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Serum-free culture conditions for serial subculture of undifferentiated PC12 cells
Journal of Neuroscience Methods 151 (2006) 250–261
Serum-free culture conditions for serial subculture of undifferentiated PC12 cells Kiyoshi Ohnuma a , Yohei Hayashi b , Miho Furue c , Kunihiko Kaneko a , Makoto Asashima b,d,∗ b
a Department of Basic Science, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan c Department of Biochemistry and Molecular Biology, Kanagawa Dental College, Yokosuka, Kanagawa 238-8580, Japan d ICORP project, Japan Science and Technology Corporation (JST), Tokyo, Japan
Received 14 April 2005; received in revised form 24 July 2005; accepted 3 August 2005
Abstract PC12 cells, a widely used model neuronal cell line, are usually cultured in serum-supplemented medium. This report describes a serum-free medium for the culture of PC12 cells. PC12 cells grown in the two media types had similar growth rates and released dopamine in response to high potassium-induced calcium elevation. However, the levels of dopamine and of dopamine release in cells cultured in the serum-free medium were less than 10% of that in cells cultured in serum-supplemented medium. Dopamine levels recovered within 10 days if cells were returned to serum-supplemented medium, but dopamine release could not be recovered. Nerve growth factor (NGF) induced similar responses in PC12 cells cultured in both media, including phosphorylation of extracellular signal-regulated protein kinases and neurite extension. Transferrin was necessary for survival of neurite-bearing PC12 cells subcultured in serum-free medium and insulin promoted the cells proliferation. Ten days culture with NGF produced a similar increase in neurofilament expression and acetylcholinesterase activity in both media. These results suggest that PC12 in the hormonally defined serum-free media are qualitatively the same as those cultured in serum-supplemented media, and therefore this new culture protocol should enable more precise studies of PC12 cells culture in the absence of confounding unknown factors. © 2005 Elsevier B.V. All rights reserved. Keywords: PC12; Hormonally-defined serum-free medium; Dopamine; Extracellular signal-regulated protein kinase (ERK); Neurofilament; Acetylcholinesterase; Heparin; Flow cytometry
1. Introduction The rat adrenal pheochromocytoma PC12 cell line (Greene and Tischler, 1976) is widely used as a model neuronal cell line because the cells behave like neural progenitor cells: they proliferate in growth media, but stop proliferating and differentiate into sympathetic neuron-like cells following treatment with nerve growth factor (NGF) (Greene and Tischler, 1982). PC12 cells are also used to study signal transduction, differentiation, survival and proliferation mechanisms as they respond to many growth factors, neurotrophins and hormones (Greene and Tischler, 1976; Rukenstein et al., 1991; Togari et al., 1985). PC12 cells also provide a valuable model for studying the mechanism of synthesis and secre∗
Corresponding author. Tel.: +81 3 5454 6632; fax: +81 3 5454 4330. E-mail address: [email protected] (M. Asashima).
0165-0270/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2005.08.004
tion of neurotransmitters such as acetylcholine, dopamine and norepinephrine (Greene and Rein, 1977b; Greene and Tischler, 1976; Lucas and Kreutzberg, 1985; Schubert et al., 1977). PC12 cells are usually subcultured in medium supplemented with serum (Greene et al., 1998; Greene and Tischler, 1976). Serum may contain a wide range of growth factors, differentiation factors catecholamines, and other unknown factors that cannot be easily defined and may lead to unpredictable cell characteristics (Furue et al., 2005). For this reason, experiments using PC12 cells are often performed after and/or under short-term culture in serum-free medium to exclude undefined serum effects. Temporary serum withdrawal does not completely abrogate the effects of serum as undefined serum effects can persist for several passages (Davis, 2002). Another disadvantage of temporary serum withdrawal is the induction of other side effects and cell
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death that can occur within a few days (Greene, 1978). The present study describes the development of a novel hormonally defined serum-free medium (HD medium) that maintains PC12 cells characteristics following withdrawal of serum and loss of undefined serum effects. Previous studies have confirmed that PC12 cells can be cultured in an undifferentiated state for about 10 days in HD medium containing N2 supplements that was developed for the rat neuroblastoma B104 cell line (Bottenstein, 1984; Bottenstein and Sato, 1979). Similarly, differentiated PC12 cells can be maintained in HD medium in the presence of NGF for more than 1 month (Greene, 1978; Hatanaka, 1981; Skaper et al., 1983). Culture conditions for the serial subculture of undifferentiated PC12 cells in HD medium remain undefined (Greene et al., 1998; Greene and Tischler, 1982). The present study addresses this problem and describes the development of an HD medium for the serial subculture of PC12 cells. PC12 cells were cultured until the eighth passage, over more than 6 weeks, in HD medium after withdrawal of serum, and the characteristics of these cells (PC12-HD cells) were compared with cells subcultured in the control serum-supplemented medium (PC12-SS cells).
2. Materials and methods 2.1. Cell culture PC12 cells were obtained from the Riken Cell Bank (Cat #: RCB0009, Ibaragi, Japan). The cells were cultured in serum-supplemented medium (SS medium) or HD medium in a humidified incubator containing 5% CO2 at 37 ◦ C. The SS medium consisted of RD medium (Davis, 2002; Furue et al., 1994; Sato et al., 1987) supplemented with 10% heat-inactivated horse serum donor herd (Invitrogen Corporation, Carlsbad, CA, USA), and 5% heat-inactivated qualified fetal bovine serum (Invitrogen). The RD medium is an equal mixture of RPMI 1640 and DMEM medium, containing 2 g/l sodium bicarbonate, 15 mM HEPES, 5 mM l-glutamine, 10 nM sodium selenite, 110 mg/l sodium pyruvate, 3.25 g/l d-glucose and 100 mg/l kanamycin sulfate (Custom-made, Research Institute for the Functional Peptides, Yamagata, Japan). Sixty-millimeter plastic culture dishes (BD bioscience, San Jose, CA, USA) were coated with 30 g/ml type I collagen (Cellmatrix Type I-A, Nitta Gelatin, Inc., Osaka, Japan) in PBS for at least 30 min at room temperature (21–26 ◦ C) and then rinsed with PBS. For subculturing, the cells were detached from the culture dish by forceful aspiration of the medium through a plastic pipette (Greene et al., 1998). The cells were subcultured every 5–7 days and the medium was changed every 2–3 days. For storage, the cells were frozen in growth medium with 10% (v/v) dimethylsulfoxide (DMSO) (Greene et al., 1998). PC12 cells serially subcultured in this condition were referred to as PC12-SS cells.
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Table 1 Supplements for serum-free culture of the PC12 cells Supplement
HD proliferation Medium
HD neurite outgrowth medium
Insulin (I-5500a ) Apo-transferrin (T-1147a )
30 g/ml 10 g/ml
– 10 g/ml
3F
1 unit 10 nM 10 M 10 M
1 unit 10 nM 10 M 10 M
500 g/ml –
500 g/ml 100 ng/ml
Sodium selenite (S-9133a ) Ethanolamine (E-0135a ) -Mercaptoethanol (M-7522a ) BSA (A-6003a ) NGFb
The basal medium is RD medium. 3F is a mixture of sodium selenite, ethanolamine and -mercaptoethanol. a Sigma catalog number. b NGF 2.5S, mouse, Upstate, Waltham, MA, USA.
