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Molecular Imaging |
1 From the Experimental Neuroimaging Section, Laboratory of Diagnostic Radiology Research, Clinical Center, National Institutes of Health, Bethesda, Md. Received February 27, 2004; revision requested May 5; revision received June 2; accepted July 1. Address correspondence to A.S.A., Radiology Research, Henry Ford Health System, 1 Ford Pl, 2F, Box 82, Detroit, MI 48188 (e-mail: saali@rad.hfh.edu).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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MATERIALS AND METHODS: Twenty-five rats (group 1) received intravenous injections of labeled MSCs, and 25 additional rats (group 2) received intravenous injections of ferumoxides-PLL complex only. Complete blood counts, liver and renal function test results, and serum electrolyte and iron concentrations were measured for 42 days after the injections and compared with those measured in five control rats (group 3). To determine the duration of labeled MSCs in the circulation, venous blood was serially drawn from five additional rats (group 4) that were injected with labeled MSCs. Analyses of variance (ANOVA) followed by Fisher protected least significant difference post hoc tests were used to statistically analyze results. P < .05 was considered to indicate significance in all analyses.
RESULTS: Administration of neither labeled MSCs nor ferumoxides-PLL complex had a significant effect on hematologic or blood chemistry indicators of organ function. Of the parameters measured, only hemoglobin concentration and mean corpuscular volume (MCV) in the rats injected with labeled MSCs, as well as MCV and hemoglobin, alkaline phosphatase, aspartate aminotransferase, and direct bilirubin concentrations in the rats injected with ferumoxides-PLL complex, varied significantly during the 42-day postinjection period (P < .05, ANOVA). No other measurements, including serum electrolyte and iron concentrations, changed significantly during the test period (P > .05). Furthermore, injected labeled MSCs had cleared from the peripheral circulation by 15 minutes after injection.
CONCLUSION: Results indicate that infusing cells that are magnetically labeled with ferumoxides-PLL complex into rats does not alter biochemical or hematologic measures of organ function in a clinically relevant or preclusive manner.
© RSNA, 2005
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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The use of ferumoxides combined in a complex with transfection agents has been shown to facilitate effective and efficient labeling of stem cells and other mammalian cells (13,14). However, to our knowledge, the effect of intravenously administered magnetically labeled cells on systemic hematologic and biochemical measures has not been documented. Thus, the purpose of our study was to determine whether ferumoxides–poly-L-lysine (PLL) complex–labeled mesenchymal stem cells (MSCs) or ferumoxides-PLL complex alone intravenously infused into rats alters hematologic, blood chemistry, renal function, and/or liver function measurements.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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Cell Labeling
Human MSCs (Cambrex, Baltimore, Md) were cultured to 80% confluence in a standard MSC growth medium (MSCGM; Cambrex) at 37°C with 5% CO2. To label the MSCs, we combined ferumoxides nanoparticles (Feridex IV; Berlex Laboratories, Wayne, NJ) with PLL (molecular weight, 388 kDa; Sigma, St Louis, Mo), added this mixture (ferumoxides concentration, 50 µg/mL; PLL concentration, 1.5 µg/mL) to the standard MSC growth medium, and then mixed the medium in a rotating mixer for 1 hour. The medium containing the ferumoxides-PLL complex was then added in a 50:50 volume ratio to a standard growth medium and then incubated with the cultured MSCs in a 75-cm2 flask overnight. After overnight incubation, the medium was discarded and the cells were washed twice with sterile phosphate-buffered saline, treated with trypsin, collected, rewashed, and counted to adjust the cell concentration for injection.
Injection of MSCs or Ferumoxides-PLL Complex
The experiment rats were divided into four groups: Groups 1 and 2 consisted of 25 rats each, and groups 3 and 4 consisted of five rats each. All animals were anesthetized with 3 L/min of oxygen plus 3% isoflurane (MDS Matrix, Orchard Park, NY) in an induction chamber and then given 3 L/min of oxygen plus 1.5% isoflurane through a nose cone to maintain anesthesia. A 24-gauge catheter (JELCO, Tampa, Fla) was placed in the tail vein, and 2 x 106 ferumoxides-PLL–labeled MSCs (approximately 10 pg of iron per cell) in 0.5 mL of phosphate-buffered saline, followed by a 0.5-mL flush of sterile 0.9% saline, was injected into the group 1 and group 4 rats (by E.K.J. and A.S.A.).
