Characterization of a chip-based bioreactor for three-dimensional cell cultivation via Magnetic Resonance Imaging.

We describe the characterization of a chip-based platform (3(D)-KITChip) for the three-dimensional cultivation of cells under perfusion conditions via magnetic resonance imaging (MRI). Besides the chip, the microfluidic system is comprised of a bioreactor housing, a medium supply, a pump for generating active flow conditions as well as a gas mixing station. The closed circulation loop is ideally suited for a characterization via MRI since the small bioreactor setup with active perfusion, driven by the pump from outside the coils, not only is completely MRI-compatible but also can be transferred into the magnetic coil of an experimental animal scanner. We have found that the two halves of the chip inside the bioreactor are homogeneously perfused with cell culture medium both with and without cells inside the 3(D)-KITChip. In addition, the homogeneity of perfusion is nearly independent from the flow rates investigated in this study, and furthermore, the setup shows excellent washout characteristics after spiking with Gadolinium-DOTA which makes it an ideal candidate for drug screening purposes. We, therefore, conclude that the 3(D)-KITChip is well suited as a platform for high-density three-dimensional cell cultures, especially those requiring a defined medium flow and/or gas supply in a precisely controllable three dimensional environment, like stem cells.

T2*-relaxivity contrast imaging: first results.

In this study, T2*- relaxivity contrast imaging (RCI) is proposed for new contrast generation in MRI. The method produces images of relaxivities r*2,vasc and r*2,EES caused by susceptibility gradients across the vessel walls and cell membranes, respectively. The sensitivity to noise was assessed with a simulation study, and initial results are presented for five colorectal tumor xenografts in nude mice. Simulations show that the new relaxivity parameters are at least as accurate and precise as standard parameters such as plasma volume and interstitial volume. Mean values of both relaxivities were significantly different (r*2,vasc=10.9±2.9 mM(-1) s(-1) and r*2,EES=15.6±2.6 mM(-1) s(-1)). r*2,vasc (r=0.67) and r*2,EES (r=0.52) were weakly correlated with plasma volume and interstitial volume, respectively. Images of r*2,vasc and r*2,EES reveal a different tumor structure than plasma volume and interstitial volume maps. These results suggest that relaxivity contrast imaging is practically feasible and might offer supplementary information compared to dynamic contrast-enhanced-MRI.

Characterization and longitudinal monitoring of melanoma growth in ret-transgenic mice using a single-sequence MRI protocol.

Spontaneous melanoma models in transgenic mice are increasingly used in preclinical research as they most closely match the progression of melanoma in humans. While optical inspection only allows analysis of tumors located on the skin, the accurate measurement and growth of subcutaneous tumors have not been adequately assessed. To improve the measurement accuracy of melanoma tumors, we used a fast single-sequence MRI protocol at 9.4 Tesla for longitudinal characterization of a ret-transgenic mouse model. Repeated MRI (average acquisition time 30 min per animal) of the trunk (excluding head and distal limbs) in six siblings revealed an increase in the mean total tumor volume (TTV) from 102.0 ± 80.5 mm(3) at 35 days of age to 434.8 ± 154.9 mm(3) by 77 days. The main tumor load was located within the pelvis (>40%), followed by the proximal hind limbs and groins (>30%). The smallest detectable tumor measured 0.07 mm(3). Inter-rater reliability between a radiologist and a veterinarian analysing MRI data was 0.993 for TTV and 0.840 for number of tumors (both p < 0.001). We thus conclude that because of the high variance of TTV of same-aged mice, MRI should be used (i) to establish treatment groups matched for TTV and (ii) for longitudinal examination of the TTV in mice over the course of treatments.

Comparison of digital subtraction angiography, micro-computed tomography angiography and magnetic resonance angiography in the assessment of the cerebrovascular system in live mice.