HD proliferation medium consisted of RD medium supplemented with bovine serum albumin (BSA), 3F (sodium selenite, ethanolamine, -mercaptoethanol), insulin and apotransferrin (Table 1). Incorporation of these constituent factors into the HD medium is based on reports from previous studies (Bottenstein, 1984; Click et al., 1972; Davis, 2002; Furue et al., 1994; Sato et al., 1987). These factors were added to the RD medium immediately prior to use from 100-fold concentrated stock solutions. Culture dishes were coated with 3 g/ml type IV collagen (mouse, Cat #: 354233, BD bioscience,) in PBS for at least 30 min at room temperature and then rinsed with PBS. Cell culture procedures were the same as described above. The PC12 cells serially subcultured for 8–15 passages over 2–3 months under these conditions were referred to as PC12-HD cells. The maintenance of neurite-bearing PC12 cells for more than 10 days without subculture was achieved by culturing the cells in HD neurite outgrowth medium (Table 1). 2.2. Cell counting The tendency of PC12 cells to form clumps, even after extensive trituration, can lead to errors in cell counting. To assess cell numbers accurately, the cells were solubilized in nuclear counting solution contained 5 mg/ml ethylhexadecyldimethylammonium bromide, 0.165 mg/ml NaCl, 0.28% (v/v) acetic acid, 0.5% (v/v) Triton X-100, 2 mM MgCl2 and 10% (v/v) PBS (Greene et al., 1998; Soto and Sonnenschein, 1985), and the intact nuclei were counted using a particle counter (Coulter counter Z-1, Beckman Coulter, Inc.). Using the plateau calibration methods described in the user manual of the Coulter counter, the lower limit size setting for PC12 cell nuclei was measured to be 4 m. 2.3. Dopamine assay Depolarization-induced dopamine release and intracellular dopamine content were quantified as described previously (Aoyagi and Takahashi, 2001). Cells cultured in 60-mm plas-
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tic dishes were rinsed three times with normal bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2 , 1 mM MgCl2 , 10 mM d-glucose, 1 mg/ml BSA and 10 mM HEPES–Na at pH 7.4, and incubated in 1.4 ml of normal bath solution or high potassium bath solution for 15 min at room temperature. The high potassium bath solution contained 95 mM NaCl, 50 mM KCl, 2 mM CaCl2 , 1 mM MgCl2 , 10 mM d-glucose, 1 mg/ml BSA and 10 mM HEPES–Na at pH 7.4. The incubated bath solution was then added to solution containing 0.35 ml of 0.1N perchloric acid and 0.5 mM EDTA and stored at −80 ◦ C. The intracellular dopamine content was measured by first solubilizing the cells in 40 mM perchloric acid and 20 M EDTA followed by sonication and centrifugation at 12,600 rpm for 15 min at 4 ◦ C. Dopamine in the supernatant was quantified using HPLC-DPE methods (SRL, Inc., Tokyo, Japan). 2.4. Intracellular calcium imaging PC12-HD or PC12-SS cells were inoculated on glassbased dishes (Iwaki, Tokyo Japan) coated with 30 g/ml type IV or 3 g/ml type I collagen in HD proliferation medium or SS medium, respectively, and cultured for 3 days. After the cells were rinsed with normal bath solution, the cells were incubated in calcium green-1 loading solution consisting of 5 M calcium green-1 AM (Molecular Probes, Inc., Eugene, OR, USA), 0.02% (v/v) cremophor EL (Nacalai tesque, Inc., Kyoto, Japan), 0.5% (v/v) DMSO in normal bath solution for 1 h at room temperature. Cremophor EL, a non-ionic surfactant, was used to facilitate calcium green-1 AM solubilization. After mounting the glass-based dish onto the stage of an upright IX70 microscope (Olympus, Tokyo, Japan), the chamber was perfused with normal bath solution at 30 ◦ C. The single-wavelength fluorescence indicator calcium green-1 was excited with light at 480 nm and the resulting fluorescence images at wavelengths greater than 510 nm were acquired using an monochrome cooled CCD camera 4920 (Cohu, Inc., San Diego, CA, USA) and LG3 frame grabber (Scion Corporation, Frederick, ML, USA). Using a U-shaped tube (Fenwick et al., 1982) with gravity feed and manual switching valve, high potassium bath solution was pulsed over the cells. Background signal (large white rectangles at right side of images in Fig. 3D) was subtracted from each region of interest (small white rectangles in Fig. 3D). Relative fluorescent change (F − F0 )/F0 , were calculated, where F is fluorescence intensity and F0 is initial fluorescence intensity before puffing of high-potassium bath solution. 2.5. Flow cytometry analysis The cells were harvested using calcium- and magnesiumfree Dulbecco’s phosphate-buffered saline (PBS) containing 1 mg/ml BSA (BSA–PBS) and then fixed for 15 min with 2% paraformaldehyde at 4 ◦ C, followed by 15 min in cold 70% ethanol in PBS. The cells were then rinsed with PBS
and incubated for 30 min in blocking buffer consisting of 10 mg/ml BSA and 0.2% Triton X-100 in PBS. Cells were incubated for 30 min at room temperature with primary antibodies diluted in the blocking buffer. After two rinses with BSA–PBS, cells were incubated for 30 min at room temperature with secondary antibodies diluted in blocking buffer. The primary antibodies used included mouse anti-extracellular signal-regulated protein kinase (ERK) monoclonal antibody (Pan ERK, 1 g/ml, BD Bioscience), mouse anti-ERK1/2 (pT202/pY204) phospho-specific monoclonal antibody (1 g/ml, BD Bioscience) and rabbit anti-neurofilament 68kDa polyclonal antibody (1/1000 dilution, Chemicon International, Inc., Temecula, CA, USA). The secondary antibodies used included FITC-conjugated goat anti-mouse IgG (H + L) FITC (2 g/ml dilution, Beckman Coulter), and FITC conjugated donkey anti-rabbit IgG (H + L) (1 g/ml, Rockland, Inc., Gilbertsville, PA, USA). Flow cytometry analysis of immunofluorescently labeled cells was performed using an EPICS ALTRA (Beckman Coulter, Inc., Miami, FL, USA). 2.6. Acetylcholinesterase (AChE) activity AChE activities were measured as previously described (Ellman et al., 1961; Rieger et al., 1980). Briefly, the cells were homogenized in solubilization buffer containing 10 mM Tris–HCl (pH 7.2), 1 M NaCl, 50 mM MgCl2 and 1% (v/v) Triton X-100. Acetylthiocholine at 0.5 mM was used as a substrate. The rate of absorbance change, which is proportional to the rate of acetylthiocholine hydrolysis by extracted AChE, was measured at 412 nm in a photometer.
3. Results 3.1. HD proliferation medium for serial subculture of PC12 cells To develop an HD proliferation medium for PC12 cells, we examined the effects of insulin, transferrin and 3F (sodium selenite, ethanolamine, -mercaptoethanol) on the proliferation of PC12 cells. Cell numbers were counted after 6 days of culture in RD medium supplemented with these factors (Fig. 1). Maximum growth of the PC12 cells was achieved in RD medium supplemented with 30 g/ml insulin, 3–30 g/ml transferrin and 1 unit of 3F (10 nM sodium selenite, 10 M ethanolamine, 10 M -mercaptoethanol). The addition of BSA to medium is known to be beneficial to the proliferation and maintenance of cells in culture as BSA protects cells from mechanical stress (Davis, 2002). The ability of BSA to improve PC12 cells harvesting efficiency was assessed by comparing cell numbers following rinse and trituration in the presence and absence of BSA. We found that the number of cells was greater when cells were rinsed and triturated in the presence of BSA than without (Fig. 1B). This result suggests that BSA does protect PC12 cells from mechanical stress during the passage procedure and should
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Fig. 1. Proliferation and morphology of PC12-SS cells in HD medium. (A) PC12-SS cells were cultured for about 18 h in fresh basal RD medium without any supplements for serum starvation before the experiments, and then inoculated onto type I collagen-coated 24-well plates at a cell density of 104 cells/cm2 . The culture conditions were RD medium supplemented with 0–30 g/ml insulin (Ins), 0 (open symbol) or 1 unit (closed symbol) of 3F and 0–300 g/ml apo-transferrin (Abscissa). The cells were counted after 6 days of culture (n = 3, mean ± S.E.). (B) The effect of BSA on harvesting efficiency. PC12-HD cells at a cell density of 5 × 104 cells/cm2 were inoculated onto a 3 g/ml type IV collagen-coated 12-well plate in HD proliferation medium and cultured 4 days. The cells were rinsed three times and harvested using complete HD proliferation medium (BSA+) and HD proliferation medium without BSA (BSA−). ** Statistically significant at P < 0.001, t-test (n = 4, mean ± S.E.). (C–I) Phase-contrast photomicrographs of PC12 cells. (C) PC12-SS cells were inoculated in SS medium onto dishes coated with 30 g/ml type I collagen. (D–I) PC12-SS cells were cultured in HD proliferation medium on dishes coated with 30 g/ml type I collagen for 6 days and subcultured for one day in HD medium on dishes coated with 30 (D) or 300 (E) g/ml type I collagen, with 3 (F) or 30 (G) g/ml type IV collagen or with 10 (H) or 100 (I) g/ml poly-l-lysine. Insets show high magnification views of the cells (arrow). Scale bar = 20 m.
be included in the medium. Taken together, these results were used to determine the most efficient composition of the HD proliferation medium (Table 1). 3.2. Coating conditions for HD medium After a few subcultures in HD proliferation medium in a type I collagen-coated dish, the cells were smaller and brighter by phase-contrast microscopy than cells cultured in SS medium (Fig. 1B and C). This observation indicates that the cells were rounded when grown in HD proliferation medium but became more spread out in SS medium. This
morphological difference suggests that the cells attached only weakly to the type I collagen-coated dish in HD proliferation medium and were easily detached from the dish during culture. Previous studies (Turner et al., 1987; Vlodavsky et al., 1982) found that type IV collagen as a substrate produced a higher percentage of PC12 cells that were more spread out and more firmly attached than did type I collagen as a substrate. PC12 cells are also known to attach well to poly-l-lysine-coated dishes in SS medium (Greene et al., 1998). In the present study, we tested type IV collagen and poly-l-lysine as a coating substrate. PC12-SS cells that had been subcultured twice in HD proliferation
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medium were inoculated in HD proliferation medium onto 30 or 300 g/ml type I collagen-coated dishes, 3 or 30 g/ml type IV collagen-coated dishes and 10 or 100 g/ml polyl-lysine-coated dishes. Cells were cultured for 1 day and phase-contrast micrograph images were taken. On dishes coated with 30 or 300 g/ml type I collagen, cells were small and bright (Fig. 1D and E). Cells were also small and bright on poly-l-lysine-coated dish (Fig. 1H and I). By comparison, the cells cultured on type IV collagen-coated dishes were dark and spread out, even when the concentration of collagen was decreased to 3 g/ml (Fig. 1F and G), and cells had a morphology similar to PC12-SS cells on 30 g/ml type I collagen-coated dishes (Fig. 1C). These results suggest that in HD proliferation medium, PC12 cells attach more firmly on dishes coated with 3 or 30 g/ml type IV collagen than on dishes coated with 30 or 300 g/ml type I collagen or 10 or 100 g/ml poly-l-lysine (see also Supplementary data). Based on these observations, 3 g/ml type IV collagen was
chosen as the most appropriate substrate for coating dishes for the serial subculture of PC12 cells in HD proliferation medium. 3.3. Growth of PC12-HD and PC12-SS cells Cells could be successfully serially subcultured for more than eight passages over about 2 months in HD proliferation medium on dishes coated with 3 g/ml type IV collagen. We refer to these cells as PC12-HD cells. The PC12-HD cells spread well and were an equivalent size to PC12-SS cells under phase-contrast microscopy (Fig. 2B and C). To assess growth curves for the PC12-HD and PC12-SS cells, cell counts were performed every 2 days (Fig. 2A). Cells grown under both conditions were in log-growth phase within 21 h. The doubling times of PC12-HD cells and PC12-SS cells were 35.2 ± 2.0 and 33.2 ± 0.8 h, respectively (P > 0.05, ttest). The cell density of the PC12-HD cells was 70–87% less
Fig. 2. Cell growth of PC12-HD and PC12-SS cells. (A) Growth curve of PC12-HD cells (open circle, dotted line) and PC12-SS cells (closed circle, solid line). PC12-HD cells and PC12-SS cells at a cell density of 2 × 104 cells/cm2 were inoculated onto a 3 g/ml type IV collagen-coated 24-well plate in HD proliferation medium, and onto a 30 g/ml type I collagen-coated 24-well plate in SS medium, respectively. The cells were counted at 4 and 21 h after plating and every 48 h thereafter (n = 5, mean ± S.E.; most error bars cannot be seen due to small S.E.). (B and C) Phase-contrast photomicrographs of PC12-HD cells (B) and PC12-SS cells (C) on the fifth day. Scale bar = 50 m.
