Animals, diabetes induction, and pre-treatment

Lewis rats (260–280 g) were purchased from Harlan (Rehovot, Israel), and diabetes was induced by a single intravenous infusion of 85 mg/Kg body weight of Streptozotocin (STZ; Sigma, Israel) as previously described10. Animals had free access to food at all times and were considered diabetic when non-fasting blood glucose exceeded 450 mg/dl for 4 consecutive days or more. To prepare the diabetic animals for device implantation in a non-stressing, close-to-normal blood glucose environment, 1.5 capsules of a sustained release insulin implant (Linplant, LinShin, Toronto, Canada) were inserted under the skin of the diabetic animals, which were considered ready for implantation of the device when their non-fasted blood glucose was under 250 mg/dL for 3 consecutive days or more. The sustained-release insulin capsules were removed 48 hr after device implantation, leaving the encapsulation device as the only source for insulin. The efficacy of glycemic control was followed after implantation by assessing the functionality of the islets in the device through twice daily measurements of non-fasting blood glucose concentration. Animals were sedated, blood samples were collected from the tail, and glucose levels were measured by glucometer (Accu-Chek sensor, Roche Diagnostics GmbH). Blood glucose concentration ≤200 mg/dl was deemed to be normoglycemic. Intravenous glucose tolerance tests (IVGTT) were performed 21 and 42 days post-transplantation as follows: animals were fasted overnight. On the following morning, 1 ml of 0.7 M glucose solution was infused within 10–15 sec (dose of 500 mg/kg BW), and blood glucose samples were collected for measurement before infusion and at 10, 30, 60, 120 and 180 min following glucose infusion. Selected devices were electively explanted from normoglycemic animals for further study at times ranging from 42 to 238 days post-transplantation.

Islet isolation and culture

Pancreata from 9 to 10-week old male Lewis rats weighing 260–280 g underwent collagenase digestion following a standard procedure with slight modification, as described previously10. Briefly, each pancreas was infused with 10 ml enzymatic digestive blend containing 15 PZ units of collagenase NB8 (Serva, Heidelberg, Germany) and 1 mg/ml bovine DNAse (Sigma, Israel) dissolved in HBSS solution (Bet-HaEmek, Israel) for 14 min. Islets were purified on discontinuous Histopaque gradient (1.119/1.100/1.077 in RPMI) run for 20 min at 1,750 × g and 6 °C, washed twice, and cultured in complete CR medium (1:1, CMRL:RPMI medium, Bet HaEmek, Israel), supplemented with 10% fetal bovine serum (Bet-HaEmek, Israel) for 1 wk prior to being integrated in implantable devices. Measurement of islet size and number in a preparation, from which islet volume and number of IEQ were determined, was carried out by visual counting as previously described10. Insulin content of islets was measured with acid/ethanol extraction and ELISA (ThermoFisher Scientific). Islet purity estimated visually was always greater than 95%. Naked islet viability, assessed as the fraction of green cells measured with the Acridine Orange/Propidium Iodide membrane integrity assay was always greater than 95%.

The βAir device

The subcutaneously-implantable device, named βAir10, had an external disc-shaped housing made of clinical grade polyether ether ketone (PEEK Optima LT1R40; Invibio, Lancashire, UK) with a diameter of 31.3 mm and thickness of 7 mm (Fig. 1). The device consisted of three major components: (1) The islet compartment contained about 2,400 IEQ embedded in 500 to 600-µm thick ultrapure high guluronic acid alginate, reinforced with 100-µm thick stainless steel grids having about 80% fractional open area (top grid, Fig. 1A, inset, Suron, Maagan Michael, Israel), glued to the PEEK housing with medical epoxy adhesive (Epotek 301–2, Epoxy Technology Inc., Billerica, MA, USA). Mechanical support was provided by the bottom grid, identical to the top grid, which was placed under the gas permeable membrane and reinforced by PEEK mechanical supports (Fig. 1B). To vary islet surface density, 2,400 IEQ were immobilized in a slab with a diameter of 11.3, 9.3, or 8.0 mm, resulting in densities of 2,400, 3,600, or 4,800 IEQ/cm2 en face surface area for oxygen transport, respectively (Fig. 1B,C). (2) The gas chamber (3-ml volume) was separated from the islet compartment by a 25-µm gas-permeable silicone rubber-teflon membrane (Silon, BMS, Allentown, PA) and contained inlet and outlet gas chamber ports connected by two polyurethane tubes to subcutaneous access ports (Cat. No. PMINO-PU-C70, Instech Solomon, PA) implanted under the skin at a site remote from the device, as previously described10. (3) A 25-µm, 0.4-µm pore diameter hydrophilized microporous polytetrafluoroethylene (PTFE) membrane (Biopore, Millipore, Billerica, MA), separated the islet module from host tissue and protected the islets from the cellular part of the immune system. Devices with a diameter of 18 mm were used to give islet densities of 1,000 IEQ/cm2. Some of these devices made use of an earlier, less effective design10 and the results are provided here selectively for comparison.

