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The MR tracking of transplanted ATDC5 cells using fluorinated poly-l -lysine-CF3
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Biomaterials 28 (2007) 434–440 www.elsevier.com/locate/biomaterials
The MR tracking of transplanted ATDC5 cells using fluorinated poly-L-lysine-CF3 Junichi Makia,c, Chiaki Masudaa,d, Shigehiro Morikawab, Masahito Moritab, Toshiro Inubushib, Yoshitaka Matsusuec, Hiroyasu Taguchid, Ikuo Tooyamaa, a
Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu 520–2192, Japan b Biomedical MR Science Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu 520–2192, Japan c Department of Orthopedic Surgery, Shiga University of Medical Science, Setatsukinowa-cho, Otsu 520–2192, Japan d Graduate School of Home and Economics, Kyoto Women’s University, 35 Imakumano-Kitahiyoshi-cho, Higashiyama-ku, Kyoto 605–8501, Japan Received 1 May 2006; accepted 24 August 2006 Available online 15 September 2006
Abstract Magnetic resonance (MR) imaging using super-paramagnetic iron oxides (SPIOs) is a powerful tool to monitor transplanted cells in living animals. However, since SPIOs are negative contrast agents it is difficult to track transplanted cells in bone and cartilage that originally display low signals. In this study, we examined the feasibility of tracking with fluorescein isothiocyanate (FITC)-labeled poly-Llysine-CF3 (PLK-CF3) using mouse ATDC5 cells, a stem cell line of bone and cartilage cells. FITC-labeled PLK-CF3 was easily internalized by ATDC5 cells by adding it into culture medium. No acute or long-term toxicities were seen at less than 160 mg/ml. Labeled cells transplanted into the cranial bone of mice were detected for at least 7 days by MR images. FITC-labeled PLK-CF3 is a useful positive contrast agent for MR tracking in bone and cartilage. r 2006 Elsevier Ltd. All rights reserved. Keywords: Bone; Cartilage; Cell therapy; Molecular imaging; Magnetic resonance; Transplantation
1. Introduction Cell replacement therapy, using several kinds of cells including embryonic and adult stem cells, is applied to clinical and pre-clinical use [1,2], particularly in the field of orthopedics [3,4]. It is of great importance to be able to non-invasively track transplanted cells in vivo. Several current methods include positron emission tomography [5–7], fluorescent microscopy [8] and magnetic resonance (MR) imaging [9–13]. Magnetic labeling, commonly with super-paramagnetic iron oxides (SPIOs) [11,14] is a key step in MR tracking. One of its limitations is that although SPIOs have high sensitivity, MR signals in the surrounding areas are reduced [15]. Therefore, it is difficult to track cells in the bone and cartilage, which initially display low signals. In order to solve this issue, positive MR materials are Corresponding author. Tel.: +81 77 548 2328; fax: +81 77 548 2402.
E-mail address: [email protected] (I. Tooyama). 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.08.050
needed. Gadolinium is often used as a positive contrast agent [16–19]. However gadolinium may be toxic and can be retained in tissues for a long time after cell death. More recently, perfluoropolyether has been used as a positive contrast agent [20]. 19F displays positive signals but since it is present at a very low level in the body, the signal-to-noise ratio is very high. Therefore, we chose a candidate, N-4(trifluoromethoxy)-benzylated poly-L-lysine (PLK-CF3), as a positive contrast agent and examined the feasibility of using it for MR tracking of the cartilage stem cell line, ATDC5 cells. 2. Materials and methods 2.1. Synthesis of PLK-CF3 We used three commercially available poly-L-lysines with molecular weights 1–4, 5–15 and 15–30 kDa. We prepared the fully PLK-CF3 from poly-L-lysine of molecular weight 1–4 kDa, as follows. A preliminary experiment showed that the fully PLK-CF3 was obtained as a sole product and the material was soluble in methanol, ethanol, ethyl acetate,
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Fig. 1. Structure of PLK-CF3. 4-(trifluoromethoxy-9-benzyl bromide and/or fluoresceinisothiocyanate are bounded to n-terminal residues (Rn) of poly-Llysine.
chloroform and dimethyl sulfoxide, but was insoluble in water. Thus, we prepared benzylated poly-L-lysine with about half of the amino groups in the molecule (Fig. 1). For the synthesis of PLK-CF3, 100 mg of poly-L-lysine hydrobromide was dissolved in a mixture of water (0.5 ml) and methanol (0.5 ml). To the mixture was added a solution of 72 mg of 4-(trifluoromethoxy)benzyl bromide in methanol (0.5 ml) and 130 mg of solid potassium carbonate, in turn, and the mixture stirred for 19 h at room temperature. Ethanol (15 ml) was added to the mixture and the insoluble material removed by filtration. Ethanolic hydrogen chloride was added to the filtrate and the mixture concentrated to about 2 ml. The required PLK-CF3 was precipitated out by adding 50 ml of diethyl ether to the solution. For labeling of PLK-CF3 with fluorescein isothiocyanate (FITC), a mixture of PLK-CF3 (1 mol), FITC (0.1 mol) and tributylamine (1.2 mol) in methanol was stirred for 5 h at room temperature. The FITC labeled compound was obtained by addition of diethyl ether to the mixture. In our FITC-labeled PLK-CF3, half of the amino groups were 4-(trifluoromethoxy)benzylated and 10% of the amino groups were combined with FITC (Fig. 1). Some PLK-CF3 was labeled with Cy5.5 (Amersham Biosciences UK Limited, UK) instead of FITC because Cy5.5 displayed good permeability in tissues.
