Clark K. Colton

Implantable biohybrid artificial organs

Biohybrid artificial organs encompass all devices which substitute for an organ or tissue function and incorporate both synthetic materials and living cells. This review concerns implantable immunoisolation devices in which the tissue is protected from immune rejection by enclosure within a semipermeable membrane. Two critical areas are discussed in detail: (i) Device design and performance as it relates to maintenance of cell viability and function. Attention is focussed on oxygen supply limitation and how it is affected by tissue density and the development of materials that induce neovascularization at the host tissue-membrane interface; and (ii) Protection from immune rejection. Our current knowledge of the mechanisms that may be operative in immune rejection in the presence of a semipermeable membrane barrier is limited. Nonetheless, recent studies shed light on the role played by membrane properties in preventing immune rejection, and many studies demonstrate substantial progress towards clinically useful implantable immunoisolation devices.

Effect of Hypoxia on Insulin Secretion by Isolated Rat and Canine Islets of Langerhans

The effect of pO2s reduced below physiological levels on GSIR by isolated islets of Langerhans was investigated with a microperifusion apparatus that provided control of pO2 and rapid dynamic response. Second-phase insulin secretion was reduced substantially by hypoxia. The response to lower pO2 was rapid and reversible. Although the steady, normoxic (pO2 = 142 mmHg) second-phase secretion rate varied widely from one islet preparation to another, the ratio of Sx to S142 for each preparation could be represented by a single curve that exhibited a continuous reduction with decreasing pO2. For rat islets perifused 1 day after isolation, the secretion rate was nearly 100% of the normoxic value at a pO2 of 60 mmHg, 50% at 27 mmHg (P50, the pO2 at which the S142 is reduced by 50%), and ∼2% at 5 mmHg. Oxygen sensitivity of second-phase secretion rate declined after 1 wk of in vitro culture: P50 was 13 mmHg after 1 wk and remained at 10 mmHg after 2–5 wk of culture. Canine islets exhibited a P50 of 16 mmHg after 1 wk of culture. The reduction in insulin secretion is thought to be associated with the existence of pO2 gradients outside and inside the isolated islets, resulting in exposure of islet cells to low pO2 levels that decrease radially from the periphery to the core. We hypothesize that the effect of low pO2 on S is manifested through depletion of the energy stores of the β-cells. The effect of hypoxia on S may be an important factor in some in vitro secretion studies and may play a critical role in the effectiveness of transplanted islets before their revascularization and of immunoisolated islet implantation devices.

A CONCENTRATION POLARIZATION MODEL FOR THE FILTRATE FLUX IN CROSS-FLOW MICROFILTRATION OF PARTICULATE SUSPENSIONS

Cross-flow filtration with microporous membranes is increasingly used in the separation and concentration of particulate suspensions. Existing models for the filtrate flux are inadequate for correlating experimental observations and are based on contradictory physical mechanisms. We propose that the flux is limited by the formation of a dynamic concentration polarization boundary layer consisting of a high concentration of retained particles. A simple model is developed incorporating a shear-enhanced diffusivity of the large particles which arises from mutually induced velocity fields in the shear flow of the concentrated suspension. Predictions of the model agree well with experimental data for a variety of particulate suspensions. The model provides both a fundamental understanding of the physical phenomena governing flux and a rational basis for design of improved cross-flow filters.

The osmotic pressure of concentrated protein solutions: Effect of concentration and ph in saline solutions of bovine serum albumin

Abstract Osmotic pressure measurements are reported as a function of bovine serum albumin (BSA) concentration in 0.15 M sodium chloride at pH 4.5, 5.4, and 7.4. The measured values increased markedly with increasing BSA concentration and with increasing pH (and therefore increasing macroion charge). At a concentration of 450 g/liter solution and a pH of 7.4, osmotic pressure was nearly five atmospheres, which is more than four times the value measured at the same concentration and a pH of 4.5 and about 30 times the value expected for an ideal solution. A semi-empirical analytical expression was developed which gave good agreement between prediction and the experimental data of this and other studies. The data were also compared to the prediction of a three-term virial equation wherein the second and third virial coefficients were calculated by using McMillan-Mayer solution theory. The expression for the potential of mean force was obtained by comparing various contributions to the potential energy of interaction. The terms for electrostatic repulsion and dispersion attraction are the same as those used in the DLVO theory of colloid stability. The predicted curves are of the correct order of magnitude and follow the correct qualitative trend with pH, but they fail to display the strong pH-dependence of the data. The factors responsible for this deficiency are assessed and opportunities for developing a more realistic potential function are identified.

