Oxygen supply by photosynthesis to an implantable islet cell device.

Transplantation of islet cells is an effective treatment for type 1 diabetes with critically labile metabolic control. However, during islet isolation, blood supply is disrupted, and the transport of nutrients/metabolites to and from the islet cells occurs entirely by diffusion. Adequate oxygen supply is essential for function/survival of islet cells and is the limiting factor for graft integrity. Recently, we developed an immunoisolated chamber system for transplantation of human islets without immunosuppression. This system depended on daily oxygen supply. To provide independence from this external source, we incorporated a novel approach based on photosynthetically-generated oxygen. The chamber system was packed sandwich-like with a slab of immobilized photosynthetically active microorganisms (Synechococcus lividus) on top of a flat light source (LEDs, red light at 660 nm, intensity of 8 µE/m(2)/s). Islet cells immobilized in an alginate slab (500-1,000 islet equivalents/cm(2)) were mounted on the photosynthetic slab separated by a gas permeable silicone rubber-Teflon membrane, and the complete module was sealed with a microporous polytetrafluorethylene (Teflon) membrane (pore size: 0.4 µm) to protect the contents from the host immune cells. Upon illumination, oxygen produced by photosynthesis diffused via the silicone Teflon membrane into the islet compartment. Oxygen production from implanted encapsulated microorganisms was stable for 1 month. After implantation of the device into diabetic rats, normoglycemia was achieved for 1 week. Upon retrieval of the device, blood glucose levels returned to the diabetic state. Our results demonstrate that an implanted photosynthetic bioreactor can supply oxygen to transplanted islets and thus maintain islet viability/functionality.

Rat islet cell aggregates are superior to islets for transplantation in microcapsules.

AIMS/HYPOTHESIS: Islet transplantation is a promising treatment for type 1 diabetes but is hampered by a shortage of donor human tissue and early failure. Research on islet cell transplantation includes finding new sources of cells and immunoisolation to protect from immune assault and tumourigenic potential. Small islet cell aggregates were studied to determine if their survival and function were superior to intact islets within microcapsules because of reduced oxygen transport limitation and inflammatory mediators. METHODS: Islet cell aggregates were generated by dispersing rat islets into single cells and allowing them to re-aggregate in culture. Rat islets and islet cell aggregates were encapsulated in barium alginate capsules and studied when cultured in low (0.5% or 2%) or normal (20%) oxygen, or transplanted into mice. RESULTS: Encapsulated islet cell aggregates were able to survive and function better than intact islets in terms of oxygen-consumption rate, nuclei counts, insulin-to-DNA ratio and glucose-stimulated insulin secretion. They also had reduced expression of pro-inflammatory genes. Islet cell aggregates showed reduced tissue necrosis in an immunodeficient transplant model and a much greater proportion of diabetic xenogeneic transplant recipients receiving islet cell aggregates (tissue volume of only 85 islet equivalents) had reversal of hyperglycaemia than recipients receiving intact islets. CONCLUSIONS/INTERPRETATION: These aggregates were superior to intact islets in terms of survival and function in low-oxygen culture and during transplantation and are likely to provide more efficient utilisation of islet tissue, a finding of importance for the future of cell therapy for diabetes.

Human islet oxygen consumption rate and DNA measurements predict diabetes reversal in nude mice.

There is a need for simple, quantitative and prospective assays for islet quality assessment that are predictive of islet transplantation outcome. The current state-of-the-art athymic nude mouse bioassay is costly, technically challenging and retrospective. In this study, we report on the ability of 2 parameters characterizing human islet quality: (1) oxygen consumption rate (OCR), a measure of viable volume; and (2) OCR/DNA, a measure of fractional viability, to predict diabetes reversal in nude mice. Results demonstrate that the probability for diabetes reversal increases as the graft's OCR/DNA and total OCR increase. For a given transplanted OCR dose, diabetes reversal is strongly dependent on OCR/DNA. The OCR and OCR/DNA (the 'OCR test') data exhibit 89% sensitivity and 77% specificity in predicting diabetes reversal in nude mice (n = 86). We conclude that the prospective OCR test can effectively replace the retrospective athymic nude mouse bioassay in assessing human islet quality prior to islet transplantation.