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than that of the PC12-SS cells at every data point (P < 0.01, ttest). These results indicate that the initial number of attached PC12-HD cells was less than for the PC12-SS cells, but the growth rates of both cells were the same. 3.4. Dopamine release from PC12-HD and PC12-SS cells PC12 cells are known to synthesize, store and release dopamine (Greene and Rein, 1977a; Greene and Tischler, 1976). To examine whether PC12-HD cells retain this characteristic, we determined the amount of released dopamine in PC12-HD and PC12-SS cells. Three days after subculturing, both cell types were depolarized using a high-potassium (50 mM) bath solution to elicit dopamine release, which was quantified using HPLC. The PC12-HD cells released a significantly increased amount of dopamine in response
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to high potassium compared to the same cells incubated in a solution containing normal levels (5 mM) of potassium (P < 0.01, t-test; Fig. 3A). This result indicates that PC12HD cells not only release dopamine, but also synthesize and store dopamine because the HD medium does not contain dopamine. Although PC12-HD cells can release dopamine, the level of dopamine released from these cells is about 200-fold lower than the level released from PC12-SS cells (Fig. 3B). We then proceeded to ascertain the underlying mechanism leading to the decrease in the ability of PC12-HD cells to synthesize, store or release dopamine. We initially examine the elevation of intracellular calcium followed by membrane depolarization as elevation of intracellular calcium triggers exocytosis. The fluorescent calcium indicator, calcium green1 AM, was loaded into the cells and the high potassium bath solution was applied to the cells. In both PC12-HD and
Fig. 3. Dopamine release: (A–C) the absolute level (A) and the relative level of dopamine based on A (B, C). The PC12-HD cells (open column), PC12-HD cell cultured 10 days in SS medium (gray column) and PC12-SS cells (closed column) were incubated in a high-potassium bath solution (high K+ , 50 mM KCl) or a normal bath solution (Nrm, 5 mM KCl) for 15 min at room temperature. These cells were also solubilized to measure intracellular dopamine content (content) n = 3, mean ± S.E. Statistically significant at * P < 0.01, ** P < 0.001 and *** P < 0.0001, t-test. (B) Relative level of dopamine. Each absolute level was normalized according to the corresponding absolute level in PC12-SS cells. (C) Releasable dopamine is the percentage of dopamine released by high-potassium from the cellular dopamine level. (D and E) Intracellular calcium change evoked by a 10-s exposure to a high-potassium bath solution (horizontal bar). (D) Time course of relative fluorescence change of calcium green-1 and images (inset) in PC12-HD (upper) and PC12-SS (lower) cells. (E) Maximum fluorescence change, n = 21 (seven regions of interest in three dishes), mean ± S.E.
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PC12-SS cells, fluorescence rapidly increased then decreased following a 10 s application of high potassium bath solution (Fig. 3D). The maximum change in fluorescence for both cell types was almost the same (Fig. 3E) indicating that a high potassium level elicits an intracellular calcium rise in both cell types. We can conclude that elevation of intracellular calcium elicited by membrane depolarization is maintained in PC12-HD cells. The intracellular dopamine level of PC12-HD cells was then examined. The level of dopamine in PC12-HD cells was 10% the level in PC12-SS cells (Fig. 3B). The level of releasable dopamine in PC12HD cells, expressed as the percentage of cellular dopamine levels released by depolarization, was 0.7%, which is about 20-fold less than that observed for PC12-SS cells (Fig. 3C). These results indicate that both the ability to synthesize and/or store dopamine and ability to release dopamine following calcium elevation is provided by factors present in the serum.
3.5. Dopamine release from PC12-HD cells cultured 10 days in SS medium To clarify the effect of serum on dopamine related characteristics, PC12-HD cells were cultured for 10 days in SS medium and the intracellular dopamine content and the amount of released dopamine were measured. The intracellular dopamine level and the level released in normal and in high potassium bath solutions, increased after 10 days culture in SS medium (Fig. 3A). The relative increase in intracellular dopamine from 10 to 60% following culture of cells in SS medium (Fig. 3B) strongly suggests the presence of factors in the serum to enhance dopamine synthesis and/or storage. By comparison, the proportion of releasable dopamine did not recover after 10 days culture in SS medium (Fig. 3C). Preliminary experiments showed that the absolute level of dopamine released from PC12-SS cells cultured temporally for 1–2 weeks in HD proliferation medium decreased to 10%,
Fig. 4. Flow cytometry analysis of the effect of NGF on ERK expression and its phosphorylation in PC12-HD and PC12-SS cells. PC12-HD (A and B) and PC12-SS (C and D) cells treated with BSA–PBS (CNT, dotted line) or 100 ng/ml NGF in BSA–PBS (solid line) for three minutes at 37 ◦ C were analyzed using non-specific anti-ERK antibody, phospho-specific anti-ERK 1/2 antibody. Abscissas represent fluorescence intensity corresponding to non-specific anti-ERK antibody (A and C) and phospho-specific anti-ERK1/2 antibody (B and D). The arrows indicate the range of positive cells.
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but recovered to more than 60% after culture in SS medium for 1 week (see Supplementary data). These results suggest that the decrease in releasable dopamine by temporal culture in HD proliferation medium is recoverable. There is a possibility that long-term serial subculture in HD proliferation medium causes clonal drift in cells. 3.6. Short-term treatment with NGF enhances ERK phosphorylation Previous studies reported PC12 cells as a good model for studying NGF-related signaling through receptor tyrosine kinase A (Trk A), Raf, mitogen-activated protein kinase kinase (MEK) and ERK (for review, see Vaudry et al., 2002). ERK phosphorylation induced by NGF occurs within minutes with the maximal level reached by 5 min (Lazarovici et al., 1998; Peraldi et al., 1993). To determine ERK expression and phosphorylation in PC12-HD and PC12-SS cells treated with
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NGF, the distribution of cells that were immunopositive for non-specific ERK (pan-ERK) antibody and phospho-specific ERK 1 and 2 (ERK 1/2) antibody was measured using flow cytometry (Fig. 4). Approximately 90% of both PC12-HD and PC12-SS cells were immunoreactive for pan-ERK, and the percentage did not change following NGF treatment. By comparison, less than 3% of both cell types were positively labeled for phospho-specific ERK1/2 prior to treatment with NGF and this percentage increased to 90% with 3 min of NGF treatment. These results indicate that ERK1/2 phosphorylation was promoted by NGF in PC12-HD cells to the same extent as in PC12-SS cells, suggesting that these signaling pathways were retained under the HD culturing conditions. 3.7. Extension of neurites following NGF treatment PC12 cells have been shown to differentiate into sympathetic neuron-like cells with neurite extension in response
Fig. 5. Neurite extension and cell survival of PC12-HD cells by NGF-treatment. PC12-HD at the cell density of 2 × 104 cells/cm2 were inoculated onto a 3 g/ml type IV collagen-coated 24-well plate in complete HD proliferation medium (A, E and I), without insulin (B, F, J and M), without transferrin (C, Q and K), and without insulin and transferrin (D, H and L) supplemented with 100 ng/ml NGF (E–M). Phase-contrast photomicrographs taken at day 4 (A–H), day 10 (I–L) and day 32 (M) are shown. Scale bar = 50 m.