2.2. Fluorescent labeling and analysis of cultured cells Murine ATDC5 cells were obtained from the RIKEN cell bank (Tsukuba, Japan). ATDC5 cells were cultured in maintenance medium consisting of a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (Sigma, St. Louis, MO) consisting of 5% fetal bovine serum (Sigma), antibiotic–antimycotic (Nacalai Inc., Osaka, Japan) 100 units/ml penicillin, 100 mg/ml streptomycin, 10 mg/ml human transferrin (Roche Applied Science, Mannheim, Germany). Cells were maintained at 37 1C in humidified 5% CO2, 95% air atmosphere. Just before cells reached confluency, we added FITC-labeled PLK-CF3 into the culture medium at 5, 10, 20, 40, 80, 160 or 320 mg/ml. The incubation time also varied from 2 to 48 h. After incubation, we washed the culture dishes with 10 mM phosphate buffered saline (PBS), pH 7.4 and fixed cells for 10 min with 4% paraformaldehyde (PFA) in 0.1 M phosphate
buffer, pH 7.4, at room temperature. After washing the culture dishes with PBS, fluorecent signals were observed by fluorecent microscopy (IX 70, OLYMPUS Co., Tokyo, Japan). The images were analyzed by the Meta Morph imaging system (OLYMPUS Co., Tokyo, Japan). The labeling intensity was calculated using the following formula: Labeling intensity ¼ (fluorescent intensity) (labeling area). Cell toxicity was investigated using trypan blue staining after incubation for 24 h with different doses of FITC-labeled PLK-CF3. The ratio of living cells to total cells was counted. For measuring long-term toxicity, we incubated ATDC5 cells for 24 h with different doses of FITClabeled PLK-CF3. After washing with PBS, cells were recovered using 0.25% trypsin into Flacon tube, and plated in 60 mm culture dish. We counted cell numbers in each dish at days 4 and 7 of incubation.
2.3. MR imaging of PLK-CF3 in the cranial bone of living mouse Experimental procedures were approved by the Committee on Animal Care of the Shiga University of Medical Science. Five male ICR mice (40–50 g) were used. ATDC5 cells were labeled for 24 h with 80 mg/ml of FITC-labeled PLK-CF3. After washing with PBS, cells were harvested using 0.05% trypsin (Nacalai Inc., Osaka, Japan). Labeled cells (2 106–6 106) were resuspended in a mixture of Cellmatrix (Nittagelatin Co., Osaka, Japan) and culture medium at a ratio of 8:2 at a final concentration of 1 107 labeled cells/ml of the gel mixture. Cranial skins of mice were opened under anesthesia (sodium pentobarbital 50 mg/ml, i.p.). The cranial bone of each mouse was defected with a reamer and the defected area was covered with Cellmatrix containing labeled cells. ATDC5 transplanted in the cranial bone was confirmed by fluorescent microscope (Lightools Macro-Imaging System plus cooled, Lightools Research Co., Encinitas, CA, USA), then applied to MR imaging measurements. On days 1, 7 and 21 after transplantation, MR images were acquired with a 7 T Unity Inova MR scanner (Varian, Palo Alto, CA). A surface coil 20 mm in diameter, which can be tuned to both 1H and 19F frequencies (300 and 282 MHz), was used for signal acquisition. First, a gradient echo 1 H image of the mouse brain was acquired in the axial plane. MR imaging parameters were with 100 ms repetition time (TR), 5 ms echo time (TE),
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451 flip angle, 1 mm slice thickness, 35 35 mm2 field-of-view (FOV) and 256 256 matrices. Subsequently, 19F chemical shift imaging (CSI) data was acquired in 35 35 mm2 FOV, 50 ms TR and 1024–2048 acquisitions for 8 8 phase encoding steps. The raw data was processed by 3D-Fourier transformation with zero filling and converted to 32 32 spectral data sets. The FITC-labeled PLK-CF3 image was constructed by extracting the signal intensities of FITC-labeled PLK-CF3 in individual pixels. During the in vivo nuclear magnetic resonance (NMR) measurement, general anesthesia was maintained with intermittent infusion of sodium pentobarbital through a polyethylene tube inserted intraperitoneally.
2.4. Histological examination in the cranial bone After obtaining MR images, some mice were deeply anesthetized with pentobarbital (70 mg/kg) and the aorta was perfused with approximately 20 ml of 10 mM PBS followed by 50 ml of ice-cold 4% PFA in 0.1 M phosphate buffer (pH 7.4). After perfusion and fixation, the cranial bone was dissected out and post-fixed for 2–7 days with the same fixative. The cranial bone was then replaced for 7 days in ice-cold 0.1 M phosphate buffer containing 10% EDTA for de-calcification. After cryoprotection with 15% sucrose, the cranial bone was cut into 20 mm thick sections in a cryostat. The sections were then stained with hematoxyline–eosine.
3. Results 3.1. Structure of PLK-CF3 and its
19
F NMR spectra
Fig. 2A shows the 19F NMR spectrum of PLK-CF3 prepared from poly-L-lysine with molecular weights of 1–4 kDa. The 19F-NMR spectrum showed a singlet peak at d-59.3 ppm (Fig. 2A). Figs. 2B and C demonstrate the 19 F-NMR spectra of PLK-CF3 prepared from poly-Llysine with molecular weights of 5–15 and 15–30 kDa, respectively. Their 19F NMR spectra showed a singlet peak around d-59.3 ppm and a broad peak at d-59.9 ppm. The broad signal was about two times stronger than the singlet peak. The 19F-NMR spectrum of FITC-labeled PLK-CF3 (MW 1-4 kDa) showed a singlet peak at d-59.3 ppm with a broad signal around d-59.9 ppm (Fig. 2D). But the broad peak was smaller than of unlabelled PLK-CF3 (MW 5–15 or 15–30 kDa).
Fig. 2. 19F-NMR spectra of PLK-CF3 with a MW of 1–4 kDa (A, D), 5–15 kDa (B), and 15–30 kDa (C). D; FITC labeled PLK-CF3 with a MW of 1–4 kDa.