With use of the same method, the ferumoxides-PLL complex alone in 0.5 mL of saline (40 µg/mL iron, 1.2 µg/mL PLL), followed by a 0.5-mL flush of sterile 0.9% saline, was injected into the group 2 rats. The dose of ferumoxides-PLL complex was adjusted to 20:0.6 µg/mL (ferumoxides-to-PLL concentration) to match the total volume of iron injected into the group 1 and group 4 rats based on the approximate iron concentration per cell (10 pg per cell) and the total number of cells injected (2 x 106 cells). The group 3 animals were handled identically and received only a 0.5-mL injection of saline. All rats in groups 1–3 were returned to their cages and monitored (by E.K.J. and veterinary technicians) throughout the 42-day experiment for obviously abnormal behaviors or changes in body weight that could have indicated illness.
Blood Chemistry and Hematologic Measurements
For hematologic and chemistry analyses, blood was obtained (by E.K.J., A.S.A., L.B.W., and P.A.) at 1, 3, 7, 14, 28, and 42 days after injection from three to five rats that were injected with labeled MSCs (group 1) and from three to five rats that were injected with ferumoxides-PLL complex only (group 2). Blood was also drawn from the five control rats (group 3) for comparisons between experimental measurements and baseline measurements obtained on day 7 (n = 2) and day 26 (n = 3). To draw blood, we anesthetized the rats by using the protocol described for the intravenous injection of labeled MSCs or ferumoxides-PLL complex and deeply sedated the animals—such that they would have no reactions to pain stimuli—by means of intravenous injection of a 40–60 mg/kg dose of phenobarbital. The abdomens of the rats in groups 1–3 were opened by means of a midline incision, and all blood was drawn from each rat with a syringe through a 21-gauge catheter inserted into either the inferior vena cava or the terminal portion of the abdominal aorta. For total and differential blood counts, blood was collected in ethylenediaminetetraacetic acid tubes, and for chemistry and serum iron measurements, blood was collected in tubes that facilitated serum separation. Owing to the large amount of blood drawn (at least 2 mL per rat) and the invasive nature of the procedure, all rats were immediately euthanized with an additional 100–500 mg/kg dose of phenobarbital after blood collection, according to the protocol.
With use of all of the blood samples obtained from the rats in groups 1–3, the following measurements were obtained: complete blood count and differentials (of white blood cells, red blood cells, platelets, monocytes, lymphocytes, eosinophils, and basophils), hematocrit and hemoglobin levels, mean corpuscular volume (MCV), liver function (aspartate aminotransferase [AST], alanine aminotransferase, alkaline phosphatase, bilirubin, albumin, and total protein levels), renal function (electrolyte, blood urea nitrogen, creatinine, and uric acid levels), and serum iron concentrations. Personnel in the section of the clinical pathology laboratory dedicated to animal research at our institution performed the hematologic and blood chemistry analyses.
Determination of Serum Iron Content
MR imaging relaxometry was used to determine the serum iron content in the rats injected with labeled MSCs, the rats injected with ferumoxides-PLL complex only, and the control rats. To perform MR imaging relaxometry, 0.5 mL of serum was placed into glass tubes. The tubes were then put in an oven at 110°C until all liquid had evaporated. The portion of the samples remaining in the tubes was completely digested in 500 µL of a 3:1 perchloric acid–nitric acid mixture at 60°C. MR relaxation rates, 1/T1 and 1/T2 (in seconds–1), were then measured at room temperature and at 1.0 T by using a custom-designed, variable-field MR relaxometer (4) with a Carr-Purcell-Meiboom-Gill pulse sequence. Iron content was extrapolated from a standard curve obtained from FeCl3 digested in the same acid mixture at concentrations of 0.01–10.00 mmol/L.
