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Exp Mol Med. May 31, 2012; 44(5): 330–339.
Published online Feb 8, 2012. doi:  10.3858/emm.2012.44.5.037
PMCID: PMC3366326
Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system
Yoshitaka Fukaya,1 Masayuki Kuroda,corresponding author2,3 Yasuyuki Aoyagi,2,3 Sakiyo Asada,2,3 Yoshitaka Kubota,1 Yoshitaka Okamoto,2 Toshinori Nakayama,4 Yasushi Saito,5 Kaneshige Satoh,1 and Hideaki Bujo3
1Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Chiba University, Chiba 260-0856, Japan.
2Center for Advanced Medicine, Chiba University Hospital, Chiba 260-0856, Japan.
3Department of Genome Research and Clinical Application, Graduate School of Medicine, Chiba University, Chiba 260-0856, Japan.
4Department of Immunology, Graduate School of Medicine, Chiba University, Chiba 260-0856, Japan.
5Chiba University, Chiba 260-0856, Japan.
corresponding authorCorresponding author.
Corresponding author: Tel, 81-43-222-7171; Fax, 81-43-226-8130; kurodam/at/faculty.chiba-u.jp
Accepted February 7, 2012.
Auto-transplantation of adipose tissue is commonly used for the treatment of tissue defects in plastic surgery. The survival of the transplanted adipose tissue is not always constant, and one of reasons is the accelerated apoptosis of the implanted preadipocytes. We have recently established highly homogeneous preadipocytes, named ccdPAs. The aim of the current study was to evaluate the regulation of the potency of platelet-rich plasma (PRP) on the apoptosis of ccdPAs in vitro. PRP stimulated the proliferation of the preadipocytes in a dose-dependent manner, and the stimulatory activity of 2% PRP was significantly higher than that of 2% FBS or 2% platelet-poor plasma (PPP). The presence of 2% PRP significantly inhibited serum starvation- or TNF-α/cycloheximide-induced apoptosis in comparison to 2% FBS or 2% PPP. DAPK1 and Bcl-2-interacting mediator of cell death (BIM) mRNAs were reduced in the preadipocytes cultured with 2% PRP in comparison to those cultured in 2% FBS. The gene expression levels were significantly higher in cells cultured without serum in comparison to cells cultured with 2% FBS, and the levels in the cells with 2% PRP were reduced to 5-10% of those in the cells without serum. These results indicated that ccdPAs exhibit anti-apoptotic activities, in addition to increased proliferation, when cultured in 2% PRP in comparison to the same concentration of FBS, and that this was accompanied with reduced levels of DAPK1 and BIM mRNA expression in in vitro culture. PRP may improve the outcome of transplantation of adipose tissue by enhancing the anti-apoptotic activities of the implanted preadipocytes.
Keywords: adipocytes, apoptosis, Bcl-2-like protein 11, death-associated protein kinase, platelet-rich plasma, tissue transplantation
Aspirated fat is a common source of autologous tissue transplantation for the correction of tissue defects in plastic and reconstructive surgery (Billings and May, 1989; Patrick, 2000, 2001). Aspirated fat contains multipotential preadipocytes and progenitor cells, which have been utilized as a source of cell-based regenerative medicine (Stashower et al., 1999; Zuk et al., 2001; Gimble et al., 2007; Yoshimura et al., 2009; Bauer-Kreisel et al., 2010; Sterodimas et al., 2010). Although several different techniques of fat grafting have been developed, the outcomes of the transplantation vary widely. The most important factor required for successful grafting is to optimize the survival of the transplanted preadipocytes and other cells in the graft. In previous studies, we and others have shown that various cytokines are involved in the efficient cell survival of the implants (Kimura et al., 2003; Yamaguchi et al., 2005; Cho et al., 2006; Torio-Padron et al., 2007; Kuramochi et al., 2008; Ning et al., 2009).