2-Aminoethoxydiphenyl borate as a common activator of TRPV1, TRPV2, and TRPV3 channels.

2-Aminoethoxydiphenyl borate (2APB) had been depicted as a universal blocker of transient receptor potential (TRP) channels. While evidence has accumulated showing that some TRP channels are indeed inhibited by 2APB, especially in heterologous expression systems, there are other TRP channels that are unaffected or affected very little by this compound. More interestingly, the thermosensitive TRPV1, TRPV2, and TRPV3 channels are activated by 2APB. This has been demonstrated both in heterologous systems and in native tissues that express these channels. A number of 2APB analogs have been examined for their effects on native store-operated channels and heterologously expressed TRPV3. These studies revealed a complex mechanism of action for 2APB and its analogs on ion channels. In this review, we have summarized the current results on 2APB-induced activation of TRPV1-3 and discussed the potential mechanisms by which 2APB may regulate TRP channels.

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.

Pancreas oxygenation is limited during preservation with the two-layer method.

BACKGROUND: The two-layer method (TLM) for pancreas preservation reportedly improves islet yield and transplantation outcome relative to previous methods. Increased ATP concentrations support the hypothesis that these improvements are related to better oxygenation from the perfluorocarbon solution. However, there are limited direct measurements of oxygen partial pressure, (pO(2)) in pancreata preserved with the TLM. Theory predicts that only a small fraction of a human pancreas can be oxygenated externally. In this report we examine pancreas oxygenation with the TLM using theory and direct pO(2) measurements. METHODS: pO(2) profiles in cylindrical pancreata were calculated at various temperatures with a diffusion-reaction model. The pO(2) was measured using fiber optic sensors in the core of porcine pancreatic tissue preserved with the TLM in media saturated with 100% oxygen. RESULTS: The model predicts that at 8 degrees C, even in the absence of an external pO(2) gradient, oxygen penetration depth is about 1 mm and insensitive to pancreas diameter, while the oxygenated volume fraction is about 15% for a 2.5-cm-diameter pancreas. Experimental measurements verified that pO(2) is virtually zero in the core of a 1-cm-thick pancreatic piece preserved with the TLM. Penetration of solution around the sensor may be responsible for the observed lag and for the previously reported nonzero pO(2) measurements. Reoxygenation of heat-treated tissue took several hours. CONCLUSIONS: The TLM can oxygenate only a small volume fraction of a human pancreas. Pancreas oxygenation through the native vasculature should be explored to further improve yield of viable islets.

Islets in alginate macrobeads reverse diabetes despite minimal acute insulin secretory responses.

BACKGROUND: Encapsulation of islets has been widely investigated as a treatment for diabetes. The characteristics and dynamics of insulin secretion by encapsulated islets in response to glucose and other secretagogues are not well understood. METHODS: In our study, macroencapsulated syngeneic islets at 3-4 wk after transplantation were studied for insulin release in response to i.v. glucose (hyperglycemic clamps at 250 or 350 mg/dl plasma glucose), arginine (i.v. bolus, 100 mg/kg), glucagon-like peptide-1 (i.v. infusion for 20 min, 2.2 pmol/kg/min), and meal challenge. Syngeneic islets (6000 islets) were encapsulated in alginate macrobeads (2-3 mm diameter) with or without poly-L-lysine coating and transplanted into the peritoneal cavity of STZ-diabetic Lewis rats. Normal (nontransplanted) and diabetic Lewis rats transplanted with "naked" islets under the kidney capsule served as controls. RESULTS: Animals transplanted with macrobeads displayed subnormal insulin responses to glucose, arginine, and glucagon-like peptide-1 despite achieving normoglycemia faster than animals with renal subcapsular islet transplants. Plasma insulin responses to meal challenges were blunted in animals with macrobeads resulting in increased plasma glucose excursions. CONCLUSIONS: We conclude that, after transplantation into diabetic Lewis rats, macroencapsulated islets have significantly impaired insulin secretion despite achieving normal fed glycemic levels.

NMR spectroscopy in beta cell engineering and islet transplantation.

