The Use of the BD Oxygen Biosensor System to Assess Isolated Human Islets of Langerhans: Oxygen Consumption as a Potential Measure of Islet Potency

Abstract

The measurement of cellular oxygen consumption rate (OCR) is a potential tool for the assessment of metabolic potency of isolated islets of Langerhans prior to clinical transplantation. We used a commercially available 96-well plate fluoroprobe, the BD Oxygen Biosensor System (OBS), to estimate OCR in 27 human islet preparations, and compared these results to those of concurrent mouse transplantations. OCR was estimated both from the dO2 at steady state and from the transient rate of change of dO2 during the initial culture period immediately after seeding ("dO2 slope"). To demonstrate the validity of the OBS-derived values, it was shown that they scaled linearly with islet equivalent number/DNA concentration and with each other. These measurements were obtained for each preparation of islets incubated in media supplemented with either low (2.2 mM) or high (22 mM) glucose. Concurrently, one to three athymic nude mice were transplanted with 2,000 IEQs under the kidney capsule. The OCR Index, defined as the ratio of the DNA-normalized "dO2 slope" in high glucose to that in low glucose, proved highly predictive of mouse transplant results. Of the 69 mice transplanted, those receiving islets where the OCR Index exceeded 1.27 were 90% likely to reverse within 3 days, whereas those receiving islets with an OCR Index below 1.27 took significantly longer, often failing to reverse at all over a 35-day time period. These results suggest that the OBS could be a useful tool for the pretransplant assessment of islet cell potency.

Detailed protocol for evaluation of dynamic perifusion of human islets to assess β-cell function

“…The majority of the methods currently being used to assess islets are expensive or cumbersome and the data are difficult to reproduce. [23][24][25][26][27][28] The current standard method to assess islet quality is to transplant islets into diabetic immunodeficient rodents. 29,30 However, the results are typically not available until long after islets are transplanted into a patient, given the time required to reverse diabetes in animal models.…”

The Sequential Combination of a JNK Inhibitor and Simvastatin Protects Porcine Islets from Peritransplant Apoptosis and Inflammation

“…To prepare study, we showed that this method for determining the estimate of the islet in the high-glucose Krebs buffer divided by the DNA content-normalized OCR estimate β-cell viability index predicted posttransplant outcome (15). Briefly, 1,000 islet equivalents (IEQ) of porcine of the islet in the low-glucose Krebs buffer (8). islets were dispersed into single-cell suspensions in 1 ml Assessment of Islet Function In Vivo accutase solution (Innovative Cell Technologies, Inc., San Diego, CA, USA) for each stain.…”

Shipment of Human Islets for Transplantation

“…Oxygen consumption rate assessment: Cellular oxygen consumption as a marker of islet potency was performed as previously described (25). Briefly, triplicate aliquots of 500 IEQ, in low (2.2 mM) and high (22 mM) glucose Krebs buffer solution, respectively, were placed in a commercially available plate-based oxygen biosensor.…”