Platelet rich plasma (PRP) (Eppley et al., 2006; Foster et al., 2009; Redler et al., 2011) has been widely applied for practical medicine, such as aesthetic plastic surgery and the treatment of soft-tissue ulcers (Welsh, 2000; Man et al., 2001; Margolis et al., 2001; Bhanot and Alex, 2002; Martinez-Zapata et al., 2009; Sclafani, 2009). Once activated, platelets secrete various bioactive cytokines, including platelet-derived growth factor (PDGF) and transforming growth factor beta 1 (TGF-β1), which increase angiogenesis and cell proliferation relevant to soft tissue regeneration. PRP has been applied for fat grafting, and in fact, has been shown to improve the survival of implanted adipose tissue in patients (Abuzeni and Alexander, 2001; Sadati et al., 2006; Cervelli et al., 2009). Thus, the use of PRP has been broadened to the tissue-engineering field using adipose tissue-derived multi-potential cells (Anitua et al., 2006; Muller et al., 2009). PRP is also expected to function as an autologous fibrin-based scaffold for transplanted cells (Anitua et al., 2006; Wu et al., 2009; Kang et al., 2011). In fact, recent our study showed that fibrin-based scaffold decreased the apoptotic cell death of murine ccdPAs in mice transplantation model (Aoyagi et al., 2011).
We have recently identified proliferative preadipocytes, ceiling culture-derived proliferative adipocytes (ccdPAs), as homogeneous cells suitable for ex vivo gene therapy applications via autologous transplantation (Asada et al., 2011; Kuroda et al., 2011). The ccdPAs are characterized by their high proliferative capacity with spontaneous adipogenic potential in scaffold fibrin gel culture (Aoyagi et al., 2012). The establishment of a highly homogenous preadipocyte line made it possible to perform examinations to identify the optimal scaffolds and cytokines that can be used to improve the survival of transplanted preadipocytes. We herein studied the effects of PRP, and an autologous cytokine cocktail, on the apoptotic properties of preadipocytes using the ccdPAs.
PRP inhibits fibrin scaffold gel shrinkage and improves the viability of ccdPAs in 3-dimensional culture
We have recently established a 3-dimensional (3-D) culture system for ccdPAs using fibrin gel (FG) (Aoyagi et al., 2012). Using the 3-D culture system, the effects of PRP on the gel shrinkage and cell viability were analyzed in comparison to FBS. The FG/ccdPAs were formed and maintained in culture medium containing 10% FBS for 16 hr. The culture medium was replaced with fresh medium containing 2% PRP, 2% FBS or 10% FBS, or with medium without serum, and cells were subsequently incubated for an additional 24 hr.
The resulting gel sizes varied among the cultures grown in each type of medium. The gels without serum or with 2% FBS showed a drastic volume reduction, while the volumes of the gels cultured with 10% FBS or 2% PRP were not obviously reduced (Figure 1A). The culture supernatants were collected from each well and LDH activity was measured to evaluate the viability of cells. The LDH activity significantly decreased in the culture medium with 2% PRP in comparison to the medium with 2% or 10% FBS (Figure 1B). TUNEL staining of the gel sections showed the number of apoptotic cells to significantly decrease in the medium with 2% PRP in comparison to the medium with 2% FBS (1.5 ± 1.1% vs 9.8 ± 1.9%, P < 0.05). These results suggested that 2% PRP inhibits the shrinkage of FG/ccdPAs gels, and improves the cell viability in comparison to the same concentration of FBS.
Figure 1
Figure 1
The effects of platelet-rich plasma (PRP) on the 3-dimensional culture of ccdPAs. 100 µl of fibrin gels containing 1 × 107 cells/ml of ccdPAs (FG/ccdPAs) were formed in cell culture insert and incubated in DMEM/HAM with 10% FBS for 16 (more ...)
PRP has a high proliferation-inducing potential for ccdPAs in plate culture
In order to evaluate the function of PRP on cell survival in the gel, we next examined the effects of PRP on the proliferation of ccdPAs in comparison to FBS. The cells (2.5 × 105 cells) were seeded and incubated with DMEM/HAM containing 20% FBS in 10 cm dishes for 16 hr. The media was replaced with medium containing 2% PRP, 2% FBS, or 10% FBS, and the cells were then cultured for 3 days. The cell appearance was not apparently changed among the ccdPAs cultured for 3 days in plates with media containing 2% PRP, 2% FBS, or 10% FBS (Figure 2A). To examine the cell proliferation, 2 × 103 cells of ccdPAs were seeded onto 96 well plates and incubated at 37[degree celsius] for 24 hr. The media was replaced with medium with or without 2% PRP, 2% FBS, or 10% FBS (Day 0), and the cells were cultured for 3 days. The number of cells in each well was evaluated by measuring the DNA content. In contrast to the observation that the cell numbers on Day 3 were not significantly changed in comparison to those at Day 0 in the cultures incubated in medium containing 2% FBS, the cell numbers were significantly increased in cells cultured in the medium with 10% FBS or 2% PRP, and notably, the number of cells on Day 3 in the medium containing 2% PRP was significantly increased in comparison to the cells cultured with 10% FBS (Figure 1B). The cell numbers in the media with various concentrations of PRP showed a dose-dependent increase up to 5% PRP; the number of cells present in the media with 0.5-1% PRP was almost equivalent to that of the cells cultured with 5-10% FBS (Figure 2C). These results indicated that the proliferation-inducing potential of PRP for ccdPAs was higher in comparison to that of FBS.
