue cultureware (10 ml/150-mm dish;
5 ml/100-mm dish; 1 ml/35-mm dish; 0.5 ml/well of
24-well culture plates). Leave dishes uncovered to air
dry overnight in a tissue culture hood. Alternatively,
for quicker drying, dilute the collagen by a factor of
five less and add to cultureware in one-fifth the aforementioned
volumes. Spread the collagen evenly over
the surface of the culture dish with the use of an Lshaped
glass rod. Dry collagen by leaving the plates
uncovered for 1-2h in a tissue culture hood. Store
collagen-coated dishes at room temperature and use
within 1 week after preparation.
- Feed PC 12 cells three times a week with complete
growth medium. Remove approximately twothirds
of the culture1 medium from each plate and
replace with fresh complete growth medium (10 ml for
150-mm dishes; 5 ml for 100-mm dishes; 1.5 ml for 35-
mm dishes). The medium should be added gently and
from the side of the tissue culture dish. The feeding
schedule should be kept rigid for maximum cell viability.
Maintain PC12 cells in a 37°C incubator with a
water-saturated, 7.5% COs atmosphere.
- Passage PC12 cells (subcultured) when the cultures
are 80-90% confluent. Dislodge the cells from the
surface of the dish by repeated and forceful discharge
of the culture medium directly onto the cells with a
disposable glass Pasteur pipette. Forceful trituration
of the cell suspension within the Pasteur pipette also
decreases cell clumping. Mix the culture medium containing
detached PC12 cells with fresh complete
medium in a 1:3 or 1:4 ratio. Plate the PC12 cells
onto collagen-coated dishes, and increase the passage
number of the newly plated PC12 cells by one. As the
cell doubling time is 3-4 days, subculture every 7-10
days. To avoid the potential accumulation of variants
within the cultures, experiments should be carried out
with cells that have undergone no greater than 50
- Stock cultures of PC12 cells are frozen at high
density (>5 × 106 cells/ml; see later for cell counting
procedure) as follows. Dislodge cells from tissue
culture dish as described earlier, pellet by centrifugation at room temperature for 10min at 500g, and
remove the medium. Add the appropriate volume of
freezing medium (described earlier) and resuspend the
cell pellet. Aliquot into a Nunc freezing vial (1 ml/vial)
and transfer to a-80°C freezer for at least 1 day. For
high-viability, long-term storage, the vials should be
maintained in liquid nitrogen. The vials should not be
permitted to warm up during the transfer to liquid
nitrogen (e.g., transfer on dry ice).
- Thaw frozen PC12 cell stocks (in freezing vials)
rapidly in a 37°C water bath (2-3min). Immediately
transfer the cells into 9 volumes of complete growth
medium. Pellet the cells by centrifugation at room temperature
for 10min at 500g. Discard the supernatant.
Resuspend the cell pellet in fresh, complete growth
medium and plate cells on collagen-coated dishes.
: Mix 1 ml of heat-inactivated horse
serum per 100 ml of RPMI 1640 medium. Store at
C. Assessment of Survival-Promoting Actions
of NGF and Other Substances
- Dislodge PC12 cells from stock culture dishes
and triturate well using a glass Pasteur pipette to break
up cell clumps. Then plate cells at low densities (5 × 106 cells per 150-mm dish; 1-2 × 106 cells per 100-mm
dish; 2-5 × 105 cells per 35-mm dish; see cell-counting
procedure described later) on collagen-coated dishes
in medium supplemented with NGF (50-100ng NGF
final concentration/ml of medium). Dilute NGF from
the stock into the medium just before use. Because
NGF binds to glass surfaces, use plasticware. Diluted
solutions of NGF are not stable. Although neurite outgrowth
is satisfactory in complete growth medium,
low serum medium is recommended instead in order
to economize on serum as well as to reduce cell clumping.