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to NGF in SS growth medium, basal medium without any supplement and basal medium with insulin and/or transferrin (Greene, 1978; Greene and Tischler, 1976; Hatanaka, 1981; Skaper et al., 1983). To examine neurite outgrowth from PC12-HD cells, 100 ng/ml NGF was added to complete HD proliferation medium or HD proliferation medium without insulin and/or transferrin and the morphological changes were monitored by phase-contrast microscopy. Neurites began to extend from the cells within 4 days in all media (Fig. 5E–H) and more than half of the cells showed neurite extension after 10 days (Fig. 5I–L). The results suggest that PC12-HD cells maintain the ability to extend neurites. 3.8. Proliferation and cell survival following NGF treatment The proliferation of PC12 cells is inhibited by NGF (Greene and Tischler, 1976). To examine the effect of NGF on PC12-HD cell proliferation, the cells were inoculated in HD proliferation medium supplemented with various concentrations of NGF for 10 days, and then the cells were counted. NGF inhibited PC12-HD cell proliferation in a dosedependent manner (Fig. 6). It is also known that NGF supports PC12 cell survival (Greene, 1978). To examine the effect of NGF on the survival of PC12-HD cells, various concentrations of NGF were added to the HD proliferation medium without insulin and/or transferrin, which do not support cell proliferation (Fig. 1A), and photomicrographs were taken on culture days 4 and 10
(Fig. 5). Cell numbers were counted on culture day 10 (Fig. 6). By day 4, in the absence of NGF and insulin or transferring, almost all cells were shrunken and had detached from the dish (Fig. 5B–D). By comparison, in the presence of 100 ng/ml NGF, the cells were attached to the dish and had spread (Fig. 5F–H), suggesting that NGF supported PC12-HD cell survival for at least 4 days without insulin and transferrin. In medium with transferrin but without insulin, the number of cells increased with increasing doses of NGF (Fig. 6), and most cells were still viable at day 10 in the presence of 100 ng/ml NGF (Fig. 5J). In the absence of transferrin, most cells were shrunken and had detached from the dish by day 10 (Fig. 5K and L), and the cell densities of the PC12-HD cells were less than the initial plating density irrespective of the presence of the insulin and NGF (Fig. 6). These results strongly suggest that NGF can support PC12-HD cell survival for at least 4 days without insulin and transferrin, and for 10 days with transferrin. In medium with 100 ng/ml NGF and transferrin but no insulin, PC12-HD cell proliferation almost ceased and the cells remained viable with long neurites for more than 1 month (Fig. 5M). In the presence of 1–100 ng/ml NGF, cell numbers in the medium with transferrin and insulin were higher than in medium with transferrin and no insulin. These results suggest that transferrin is necessary for survival of neurally differentiated PC12-HD cells and that insulin promotes cell proliferation. As most neuronal cells do not proliferate after differentiation into neurons, medium containing NGF and transferrin, but not insulin may be suitable for studying the characteristics of differentiated PC12-HD cells. We refer to this medium as HD neurite outgrowth medium (Table 1) and used it for analysis of differentiated PC12-HD cells. 3.9. Neurofilament expression and AChE activity in undifferentiated and neuronally differentiated PC12 cells
Fig. 6. The effect of NGF on PC12-HD cell growth and survival. PC12-HD at the cell density of 2 × 104 cells/cm2 were inoculated onto a 24-well plate coated with 3 g/ml type IV collagen in complete HD proliferation medium (closed rectangle), medium without insulin (closed triangle), medium without transferrin (open rectangle), medium without both insulin and transferrin (open triangle) supplemented with various concentration of NGF. The cells were counted after 10 days of culture. The arrow and the arrowhead correspond to the HD proliferation medium and the HD neurite outgrowth medium (Table 1), respectively (n = 4, mean ± S.E.; most error bars cannot be seen due to small S.E.).
To confirm that the PC12-HD cells underwent differentiation following NGF treatment, neurofilament (NF) and AChE were measured, both of which are known to increase during culture in NGF-containing medium (Lee and Page, 1984; Rieger et al., 1980). NF expression and AChE levels in PC12-HD and PC12-SS cells cultured in the HD neurite outgrowth medium for 10 days were compared to those of control groups, which were PC12-HD and PC12-SS cells cultured in HD proliferation medium and SS medium, respectively (Fig. 7). The percentage of NF-immunoreactive cells was measured using flow cytometry (Fig. 7A–C), and the AChE activity was analyzed in cell lysates (Fig. 7D). The peak position (mode) of NF-positive (+) cells and the percentage of NF-strong positive (++) cells for both PC12-HD and PC12-SS cells cultured in the HD neurite outgrowth medium were larger than those of the control group (Fig. 7A–C). These results indicate that the expression of NF was increased in both PC12-HD and PC12-SS following treatment with NGF. The percentage of NF-positive cells (+)
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Fig. 7. The effect of NGF on NF expression and AChE activity. PC12-HD and PC12-SS cells were cultured in HD proliferation medium or SS medium, respectively, for 6 days (proliferation: dotted line in (A and B), open column in (C and D)), or both cells were cultured in HD neurite outgrowth medium for 10 days (Neurite: solid line in (A and B), filled column in (C and D)). Flow cytometry analysis of NF-immunoreactive cells was performed on PC12-HD (A) and PC12-SS (B) cells using NF antibody and second antibody (thick line) and second antibody only (control, thin line). The arrows indicate the range of NF-positive (+) and NF-strong positive (++) cells. The arrowhead represents a weak positive peak. (C) The percentage of NF-positive (+) and strong positive (++) cells based on (A and B). * Statistically significant at P < 0.00002, Z test. (D) AChE activity of PC12-HD and PC12-SS cells. AChE activity is expressed in nanomoles acetylthiocholine hydrolyzed per minute per 106 cells. * Statistically significant at P < 0.01, t-test (n = 4, mean ± S.E.).
was decreased in the PC12-HD cells cultured in HD neurite outgrowth medium (Fig. 7C) due to an increase in the number of weak positive cells (arrowhead in Fig. 7A). These weakly positive cells were not seen in the PC12-SS cultures (Fig. 7B), although the cells were also cultured in HD neurite outgrowth medium for 10 days. The DNA content of the NF-weak positive cells measured using propidium iodide was smaller than expected for the peak position of G1 phase-cells (data not shown), suggesting that the NF-weak positive cells might correspond to dying cells. Together, these results suggest that serum contains some cell survival factors that affect cell viability for at least 10 days after serum withdrawal. The AChE activity of the PC12-HD cells was significantly increased after 10 days culture in the HD neurite outgrowth medium (Fig. 7D; P < 0.01, t-test), indicating an NGF-induced increase in the synthesis of AChE. However, the absolute amount of AChE in the PC12-HD cells was 40% less than that in the PC12-SS cells. It is possible that the NF-
weak positive cells have reduced synthesis of AChE or there may be some factors in the serum that act synergistically with NGF to synthesize AChE.
4. Discussion This is the first report of a hormonally defined serum-free medium developed for the characterization and serial subculture of PC12 cells. Serial subculture in the HD medium did not change the rate of growth of these cells. We also showed that the PC12 cells grown in the HD medium retain their ability to synthesize, store and release dopamine following a rise in intracellular calcium (Greene and Rein, 1977a; Greene and Tischler, 1976). PC12 cells normally exhibit NGF-induced signaling cascades including phosphorylation of ERK, and as the result of such activation, the cells differentiate into sympathetic neuron-like cells (Greene and Tischler, 1976, 1982;
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Vaudry et al., 2002). This study demonstrated that PC12-HD cells exhibit neurite extension, growth inhibition, promotion of survival, elevation of NF expression and increased AChE activity in response to NGF. Thus, PC12-HD appears to retain the features of control PC12-SS cells. Although PC12-HD and PC12-SS cells were qualitatively the same, the cells were quantitatively different. PC12-HD cells released less dopamine compared with PC12-SS cells and those cultured in HD neurite outgrowth medium contained a population of NF-weak positive cells and synthesized less AChE. It is possible that these differences result from the clonal drift of cells cultured in HD medium. Despite the clonal drift of cells cultured in HD medium, this medium is useful to study mechanisms like the molecular basis of exocytosis (Corradi et al., 1996; Shoji-Kasai et al., 2001). The ability to recover the intracellular dopamine level of PC12-HD cells from 10 to 60% by culturing these cells in SS medium, suggests that some factors in serum, such as growth factors and hormones, appear to be necessary for developing these specific cellular characteristics. For instance, our preliminary experiment showed that addition of 30 g/ml insulin or 1 g/ml heparin into the HD neurite outgrowth medium reduced the NF-weak positive peak and increased AChE activity (see Supplementary data) and these factors are known to support the NGF effects on PC12 cells (Hatanaka, 1981; Neufeld et al., 1987; Skaper et al., 1983). Uncovering the importance of various supplements is possible when cells are cultivated in hormonally defined serum-free medium. The HD medium is also useful for analyzing and quantifying low levels of material synthesized by and secreted from the PC12 cells. In summary, we have developed a hormonally defined serum-free medium for the culture of PC12 cells, which maintains all the important characteristics of PC12 cells grown in serum-supplemented medium. By removing a number of confounding unknown factors that may be present in serum, this will facilitate more precise studies of PC12 cells. These cells are increasingly being used to test new materials (Okuyama et al., 2003), and have been used in brain implants to treat Parkinson’s disease (Yoshida et al., 2003). Therefore, this new culture protocol may, in addition to advancing cell biology including biophysics and bioengineering studies, contribute to clinical advances in the treatment of neurological diseases.
Acknowledgements This work was supported by a research fellowship (1383203) from the Japan Society for the Promotion of Science for Young Scientists, a 21st century COE grant from the Japan Society for the Promotion of Science. The authors would like to thank Mr. Yasuhiko Nagasaka from Beckman Coulter, Inc. for his technical assistance and helpful advice regarding flow cytometry, and Dr. Kyota Aoyagi from Kitazato University for his helpful advice regarding dopamine assay.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jneumeth. 2005.08.004.