Figure 2
Figure 2
Platelet-rich plasma (PRP) promotes the proliferation of ccdPAs. (A) Photographs of ccdPAs were taken after 3 days in culture containing DMEM/F12-HAM in the absence of serum (a) or the presence of 2% (b) or 10% (c) FBS or 2% (d) PRP. The scale bar indicates (more ...)
PRP inhibits the apoptosis of ccdPAs
The high proliferation-inducing property of PRP for ccdPAs in culture prompted us to further analyze PRP for protective effects against apoptosis in ccdPAs, since PRP is rich in cytokines and proteinases involved not only in proliferation, but also in regulating apoptosis (Eppley et al., 2004, 2006; Foster et al., 2009; Redler et al., 2011). The protection of the cells from death may contribute to their longer survival after transplantation, together with a high potential for proliferation. To investigate the possibility, the ccdPAs (1 × 106 cells) were seeded and incubated in a 10 cm dish with DMEM/HAM medium containing 20% FBS for 16 hr, and subsequently incubated with the medium with or without 2% FBS, 2% PRP, or 2% PPP. After incubation for 8 hr, the cells were collected and stained with Annexin V-FITC. The flow cytometric analysis showed that 5.5% of cells were identified as Annexin V positive in the medium without serum (Figure 3A). The number of apoptotic cells was significantly decreased in the media with 2% FBS, 2% PRP, or 2% PPP in comparison to that in the cells cultured in serum-free medium. Among the various supplements, 2% PRP drastically reduced the number of apoptotic cells in comparison to 2% FBS or 2% PPP. ERK1/2 phosphorylation was examined to further analyze the protective effect of PRP against apoptosis, because the activation of the cascade is important for apoptosis via various intracellular signals including TNF-α (Cawthorn and Sethi, 2008; Mebratu and Tesfaigzi, 2009; Cagnol and Chambard, 2010). The cells were incubated with 2% PRP, FBS, or PPP for 2 hr, and apoptosis was induced by TNF-α and cycloheximide. Phosphorylation was detected following treatment with TNF-α and cycloheximide for 5 min in the cells cultured with 2% FBS. A densitometric analysis showed the amount of phosphorylated ERK2, and not phosphorylated ERK1, to significantly decrease in the medium with 2% PRP in comparison to the medium with 2% FBS (Figure 3B). The caspase-3 activity induced by TNF-α and cycloheximide for 3 hr were also significantly decreased in ccdPAs in the medium with 2% PRP in comparison to the cells cultured with 2% FBS or 2% PPP (Figure 3C). Thus, the apoptosis of ccdPAs was inhibited by culturing them in the medium with 2% PRP in vitro.
Figure 3
Figure 3
PRP decreased the apoptotic cell death and caspase 3 activity induced by TNF-α and cycloheximide in ccdPAs. (A) ccdPAs were seeded and incubated for 16 hr with medium containing 20% FBS, and the medium was then replaced with fresh medium without (more ...)
PRP almost completely inhibits the expression of the pro-apoptotic genes, DAPK1 and BIM, in ccdPAs after serum starvation
In order to identify the molecules involved in the anti-apoptotic effects of PRP on ccdPAs in culture, the expression profiles of representative apoptosisrelated genes were examined using a PCR array profiler. The cells (2.5 × 105 cells) were seeded into 10 cm dishes and incubated in DMEM/HAM containing 20% FBS for 16 hr. The media was replaced with DMEM/HAM containing 2% PRP or FBS, the cells were incubated for 3 days, and the total RNA was isolated from the cultured cells to analyze the expression of apoptosis-related genes. Two independent experiments showed that, among the 84 genes examined, there were 8 genes with a more than 2-fold increase in expression, and 9 genes with a more than 2-fold decrease in expression in the cells cultured in the medium with 2% PRP compared with those cultured in the medium with 2% FBS (Table 1). We focused our interest on two genes, DAPK1 (reduced to 7.4% of the expression level observed with FBS) and BCL2L11 (also called BIM, reduced to 18.9% of the level observed with FBS), as representative genes with the obvious downregulation in the medium with 2% PRP (Figure 4). DAPK1 and BIM have been shown to be one of master regulators of cell death (Gozuacik and Kimchi, 2006), and is essential for BAX-dependent cell death (Kim et al., 2009; Ren et al., 2010), respectively.