Device assembly

An average dose of 2,400 ± 200 IEQ (range 1,700–3,300 IEQ) was collected by 5-min sedimentation. The pellet was gently mixed with 2.2% (w/v) ultrapure high-guluronic acid (68%) alginate (Pronova UPMVG, Novamatrix; Sandvika, Norway). The mixture was placed in the islet module compartment and spread through the openings of the top grid (Fig. 1A, inset) with the tip of a long-nosed Pasteur pipette. The microporous PTFE (Biopore) membrane was then fixed onto the device using a Viton O-Ring (hardness 75 Shore and outer diameter 27 mm, McMaster Carr, Aurora, OH) and sealed to the plastic housing with medical silicone adhesive (MED 2000, Polytek Easton, PA). The alginate was cross-linked by applying a flat sintered glass disc (Pyrex, UK) saturated with strontium chloride dissolved in RPMI medium for a final concentration of 70 mM. The device and sintered glass were immersed in the RPMI-strontium medium for 16 min, resulting in a 500- to 600-µm thick coin-like slab. The thickness variations originated with variation in glue thickness. The device was washed for an additional 5 min at 37 °C in complete CR medium (Beit HaEmek, Israel). Fully fabricated devices were washed in complete CR medium at 37 °C with agitation for 2 h before implantation.

Device implantation

All animal experiments were performed in strict accordance with the Institutional Animal Care and Use Committee Guidebook. The study was approved by The Council for Experiments on Animal Subjects, Ministry of Health, Israel (Permit No. IL05-05-012). All efforts were made to minimize animal suffering. Rats were anesthetized by intraperitoneal injection of 90 mg/Kg Ketamine and 10 mg/Kg Xylazine followed by isoflurane inhalation. A 3-cm incision was made for the device on the dorsal skin, and muscles were separated from the hypodermis. A second incision was made in the skin between the shoulder blades, and two channels connecting this site with the device implantation site were created by traversing 3-mm wide stainless steel needles under the skin. The device was inserted under the dorsal skin incision with the islet module facing the fascia, and the gas chamber ports were connected to the remote subcutaneous access ports. The skin was sutured and fixed with a tissue adhesive (Histoacryl, Tufflingen, Germany). Devices were implanted into 141 animals. Data was discarded from four animals due to mechanical problems with the device.

Gas mixture replacement

Every 24 h the animal was sedated with isoflurane inhalation. A 27G needle was inserted into each of the two implanted access ports, and the gas chamber was purged with 20 ml (about 6.7 chamber volumes) of gas mixture containing the specified pO 2 , 40 mmHg CO 2, and balance N 2 . Final total pressure in the gas chamber was equal to ambient atmospheric pressure. To obtain the different oxygen mixtures, prefilled cylinders were used (Maxima, Israel).