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Serum-free culture conditions for serial subculture of undifferentiated PC12 cells Kiyoshi Ohnuma a , Yohei Hayashi b , Miho Furue c , Kunihiko Kaneko a , Makoto Asashima b,d,∗ b
a Department of Basic Science, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan c Department of Biochemistry and Molecular Biology, Kanagawa Dental College, Yokosuka, Kanagawa 238-8580, Japan d ICORP project, Japan Science and Technology Corporation (JST), Tokyo, Japan
Received 14 April 2005; received in revised form 24 July 2005; accepted 3 August 2005
Abstract PC12 cells, a widely used model neuronal cell line, are usually cultured in serum-supplemented medium. This report describes a serum-free medium for the culture of PC12 cells. PC12 cells grown in the two media types had similar growth rates and released dopamine in response to high potassium-induced calcium elevation. However, the levels of dopamine and of dopamine release in cells cultured in the serum-free medium were less than 10% of that in cells cultured in serum-supplemented medium. Dopamine levels recovered within 10 days if cells were returned to serum-supplemented medium, but dopamine release could not be recovered. Nerve growth factor (NGF) induced similar responses in PC12 cells cultured in both media, including phosphorylation of extracellular signal-regulated protein kinases and neurite extension. Transferrin was necessary for survival of neurite-bearing PC12 cells subcultured in serum-free medium and insulin promoted the cells proliferation. Ten days culture with NGF produced a similar increase in neurofilament expression and acetylcholinesterase activity in both media. These results suggest that PC12 in the hormonally defined serum-free media are qualitatively the same as those cultured in serum-supplemented media, and therefore this new culture protocol should enable more precise studies of PC12 cells culture in the absence of confounding unknown factors. © 2005 Elsevier B.V. All rights reserved. Keywords: PC12; Hormonally-defined serum-free medium; Dopamine; Extracellular signal-regulated protein kinase (ERK); Neurofilament; Acetylcholinesterase; Heparin; Flow cytometry
1. Introduction The rat adrenal pheochromocytoma PC12 cell line (Greene and Tischler, 1976) is widely used as a model neuronal cell line because the cells behave like neural progenitor cells: they proliferate in growth media, but stop proliferating and differentiate into sympathetic neuron-like cells following treatment with nerve growth factor (NGF) (Greene and Tischler, 1982). PC12 cells are also used to study signal transduction, differentiation, survival and proliferation mechanisms as they respond to many growth factors, neurotrophins and hormones (Greene and Tischler, 1976; Rukenstein et al., 1991; Togari et al., 1985). PC12 cells also provide a valuable model for studying the mechanism of synthesis and secre∗
Corresponding author. Tel.: +81 3 5454 6632; fax: +81 3 5454 4330. E-mail address: [email protected] (M. Asashima).
0165-0270/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2005.08.004
tion of neurotransmitters such as acetylcholine, dopamine and norepinephrine (Greene and Rein, 1977b; Greene and Tischler, 1976; Lucas and Kreutzberg, 1985; Schubert et al., 1977). PC12 cells are usually subcultured in medium supplemented with serum (Greene et al., 1998; Greene and Tischler, 1976). Serum may contain a wide range of growth factors, differentiation factors catecholamines, and other unknown factors that cannot be easily defined and may lead to unpredictable cell characteristics (Furue et al., 2005). For this reason, experiments using PC12 cells are often performed after and/or under short-term culture in serum-free medium to exclude undefined serum effects. Temporary serum withdrawal does not completely abrogate the effects of serum as undefined serum effects can persist for several passages (Davis, 2002). Another disadvantage of temporary serum withdrawal is the induction of other side effects and cell
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death that can occur within a few days (Greene, 1978). The present study describes the development of a novel hormonally defined serum-free medium (HD medium) that maintains PC12 cells characteristics following withdrawal of serum and loss of undefined serum effects. Previous studies have confirmed that PC12 cells can be cultured in an undifferentiated state for about 10 days in HD medium containing N2 supplements that was developed for the rat neuroblastoma B104 cell line (Bottenstein, 1984; Bottenstein and Sato, 1979). Similarly, differentiated PC12 cells can be maintained in HD medium in the presence of NGF for more than 1 month (Greene, 1978; Hatanaka, 1981; Skaper et al., 1983). Culture conditions for the serial subculture of undifferentiated PC12 cells in HD medium remain undefined (Greene et al., 1998; Greene and Tischler, 1982). The present study addresses this problem and describes the development of an HD medium for the serial subculture of PC12 cells. PC12 cells were cultured until the eighth passage, over more than 6 weeks, in HD medium after withdrawal of serum, and the characteristics of these cells (PC12-HD cells) were compared with cells subcultured in the control serum-supplemented medium (PC12-SS cells).
2. Materials and methods 2.1. Cell culture PC12 cells were obtained from the Riken Cell Bank (Cat #: RCB0009, Ibaragi, Japan). The cells were cultured in serum-supplemented medium (SS medium) or HD medium in a humidified incubator containing 5% CO2 at 37 ◦ C. The SS medium consisted of RD medium (Davis, 2002; Furue et al., 1994; Sato et al., 1987) supplemented with 10% heat-inactivated horse serum donor herd (Invitrogen Corporation, Carlsbad, CA, USA), and 5% heat-inactivated qualified fetal bovine serum (Invitrogen). The RD medium is an equal mixture of RPMI 1640 and DMEM medium, containing 2 g/l sodium bicarbonate, 15 mM HEPES, 5 mM l-glutamine, 10 nM sodium selenite, 110 mg/l sodium pyruvate, 3.25 g/l d-glucose and 100 mg/l kanamycin sulfate (Custom-made, Research Institute for the Functional Peptides, Yamagata, Japan). Sixty-millimeter plastic culture dishes (BD bioscience, San Jose, CA, USA) were coated with 30 g/ml type I collagen (Cellmatrix Type I-A, Nitta Gelatin, Inc., Osaka, Japan) in PBS for at least 30 min at room temperature (21–26 ◦ C) and then rinsed with PBS. For subculturing, the cells were detached from the culture dish by forceful aspiration of the medium through a plastic pipette (Greene et al., 1998). The cells were subcultured every 5–7 days and the medium was changed every 2–3 days. For storage, the cells were frozen in growth medium with 10% (v/v) dimethylsulfoxide (DMSO) (Greene et al., 1998). PC12 cells serially subcultured in this condition were referred to as PC12-SS cells.
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Table 1 Supplements for serum-free culture of the PC12 cells Supplement
HD proliferation Medium
HD neurite outgrowth medium
Insulin (I-5500a ) Apo-transferrin (T-1147a )
30 g/ml 10 g/ml
– 10 g/ml
3F
1 unit 10 nM 10 M 10 M
1 unit 10 nM 10 M 10 M
500 g/ml –
500 g/ml 100 ng/ml
Sodium selenite (S-9133a ) Ethanolamine (E-0135a ) -Mercaptoethanol (M-7522a ) BSA (A-6003a ) NGFb
The basal medium is RD medium. 3F is a mixture of sodium selenite, ethanolamine and -mercaptoethanol. a Sigma catalog number. b NGF 2.5S, mouse, Upstate, Waltham, MA, USA.
