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.

Commercially available gas-permeable cell culture bags may not prevent anoxia in cultured or shipped islets.

Prolonged anoxia has deleterious effects on islets. Gas-permeable cell culture devices can be used to minimize anoxia during islet culture and especially during shipment when elimination of gas-liquid interfaces is required to prevent the formation of damaging gas bubbles. Gas-permeable bags may have several drawbacks, such as propensity for puncture and contamination, difficult islet retrieval, and significantly lower oxygen permeability than silicone rubber membranes (SRM). We hypothesized that oxygen permeability of bags may be insufficient for islet oxygenation. We measured oxygen transmission rates through the membrane walls of three different types of commercially available bags and through SRM currently used for islet shipment. We found that the bag membranes have oxygen transmission rates per unit area about 100-fold lower than SRM. We solved the oxygen diffusion-reaction equation for 150-microm diameter islets seeded at 3000 islet equivalents per cm2, a density adequate to culture and ship an entire human or porcine islet preparation in a single gas-permeable device, predicting that about 40% of the islet volume would be anoxic at 22 degrees C and about 70% would be anoxic at 37 degrees C. Islets of larger size or islets accumulated during shipment would be even more anoxic. The model predicted no anoxia in islets similarly seeded in devices with SRM bottoms. We concluded that commercially available bags may not prevent anoxia during islet culture or shipment; devices with SRM bottoms are more suitable alternatives.

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.

High-density culture of human islets on top of silicone rubber membranes.

Islet culture has emerged as a standard practice prior to clinical transplantation. However, culturing large numbers of islets requires low islet density (number of islets per unit surface area) and, consequently, 20 to 30 flasks per pancreas in order to avoid hypoxia-induced death (HID). There is a need for a simple, practical, small-footprint culture vessel that will accommodate aseptic maintenance of entire human islet isolations while avoiding HID. In this communication, we examine the hypothesis that by improving oxygen transfer through culture of islets on silicone rubber membranes (SRM), we may increase islet surface coverage and reduce the number of flasks required while avoiding HID. Our results demonstrate that islets cultured for up to 48 hours in vessels with SRM bottoms at 2000 to 4000 islet equivalents (IE)/cm(2), a surface coverage 10- to 20-fold higher than the standard culture protocol, displayed no significant loss of viability. In contrast, islets cultured for 48 hours at 4000 IE/cm(2) in flasks with gas-impermeable bottoms suffered a 60% to 70% reduction in viability. The data suggest that it is possible to culture all islets isolated from a human pancreas on SRM in a single, standard-sized vessel while maintaining the same viability as with the current, standard culture protocols that require 20 to 30 flasks. This approach may lead to substantial improvements in islet culture for research and clinical transplantation.

Quantitating staphylococcal enterotoxin B in diverse media using a portable fiber-optic biosensor.

A new, portable fiber-optic biosensor has been used to detect staphylococcal enterotoxin B, a causative agent of food poisoning, at levels as low as 0.5 ng/ml in buffer. The toxin (SEB) can also be detected and quantitated in other relevant media: human serum, urine, and aqueous extract of ham. The level of toxin, from 5 to 200 ng/ml, can be accurately predicted in these media by calibrating each fiber and by comparing results to a single standard curve based on toxin in buffer. The quantitative fluorescent sandwich immunoassay provides results in 45 min; qualitative results are provided in 15-20 min. Using a blender and a benchtop centrifuge, fast, simple aqueous extracts of contaminated ham samples were prepared and tested. Ham spiked with 5 or 40 micrograms SEB per 100 g food resulted in biosensor readings indicative of 11 or 69% recovery of the toxin, respectively. Finally, the SEB assay is highly specific; SEA and SED give only 2-3% of the signal at 5000 ng/ml as SEB gives at 1000 ng/ml. This specific, sensitive assay for SEB on the portable fiber-optic biosensor permits easy monitoring of clinical samples or on-site analysis of suspect food samples.

Receptor-mediated binding of IgE-sensitized rat basophilic leukemia cells to antigen-coated substrates under hydrodynamic flow.