PURPOSE: Mice are often used as small animal models of brain ischemia, venous thrombosis, or vasospasm. This article aimed at providing an overview of the currently available methodologies for in vivo imaging of the murine cerebrovasculature and comparing the capabilities and limitations of the different methods. METHODS: Micro-computed tomography angiography (CTA) was performed during intra-arterial and intravenous administration of a contrast agent bolus. Digital subtraction angiography (DSA) was performed during intra-arterial administration of contrast agent using the micro-CT scanner. Time-of-flight (ToF) magnetic resonance (MR) angiography was performed using a small animal scanner (9.4 T) equipped with a cryogenic transceive quadrature coil. Datasets were compared for scan time, contrast-to-noise ratio (CNR), temporal and spatial resolution, radiation dose, contrast agent dose and detailed recognition of cerebrovascular structures. RESULTS: Highest spatial resolution was achieved using micro-CTA (16 x 16 x 16 µm) and DSA (14 x 14 µm). Compared to micro-CTA (20-40 s) and ToF-MRA (57 min), DSA provided highest temporal resolutions (30 fps) allowing analyses of the cerebrovascular blood flow. Highest mean CNR was reached using ToF-MRA (50.7 ± 15.0), while CNR of micro-CTA depended on the intra-arterial (19.0 ± 1.0) and intravenous (1.3 ± 0.4) use of agents. The CNR of DSA was 10.0 ± 1.8. CONCLUSIONS: The use of dedicated small animal scanners allows cerebrovascular imaging in live animals as small as mice. As each of the methods analyzed has its advantages and limitations, choosing the best suited imaging modality for a defined question is of great importance. By this means the aforementioned methods offer a great potential for future projects in preclinical cerebrovascular research including ischemic stroke or vasospasm.

Quantification of glomerular number and size distribution in normal rat kidneys using magnetic resonance imaging.

BACKGROUND: Glomerular number and size are important risk factors for chronic kidney disease (CKD) and cardiovascular disease and have traditionally been estimated using invasive techniques. Here, we report a novel technique to count and size every glomerulus in the rat kidney using magnetic resonance imaging (MRI). METHODS: The ferromagnetic nature of cationized ferritin allowed visualization of single glomeruli in high-resolution susceptibility-weighted MRI. A segmentation algorithm was used to identify and count all glomeruli within the whole kidney. To prove our concept, we estimated total glomerular number and mean glomerular volume of each kidney using design-based stereology. RESULTS: The glomerular counts obtained with MRI agreed well with estimates obtained using traditional methods [MRI, 32 785 (3117); stereology, 35 132 (3123)]. For the first time, the glomerular volume distribution for the entire kidney is shown. Additionally, the method is substantially faster than the current methods. CONCLUSIONS: MRI provides a new method for measuring these important microanatomical markers of disease risk and leads the way to in vivo analysis of these parameters, including longitudinal studies of animal models of CKD.

Susceptibility weighted imaging (SWI) of the kidney at 3T--initial results.

Susceptibility weighted imaging provides diagnostic information in strokes, hemorrhages, and cerebral tumors and has proven to be a valuable tool in imaging venous vessels in the cerebrum. The SWI principle is based on the weighting of T(2)* weighted magnitude images with a phase mask, therewith improving image contrast of veins or neighbouring structures of different susceptibility, in general. T(2)* weighted MRI is already used for assessment of kidney function. In this paper, the feasibility of SWI on kidneys was investigated. Translation of SWI from the brain to the kidneys comes along with two main challenges: (i) organ motion due to breathing and (ii) a higher oxygenation level of renal veins compared to the brain. To handle these problems, the acquisition time has been cut down to allow for breath-hold examinations, and different post-processing methods including a new phase mask were investigated to visualize renal veins. Results showed that by a new post-processing strategy SWI contrast was enhanced on average by a factor of 1.33 compared to the standard phase mask. In summary, initial experiences of SWI on the kidneys demonstrated the feasibility. However, further technical developments have to be performed to make this technology applicable in clinical abdominal MRI.

2D and 3D radial multi-gradient-echo DCE MRI in murine tumor models with dynamic R*2-corrected R1 mapping.

