C. K. Colton E. S. Avgoustiniatos Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139

Bioengineering in Dewelopment 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.

Introduction Diabetes is a chronic disease that constitutes a major national health problem. The inadequacy of conventional therapy has stimulated research on development of alternative therapeutic modalities. This paper concerns one such approach, the hybrid artificial pancreas, which is fabricated from both synthetic materials and living tissue. We begin with a brief discussion of the characteristics and costs of diabetes and the alternative approaches suggested for improved therapy. The remainder of the paper, devoted to the hybrid approach, is divided into three major sections: (1) The technical requirements and problems that must be addressed to bring this approach to clinical reality; (2) a review of previous work in these various areas; and (3) a discussion of critical unresolved problems. The pancreas is located in the abdomen adjacent to the liver and beneath the stomach. It consists mostly of acinar cells which secrete digestive enzymes into the small intestine. About 1 to 2 percent of the mass is composed of islets of Langerhans which secrete the hormones insulin, glucagon, and somatostatin into the portal vein that drains into the liver. In response to increasing blood glucose concentration, the beta cells in the islets secrete insulin, a small protein of about 6 kD molecular weight. Insulin acts on cell receptors throughout the body to increase the uptake and utilization of glucose. Insulin secretion is either impaired or destroyed entirely in diabetes, as a result of which the blood glucose concentration climbs to levels far exceeding its normal range. There were 5.7 million diagnosed diabetics in the United States as of 1985 and about 5 million more as yet undiagnosed (Table 1). The total of nearly 11 million is increasing at about 5 percent per year. Diabetes and its complications are the third leading cause of death in the United States, accounting for 34 thousand directly related to diabetes and 260 thousand attributable to its complications. These complications include blindness, kidney disease, gangrene, heart disease, and stroke, all of which occur at an incidence substantially greater than that in the normal population. Diabetes is the leading cause of Contributed by the Bioengineering Division for publication in the JOURNAL OF BIOMECHANICAL ENGINEERINO. Manuscript received by the Bioengineering

Division February 12, 1991; revised manuscript received February 12, 1991.

blindness in adults, and it leads to 20 thousand lower extremity amputations per year. A complication not found in the general population is ketoacidosis, which is the leading cause of the death among diabetics between the ages of 10 and 19. The death rate among diabetics is 8 times higher than that of normals, and they have a substantially reduced life expectancy. There is a 50 percent mortality by age 50 for diabetics with onset before age 20. There are two major types of diabetes. Type I occurs in about 5 to 10 percent of cases of diabetes. Thus, there were about 0.5 to 1 million Type I diabetes in the United States in Table 1 Diabetes statistics (USA, 1985)" Incidence 1 in 40 Diagnosed diabetes 5,700,000 Undiagnosed diabetes 5,000,000 1 in 20 Estimated total 10,700,000 Increasing 5%/year Mortality Diabetes and its complications are third leading cause of death Deaths each year: 34,000 directly related to diabetes 260,000 due to its complications Incidence X Normal Complications Blindness Leading cause of new blindness in adults 25 (5,000/year) 17 Kidney disease 5 Gangrene 20,000 lower extremity amputations/year Heart disease/stroke 75% of diabetic deaths due to atherosclerosis Ketoacidosis 15,000 episodes/year 42% of diabetic mortality between ages 10-19 Mortality Life expectancy 1/3 less 50% mortality by age 50 for diabetes with onset before age 20 "Adapted from Marble et al., 1985 [1].

1 5 2 / V o l . 113, MAY 1991

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Table 2 The cost of diabetes (1987)' Individual patient (for diabetes only) Type II diabetic on oral drugs Type I diabetic on insulin injections National cost (USA)

Annual cost $1200 $1600-3400

Diagnostic and therapeutic care Inpatient hospital (including complications) Nursing home Outpatient care Physician visits Pharmaceuticals/supplies/tests Total direct costs Work losses due to disability Productivity losses due to premature death Total indirect costs Total cost

