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Experimental Studies |
1 From the Experimental Neuroimaging Section, Laboratory of Diagnostic Radiology Research, Clinical Center, National Institutes of Health, Bldg 10, Rm B1N256, 10 Center Dr, MSC 1074, Bethesda, MD 20892-1074. Received May 30, 2002; revision requested July 29; final revision received October 9; accepted November 5. Address correspondence to J.A.F. (e-mail: jafrank@helix.nih.gov).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONADDENDUMREFERENCES |
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MATERIALS AND METHODS: Three TAs were incubated with ferumoxides and MION-46L in cell culture medium at various concentrations. Human mesenchymal stem cells, mouse lymphocytes, rat oligodendrocyte progenitor CG-4 cells, and human cervical carcinoma cells were incubated 2–48 hours with 25 µg of iron per milliliter of combined TAs and SPIO. Cellular labeling was evaluated with T2 relaxometry, MR imaging of labeled cell suspensions, and Prussian blue staining for iron assessment. Proliferation and viability of mesenchymal stem cells and human cervical carcinoma cells labeled with a combination of TAs and ferumoxides were evaluated.
RESULTS: When ferumoxides-TA or MION-46L–TA was used, intracytoplasmic particles stained with Prussian blue stain were detected for all cell lines with a labeling efficiency of nearly 100%. Limited or no uptake was observed for cells incubated with ferumoxides or MION-46L alone. For TA-SPIO–labeled cells, MR images and relaxometry findings showed a 50%–90% decrease in signal intensity and a more than 40-fold increase in T2s. Cell viability varied from 103.7% ± 9 to 123.0% ± 9 compared with control cell viability at 9 days, and cell proliferation was not affected by endosomal incorporation of SPIO nanoparticles. Iron concentrations varied with ferumoxides-TA combinations and cells with a maximum of 30.1 pg ± 3.7 of iron per cell for labeled mesenchymal stem cells.
CONCLUSION: Magnetic labeling of mammalian cells with use of ferumoxides and TAs is possible and may enable cellular MR imaging and tracking in experimental and clinical settings.
© RSNA, 2003
Index terms: Cell labeling • Experimental study • Iron • Magnetic resonance (MR), experimental studies • Stem cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONADDENDUMREFERENCES |
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Dextran-coated SPIO nanoparticles are MR contrast agents approved by the Food and Drug Administration (FDA) for use in hepatic reticuloendothelial cell imaging, and ultrasmall SPIOs (USPIOs) are in phase III clinical trials for use as blood pool agents or for use with lymphography (15–17). These contrast agents cannot be used to efficiently label stem cells or other mammalian cells (18,19) in vitro in their native unmodified form (6–8,10,20). By conjugating antigen-specific internalizing monoclonal antibodies to the surface dextran coating, however, cells can be magnetically labeled during their normal expansion in culture medium (6). This magnetic labeling approach is limited, however, because it requires the availability of an internalizing monoclonal antibody that recognizes a specific cellular surface antigen.
Other approaches have involved the synthesis and modification of USPIO particles such as MION-46L (CMIR, Charleston, Mass) by cross-linking the dextran (CLIO) and conjugating with short human immunodeficiency virus–transactivator transcription (Tat) proteins (MION-Tat or CLIO-Tat), thereby facilitating incorporation into the cells (7,8,10). Ex vivo MR imaging performed within 3 days following transplantation with CLIO-Tat–labeled cells has demonstrated in vivo migration in tissue (10). However, since the Tat protein has an affinity for the nucleus, it is possible that during biodegradation of the CLIO-Tat particle, reactive iron species may be released temporarily. Reactive iron species could then catalyze the formation of hydroxyl free radicals through a Haber-Weiss reaction (21). Subsequent reactions may initiate lipid peroxidation, the formation of lipid hydroperoxides, which can destroy membrane structure and function, as well as lead to damage of proteins and DNA within the nucleus (21).
