Glucose-Stimulated Insulin Response of Silicon Nanopore-Immunoprotected Islets under Convective Transport

Abstract

Major clinical challenges associated with islet transplantation for type 1 diabetes include shortage of donor organs, poor engraftment due to ischemia, and need for immunosuppressive medications. Semipermeable membrane capsules can immunoprotect transplanted islets by blocking passage of the host's immune components while providing exchange of glucose, insulin, and other small molecules. However, capsules-based diffusive transport often exacerbates ischemic injury to islets by reducing the rate of oxygen and nutrient transport. We previously reported the efficacy of a newly developed semipermeable ultrafiltration membrane, the silicon nanopore membrane (SNM) under convective-driven transport, in limiting the passage of pro-inflammatory cytokines while overcoming the mass transfer limitations associated with diffusion through nanometer-scale pores. In this study, we report that SNM-encapsulated mouse islets perfused in culture solution under convection outperformed those under diffusive conditions in terms of magnitude (1.49-fold increase in stimulation index and 3.86-fold decrease in shutdown index) and rate of insulin secretion (1.19-fold increase and 6.45-fold decrease during high and low glucose challenges), respectively. Moreover, SNM-encapsulated mouse islets under convection demonstrated rapid glucose-insulin sensing within a physiologically relevant time-scale while retaining healthy islet viability even under cytokine exposure. We conclude that encapsulation of islets with SNM under convection improves islet in vitro functionality. This approach may provide a novel strategy for islet transplantation in the clinical setting.

Keywords: convection; diffusion; glucose-insulin kinetics; immunoisolation; silicon nanopore membranes (SNM).

Figure 1

Diagrams of the experimental conditions. (a) Schematic diagram of the perfusion device for in vitro assessment of glucose-stimulated insulin secretion from membrane-encapsulated mouse islets. A peristaltic pump circulated media through the upper compartment of a two-layered flow cell separated by a membrane (SNM or SμM). Islets were encapsulated in the bottom chamber. A 3-way valve was incorporated into the system to establish transmembrane pressure to create either diffusive or convective condition. A set of 75 perfusate samples was collected from the bottom chamber at 10 min intervals for up to 1.5 h. The bottom chamber was replenished with media following each collection and correction for dilution was made in calculation of the insulin concentration. (b) Schematic of all experimental conditions including the membrane-encapsulated islets under diffusion and convection (device, diffusion vs device, convection), and static culture as control (no device, no perfusion). A mixture of cytokines was also added to examine the effect on each condition (+Ck). The glucose-stimulated insulin kinetics and viability were analyzed for all conditions.

Figure 2

Glucose—insulin kinetics of membrane-encapsulated islets under convection and diffusion without cytokine exposure. (a) Insulin release kinetics of membrane-encapsulated mouse islets during 90 min low-high- low (1.6, 16.6, and 1.6 mM) glucose stimulation under convective (2 psi) (Conv) and diffusive transport (Diff) without subjection to cytokines. The naked islets cultured under static conditions served as controls (Control). The SNM-and SμM- encapsulated islets under convective transport (SNM, Conv & SμM, Conv) exhibited higher insulin secretion following stimulation at high glucose concentration and faster insulin release kinetics in response compared to those under diffusive transport (Control & SNM, Diff) (,ean ± SEM, n ≥ 3). (b) Stimulation index (SI) was calculated as the ratio of (1) the first insulin collection in the high glucose phase at 40 min to the last insulin collection point of the previous low glucose phase at 30 min (Immediate Stimulation), and (2) the highest insulin secretion in the high glucose phase to the last insulin collection point of the previous low glucose phase at 30 min (Maximum Stimulation). The SI indicates the magnitude of insulin released as stimulated by a higher concentration of glucose. Without cytokine exposure, SNM- encapsulated islets under convection (SNM, Conv) and diffusion (SNM, Diff), SμM-encapsulated islets under convective transport (SμM, Conv), and the naked islets cultured under static conditions (Control) all exhibited similar magnitude of glucose-induced insulin secretion when transitioning from low glucose to high glucose (Immediate Stimulation). However, the SI of SNM- and SμM-encapsulated islets under convection (SNM, Conv & SμM, Conv) was the highest compared to that under diffusion (SNM, Diff) and the naked islets cultured under static conditions (Control) when the highest insulin secretion in the high glucose phase was used (Maximum Stimulation) (mean ± SEM, n ≥ 3). (c) Shut-down index (SDI) was the ratio of (1) the first insulin collection point in the subsequent low glucose phase at 70 min to the last insulin collection point in the high glucose phase at 60 min (Immediate Shutdown), and (2) the lowest insulin secretion in the subsequent low glucose phase to the last insulin collection point in the high glucose phase at 60 min (Maximum Shutdown). The SDI reflects the magnitude of cessation in insulin production once glucose concentration returns to normal. Without cytokine exposure, SNM- and SμM-encapsulated islets under convection (SNM, Conv & SμM, Conv) exhibited the highest magnitude of insulin reduction compared to the diffusive condition (SNM, Diff) and the naked islet culture (Control) as glucose dropped low (Immediate Shutdown & Maximum Shutdown) (mean ± SEM, n ≥ 3, *p < 0.05).

