Artificial pancreas using living beta cells:. effects on glucose homeostasis in diabetic rats.

An artificial pancreas consisting of beta cells cultured on synthetic semipermeable hollow fibers was tested in rats with alloxan-induced diabetes. When implanted ex vivo as arteriovenous shunts in the circulatory system these devices lowered concentrations of plasma glucose from 533 to between 110 and 130 milligrams per 100 milliliters, increased concentrations of plasma insulin, and restored intravenous glucose tolerance tests essentially to normal.

Ultrafiltration of lipoproteins through a synthetic membrane. Implications for the filtration theory of atherogenesis.

To investigate the interaction of lipoproteins with semipermeable membranes, solutions of low density lipoproteins (LDL), very low density lipoproteins (VLDL), mixtures of the two, and diluted, normal, and hyperlipidemic serum were ultrafiltered through a synthetic membrane (500 A nominal pore diameter) using a stirred laboratory ultrafiltration cell. The pressure dependence of ultrafiltrate flux showed that a concentrated layer of lipoproteins was built up at the membrane surface (concentration polarization) and that VLDL was more subject to polarization than LDL. This phenomenon controlled the observed lipoprotein transport behavior. Whereas true membrane rejection (the fraction of the solute on the membrane surface which does not pass through the membrane) was greater than 0.95 for both LDL and VLDL, observed solute rejection varied from nearly 0 to 1.0, depending upon experimental conditions. If concentration polarization occurs in the arterial system, these results suggest that lipoprotein transport into arterial wall may be influenced not only by arterial blood pressure and the properties of the arterial wall, but also by local hemodynamic conditions and by the relative as well as absolute magnitudes of LDL and VLDL concentration.

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.

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.

Time course of membrane microarchitecture-driven neovascularization.

The host response to a microporous material that induces neovascularization at the material-tissue interface was studied in terms of the number and types of cells invading the membrane, the degree of vascularization at the material-tissue interface, and the characteristics of the surrounding connective tissue as a function of time following implantation. Millipore-MF mixed esters of cellulose membranes with a nominal pore diameter of 8.0 microns were implanted subcutaneously into male Sprague-Dawley rats and explanted at 3, 5, 7, 10, 21 and 329 days post-implantation. Two samples from each of two devices at each implantation time were embedded in paraffin, sectioned to a thickness of 5 microns, and stained with haematoxylin and eosin for light microscopic observation. The density of cells in the membrane increased up to 7 days following implantation, then remained roughly constant through 21 days and decreased at the 329 day time point. The vascularity of the material-tissue interface increased up to 10 days and remained at this level even at 329 days post-implantation. The connective tissue was disorganized, loose and avascular at 3 days, resembled granulation tissue at 5 days, and underwent fibrous capsule formation and maturation starting at 7 days following implantation.

Analysis of membrane processes for blood purification.

Physical phenomena play an important role in membrane processes for blood purification. They largely determine the separation performance of these devices and they interact with chemical and biological phenomena to determine their biocompatibility, or lack thereof, in the clinical setting. In the first part of this paper, analyses of physical phenomena which determine the separation and purification characteristics are reviewed for several processes, including hemodialysis, hemofiltration, combined hemodialysis and ultrafiltration, and membrane plasmapheresis with cross-flow microfiltration. Special attention is given to transport of high-molecular weight solutes in hemodialysis, for use in subsequent analyses, and to the factors which determine filtrate flux in membrane plasmapheresis, because recent findings in this area provide an understanding of filtration processes in general. The second part concerns the problem of biocompatibility, especially as manifested in renal prostheses. After reviewing some of the pathways to bioincompatibility, exploratory analyses are presented using relatively simple models. The objective of these analyses is to provide an initial quantitative framework for examining the likelihood of monocyte secretion of interleukin-1 being stimulated by various routes. Issues examined, for which illustrative calculations are presented, include (1) transport of endotoxin fragments across regenerated cellulose and other membranes, (2) anaphylatoxin C5a concentrations in conventional hemodialysis and (3) the effects of equilibrium and reaction phenomena, ultrafiltration, diffusive membrane permeation and membrane adsorption on the disposition of C5a which is generated at the membrane surface.

Bioengineering in development of the hybrid artificial pancreas.