Determining Duration of MSCs in the Circulation
After anesthesia was induced, as described earlier, a 27-gauge cannula was introduced into the tail veins of the group 4 rats. A total of 2 x 106 magnetically labeled MSCs in 0.5 mL of phosphate-buffered saline were injected (by E.K.J., A.S.A., and G.T.Y.) through the tail vein to determine the circulating duration of the ferumoxides-PLL–labeled MSCs. During the remaining experiments, these rats remained anesthetized with isoflurane. Just before injection of the labeled MSCs, their abdomens were opened for access to the inferior vena cava and a 27-gauge catheter was introduced. One hundred microliters of blood was collected from the inferior vena cava before injection of the cells and at 1, 5, 15, 30, 60, and 120 minutes after injection and placed in a tube with 1 mL of ethylenediaminetetraacetic acid.
The group 4 rats were immediately euthanized with 100–500 mg/kg of phenobarbital after the blood collection at 120 minutes. Red blood cells were lysed by means of adding 2 mL of lysing buffer, the samples were centrifuged, and the supernatant was poured off to remove the lysed red blood cells. Cytospin slides were then created from the remaining cells in each sample, stained with Prussian blue for detection of iron-positive cells, and counterstained with nuclear fast red. On each slide, the iron-positive cells in five randomly selected x40-magnified fields of view were counted manually to determine the relative number of labeled MSCs in the circulating blood at 1, 5, 15, 30, 60, and 120 minutes after injection.
Statistical Analyses
All data are presented as means ± 1 standard deviation. Analysis of variance (ANOVA) was performed with StatView 4.51 software (Abacus Concepts, Berkeley, Calif) to determine if significant differences existed among any blood chemistry and hematologic measurements obtained during the 42-day postinjection period in the group 1 and group 2 rats. P < .05 was considered to indicate significance, and all analyses were followed by a Fisher protected least significant difference post hoc test to compare individual postinjection data points in group 1 with the corresponding data points in group 2 and to compare each data point in groups 1 and 2 with those in group 3 (the control group). Hematologic or blood chemistry values that were obtained from fewer than three samples, as a result of blood clotting and hemolysis, were omitted from statistical analysis and are not reported in this article. All results presented were evaluated by G.T.Y., J.A.F., and A.S.A., and J.A.F. was consulted regarding the statistical methods used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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Intravenous injection of labeled MSCs or ferumoxides-PLL complex appears to have little effect on hematologic and serum biochemical measurements. However, in the current study, some sustained deviations from the control values were seen in certain marker values, specifically hemoglobin concentration and MCV in both the rats injected with labeled MSCs and those injected with ferumoxides-PLL complex, as well as AST, alanine aminotransferase, and alkaline phosphatase in the rats injected with ferumoxides-PLL complex. These results might be explained by the limited sample sizes: Some of the mean values reported were calculated by using samples obtained from only three rats. Furthermore, in an attempt to limit the number of rats euthanized, the control values were obtained only on days 7 and 26 and averaged. Therefore, we were not able to compare each experimental value with a control value obtained on the same day, and, consequently, some sample age–related variability was introduced during the somewhat long 42-day experimentation period. The control values available from the animal supplier were not used in this study because of variabilities resulting from differences in the diet and environment of the rats and in the clinical pathologic analysis techniques used between the supplier setting and our experimental setting. Furthermore, we used only 4–6-week-old (aged 10–16 weeks by the end of study) female rats in this study, whereas the supply company’s control values applied to 11–12-week-old male rats. This difference in sex and substantial difference in age—given the life span of rats—between the animals used to establish the control values and the animals that we used in our experiments led us to discount the supply company’s control data.
Although no AST control value was available for comparative analyses, the AST concentrations for ferumoxides-PLL– injected rats on days 1 and 3 were about 40–60 U/L lower than those on later days and of the MSC-injected rats on all days tested. Therefore, it appears that ferumoxides-PLL injection leads to a transient decrease in AST, but the nature of any correlation between ferumoxides-PLL injection and AST decrease is not clear. It should be noted that although we felt compelled to disregard the supply company’s normal ranges, normal AST concentrations for female Harlan-Sprague-Dawley rats are reported in an experimental animal text (16) to range from 44.5 to 153.5 U/L. Considering this text reports a normal range span of over 100 U/L, the difference of 40–60 U/L that we found in our results is not necessarily clinically important. Moreover, this result is not clinically alarming, as increases in AST—not moderate decreases—are typically associated with acute liver dysfunction, and we observed no significant differences in AST concentrations during the experimentation period in the rats injected with labeled MSCs.