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3.2. Labeling of ATDC5 cells with PLK-CF3 When FITC-labeled PLK-CF3 was added to the culture medium, the peptide was easily internalized by mouse ATDC5 cells (Fig. 3). Confocal microscopy revealed that most FITC signals were observed mainly in the cytoplasm. The labeling intensity of ATDC5 cells increased with increasing FITC dose and reached a maximum at 80 mg/ml (Fig. 4). At this concentration, the ratio of labeled cells per total cells was more than 99%. The labeling intensity was decreased at a dose of 320 mg/ml (Po0:01, Fig. 4), suggesting that the label was toxic at this dose. Examination of cell viability confirmed this, with a significant
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decrease in the number of living cells to 77.2% of the control at 320 mg/ml (Po0:01, Fig. 5). In order to assess long-term toxicity, we examined proliferation rates of ATDC5 cells after incubation with 0, 20, 40, 80, 160 and 320 mg/ml of FITC labeled PLK-CF3 (Fig. 6). Compared with unlabeled cells, cell number was significantly decreased at 320 mg/ml (Po0:05, Fig. 6). When the concentration of FITC labeled PLK-CF3 was adjusted to 20 mg/ml, the labeling intensity also increased time-dependently and reached a maximum at 24 h (Fig. 7). 3.3. MR imaging of transplanted ATDC5 cells Cultured ATDC5 cells were labeled for 24 h with 80 mg/ml of FITC-labeled PLK-CF3 or Cy5.5-labeled PLKCF3. Then, 3 106 cells were transplanted into the cranial bone of mice (Fig. 8A), respectively. 19F-MRI successfully detected 19F signals in the transplanted area in the mouse
Fig. 3. Confocal microscopic observations of ATDC5 cells incubated for 24 h with 20 mg/ml of FITC-labeled PLK-CF3 (MW 1–4 kDa).
Fig. 4. Relative fluorescent labeling intensity of ATDC5 cells incubated for 24 h with different doses of FITC-labeled PLK-CF3 (MW 1–4 kDa), where the value at 80 mg/ml was taken as 100%. Bars indicate standard deviations. Po0:05 compared with the value at 80 mg/ml.
Fig. 5. Cell toxicity of PLK-CF3. The ratio of living cells per total cells incubated for 24 h with a various concentration of PLK-CF3 (MW 1–4 kDa). Bars indicate standard deviations. Po0:01 compared with any other concentrations.
Fig. 6. Longitudinal proliferative study of labeled and unlabeled cells. ATDC5 cells were incubated for 24 h with various concentrations of PLKCF3 (MW 1–4 kDa) and cell number was counted at 4 and 7 days after treatment. Po0:05 compared with the value of unlabeled cells.
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cranial bone (Fig. 8A). On the day of transplantation, the transplanted cells were clearly detected by 19F-MRI (Fig. 8B) and fluorescent microscopy (Fig. 8C) in living animals. But MR signals gradually decreased with time, and were able to be detected until 7 days after transplantation (Fig. 9). Figs. 10A and B show the chemical shift of
19
F-NMR spectra obtained from ATDC5 cells in a cranial bone at 1 day and 7 days after the implantation, respectively. The signal intensity seems to be decreased during the 7-day period. Histological examination revealed that ATDC5 cells were maintained in transplanted gel tissue (Fig. 11). 4. Discussion 4.1. Structure of PLK-CF3 for
Fig. 7. Relative fluorescent intensity of ATDC5 cells incubated for different times with 20 mg/ml of FITC-labeled PLK-CF3 (MW 1–4 kDa). The value at 24 h was taken as 100%.
19
F-MRI
In this study, we examined PLK-CF3 and FITC-labeled PLK-CF3 as contrast agents for MR imaging. Signals of 19 F-MRI became clearly as more fluorines combined with PLK. However, if all animo groups of the compound were combined with 19F, then the compound was not watersoluble. We investigated various ratios and determined that 50% and 10% of all amino groups should be combined with CF3 and FITC. As the molecular weight of PLK increased the sharpness of the singlet peak assigned to the trifluoromethyl group decreased. This indicates that the free rotation of the
Fig. 8. Comparison of implanted area (black arrow in A) in a mouse cranial bone and 19F-MRI signals (white arrow in B) and Cy5.5 fluorescent images (C).
Fig. 9. Time curse of proton image (A, D), 19F-MRI signals (B, E) and the merged images (C, F) of ATDC5 cells in the cranial bone implanted after 1 day (A, B, C) and 7 days (D, E, F).
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agents that may have some adverse effects. The labeling efficiency of FITC labeled PLK-CF3 was the maximal at a concentration of 80 mg/ml for a 24 h incubation. No toxicity was seen under these conditions, which are also suitable for other cells such as mesenchymal stem cells and neural cells (data not shown). In order to examine long-term toxicity, we followed proliferation 7 days after incubating cells with various concentration of FITC labeled PLK-CF3. Cells incubated with less than 160 mg/ml FITC labeled PLK-CF3 displayed the same proliferation profile as unlabeled cells. However, there was a significant decrease in the rate of proliferation at 320 mg/ml of FITC labeled PLK-CF3 as well as signs of acute toxicity. Thus, FITC labeled PLK-CF3 should be used at a concentration of less than 160 mg/ml. 4.3. Cell tracking Fig. 10. Chemical shift spectra of 19F-NMR obtained from ATDC5 cells in the cranial bone at 1 day (A) and 7 days (B) after the implantation. The same mouse as shown in Fig. 9.