The distribution of labeled low-density lipoproteins across the rabbit thoracic aorta in vivo☆

Abstract Transmural concentration profiles of 125 I-labeled low-density lipoproteins (LDL) within the descending thoracic aorta were determined as a function of time following intravenous injection into normal conscious rabbits. The animals were sacrificed after 10 min to 67 h, and the descending thoracic aorta was immediately excised, opened longitudinally, rinsed and frozen. Samples of frozen aorta were sectioned parallel to the intimal surface and washed with trichloroacetic acid (TCA) prior to counting. Up to 4 h, transmural concentration profiles of TCA-precipitable radioactivity had steep gradients near the intimal surface, moderate gradients near the medial-adventitial border, and were relatively flat in the middle of the media. After 24 h, the steep intimal gradient had disappeared. Concentration levels were otherwise comparable to those at 4 h. All gradients disappeared and concentration levels were lower after 67 h. The rate of accumulation of TCA-precipitable radioactivity was initially rapid (measurable concentrations were found throughout the media after only 10 min), although it was less than that of [ 125 I] albumin observed in a previous study. The results are consistent with entry of [ 125 I]LDL into the media from both the luminal and adventitial sides and with gradual degradation of the labeled LDL within the aortic wall. Approximate calculations indicate that the LDL mass transfer resistance associated with the intimal endothelium is about an order of magnitude greater than that associated with the media.

Transplantation of human islets without immunosuppression

Significance Diabetes mellitus type 1 is an autoimmune disease that results in irreversible destruction of insulin-producing beta cells. Substantial advances have been made in beta cell replacement therapies over the last decades. However, lack of eligible donor organs and the need for chronic immunosuppression to prevent rejection critically limit a widespread application of these strategies. In this paper we present the clinical success of using a bioartificial pancreas for the transplantation of insulin-producing islets without affecting the immune system. In a patient with long-standing type-1 diabetes we could demonstrate persistent graft function and regulated insulin secretion without the need for immune-modulating medication. This strategy opens up avenues for more widespread and safe application of various cell-based therapies. Transplantation of pancreatic islets is emerging as a successful treatment for type-1 diabetes. Its current stringent restriction to patients with critical metabolic lability is justified by the long-term need for immunosuppression and a persistent shortage of donor organs. We developed an oxygenated chamber system composed of immune-isolating alginate and polymembrane covers that allows for survival and function of islets without immunosuppression. A patient with type-1 diabetes received a transplanted chamber and was followed for 10 mo. Persistent graft function in this chamber system was demonstrated, with regulated insulin secretion and preservation of islet morphology and function without any immunosuppressive therapy. This approach may allow for future widespread application of cell-based therapies.

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 grafts 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.

Improvement of islet function in a bioartificial pancreas by enhanced oxygen supply and growth hormone releasing hormone agonist

Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients. Here we present a promising approach to address these limitations by utilization of a macrochamber specially engineered for islet transplantation. The s.c. implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. The minimally invasive implantable chamber normalized blood glucose in streptozotocin-induced diabetic rodents for up to 3 mo. Sufficient graft function depended on oxygen supply. Pretreatment with the growth hormone-releasing hormone (GHRH) agonist, JI-36, significantly enhanced graft function by improving glucose tolerance and increasing β-cell insulin reserve in rats thereby allowing for a reduction of the islet mass required for metabolic control. As a result of hypervascularization of the tissue surrounding the device, no relevant delay in insulin response to glucose changes has been observed. Consequently, this system opens up a fundamental strategy for therapy of diabetes and may provide a promising avenue for future approaches to xenotransplantation.

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.