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

OCR Number of viable cells Amount of good tissue Volume of viable tissue DNA Number of cells Total amount of tissue Total tissue volume Quality of the tissue = Fractional viability

Oxygen Consumption Rate Assessment Parameters

OCR DNA Viable tissue volume Total tissue volume

Parameter Proportional To Measure of

OCR/DNA (OCR/DNA)v

Normalized Oxygen Consumption Rate, OCR/DNA (nmol/min•mgDNA)

Oxygen Consumption Rate, OCR (nmol/min) Fractional Viability (%) Viable Islet Equivalents, VIEQ

Response to Rat Islet Transplants in Diabetic Balb/C Mice (Anti-CD4)

4 5 6 50 100 150 200 250 300 350 400 450 100 800 700 600 500 400 300 200 100 50 40 20 10 60 30 80 70 90 1 2 3

4500 IEQ 900 IEQ Viable Dead 500 VIEQ 100 VIEQ FV = 10% 50% 90%

Response to Human Islet Transplants in Diabetic Nude Mice

Fractional Viability (%) Oxygen Consumption Rate, OCR (nmol/min) Normalized Oxygen Consumption Rate, OCR/DNA (nmol/min•mgDNA)

50 100 150 200 250 300 350 400 450 20 10 80 90 60 30 70 50 40

Viable Islet Equivalents, VIEQ

100 800 700 600 500 400 300 200 100

Group all cure mixed all fail Human

6 5 4 3 2 1

None Cure All Cure Some Cure

Response to Human Islet Transplants in Diabetic Immunodeficient Mice

50 100 150 200 Normalized Oxygen Consumption Rate (nmol/min mg DNA) 1 2 3 4 5 6 7 8 Oxygen Consumption Rate (nmol/min)

Human islets were taken from the highest purity fraction (>90% by DTZ)

Transplantation Studies

OCR/DNA = 142 nmoles/min/mgDNA OCR Dose Transplanted = 1.0 - 7.5 nmoles/min

100 200 300 400 500 600 700

• 5

5 10 15 20 25 30 Time (days) Blood Glucose (mg/dl) OCR = 5.0 OCR = 5.0 OCR = 5.0 100 200 300 400 500 600 700

• 5

5 10 15 20 25 30 Time (days) Blood Glucose (mg/dl) OCR = 1.5 OCR = 1.5 OCR = 1.5 100 200 300 400 500 600 700

• 5

5 10 15 20 25 30 Time (days) Blood Glucose (mg/dl) OCR = 1 OCR = 1 OCR = 1 100 200 300 400 500 600 700

• 5

5 10 15 20 25 30 Time (days) Blood Glucose (mg/dl) OCR = 7.5 OCR = 7.5 OCR = 7.5 100 200 300 400 500 600 700

• 5

5 10 15 20 25 30 Time (days) Blood Glucose (mg/dl) OCR = 2.5 OCR = 2.5 OCR = 2.5

Transplantation Studies

OCR/DNA = 109 nmoles/min/mgDNA OCR Dose Transplanted = 1.0 - 7.5 nmoles/min

100 200 300 400 500 600 700

• 5

5 10 15 20 25 30 Time (days) Blood Glucose (mg/dl) OCR = 5.0 OCR = 5.0 OCR = 5.0 100 200 300 400 500 600 700

• 5

5 10 15 20 25 30 Time (days) Blood Glucose (mg/dl) OCR = 1.5 OCR = 1.5 OCR = 1.5 100 200 300 400 500 600 700

• 5

5 10 15 20 25 30 Time (days) Blood Glucose (mg/dl) OCR = 1 OCR = 1 OCR = 1 100 200 300 400 500 600 700

• 5

5 10 15 20 25 30 Time (days) Blood Glucose (mg/dl) OCR = 7.5 OCR = 7.5 OCR = 7.5 100 200 300 400 500 600 700

• 5

5 10 15 20 25 30 Time (days) Blood Glucose (mg/dl) OCR = 2.5 OCR = 2.5 OCR = 2.5

None Cure All Cure Some Cure

Response to Human Islet Transplants in Diabetic Immunodeficient Mice

50 100 150 200 Normalized Oxygen Consumption Rate (nmol/min mg DNA) 1 2 3 4 5 6 7 8 Oxygen Consumption Rate (nmol/min)

Human islets were taken from the highest purity fraction (>90% by DTZ)

Statistical Analysis by City of Hope ABCC

OCR/DNA OCR 80 100 120 140 160 180 1 2 3 4 5 6 7

x x x xx

Sensitivity of 91.7% and a specificity of 90.5%

Conclusion

• Human islet potency assay based on OC

and DNA measurements is highly sensitive and highly specific to the mouse transplantation outcome