Islet Quality Assessment Clark K. Colton Department of Chemical Engineering Massachusetts Institute of Technology Cambridge, MA Introductory overview Quantity and composition of islet preparations Quantitative membrane integrity measurements Oxygen consumption rate measurements Stirred chamber methods and characteristics prediction of transplantation outcome Oxygen biosensor system
Islets Are Damaged During Isolation from Human Pancreas Enzymatic Digestion and Mechanical Disruption Distension with Collagenase/Protease solution Shaker Density Gradient Exocrine Centrifugation Tissue Ischaemic Conditions Islet Preparation 150 µm 1-2% original pancreas volume
What Do We Want To Know? For a given islet preparation: What is the “potency” or “dose?” Can we predict transplantation outcome? Goals for Islet Quality Assessment Quantity Viability How much tissue is there? For (1) total tissue and (2) islets • Volume • How much is viable? • Number of cells • What fraction is viable? What is the tissue composition? • Islet – β cells, other What does viability mean, anyway? • Dead • Exocrine– acinar, duct • Live • Live now, dead later because Function of irreversible commitment What is the insulin secretory capacity? to the cell death process
Why Are Islet Preparations So Difficult To Characterize? 4. Many techniques for cells 1. Islets are cellular aggregates. Variety of shapes and sizes are inapplicable to islets Visual size estimation is because the islets cannot be • prone to error usefully dissociated into cells. • operator dependent • large uncertainty agitation Dispersed Islet Cells Serine proteases 2. Human preparations have (trypsin) varying amounts of impurities. Distinguishing properties of • Cells are damaged: anoikis islets/exocrine tissue difficult • Cells are lost 3. The islet is a moving target. • Recovered cells are likely not Damage occurs during • isolation representative of original islet • culture • shipment
What Tools Are Available? • Safety • Identity • Quantity of tissue Volume Number of Cells Composition • Viability Membrane Integrity Mitochondrial Function Apoptosis • Potency Glucose Stimulated Insulin Release Immunodeficient Mouse Transplant • Other Gene Expression Profiling
Quantity of Tissue Tissue Parameter Type of Quantity Method Assayed Measured Volume Islet Tissue volume ● Packed cell volume of tissue pellet Preparation ● Ultrasound scattering Islet volume ● Insulin content ● Dithizone (DTZ) staining Visual counting Enumeration of islet Image analysis equivalents (IEQ) Number of Cells Islet Total DNA ● DNA content Preparation Total intact ● Nuclei counting cell nuclei Cell Composition Islet Volume fraction ● DTZ staining Preparation islets ● Morphology (light microscopy) Individual cell ● Ultrastructural analysis (electron microscopy) types Dispersed Individual cell ● Differential staining Cells types (laser scanning cytometry)
Viability of Tissue Tissue Type of Assay Method Assayed Cell Membrane Islet Live/Dead (Membrane Permeable) Preparation Fluorescein Diacetate (FDA)/Propidium Iodide (PI) Integrity SYTO 13/Ethidium Bromide (EB) All/Dead LDS 751/Sytox Orange Dead Trypan Blue Quantitative assay via Nuclei Counting- 7- AAD Islet Mitochondrial Redox state of the cell-Tetrazolium salts MTT, MTS Preparation Function Oxidative phosphorylation-Oxygen consumption rate Energetic State-[ATP], [ATP]/[ADP], ATP production rate Dispersed Mitochondrion membrane potential (MMP)-Fluorescent Cells dyes JC-1, TMRE (Flow Cytometry) Islet Apoptotic Magic angle spinning 1H-NMR spectroscopy Preparation Events Early: Signaling pathway – Caspase activation Disrupted Late: Nucleosome DNA fragmentation Cells Phosphatidyl serine translocation – Annexin V Fixed Tissue DNA fragmentation – TUNEL or Cells
Viability of Tissue Tissue Type of Assay Method Assayed Cell Membrane Islet Live/Dead (Membrane Permeable) Preparation Fluorescein Diacetate (FDA)/Propidium Iodide (PI) Integrity SYTO 13/Ethidium Bromide (EB) All/Dead LDS 751/Sytox Orange Dead Trypan Blue Quantitative assay via Nuclei Counting- 7- Do These Assays Give Equivalent Results? aminoactinomycin D Islet Mitochondrial Redox state of the cell-Tetrazolium salts MTT, MTS Preparation Function Oxidative phosphorylation-Oxygen consumption rate (OCR) Dispersed Energetic State-[ATP], [ATP]/[ADP], ATP production rate Cells Mitochondrion membrane potential (MMP)-Fluorescent dyes Islet Apoptotic JC-1, TMRE Preparation Events Magic angle spinning 1H-NMR spectroscopy Dispersed Cells Early: Signaling pathway – Caspase activation Fixed Tissue Late: Nucleosome DNA fragmentation or Cells Phosphatidyl serine translocation – Annexin V