Dynamic contrast-enhanced MRI is extensively studied to define and evaluate biomarkers for early assessment of vasculature-targeting therapies. In this study, two-dimensional and three-dimensional radial multi-gradient-echo techniques for dynamic R*(2)-corrected R(1) mapping based on the spoiled gradient recalled signal equation were implemented and validated at 4.7 T. The techniques were evaluated on phantoms and on a respiratory motion animated tumor model. R(1) measurements were validated with respect to a standard inversion-recovery spin-echo sequence in a four-compartment phantom covering a range of relaxation rates typically found in tumor tissue. In the range of [0.4, 3] sec(-1), R(1) differences were less than 10% for both two-dimensional and three-dimensional experiments. A dynamic contrast-enhanced MRI pilot study was performed on a colorectal tumor model subcutaneously implanted in mice at the abdominal level. Low motion sensitivity of radial acquisition allowed image recording without respiratory triggering. Three-dimensional K(trans) maps and significantly different mean K(trans) values were obtained for two contrast agents with different molecular weights. The radial multi-gradient-echo approach should be most useful for preclinical experimental conditions where the tissue of interest experiences physiologic motion, like spontaneous extracerebral tumors developed by transgenic mice, and where dynamic contrast-enhanced MRI is performed with high-relaxivity contrast agents.

In vitro setup to study permeability characteristics of contrast agents by MRI.

An experimental setup consisting of a hollow fiber module (HFM) was developed for the in vitro study of contrast agent (CA) permeability. Controllable flow and known fiber characteristics allowed permeability studies under well-defined conditions with CAs of different molecular weight (MW). In the MRI experiments performed at 4.7 T, the system was perfused at a constant flow rate (5 ml/min) with water and four CA of different MW: Gd-DOTA (MW=0.6 kDa), P846 (3.5 kDa), P792 (6.5 kDa) and P717 (50.5 kDa). R(1) time courses were measured with a saturation-recovery multi-gradient-echo snapshot sequence in the fiber-free HFM input and the fiber-filled center. Concentration time courses were calculated, and CA extravasation was analyzed with a pharmacokinetic model yielding exchange rate constant k(ie). Only Gd-DOTA (k(ie)=2.37+/-0.16 min(-1)) and P846 (k(ie)=0.58+/-0.17 min(-1)) showed quantifiable extravasation. P717 perfusion yielded an intra-capillary volume fraction of 15.6+/-2.7% compared with 12% estimated from the HFM manufacturer's specifications. In conclusion, the experimental setup allowed classification of in vitro permeability characteristics for CAs with different MW and therefore holds potential for systematic comparison of CAs currently under development.

Simultaneous dynamic T1 and T2* measurement for AIF assessment combined with DCE MRI in a mouse tumor model.

A double-delay SR-MGE-SNAP sequence allowing simultaneous T1 and T2* measurement was developed for integrating arterial input function (AIF) measurement into DCE MRI. Implemented on a 4.7-T animal MR system, this technique was applied to mice with colorectal tumor xenografts. AIF, measured in the mouse heart, was modeled by a bi-exponential function, whereas tumor K(trans) and v(e) parameter maps were obtained from analysis with a two- compartment model using an individually measured AIF. AIF analysis of T2*-corrected data yielded A1 = 9.2 +/- 4.3 kg/l, A(2) = 4.2 +/- 0.8 kg/l, m1 = 2.3 +/- 1.1 min(-1), and m2 = 0.05 +/- 0.02 min(-1). The mean initial plasma concentration C ( p )(t = 0) = 8.0 +/- 2.7 mM was compatible with estimated 8.6 mM. Without T2*-correction distribution phase parameters A1, m1, and C(p)(t = 0) were underestimated. In tumors, neglect of T2* effects yielded mean K(trans) values which were reduced by 14% (P < 0.05), whereas v(e) showed only a slight non-significant reduction. Simultaneous measurement of DeltaR1 and DeltaR2* studied in highly and poorly vascularized and (pre-)necrotic tumor regions revealed complementary behavior of both parameters with respect to vascular properties. In conclusion, the presented measurement technique is a promising tool for dynamic MRI applications studied in animal models at high field strengths and/or with CA of high relaxivities, as it combines classical DCE MRI integrating AIF assessment with dynamic T2* measurement.