$ 7.0 billion $ 0.9 billion $ 0.4 $ 1.3 $ 9.6 $ 3.3 $ 7.5 $10.8 $20.4

billion billion billion billion billion billion billion

"Adapted from Jewler, 1988 [3]. Table 3 Existing and proposed alternatives for treatment of diabetes 1. Conventional treatment Type II—diet, oral drugs Type I—insulin injection Single daily dose Multiple daily doses and self-monitoring of blood glucose concentration 2. Transplantation Whole or segmental pancreas Islets of Langerhans (allograft, xenograft) 3. Implantable devices Open loop systems Polymeric matrix-controlled release Pump and insulin reservoir Implantable or wearable Constant or programmable rate Closed loop systems Bioresponsive membrane (glucose-modulated permeability) Pump, insulin reservoir, and glucose sensor Hybrid artificial pancreas Transplanted tissue immunoisolated from immune system with semipermeable membrane Intravascular Extravascular 4. Prevention Immunomodulation 1985 [2]. These individuals are dependent upon exogenous insulin for life. Onset usually occurs during youth. The remaining 80 percent are Type II or noninsulin dependent diabetics, only a small fraction of whom require insulin to control hyperglycemia. Onset usually occurs after age 40. Costs for treatment of diabetes ranged from $1000 to $3000 per patient per year in 1987 (Table 2). The total cost to the nation, including costs associated with complications and indirect costs, is about $20 billion annually. The causes of diabetes include genetic factors, toxic chemicals, destruction of the pancreas through disease, and possibly virus infections. The initial event in Type I diabetes is an autoimmune process mediated by islet cell antibodies and/or lymphocytes that selectively destroy the islets [4]. Susceptibility to this process is hereditary and may be triggered by chemical insults or viruses. Diabetes is manifested primarily as hyperglycemia (increased blood glucose concentration). The first detectable sign is an abnormal intravenous glucose tolerance test (IVGTT), which is observed only after two-thirds of the beta cells are gone. With more than 90 percent gone, an abnormal oral glucose tolerance test (OGTT) is observed, and the overt symptoms of diabetes appear after 99 percent of the beta cells are destroyed [5]. There is increasing evidence that the complications of diabetes arise from hyperglycemia [6] and that they are mediated by non-enzymatic glycosylation of proteins which damages many tissues in the body. For this reason, the American Diabetes Association has stated that "the goals of appropriate therapy for those with diabetes should include a serious effort

to achieve levels of blood glucose as close to those in the nondiabetic as feasible." Conventional therapy consists of one insulin injection per day, but this does not prevent hyperglycemia. In normal man, insulin is secreted as required so as to keep blood glucose concentration between about 80 and 120 mg/dl. Conversely, in a diabetic with a single insulin injection, blood glucose concentration is still abnormally high throughout the day. One current approach to improving therapy (Table 3) is intensive blood glucose monitoring by the patient, combined with multiple daily insulin injections, with the dosage based upon the measured glucose concentration. A variety of instruments are now available for patient monitoring of blood glucose in the home. However, these techniques require highlymotivated and well-trained patients. The margin of error in patient monitoring can be high, and even with multiple daily injections the excursions of blood glucose concentration can be substantial, especially in unstable juvenile diabetics. Alternative Therapies for Treatment of Diabetes The most physiological alternative to insulin injections is transplantation of the whole pancreas or portions thereof. Because chronic immunosuppression required to prevent rejection of the foreign tissue by the immune system causes serious side effects, pancreatic transplantation is only carried out in patients who have already had a kidney transplant and are taking immunosuppressive drugs. Furthermore, the scarcity of transplantable pancreatic tissue from human cadaver limits widespread application. An alternative is to use the islets of Langerhans [7]. Human islets have a diameter of about 150 jtim, and about 60 percent of the islet mass is beta cells that secrete insulin. There are about 1 million islets in a human pancreas; in principle, about lOpercent, or 100thousand islets, which comprise a volume substantially less than 1 ml, should be enough to provide normoglycemia in a 70 kg patient. The reduced mass and elimination of major surgery make islets attractive in comparison to whole organ transplantation. Furthermore, techniques have been developed for immunoalteration of islets to prevent cell-mediated rejection [8J. The first successful allograft (donor and recipient same species) islet transplantation into a Type I diabetic patient has recently been reported [9] using roughly eight times the minimum number of islets identified above. Even if this approach proves successful, the limited availability of human islets still applies. Use of xenogeneic tissue (donor and recipient different species) could make transplantation available to all who need it, but successful methods for immunosuppression for xenogeneic transplantation have yet to be developed [10, 11]. All of the remaining approaches involve synthetic materials. In one category are polymeric matrices which provide controlled release of insulin [12] and electromechanical systems consisting of an insulin pump and reservoir [13]. The latter is available in the form of implantable or wearable systems that