Table 1
Table 1
Apoptosis-related genes affected by PRP
Figure 4
Figure 4
The results of the analysis of the expression of apoptosis-related genes affected by PRP. Total RNA was extracted from the cells cultured in the presence of 2% FBS or 2% PRP for 72 hr. The total RNA was subjected to cDNA synthesis and subsequent quantification (more ...)
The role of DAPK1 and BIM genes in apoptosis of preadipocytes was investigated by examining the effect of serum starvation of cells incubated with PRP on the expressions of these genes. The ccdPAs (2.5 × 105 cells) were seeded into 10 cm dishes and incubated in DMEM/HAM containing 20% FBS for 16 hr. The medium was replaced by medium containing 10% FBS (control), 2% FBS, 2% PRP, or 2% PPP, the cells were cultured for 3 days and the expression of DAPK1 and BIM were analyzed. The DAPK1 mRNA level was increased 10.6-fold by serum starvation in comparison to the level in the cells cultured with 10% FBS, and the mRNA level was decreased to that of 10% FBS by the presence of 2% PRP. It is worth noting that, although the mRNA levels of DAPK1 in the cells cultured with 2% FBS and 2% PPP significantly decreased in comparison to those cultured without serum, the reductions were by 20.4% and 11.9%, respectively, which were less than that (93.6%) induced by culture in 2% PRP. The BIM mRNA levels were also drastically increased in the cells cultured in serum free medium in comparison to those cultured in the medium with 10% FBS (Figure 2C). The mRNA levels were reduced to those observed in the cells cultured with 10% FBS by the addition of 2% PRP. Again, the potential of 2% PRP to inhibit the mRNA expression of the target gene (by 87.2%) was significantly higher than that of the 2% FBS (54.1%) or 2% PPP (72.2%). Thus, PRP almost completely inhibited the expression of apoptosis-related genes induced by serum starvation.
PRP inhibited the volume reduction of the 3D gels embedded with ccdPAs, the homogeneous preadipocytes, in comparison to the same concentration of FBS, and this was accompanied by increased cell viability in the gel. These observations prompted us to analyze the effects of PRP on the apoptosis and proliferation of the ccdPAs. The results showed that 2% PRP had a higher inhibitory effect on the apoptotic cell death of ccdPAs than 2% FBS or 2% PPP (Figure 3). A comparison between 2% PRP and 2% FBS by a gene expression profile analysis revealed that PRP downregulated 11% of the 84 representative apoptosis-related genes and upregulated 10% of the 84 representative apoptosis-related genes (Figure 4 and Table 1). The most drastically reduced genes were DAPK1, the protein product of which plays important roles in a wide range of signal transduction pathways with diverse outcomes, such as apoptosis, autophagy and immune responses (Lin et al., 2010), and BIM, encoding one of the BH3-only proteins, which is a critical regulator of apoptosis in many cell types (Ramesh et al., 2009). The induction of these genes by apoptotic stimuli was almost completely prevented in the presence of PRP (Figure 5).
Figure 5
Figure 5
Serum starvation induced the expression of the DAPK1 and BIM genes, which was prevented by culture with 2% PRP. The ccdPAs were seeded and incubated for 16 hr in DMEM/HAM containing 20% FBS, and the culture medium was then replaced with medium without (more ...)
