Assessment of tissue-engineered islet graft viability by fluorine magnetic resonance spectroscopy.

INTRODUCTION: Despite significant progress in the last decade, islet transplantation remains an experimental therapy for a limited number of patients with type 1 diabetes. Tissue-engineered approaches may provide promising alternatives to the current clinical protocol and would benefit greatly from concurrent development of graft quality assessment techniques. This study was designed to evaluate whether viability of tissue-engineered islet grafts can be assessed using fluorine magnetic resonance spectroscopy ((19)F-MRS), by the noninvasive measurement of oxygen partial pressure (pO(2)) and the subsequent calculation of islet oxygen consumption rate (OCR). METHODS: Scaffolds composed of porcine plasma were seeded with human islets and perfluorodecalin. Each graft was covered with the same volume of culture media in a Petri dish. Four scaffolds were seeded with various numbers (0-8000) of islet equivalents (IE) aliquoted from the same preparation. After randomizing run order, grafts were examined by (19)F-MRS at 37°C using a 5T spectrometer and a single-loop surface coil placed underneath. A standard inversion recovery sequence was used to obtain characteristic (19)F spin-lattice relaxation times (T1), which were converted to steady-state average pO(2) estimates using a previously determined linear calibration (R(2) = 1.000). Each condition was assessed using replicate (19)F-MRS measurements (n = 6-8). RESULTS: Grafts exhibited IE dose-dependent increases in T1 and decreases in pO(2) estimates. From the difference between scaffold pO(2) estimates and ambient pO(2), the islet preparation OCR was calculated to be 95 ± 12 (mean ± standard error of the mean) nmol/(min·mg DNA) using theoretical modeling. This value compared well with OCR values measured using established methods for human islet preparations. CONCLUSIONS: (19)F-MRS can be used for noninvasive pre- and possibly posttransplant assessment of tissue-engineered islet graft viability by estimating the amount of viable, oxygen-consuming tissue in a scaffold.

Surgical protocol involving the infusion of paramagnetic microparticles for preferential incorporation within porcine islets.

INTRODUCTION: Despite significant advances, widespread applicability of islet cell transplantation remains elusive. Refinement of current islet isolation protocols may improve transplant outcomes. Islet purification by magnetic separation has shown early promise. However, surgical protocols must be optimized to maximize the incorporation of paramagnetic microparticles (MP) within a greater number of islets. This study explores the impact of MP concentration and infusion method on optimizing MP incorporation within islets. METHODS: Five porcine pancreata were procured from donors after cardiac death. Splenic lobes were isolated and infused with varying concentrations of MP (8, 16, and 32 × 10(8) MP/L of cold preservation solution) and using one of two delivery techniques (hanging bag versus hand-syringe). After procurement and infusion, pancreata were stored at 0°C to 4°C during transportation (less than 1 hour), fixed in 10% buffered formalin, and examined by standard magnetic resonance imaging (MRI) and histopathology. RESULTS: T2*-weighted MRI showed homogeneous distribution of MP in all experimental splenic lobes. In addition, histologic analysis confirmed that MP were primarily located within the microvasculature of islets (82% to 85%), with few MP present in acinar tissue (15% to 18%), with an average of five to seven MP per islet (within a 5-µm thick section). The highest MP incorporation was achieved at a concentration of 16 × 10(8) MP/L using the hand-syringe technique. CONCLUSION: This preliminary study suggests that optimization of a surgical protocol, MP concentrations, and applied infusion pressures may enable more uniform distribution of MP in the porcine pancreas and better control of MP incorporation within islets. These results may have implications in maximizing the efficacy of islet purification by magnetic separation.

Pancreas oxygen persufflation increases ATP levels as shown by nuclear magnetic resonance.

BACKGROUND: Islet transplantation is a promising treatment for type 1 diabetes. Due to a shortage of suitable human pancreata, high cost, and the large dose of islets presently required for long-term diabetes reversal; it is important to maximize viable islet yield. Traditional methods of pancreas preservation have been identified as suboptimal due to insufficient oxygenation. Enhanced oxygen delivery is a key area of improvement. In this paper, we explored improved oxygen delivery by persufflation (PSF), ie, vascular gas perfusion. METHODS: Human pancreata were obtained from brain-dead donors. Porcine pancreata were procured by en bloc viscerectomy from heparinized donation after cardiac death donors and were either preserved by either two-layer method (TLM) or PSF. Following procurement, organs were transported to a 1.5-T magnetic resonance (MR) system for (31)P nuclear magnetic resonance spectroscopy to investigate their bioenergetic status by measuring the ratio of adenosine triphosphate to inorganic phosphate (ATP:P(i)) and for assessing PSF homogeneity by MRI. RESULTS: Human and porcine pancreata can be effectively preserved by PSF. MRI showed that pancreatic tissue was homogeneously filled with gas. TLM can effectively raise ATP:P(i) levels in rat pancreata but not in larger porcine pancreata. ATP:P(i) levels were almost undetectable in porcine organs preserved with TLM. When human or porcine organs were preserved by PSF, ATP:P(i) was elevated to levels similar to those observed in rat pancreata. CONCLUSION: The methods developed for human and porcine pancreas PSF homogeneously deliver oxygen throughout the organ. This elevates ATP levels during preservation and may improve islet isolation outcomes while enabling the use of marginal donors, thus expanding the usable donor pool.