Islet oxygen consumption rate (OCR)

OCR of naked islets was measured as described previously10. Measurements were also made with an immobilized aliquot of 250 IEQ from the preparation to be transplanted in the device and with the islet alginate slab after device explantation. The preimplant islet sample was immobilized in 30 μL of high guluronic acid alginate shaped as a coin with a thickness of 500 μm and diameter of 8.7 mm. After elective explantation of a device, the alginate slab containing the islets was carefully removed. The number of IEQ (≥200 islets) in a small defined surface area was quickly determined by manual counting under a microscope. The slab having the largest diameter was cut into a smaller piece that fit into the measurement chamber. The pre- or post-implant slab was placed on a glass slide, a 5-mm diameter magnetic stirrer bar was placed on top of the slab, and the assembly was covered with a conical OCR measurement chamber (Fig. 2). The conical chamber was filed with 1:1, CMRL:RPMI medium containing 1% (v/v) fetal bovine serum to a final volume of 620 µl. The chamber was equipped with Clark-type oxygen electrode of 500-µm diameter connected to a picoammeter controller (Cat No. PA2000, Unisense, Arhaus, Denmark). The O 2 measurement chamber was placed within a Perspex box with the air temperature maintained at 37 ± 1 °C using a temperature control unit (Eurotherm 808; Eurotherm Worthing, UK). The stirring speed was increased until measured OCR did not change (about 70 rpm), thereby assuring minimal effects associated with mass transfer boundary layers around the islets and the O 2 electrode. No damage to the alginate slab or the islets was observed as assessed by islet and slab morphology and stable OCR readings. The electrode was calibrated using medium equilibrated with gas containing zero or ambient air oxygen concentrations. The O 2 level in both phases are reported here as oxygen partial pressure pO2, in units of mmHg, which is related to oxygen concentration c by the equation c = αpO 2 , where α is the Bunsen solubility coefficient, 1.34 × 10−9 mole/(cm3mmHg) for oxygen in medium at 37 °C. Consequently, for example, at steady-state ambient O 2 partial pressure of 160 mmHg (21% O 2 , 1 atm), dissolved O 2 concentration is 215 µM in the medium at 37 °C. As a result of the O 2 consumption by the islets, the O 2 concentration in the medium within the conical measurement chamber decreased with time from its initial value in equilibrium with ambient air. The data for O 2 concentration versus time was fitted by linear regression, and the slope was used to estimate OCR of the islets38. Data was used between 160 and 80 mmHg, which yielded the highest slope. Estimation of OCR by this method required that the pO 2 profiles within the slab were in quasi-steady state and that the pO2 difference between the medium and the slab interior was small. Approximate theoretical analysis similar to that previously used for OCR measurement with microencapsulated islets39 indicated that these conditions were met for the measurements made in this study.

Oxygen gas measurements

To measure O 2 concentration in the gas chamber within the implanted devices, a 27G needle connected to 1.0 ml syringe was inserted into one of the implanted subcutaneous access ports, and a 250-µl sample was taken from the gas chamber 24 hr after the last O 2 replenishment and injected into the conical measurement chamber. The change in the electrode measurement was used to calculate the oxygen concentration in the sample from the gas chamber. The O 2 electrode was calibrated with gas mixtures having pO 2 of zero (pure N 2 ), 160 mmHg (ambient air), and 304 mmHg.

Oxygen profile across the islet slab

About 2,400 IEQ were immobilized at various densities as described for βAir device assembly, but without the PTFE (Biopore) membrane and without the metal grid on top. The device was placed in a covered 90 mm Petri-dish, overlain with RPMII medium so as to create a layer of minimal depth on top of the device, and the space above the slab was purged with a gas stream having 40 mmHg O 2 , 40 mmHg CO 2, and 680 mmHg N 2 (Fig. 3), which simulated the gas composition in the subcutis. The gas chamber was purged with oxygen concentrations varying between 152 and 305 mmHg. An O 2 electrode with a diameter of 500 µm, attached to a micromanipulator, was inserted into the islet-containing slab and advanced at 100 µm increments from the distal side of the islet slab downwards toward the gas permeable membrane. At each step, the O 2 electrode readings reached a steady-state level before moving to the next step. The entire measurement system was located in a 37 °C chamber. Although the presence of the electrode would disturb the O 2 field between the surface of the oxygen-permeable membrane and the face of the electrode, the pO 2 measurement of primary interest here was the minimum value farthest from the gas chamber where the electrode first entered the slab. Under these conditions, the error incurred in determining if the pO 2 is about 50 mmHg is expected to be small.