Determination of pharmacokinetic parameters in DCE MRI: Consequence of nonlinearity between contrast agent concentration and signal intensity.

OBJECTIVES: We sought to compare pharmacokinetic modeling of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) data by assuming a linear and nonlinear relationship between signal intensity (SI) and contrast agent (CA) concentration. MATERIALS AND METHODS: Data sets were generated by computer-based simulation studies and DCE-MRI examination of 5 tumor-bearing mice using a 1.5 T MR-scanner. Two approaches were investigated: a linear and nonlinear relationship between SI and CA concentration before pharmacokinetic analysis. In a pharmacokinetic 2-compartment model, values of exchange rate constant kep and amplitude A were compared for both assumptions. RESULTS: In the linear approach, A was as much as 30% less for kep values between 1.0 and 5.0 min, whereas kep was as much as 60% greater, for kep between 0.2 and 5.0 min compared with the nonlinear one, as demonstrated in simulations and animal studies. CONCLUSIONS: Nonlinearity between SI and CA concentration has to be considered for accurate parameter calculation in DCE-MRI studies.

Synthesis and characterization of HE-24.8: a polymeric contrast agent for magnetic resonance angiography.

The physical and biological properties of a water-soluble polymeric contrast agent based on a complex of N-(2-hydroxypropyl)methacrylamide copolymer with gadolinium (HE-24.8) were investigated, and its potential for experimental magnetic resonance (MR) angiography was assessed. Relaxivities of Gd-DTPA-BMA, Gd-DTPA-HSA (human serum albumin), and HE-24.8 were determined at 1.5 T. Thermic stability and biocompatibility of HE-24.8 were assessed in vitro and by analyzing kinetics and organ distribution in rats for up to 2 weeks. For comparison, HE-24.8- and Gd-DTPA-HSA-enhanced micro-MR angiographies of brain, chest, and subcutaneous tumors in rats were performed. T1 relaxivity of HE-24.8 (21.3 +/- 1.1 mM(-1) s(-1)) was 5-fold higher than that of Gd-DTPA-BMA (4.1 +/- 0.1 mM(-1) s(-1)) and twice as high as that of Gd-DTPA-HSA (12.4 +/- 0.2 mM(-1) s(-1)). Varying the molecular weight of the polymer (15-46 kDa) did not significantly change the T1 relaxivity. In rats, 20 and 10% of the injected dose of HE-24.8 was detected at 24 and 168 h postinjection, respectively. Upon a relatively rapid initial renal clearance, no specific retention in any organ was noted, with some exception for the reticulo-endothelial system. No measurable release of gadolinium from the polymer-Gd complex or cell toxicity was observed during its incubation in aqueous environment. Excellent display of rat and tumor vascularization was achieved with Gd-DTPA-HSA and HE-24.8; however, contrast of vessels was higher in HE-24.8-enhanced scans. HE-24.8 is considered a macromolecular contrast agent highly suited for experimental MR studies.

Simple models improve the discrimination of prostate cancers from the peripheral gland by T1-weighted dynamic MRI.

Evaluation of the accuracy of descriptive and physiological parameters calculated from signal intensity-time curves using T1-weighted dynamic contrast enhanced MRI (DCE MRI) to differentiate prostate cancers from the peripheral gland. Twenty-seven patients with prostate cancers were examined with DCE MRI prior radical prostatectomy. Regions of interest were defined in tumors and non-affected areas in the peripheral zone. Dynamic data were parameterized in amplitude and exchange rate constant (kep) using a two-compartment model. Additionally, relative slope during 26, 39, 52 and 65 s, areas under the curve (AUC) and time to start of signal intensity increase (tlag) were determined. Vessel density (VD) of excised prostates was quantified in tumor areas using a CD34 stain. The parameter slope52 showed 20% higher values (P<0.001) in tumors than in the peripheral gland and compared with the other parameters the largest area under the ROC curve (0.81). The minimum total error rate was attained at a cut-point of 0.021, yielding a sample value of sensitivity and specificity of 70% and 88%, respectively, and a bias-corrected sum of sensitivity and specificity of 1.54. In addition, amplitude (P<0.001), kep (P=0.03) and AUC (P<0.001) were significantly higher in tumors. tlag did not discriminate carcinomas from glandular tissue. VD was higher in tumors than in the non-affected peripheral prostate (P=0.05). However, none of the dynamic parameters in carcinomas showed a significant correlation with VD or Gleason score. Although pharmacokinetic modeling in DCE MRI showed potential to discriminate prostate cancers from peripheral prostate tissue, descriptive parameters of the early signal enhancement after contrast media injection reached higher sensitivity and specificity.