HD proliferation medium consisted of RD medium supplemented with bovine serum albumin (BSA), 3F (sodium selenite, ethanolamine, -mercaptoethanol), insulin and apotransferrin (Table 1). Incorporation of these constituent factors into the HD medium is based on reports from previous studies (Bottenstein, 1984; Click et al., 1972; Davis, 2002; Furue et al., 1994; Sato et al., 1987). These factors were added to the RD medium immediately prior to use from 100-fold concentrated stock solutions. Culture dishes were coated with 3 g/ml type IV collagen (mouse, Cat #: 354233, BD bioscience,) in PBS for at least 30 min at room temperature and then rinsed with PBS. Cell culture procedures were the same as described above. The PC12 cells serially subcultured for 8–15 passages over 2–3 months under these conditions were referred to as PC12-HD cells. The maintenance of neurite-bearing PC12 cells for more than 10 days without subculture was achieved by culturing the cells in HD neurite outgrowth medium (Table 1). 2.2. Cell counting The tendency of PC12 cells to form clumps, even after extensive trituration, can lead to errors in cell counting. To assess cell numbers accurately, the cells were solubilized in nuclear counting solution contained 5 mg/ml ethylhexadecyldimethylammonium bromide, 0.165 mg/ml NaCl, 0.28% (v/v) acetic acid, 0.5% (v/v) Triton X-100, 2 mM MgCl2 and 10% (v/v) PBS (Greene et al., 1998; Soto and Sonnenschein, 1985), and the intact nuclei were counted using a particle counter (Coulter counter Z-1, Beckman Coulter, Inc.). Using the plateau calibration methods described in the user manual of the Coulter counter, the lower limit size setting for PC12 cell nuclei was measured to be 4 m. 2.3. Dopamine assay Depolarization-induced dopamine release and intracellular dopamine content were quantified as described previously (Aoyagi and Takahashi, 2001). Cells cultured in 60-mm plas-
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tic dishes were rinsed three times with normal bath solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl2 , 1 mM MgCl2 , 10 mM d-glucose, 1 mg/ml BSA and 10 mM HEPES–Na at pH 7.4, and incubated in 1.4 ml of normal bath solution or high potassium bath solution for 15 min at room temperature. The high potassium bath solution contained 95 mM NaCl, 50 mM KCl, 2 mM CaCl2 , 1 mM MgCl2 , 10 mM d-glucose, 1 mg/ml BSA and 10 mM HEPES–Na at pH 7.4. The incubated bath solution was then added to solution containing 0.35 ml of 0.1N perchloric acid and 0.5 mM EDTA and stored at −80 ◦ C. The intracellular dopamine content was measured by first solubilizing the cells in 40 mM perchloric acid and 20 M EDTA followed by sonication and centrifugation at 12,600 rpm for 15 min at 4 ◦ C. Dopamine in the supernatant was quantified using HPLC-DPE methods (SRL, Inc., Tokyo, Japan). 2.4. Intracellular calcium imaging PC12-HD or PC12-SS cells were inoculated on glassbased dishes (Iwaki, Tokyo Japan) coated with 30 g/ml type IV or 3 g/ml type I collagen in HD proliferation medium or SS medium, respectively, and cultured for 3 days. After the cells were rinsed with normal bath solution, the cells were incubated in calcium green-1 loading solution consisting of 5 M calcium green-1 AM (Molecular Probes, Inc., Eugene, OR, USA), 0.02% (v/v) cremophor EL (Nacalai tesque, Inc., Kyoto, Japan), 0.5% (v/v) DMSO in normal bath solution for 1 h at room temperature. Cremophor EL, a non-ionic surfactant, was used to facilitate calcium green-1 AM solubilization. After mounting the glass-based dish onto the stage of an upright IX70 microscope (Olympus, Tokyo, Japan), the chamber was perfused with normal bath solution at 30 ◦ C. The single-wavelength fluorescence indicator calcium green-1 was excited with light at 480 nm and the resulting fluorescence images at wavelengths greater than 510 nm were acquired using an monochrome cooled CCD camera 4920 (Cohu, Inc., San Diego, CA, USA) and LG3 frame grabber (Scion Corporation, Frederick, ML, USA). Using a U-shaped tube (Fenwick et al., 1982) with gravity feed and manual switching valve, high potassium bath solution was pulsed over the cells. Background signal (large white rectangles at right side of images in Fig. 3D) was subtracted from each region of interest (small white rectangles in Fig. 3D). Relative fluorescent change (F − F0 )/F0 , were calculated, where F is fluorescence intensity and F0 is initial fluorescence intensity before puffing of high-potassium bath solution. 2.5. Flow cytometry analysis The cells were harvested using calcium- and magnesiumfree Dulbecco’s phosphate-buffered saline (PBS) containing 1 mg/ml BSA (BSA–PBS) and then fixed for 15 min with 2% paraformaldehyde at 4 ◦ C, followed by 15 min in cold 70% ethanol in PBS. The cells were then rinsed with PBS
and incubated for 30 min in blocking buffer consisting of 10 mg/ml BSA and 0.2% Triton X-100 in PBS. Cells were incubated for 30 min at room temperature with primary antibodies diluted in the blocking buffer. After two rinses with BSA–PBS, cells were incubated for 30 min at room temperature with secondary antibodies diluted in blocking buffer. The primary antibodies used included mouse anti-extracellular signal-regulated protein kinase (ERK) monoclonal antibody (Pan ERK, 1 g/ml, BD Bioscience), mouse anti-ERK1/2 (pT202/pY204) phospho-specific monoclonal antibody (1 g/ml, BD Bioscience) and rabbit anti-neurofilament 68kDa polyclonal antibody (1/1000 dilution, Chemicon International, Inc., Temecula, CA, USA). The secondary antibodies used included FITC-conjugated goat anti-mouse IgG (H + L) FITC (2 g/ml dilution, Beckman Coulter), and FITC conjugated donkey anti-rabbit IgG (H + L) (1 g/ml, Rockland, Inc., Gilbertsville, PA, USA). Flow cytometry analysis of immunofluorescently labeled cells was performed using an EPICS ALTRA (Beckman Coulter, Inc., Miami, FL, USA). 2.6. Acetylcholinesterase (AChE) activity AChE activities were measured as previously described (Ellman et al., 1961; Rieger et al., 1980). Briefly, the cells were homogenized in solubilization buffer containing 10 mM Tris–HCl (pH 7.2), 1 M NaCl, 50 mM MgCl2 and 1% (v/v) Triton X-100. Acetylthiocholine at 0.5 mM was used as a substrate. The rate of absorbance change, which is proportional to the rate of acetylthiocholine hydrolysis by extracted AChE, was measured at 412 nm in a photometer.
3. Results 3.1. HD proliferation medium for serial subculture of PC12 cells To develop an HD proliferation medium for PC12 cells, we examined the effects of insulin, transferrin and 3F (sodium selenite, ethanolamine, -mercaptoethanol) on the proliferation of PC12 cells. Cell numbers were counted after 6 days of culture in RD medium supplemented with these factors (Fig. 1). Maximum growth of the PC12 cells was achieved in RD medium supplemented with 30 g/ml insulin, 3–30 g/ml transferrin and 1 unit of 3F (10 nM sodium selenite, 10 M ethanolamine, 10 M -mercaptoethanol). The addition of BSA to medium is known to be beneficial to the proliferation and maintenance of cells in culture as BSA protects cells from mechanical stress (Davis, 2002). The ability of BSA to improve PC12 cells harvesting efficiency was assessed by comparing cell numbers following rinse and trituration in the presence and absence of BSA. We found that the number of cells was greater when cells were rinsed and triturated in the presence of BSA than without (Fig. 1B). This result suggests that BSA does protect PC12 cells from mechanical stress during the passage procedure and should
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Fig. 1. Proliferation and morphology of PC12-SS cells in HD medium. (A) PC12-SS cells were cultured for about 18 h in fresh basal RD medium without any supplements for serum starvation before the experiments, and then inoculated onto type I collagen-coated 24-well plates at a cell density of 104 cells/cm2 . The culture conditions were RD medium supplemented with 0–30 g/ml insulin (Ins), 0 (open symbol) or 1 unit (closed symbol) of 3F and 0–300 g/ml apo-transferrin (Abscissa). The cells were counted after 6 days of culture (n = 3, mean ± S.E.). (B) The effect of BSA on harvesting efficiency. PC12-HD cells at a cell density of 5 × 104 cells/cm2 were inoculated onto a 3 g/ml type IV collagen-coated 12-well plate in HD proliferation medium and cultured 4 days. The cells were rinsed three times and harvested using complete HD proliferation medium (BSA+) and HD proliferation medium without BSA (BSA−). ** Statistically significant at P < 0.001, t-test (n = 4, mean ± S.E.). (C–I) Phase-contrast photomicrographs of PC12 cells. (C) PC12-SS cells were inoculated in SS medium onto dishes coated with 30 g/ml type I collagen. (D–I) PC12-SS cells were cultured in HD proliferation medium on dishes coated with 30 g/ml type I collagen for 6 days and subcultured for one day in HD medium on dishes coated with 30 (D) or 300 (E) g/ml type I collagen, with 3 (F) or 30 (G) g/ml type IV collagen or with 10 (H) or 100 (I) g/ml poly-l-lysine. Insets show high magnification views of the cells (arrow). Scale bar = 20 m.
be included in the medium. Taken together, these results were used to determine the most efficient composition of the HD proliferation medium (Table 1). 3.2. Coating conditions for HD medium After a few subcultures in HD proliferation medium in a type I collagen-coated dish, the cells were smaller and brighter by phase-contrast microscopy than cells cultured in SS medium (Fig. 1B and C). This observation indicates that the cells were rounded when grown in HD proliferation medium but became more spread out in SS medium. This
morphological difference suggests that the cells attached only weakly to the type I collagen-coated dish in HD proliferation medium and were easily detached from the dish during culture. Previous studies (Turner et al., 1987; Vlodavsky et al., 1982) found that type IV collagen as a substrate produced a higher percentage of PC12 cells that were more spread out and more firmly attached than did type I collagen as a substrate. PC12 cells are also known to attach well to poly-l-lysine-coated dishes in SS medium (Greene et al., 1998). In the present study, we tested type IV collagen and poly-l-lysine as a coating substrate. PC12-SS cells that had been subcultured twice in HD proliferation
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medium were inoculated in HD proliferation medium onto 30 or 300 g/ml type I collagen-coated dishes, 3 or 30 g/ml type IV collagen-coated dishes and 10 or 100 g/ml polyl-lysine-coated dishes. Cells were cultured for 1 day and phase-contrast micrograph images were taken. On dishes coated with 30 or 300 g/ml type I collagen, cells were small and bright (Fig. 1D and E). Cells were also small and bright on poly-l-lysine-coated dish (Fig. 1H and I). By comparison, the cells cultured on type IV collagen-coated dishes were dark and spread out, even when the concentration of collagen was decreased to 3 g/ml (Fig. 1F and G), and cells had a morphology similar to PC12-SS cells on 30 g/ml type I collagen-coated dishes (Fig. 1C). These results suggest that in HD proliferation medium, PC12 cells attach more firmly on dishes coated with 3 or 30 g/ml type IV collagen than on dishes coated with 30 or 300 g/ml type I collagen or 10 or 100 g/ml poly-l-lysine (see also Supplementary data). Based on these observations, 3 g/ml type IV collagen was
chosen as the most appropriate substrate for coating dishes for the serial subculture of PC12 cells in HD proliferation medium. 3.3. Growth of PC12-HD and PC12-SS cells Cells could be successfully serially subcultured for more than eight passages over about 2 months in HD proliferation medium on dishes coated with 3 g/ml type IV collagen. We refer to these cells as PC12-HD cells. The PC12-HD cells spread well and were an equivalent size to PC12-SS cells under phase-contrast microscopy (Fig. 2B and C). To assess growth curves for the PC12-HD and PC12-SS cells, cell counts were performed every 2 days (Fig. 2A). Cells grown under both conditions were in log-growth phase within 21 h. The doubling times of PC12-HD cells and PC12-SS cells were 35.2 ± 2.0 and 33.2 ± 0.8 h, respectively (P > 0.05, ttest). The cell density of the PC12-HD cells was 70–87% less
Fig. 2. Cell growth of PC12-HD and PC12-SS cells. (A) Growth curve of PC12-HD cells (open circle, dotted line) and PC12-SS cells (closed circle, solid line). PC12-HD cells and PC12-SS cells at a cell density of 2 × 104 cells/cm2 were inoculated onto a 3 g/ml type IV collagen-coated 24-well plate in HD proliferation medium, and onto a 30 g/ml type I collagen-coated 24-well plate in SS medium, respectively. The cells were counted at 4 and 21 h after plating and every 48 h thereafter (n = 5, mean ± S.E.; most error bars cannot be seen due to small S.E.). (B and C) Phase-contrast photomicrographs of PC12-HD cells (B) and PC12-SS cells (C) on the fifth day. Scale bar = 50 m.