Once plated, the cultures should be maintained in
a 37~ incubator with a water-saturated, 7.5% CO2 atmosphere and exchange the medium three times per
week as described earlier. Neurite-bearing cells should
be noticeable within 1-3 days of NGF treatment, and
the number of PC12 cells with neurites should increase
progressively with time of NGF exposure. By 7-10
days of treatment, at least 90% of the cells should generate
- To determine the proportions of neurite-bearing
PC12 cells after NGF treatment, observe cultures with
a phase-contrast microscope under high magnification
(e.g., 200×). Within random fields, score the proportions
of cells that possess at least one neurite greater
than 20µm (about two cell body diameters) in length.
Continue counting until the total number of cells
assessed exceeds 100. For consistent results, count only
discernible and/or single cells, but not cell clumps.
To determine mean neurite lengths and rates of
neurite elongation, observe cultures using an eyepiece
equipped with a calibrated micrometer. The latter is
used to measure the entire length of randomly chosen
neurites. At least 20-25 neurites are measured per
- Neurite regeneration experiments are carried out
with NGF-pretreated PC12 cell cultures. This permits
study of rapid neurite growth as well as a quantitative
bioassav for NGE Treat the cells first with NGF for
7-10 days as described in step 1. Then rinse the cultures
five times with medium (without NGF) while the
cells are still attached to the substrate. Mechanically
dislodge the cells from the dish by trituration through
a Pasteur pipette and wash them an additional five
times in medium (without NGF) by repeated centrifugation
at 500g for 10min at room temperature. Plate
the washed cells at low density (about 105 cells/35-mm
dish) in medium (complete or low serum) in the presence
or absence of NGF (see step 1). Examine the
cultures 24h later and score for percentage of
neurite-bearing cells or cell clumps. Because NGFtreated
PC12 cells tend to aggregate, it is often necessary
to score clumps rather than single cells. The
ability of NGF to induce neurite regeneration from
PC12 cells is determined by subtracting the number of
neurite-bearing cells in culture medium without NGF
from the number of neurite-bearing cells in NGFcontaining
culture medium. For well-washed cultures,
80-100% of the cells or cell clumps should regenerate
neurites with NGF, whereas no more than 10-20%
should regenerate neurites in the absence of NGE The
regeneration protocol can be used as a quantitative
bioassay for NGF (Greene et al., 1987).
- Nuclei counting solution stock (Soto and Sonnenschein,
1985): Dissolve 5g ethylhexadecyldimethylammonium
bromide and 0.165g NaCl in 80ml of reverse
osmosis/Milli-Q water. Add 2.8 ml glacial acetic acid
and I drop bromphenol blue. Bring final volume to 100
ml and filter through a 0.45-µm filter unit. Store the
solution at room temperature.
- Working nuclei counting solution (Soto and Sonnenschein,
1985): Mix phosphate-buffered saline (10ml), 10% Triton X-100 (5 ml), 1M MgCl2 (200 µl), and nuclei-
counting solution stock (10 ml) with enough
reverse osmosis/Milli-Q water so that the final volume
is 100ml. Pass through a 0.45µm filter unit.
Store the working nuclei-counting solution at room
D. Cationic Lipid-Based Transfection Protocol
for PC 12 Cells
OptiMEM-I and Lipofectamine2000
- To determine the numbers of PC12 cells suspended
in culture or other medium, pellet the cells by
centrifugation, aspirate to remove the medium, and
resuspend the cells in a known volume of the working
nuclei-counting solution. This solution provides a
homogeneous suspension of intact nuclei, which are
quantified using a hemocytometer. To count cells
attached to a substrate, remove the medium and
replace with a known volume of working nucleuscounting
solution. Resuspend nuclei by trituration and
quantify with a hemocytometer.