Decreased hemoglobin concentrations were seen in the first week after injection in the group 1 and group 2 rats. Although the cause of this decrease is not known for certain, it is unlikely that ferumoxides administration in either injection group had any effect on hemoglobin concentration during this week, as study results have shown that ferumoxides administered intravenously takes approximately 30–45 days to become available for heme incorporation (17). This may be why on day 28, the hemoglobin concentrations in the rats injected with ferumoxides-PLL complex were significantly higher (P < .05) than the control value. However, there appears to be no extended alteration: The measurement returned to the control value by day 42. The hemoglobin concentration never increased above the control value during the test period, even beyond 30 days, in the rats injected with labeled MSCs, probably because labeled MSCs maintain their iron load for well over a month (18).
Although significant decreases in MCV were seen in the rats after the labeled MSC and ferumoxides-PLL complex injections, the decreases were not clinically important. In fact, the decreases were very small in both groups at all time points (never varying by more than 5.2% from the control value), and there was no significant difference in MCV between the group 1 and group 2 rats at any time point. Therefore, it is unlikely that these changes were the result of an adverse effect of the labeled MSC or ferumoxides-PLL complex injection.
Beyond these considerations, it is not clear why some differences in organ function measurements existed between the rats injected with labeled MSCs, the rats injected with ferumoxides-PLL complex only, and the control rats. Free ferumoxides-PLL complex or PLL may lead to these effects since a greater deviation from the control value was observed in the rats injected with ferumoxides-PLL complex (group 2) than in those injected with labeled MSCs (group 1). The amount of ferumoxides-PLL complex injected into the group 2 rats was the same amount of iron that would be either attached to the surface of the labeled cells or internalized in the endosomes of these cells injected into but free in the circulation of the group 2 rats, so it may be possible to observe greater alterations in the hematologic and serum biochemical measurements in subjects injected with ferumoxides-PLL complex.
Indicators of renal function in the group 1 and group 2 rats revealed some significant alterations during the test period, but the changes were minor and did not indicate dysfunction. Alterations in serum iron concentrations also were seen in the group 1 and group 2 rats, but they were clinically unimportant as well.
The rapid clearance of the labeled MSCs from the circulation was probably due to their early homing to the liver and spleen. MSCs then might be released and migrate to other tissue with proper signaling. Early homing to the liver was also observed in our previous studies (19).
In terms of practical applications, despite the great promise of cellular labeling with superparamagnetic iron oxide particles, the advantages of this technique will be largely dependent on clinical approval. Animal studies are extremely valuable for furthering medical knowledge and developing clinical treatments, but the ultimate goal in cellular labeling is to aid in clinical diagnostic examinations and therapies. The preclinical results presented in this article can provide a basis for the future application of techniques for tracking superparamagnetic iron oxide–labeled stem cells in clinical trials. In summary, the administration of ferumoxides-PLL–labeled cells does not affect serum hematologic or chemical markers of organ function in a clinically important or preclusive manner.
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FOOTNOTES |
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Authors stated no financial relationship to disclose.
Author contributions: Guarantors of integrity of entire study, A.S.A., J.A.F.; study concepts, G.T.Y., A.S.A., J.A.F.; study design, G.T.Y., E.K.J., J.A.F., A.S.A.; literature research, G.T.Y.; experimental studies, G.T.Y., L.B.W., P.A., E.K.J.; data acquisition, G.T.Y., L.B.W., P.A., A.S.A., E.K.J.; data analysis/interpretation, all authors; statistical analysis, G.T.Y.; manuscript preparation, definition of intellectual content, and editing, G.T.Y., J.A.F., A.S.A.; manuscript revision/review, all authors; manuscript final version approval, A.S.A., J.A.F.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODS RESULTS DISCUSSIONREFERENCES |
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