Over the past 10 years, there has been substantial research in the development of new TAs, including cationic peptides, dendrimers, polyamines, and lipids for nonviral transfection of DNA into the nucleus (13,14,22,23). These TAs are developed to overcome the problem of the endosomal capture of the TA-DNA complex and inefficient release of the targeted material into the nucleus. TAs are macromolecules with molecular weights from 1 to well over 100 kd that possess an electrostatic charge. On the basis of efficient labeling of mammalian cells with the dendrimer-coated MD-100 (9), we hypothesized that commercially available TAs would coat by means of electrostatic interaction (24) with dextran-coated SPIO MR contrast agents and chaperon these nanoparticles into cells. The purpose of our study was to label both mammalian and stem cells by combining commercially available TAs with the dextran-coated SPIO MR contrast agents ferumoxides (Feridex; Berlex, Wayne, NJ) (FDA approved) or MION-46L (an experimental contrast agent).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONADDENDUMREFERENCES |
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The TAs used in this study were Superfect (Qiagen, Valencia, Calif), a low-generation heat-activated dendrimer; Lipofectamine Plus (Invitrogen Life Technologies, Gaithersburg, Md), a liposomal agent; and poly-L-lysine (PLL) (Sigma, St Louis, Mo), a polyamine. All TAs were mixed with ferumoxides and MION-46L for 60 minutes in cell culture medium at room temperature on a rotating shaker. For PLUS/lipofectamine, lipofectamine was added to the media only for the final 15 minutes of the 1 hour in which PLUS was combined with ferumoxides. PLL at 1.5 mg/mL or various dilutions of TAs from stock solutions as supplied by the manufacturer was mixed with SPIO nanoparticles to label cells. Optimal protocols for magnetic labeling varied, depending on the type of cell and TA. Since MION-46L is neither FDA approved nor commercially available, use of this agent was studied only for HeLa cells. Cells were washed three times with phosphate-buffered saline to remove ferumoxides-TA or MION-46L–TA and were then trypsinized before making cytospin slides. For Prussian blue staining, cells were fixed with 4% glutaraldehyde, washed, incubated for 30 minutes with 2% potassium ferrocyanide (Perls reagent) in 6% hydrochloric acid, washed, and counterstained with nuclear fast red.
For diaminobenzide-enhanced Prussian blue staining, slides were reacted with unactivated and activated (containing 0.03% hydrogen peroxide) 0.014% diaminobenzide for 15 minutes each, washed three times with Prussian blue stain, and then counterstained with nuclear fast red. To sample a broad range of cellular properties, thereby demonstrating the nonspecific nature of the ferumoxides-TA labeling technique, cell lines were chosen with the following characteristics: predominantly nonphagocytic, different species (human, rat, and mouse), cell type (stem cells, hematopoietic-derived cells, and commonly used tumor cells), cell size, and culture properties (growth in confluent or in suspension). Three TAs (dendrimer, liposomal agent, and polyamine) were selected to show the nonspecific electrostatic interaction with the SPIO or USPIO that was used for cell labeling by means of endocytosis.
Assessment of Toxicity and Proliferation
For assessment of toxicity and normal proliferation of ferumoxides-TA–labeled cells, MSC and HeLa cells were evaluated by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Roche Molecular Biochemicals, Indianapolis, Ind). MSC and HeLa cells were labeled in triplicate with ferumoxides and ferumoxides-TA combinations (PLL, PLUS/lipofectamine, and Superfect) in 96 flat-bottom well plates with 5 x 103 cells per well (for MSCs) and in 24 well plates with 2 x 104 cells per well (for HeLa cells). After cells were labeled and washed three times with Prussian blue stain, MTT was added at a final concentration of 0.5 mg/mL of medium, and cells were incubated for 4 hours at 37°C and 5% CO2. An equal volume of solubilized solution was then added (according to the manufacturer), followed by overnight incubation at 37°C and 5% CO2. The absorbance of the formazan product was then measured at a wavelength of 570 nm, with 750 nm as the (subtracted) reference (9).