Persufflation improves pancreas preservation when compared with the two-layer method.

Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the large dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in low islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from donors after cardiac death and preserved by either TLM or persufflation for 24 hours and subsequently fixed. Biopsies collected from several regions of the pancreas were sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries and significantly less autolysis/cell death relative to regions not exposed to persufflation or to tissues preserved with TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized.

Real-time noninvasive assessment of pancreatic ATP levels during cold preservation.

31P-NMR spectroscopy was utilized to investigate rat and porcine pancreatic ATP:P(i) ratios to assess the efficacy of existing protocols for cold preservation (CP) in maintaining organ quality. Following sacrifice, rat pancreata were immediately excised or left enclosed in the body for 15 minutes of warm ischemia (WI). After excision, rat pancreata were stored at 6 degrees C to 8 degrees C using histidine-tryptophan-ketoglutarate solution (HTK) presaturated with air (S1), HTK presaturated with O2 (S2), or the HTK/perfluorodecalin two-layer method (TLM) with both liquids presaturated with O2 (S3). 31P-NMR spectra were sequentially collected at 3, 6, 9, 12, and 24 hours of CP from pancreata stored with each of the three protocols examined. The ATP:Pi ratio for rat pancreata exposed to 15 minutes of WI and stored with S3 increased during the first 9 hours of CP, approaching values observed for organs procured with no WI. A marked reduction in the ATP:Pi ratio was observed beyond 12 hours of CP with S3. After 6 hours of CP, the ATP:Pi ratio was highest for S3, substantially decreased for S2, and below detection for S1. In sharp contrast to the rat model, ATP was barely detectable in porcine pancreata exposed to minimal warm ischemia (<15 minutes) stored with the TLM regardless of CP time. We conclude that 31P-NMR spectroscopy is a powerful tool that can be used to (1) noninvasively evaluate pancreata prior to islet isolation, (2) assess the efficacy of different preservation protocols, (3) precisely define the timing of reversible versus irreversible damage, and (4) assess whether intervention will extend this timing.

Water content in an engineered dermal replacement during permeation of Me2SO solutions using rapid MR imaging.

The successful cryopreservation of cell and tissues typically requires the use of specialized solutions containing cryoprotective agents. At room temperature, the introduction of a cryopreservation solution can result in cell damage/death resulting from osmotic stresses and/or biochemical toxicity of the solution. For tissues, the permeation and equilibration of a cryoprotective solution throughout the tissue is important in enhancing the uniformity and consistency of the postthaw viability of the tissue. Magnetic resonance (MR) is a common nondestructive technique that can be used to quantitate the temporal and spatial composition of water and cryoprotective agents in a three-dimensional system. We have applied a recently developed rapid NMR imaging technique to quantify the transport of water in an artificial dermal replacement upon permeation of dimethyl sulfoxide (Me2SO) solutions. Results indicate that the rate of water transport is slower in the presence of Me2SO molecules. Furthermore, the transport is concentration-dependent, suggesting that Me2SO tends to retain bound water molecules in the tissue. Moreover, water transport decreases with decreasing temperature, and the presence of cells tends to increase water transport.

Near-infrared and nuclear magnetic resonance spectroscopic assessment of tissue energetics in an isolated, perfused canine hind limb model of dysoxia.

This controlled laboratory study examined the efficacy of near-infrared spectroscopy (NIRS) and 31P-nuclear magnetic resonance (NMR) spectroscopy in measuring regional tissue oxygenation in a isolated, perfused hind limb model of tissue dysoxia. Isolated hind limb perfusion was carried out in 20 mongrel dogs and oxygen delivery was varied by manipulating either hemoglobin concentration, oxygen saturation, or flow. Hind limbs from anesthetized mongrel dogs (n = 20) were separated and isolated perfusion performed. NIRS probes for recording relative O2 saturation of tissue hemoglobin (HbO2) and cytochrome a,a3 and NMR probes for measuring 31P-high energy phosphates were placed over the limb. Measurements of physiologic parameters, blood gases, lactate, NIRS values for HbO2 and cytochrome a,a3 redox state, and 31P-phosphate levels were recorded at set intervals throughout the experiment. Measures of tissue oxygen consumption (VO2) correlated with tissue oxygenation as measured by HbO2 and cytochrome a,a3 redox state (NIRS), as well as by 31P-high energy phosphate levels (NMR) throughout the experiment. Delivery-dependent tissue oxygenation was detected at a higher DO2 by NIRS than by VO2 or NMR. Tissue oxygenation as measured by NIRS and NMR shows excellent correlation with oxygen delivery in an isolated, perfused model of shock. NIRS may allow early detection of tissue dysoxia using rapid non-invasive techniques.