Mathematical model of oxygen diffusion and consumption in islet-alginate slab

We consider a one-dimensional slab of thickness L containing a heterogeneous medium of islets dispersed in alginate. Oxygen concentration c is linearly proportional to oxygen partial pressure p according to c = αp, where α is the Bunsen solubility coefficient. At the slab face next to the gas chamber, x = 0 (the small effect of the Silon membrane is ignored), the oxygen partial pressure is set at p 0 . At the slab surface that interfaces with host tissue, we assume the most conservative case that all oxygen is supplied by the gas chamber, and the interface is taken to be impermeable to oxygen. We set the oxygen partial pressure at this surface, x = L, as p L = 50 mmHg, to be consistent with experimental conditions, in which case the oxygen consumption rate can be assumed constant at V max 40. Under these conditions, a steady state oxygen mass balance equating the rate of diffusion to the rate of consumption across a differentiated slice of the slab can be written as

$${({\rm{\alpha }}{\rm{D}})}_{{\rm{eff}}}\frac{{{\rm{d}}}^{2}{\rm{p}}}{{{\rm{dx}}}^{2}}={{\rm{V}}}_{{\rm{\max }}}\varphi $$ (1)

where (αD) eff is the effective permeability of oxygen in the slab given by

$$\frac{{({\rm{\alpha }}{\rm{D}})}_{{\rm{eff}}}}{{({\rm{\alpha }}{\rm{D}})}_{{\rm{c}}}}=\frac{2-2\varphi +\rho (1+2\varphi )}{2+\varphi +\rho (1-\varphi )}$$ (2)

ϕ is the volume fraction of the dispersed phase, and ρ is a permeability ratio

$$\rho =\frac{{({\rm{\alpha }}{\rm{D}})}_{{\rm{d}}}}{{({\rm{\alpha }}{\rm{D}})}_{{\rm{c}}}}$$ (3)

The solution is given by41

$$\frac{{\rm{p}}({\rm{x}})}{{{\rm{p}}}_{0}}=\frac{{{\rm{V}}}_{{\rm{\max }}}\varphi {{\rm{L}}}^{2}}{{({\rm{\alpha }}{\rm{D}})}_{{\rm{eff}}}{{\rm{p}}}_{0}}\{\frac{1}{2}{(\frac{{\rm{x}}}{{\rm{L}}})}^{2}-(\frac{{\rm{x}}}{{\rm{L}}})\}+1$$ (4)

Evaluating Equation (4) at x = L yields a relation between the required oxygen partial pressure difference and the other variables.

$${{\rm{p}}}_{0}-{{\rm{p}}}_{{\rm{L}}}=\frac{{{\rm{V}}}_{{\rm{\max }}}\varphi {{\rm{L}}}^{2}}{{2({\rm{\alpha }}{\rm{D}})}_{{\rm{eff}}}}$$ (5)

or an expression for the maximum thickness possible for prescribed operating conditions.

$${\rm{L}}={[\frac{{2({\rm{p}}}_{0}-{{\rm{p}}}_{{\rm{L}}}{)({\rm{\alpha }}{\rm{D}})}_{{\rm{eff}}}}{{{\rm{V}}}_{{\rm{\max }}}\varphi }]}^{1/2}$$ (6)

Noting that islet surface density S can be expressed by

$${\rm{S}}\,\,\,=\,\,\,\frac{{\rm{L}}{\varphi }}{{{\rm{V}}}_{{\rm{IE}}}}$$ (7)

where V IE is the volume of an IEQ (1.77 × 10−6 cm3/IEQ), Equation (6) can be rearranged to express the maximum surface density attainable

$${\rm{S}}=\frac{1}{1.77\times {10}^{-6}}={[\frac{{2({\rm{p}}}_{0}-{{\rm{p}}}_{{\rm{L}}}{)({\rm{\alpha }}{\rm{D}})}_{{\rm{eff}}}{\varphi }}{{{\rm{V}}}_{{\rm{\max }}}}]}^{1/2}$$ (8)

Statistics

Data are expressed as mean + standard deviation. Statistical significance (p < 0.05) was determined by Student’s t-test.