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Table 4 Large scale islet isolation 1. Donor species Secretagogue sensitivity Insulin immunogenicity Tissue immunogenicity Insulin secretory capacity Ethical considerations Adult vs. fetal/neonatal Availability 2. Islet mass required 3. Isolation and purification process design Yield Selectivity Cost Preservation 4. Form of preparation Whole islets Islet cell aggregates 5. Functional characteristics Minimization of damage Viability Insulin secretion characteristics Secretion rate/tissue volume Secretion kinetics Purity Tissue components Fibroblasts Pyrogens Pathogens Immunogenicity Table 5 Device design 1. Configuration Intravascular Thin channels (hollow fibers, flat sheet) Wide-bore tube Diffusion vs. convection Extravascular Planar, tubular, spherical Dimensions of cell compartment Volume Diffusion distances Suitability for retrieval 2. Membrane/Encapsulant Mechanical properties Morphological and chemical properties Transport properties Permeability requirements Microsolutes-nutrients, 0 2 , wastes Macrosolutes-insulin, essential proteins Insulin inhibition of insulin secretion Molecular size cutoff Immune cells Proteins of the humoral immune response 3. Manufacturing Ease of fabrication Reliability Cost 4. Functional characteristics Tissue viability Insulin secretion rate Basal and glucose stimulated Dynamic response can provide constant or programmable rates. These open loop systems do not make use of information on blood glucose concentration and therefore cannot reliabily provide good blood glucose control. Closed-loop systems with feedback can in principle provide good control. These include bioresponsive membranes in which permeability to insulin is modulated by glucose [14] and electromechanical systems that incorporate an implantable glucose sensor. Glucose sensors based upon electrochemical [15] and enzymatic [16] methods have been described. Ex vivo systems incorporating an insulin pump and reservoir, glucose sensor, and appropriate computer algorithms can provide very good blood glucose control [17]. The recent demonstration of successful chronic intravascular implantation of a blood glucose sensor in dogs [16] suggests the future possibility of a fully-implantable electromechanical artificial pancreas suitable for long-term operation. Lastly, the hybrid artificial pancreas is in principle capable of providing

Table 6 Interaction with host and tissue 1. Implantation site Intravascular Anatomical arrangement Hemodynamics Extravascular Accessibility to bloodstream Retrievability Portal vs. peripheral insulin delivery Effect on insulin concentration Extent of surgical risk Oxygen supply 2. Biocompatibility Toxicity Complement activation Intravascular Vascular thrombosis Extravascular Fibrotic tissue encapsulation Local microvascularization Transport limitations 3. Functional characteristics Long-term viability Blood glucose control Fasting and postprandial Oral glucose tolerance test (OGTT) Intravenous tolerance test (IVGTT) 4. Prevention of Immune Rejection Allograft, xenograft Immune cells, humoral components excellent blood-glucose control because it makes use of the physiological feedback inherent in pancreatic islets. This imputed capability, coupled with the notion that prevention of hyperglycemia may prevent diabetic complications if normoglycemia is achieved prior to the onset of complications, is responsible for current enthusiasm for the hybrid approach [18]. In a hybrid device, transplanted tissue is immunoisolated from the immune system with a semipermeable membrane that permits passage of glucose and insulin but retains components of the immune system that can cause rejection. This method has potential for transplantation of xenogeneic tissue without need for immunosuppression. In the form of an intravascular implant, the islets are typically cultured on the outside of a semipermeable tubular membrane, and the device is implanted as a shunt in the cardiovascular system. In the form of an extravascular implant, the islets are surrounded by a semipermeable membrane or encapsulant. The ultimate goal of much basic research in diabetes is prevention of the disease. Development of a fundamental and thorough understanding of mechanisms involved in the autoimmune process may some day lead to immunotherapeutic techniques that prevent islet destruction in Type I diabetes [19]. Technical Requirements of the Hybrid Artificial Pancreas This section is divided into three parts dealing broadly with large-scale islet isolation, device design, and interaction of the device with the host and tissue (Tables 4-6). Although these requirements are directed toward islet transplantation for diabetes, most of these same requirements also apply to other applications of immunoisolation involving, for example, hepatic [20], renal epithelial [21], thymic [22], pituitary [23], and neural [24] tissues. Immunoisolation techniques may also apply to gene therapy [25, 26] in situations where it is desirable to isolate the transformed cells from the rest of the body or when allogeneic or xenogeneic cells are employed. Islet Isolation. The first issue in islet isolation (Table 4) is selection of the donor species. The donor islets should as closely as possible mimic the sensitivity to secretagogues found in human islets. The secreted insulin should cause minimal immune response. Equally important, islets from different species may display different immunogenic properties which could