Dynamic contrast-enhanced magnetic resonance imaging rapidly indicates vessel regression in human squamous cell carcinomas grown in nude mice caused by VEGF receptor 2 blockade with DC101.

The purpose of our study was the investigation of early changes in tumor vascularization during antiangiogenic therapy with the vascular endothelial growth factor (VEGF) receptor 2 antibody (DC101) using dynamic contrast-enhanced magnetic resonance imaging (DCE MRI). Subcutaneous heterotransplants of human skin squamous cell carcinomas in nude mice were treated with DC101. Animals were examined before and repeatedly during 2 weeks of antiangiogenic treatment using Gd-DTPA-enhanced dynamic T1-weighted MRI. With a two-compartment model, dynamic data were parameterized in "amplitude" (increase of signal intensity relative to precontrast value) and k(ep) (exchange rate constant). Data obtained by MRI were validated by parallel examinations of histological sections immunostained for blood vessels (CD31). Already 2 days after the first DC101 application, a decrease of tumor vascularization was observed, which preceded a reduction of tumor volume. The difference between treated tumors and controls became prominent after 4 days, when amplitudes of treated tumors were decreased by 61% (P =.02). In line with change of microvessel density, the decrease in amplitudes was most pronounced in tumor centers. On day 7, the mean tumor volumes of treated (153 +/- 843 mm(3)) and control animals (596 +/- 384 mm(3)) were significantly different (P =.03). After 14 days, treated tumors showed further growth reduction (83 +/- 93 mm(3)), whereas untreated tumors (1208 +/- 822 mm(3)) continued to increase (P =.02). Our data underline the efficacy of DC101 as antiangiogenic treatment in human squamous cell carcinoma xenografts in nude mice and indicate DCE MRI as a valuable tool for early detection of treatment effects before changes in tumor volume become apparent.

Evaluation of chest motion and volumetry during the breathing cycle by dynamic MRI in healthy subjects: comparison with pulmonary function tests.

RATIONALE AND OBJECTIVES: To investigate diaphragm and chest wall motion during the whole breathing cycle using magnetic resonance imaging (MRI) and a volumetric model in correlation with spirometry. MATERIALS AND METHODS: Breathing cycles of 15 healthy volunteers were examined using a trueFISP sequence (5 slices in 3 planes, 3 images per second). Time-distance curves were calculated and correlated to spirometry. A model for vital capacity (VC), continuous time-dependent vital capacity (tVC), and investigating the influence of horizontal and vertical parameters on tVC was introduced. RESULTS: Time-distance curves of the breathing cycle using MRI correlated highly significant with spirometry (P < 0.0001). VC calculated by the model was similar to VC measured in spirometry (5.00 L vs. 5.15 L). tVC correlated highly significantly with spirometry (P < 0.0001). Vertical parameters had a more profound influence on tVC change than horizontal parameters. CONCLUSIONS: Dynamic MRI is a simple noninvasive method to evaluate local chest wall motion and respiratory mechanics. It widens the repertoire of tools for lung examination with a high temporal resolution.

Dynamic magnetic resonance tomography and proton magnetic resonance spectroscopy of prostate cancers in rats treated by radiotherapy.