PRP, a concentrate of physiological cytokines, has been widely utilized as an injectable material in the clinic since the 1970s to enhance soft and hard tissue healing (Andia et al., 2010; Lopez-Vidriero et al., 2010; Redler et al., 2011; Yu et al., 2011), mainly by stimulating cell proliferation and angiogenesis in the injured tissues. PRP promotes the growth of various cells, including tissue-derived progenitor cells (Liu et al., 2002; Lucarelli et al., 2003; Doucet et al., 2005; Frechette et al., 2005; Vogel et al., 2006; Kakudo et al., 2008; Kurita et al., 2008; Cervelli et al., 2009; Chieregato et al., 2011), and increases the revascularization of the transplanted tissues (Bir et al., 2009). Based on these findings, the clinical applications of PRP have been broadened to recommend its use as an additive to tissue/cell transplantation therapies in plastic and reconstructive surgeries, and more recently in regenerative medicine. In fact, PRP has been shown to improve the fat graft survival (Abuzeni and Alexander, 2001; Sadati et al., 2006; Cervelli et al., 2009; Nakamura et al., 2010; Pires Fraga et al., 2010; Oh et al., 2011) and bone and periodontal regenerations via cell transplantation (Tobita et al., 2008; Chen et al., 2010; Yamada et al., 2010; Arvidson et al., 2011).
In order to apply PRP for clinical transplantation therapy using preadipocytes cultured in vitro, it is necessary to elucidate the effects of PRP on cell survival in the grafts. However, the mechanisms by which PRP increases graft survival have not been well-characterized so far. The current study showed that PRP strongly induces the proliferation of ccdPAs, preadipocytes which were previously shown to be more adipogenic than ASCs (Asada et al., 2011), compared with FBS at an equivalent concentration. Vogel et al. (2006) described that, because the addition of 2% PRP did not result in sufficient thrombocyte-clot formation to maintain a clot in the medium, a higher concentration of PRP, 3%, was evaluated for the stimulation of the MSC proliferation. In this study, to evaluate the efficacy of lower concentrations of PRP, the PRP was activated by thrombin to release cytokines (Aiba-Kojima et al., 2007) prior to the experiments. As a result, 2% PRP showed almost the same effects on proliferation as 10% PRP, indicating its usability as a substitute for FBS in the expansion of preadipocytes for clinical applications. Finally, 2% PRP showed anti-apoptotic activities on the preadipocytes, providing evidence that it can be used as an efficacious additive in the cell transplantation cocktail.
We observed that the expression of the DAPK1 and BIM genes was substantially upregulated by serum starvation in ccdPAs. However, the addition of PRP in the growth media effectively inhibited the apoptosis and downregulated the expression of these genes. TGF-β has been shown to induce the expression of DAPK1 and BIM, and to lead to subsequent apoptosis in other cell types (Jang et al., 2002; Wildey et al., 2003; Ramjaun et al., 2007; Yu et al., 2008). PRP may therefore inhibit the TGF-β-induced apoptosis cascade(s) during serum starvation in ccdPAs. Further analyses are needed to elucidate the mechanism(s) underlying the inhibitory potential of PRP on the expression levels of the representative apoptotic genes. The gene expression analysis also showed that PRP regulated the expression levels of genes involved in TNF signaling (TNFRSF10A, TNFRSF25, TNFSF10, TNFRSF9, and TRAF2), and of the Bcl protein superfamily, with its related proteins (HRK, BNIP1, and BAG4) (Table 1). The changes in the expression of these genes may also improve the survival of ccdPAs by modulating the apoptotic stimuli, considering that TNF-α signaling plays an important role in the regulation of the adipose tissue mass (Warne, 2003).
In conclusion, PRP inhibits cell apoptosis as well as or better than FBS, and also promotes the proliferation of the ccdPAs. The gene expression analyses identified that the DAPK1 and BIM genes were the most highly downregulated apoptosis-related genes by PRP treatment in the preadipocytes. The identified characteristics of PRP with regard to the preadipocytes have advantages including increases in the cell number and improved cell survival in the transplanted grafts. Together with our findings for the efficacies of fibrin scaffold in transplantation of ccdPAs (Aoyagi et al., 2011), the use of PRP for cell preparation and implantation of fat tissues and/or propagated cells may provide the graft with stable long-term survival after auto-transplantation.
Cell culture
Subcutaneous adipose tissues were obtained from healthy donors after informed consent was obtained, with approval from the ethics committee of Chiba University School of Medicine, and all studies were performed according to the guidelines of the Declaration of Helsinki. The preparation of the ceiling culture-derived proliferative adipocytes (ccdPAs) was performed as described previously (Kuroda et al., 2011). Dulbecco's modified Eagle's medium/F12-HAM (DMEM/HAM, Sigma-Aldrich, St. Louis, MO) supplemented with 20% fetal bovine serum (FBS, SAFC Biosciences, Lenexa, KS) and 40 µg/ml gentamicin (GENTACIN, Schering-Plough Co., Kenilworth, NJ) was used as the culture media, unless otherwise noted in the text.