Islet transplantation is a promising method for restoring normoglycemia and alleviating the long term complications of diabetes. Widespread application of islet transplantation is hindered by the limited supply of human islets and requires a large increase in the availability of suitable insulin secreting tissue as well as robust quality assessment methodologies that can ensure safety and in vivo efficacy. We explore the application of nuclear magnetic resonance (NMR) spectroscopy in two areas relevant to beta cell engineering and islet transplantation: (1) the effect of genetic alterations on glucose metabolism, and (2) quality assessment of islet preparations prior to transplantation. Results obtained utilizing a variety of NMR techniques demonstrate the following: (1) Transfection of Rat1 cells with the c-myc oncogene (which may be involved in cell proliferation and cell cycle regulation) and overexpression of Bcl-2 (which may protect cells from stresses such as hypoxia and exposure to cytokines) introduce a wide array of alterations in cellular biochemistry, including changes in anaerobic and oxidative glucose metabolism, as assessed by 13C and 31P NMR spectroscopy. (2) Overnight incubation of islets and beta cells in the bottom of centrifuge tubes filled with medium at room temperature, as is sometimes done in islet transportation, exposes them to severe oxygen limitations that may cause cell damage. Such exposure, leading to reversible or irreversible damage, can be observed with NMR-detectable markers using conventional 13C and 31P NMR spectroscopy of extracts. In addition, markers of irreversible damage (as well as markers of hypoxia) can be detected and quantified without cell extraction using high-resolution magic angle spinning 1H NMR spectroscopy. Finally, acute ischemia in a bed of perfused beta cells leads to completely reversible changes that can be followed in real time with 31P NMR spectroscopy.

Improved vascularization of planar membrane diffusion devices following continuous infusion of vascular endothelial growth factor.

Improving blood vessel formation around an immunobarrier device should improve the survival of the encapsulated tissue. In the present study we investigated the formation of new blood vessels around a planar membrane diffusion device (the Baxter Theracyte System) undergoing a continuous infusion of vascular endothelial growth factor through the membranes and into the surrounding tissue. Each device (20 microl) had both an inner immunoisolation membrane and an outer vascularizing membrane. Human recombinant vascular endothelial growth factor-165 was infused at 100 ng/day (low dose: n = 6) and 500 ng/day (high dose: n = 7) for 10 days into devices implanted s.c. in Sprague-Dawley rats; noninfused devices transplanted for an identical period were used as controls (n = 5). Two days following the termination of VEGF infusion, devices were loaded with 20 microl of Lispro insulin (1 U/kg) and the kinetics of insulin release from the lumen of the device was assessed. Devices were then explanted and the number of blood vessels (capillary and noncapillary) was quantified using morphometry. High-dose vascular endothelial growth factor infusion resulted in two- to threefold more blood vessels around the device than that obtained with the noninfused devices and devices infused with low-dose vascular endothelial growth factor. This increase in the number of blood vessels was accompanied by a modest increase in insulin diffusion from the device in the high-dose vascular endothelial growth factor infusion group. We conclude that vascular endothelial growth factor can be used to improve blood vessel formation adjacent to planar membrane diffusion devices.

In situ electrochemical oxygen generation with an immunoisolation device.

The viability and function of transplanted tissue encapsulated in immunobarrier devices is subject to oxygen transport limitation. In this study, we have designed and used an in situ electrochemical oxygen generator which decomposes water electrolyticaly to provide oxygen to the adjacent planer immunobarrier diffusion chamber. The rate of oxygen generation, which increases linearly with electrical current, was accurately controlled. A theoretical model of oxygen diffusion was also developed and was used to calculate the oxygen profiles in some of the experimental systems. In vitro culture experiments were carried out with beta TC3 cells encapsulated in titanium ring devices. The growth and viability of cells with or without in situ oxygen generation was studied. We found that under otherwise similar culturing conditions, the thickness of the cell layer and the viability of cells was the highest in devices cultured in stirred media with oxygen generation, even though the thickness had not reached the theoretically predicted value, and lowest in those unstirred and without oxygen generation.

Reversal of hyperglycemia in mice after subcutaneous transplantation of macroencapsulated islets.