- Wash PC12 cells (either naive cells growing with
serum or neuronally differentiated cells growing with
serum and NGF) with serum-free RPMI medium five
times while still attached to culture dishes and then,
after detachment by trituration, wash another five
times in serum-free RPMI medium by centrifugation/
resuspension. Resuspend the cells in RPMI 1640
medium with or without NGF or other potential
trophic agents. Plate the cells in collagen-coated, 24-
well culture plates in 0.5ml of medium (0.5-2 × 105 cells/well). Exchange the serum-free culture medium
three times per week. Carry out cell counts by removing
the medium, adding working nuclei-counting
solution, and counting intact nuclei. Typically, without
trophic substances such as NGF, 50% of the cell die by
24h of serum deprivation and 90% by 3-4 days.
: Both reagents are
purchased from Invitrogen. OptiMEM-I is used for
transfection without antibiotics.
|FIGURE 1 PC12 cells expressing green
fluorescent protein (GFP).
non-NGF-treated (naive) PC12 cells transfected
with plasmid expressing GFP (green), followed
by DAPI staining
(blue). Bar: 50µm.
- As noted in Section I, an advantage of PC12 cells
is that they can be subjected to genetic manipulation.
A relatively high transient transfection efficiency (at
least 20-30%) of PC12 cells is attainable by the use of
lipid-based transfection reagents (Fig. 1). Following
transfection, stable lines of PC12 transfectants can
also be obtained by applying the appropriate selection
pressure (e.g., G418 for mammalian expression plasmid carrying the neomycin resistance gene). The
day before transfection, seed PC12 cells at high density
(at least 90-95% confluency) on collagen-coated tissue
culture plates and maintain in complete growth
- For PC12 cells plated on a 100-mm dish, add 8 µg
DNA to 500µl OptiMEM-I in one tube (tube 1) and
add 20µl Lipofectamine2000 to 500µl OptiMEM-I in
another (tube 2). The contents of tube 2 should be incubated
at room temperature for at least 5min before
dropwise addition to tube 1. After which, incubat the
mixture further at room temperature for 20min.
- During this time, remove complete growth
medium from PC12 cells and replace with 4ml of
- Add the Lipofectamine2000/DNA mixture to the
PC12 cells and return the culture to the incubator. After
4-6 h, aspirate the Lipofectamine2000/DNA mixture
(in OptiMEM-I) off completely and replace with complete
growth medium. Expression of the transgene can
be monitored 24-48 h afterward.
- Note that the amount of DNA and Lipofectamine2000,
as well as the volume of OptiMEM-I, can be
adjusted proportionally to accommodate PC12 cells
that are plated on smaller/larger size tissue culture
plates. The aforementioned procedure can also been
used to transfect PC12 cells that were already differentiated
by NGF treatment. However, the transfection
efficiency is significantly lower (1-2%) due to the need to culture neuronally differentiated PC12 cells at lower
By adhering to the aforementioned protocols, our
laboratory has been able to maintain (since 1977) PC12
cell stocks that are consistently responsive to NGF and
that present a stable phenotype. However, a survey
of the literature concerning the use of PC12 cells
occasionally reveals conflicting or inconsistent results
between laboratories. One possible cause for this may
be the generation of variant "PC12 cell" lines. Like
other continuous cell lines, PC12 cells are subjected to
spontaneous mutations, and clonal PC12 cell variants
have been identified from past studies. The introduction
of nonstandard culture methods (e.g., changing
sera, medium, substrate) can favor the selection of
such variants over the wild-type population. Although
the use of "variant" PC12 cell lines does not necessarily
undermine the validity of data generated with
them, it can give rise to uncertainty or confusion when
one attempts to integrate/reproduce the finding from
various reports. It is therefore our suggestion that a
uniform standard of culturing PC12 cells be adopted
for studies with this cell line.
Another cautionary note on the use of the PC12 cell
line is that although it is a convenient model system
for studying neuronal development and function, it is
not a full substitute for "bona fide" nerve cells. Therefore,
whenever feasible, experimental results obtained
with PC12 cells should be verified or compared with
- Poor survival or growth of stock cultures has
three probable causes: (i) the initial plating density is
too low, (ii) the horse serum is not properly heat inactivated
or is of insufficient quality (the latter is the
usual cause for failure to thrive), and (iii) the culture
medium is outdated (the glutamine has degraded).