The hybrid artificial pancreas for treatment of diabetes consists of insulin-secreting pancreatic tissue which is surrounded by a membrane that protects the tissue from rejection by the immune system following implantation. In this paper, we review the alternative therapeutic approaches for diabetes under study and then discuss the technical requirements that must be met by a hybrid device useful to humans. Previous work on intravascular and extravascular immunoisolation devices is reviewed from the standpoint of these requirements, and three critical unresolved issues are discussed: biocompatibility, oxygen supply limitations, and prevention of immune rejection.

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.

Kinetics of hemodiafiltration. II. Clinical characterization of a new blood cleansing modality.

Hemodiafiltration, a process in which whole blood is first diluted with a physiologic electrolyte solution and then ultrafiltered across a membrane to convectively remove solutes and excess water, has been applied clinically for the first time. Six-hour hemodiafiltration with a 1.6 m.2 hollow-fiber ultrafilter was applied intermittently to an anephric patient as an alternative to 6-hour hemodialysis using a 1.45 m.2 coli. A quantitative basis for evaluating clinical hemodiafiltration kinetics was developed, and the results were compared with data from prototype devices. With blood, diluting fluid, and ultrafiltrate flow rates of 200 ml. per minute, removal rates of urea and creatinine by both hemodiafiltration and dialysis were comparable, but for solutes of larger molecular weight (uric acid, phosphate, and inulin) removal rate was significantly greater for hemodiafiltration. The observed ultrafiltrate flux was similar to values predicted from in vitro studies. With the present membrane formulation the measured sieving coefficients for inulin, creatinine, and urea were not significantly different from one, and whole blood clearances for these solutes were 117, 108, and 101 ml. per minute, respectively. This solute clearance pattern is very similar to the human kidney and in sharp contrast to standard coil hemodialysis.

Kinetics of hemodiafiltration. I. In vitro transport characteristics of a hollow-fiber blood ultrafilter.

The transport characteristics of a hollow-fiber blood ultrafilter were studied in vitro to provide an understanding of the factors which control solute removal rate and to permit design of a full-scale clinical device. The dependence of ultrafiltrate flux on transmembrane pressure difference, protein concentration, flow rate, and fiber geometry was correlated in terms of available theoretical analyses. Solute rejection was 1.0 for albumin and decreased to nearly zero for solutes of several thousand molecular weight. An analysis of overall hemodiafilter behavior showed that blood and ultrafiltrate flow rates of 200 ml. per minute can be attained with a device of reasonable size which would provide an inulin whole blood clearance of about 100 ml. per minute.

Permeability of dialyzer membranes to TNF alpha-inducing substances derived from water bacteria.

Pro-inflammatory cytokine-inducing substances derived from cultured E. coli have previously been shown to pass across low-flux regenerated cellulosic dialyzer membranes. In the present study, a sterile filtrate of Pseudomonas maltophilia grown from standard bicarbonate dialysis fluid was used to test the permeability of various dialyzer membranes (regenerated cellulose, cellulose triacetate, polyacrylonitrile, polysulfone and polyamide) to TNF alpha-inducing bacterial substances. Pyrogen-free tissue culture medium (MEM) was recirculated for 60 minutes in the dialysate compartment of a closed-loop dialysis system, then P. maltophilia filtrate was added and recirculation was continued for a further hour. Samples from the dialysate (MEM) and the blood side (containing 10% human plasma in MEM) were incubated with donor mononuclear cells (MNC) for 18 hours and TNF alpha release was measured in MNC supernatants by radioimmunoassay. Five minutes after the addition of P. maltophilia filtrate, mean TNF alpha-inducing activity in the dialysate increased from (mean +/- SEM) 0.10 +/- 0.02 to 18.2 +2- 1.5 (ng/2.5 x 10(6) MNC/18 hr). TNF alpha-inducing activity in the blood side increased with regenerated cellulose from 0.10 +/- 0.01 to 4.57 +/- 1.55 (N = 8; P less than 0.001); with cellulose triacetate from 0.20 +/- 0.05 to 0.44 +/- 0.10 (N = 5; P less than 0.05), and with polyacrylonitrile from 0.10 +/- 0.02 to 1.16 +/- 0.45 (N = 5; P less than 0.03). No increased TNF alpha-inducing activity was observed in the blood side of polysulfone (N = 5) or polyamide dialyzers (N = 5).(ABSTRACT TRUNCATED AT 250 WORDS)