Pulse chase experiments were performed with ferumoxides-TA–labeled MSC and HeLa cells to determine long-term cell ability to continue to proliferate. MSCs were incubated for 4 hours and HeLa cells for 24 hours with various ferumoxides-TA complexes, washed three times with Prussian blue stain, and then incubated in fresh culture media for 9 days. MTT analysis of labeled and unlabeled control cells was then determined as described above. To determine if ferumoxides-TA would induce apoptosis (programmed cell death), labeled MSC and HeLa cells and corresponding unlabeled control cells were harvested at day 3 (MSC) and day 24 (HeLa) following a pulse chase study and washed twice with ice-cold phosphate-buffered saline (Prussian blue stain) and then resuspended in annexin-binding buffer (Vybrant apoptosis assay kit; Molecular Probes, Eugene, Ore) at 1 x 106 cells per milliliter. Five microliters of Alexa fluor 488 annexin V and 1 µL of red fluorescent propidium iodide were added to 100 µL of cell suspension and incubated for 15–20 minutes at room temperature in the dark. After incubation, 400 µL of annexin-binding buffer was added to the cell suspension, and the cells were analyzed with a fluocytometer (FACSCalibur; BD, Palo Alto, Calif) by using emission at 530 nm (FL1) and 575 nm (FL3). CellQuest software (BD) was used to analyze the percentage of apoptotic cells.
MR Relaxometry
MR relaxometry was performed (H.A.Z., B.R.M.) to assess ferumoxides-TA cellular uptake. Ferumoxides-TA–labeled cells or cells incubated with ferumoxides alone were suspended in 500 µL 4% gelatin in Prussian blue stain at 60°C and then chilled at 3°C to allow the suspensions to solidify. T2 relaxometry was performed by using a custom-designed variable-field MR relaxometer at 1.0 T at 23°C. T2s were collected by using a Carr-Purcell-Meiboom-Gill pulse sequence with 500 echoes and an interecho time (tau) of 1 and 5 msec.
MR Imaging
MR imaging of cell suspensions in gelatin was performed at 1.5 T (Signa, Echospeed; GE Medical Systems, Milwaukee, Wis) by using a 5-inch (12.7-cm) receive-only surface coil. MR imaging was performed by using a fast spin-echo (repetition time msec/echo time msec, 3,000/45; echo train length, eight) sequence and a multisection gradient-echo (300/20; flip angle, 20°) sequence. All images were obtained with a matrix size of 256 x 160 with two signals acquired, a section thickness of 2 mm, and a 6–8-cm field of view. Signal intensities of the unlabeled and ferumoxides-TA–labeled cells were measured separately by two authors (B.K.L., J.A.F.) by using a circular 35-mm2 region of interest and the software available on the MR unit. Percentage change in signal intensity was normalized to the unlabeled cells by using the following equation:
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Iron Content
The iron content of the labeled cells was assessed by using a Ferrozine (J.T. Baker, Phillipsburg, NJ)-based spectrophotometric assay performed by using a UV spectrophotometer (model 1601; Shimadzu, Columbia, Md) and MR relaxometry, as described previously (9,25). In brief, cell suspensions were first dried at 110°C overnight and then completely digested in a mixture (500 µL) of perchloric and nitric acid at a 3:1 ratio. The samples were digested for at least 3 hours at 60°C by using a heating block. For these 500-µL samples, 1/T1 and 1/T2 were then measured at room temperature and at 0.1 and 1.0 T by using an MR relaxometer as described above. Iron concentration in the sample was calculated from a standard curve, which was derived from calibration standards of ferrous chloride containing 0.01–10 mmol/L iron in the same acid mixture. The iron content was further determined and validated with use of a Ferrozine-based spectrophotometric assay by using triplicate 50-µL samples from the acid-digested cell suspension. Average iron content per cell (mean ± SD) was obtained from the mean values determined from relaxometry and Ferrozine-based assays, divided by the cell number in each sample.