Rapid MR imaging of cryoprotectant permeation in an engineered dermal replacement.

Magnetic resonance (MR) imaging is a powerful technique for monitoring the permeation of cryoprotective agents (CPAs) inside tissues. However, the techniques published until now suffer from inherently long imaging times, limiting the application of these techniques to slow diffusion processes and large CPA concentrations. In this study, we present a rapid MR imaging technique based on a CHESS-FLASH scheme combined with Keyhole image acquisition. This technique can image the fast permeation of Me(2)SO solutions into freeze-dried artificial dermal replacements for concentrations down to 10% v/v. Special attention is given to evaluating the technique for quantitative analysis.

Effect of harvesting protocol on performance of a hollow fiber bioreactor.

In this study, a bioreactor subject to Starling flow in closed shell batch harvest mode was compared to two forms of additional forced extracapillary (EC) space convection including EC circulation and EC cycling. Despite the presence of Starling flow as the dominant EC convection mechanism in the batch harvest system, the bioreactor start up was fairly good. However, the antibody productivity of the batch harvest system fell off rapidly after day 20 resulting in only 4.5 g of antibody produced. EC circulation with flow parallel to the fibers had a slightly better start up than the batch harvest. However, the antibody productivity also dropped after day 20 with EC circulation, resulting in only 7.5 g of antibody produced. EC cycling with flow both parallel and perpendicular to the fibers resulted in a start up similar to that of EC circulation. However, in contrast to the other two systems, antibody productivity in the EC cycling system was stable over the 60-day experiment resulting in the production of 23 g of antibody. These results demonstrate the importance of inducing the proper flow distribution in the EC space to allow consistent and stable production in hollow fiber bioreactors.

MRI compatibility and visibility assessment of implantable medical devices.

We have developed a protocol to evaluate the magnetic resonance (MR) compatibility of implantable medical devices. The testing protocol consists of the evaluation of magnetic field-induced movement, electric current, heating, image distortion, and device operation. In addition, current induction is evaluated with a finite element analysis simulation technique that models the effect of radiofrequency fields on each device. The protocol has been applied to several implantable infusion pumps and neurostimulators with associated attachments. Experiments were performed using a 1.5-T whole-body MR system with parameters selected to approximate the intended clinical and worst case configuration. The devices exhibited moderate magnetic field-induced deflection and torque but had significant image artifacts. No heating was detected for any of the devices. Pump operation was halted in the magnetic field, but resumed after removed. Exposure to the magnetic field activated some of the neurostimulators.

Paramagnetic tracer concentration evolution by NMR relaxation time mapping: application to Aris-Taylor dispersion.

A procedure to study tracer dispersion was proposed and tested for the case of tracer spreading in tube flow. Concentration maps of paramagnetic tracers Gd3+ were measured in time through direct measurements of spin lattice relaxation time T1 obtained by using a two-point stimulated echo pulse sequence. The procedure was used to test the linear dependence of Peclet number on inverse velocity in the range of flow rates 0.3-1.2 cc/min.

Dispersion of paramagnetic tracers in bead packs by T1 mapping: experiments and simulations.

NMR imaging was used to study dispersion in 6 mm bead pack. T1 maps were employed to measure the rate of axial spreading of paramagnetic tracers (GdCl3) inside the bead pack in the range of flow rate from 0.015 mL/s to 0.175 mL/s. From the T1 maps, tracer concentration profiles were obtained, which yielded dimensionless axial dispersion coefficient and mean transit time. Spatial variations in the dispersion coefficient were observed at flow rates above 0.08 mL/s. We hypothesized that the observed spatial oscillations in the dispersion coefficient arise from the spatial variations of the velocity distribution. To validate this mechanism we showed by simulation that similar dispersion coefficient variation occur in a layered network.

Positron emission tomography within a magnetic field using photomultiplier tubes and lightguides.