Fig. 11. Histological examination of transplanted cells 7 days after transplantation. Transplanted cells were observed in the cranial bone (arrow). Bar ¼ 50 mm.
trifluoromethyl group of the smaller PLK-CF3 is entirely free, but some of the trifluoromethyl group of the larger PLK-CF3 cannot rotate freely inside the molecule. From our results, we suggest that CF3 and FITC should be combined to 50% and 10% of amino groups, respectively, of PLK with molecular weights of 1–4 kDa. 4.2. The labeling efficiency and toxicity of FITC-labeled PLK-CF3 This study demonstrated that ATDC5 cells were easily labeled with FITC-labeled PLK-CF3 by just adding the peptides into the culture medium. This is beneficial for possible clinical and non-clinical applications because it is a simple procedure, which does not requires any transfection
In this study, 19F MRI successfully detected ATDC5 cells transplanted into the cranial bone for 1 week. So far, only perfluoropolyether has been reported as a material for cell tracking with 19F MRI [20]. Thus FITC-labeled PLKCF3 is the second material to be described for cell tracking with 19F MRI. Transplanted cells labeled with FITC-labeled PLK-CF3 were also detected by fluorescent microscope. Since fluorescent materials were easily bound to amino-group of PLK-CF3, this reagent has the potential to be used not only for MR tracking but also photo tracking. The signal intensity of FITC-labeled PLK-CF3 gradually decreased after transplantation. Chemical shift spectra of 19 F-NMR obtained from ATDC5 cells implanted in the cranial bone showed that the signal intensity seemed to decrease by about half over 7 days. It is possible that this may be due to the death and subsequent removal of transplanted cells from the cranial bone. However, this seems unlikely as histological examination showed that transplanted cells remained in gels at even 7 days after transplantation. The signal intensity in a single cell diminishes when cells divide. However, since the cells were packed in a gel, the effect of cell division should be minimal. FITC-labeled PLK-CF3 may have been metabolically degraded in the transplanted cells. Compared with SPIOs, FITC-labeled PLK-CF3 is useful for cell tracking for a short time and is not appropriate for cell tracking for longer periods. This disadvantage is also an advantage because the effect of FITC-labeled PLK-CF3 on cells is only for a short time. Further study will be needed to improve the tracking time with FITC-labeled PLK-CF3. 4.4. Clinical utility of cell tracking with FITC-labeled PLK-CF3 Tissue engineering techniques including stem cell therapy have been explored to repair cartilage and bone defects [21]. However, it is difficult to observe transplanted cells
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non-invasively. MR tracking using SPIOs is a powerful tool for cell tracking, but it is difficult to track cells in bone and cartilage, which initially display low signals. Since PLK-CF3 is a positive contrast agent, it is suitable for MR tracking in bone and cartilage cells. In addition, unlike with SPIOs, MR signals can be obtained from surrounding areas when using PLK-CF3. Thus, we can collect information about cell localization as well as environment alterations such as temperature, pH and some metabolites. Culture cells can easily be labeled by adding PLK-CF3 into the medium. This is a tremendous advantage for clinical use as it dose not require the use of transfection agents. So far, the sensitivity of PLK-CF3 is low compared with SPIOs. However, if the sensitivity can be increased to detect a single or a few cells, MR tracking can be used to observe transplanted cells distributed in the body including bone, cartilage and bone marrow after administration of therapeutic cells into vessels. Further study will be needed to increase the sensitivity. 5. Conclusion We successfully obtained MR images of ATDC5 cells transplanted in the cranial bone using PLK-CF3 and 19F MRI. ATDC5 cells were easily labeled with PLK-CF3 just by adding the peptides into the culture medium. Since fluorine shows a positive MR signal, it is suitable for tissues such as bone, cartilage and liver that originally display low proton signals. References [1] Conrad C, Huss R. Adult stem cell lines in regenerative medicine and reconstructive surgery. J Surg Res 2005;124:201–8. [2] Sylvester KG, Longaker MT. Stem cells: review and update. Arch Surg 2004;139:93–9. [3] Christophel JJ, Chang JS, Park SS. Transplanted tissue-engineered cartilage. Arch Facial Plast Surg 2006;8:117–22. [4] Gimeno MJ, Maneiro E, Rendal E, Ramallal M, Sanjurjo L, Blanco FJ. Cell therapy: a therapeutic alternative to treat focal cartilage lesions. Transplant Proc 2005;37:4080–3. [5] Bengel FM, Schachinger V, Dimmeler S. Cell-based therapies and imaging in cardiology. Eur J Nucl Med Mol Imaging 2005; 32(Suppl. 2):S404–16. [6] Kirik D, Breysse N, Bjorklund T, Besret L, Hantraye P. Imaging in cell-based therapy for neurodegenerative diseases. Eur J Nucl Med Mol Imaging 2005;32(Suppl. 2):S417–34.
[7] Wu JC, Spin JM, Cao F, Lin S, Xie X, Gheysens O, et al. Transcriptional profiling of reporter genes used for molecular imaging of embryonic stem cell transplantation. Physiol Genomics 2006;25:29–38. [8] Tanaka M, Swijnenburg RJ, Gunawan F, Cao YA, Yang Y, Caffarelli AD, et al. In vivo visualization of cardiac allograft rejection and trafficking passenger leukocytes using bioluminescence imaging. Circulation 2005;112:I105–10. [9] Bulte JW, Duncan ID, Frank JA. In vivo magnetic resonance tracking of magnetically labeled cells after transplantation. J Cereb Blood Flow Metab 2002;22:899–907. [10] Bulte JW, Ben-Hur T, Miller BR, Mizrachi-Kol R, Einstein O, Reinhartz E, et al. MR microscopy of magnetically labeled neurospheres transplanted into the Lewis EAE rat brain. Magn Reson Med 2003;50:201–5. [11] Frank JA, Miller BR, Arbab AS, Zywicke HA, Jordan EK, Lewis BK, et al. Clinically applicable labeling of mammalian and stem cells by combining superparamagnetic iron oxides and transfection