Figure 3

Glucose—insulin kinetics of membrane-encapsulated islets under convection and diffusion with cytokine exposure. (a) Insulin release kinetics of membrane-encapsulated mouse islets during 90 min low—high—low (1.6, 16.6, and 1.6 mM) glucose stimulation under convective (2 psi) (Conv) and diffusive transport (Diff) with subjection to cytokines (+Ck). Experiments with cytokine exposure (+Ck) consisted of media containing TNF-α (2000 U/mL), IFN-γ (1000 U/mL), and IL-1β (10 000 U/mL). The naked islets cultured under static conditions served as controls (Control, + Ck). The SNM- and SμM-encapsulated islets under convective transport (SNM, Conv, + Ck & SμM, Conv, + Ck) exhibited higher insulin secretion following stimulation at high glucose concentration and faster insulin release kinetics in response compared to those under diffusive transport (SNM, Diff, + Ck) and naked islets cultured under static conditions (Control, + Ck) (mean ± SEM, n ≥ 3). (b) Stimulation index (SI) calculated as the ratio of (1) the first insulin collection in the high glucose phase at 40 min to the last insulin collection point of the previous low glucose phase at 30 min (Immediate Stimulation), and (2) the highest insulin secretion in the high glucose phase to the last insulin collection point of the previous low glucose phase at 30 min (Maximum Stimulation). The SI indicates the magnitude of insulin released as stimulated by a higher concentration of glucose. With cytokine exposure (+Ck), SμM-encapsulated islets under convective transport (SμM, Conv) showed the highest magnitude in glucose-induced insulin secretion compared to other conditions (Immediate Stimulation). However, when using the highest insulin secretion in the high glucose phase (Maximum Stimulation), the calculated SI was the highest for SNM-and SμM-encapsulated islets under convection (SNM, Conv & SμM, Conv) compared to that under diffusion (SNM, Diff) and naked islets cultured under static conditions (Control) (mean ± SEM, n ≥ 3, *p < 0.05). (c) Shut-down index (SDI) calculated as the ratio of (1) the first insulin collection point in the subsequent low glucose phase at 70 min to the last insulin collection point in the high glucose phase at 60 min (Immediate Shutdown), and (2) the lowest insulin secretion in the subsequent low glucose phase to the last insulin collection point in the high glucose phase at 60 min (Maximum Shutdown). The SDI reflects the magnitude of cessation in insulin production once glucose concentration returns to normal. With cytokine exposure (+Ck), the SNM- and SμM-encapsulated islets under convection (SNM, Conv & SμM, Conv) exhibited the highest magnitude of insulin reduction compared to the diffusive condition (SNM, Diff) and the naked islet culture (Control) as glucose dropped low (Immediate Shutdown & Maximum Shutdown) (mean ± SEM, n ≥ 3, *p < 0.05).

Figure 4

In vitro viability of mouse islets. (a) Viability of mouse islets was measured following the 90 min low−high−low (1.6, 16.6, and 1.6 mM) glucose stimulation in which islets were subjected to the mock-loop circuit with (+Ck) or without cytokine exposure for SNM- and SμM-encapsulation under convection (SNM, C & SμM, C). The naked islet culture under static culture with cytokine exposure (Control,+Ck) showed significantly less viability compared to all other conditions (mean ± SEM, n ≥ 3, *p < 0.05). (b) Viable (green) and dead (red) cells were stained for control static culture without cytokines (A: Control), control static culture with cytokines (B: Control, + Ck), SNM-encapsulated mouse islets under convection without cytokines (C: SNM, C), SNM-encapsulated mouse islets under convection with cytokines (D: SNM, C, + Ck), SμM-encapsulated mouse islets under convection without cytokines (E: SμM, C), and SμM-encapsulated mouse islets under convection with cytokines (F: SμM, C,+Ck). Experiments with cytokine exposure (indicated by+Ck) consisted of media containing TNF-α, IFN-γ, and IL-1β. Both control static culture with cytokines (B: Control, + Ck) and SμM-encapsulated mouse islets under convection with cytokines (F: SμM, C, + Ck) showed a higher level of islet damage compared to other groups, however, the viability of SμM-encapsulated mouse islets under convection with cytokines (F: SμM, C, + Ck) was not statistically significant (n.s.).