Statistical Analysis
Data are expressed as mean ± SD from three to nine data samples, unless stated otherwise. MTT-based toxicity and proliferation data are expressed as percentage of the corresponding unlabeled control cells, and analysis of variance (Statview version 4.51; Abacus, Berkeley, Calif) was performed to test for statistical significance. Comparisons of iron concentrations between unlabeled and labeled cells were performed by using the unpaired Student t test. Correlation between signal intensity on MR images and 1/T2 was performed with use of nonlinear regression analysis by using a standard spreadsheet program (Ca-Cricket Graph III; CA Associates, Islandia, NY). A P value of <.05 was considered to indicate a statistically significant difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONADDENDUMREFERENCES |
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Although there was increased formazan absorbance value observed in labeled cells, no significant (P = .33) difference was found among the unlabeled control cells and labeled cells. There was no difference in the rate of apoptosis for ferumoxides-PLL–labeled (1:1,250) MSCs and unlabeled cells (labeled, 1.4% vs unlabeled, 3.1%) or for ferumoxides-PLL–labeled (1:1,250) and unlabeled HeLa cells (labeled, 18.6%, vs unlabeled, 20.8%) evaluated on day 24.
MR Relaxometry
The 1/T2s for unlabeled control MSCs, HeLa cells, mouse lymphocytes, and CG-4 cell suspensions incubated with ferumoxides-TA for 2–48 hours are shown in Figure 3. Overall, a dramatic increase was observed in the 1/T2 of ferumoxides-TA–labeled cell lines compared with that of unlabeled control cells (see Table for cell densities in suspensions). The maximal change in 1/T2 was as follows: HeLa cells, from 1.28 sec-1 (unlabeled) to 87.2 sec-1 (ferumoxides-Superfect [1:2,000]); CG-4 cells, from 1.93 sec-1 (unlabeled) to 23.25 sec-1 (ferumoxides-PLUS/lipofectamine [1:125/1:250]); mouse lymphocytes, from 0.9 sec-1 (unlabeled) to 10.2 sec-1 (ferumoxides-PLL [1:2,500]); and MSCs, 2.77 sec-1 (unlabeled) to 80.6 sec-1 (ferumoxides-Superfect [1:1,250]). In addition, there was clearly an echo time dependence in the T2 as tau increased from 1 to 5 msec. These results indicate that the iron nanoparticles are clustered within the endosomes and cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONADDENDUMREFERENCES |
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Recently, Frank et al (26) observed a decrease in T1s and T2s with ferumoxides and, to a lesser extent, with MION-46L, when these agents are combined at various dilutions with PLL, PLUS/lipofectamine, or Superfect. These authors reported up to a 30%–90% decrease in T1s and T2s for various ferumoxides-TA or TA-MION-46L in solution with TA shielding the normal water exchange with the SPIO nanoparticle for effective dipole-dipole T1. The increase in 1/T2 indicates that there is an appreciable inner sphere effect, presumably exerted by TA, coating the nanoparticle and shielding the water from its surface (26). These results indicate that under appropriate conditions, highly surface-charged macromolecular TAs undergo electrostatic interactions with the existing surface charges on ferumoxides or MION-46L nanoparticles and then self-assemble into complexes (26). However, the shielding of the water from the surface of the dextran-coated SPIO nanoparticles does not necessarily translate into cellular magnetic labeling efficiency. The structure and stability of the self-assembling ferumoxides-TA or MION-46L–TA complexes, as with DNA-TA, will vary according to the surface charge of the macromolecules, concentration of the nanoparticle and TA, pH of the solution, and time (27). We observed variability in intracytoplasmic uptake of the SPIO particles into endosomes, depending on the class and relative ratio of TA to USPIO.