BACKGROUND: Macroencapsulated islets can reverse hyperglycemia in diabetic animals when transplanted i.p. or into the fat pad. The s.c. space is an attractive site for such transplantation because macrocapsules can be implanted with local anesthesia and be easily removed or reloaded with fresh islets. METHODS: Immunoprotective 20 microl ported devices were transplanted under the skin of Streptozocin-diabetic nude mice. Devices were loaded with 1200 rat islets in culture medium or in alginate. Empty devices were implanted for 2 weeks and then loaded with islets. Normal mice and mice with islets transplanted under the renal capsule or under the skin were used as controls. Seven weeks after transplantation, an intraperitoneal glucose tolerance test (IPGTT) was performed, followed by implant removal. RESULTS: Three weeks after transplantation, normal blood glucose levels were observed in all animals. Compared with those of normal controls, IPGTTs showed accelerated blood glucose clearance in mice transplanted with islets either within devices or beneath the kidney capsule. Fasted transplanted mice were hypoglycemic before glucose injection and 2 hr later. After removal of the implants, all recipient mice returned to hyperglycemia. Histological evaluation revealed viable islet cells and a network of close vascular structures outside the devices. CONCLUSIONS: Macroencapsulated islets transplanted into the s.c. space were able to survive and regulate blood glucose levels in mice. The observed differences in glucose metabolism between normal and transplanted mice may be attributed to the site of transplantation and to the use of rat islets, which have a different set point for glucose induced insulin release.

Characterization of cell detachment from hollow fiber affinity membranes.

Hollow fiber affinity membranes have potential for use in cell separations because they offer many advantages over currently available techniques. An understanding of the parameters controlling cell attachment and detachment from the surface is vital to the success of the separation. Cell attachment was probed with transmission electron microscopy (TEM), which revealed that gravity settling must be used to bring the cells to the surface of the membrane, because forward pressurization caused cells to infiltrate the pores of the membranes. Fluorescence microscopy studies showed that viable target cells could be recovered from the surface through the use of either back pressurization or liquid drainage and that 98% of the cells could be recovered from the surface through the sequential use of both. This ability to reproducibly detach target cells from the surface suggests that cell recovery will not be a limiting factor for cell separations with hollow fiber membranes.

Estimating number and volume of islets transplanted within a planar immunobarrier diffusion chamber.

Cell therapy involving the transplantation of cells or tissues with specific differentiated functions has potential in the treatment of human disease. However, the need for immunosuppressive drugs may lead to a variety of serious side effects. One approach to minimizing or eliminating systemic immunosuppression is immunoisolation, in which the transplanted tissue is enclosed in a semipermeable barrier or membrane in order to protect it from immune rejection, thereby creating what has been termed an implantable biohybrid artificial organ. Devices of this type are under study for the treatment of a wide variety of diseases, for example, diabetes.

Function and survival of macroencapsulated syngeneic islets transplanted into streptozocin-diabetic mice.

BACKGROUND: Macroencapsulation is a strategy to protect transplanted islets from rejection and autoimmune attack. This study addresses questions about the survival and function of macroencapsulated syngeneic islets. METHODS: Planar immunobarrier membrane diffusion devices were used for syngeneic islet transplantation. After being mixed with a 1% alginate solution, a total of 250, 500, 750 or 1000 islets were loaded into the devices, which were implanted into the epididymal fat pad(s) of streptozocin diabetic mice. RESULTS: The success rate for restoration of normoglycemia at week 4 was highest for the recipients receiving two devices, each with 500 islets. Loading 750 or 1000 islets provided no improvement over loading 500 islets in a single device. Devices containing 250 islets were rarely successful. There was a striking tendency of transplants to either bring glucose levels into the near normal range or to fail with marked hyperglycemia. After an overnight fast at 1 and 4 weeks, but not at 12 weeks, hypoglycemia was found. The insulin content of devices from animals with normalized glucose values was higher than the insulin content in failed devices. Islet volume was maintained for 12 weeks, and fibrosis did not increase. CONCLUSIONS: A relatively small mass of macroencapsulated islet tissue can survive and function well enough to normalize glucose levels for at least 12 weeks. Maintenance of glucose levels in the near-normal range seems to have a beneficial influence on graft success. The finding of fasting hypoglycemia raises important clinical questions about islet dysfunction. Important limitations in the requirements for islet packing density in macroencapsulation have been defined. New approaches for improving islet packing density must be developed to make diffusion-dependent macroencapsulation more practical.