agents. Radiology 2003;228:480–7. [12] Franklin RJ, Blaschuk KL, Bearchell MC, Prestoz LL, Setzu A, Brindle KM, et al. Magnetic resonance imaging of transplanted oligodendrocyte precursors in the rat brain. Neuroreport 1999;10: 3961–5. [13] Heckl S, Pipkorn R, Nagele T, Vogel U, Kuker W, Voight K. Molecular imaging: bridging the gap between neuroradiology and neurohistology. Histol Histopathol 2004;19:651–68. [14] Toyoda K, Tooyama I, Kato M, Sato H, Morikawa S, Hisa Y, et al. Effective magnetic labeling of transplanted cells with HVJ-E for magnetic resonance imaging. Neuroreport 2004;15:589–93. [15] Song Y, Morikawa S, Morita M, Inubushi T, Takada T, Torii R, et al. Magnetic resonance imaging using hemagglutinating virus of Japan-envelope vector successfully detects localization of intracardially administered microglia in normal mouse brain. Neurosci Lett 2006;395:42–5. [16] Anderson SA, Lee KK, Frank JA. Gadolinium-fullerenol as a paramagnetic contrast agent for cellular imaging. Invest Radiol 2006;41:332–8. [17] Modo M, Cash D, Mellodew K, Williams SC, Fraser SE, Meade TJ, et al. Tracking transplanted stem cell migration using bifunctional, contrast agent-enhanced, magnetic resonance imaging. Neuroimage 2002;17:803–11. [18] Modo M, Mellodew K, Cash D, Fraser SE, Meade TJ, Price J, et al. Mapping transplanted stem cell migration after a stroke: a serial, in vivo magnetic resonance imaging study. Neuroimage 2004; 21:311–7. [19] Vuu K, Xie J, McDonald MA, Bernardo M, Hunter F, Zhang Y, et al. Gadolinium–rhodamine nanoparticles for cell labeling and tracking via magnetic resonance and optical imaging. Bioconjug Chem 2005;16:995–9. [20] Ahrens ET, Flores R, Xu H, Morel PA. In vivo imaging platform for tracking immunotherapeutic cells. Nat Biotechnol 2005;23:983–7. [21] Ochi M, Uchio Y, Kawasaki K, Wakitani S, Iwasa J. Transplantation of cartilage-like tissue made by tissue engineering in the treatment of cartilage defects of the knee. J Bone Jt Surg (Br) 2002;84-B:571–8.
Biomaterials 28 (2007) 434–440 www.elsevier.com/locate/biomaterials
The MR tracking of transplanted ATDC5 cells using fluorinated poly-L-lysine-CF3 Junichi Makia,c, Chiaki Masudaa,d, Shigehiro Morikawab, Masahito Moritab, Toshiro Inubushib, Yoshitaka Matsusuec, Hiroyasu Taguchid, Ikuo Tooyamaa, a
Molecular Neuroscience Research Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu 520–2192, Japan b Biomedical MR Science Center, Shiga University of Medical Science, Setatsukinowa-cho, Otsu 520–2192, Japan c Department of Orthopedic Surgery, Shiga University of Medical Science, Setatsukinowa-cho, Otsu 520–2192, Japan d Graduate School of Home and Economics, Kyoto Women’s University, 35 Imakumano-Kitahiyoshi-cho, Higashiyama-ku, Kyoto 605–8501, Japan Received 1 May 2006; accepted 24 August 2006 Available online 15 September 2006
Abstract Magnetic resonance (MR) imaging using super-paramagnetic iron oxides (SPIOs) is a powerful tool to monitor transplanted cells in living animals. However, since SPIOs are negative contrast agents it is difficult to track transplanted cells in bone and cartilage that originally display low signals. In this study, we examined the feasibility of tracking with fluorescein isothiocyanate (FITC)-labeled poly-Llysine-CF3 (PLK-CF3) using mouse ATDC5 cells, a stem cell line of bone and cartilage cells. FITC-labeled PLK-CF3 was easily internalized by ATDC5 cells by adding it into culture medium. No acute or long-term toxicities were seen at less than 160 mg/ml. Labeled cells transplanted into the cranial bone of mice were detected for at least 7 days by MR images. FITC-labeled PLK-CF3 is a useful positive contrast agent for MR tracking in bone and cartilage. r 2006 Elsevier Ltd. All rights reserved. Keywords: Bone; Cartilage; Cell therapy; Molecular imaging; Magnetic resonance; Transplantation
1. Introduction Cell replacement therapy, using several kinds of cells including embryonic and adult stem cells, is applied to clinical and pre-clinical use [1,2], particularly in the field of orthopedics [3,4]. It is of great importance to be able to non-invasively track transplanted cells in vivo. Several current methods include positron emission tomography [5–7], fluorescent microscopy [8] and magnetic resonance (MR) imaging [9–13]. Magnetic labeling, commonly with super-paramagnetic iron oxides (SPIOs) [11,14] is a key step in MR tracking. One of its limitations is that although SPIOs have high sensitivity, MR signals in the surrounding areas are reduced [15]. Therefore, it is difficult to track cells in the bone and cartilage, which initially display low signals. In order to solve this issue, positive MR materials are Corresponding author. Tel.: +81 77 548 2328; fax: +81 77 548 2402.
E-mail address: [email protected] (I. Tooyama). 0142-9612/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2006.08.050
needed. Gadolinium is often used as a positive contrast agent [16–19]. However gadolinium may be toxic and can be retained in tissues for a long time after cell death. More recently, perfluoropolyether has been used as a positive contrast agent [20]. 19F displays positive signals but since it is present at a very low level in the body, the signal-to-noise ratio is very high. Therefore, we chose a candidate, N-4(trifluoromethoxy)-benzylated poly-L-lysine (PLK-CF3), as a positive contrast agent and examined the feasibility of using it for MR tracking of the cartilage stem cell line, ATDC5 cells. 2. Materials and methods 2.1. Synthesis of PLK-CF3 We used three commercially available poly-L-lysines with molecular weights 1–4, 5–15 and 15–30 kDa. We prepared the fully PLK-CF3 from poly-L-lysine of molecular weight 1–4 kDa, as follows. A preliminary experiment showed that the fully PLK-CF3 was obtained as a sole product and the material was soluble in methanol, ethanol, ethyl acetate,
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Fig. 1. Structure of PLK-CF3. 4-(trifluoromethoxy-9-benzyl bromide and/or fluoresceinisothiocyanate are bounded to n-terminal residues (Rn) of poly-Llysine.