- The most probable cause of poor cell adherence
is an insufficient level of collagen as the substrate.
- A poor NGF response is indicated by the continuous
proliferation of PC12 cells in NGF-containing
medium and by the lack of neurite-bearing cells even
after long-term NGF treatment. A possible cause is that
the initial plating density is too high. Another is that
the NGF may be inactive. Alternatively, the collagen
concentration on the dishes is too high or too low or
the collagen has deteriorated. Finally, the cultures may
contain a high proportion of nonresponsive variants
(in this case, start with a new cell stock of lower
- Spontaneously arising PC12 cell variants are
indicated by the presence of flat (phase-dark), rapidly
dividing, non-NGF responsive cells. Alternatively,
contaminating variants may appear spiky in morphology
even in the absence of NGE The best solution to
this is to replace the entire stock with PC12 cells from
an earlier passage and to adopt culture conditions that
do not favor selection of variants.
- More than 50% of PC12 cells in serum-free
medium without NGF should die within 24h after
plating. However, PC12 cells at high density are
capable of conditioning the culture medium, retarding
death. Therefore, if cultures exhibit a delay in serumfree
cell death, the experiment should be repeated with
a lower density of cells. Alternatively, a delay of cell
death could be due to an insufficient washout of serum
or, for neuronally differentiated cultures, of NGE In
this case, a more stringent washing procedure should
- Generally, it is prudent to discard contaminated
cultures and replace with fresh PC12 cell stock.
However, if it is necessary to rescue a nonreplaceable
culture (such as cell line established from transfection
experiments), the following treatments may be effective
in removing common sources of contamination. (i)
Yeast: treat the culture with 1% fungizone (final concentration)
in complete medium. (ii) Mold: remove the
contaminant by aspiration. Alternatively, use a Pasteur
pipette to remove some of the PC12 cells from a small
unaffected area of the dish and replate the cells onto a
new dish. Treat the cells with 1% fungizone in complete
medium. (iii) Bacteria: a combination of antibiotics
and bacterial static agents [see Sambrook et al. (1989) for appropriate doses] may be added to the
culture. PC12 cells can tolerate ampicillin, kanamycin,
spectinomycin, tetracycline, and chloramphenicol.
Greene, L. A., Aletta, J. M., Rukenstein, A., and Green, S. H. (1987).
PC12 pheochromocytoma cells: Culture, nerve growth factor
treatment, and experimental exploitation. Methods Enzymol
Greene, L. A., Sobeih, M. M., and Teng, K. K. (1991). Methodologies
for the culture and experimental use of the PC12 rat pheochromocytoma
cell line. In "Culturing Nerve Cells"
(G. Banker and K.
Goslin, eds.), pp. 207-226. MIT Press
, Cambridge, MA.
Greene, L. A., and Tischler, A. S. (1976). Establishment of a noradrenergic
clonal line of rat adrenal pheochromocytoma cells
which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73
Greene, L. A., and Tischler, A. S. (1982). PC12 pheochromocytoma
cultures in neurobiological research. Adv. Celt. Neurobiol
Kleitman, N., Wood, P. M., and Bunge, R. P. (1991). Tissue culture
methods for the study of myelination. In "Culturing Nerve Cells"
(G. Banker and K. Goslin, eds.), pp 337-377. MIT Press,
Levi, A., and Alemái, S. (1991). The mechanism of action of nerve
growth factor. Annu. Rev. Pharmacol. Toxicol
Levi-Montalcini, R., and Angeletti, P. U. (1968). Nerve growth factor. Physiol. Rev
Mobley, W. C., Schenker, A., and Shooter, E. M. (1976). Characterization
and isolation of proteolytically modified nerve growth
factor. Biochemistry 15
Sambrook, J., Fritsch, E. E, and Maniatis, T. (1989). "Molecular
Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY.
Soto, A. M., and Sonnenschein, C. (1985). The role of estrogen on the
proliferation of human breast tumor cells (MCF-7). J. Steroid