TAs are commonly used to shuttle DNA into cells for incorporation into the host cell nucleus. TAs are mixed with the DNA, and under appropriate conditions (ie, temperature, concentration, pH), through electrostatic interactions, the two self-assemble into DNA-TA complexes (27). One reason why there are a variety of TAs and protocols used for DNA incorporation into cells is the need to overcome the problem of the endosomal capture of the DNA-TA complex and inefficient release of the targeted material into the nucleus. However, this undesirable result of DNA-TA complexes being sequestered in endosomes is actually the preferred outcome for labeling cells with MR contrast agents. It is believed that the mechanism of endosomal uptake of the SPIO-TA is similar to MD-100 uptake, a TA-coated SPIO nanoparticle (9). Magnetodendrimers are thought to interact with the cell surface through electrostatic (van der Waals) bonds, resulting in a bending of the membrane that initiates endocytosis (9,14).
It was not the intent of this work to survey and evaluate all possible protocols for combining all types of commercially available TAs with ferumoxides or MION-46L. We have demonstrated effective labeling (ie, ability to alter signal intensity and T2 of cells in suspension) of stem (ie, mesenchymal stem) cells and other cells (ie, lymphocytes, CG-4 cells, and HeLa cells) derived from humans, rats, and mice by means of Prussian blue staining, relaxometry, and MR imaging. The results indicate that with relatively low concentrations of iron (ie, 25 µg of iron per milliliter) in culture media, cellular labeling with SPIO and USPIO particles can be accomplished. Although there are other reports of using nonmodified ferumoxides or MION-46L to label cells, in general, most nonphagocytic cells do not take up SPIO nanoparticles efficiently or require cells to be exposed to high amounts of iron in culture (19,20,28).
Validation of intracytoplasmatic iron in labeled cells by using simple Prussian blue staining has not been shown in these studies, presumably as a result of lower uptake that is below the threshold of detection with Prussian blue stain. In this study, HeLa cells were incubated with ferumoxides-PLL and MION-46L-PLL under the same conditions (ie, iron concentration, incubation time with cells), and we observed larger numbers (qualitatively) of intracytoplasmic Prussian blue–positive particles in cells labeled with ferumoxides versus those labeled with MION-46L. Although there has been a tendency toward using MION-46L with or without covalently attached monoclonal antibodies or exogenous proteins for cell labeling, it is clear that effective labeling of cells was achieved by using the larger (ie, ferumoxides) dextran-coated SPIO. Because of its larger size and oligocrystallinity, ferumoxides is more magnetic and has a higher 1/T2 than that of MION-46L (29), making it better suited for use as a T2 cellular contrast agent. Ferumoxides-TA did produce qualitatively identical results in HeLa cells, CG-4 cells, and MSCs with intracytoplasmic Prussian blue–positive particles as observed with MD-100 (9). The major advantage of using ferumoxides for labeling cells is that it is an FDA-approved MR contrast agent, and therefore, quality control, sterility, and stability have all been well documented.