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effect the relative difficulty with which immune protection can be achieved. Large differences in insulin secretory capacity may favor one species over another. Some species, such as nonhuman primates and animals domesticated as pets, may be ruled out on ethical grounds. Fetal or neonatal islets, which replicate in culture, may offer an advantage over adult islets that do not replicate. Although rodent, bovine, and canine islets have been evaluated, porcine islets are now receiving greatest attention since they likely provide the best compromise amongst these requirements, and pigs are easily bred in large numbers. The mass of islets required to produce normoglycemia is an extremely important parameter because it will have substantial impact on the ultimate cost of the device and because the problems of achieving a satisfactory implantable design are magnified as the mass of tissue increases. Currently available data are fragmentary, and the islet requirement has an uncertainty of at least a factor of three. On the basis of the fraction of the human pancreas remaining when the OGTT becomes abnormal, a minimum estimate would be the equivalent of about 1500 human islets/kg body weight. One group [27] has estimated a requirement of about 3500 human islets/kg. Studies of allograft transplantations with canine islets [28] indicate that 6000-8000 canine islets/kg are required to insure normoglycemia in dogs. This number is deceptively high because the canine islet has a diameter of only about 100 fim. Consequently, on a volume basis, these results translate into about 2500 human equivalent islets/kg. In the one successful human islet allograft transplantation reported [9], 12,000 islets/kg were employed, but the minimum number required for normoglycemia without exogenous insulin was not evaluated. In an extensive immunoisolation study using canine islet allografts, as many as 22,000 canine islets/kg (about 7500 human islet equivalent/kg) produced varying degrees of blood glucose control. The large disparity between these data and those obtained with transplantation suggests that the islet viability and/ or secretory capacity may have been compromised in the immunoisolated state. Processes for isolating large numbers of islets from a single pancreas have been described for human [29, 30], porcine [31, 32], rat, and dog [33] islets. All have in common the steps of collagenase enzymatic digestion and centrifugal separation on the basis of size and density. There is room for further improvement in yield and purity by use of more selective cell separation methods. Furthermore, the economy of scale associated with a larger process may ultimately reduce cost, especially since islets can be isolated at a central location and preserved by cryogenic storage [34, 35] until needed. Although most immunoisolation work to date has been carried out with whole islets, the use of islet cell aggregates may provide attractive advantages. These aggregates form during culture of dispersed islet cells produced by enzymatic islet digestion [36, 37]. Because of their reduced size as compared to whole islets, they may offer increased flexibility in the design and loading of tissue chambers. Immunogeneticity of the aggregates may also be reduced as compared to whole islets [38]. Preservation of the functional characteristics of the islets is essential. The isolation process should minimize tissue damage, including loss of viability and of insulin secretion characteristics (secretion rate per unit tissue volume and dynamic response to glucose stimulation). Purity is essential. Extraneous tissue components (acinar and connective tissue) can damage islets through the presence of proteolytic enzymes and can also increase the potentially immunogenic load on the recipient. The rapid proliferation of fibroblasts that invariably accompany crude cell preparations may inhibit the survival of islet tissue. Fortunately, techniques are available for fibroblast elimination [39] during culture. Especially dangerous are pathogens which may be carried with animal tissue. These include microorganisms, viruses, and exotic and poorly characterized