The spatial resolution of positron emission tomography (PET) improves when positron annihilation takes place in a strong magnetic field. In a magnetic field, the Lorentz force restricts positron range perpendicular to the field. Since positron annihilation occurs closer to its point of origin, the positron annihilation point spread function decreases. This was verified experimentally by measuring the spread function of positron annihilation from a 500 mm 68Ge bead imbedded in tissue-equivalent wax. At 5 T the spread function full width at half maximum (FWHM) and the full width at tenth maximum (FWTM) decrease by a factor of 1.42 and 2.09, respectively. Two NaI(Tl) scintillation crystals that interface to a pair of photomultiplier tubes (PMTS) through long lightguides detect positron annihilation at zero field and 5.0 T. Photomultiplier tubes, inoperable in strong magnetic fields, are functional if lightguides bring the photons produced by scintillators within the field to a minimal magnetic field. These tests also demonstrate techniques necessary for combining magnetic resonance imaging (MRI) and PET into one scanner.

Use of a magnetic field to increase the spatial resolution of positron emission tomography.

Detector geometry, spatial sampling, and more fundamentally, positron range and noncollinearity of annihilation photon emission define Positron Emission Tomography (PET) spatial resolution. In this paper, a strong magnetic field is used to constrain positron travel transverse to the field. Measurement of the spread function from a 500 microns diameter 68Ga impregnated resin bead shows a squeezing of the full width at half maximum (FWHM) by a factor of 1.0, 1.22, 1.42, and 2.05, at 0, 4.0, 5.0, and 9.4 Tesla, respectively. The full width at tenth maximum (FWTM) decreases by a factor of 1.0, 1.73, 2.09, and 3.20, at 0, 4.0, 5.0, and 9.0 Tesla, respectively. Acquiring a PET image in a magnetic field should significantly reduce resolution loss due to positron range.

Quantitative flow measurements in bioreactors by nuclear magnetic resonance imaging.

We have developed nuclear magnetic resonance (NMR) flow imaging techniques to measure fluid flow in a cell-free hollow fiber bioreactor (HFBR). Using 1H NMR we track the motion of protons and obtain velocity distributions as a function of position and time. These measurements enable the visualization of flow patterns needed for module design and for establishing desired operating conditions. Uneven flow in the cell-containing region of an HFBR can result in concentration gradients and uneven cell distribution that may lead to reduced cell viability. Results from this non-invasive method could be used to design more efficient cell bioreactors or membrane separation devices.

Design of a 13C (1H) RF probe for monitoring the in vivo metabolism of [1-13C]glucose in primate brain.

The design of an RF probe suitable for obtaining proton-decoupled 13C spectra from a subhuman primate brain is described. Two orthogonal saddle coils, one tuned to the resonant frequency of 13C and the other to the resonant frequency of 1H, were used to monitor the in vivo metabolism of [1-13C]glucose in rhesus monkey brain at 2.1 T. Difference spectra showed the appearance of 13C-enriched glutamate and glutamine 30 to 40 min after a bolus injection of [1-13C]glucose.

Proton decoupled 13C NMR imaging.

Proton decoupled 13C images were obtained at 2.1 Tesla. 13C[1H] images showed an increase in sensitivity over nondecoupled 13C images because of the nuclear Overhauser effect and elimination of multiple lines from scalar 13C-1H spin-spin couplings. The improvement in S/N for 13C[1H] images was smaller than expected because of a significant decrease in decoupling efficiency when 13C spin echoes were acquired in a readout gradient. Images of 13C compounds that had a wide range of chemical shifts showed separated and/or overlapping images, which is consistent with chemical shift imaging artifacts seen in 1H images. This work examines the technical constraints of acquiring and the difficulties of interpreting 13C[1H] images.

13C nuclear magnetic resonance study of the complexation of calcium by taurine.

13C Nuclear magnetic resonance chemical shifts, 1JC-C scalar coupling constants, spin-lattice relaxation times, and nuclear Overhauser effects were determined for taurine-[1,2 13C] and a taurine-[1 13C] and taurine-[2 13C] mixture in the presence and absence of calcium. Ionization constants for taurine amino and sulfonic acid groups and chemical shifts of N-methylene and S-methylene carbons of the taurine cation, zwitterion, and anion were obtained from simultaneous least squares analysis of 13C titration curves of both taurine carbons. Comparison of taurine titration shifts to values for related compounds reveals some unusual electronic properties of the taurine molecule. Stability constants of 1:1 calcium complexes with taurine zwitterions and anions, as well as their 13C chemical shifts, were obtained by least squares analysis of titration curves measured in the presence of calcium. The stability constants of calcium-taurine complexes were significantly lower than previous values and led to estimates that only approximately one percent of intracellular calcium of mammalian myocardial cells would exist in a taurine complex. The implications of these results with respect to the effect of taurine on calcium ion flux are discussed.