chloroform and dimethyl sulfoxide, but was insoluble in water. Thus, we prepared benzylated poly-L-lysine with about half of the amino groups in the molecule (Fig. 1). For the synthesis of PLK-CF3, 100 mg of poly-L-lysine hydrobromide was dissolved in a mixture of water (0.5 ml) and methanol (0.5 ml). To the mixture was added a solution of 72 mg of 4-(trifluoromethoxy)benzyl bromide in methanol (0.5 ml) and 130 mg of solid potassium carbonate, in turn, and the mixture stirred for 19 h at room temperature. Ethanol (15 ml) was added to the mixture and the insoluble material removed by filtration. Ethanolic hydrogen chloride was added to the filtrate and the mixture concentrated to about 2 ml. The required PLK-CF3 was precipitated out by adding 50 ml of diethyl ether to the solution. For labeling of PLK-CF3 with fluorescein isothiocyanate (FITC), a mixture of PLK-CF3 (1 mol), FITC (0.1 mol) and tributylamine (1.2 mol) in methanol was stirred for 5 h at room temperature. The FITC labeled compound was obtained by addition of diethyl ether to the mixture. In our FITC-labeled PLK-CF3, half of the amino groups were 4-(trifluoromethoxy)benzylated and 10% of the amino groups were combined with FITC (Fig. 1). Some PLK-CF3 was labeled with Cy5.5 (Amersham Biosciences UK Limited, UK) instead of FITC because Cy5.5 displayed good permeability in tissues.
2.2. Fluorescent labeling and analysis of cultured cells Murine ATDC5 cells were obtained from the RIKEN cell bank (Tsukuba, Japan). ATDC5 cells were cultured in maintenance medium consisting of a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (Sigma, St. Louis, MO) consisting of 5% fetal bovine serum (Sigma), antibiotic–antimycotic (Nacalai Inc., Osaka, Japan) 100 units/ml penicillin, 100 mg/ml streptomycin, 10 mg/ml human transferrin (Roche Applied Science, Mannheim, Germany). Cells were maintained at 37 1C in humidified 5% CO2, 95% air atmosphere. Just before cells reached confluency, we added FITC-labeled PLK-CF3 into the culture medium at 5, 10, 20, 40, 80, 160 or 320 mg/ml. The incubation time also varied from 2 to 48 h. After incubation, we washed the culture dishes with 10 mM phosphate buffered saline (PBS), pH 7.4 and fixed cells for 10 min with 4% paraformaldehyde (PFA) in 0.1 M phosphate
buffer, pH 7.4, at room temperature. After washing the culture dishes with PBS, fluorecent signals were observed by fluorecent microscopy (IX 70, OLYMPUS Co., Tokyo, Japan). The images were analyzed by the Meta Morph imaging system (OLYMPUS Co., Tokyo, Japan). The labeling intensity was calculated using the following formula: Labeling intensity ¼ (fluorescent intensity) (labeling area). Cell toxicity was investigated using trypan blue staining after incubation for 24 h with different doses of FITC-labeled PLK-CF3. The ratio of living cells to total cells was counted. For measuring long-term toxicity, we incubated ATDC5 cells for 24 h with different doses of FITClabeled PLK-CF3. After washing with PBS, cells were recovered using 0.25% trypsin into Flacon tube, and plated in 60 mm culture dish. We counted cell numbers in each dish at days 4 and 7 of incubation.
2.3. MR imaging of PLK-CF3 in the cranial bone of living mouse Experimental procedures were approved by the Committee on Animal Care of the Shiga University of Medical Science. Five male ICR mice (40–50 g) were used. ATDC5 cells were labeled for 24 h with 80 mg/ml of FITC-labeled PLK-CF3. After washing with PBS, cells were harvested using 0.05% trypsin (Nacalai Inc., Osaka, Japan). Labeled cells (2 106–6 106) were resuspended in a mixture of Cellmatrix (Nittagelatin Co., Osaka, Japan) and culture medium at a ratio of 8:2 at a final concentration of 1 107 labeled cells/ml of the gel mixture. Cranial skins of mice were opened under anesthesia (sodium pentobarbital 50 mg/ml, i.p.). The cranial bone of each mouse was defected with a reamer and the defected area was covered with Cellmatrix containing labeled cells. ATDC5 transplanted in the cranial bone was confirmed by fluorescent microscope (Lightools Macro-Imaging System plus cooled, Lightools Research Co., Encinitas, CA, USA), then applied to MR imaging measurements. On days 1, 7 and 21 after transplantation, MR images were acquired with a 7 T Unity Inova MR scanner (Varian, Palo Alto, CA). A surface coil 20 mm in diameter, which can be tuned to both 1H and 19F frequencies (300 and 282 MHz), was used for signal acquisition. First, a gradient echo 1 H image of the mouse brain was acquired in the axial plane. MR imaging parameters were with 100 ms repetition time (TR), 5 ms echo time (TE),
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451 flip angle, 1 mm slice thickness, 35 35 mm2 field-of-view (FOV) and 256 256 matrices. Subsequently, 19F chemical shift imaging (CSI) data was acquired in 35 35 mm2 FOV, 50 ms TR and 1024–2048 acquisitions for 8 8 phase encoding steps. The raw data was processed by 3D-Fourier transformation with zero filling and converted to 32 32 spectral data sets. The FITC-labeled PLK-CF3 image was constructed by extracting the signal intensities of FITC-labeled PLK-CF3 in individual pixels. During the in vivo nuclear magnetic resonance (NMR) measurement, general anesthesia was maintained with intermittent infusion of sodium pentobarbital through a polyethylene tube inserted intraperitoneally.
2.4. Histological examination in the cranial bone After obtaining MR images, some mice were deeply anesthetized with pentobarbital (70 mg/kg) and the aorta was perfused with approximately 20 ml of 10 mM PBS followed by 50 ml of ice-cold 4% PFA in 0.1 M phosphate buffer (pH 7.4). After perfusion and fixation, the cranial bone was dissected out and post-fixed for 2–7 days with the same fixative. The cranial bone was then replaced for 7 days in ice-cold 0.1 M phosphate buffer containing 10% EDTA for de-calcification. After cryoprotection with 15% sucrose, the cranial bone was cut into 20 mm thick sections in a cryostat. The sections were then stained with hematoxyline–eosine.