Number and volume of islets transplanted in immunobarrier devices.

Immunobarrier devices may prevent immune destruction of transplanted islets, but there are concerns about survival within such devices. Islets were transplanted in diffusion chambers that employed two laminated polytetrafluoroethylene membranes held together with titanium rings. Five hundred syngeneic mouse islets placed in devices were transplanted into the epididymal fat pads of streptozotocin (STZ) diabetic mice (B6AF1). After 2 wk the devices were removed. Sections were made parallel to the membrane surface. Eight to 13 systematically selected sections of each device were analyzed by planimetry to determine the area of the device space and of the islets within that space. From these data we estimated total volume of the device, volume of islets, and number of islets in a device. The data were segregated into two groups: group I (blood glucose less than 100 mg/dL 2 wk after implantation), and group II (over 150 mg/dL). The volume (mean +/- SE) of devices implanted for 2 wk was 2.1 +/- 0.4 microL in group I and 2.2 +/- 0.2 microL in group II. The islet volume and number within devices were 0.30 +/- 0.06 and 0.17 +/- 0.01 microL, or 340 +/- 50 and 230 +/- 20 islets in group I and group II, respectively. The volume of fibrous tissue in devices was about 0.50 microL. About 10% of the islet tissue had central necrosis. The beta cell volume in a membrane device needed for cure is comparable to that required with islets under the kidney capsule (0.25-0.80 microL). The mass of islets contained within membrane devices needed to cure diabetes is equivalent to that of a graft in an optimal transplant site such as under the kidney capsule.

Microcinematographic studies of flow patterns in the excised rabbit aorta and its major branches.

Arterial fluid mechanics may play a role as a localizing factor for early atherosclerosis. Flow patterns in natural rabbit aortas rendered transparent were studied using a microcinematographic visualization technique. The aortic arch exhibited a single cell of clockwise-rotating helical secondary flow along the ventral and inner walls. Flow separation occurred proximal to the two arch branches with flow reversal proximal to the brachiocephalic artery. Sinusoidal flow rendered the helical motion more pronounced in systole, while the reverse flow zone periodically expanded and contracted. Steady flow in the abdominal aorta revealed streamlines which follow slow looping trajectories lateral to ostia before tracing helical paths into the branches. Flow separation was present along the dorsal wall of the aorta opposite the superior mesenteric artery. With the exception of the left renal artery, steady flow wall shear stresses were higher distal to ostia than proximal. Spatial gradients of wall shear stress were larger around branches than elsewhere. Similar to observed flow patterns, sites of enhanced macromolecular permeability, as observed previously in the normal rabbit aorta, follow a clockwise helical pattern in the arch and exhibit a distribution around ostia that correlates to some degree with regions of elevated shear stress gradients.

Measurement of flow rates through aortic branches in the anesthetized rabbit.

The volumetric flow rates, mean and pulsatile, in the aorta and its major branches were measured in nonfed, anesthetized rabbits, using a transit time Doppler ultrasonic flowmeter. Anesthesia was maintained with isoflurane, and a vasodilator was applied topically during the measurements to avoid introducing additional flow resistance due to vasoconstriction. The cranial mesenteric and celiac arteries received the bulk of the aortic flow, (mean +/- SD) 29.5 +/- 6.6% and 23.3 +/- 5.8%, respectively, for mean flow. The brachiocephalic artery received as much as 14.7 +/- 3.2%, while each of the other branches received a considerably smaller fraction: 7.1 +/- 2.5% for the left subclavian artery, 6.2 +/- 2.6% and 5.1 +/- 2.2%, respectively, for the right and left renal arteries, and 6.0 +/- 2.5% for each of the two iliac arteries. Flow divisions were nearly the same in paired vessels. Peak pulsatile flow divisions were similar to their steady flow counterparts in the brachiocephalic, left subclavian, celiac, and cranial mesenteric arteries, but were smaller in the renal and iliac arteries, although the difference was not statistically significant. Reverse flow from one or more of the branches back into the aorta occurred in diastole in seven of eight rabbits studied.