The physiological function of many cells is dependent on their ability to adhere via receptors to ligand-coated surfaces under fluid flow. We have developed a model experimental system to measure cell adhesion as a function of cell and surface chemistry and fluid flow. Using a parallel-plate flow chamber, we measured the binding of rat basophilic leukemia cells preincubated with anti-dinitrophenol IgE antibody to polyacrylamide gels covalently derivatized with 2,4-dinitrophenol. The rat basophilic leukemia cells' binding behavior is binary: cells are either adherent or continue to travel at their hydrodynamic velocity, and the transition between these two states is abrupt. The spatial location of adherent cells shows cells can adhere many cell diameters down the length of the gel, suggesting that adhesion is a probabilistic process. The majority of experiments were performed in the excess ligand limit in which adhesion depends strongly on the number of receptors but weakly on ligand density. Only 5-fold changes in IgE surface density or in shear rate were necessary to change adhesion from complete to indistinguishable from negative control. Adhesion showed a hyperbolic dependence on shear rate. By performing experiments with two IgE-antigen configurations in which the kinetic rates of receptor-ligand binding are different, we demonstrate that the forward rate of reaction of the receptor-ligand pair is more important than its thermodynamic affinity in the regulation of binding under hydrodynamic flow. In fact, adhesion increases with increasing receptor-ligand reaction rate or decreasing shear rate, and scales with a single dimensionless parameter which compares the relative rates of reaction to fluid shear.

Motion of model leukocytes near a wall in simple shear flow.

Receptor-mediated cell adhesion to surfaces depends on the motion of the cell prior to adhesion. We studied the hydrodynamic behavior of cells near a wall in simple shear flow using a model leukocyte system that we have also used extensively in cell-substrate adhesion studies. Specifically, we measured the velocity of rat basophilic leukemia (RBL) cells near a surface in a parallel-plate flow chamber and compared it to the motion of polystyrene beads and glutaraldehyde-fixed RBL cells. We found that RBL cells (13 microns diameter) travel 25% faster than polystyrene beads of 14.5 microns diameter for a wide range of shear rates (20-180 s-1); this suggests that RBL cells would travel 39% faster than polystyrene beads of equivalent size. Glutaraldehyde-fixed RBL cells travel at a velocity between those of live cells and 14.5-microns beads. These differences in velocities have been observed over both polyacrylamide gel and glass substrates. Application of a theory for hard sphere motion near a wall in simple shear flow at low Reynolds number [Goldman, A.J.; Cox, R.G.; Brenner, H. Chem. Eng. Sci. 1967b, 22, 653-660] to our measured cell velocities suggests that cells are separated from the wall by > or = 550 nm. Such large separation distances have also been predicted by others who have used hard sphere theory to analyze the effect of shear flow on cell motion near walls. However, the extensive receptor-mediated cell adhesion seen in these systems is inconsistent with these separation distances, which are approximately 30 times greater than the distance required for receptor-ligand contact. Instead, we propose that, because of cell deformability and cell surface roughness, cells remain within a separation distance that allows for molecular contact while they travel faster than the hard sphere theory predicts. Therefore, the theory of Goldman and co-workers, while adequate for hard sphere motion, is likely not accurate for cellular motion.

Statistics of cell adhesion under hydrodynamic flow: simulation and experiment.

Adhesion under hydrodynamic flow is a step in many complicated physiological processes such as the neutrophil-mediated inflammatory response and cancer cell metastasis. We use a combination of computer simulation and experiment to explore how a population of cells interacts with a ligand-coated substrate under shear flow. To simulate the binding of a single cell to a surface, we use a microvilli-hard sphere model in which receptor-ligand bonds are treated as springs, and the net motion of the cell is determined from a force balance involving hydrodynamic, bonding, and colloidal forces. We show that the adhesive phenotype of a cell depends strongly on the fractional spring slippage of receptor-ligand bonds, which relates the extension of a bond to its rate of breakage; a lower spring slippage indicates bonds can withstand a great deal of extension without a significant increase in the breakage rate, and hence leads to more strongly adherent cells. We construct the behavior of a population of cells by simulating many cells using this algorithm. We show that a homogeneous population of cells with identical numbers of receptors, modeled with parameters suitable to recreate neutrophil rolling, will display a distribution of translational velocities. In addition, we calculate the average velocity for a heterogeneous population of cells which has a Gaussian distribution in receptor number. As the standard deviation of this distribution increases, the average observed velocity for the population increases. Although the homogeneous and heterogeneous populations have the same average number of receptors (10(5)) per cell, there is a significant difference in their average velocity when the standard deviation of receptor number in the heterogeneous population is as little as 25% of the average receptor number. We also present experimental evidence that not all cells exhibit the slow rolling characteristic of neutrophil-endothelial interaction, but rather appear to exist in a "binary" state in which cells are either adherent or noninteracting. We have developed an experimental model system for studying adhesion under hydrodynamic flow, using the rat basophilic leukemia (RBL) derivatized polyacrylamide gels in a flow chamber. Cells are injected into a portion of the flow chamber in which the substrate is not coated with antigen, and allowed to flow over the antigen-coated portion of the gel. We have measured the spatial distribution of cell binding for a population of cells at different flow rates, and have shown that cell binding decreases as shear rate increases.(ABSTRACT TRUNCATED AT 400 WORDS)