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than that of the PC12-SS cells at every data point (P < 0.01, ttest). These results indicate that the initial number of attached PC12-HD cells was less than for the PC12-SS cells, but the growth rates of both cells were the same. 3.4. Dopamine release from PC12-HD and PC12-SS cells PC12 cells are known to synthesize, store and release dopamine (Greene and Rein, 1977a; Greene and Tischler, 1976). To examine whether PC12-HD cells retain this characteristic, we determined the amount of released dopamine in PC12-HD and PC12-SS cells. Three days after subculturing, both cell types were depolarized using a high-potassium (50 mM) bath solution to elicit dopamine release, which was quantified using HPLC. The PC12-HD cells released a significantly increased amount of dopamine in response
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to high potassium compared to the same cells incubated in a solution containing normal levels (5 mM) of potassium (P < 0.01, t-test; Fig. 3A). This result indicates that PC12HD cells not only release dopamine, but also synthesize and store dopamine because the HD medium does not contain dopamine. Although PC12-HD cells can release dopamine, the level of dopamine released from these cells is about 200-fold lower than the level released from PC12-SS cells (Fig. 3B). We then proceeded to ascertain the underlying mechanism leading to the decrease in the ability of PC12-HD cells to synthesize, store or release dopamine. We initially examine the elevation of intracellular calcium followed by membrane depolarization as elevation of intracellular calcium triggers exocytosis. The fluorescent calcium indicator, calcium green1 AM, was loaded into the cells and the high potassium bath solution was applied to the cells. In both PC12-HD and
Fig. 3. Dopamine release: (A–C) the absolute level (A) and the relative level of dopamine based on A (B, C). The PC12-HD cells (open column), PC12-HD cell cultured 10 days in SS medium (gray column) and PC12-SS cells (closed column) were incubated in a high-potassium bath solution (high K+ , 50 mM KCl) or a normal bath solution (Nrm, 5 mM KCl) for 15 min at room temperature. These cells were also solubilized to measure intracellular dopamine content (content) n = 3, mean ± S.E. Statistically significant at * P < 0.01, ** P < 0.001 and *** P < 0.0001, t-test. (B) Relative level of dopamine. Each absolute level was normalized according to the corresponding absolute level in PC12-SS cells. (C) Releasable dopamine is the percentage of dopamine released by high-potassium from the cellular dopamine level. (D and E) Intracellular calcium change evoked by a 10-s exposure to a high-potassium bath solution (horizontal bar). (D) Time course of relative fluorescence change of calcium green-1 and images (inset) in PC12-HD (upper) and PC12-SS (lower) cells. (E) Maximum fluorescence change, n = 21 (seven regions of interest in three dishes), mean ± S.E.
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PC12-SS cells, fluorescence rapidly increased then decreased following a 10 s application of high potassium bath solution (Fig. 3D). The maximum change in fluorescence for both cell types was almost the same (Fig. 3E) indicating that a high potassium level elicits an intracellular calcium rise in both cell types. We can conclude that elevation of intracellular calcium elicited by membrane depolarization is maintained in PC12-HD cells. The intracellular dopamine level of PC12-HD cells was then examined. The level of dopamine in PC12-HD cells was 10% the level in PC12-SS cells (Fig. 3B). The level of releasable dopamine in PC12HD cells, expressed as the percentage of cellular dopamine levels released by depolarization, was 0.7%, which is about 20-fold less than that observed for PC12-SS cells (Fig. 3C). These results indicate that both the ability to synthesize and/or store dopamine and ability to release dopamine following calcium elevation is provided by factors present in the serum.
3.5. Dopamine release from PC12-HD cells cultured 10 days in SS medium To clarify the effect of serum on dopamine related characteristics, PC12-HD cells were cultured for 10 days in SS medium and the intracellular dopamine content and the amount of released dopamine were measured. The intracellular dopamine level and the level released in normal and in high potassium bath solutions, increased after 10 days culture in SS medium (Fig. 3A). The relative increase in intracellular dopamine from 10 to 60% following culture of cells in SS medium (Fig. 3B) strongly suggests the presence of factors in the serum to enhance dopamine synthesis and/or storage. By comparison, the proportion of releasable dopamine did not recover after 10 days culture in SS medium (Fig. 3C). Preliminary experiments showed that the absolute level of dopamine released from PC12-SS cells cultured temporally for 1–2 weeks in HD proliferation medium decreased to 10%,
Fig. 4. Flow cytometry analysis of the effect of NGF on ERK expression and its phosphorylation in PC12-HD and PC12-SS cells. PC12-HD (A and B) and PC12-SS (C and D) cells treated with BSA–PBS (CNT, dotted line) or 100 ng/ml NGF in BSA–PBS (solid line) for three minutes at 37 ◦ C were analyzed using non-specific anti-ERK antibody, phospho-specific anti-ERK 1/2 antibody. Abscissas represent fluorescence intensity corresponding to non-specific anti-ERK antibody (A and C) and phospho-specific anti-ERK1/2 antibody (B and D). The arrows indicate the range of positive cells.
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but recovered to more than 60% after culture in SS medium for 1 week (see Supplementary data). These results suggest that the decrease in releasable dopamine by temporal culture in HD proliferation medium is recoverable. There is a possibility that long-term serial subculture in HD proliferation medium causes clonal drift in cells. 3.6. Short-term treatment with NGF enhances ERK phosphorylation Previous studies reported PC12 cells as a good model for studying NGF-related signaling through receptor tyrosine kinase A (Trk A), Raf, mitogen-activated protein kinase kinase (MEK) and ERK (for review, see Vaudry et al., 2002). ERK phosphorylation induced by NGF occurs within minutes with the maximal level reached by 5 min (Lazarovici et al., 1998; Peraldi et al., 1993). To determine ERK expression and phosphorylation in PC12-HD and PC12-SS cells treated with
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NGF, the distribution of cells that were immunopositive for non-specific ERK (pan-ERK) antibody and phospho-specific ERK 1 and 2 (ERK 1/2) antibody was measured using flow cytometry (Fig. 4). Approximately 90% of both PC12-HD and PC12-SS cells were immunoreactive for pan-ERK, and the percentage did not change following NGF treatment. By comparison, less than 3% of both cell types were positively labeled for phospho-specific ERK1/2 prior to treatment with NGF and this percentage increased to 90% with 3 min of NGF treatment. These results indicate that ERK1/2 phosphorylation was promoted by NGF in PC12-HD cells to the same extent as in PC12-SS cells, suggesting that these signaling pathways were retained under the HD culturing conditions. 3.7. Extension of neurites following NGF treatment PC12 cells have been shown to differentiate into sympathetic neuron-like cells with neurite extension in response
Fig. 5. Neurite extension and cell survival of PC12-HD cells by NGF-treatment. PC12-HD at the cell density of 2 × 104 cells/cm2 were inoculated onto a 3 g/ml type IV collagen-coated 24-well plate in complete HD proliferation medium (A, E and I), without insulin (B, F, J and M), without transferrin (C, Q and K), and without insulin and transferrin (D, H and L) supplemented with 100 ng/ml NGF (E–M). Phase-contrast photomicrographs taken at day 4 (A–H), day 10 (I–L) and day 32 (M) are shown. Scale bar = 50 m.