potentially infectious agents such as prions [40]. The possibility that implantation of a device to cure a disease may lead to infection by exogenous agents is a serious, issue that ultimately may require breeding of pathogen free donor animals specifically for immunoisolation. Lastly, application of immunoalteration culturing techniques to the purified islet preparation might be useful for reducing immunogenicity of the implanted tissue. Device Design. Issues related to device design are summarized in Table 5. A large diversity of configurations are possible, and virtually all have been examined. Initial studies with intravascular devices employed thin channels in the form of hollow fibers, although a flat sheet configuration is in principle possible. Experience with extracorporeal blood flow has demonstrated that blood clots can form in hollow fiber devices, especially at inlet and outlet headers where secondary flows occur, even in the presence of extensive anticoagulation [41]. Observation of thrombus formation in early intravascular hollow fiber devices led to the use of semipermeable tubular membranes with larger diameter in the hope of minimizing or eliminating clot formation without the need for systemic anticoagulation. Substantial experimental and theoretical investigation has been carried out with intravascular devices to assess the relative contributions of diffusive and convective transport mechanisms and their impact on insulin secretory response as well as for the purpose of developing designs in which the more efficient convective mode is utilized. Extravascular devices have been employed using planar, tubular, and spherical geometries, the latter involving microencapsulation of individual islets. The dimensions of the cell compartment or capsule, especially diffusion distances, are critically important because they can lead to large concentration gradients that influence both microsolute and macrosolute transport. For example, large diffusion distances can degrade the observed kinetics of the insulin secretory response and can also establish oxygen concentration gradients that may expose the islet tissue to hypoxic or even anoxic conditions. It may be essential to be able to retrieve the device from a patient in case of failure, or to remove and replace the islet tissue if it loses insulin secretion function, and it is often desirable to do so for research purposes. In this connection, retrieval of intravascular and some extravascular devices is possible, whereas anything close to quantitative retrieval of microcapsules is not. Development of a permanently implanted receptacle suitable for tissue replacement [42] remains a challenge. A variety of properties are essential for adequate performance of the semipermeable membrane or encapsulant. It must have sufficient mechanical strength to maintain its integrity in the presence of stresses to which it is exposed in the in vivo environment. Failure of the membrane, or even the development of isolated defects, may lead to catastrophic consequences. Morphological and chemical properties are important primarily with respect to biocompatibility at the interface between the device and the host tissue. Membrane transport properties relate to the essential functions of the device. Membrane permeability must satisfy disparate and potentially conflicting requirements. Microsolutes such as nutrients, oxygen, and wastes, and macrosolutes such as insulin and essential proteins must be able to pass to and from the cell chamber at rates compatible with tissue viability and satisfactory insulin secretory dynamics. Too low a membrane permeability to insulin would lead to a substantial increase in the insulin concentration to which the islet tissue is exposed, thereby leading to the possibility of inhibition of insulin secretion. Whether insulin does inhibit its own secretion is the subject of much debate [43, 44]. One essential protein is transferrin (77 kD molecular weight) which complexes with iron and transports it to the cells. The requirement of islet cells for transferrin is unknown, making it difficult to establish quantitative criteria