Time Dependence of Cell Death and Cell Viability Assays Jurkat Cells INS-1 Cells Assays performed: Suspension Culture Surface-attached culture 1 µM Camptothecin 5 mM Streptozotocin Mitochondrial Function OCR, ATP, MTT, MTS Fraction Apoptotic Apoptosis Events Cells ( ) Annexin V, Multi-caspase activation Membrane integrity Apoptosis Trypan blue, FDA/PI, 7-AAD, Events LDS 751/Sytox Orange Rat, Human Islets Anoxia, 37 o C with Intact Membranes ( ) Relative Fraction of Cells Membrane Relative Fraction of Viable Cells ( ) Integrity Mitochondrial function Time of Stress Exposure (hr) Membrane Integrity measurements (7-AAD) lag other measures of cell viability
How Can Oxygen Consumption Rate (OCR) Be Measured? Hardware Perfusion Systems Stagnant Liquid Film Stirred Tank ∆ pO 2 across tissue ∆ pO 2 Measured Sensor pO 2 beneath cells rate of bulk liquid flow rate ∆ t pO 2 decrease Variables Source custom-made BD Oxygen Biosensor Instech Micro Oxygen System (BD OBS) Uptake System Pros elegant simple accurate flexible research tool inexpensive precise follow transient dynamics rapid rapid Direct measurement of Direct measurement of OCR OCR Cons very complex measurement is complex time consuming inaccurate Requires mathematical model to calculate OCR
How Can Oxygen Consumption Rate (OCR) be Measured? Hardware Perfusion Systems Stagnant Liquid Film Stirred Tank ∆ pO 2 across tissue ∆ pO 2 Measured pO 2 beneath cells rate of bulk liquid flow rate ∆ t pO 2 decrease Variables Source custom-made BD oxygen biosensor Instech micro oxygen system uptake system Pros elegant simple accurate flexible inexpensive precise follow transient dynamics rapid rapid little training required Dionne, K.E., et al. A microperifusion system with environmental control for Cons very complex precision is poor moderately expensive studying insulin secretion by pancreatic tissue, Biotechnol. Prog. , 7 , 1991, research tool accuracy is questionable 359-368 not for routine use limited experience
How Can Oxygen Consumption Rate (OCR) Be Measured? Hardware Perfusion Systems Stagnant Liquid Film Stirred Tank ∆ pO 2 across tissue ∆ pO 2 Measured Sensor pO 2 beneath cells rate of bulk liquid flow rate ∆ t pO 2 decrease Variables Source custom-made BD Oxygen Biosensor Instech Micro Oxygen System (BD OBS) Uptake System Pros elegant simple accurate flexible research tool inexpensive precise follow transient dynamics rapid rapid Direct measurement of Direct measurement of OCR OCR Cons very complex measurement is complex time consuming inaccurate Requires mathematical model to calculate OCR
How Can Oxygen Consumption Rate (OCR) be Measured? Hardware Perfusion Systems Stagnant Liquid Film Stirred Tank ∆ pO 2 across tissue ∆ pO 2 Measured pO 2 beneath cells rate of bulk liquid flow rate ∆ t pO 2 decrease Variables Source custom-made BD oxygen biosensor Instech micro oxygen system uptake system Pros elegant simple accurate flexible inexpensive precise follow transient dynamics rapid rapid little training required Sweet I.R., et al. Regulation of ATP/ADP in Pancreatic Islets, Diabetes , 53 , 2004, 401-409 Cons very complex precision is poor moderately expensive research tool accuracy is questionable not for routine use limited experience
How Can Oxygen Consumption Rate (OCR) Be Measured? Hardware Perfusion Systems Stagnant Liquid Film Stirred Tank ∆ pO 2 across tissue ∆ pO 2 Measured Sensor pO 2 beneath cells rate of bulk liquid flow rate ∆ t pO 2 decrease Variables Source custom-made BD Oxygen Biosensor Instech Micro Oxygen System (BD OBS) Uptake System Pros elegant simple accurate flexible research tool inexpensive precise follow transient dynamics rapid rapid Direct measurement of Direct measurement of OCR OCR Cons very complex measurement is complex time consuming inaccurate Requires mathematical model to calculate OCR
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