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to NGF in SS growth medium, basal medium without any supplement and basal medium with insulin and/or transferrin (Greene, 1978; Greene and Tischler, 1976; Hatanaka, 1981; Skaper et al., 1983). To examine neurite outgrowth from PC12-HD cells, 100 ng/ml NGF was added to complete HD proliferation medium or HD proliferation medium without insulin and/or transferrin and the morphological changes were monitored by phase-contrast microscopy. Neurites began to extend from the cells within 4 days in all media (Fig. 5E–H) and more than half of the cells showed neurite extension after 10 days (Fig. 5I–L). The results suggest that PC12-HD cells maintain the ability to extend neurites. 3.8. Proliferation and cell survival following NGF treatment The proliferation of PC12 cells is inhibited by NGF (Greene and Tischler, 1976). To examine the effect of NGF on PC12-HD cell proliferation, the cells were inoculated in HD proliferation medium supplemented with various concentrations of NGF for 10 days, and then the cells were counted. NGF inhibited PC12-HD cell proliferation in a dosedependent manner (Fig. 6). It is also known that NGF supports PC12 cell survival (Greene, 1978). To examine the effect of NGF on the survival of PC12-HD cells, various concentrations of NGF were added to the HD proliferation medium without insulin and/or transferrin, which do not support cell proliferation (Fig. 1A), and photomicrographs were taken on culture days 4 and 10
(Fig. 5). Cell numbers were counted on culture day 10 (Fig. 6). By day 4, in the absence of NGF and insulin or transferring, almost all cells were shrunken and had detached from the dish (Fig. 5B–D). By comparison, in the presence of 100 ng/ml NGF, the cells were attached to the dish and had spread (Fig. 5F–H), suggesting that NGF supported PC12-HD cell survival for at least 4 days without insulin and transferrin. In medium with transferrin but without insulin, the number of cells increased with increasing doses of NGF (Fig. 6), and most cells were still viable at day 10 in the presence of 100 ng/ml NGF (Fig. 5J). In the absence of transferrin, most cells were shrunken and had detached from the dish by day 10 (Fig. 5K and L), and the cell densities of the PC12-HD cells were less than the initial plating density irrespective of the presence of the insulin and NGF (Fig. 6). These results strongly suggest that NGF can support PC12-HD cell survival for at least 4 days without insulin and transferrin, and for 10 days with transferrin. In medium with 100 ng/ml NGF and transferrin but no insulin, PC12-HD cell proliferation almost ceased and the cells remained viable with long neurites for more than 1 month (Fig. 5M). In the presence of 1–100 ng/ml NGF, cell numbers in the medium with transferrin and insulin were higher than in medium with transferrin and no insulin. These results suggest that transferrin is necessary for survival of neurally differentiated PC12-HD cells and that insulin promotes cell proliferation. As most neuronal cells do not proliferate after differentiation into neurons, medium containing NGF and transferrin, but not insulin may be suitable for studying the characteristics of differentiated PC12-HD cells. We refer to this medium as HD neurite outgrowth medium (Table 1) and used it for analysis of differentiated PC12-HD cells. 3.9. Neurofilament expression and AChE activity in undifferentiated and neuronally differentiated PC12 cells
Fig. 6. The effect of NGF on PC12-HD cell growth and survival. PC12-HD at the cell density of 2 × 104 cells/cm2 were inoculated onto a 24-well plate coated with 3 g/ml type IV collagen in complete HD proliferation medium (closed rectangle), medium without insulin (closed triangle), medium without transferrin (open rectangle), medium without both insulin and transferrin (open triangle) supplemented with various concentration of NGF. The cells were counted after 10 days of culture. The arrow and the arrowhead correspond to the HD proliferation medium and the HD neurite outgrowth medium (Table 1), respectively (n = 4, mean ± S.E.; most error bars cannot be seen due to small S.E.).
To confirm that the PC12-HD cells underwent differentiation following NGF treatment, neurofilament (NF) and AChE were measured, both of which are known to increase during culture in NGF-containing medium (Lee and Page, 1984; Rieger et al., 1980). NF expression and AChE levels in PC12-HD and PC12-SS cells cultured in the HD neurite outgrowth medium for 10 days were compared to those of control groups, which were PC12-HD and PC12-SS cells cultured in HD proliferation medium and SS medium, respectively (Fig. 7). The percentage of NF-immunoreactive cells was measured using flow cytometry (Fig. 7A–C), and the AChE activity was analyzed in cell lysates (Fig. 7D). The peak position (mode) of NF-positive (+) cells and the percentage of NF-strong positive (++) cells for both PC12-HD and PC12-SS cells cultured in the HD neurite outgrowth medium were larger than those of the control group (Fig. 7A–C). These results indicate that the expression of NF was increased in both PC12-HD and PC12-SS following treatment with NGF. The percentage of NF-positive cells (+)
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Fig. 7. The effect of NGF on NF expression and AChE activity. PC12-HD and PC12-SS cells were cultured in HD proliferation medium or SS medium, respectively, for 6 days (proliferation: dotted line in (A and B), open column in (C and D)), or both cells were cultured in HD neurite outgrowth medium for 10 days (Neurite: solid line in (A and B), filled column in (C and D)). Flow cytometry analysis of NF-immunoreactive cells was performed on PC12-HD (A) and PC12-SS (B) cells using NF antibody and second antibody (thick line) and second antibody only (control, thin line). The arrows indicate the range of NF-positive (+) and NF-strong positive (++) cells. The arrowhead represents a weak positive peak. (C) The percentage of NF-positive (+) and strong positive (++) cells based on (A and B). * Statistically significant at P < 0.00002, Z test. (D) AChE activity of PC12-HD and PC12-SS cells. AChE activity is expressed in nanomoles acetylthiocholine hydrolyzed per minute per 106 cells. * Statistically significant at P < 0.01, t-test (n = 4, mean ± S.E.).
was decreased in the PC12-HD cells cultured in HD neurite outgrowth medium (Fig. 7C) due to an increase in the number of weak positive cells (arrowhead in Fig. 7A). These weakly positive cells were not seen in the PC12-SS cultures (Fig. 7B), although the cells were also cultured in HD neurite outgrowth medium for 10 days. The DNA content of the NF-weak positive cells measured using propidium iodide was smaller than expected for the peak position of G1 phase-cells (data not shown), suggesting that the NF-weak positive cells might correspond to dying cells. Together, these results suggest that serum contains some cell survival factors that affect cell viability for at least 10 days after serum withdrawal. The AChE activity of the PC12-HD cells was significantly increased after 10 days culture in the HD neurite outgrowth medium (Fig. 7D; P < 0.01, t-test), indicating an NGF-induced increase in the synthesis of AChE. However, the absolute amount of AChE in the PC12-HD cells was 40% less than that in the PC12-SS cells. It is possible that the NF-
weak positive cells have reduced synthesis of AChE or there may be some factors in the serum that act synergistically with NGF to synthesize AChE.
4. Discussion This is the first report of a hormonally defined serum-free medium developed for the characterization and serial subculture of PC12 cells. Serial subculture in the HD medium did not change the rate of growth of these cells. We also showed that the PC12 cells grown in the HD medium retain their ability to synthesize, store and release dopamine following a rise in intracellular calcium (Greene and Rein, 1977a; Greene and Tischler, 1976). PC12 cells normally exhibit NGF-induced signaling cascades including phosphorylation of ERK, and as the result of such activation, the cells differentiate into sympathetic neuron-like cells (Greene and Tischler, 1976, 1982;
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Vaudry et al., 2002). This study demonstrated that PC12-HD cells exhibit neurite extension, growth inhibition, promotion of survival, elevation of NF expression and increased AChE activity in response to NGF. Thus, PC12-HD appears to retain the features of control PC12-SS cells. Although PC12-HD and PC12-SS cells were qualitatively the same, the cells were quantitatively different. PC12-HD cells released less dopamine compared with PC12-SS cells and those cultured in HD neurite outgrowth medium contained a population of NF-weak positive cells and synthesized less AChE. It is possible that these differences result from the clonal drift of cells cultured in HD medium. Despite the clonal drift of cells cultured in HD medium, this medium is useful to study mechanisms like the molecular basis of exocytosis (Corradi et al., 1996; Shoji-Kasai et al., 2001). The ability to recover the intracellular dopamine level of PC12-HD cells from 10 to 60% by culturing these cells in SS medium, suggests that some factors in serum, such as growth factors and hormones, appear to be necessary for developing these specific cellular characteristics. For instance, our preliminary experiment showed that addition of 30 g/ml insulin or 1 g/ml heparin into the HD neurite outgrowth medium reduced the NF-weak positive peak and increased AChE activity (see Supplementary data) and these factors are known to support the NGF effects on PC12 cells (Hatanaka, 1981; Neufeld et al., 1987; Skaper et al., 1983). Uncovering the importance of various supplements is possible when cells are cultivated in hormonally defined serum-free medium. The HD medium is also useful for analyzing and quantifying low levels of material synthesized by and secreted from the PC12 cells. In summary, we have developed a hormonally defined serum-free medium for the culture of PC12 cells, which maintains all the important characteristics of PC12 cells grown in serum-supplemented medium. By removing a number of confounding unknown factors that may be present in serum, this will facilitate more precise studies of PC12 cells. These cells are increasingly being used to test new materials (Okuyama et al., 2003), and have been used in brain implants to treat Parkinson’s disease (Yoshida et al., 2003). Therefore, this new culture protocol may, in addition to advancing cell biology including biophysics and bioengineering studies, contribute to clinical advances in the treatment of neurological diseases.
Acknowledgements This work was supported by a research fellowship (1383203) from the Japan Society for the Promotion of Science for Young Scientists, a 21st century COE grant from the Japan Society for the Promotion of Science. The authors would like to thank Mr. Yasuhiko Nagasaka from Beckman Coulter, Inc. for his technical assistance and helpful advice regarding flow cytometry, and Dr. Kyota Aoyagi from Kitazato University for his helpful advice regarding dopamine assay.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jneumeth. 2005.08.004.
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