Real Time Human Islet Potency Assay Based on Oxygen Consumption - PowerPoint PPT Presentation

Real Time Human Islet Potency Assay Based on Oxygen Consumption Rate Klearchos Papas, PhD University of Minnesota Medical School

Oxygen Consumption Rate Assessment Parameters Parameter Proportional To Measure of OCR Number of viable cells Amount of good tissue Volume of viable tissue DNA Number of cells Total amount of tissue Total tissue volume OCR Viable tissue volume DNA Total tissue volume Quality of the tissue OCR/DNA = Fractional viability (OCR/DNA) v

Response to Rat Islet Transplants in Diabetic Balb/C Mice (Anti-CD4) Fractional Viability (%) 0 10 20 30 40 50 60 70 80 90 100 6 800 Oxygen Consumption Rate, OCR (nmol/min) 4500 IEQ Viable 700 Dead 5 Viable Islet Equivalents, VIEQ 600 4 500 500 VIEQ 400 3 300 2 900 IEQ 200 1 100 100 VIEQ FV = 10% 50% 90% 0 0 0 50 100 150 200 250 300 350 400 450 Normalized Oxygen Consumption Rate, OCR/DNA (nmol/min • mgDNA)

Response to Human Islet Transplants in Diabetic Nude Mice Fractional Viability (%) 0 10 20 30 40 50 60 70 80 90 100 Oxygen Consumption Rate, OCR (nmol/min) 6 800 Group Human 700 5 all cure Viable Islet Equivalents, VIEQ mixed 600 all fail 4 500 400 3 300 2 200 1 100 0 0 350 400 450 0 50 100 150 200 250 300 Normalized Oxygen Consumption Rate, OCR/DNA (nmol/min•mgDNA)

Response to Human Islet Transplants in Diabetic Immunodeficient Mice 8 Human islets were taken from the Oxygen Consumption Rate (nmol/min) highest purity fraction (>90% by DTZ) 7 Some All Cure Cure 6 5 4 None Cure 3 2 1 0 0 50 100 150 200 Normalized Oxygen Consumption Rate (nmol/min mg DNA)

Transplantation Studies OCR/DNA = 142 nmoles/min/mgDNA OCR Dose Transplanted = 1.0 - 7.5 nmoles/min 700 700 700 OCR = 1 OCR = 1.5 OCR = 2.5 600 600 600 OCR = 1 OCR = 1.5 OCR = 2.5 Blood Glucose (mg/dl) Blood Glucose (mg/dl) Blood Glucose (mg/dl) OCR = 1 500 OCR = 1.5 500 500 OCR = 2.5 400 400 400 300 300 300 200 200 200 100 100 100 0 0 0 -5 0 5 10 15 20 25 30 -5 0 5 10 15 20 25 30 -5 0 5 10 15 20 25 30 Time (days) Time (days) Time (days) 700 700 OCR = 5.0 OCR = 7.5 600 600 OCR = 5.0 OCR = 7.5 Blood Glucose (mg/dl) Blood Glucose (mg/dl) 500 500 OCR = 5.0 OCR = 7.5 400 400 300 300 200 200 100 100 0 0 -5 0 5 10 15 20 25 30 -5 0 5 10 15 20 25 30 Time (days) Time (days)

Transplantation Studies OCR/DNA = 109 nmoles/min/mgDNA OCR Dose Transplanted = 1.0 - 7.5 nmoles/min 700 700 700 OCR = 1.5 OCR = 1 OCR = 2.5 600 600 600 OCR = 1.5 OCR = 1 OCR = 2.5 Blood Glucose (mg/dl) Blood Glucose (mg/dl) Blood Glucose (mg/dl) 500 500 500 OCR = 1.5 OCR = 1 OCR = 2.5 400 400 400 300 300 300 200 200 200 100 100 100 0 0 0 -5 0 5 10 15 20 25 30 -5 0 5 10 15 20 25 30 -5 0 5 10 15 20 25 30 Time (days) Time (days) Time (days) 700 700 OCR = 5.0 OCR = 7.5 600 600 OCR = 5.0 OCR = 7.5 Blood Glucose (mg/dl) Blood Glucose (mg/dl) 500 OCR = 5.0 500 OCR = 7.5 400 400 300 300 200 200 100 100 0 0 -5 0 5 10 15 20 25 30 -5 0 5 10 15 20 25 30 Time (days) Time (days)

Response to Human Islet Transplants in Diabetic Immunodeficient Mice 8 Human islets were taken from the Oxygen Consumption Rate (nmol/min) highest purity fraction (>90% by DTZ) 7 Some All Cure Cure 6 5 4 None Cure 3 2 1 0 0 50 100 150 200 Normalized Oxygen Consumption Rate (nmol/min mg DNA)

Statistical Analysis by City of Hope ABCC Sensitivity of 91.7% and a specificity of 90.5% xx 7 6 5 x x OCR 4 3 2 x 1 80 100 120 140 160 180 OCR/DNA

Conclusion - Human islet potency assay based on OC and DNA measurements is highly sensitive and highly specific to the mouse transplantation outcome