RATIONALE AND OBJECTIVES: To establish an experimental setting for monitoring perfusion and metabolism in orthotopic prostate cancer at 1.5 T using dynamic contrast-enhanced magnetic resonance imaging (DCE MRI) and 1H-MR spectroscopy (MRS). METHODS: Dunning rat prostate cancer cells were injected into the prostate by open surgery. Twelve tumor-bearing rats (5 of these irradiated) and 6 healthy controls were followed up using gadolinium-diethylenetriaminepentaacetic acid -enhanced dynamic MRI and 1H-MRS. Amplitude and the exchange rate constant kep were calculated (2-compartment model). From 1H-MR spectra, ratios of choline (Cho) and creatine (tCr) were calculated. All tumors were examined histologically. RESULTS: On DCE MRI parameter maps, tumors showed increased vascularization. kep and microvessel density were correlated (r = 0.97). Tumors showed elevated Cho/tCr and an unexpected lipid fraction (2.0-2.2 parts per million). Irradiation slowed tumor growth significantly. Changes of perfusion and metabolism could be detected in all tumors during follow up. CONCLUSION: DCE MRI and 1H-MRS has potential to characterize orthotopic Dunning prostate cancer in rats, which is a promising model similar to human prostate carcinomas.

Comparing dynamic parameters of tumor vascularization in nude mice revealed by magnetic resonance imaging and contrast-enhanced intermittent power Doppler sonography.

RATIONALE: Angiogenesis is essential for spread and growth of malignant tumors. Because noninvasive methods for observing tumor vascularization are limited, most of previous results were based on histologic findings alone. In this study, dynamic parameters obtained using intermittent contrast-enhanced Doppler sonography and dynamic MRI were compared and correlated with microvessel density. METHODS: Eleven tumor-bearing nude mice were examined with dynamic T(1)-weighted sequences using Gd-DTPA in a 1.5 T magnetic resonance (MR) scanner and with intermittent power Doppler sonography after a single bolus of galactose based contrast agent. After examination 6 tumors were harvested for immunofluorescence microscopy using a CD31 stain. Using a 2-compartment model, the MR parameters amplitude (reflecting plasma volume) and k(ep) (influenced by the vessel permeability) were calculated and compared with maximal enhancement (max) and perfusion P measured with ultrasound. RESULTS: The MR amplitude correlated with the ultrasound parameter max significantly (r = 0.61; P = 0.01). Max (r = 0.67; P = 0.01), amplitude (r = 0.72; P = 0.01), and perfusion (r = 0.62; P = 0.05) correlated with the microvessel density. k(ep) moderately correlated with max, but not with perfusion and microvessel density. CONCLUSIONS: Dynamic MRI and contrast enhanced ultrasound are supplementing methods for examining perfusion and vascularity of experimental tumors.

Dynamic T1-weighted monitoring of vascularization in human carcinoma heterotransplants by magnetic resonance imaging.

Studies on tumor angiogenesis and antiangiogenic therapies are commonly performed with tumor heterotransplants in nude mice. To monitor therapeutic effects, improved noninvasive analyses of functional data are required, in addition to the assessment of tumor volume and histology. Here, we report on sequential monitoring of vascularization of human squamous cell carcinomas growing as heterotransplants in nude mice using MRI. Using a custom-developed animal coil in a conventional whole-body 1.5 T MRI scanner, dynamic T1w sequences were recorded after i.v. injection of Gd-DTPA in tumors grown for 17, 21, 25, 29 and 33 days. Amplitude and the exchange rate constant (k(ep)) were calculated according to a 2-compartment model, discriminating intravascular and interstitial spaces, and correlated with tumor size and histology. High-resolution imaging of small heterotransplants from 100 to 1,000 mm(3) was achieved, clearly discriminating vital and necrotic areas. Preceding the development of necroses, which were hyperintense in T2w images and confirmed with histology, a local decrease of amplitude and k(ep) values was observed. Significantly higher amplitudes were found in tumor periphery than in central parts, correlating well with the vascular pattern obtained by immunocytochemistry. Tumor size correlated negatively with amplitude, probably as a result of increasing necrotic areas, whereas the reason for the observed increase of k(ep) value with tumor size remains unclear. These data demonstrate that dynamic MRI is an excellent method for noninvasive assessment of tumor vascularization in small animals using a clinical whole-body scanner with little technical modifications. This technique provides functional data characterizing essential features of tumor biology and is thus appropriate for monitoring antiangiogenic therapies.