The method of magnetic cell labeling with ferumoxides-TA increased the 1/T2s to values comparable to those obtained by using MD-100–labeled cells. Bulte et al (9) reported 1/T2 increases of 1–50 sec-1 and up to 150 sec-1 for CG-4 and HeLa cells, respectively, when labeled with magnetodendrimer. By using approximately the same cell densities, the 1/T2s increased from about 6.0 to 87.2 sec-1 for HeLa cells and from 1.9 to 23.3 sec-1 for CG-4 cells when both cell lines were labeled with ferumoxides-PLUS/lipofectamine. Similar increases in 1/T2s for MSC and mouse lymphocytes were observed. The increase in 1/T2s is clearly reflected in the decrease of signal intensities observed for the cell suspensions on both fast spin-echo and gradient-echo MR images. In most combinations of ferumoxides-TA and cell lines, there is a more than 90% difference between magnetically labeled and unlabeled cells. This level of image contrast is clearly depicted on MR images. Incubation of ferumoxides-TA with the HeLa and CG-4 cell lines resulted in iron concentrations per cell that were similar (ie, 10–20 pg of iron per cell) to those obtained when incubating the cells with either MD-100 or MION-Tat nanoparticles (8,9). As expected, the labeling efficiency as determined by the amount of iron per viable cell varied depending on the experimental protocol used (ie, incubation time, cell type, cell number, and type of TA). The variability in iron concentration per cell in the current study may be due to the initial number of cells in the cultured medium, size of the cells, volume of the cytoplasm in each cell, electrostatic charge of SPIO-TA complexes, and surface charge of cell membranes. Of note, since the iron in the SPIO is biologically available, the ferumoxides-TA complex within the endosomes should be released over time and reused in normal iron metabolic pathways. In addition, the MTT and apoptosis analysis findings demonstrated no effect on cell proliferation and viability when using various complexes for ferumoxides-TA with MSCs or HeLa cells after labeling for 4 hours and incubating cells in fresh media in pulse chase experiments. These findings indicate that even with the incorporation of higher levels of iron into the endosome, the iron was not released into the cytoplasm as a free radical form, which would have been toxic to the cells and increased cell death compared with that in unlabeled cells. These results are similar to those reported for MD-100 (9).
It is important to appreciate that the cell labeling technique described above should serve as a basic protocol for incorporating SPIO into cells for cellular imaging. Experience indicates that optimization of this labeling approach is required, as with other protocols used in molecular biology. The refinements to the protocols depend on experimental conditions and cells being used. We recommend that investigators interested in using this approach for magnetic cell labeling start with ferumoxides-PLL (1:1,000–2,000 dilution of PLL) and co-incubate with cells for at least 6–12 hours, wash cells, and evaluate cell labeling by using Prussian blue stain.
Practical applications: In summary, the combination of FDA-approved dextran-coated SPIO MR contrast agents with TAs resulted in the formation of ferumoxides-TA and MION-46L–TA complexes that allow a universal nonspecific method of magnetically labeling mammalian (stem) cells, regardless of animal origin or species (ie, mouse, rat, or human). This approach does not require novel synthesis or covalent binding of proteins or antibodies to the dextran coating and therefore will provide researchers with a simple and straightforward method of magnetically labeling (stem) cells to track the extent of migration in vivo following implantation or systemic injection. Moreover, since an FDA-approved agent can be used, this method of labeling cells may facilitate the introduction of MR monitoring of the biodistribution of magnetically labeled cells in a clinical setting.
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ADDENDUM |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONADDENDUMREFERENCES |
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FOOTNOTES |
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Author contributions: Guarantor of integrity of entire study, J.A.F.; study concepts, J.A.F.; study design, A.S.A., J.A.F., J.W.M.B.; literature research, J.A.F.; experimental studies, J.A.F., B.R.M., A.S.A., H.A.Z., E.K.J., L.H.B., B.K.L.; data acquisition, B.R.M., A.S.A., H.A.Z., B.K.L.; data analysis/interpretation, A.S.A., J.A.F., J.W.M.B.; manuscript preparation and definition of intellectual content, J.A.F., J.W.M.B.; manuscript editing, J.A.F., J.W.M.B., A.S.A., B.R.M.; manuscript revision/review, J.A.F., J.W.M.B., A.S.A., L.H.B.; manuscript final version approval, J.A.F.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONADDENDUMREFERENCES |
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) and gradient-echo (
) MR images of the gel suspension of unlabeled control cells and ferumoxides-TA-labeled MSCs, HeLa cells, mouse lymphocytes, and CG-4 cells versus the 1/T2s for the suspension. The equations for the nonlinear regression analysis fit and regression r2 values are shown.