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for the necessary membrane permeability to transferrin. In contrast to the need for passage of a variety of solutes, the membrane must have sharp molecular size cutoff properties so that damaging components of the immune system can not reach the islet cells. Retention of immune cells is an absolute requirement. Some immune cells can squeeze through extremely small spaces, as evidenced by the phenomenon of monocyte diapedesis in vivo. Even when the entire cell can not pass through a certain opening, cell processes can be extended through small holes over large distances. As a rough rule of thumb, the maximum size of a continuous passage to prevent cell intrusion is on the order of 0.1 /im. Passage of proteins of the humoral immune system must be similarly restricted. A quantitative assessment of this requirement is deferred until the end of this paper. The difficulty of designing and fabricating a membrane that meets these disparate requirements has not been widely appreciated. The design of the immunoisolation device and its method of fabrication must permit manufacture with extraordinarily high reliability at a cost that can be reasonably born by the health care system. Lastly, the islets contained within the device must maintain their functional characteristics (viability and insulin secretion rate) in a state comparable to that of the isolated preparation. The glucose-stimulated insulin secretion properties can most effectively be evaluated by in vitro measurements prior to implantation. Interaction With Host. Table 6 identifies a number of important factors concerning interaction of the device with the host. Selection of implantation site is dependent upon the type of immunoisolation device employed. Intravascular devices can be connected as a shunt between two arteries, two veins, or an artery and a vein. The blood vessels must be of sufficient size to accommodate the connections and have close proximity so that complicated internal plumbing is not needed. Their location in the cardiovascular system will determine the inlet pressure and blood flow rate through the device which, in turn, may influence device performance. Extravascular devices have the advantage of less invasive surgery because they need be only injected or placed in tissue or a physiological body space (e.g., peritoneal cavity). However, accessibility to the bloodstream may be a problem if the implantation site is poorly perfused. The implantation site may also influence the extent to which an extravascular device can be retrieved. The location may also affect insulin metabolism. Insulin is normally secreted by the pancreas into the portal vein which feeds the liver where about 50 percent of the insulin is extracted, thereby leading to a reduced plasma insulin concentration in the peripheral circulation. Implantation of an immunoisolation device in or near the portal vein may carry high surgical risk. The peritoneal cavity is an attractive site, not only because of its lower surgical risk, but also because intraperitoneal insulin is almost entirely absorbed by the portal circulation [45]. Although this site provides anatomically normal insulin delivery, insulin uptake from the peritoneal cavity is slow [45, 46], and the rate of insulin appearance in the plasma is much slower than the normal physiological response. Delivery of insulin to the peripheral circulation reverses the normal portal-peripheral difference and is associated with reduced hepatic insulin extraction and elevated circulating insulin concentration in the peripheral circulation [47]. Peripheral hyperinsulinemia causes increased microvascular permeability which has been implicated in the pathogenesis of certain diabetic complications [48]. This unsettling scenario offers the prospect of an acceleration, rather than a reduction, in the development of diabetic complications consequent to device implantation. Clearly, intra-portal delivery would be most advantageous if it could be done without substantial risk. Another problem associated with implantation site is adequacy of the oxygen supply. Implantation of an intravascular device in the arterial system has the advantage 156 /Vol. 113, MAY 1991

of bringing arterial blood directly to the membrane surface where it can provide oxygen at a partial pressure (p02) of about 100 torr (mm Hg). By contrast, an extravascular implant in tissue or the peritoneal cavity would be exposed to the microvasculature that normally delivers oxygen at p0 2 « 40 torr [49], making such an implant more susceptible to oxygen diffusion limitations. Biocompatibility requirements span a wide range of phenomena. At its most elementary level, device components must not contain tissue-extractable toxic agents. Complement activation, which is determined largely by surface chemical composition, must be avoided because it elicits a reaction akin to the inflammatory response that has been well documented in connection with studies on extracorporeal devices such as hemodialyzers [50]. With intravascular implants, clot formation is the most serious issue because it could lead to poor performance or even failure of the device and could threaten the patient if thrombi are shed into the bloodstream. With extravascular implants, the most serious threat is the development of fibrotic tissue encapsulation over the membrane or encapsulant surface, a phenomenon often observed with implanted biomaterials [51] and well-recognized to be critical for advancing the hybrid artificial pancreas [52]. There are two problems associated with development of the fibrotic capsule. First, the fibrotic tissue is avascular, and the nearest capillaries are separated from the membrane by a distance corresponding to the fibrotic tissue capsule thickness, which can be as much as 100 jum. Secondly, the tissue capsule may have poor transport properties. For example, measurements of glucose permeation through fibrotic tissue capsules formed on silicone rubber implanted subcutaneously in rats gave estimates of the effective diffusion coefficient that are one to two orders of magnitude lower than the value in water [53]. Whether such poor transport properties are characteristic of the fibrotic capsule which forms on other materials is unknown. In any event, the development of a substantial tissue mass transfer resistance adjacent to the membrane would seriously compromise performance. In contrast to the development of a fibrotic layer, the ideal situation would be the development of extensive microvascularization immediately adjacent to the membrane. Once implanted, the islets must remain viable and functional for a time period sufficient to justify the cost and risk associated with the therapy. Retention of function means that blood glucose control is close to normal. Blood glucose concentrations measured after an overnight fast are insufficient to determine whether normoglycemia has been achieved. One simple way to assess blood glucose control is to measure postprandial (following a meal) glucose excursions and compare them to normal controls. A more systematic measurement can be achieved with an OGTT in which a specified oral dose of glucose is given in the fasted state, and the rise and fall of blood glucose concentration is monitored as a function of time. The most rigorous test is an IVGTT, in which an intravenous bolus of glucose solution is substituted for the oral dose in an OGTT. However, if the OGTT is suitably close to a normal response, it is not necessary that the IVGTT also be successful because human beings are not normally exposed to the rapid step change in blood glucose concentration that occurs in an IVGTT. It is reasonable to ask, what kind of dynamic response is required of a hybrid artificial pancreas in order for a nearnormal OGTT to be obtained in a diabetic patient with no endogenous insulin secretion? No experimental data have been reported from which an unequivocal answer can be obtained. However, simulations with a physiological pharmacokinetic model of glucose and insulin metabolism suggest that a delay in the insulin secretory response following glucose stimulation of as much as 10 to 15 min is tolerable without causing serious degradation in the OGTT [54, 55]. The last major requirement of the immunoisolated device Transactions of the ASME

