Improved Outcomes in Islet Isolation and Transplantation by the Use of a Novel Hemoglobin-based O2 Carrier

Abstract

During isolation, islets are exposed to warm ischemia. In this study, intraductal administration of oxygenated polymerized, stroma-free hemoglobin-pyridoxalated (Poly SFH-P) was performed to improve O 2 delivery. Rat pancreata subjected to 30-min warm ischemia were perfused intraductally with collagenase in oxygenated Poly SFH-P/RPMI or RPMI (control). PO 2 was increased by Poly SFH-P (381.7 ± 35.3 mmHg vs. 202.3 ± 28.2, p = 0.01) and pH maintained within physiological range (7.4-7.2 vs. 7.1-6.6, p = 0.009). Islet viability (77% ± 4.6 vs. 63% ± 4.7, p = 0.04) was improved and apoptosis lower with Poly SFH-P (caspase-3: 34,714 ± 2167 vs. 45,985 ± 1382, respectively, p = 0.01). Poly SFH-P improved islet responsiveness to glucose as determined by increased intracellular Ca 2+ levels and improved insulin secretion (SI 5.4 ± 0.1 vs. 3.1 ± 0.2, p = 0.03). Mitochondrial integrity was improved in Poly SFH-P-treated islets, which showed higher percentage change in membrane potential after glucose stimulation (14.7% ± 1.8 vs. 9.8 ± 1.4, respectively, p < 0.05). O 2 delivery by Poly SFH-P did not increase oxidative stress (GSH 7.1 ± 2.9 nm/mg protein for Poly SFH-P vs. 6.8 ± 2.4 control, p = 0.9) or oxidative injury (MDA 1.8 ± 0.9 nmol/mg protein vs. 6.2 ± 2.4, p = 0.19). Time to reach normoglycemia in transplanted diabetic nude mice was shorter (1.8 ± 0.4 vs. 7 ± 2.5 days, p = 0.02), and glucose tolerance improved in the Poly SFH-P group (AUC 8106 ± 590 vs. 10,863 ± 946, p = 0.03). Oxygenated Poly SFH-P improves islet isolation and transplantation outcomes by preserving mitochondrial integrity.

Microfluidic device for multimodal characterization of pancreatic islets

“…16, 18, 22, 28, 31, 43, 44 The mouse islets show a clear dose-response illustrated with a marked increase in the insulin secretion with increasing stimulatory glucose concentrations, and is consistent with previous reports. 25 There is slight delay in the onset of the peak after the islets are exposed to the stimulatory glucose consistent with previous reults. 18, 44 …”

“…25 In brief, the pancreata of 8–10 week-old C57/B6 or BALB/c (Jackson Laboratory) were perfused with 0.22 mg/mL Collagenase (Sigma, MO) and then digested for 11 minutes at 37 °C. The digested pancreata were shaken vigorously for 15 seconds and further purified through discontinuous Ficoll gradient.…”

“…Hyper-polarization of the mitochondrial membrane causes uptake of Rh123 into mitochondria and a decrease in fluorescence due to intermolecular crowding and quenching. 25 Islets were incubated in KRB2 supplemented with 10 µg/mL Rh123 for 20 minutes at 37 °C, then loaded into the microfluidic perfusion device and mounted on an inverted epifluorescence microscope equipped with a heating stage and perfused by a continuous flow (rate 1 mL/min) of KRB2 at 37 °C (pH 7.4). KRB containing different glucose concentrations were administered to the islets after rinsing in KRB2 for 10–15 minutes.…”

Islet amyloid deposition limits the viability of human islet grafts but not porcine islet grafts

“…The long-term success of human islet transplants has been limited, however, with 75% of patients who achieved insulin independence requiring insulin within 2 years posttransplant (1). Although immune rejection of islet allografts certainly plays a role in graft failure, nonimmune-mediated beta cell loss is also likely to play an important role (2)(3)(4)(5)(6).…”

Diazoxide, a KATP Channel Opener, Prevents Ischemia–Reperfusion Injury in Rodent Pancreatic Islets

“…Mice with blood glucose levels higher than 20 mM or 360 mg/dL were considered recipients for islet transplantation. After overnight incubation, 350 islets were transplanted under the left kidney capsule in each mouse under isoflurane anesthetization, as previously described [4]. Blood glucose levels were monitored every day during the 1 st week and every other day thereafter.…”