3. Results 3.1. Structure of PLK-CF3 and its
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F NMR spectra
Fig. 2A shows the 19F NMR spectrum of PLK-CF3 prepared from poly-L-lysine with molecular weights of 1–4 kDa. The 19F-NMR spectrum showed a singlet peak at d-59.3 ppm (Fig. 2A). Figs. 2B and C demonstrate the 19 F-NMR spectra of PLK-CF3 prepared from poly-Llysine with molecular weights of 5–15 and 15–30 kDa, respectively. Their 19F NMR spectra showed a singlet peak around d-59.3 ppm and a broad peak at d-59.9 ppm. The broad signal was about two times stronger than the singlet peak. The 19F-NMR spectrum of FITC-labeled PLK-CF3 (MW 1-4 kDa) showed a singlet peak at d-59.3 ppm with a broad signal around d-59.9 ppm (Fig. 2D). But the broad peak was smaller than of unlabelled PLK-CF3 (MW 5–15 or 15–30 kDa).
Fig. 2. 19F-NMR spectra of PLK-CF3 with a MW of 1–4 kDa (A, D), 5–15 kDa (B), and 15–30 kDa (C). D; FITC labeled PLK-CF3 with a MW of 1–4 kDa.
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3.2. Labeling of ATDC5 cells with PLK-CF3 When FITC-labeled PLK-CF3 was added to the culture medium, the peptide was easily internalized by mouse ATDC5 cells (Fig. 3). Confocal microscopy revealed that most FITC signals were observed mainly in the cytoplasm. The labeling intensity of ATDC5 cells increased with increasing FITC dose and reached a maximum at 80 mg/ml (Fig. 4). At this concentration, the ratio of labeled cells per total cells was more than 99%. The labeling intensity was decreased at a dose of 320 mg/ml (Po0:01, Fig. 4), suggesting that the label was toxic at this dose. Examination of cell viability confirmed this, with a significant
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decrease in the number of living cells to 77.2% of the control at 320 mg/ml (Po0:01, Fig. 5). In order to assess long-term toxicity, we examined proliferation rates of ATDC5 cells after incubation with 0, 20, 40, 80, 160 and 320 mg/ml of FITC labeled PLK-CF3 (Fig. 6). Compared with unlabeled cells, cell number was significantly decreased at 320 mg/ml (Po0:05, Fig. 6). When the concentration of FITC labeled PLK-CF3 was adjusted to 20 mg/ml, the labeling intensity also increased time-dependently and reached a maximum at 24 h (Fig. 7). 3.3. MR imaging of transplanted ATDC5 cells Cultured ATDC5 cells were labeled for 24 h with 80 mg/ml of FITC-labeled PLK-CF3 or Cy5.5-labeled PLKCF3. Then, 3 106 cells were transplanted into the cranial bone of mice (Fig. 8A), respectively. 19F-MRI successfully detected 19F signals in the transplanted area in the mouse
Fig. 3. Confocal microscopic observations of ATDC5 cells incubated for 24 h with 20 mg/ml of FITC-labeled PLK-CF3 (MW 1–4 kDa).
Fig. 4. Relative fluorescent labeling intensity of ATDC5 cells incubated for 24 h with different doses of FITC-labeled PLK-CF3 (MW 1–4 kDa), where the value at 80 mg/ml was taken as 100%. Bars indicate standard deviations. Po0:05 compared with the value at 80 mg/ml.
Fig. 5. Cell toxicity of PLK-CF3. The ratio of living cells per total cells incubated for 24 h with a various concentration of PLK-CF3 (MW 1–4 kDa). Bars indicate standard deviations. Po0:01 compared with any other concentrations.
Fig. 6. Longitudinal proliferative study of labeled and unlabeled cells. ATDC5 cells were incubated for 24 h with various concentrations of PLKCF3 (MW 1–4 kDa) and cell number was counted at 4 and 7 days after treatment. Po0:05 compared with the value of unlabeled cells.
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cranial bone (Fig. 8A). On the day of transplantation, the transplanted cells were clearly detected by 19F-MRI (Fig. 8B) and fluorescent microscopy (Fig. 8C) in living animals. But MR signals gradually decreased with time, and were able to be detected until 7 days after transplantation (Fig. 9). Figs. 10A and B show the chemical shift of
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F-NMR spectra obtained from ATDC5 cells in a cranial bone at 1 day and 7 days after the implantation, respectively. The signal intensity seems to be decreased during the 7-day period. Histological examination revealed that ATDC5 cells were maintained in transplanted gel tissue (Fig. 11). 4. Discussion 4.1. Structure of PLK-CF3 for
Fig. 7. Relative fluorescent intensity of ATDC5 cells incubated for different times with 20 mg/ml of FITC-labeled PLK-CF3 (MW 1–4 kDa). The value at 24 h was taken as 100%.
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F-MRI
In this study, we examined PLK-CF3 and FITC-labeled PLK-CF3 as contrast agents for MR imaging. Signals of 19 F-MRI became clearly as more fluorines combined with PLK. However, if all animo groups of the compound were combined with 19F, then the compound was not watersoluble. We investigated various ratios and determined that 50% and 10% of all amino groups should be combined with CF3 and FITC. As the molecular weight of PLK increased the sharpness of the singlet peak assigned to the trifluoromethyl group decreased. This indicates that the free rotation of the
Fig. 8. Comparison of implanted area (black arrow in A) in a mouse cranial bone and 19F-MRI signals (white arrow in B) and Cy5.5 fluorescent images (C).
Fig. 9. Time curse of proton image (A, D), 19F-MRI signals (B, E) and the merged images (C, F) of ATDC5 cells in the cranial bone implanted after 1 day (A, B, C) and 7 days (D, E, F).