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one in which heparin was covalently bonded to the membrane surface [72]. A membrane prepared from collodion and modified with N-chlorosulfonyl isocyanate has been claimed to have anticoagulant activity and has been used in preliminary islet immunoisolation experiments [83]. Most studies used a multiplicity of hollow fibers or a single fiber or tubular membrane with internal diameters from 0.2 mm to 1 cm. One group [74, 75, 78] employed various flat-plate designs that incorporated blood ultrafiltration. Only a few studies reported the average thickriess of the islet compartment or design parameters (e.g., membrane area, islet compartment volume) from which it can be calculated, leading to estimates of about 0.4 mm [78] (data from [84]), 1 mm [66, 67, 69], and 3 mm [80]. Intravascular Devices Islet loading, where reported, ranged from 400 to 11,000 The concept of mammalian cell culture on the outside of artificial capillaries perfused internally with culture medium islets/kg in studies aimed at achieving normoglycemia. Diawas introduced in 1972 by Knazek et al. [57]. They used units betes was induced in early studies by chemical means; partial composed of cellulose acetate hollow fibers and of a mixture and total pancreatectomy later became standard when it beof silicone polycarbonate and Amicon XM-50 hollow fibers. came apparent that endogenous insulin secretion could return High hormone secretion rates and tissue-like cell densities of with chemical induction [81, 85]. All studies reported control human choriocarcinoma cells were obtained with the latter of fasting glucose. In one case [77], connection to the portal system. This spectacular initial result established the XM-50 vein required more islets to achieve normoglycemia than did membranes for some time as the system of choice for mem- connection to the systemic circulation because of the peripheral brane bioreactors. The XM-50 membranes (nominal cutoff 50 hyperinsulinemia in the latter case. No OGTT data were rekD) were (and still are) fabricated from a polyacrylonitrile- ported. In only one study were feeding and fasting periods polyvinyl chloride (PAN-PVC) copolymer and had been de- indicated; hypoglycemia oscillations occurred during one fastveloped only recently for blood ultrafiltration [58]. They be- ing period. IVGTT data ranged from normal to moderately long to a class of anisotropic membranes consisting of a thin delayed; the best results were obtained with capillary hollow retentive skin supported on a porous spongy matrix. The hy- fibers, thin immunoisolation membranes, and small diffusion draulic permeability and retention properties are determined distances in the islet compartment. Complications invariably by the pores in the skin; the diffusive permeability is determined were associated with blood coagulation in the device or with vascular access problems, i.e., thrombosis at the anastomosis by the thickness of the spongy matrix [59]. between the device and the blood vessel. Many studies were terminated after short periods. The longest durations were 11 In Vitro Studies. In 1975, a similar system composed of XM- days with an allograft and 8 days with xenografts [70]. No 50 fibers was used to culture rat islet tissue in vitro [60], and immune rejection was reported, suggesting that hyperacute the term hybrid artificial pancreas was coined [61]. The cells rejection did not occur; no duration was long enough to test continued to release insulin at appropriate rates and remained for chronic rejection. responsive to glucose for the duration of experiments lasting In a dramatic advance over all prior studies, Maki et al. [81, up to one month. In subsequent studies with 50,000 canine islets seeded in larger capillary units [62], glucose-stimulated 82] have recently reported the first extensive, long-term in vivo insulin response was maintained for three months, stabilizing implantations of immunoisolated allografts in severely diabetic at about 20 U/day (roughly one-third to one-half of the human pancreatectomized dogs without any systemic anticoagulation. Their device consisted of a single, coiled tubular membrane requirement). 