Mold-casted non-degradable, islet macro-encapsulating hydrogel devices for restoration of normoglycemia in diabetic mice

Abstract

Islet transplantation is a potential cure for diabetic patients, however this procedure is not widely adopted due to the high rate of graft failure. Islet encapsulation within hydrogels is employed to provide a three-dimensional microenvironment conducive to survival of transplanted islets to extend graft function. Herein, we present a novel macroencapsulation device, composed of PEG hydrogel, that combines encapsulation with lithography techniques to generate polydimethylsiloxane (PDMS) molds. PEG solutions are mixed with islets, which are then cast into PDMS molds for subsequent crosslinking. The molds can also be employed to provide complex architectures, such as microchannels that may allow vascular ingrowth through pre-defined regions of the hydrogel. PDMS molds allowed for the formation of stable gels with encapsulation of islets, and in complex architectures. Hydrogel devices with a thickness of 600 μm containing 500 islets promoted normoglycemia within 12 days following transplantation into the epididymal fat pad, which was sustained over the two-month period of study until removal of the device. The inclusion of microchannels, which had a similar minimum distance between islets and the hydrogel surface, similarly promoted normoglycemia. A glucose challenge test indicated hydrogel devices achieved normoglycemia 90 min post-dextrose injections, similar to control mice with native pancreata. Histochemical staining revealed that transplanted islets, identified as insulin positive, were viable and isolated from host tissue at 8 weeks post-transplantation, yet devices with microchannels had tissue and vascular ingrowth within the channels. Taken together, these results demonstrate a system for creating non-degradable hydrogels with complex geometries for encapsulating islets capable of restoring normoglycemia, which may expand islet transplantation as a treatment option for diabetic patients. Biotechnol. Bioeng. 2016;113: 2485-2495. © 2016 Wiley Periodicals, Inc.

Keywords: encapsulation; hydrogel; macroencapsulation device; microchannels; polydimethylsiloxane (PDMS); polyethylene glycol (PEG).

Figure 1.

Macroencapsulating PEG hydrogel devices without or with microchannels. (A) Macroview of a hydrogel without microchannels. (B) A PDMS mold that contained column-like features was employed to form hydrogels with microchannels. (C) Macroview of gel device with microchannels. (D) Close-up view of micropores that form microchannels. Microchannels are ~200 μm in diameter with 500 μm spacing between pore edges. Gels were stained with sirius red for visualization. (E) Encapsulated islets surrounding a microchannel in a hydrogel. Islets appear opaque and a white arrow indicates a representative islet. (F) Representative images of hydrogel devices with encapsulated islets.

Figure 2.

Islet viability confirmed in vitro prior to transplantation. After islet isolation, samples of 50 islets were transferred to a 48-well plate (2D control, unencapsulated or “free” islets) or encapsulated in 30 μL bulk PEG hydrogels (10% wt/vol). Islet viability was assessed using a live (green)/dead (red) stain. Islets remained viable post-isolation in the 2D control (A,B) and hydrogel condition with a 5 min incubation period (C,D). Scale bar: 200 μm.

Figure 3.

Hydrogel macroencapsulation device in vascularized fat pad transplantation site. (A) Gross vascularization of the native fat pad in a diabetic recipient mouse. White arrow indicates a blood vessel. (B) Hydrogel devices, with or without microchannels, were transplanted into the intraperitoneal fat pad of diabetic mice. This image depicts a hydrogel with microchannels wrapped into the fat pad. (C) The hydrogel device remains intact and undegraded upon removal after 2 months post-transplantation. Arrows indicate gel location.

Figure 4.

Device function monitored in diabetic mice. (A) Hydrogel devices with 500 islets with microchannels (±SEM, n = 6), or (B) without micro channels (±SEM, n = 3), reversed diabetes in all recipient mice. Normoglycemic levels (<200 mg/dL) were achieved within 2 weeks post-transplantation and maintained over a 2 month period in both groups. Upon removal of hydrogel devices between Day 60–61 (indicated by an arrow), all mice reverted to a diabetic state (>300 mg/dL) within 2 days. Statistical differences were not seen in engraftment between these groups (P = 0.10) according to an unpaired t-test. (B) Devices with microchannels with 300 islets only reversed diabetes in 25% of recipient mice (n = 4), which indicates 300 islets is an insufficient number to reverse diabetes with this device.

Figure 5.

Glucose responsiveness of hydrogel devices. (A) An intraperitoneal glucose tolerance test (IPGTT) confirms both experimental hydrogel groups (with and without microchannels) and control mice achieved normoglycemia 90 min post-dextrose injection. (B) Area under the curve (AUC) indicates the mice with hydrogel devices, with or without microchannels, are not statistically significant compared to control mice according to a one-way ANOVA (±SEM, P = 0.12; n, +Microchannels (n = 6), −Microchannels (n = 3), Control (n = 9). Mice in all groups achieved normoglycemic levels (~200 mg/dL) 90 min post-injection of dextrose.

Figure 6.

Hydrogel devices explanted day 60 post-transplantation retain islet morphology and viability. (A) Insulin-positive islets were identified throughout the devices with microchannels and (B) near microchannels (indicated with a dashed white line) (10× magnification). (C) Higher magnification images confirmed islet morphology was maintained (20× magnification) in microchanneled devices. (D) Presence of insulin-positive islets was also confirmed in hydrogel devices without microchannels.

Figure 7.

Cellular growth and vessel infiltration of hydrogel devices at day 60 post-transplantation. (A) Cellular growth (indicated by dashed line) was confined to the perimeter of hydrogel devices without microchannels. Encapsulated islets (denoted by a black arrow) were identified in (−) microchannels and (B) (+) microchannel devices. (C) Cellular ingrowth (indicated by a white arrow) occurred into the specific microchannel regions of hydrogels relative to the surrounding hydrogel. (D) CD31-positive cells are identified in the microchannels of the gel which demonstrates vessels (indicated by a white arrow) are available to nearby encapsulated islets. (E) CD31-positive cells are present in the microchannels with a nuclear counterstain. Scale bar: 100 μm.

Figure 8.

Absence of fibrosis around hydrogel devices confirmed at hydrogel-adipose tissue interface. Thin layers of connective tissue at the hydrogel–adipose tissue interface (indicated by triple arrows) demonstrates no foreign body response to the hydrogel material, either without (A) or with microchannels (B). Scale bar: 100 μm.