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agents that may have some adverse effects. The labeling efficiency of FITC labeled PLK-CF3 was the maximal at a concentration of 80 mg/ml for a 24 h incubation. No toxicity was seen under these conditions, which are also suitable for other cells such as mesenchymal stem cells and neural cells (data not shown). In order to examine long-term toxicity, we followed proliferation 7 days after incubating cells with various concentration of FITC labeled PLK-CF3. Cells incubated with less than 160 mg/ml FITC labeled PLK-CF3 displayed the same proliferation profile as unlabeled cells. However, there was a significant decrease in the rate of proliferation at 320 mg/ml of FITC labeled PLK-CF3 as well as signs of acute toxicity. Thus, FITC labeled PLK-CF3 should be used at a concentration of less than 160 mg/ml. 4.3. Cell tracking Fig. 10. Chemical shift spectra of 19F-NMR obtained from ATDC5 cells in the cranial bone at 1 day (A) and 7 days (B) after the implantation. The same mouse as shown in Fig. 9.
Fig. 11. Histological examination of transplanted cells 7 days after transplantation. Transplanted cells were observed in the cranial bone (arrow). Bar ¼ 50 mm.
trifluoromethyl group of the smaller PLK-CF3 is entirely free, but some of the trifluoromethyl group of the larger PLK-CF3 cannot rotate freely inside the molecule. From our results, we suggest that CF3 and FITC should be combined to 50% and 10% of amino groups, respectively, of PLK with molecular weights of 1–4 kDa. 4.2. The labeling efficiency and toxicity of FITC-labeled PLK-CF3 This study demonstrated that ATDC5 cells were easily labeled with FITC-labeled PLK-CF3 by just adding the peptides into the culture medium. This is beneficial for possible clinical and non-clinical applications because it is a simple procedure, which does not requires any transfection
In this study, 19F MRI successfully detected ATDC5 cells transplanted into the cranial bone for 1 week. So far, only perfluoropolyether has been reported as a material for cell tracking with 19F MRI [20]. Thus FITC-labeled PLKCF3 is the second material to be described for cell tracking with 19F MRI. Transplanted cells labeled with FITC-labeled PLK-CF3 were also detected by fluorescent microscope. Since fluorescent materials were easily bound to amino-group of PLK-CF3, this reagent has the potential to be used not only for MR tracking but also photo tracking. The signal intensity of FITC-labeled PLK-CF3 gradually decreased after transplantation. Chemical shift spectra of 19 F-NMR obtained from ATDC5 cells implanted in the cranial bone showed that the signal intensity seemed to decrease by about half over 7 days. It is possible that this may be due to the death and subsequent removal of transplanted cells from the cranial bone. However, this seems unlikely as histological examination showed that transplanted cells remained in gels at even 7 days after transplantation. The signal intensity in a single cell diminishes when cells divide. However, since the cells were packed in a gel, the effect of cell division should be minimal. FITC-labeled PLK-CF3 may have been metabolically degraded in the transplanted cells. Compared with SPIOs, FITC-labeled PLK-CF3 is useful for cell tracking for a short time and is not appropriate for cell tracking for longer periods. This disadvantage is also an advantage because the effect of FITC-labeled PLK-CF3 on cells is only for a short time. Further study will be needed to improve the tracking time with FITC-labeled PLK-CF3. 4.4. Clinical utility of cell tracking with FITC-labeled PLK-CF3 Tissue engineering techniques including stem cell therapy have been explored to repair cartilage and bone defects [21]. However, it is difficult to observe transplanted cells
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non-invasively. MR tracking using SPIOs is a powerful tool for cell tracking, but it is difficult to track cells in bone and cartilage, which initially display low signals. Since PLK-CF3 is a positive contrast agent, it is suitable for MR tracking in bone and cartilage cells. In addition, unlike with SPIOs, MR signals can be obtained from surrounding areas when using PLK-CF3. Thus, we can collect information about cell localization as well as environment alterations such as temperature, pH and some metabolites. Culture cells can easily be labeled by adding PLK-CF3 into the medium. This is a tremendous advantage for clinical use as it dose not require the use of transfection agents. So far, the sensitivity of PLK-CF3 is low compared with SPIOs. However, if the sensitivity can be increased to detect a single or a few cells, MR tracking can be used to observe transplanted cells distributed in the body including bone, cartilage and bone marrow after administration of therapeutic cells into vessels. Further study will be needed to increase the sensitivity. 5. Conclusion We successfully obtained MR images of ATDC5 cells transplanted in the cranial bone using PLK-CF3 and 19F MRI. ATDC5 cells were easily labeled with PLK-CF3 just by adding the peptides into the culture medium. Since fluorine shows a positive MR signal, it is suitable for tissues such as bone, cartilage and liver that originally display low proton signals. References [1] Conrad C, Huss R. Adult stem cell lines in regenerative medicine and reconstructive surgery. J Surg Res 2005;124:201–8. [2] Sylvester KG, Longaker MT. Stem cells: review and update. Arch Surg 2004;139:93–9. [3] Christophel JJ, Chang JS, Park SS. Transplanted tissue-engineered cartilage. Arch Facial Plast Surg 2006;8:117–22. [4] Gimeno MJ, Maneiro E, Rendal E, Ramallal M, Sanjurjo L, Blanco FJ. Cell therapy: a therapeutic alternative to treat focal cartilage lesions. Transplant Proc 2005;37:4080–3. [5] Bengel FM, Schachinger V, Dimmeler S. Cell-based therapies and imaging in cardiology. Eur J Nucl Med Mol Imaging 2005; 32(Suppl. 2):S404–16. [6] Kirik D, Breysse N, Bjorklund T, Besret L, Hantraye P. Imaging in cell-based therapy for neurodegenerative diseases. Eur J Nucl Med Mol Imaging 2005;32(Suppl. 2):S417–34.
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