Preparation of PRP
Human PRP and PPP were prepared from healthy donors as follows; 52 ml of blood was obtained from the donors and mixed with 8 ml of Anticoagulant Citrate Dextrose Solution Formula A (ACD-A, TERUMO, Tokyo, Japan) solution, and transferred to 15 ml tubes. The tubes were centrifuged at 300 × g for 15 min at 20[degree celsius]. The plasma and the buffy coat below the plasma were collected and transferred to new tubes. Secondary centrifugation was performed at 2000 × g for 15 min at 20[degree celsius]. The clear supernatant (plasma) was decanted off until 6 ml was left and the middle portion of supernatant (plasma) was taken to be used as PPP. Finally, the remaining supernatant including the buffy coat was taken to be used as PRP. The platelet number of each product was automatically measured (XS 800i, sysmex Japan). The PRP utilized in this study contained 8.6 × 106 platelets/µl, which was approximately 7-fold concentrated from the original concentration in whole human plasma. Preparations of serum lysates containing platelet-released growth factors were essentially performed according to the method described by Aiba-Kojima et al. (2007). In brief, 2 U/ml of thrombin (Astellas Pharma Inc. Tokyo, Japan) was added to PRP and PPP, and the samples were agitated for 1 hr at 37[degree celsius] and then incubated overnight at 4[degree celsius]. Platelet bodies and any remaining fibrin were eliminated by centrifugation (2000 × g for 10 min), and the supernatants were obtained for the PRP and PPP. The serum samples were frozen at -20[degree celsius] and thawed at 37[degree celsius] before use. The growth medium was supplemented with 2 U/ml of heparin (Novo-Heparin, 5,000 units/5 ml for Injection, Mochida Pharm. Co. Tokyo, Japan).
Culture on fibrin scaffolds
Bolheal (The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan) was used as a clinically available material to generate the fibrin gel. Fibrinogen and thrombin solutions were diluted with Ringer's Solution (Fuso Pharmaceutical Industries, Osaka, Japan) containing 0.5% human serum albumin (Mitsubishi Tanabe Pharma., Tokyo, Japan). The ccdPAs were suspended at 1 × 107 cells/ml by the diluted fibrinogen and thrombin solution. The final concentration of fibrinogen was 4 mg/ml and the thrombin solution was used at 1 U/ml. To form fibrin clots, 50 µl of the cell-fibrinogen suspension was added to each cell culture insert (Falcon 3104; Becton Dickinson, Franklin Lakes, NJ), then shortly thereafter, 50 µl of the cell-thrombin suspension was added into the insert, mixed by pipetting, and incubated at room temperature for 2 h. The inserts with fibrin clots were put on 12 well culture plates, and culture media were added to the inserts and wells. The plates were incubated at 37[degree celsius] for 12 h in a 5% CO2 incubator, and the media were replaced by fresh media containing FBS or PRP.
LDH assay
LDH released into the culture supernatant from the FG/ccdPA was measured using the CytoTox-One Homogeneous Membrane Integrity Assay kit (Promega, Madison, WI). A 100 µl sample of each culture supernatant was collected and added into to a 96-well plate. An equal volume of CytoTox-One Reagent was added and incubated for 10 min. Fifty µl of Stop Solution was added and the sample fluorescence was measured on a fluorescence microplate reader (SPECTRA max GEMINI XPS, Molecular Devices, Carlsbad, CA) using a wavelength of 560 nm/590 nm for excitation/emission. The original culture medium before the serum concentration was changed served as a pre-treatment control sample and the control value was subtracted from the value obtained after incubation with the medium containing different concentrations of serum.