80 kD) of 5-6 mm id and total It was recognized at the outset [61] that capillary hollow (PAN-PVC, nominal cutoff 2 fiber devices were prone to clotting and that chronic systemic surface area A = 60 cm in a disk-shaped plastic housing anticoagulation (e.g., with heparin) should be avoided. Hollow connected to a polytetrafluoroethylene vascular graft of the fibers and tubular membranes with inside diameters of 200, same inner diameter. The islet chamber was an annular cavity 450, and 1100 /jm and corresponding wall thicknesses of 75, surrounding the membrane with volume V = 5 ml. The average 200, and 300 /xm, respectively, were initially implanted ex vivo thickness of the islet chamber is estimated as V/A = 0.8 mm. Unseeded devices were implanted in twenty dogs (16-20 kg) as unseeded arteriovenous (AV) shunts for short periods to study biocompatibility. Subsequent long term studies [63] dem- as shunts between the common iliac artery and vein. In the onstrated that 2.7 mm id tubular membranes as AV shunts first set, 2/12 shunts were patent and ongoing for more than remained consistently patent for 7 weeks without anticoagu- one year; the remaining 10/12 failed earlier because of thromlation and that details of construction and avoidance of flow bosis or membrane rupture. In the second set, low-dose aspirin discontinuities were important. Associated in vitro studies of therapy and an improved anastomotic technique led to 8/8 insulin secretory dynamics with seeded wide-bore devices [59, ongoing patient shunts (280-311 days). Devices were seeded with 12-22,000 islets/kg (mean 15,000) 64] demonstrated that glucose-stimulated insulin secretory dynamics were slower than with hollow fibers but could be main- and implanted into 19 dogs. Companion controls seeded from tained at a reasonable level (10-min delay as compared to the same preparations and studied in vitro released insulin at intrinsic islet kinetics) by reducing membrane wall thickness different rates (2-19 U/day). Insulin secretion following glucose stimulation was delayed 15-20 min. to the minimum consistent with mechanical strength. In the first set of seeded device implantations, 6/6 gave no Implantations. The results of ex vivo and in vivo implan- long term function. Failure was associated with poor islet functations are summarized in Table 7. We first consider together tion, thrombosis, collapse of the membrane and vascular graft, all prior work except the most recently reported studies [81, and infection. In the second set (aspirin, improved anasto82]. All were ex vivo implantations into the peripheral circu- mosis), 6/13 gave little or no function; 4/13 produced a dilation except for part of one study in which the device outlet minished exogenous insulin requirement to keep fasting blood was connected to the portal vein [77]. In addition to PAN- glucose in the range 100-250 mg/dl. In one example, exogenous PVC, membranes were made from polysulfone, sulfonated insulin requirement was reduced from 24 to 6 U/day. Histopolyacrylonitrile, ethylene vinyl alcohol copolymer, and po- logical examination upon explantation after 175 days revealed lyamide. Systemic heparinization was used in all studies except that 17 percent of the islets originally loaded remained viable. is prevention of immune rejection. This important topic will be discussed in more detail below. The concept of immunoisolation can be traced back at least nearly sixty years to a study of human insulinoma tissue placed in membrane bags and transplanted into rats [56]. However, only in the past two decades have efforts been directed specifically at a hybrid artificial pancreas. The next two sections deal with previous work with intravascular and extravascular devices. Where data are available, these works are examined with respect to the issues discussed in this last section.

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MAY 1991, Vol. 113/157

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