Long-term viability and function of transplanted islets macroencapsulated at high density are achieved by enhanced oxygen supply

Transplantation of encapsulated islets can cure diabetes without immunosuppression, but oxygen supply limitations can cause failure. We investigated a retrievable macroencapsulation device wherein islets are encapsulated in a planar alginate slab and supplied with exogenous oxygen from a replenishable gas chamber. Translation to clinically-useful devices entails reduction of device size by increasing islet surface density, which requires increased gas chamber pO Here we show that islet surface density can be substantially increased safely by increasing gas chamber pO to a supraphysiological level that maintains all islets viable and functional. These levels were determined from measurements of pO profiles in islet-alginate slabs. Encapsulated islets implanted with surface density as high as 4,800 islet equivalents/cm in diabetic rats maintained normoglycemia for more than 7 months and provided near-normal intravenous glucose tolerance tests. Nearly 90% of the original viable tissue was recovered after device explantation. Damaged islets failed after progressively shorter times. The required values of gas chamber pO were predictable from a mathematical model of oxygen consumption and diffusion in the device. These results demonstrate feasibility of developing retrievable macroencapsulated devices small enough for clinical use and provide a firm basis for design of devices for testing in large animals and humans

Enhanced Oxygen Supply Improves Islet Viability in a New Bioartificial Pancreas

<p>The current epidemic of diabetes with its overwhelming burden on our healthcare system requires better therapeutic strategies. Here we present a promising novel approach for a curative strategy that may be accessible for all insulin-dependent diabetes patients. We designed a subcutaneous implantable bioartificial pancreas (BAP)-the "beta-Air" that is able to overcome critical challenges in current clinical islet transplantation protocols: adequate oxygen supply to the graft and protection of donor islets against the host immune system. The system consists of islets of Langerhans immobilized in an alginate hydrogel, a gas chamber, a gas permeable membrane, an external membrane, and a mechanical support. The minimally invasive implantable device, refueled with oxygen via subdermally implanted access ports, completely normalized diabetic indicators of glycemic control (blood glucose intravenous glucose tolerance test and HbA1c) in streptozotocin-induced diabetic rats for periods up to 6 months. The functionality of the device was dependent on oxygen supply to the device as the grafts failed when oxygen supply was ceased. In addition, we showed that the device is immuno-protective as it allowed for survival of not only isografts but also of allografts. Histological examination of the explanted devices demonstrated morphologically and functionally intact islets; the surrounding tissue was without signs of inflammation and showed visual evidence of vasculature at the site of implantation. Further increase in islets loading density will justify the translation of the system to clinical trials, opening up the potential for a novel approach in diabetes therapy.</p>

Oxygen partial pressure in the gas chamber.

<p>Levels of oxygen at the end of successive 24 h cycles were monitored in the central cavity and in the 2 side chambers. Solid black line, central cavity; solid grey line, side chambers. Data are presented as the 3^rd grade polynomial curves.</p

Morphologic assessment of recipient pancreas and pancreatic islets following 90 days of implantation period.

<p>(<b>A</b>) A device after explanation. (<b>B</b>) The tissue surrounding the device at explantation. (<b>C–F</b>) Representative images of alginate/islet slabs (C,D - 10×; E, F - 40×). Left: HE; islets displayed an intact structure and no signs of disintegration within the alginate. Right: Immunohistochemistry for insulin showed intense cytoplasmic staining as typically seen in intact rat islets. (<b>G</b>) Representative image (2×) of a pancreas of a recipient animal at autopsy.</p

Determination of the barrier function of the chamber system.

<p>(<b>A</b>) Biopsies from alginate/islet slabs were taken at 30 days (left panel, slabs 1 and 2) and at 90 days (right panel, slab 3) to test for porcine DNA contamination inside the chamber. At both time points, no porcine DNA was detectable in any of the samples. Non-tissue samples were used as negative controls (NC). Tissue extracts from porcine muscle (M), spleen (S), and liver (L) served as positive controls (PC). (<b>B</b>) Serum samples of rat islet graft recipients were taken at various time points throughout the observation period of up to 90 days to test for development of anti-rat immunoglobulin. The 2 graphs show data of 2 individual recipients (left, 1709; right, 1736). Squares: positive control (sensitized animal); circles: negative control (healthy animal); triangles: diabetic minipig transplanted with encapsulated rat islets.</p

Chamber system for islet macroencapsulation.

<p>(<b>A</b>) Schematic view of the chamber composition. The core of the device is built as a gas module, connected to access ports for exogenous oxygen refueling. Active transport of solutes is achieved via a membrane impregnated with alginate (left, virgin; right, ready to use). Separated by gas permeable membranes, 2 compartments surround the central gas cavity that houses alginate-immobilized pancreatic islets. The plastic housing of the chamber has a latticelike design at both external surfaces and covered by hydrophylized PTFE porous membranes. (<b>B</b>) Photographic image of a completely assembled chamber with connected access ports. (<b>C</b>) X-ray image of an implanted recipient.</p

Barrier function of the membrane system for viral transmission.

<p>MRC-5 primary fibroblasts cultured in 24-transwell plates were separated from suspension containing reporter Lentiviruses by the membrane system for a period of 72 h. Infection rates were quantified by FACS analyses. Regardless of the MOI level, no relevant infection was detected in the membrane group (squares). In the control group (naïve membrane; circles), infection levels corresponded to increasing MOI levels from 40% up to 99.5%.</p

Diffusion of large molecules across the membrane system is impeded.

<p>Diffusion of mouse IgG across naïve membrane (squares) or diffusions of IgG (circles) or C1q (triangles) across reconstituted membranes. Values represent the relative amount of solute retained in the loaded chamber as a function of time.</p

Blood markers in healthy, diabetic and device carrying mini-pigs.

<p>Data presented are mean ± standard deviation.</p><p>SGOT, serum glutamic oxaloacetic transaminase.</p

Function of macroencapsulated rat islet grafts.

<p>STZ-induced diabetic minipigs were transplanted with 6,730±475 IEQ/kg BW of rat islets immobilized and integrated into the macrochamber system. ivGTT was performed prior to graft implantation, 2 weeks after implantation, and after retrieval of the graft. BW was recorded daily throughout the observation period. (<b>A</b>) Fasting blood glucose levels (black) of group one transplanted minipigs (n = 5). The graft was removed on day 30 and hyperglycemia recurred demonstrating that graft function was responsible for normoglycemia during the implantation period. Rat C-peptide levels (grey) are presented as 4^th grade polynomial curves. Error bars represent SD. <i>P</i><0.001 for comparing glucose levels during the implantation period vs the pre- and post-implantation periods (t-test). (<b>B</b>) Blood glucose levels during ivGTT of transplanted animals at two weeks (black circles) and after retrieval of the graft-containing device (black triangles). Diabetic minipigs (n = 24; double grey), naïve healthy mini-pigs (n = 11; black dashed) and naïve healthy rats (n = 36; grey dashed) served as controls. Error bars represent SD. <i>P</i><0.001 for comparing AUCs (diabetic minipigs) in the implantation period vs the pre- and post-implantation periods (t-test). (<b>C</b>) Two-day continuous glucose monitoring records of an implanted animal during week 2. (<b>D</b>) Fasting blood glucose levels (black) of group 2 transplanted minipigs (n = 3) and corresponding BW (grey). Normoglycemia was achieved rapidly after transplantation and was retained until the BW increased to a critical level of >160% of the initial BW. Data are presented as 5^th grade polynomial curves. <i>P</i><0.001 for comparing glucose levels during the first 75 days of the implantation period vs the pre-implantation period (t-test).</p