Cell proliferation assay
The cell proliferation was examined using the CyQUANTR Cell Proliferation Assay Kit (Life Technologies, Carlsbad, CA). Cells were seeded into 96 well plates at a density of 2 × 103 cells per well with DMEM/HAM/20% FBS. After 24 h, the culture medium was removed and changed to fresh DMEM/HAM without serum, or with FBS, PRP or PPP. After 3 days, the microplates were gently inverted and blotted onto paper towels to remove the medium from the wells. The microplates were then frozen and stored at -80[degree celsius] and thawed at room temperature prior to analysis. CyQUANT GR dye/cell-lysis buffer was added to each well. Cells were incubated at room temperature for 5 min and the sample fluorescence was measured on a fluorescence microplate reader (SPECTRA max GEMINI XPS, Molecular Devices) using wavelength of 480 nm/520 nm for excitation/emission.
Induction of apoptosis, and the annexin V binding and caspase 3 activity assays
The cells were seeded into 10 cm dishes at a density of 1 × 106 cells per well with DMEM/HAM/20%FBS. After 24 h, the culture medium was removed and changed to fresh DMEM/HAM with 2% FBS, 2% PRP or 2%PPP. After 2 h, apoptosis was induced by the addition of 100 ng/ml TNF-α (Peprotech, Rocky Hill, NJ) and 100 µg/ml cycloheximide (CHX, Sigma-Aldrich). After 3 h, the culture supernatant was collected, and the cells were detached by TrypZean treatment. The detached cells were suspended in the collected culture supernatant. Subsequently, the cells were stained with Annexin V-FITC using a Tali™ Apoptosis kit (Life Technologies). Stained cells were analyzed by a Tali™ Image Based Cytometer (Life technologies). Cell lysates at the concentration of 1 × 104 cells/µl were prepared from the cells treated to induce apoptosis, and the caspase 3 activity levels were measured by a caspase-3/CPP32 Fluorometric Assay Kit (Biovision, Mountain View, CA). To examine the phosphorylation status of ERK1/2, the cells were scraped off at each time point in PBS and washed. The cells were pelleted and lysed by RIPA buffer (Wako Pure Chemical Industries, Ltd. Osaka, Japan), and the protein concentration was determined by Quant-iT Protein Assay Kit (Life technologies), and 5 µg of protein were analyzed by Western blotting using anti-ERK1 and anti-ERK1/2 (pT202/pY204) as primary antibodies (BD Biosciences, Franklin Lakes, NJ). Mouse TrueBlot ULTRA HRP-conjugated Anti-Mouse IgG (eBioscience, Inc. San Diego, CA) was used as a secondary antibody, and the signals were detected by SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific Inc.) with LAS1000 apparatus (FUJI film, Tokyo, Japan). ERK signals were normalized using Actin signals detected by Anti-Actin monoclonal antibody (clone AC-40, Sigma-Aldrich).
Gene expression analysis
Cells (2.5 × 105 cells) were seeded into 10 cm dish with DMEM/HAM/20% FBS and cultured for 16 h. The culture medium was changed to fresh DMEM/HAM supplemented with 2% FBS or 2% PRP, and the cells were further incubated for 72 h. The total RNA from cultured cells was extracted using a RT2 RNA Isolation Kit (SA Bioscience, Frederick, MD). Complementary DNA was generated from 1 µg of total RNA using the RT2 First Strand Kit. An Apoptosis Reverse Transcriptase RT2 profiler PCR array and RT2 Real-Time SYBR Green/ROX PCR Mix (SA Bioscience, Frederick, MD) were used to identify the genes affected by PRP according to manufacturer's instruction. The data were analyzed by web-based data analysis software provided by the manufacturer. The probe and primer sets used to quantify the mRNA for the DAPK1 and BIM genes were purchased from Applied Biosystems (Life Technologies). The quantification of given genes was expressed as the relative mRNA level compared with a control after normalization to 18S RNA. All the real-time PCR were performed using an ABI 7500 real-time PCR apparatus.
Statistical analysis
The data are presented as the means ± S.D. Statistical comparisons were made by Student's t-test or an ANOVA followed by the post-hoc Tukey test using the SPSS software program. In all cases, P-values < 0.05 were considered to be significant.
Acknowledgements
We thank Dr. Fumiaki Matsumoto, Mr. Shunichi Konno, Dr. Shigeaki Tanaka, and Mr. Masayuki Aso for helpful and valuable suggestions about our research. This study was supported by Health and Labour Sciences Research Grants for Translational Research, Japan (H. B.), and in part by the Global COE Program (Global Center for Education and Research in Immune System Regulation and Treatment), MEXT, Japan (Y.O., Y.N., and H.B.).
Abbreviations
BIMBcl-2-interacting mediator of cell death

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