Type-1 Diabetes: Methods and Protocols

✍ Scribed by Anna Moore, Ping Wang

• Publisher: Humana Press
• Year: 2023
• Tongue: English
• Leaves: 259
• Series: Methods in Molecular Biology, 2592
• Category: Library

✦ Synopsis

This volumes details methods focusing on technological innovation and recent advances in diabetes management. Chapters will guide readers through recent advances, beta-cell regeneration, non-invasive imaging of endogenous, transplanted islets theranostics, microRNA profiling of beta-cells and artificial intelligence, and deep learning algorithms in diabetes. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.

 

Authoritative and cutting-edge, Type-1 Diabetes: Methods and Protocols is for a broad audience including basic researches, clinicians, and physician scientists whose major focus is in diabetes.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: 3D Optical Molecular Imaging of the Rodent Pancreas by OPT and LSFM
1 Introduction
2 Materials
2.1 4% Paraformaldehyde (PFA)
2.2 15% Bleaching Solution
2.3 Wash Buffer: Tris-Buffered Saline with Triton X-100 (TBST)
2.4 Blocking Solution
2.5 1.5% Low-Melting Agarose (LMA)
2.6 Clearing Solution: Benzyl Alcohol and Benzyl Benzoate (BABB)
2.7 3D Imaging Equipment
2.8 Software and Recommended Links
3 Methods
3.1 Tissue Preparation for Optical 3D Imaging
3.2 3D Molecular Imaging of the Pancreas
3.3 3D Image Processing for OPT Scanned Samples
3.4 3D Rendering and Quantification
4 Notes
References
Chapter 2: Uncommon Transplantation Sites: Transplantation of Islets and Islet Organoids in the Anterior Chamber of the Eye of...
1 Introduction
2 Materials and Equipment
2.1 Materials and Equipment for Transplantation into the Anterior Chamber of the Eye of Mice or Rats
2.2 Materials for Transplantation into the Anterior Chamber of the Eye in Nonhuman Primates
3 Methods
3.1 Equipment Preparation for Transplantation into the Anterior Chamber of the Eye of Mice or Rats
3.2 Preparation of Transplantation Material for Transplantation into the Anterior Chamber of the Eye of Mice or Rats
3.3 Preparation of the Recipient Animal for Transplantation into the Anterior Chamber of the Eye of Mice or Rats
3.4 Transplantation to the Anterior Chamber of the Eye of Mice or Rats
3.5 Preparation of the Animal for Transplantation into the Anterior Chamber of the Eye in Nonhuman Primates
3.6 Preparation of the Pancreatic Islets for Transplantation into the Anterior Chamber of the Eye in Nonhuman Primates
3.7 Transplantation of Islets into the Anterior Chamber of the Eye
4 Notes
References
Chapter 3: Generation of Insulin-Producing Multicellular Organoids
1 Introduction
2 Materials
2.1 Biological Materials
2.2 Disposables
2.3 Equipment
2.4 Cell Collection and Organoid Generation
2.5 Histological Assessment
2.6 Functional Assessment
2.7 Reagent Setup
3 Methods
3.1 Islet Dissociation
3.1.1 Transfer Islets from Culture Flask into the Precoated 50-mL Conical Tube
3.2 Generation of Organoids Using MicroTissues 3D Petri Dish
3.3 Generation of Organoids Using the Sphericalplate 5D (Sp5D)
3.4 Aggregation Tracking (Dil/DiO) (See Note 15)
3.5 Viability Assessment
3.6 In Vitro Functional Characterization
3.7 Fixation of the Organoids
3.8 Histological Characterization of Paraffin-Embedded Organoids (See Note 23)
3.9 Whole-Mount Staining of the Organoids in Suspension (See Note 29)
4 Notes
References
Chapter 4: Noninvasive Quantitative PET Imaging in Humans of the Pancreatic Beta-Cell Mass Biomarkers VMAT2 and Dopamine D2/D3...
1 Introduction
2 Materials
2.1 For the Synthesis of Radioligand, Materials Have Been Described in Detail Previously
2.2 To Perform Injection of the Radioligand and Whole Blood, Plasma, and Metabolite Analysis
2.3 For Placement of the Arterial Catheter
2.4 For Acquisition and Analysis of Dynamic PET
3 Methods
3.1 Preparation of the Individual for PET/CT Imaging
3.2 Placement of the Individual on the Bed for PET/CT Scanning
3.3 CT Acquisition
3.4 Radioligand Synthesis and Preparation
3.5 Arterial Input Function and Radioligand Metabolite Measurements
3.6 Radioligand Injection and PET Image Acquisition
3.7 Generation of Region-of-Interest Time-Activity Curves
3.8 Quantification of Pancreas Radioligand Uptake
3.8.1 Kinetic Modeling
3.8.2 Simplified Reference Model Approaches
4 Notes
References
Chapter 5: In Vivo Imaging of Naked and Microencapsulated Islet Cell Transplantation
1 Introduction
2 Materials
2.1 Cells and Animals
2.2 Reagents
2.3 Buffers and Solutions
2.4 Imaging Agents
2.5 Other Materials
2.6 Tools
2.7 Equipment
3 Methods
3.1 Labeling Naked Islet Cells with SPIOs or Perfluorocarbon Emulsions
3.2 Luciferase Transduction of Islet Cells for BLI
3.3 Synthesis and Labeling of Microencapsulated Islet Cells
3.4 Subcutaneous Transplantation of Microencapsulated Cells
3.5 Subcutaneous Transplantation of Naked (Unencapsulated) Islet Cells
3.6 I.p. Transplantation of Naked or Microencapsulated Islet Cells
3.7 1H and 19F MRI of Labeled Naked or Microencapsulated Cells
3.8 CT Imaging of Labeled Naked or Microencapsulated Cells
3.9 US Imaging of Labeled Naked or Microencapsulated Cells
3.10 BLI
4 Notes
References
Chapter 6: Diabetes Retinopathy: New Ways to Detect and Treat
1 Introduction
2 Materials
2.1 Extraction Solvents
2.2 Internal Standards
2.3 Solutions for LC Gradient
2.4 Glass and Plasticware
2.5 Equipment
3 Methods
3.1 Extraction of Sterols from Retina
3.2 Hydrolysis of Cholesteryl Esters
3.3 Liquid Chromatography Gradient
3.4 Mass Spectrometer Setup and Analysis Conditions
3.5 Peak Finding, Analyte Identification, and Quantification
4 Notes
References
Chapter 7: MRI Methods for Imaging Beta-Cell Function in the Rodent Pancreas
1 Introduction
2 Materials
2.1 Animal Holder
2.2 Intravenous Injection of Imaging Agent and Glucose
2.3 Zinc-Responsive MR Contrast Agent
3 Methods
3.1 Preparing Rodents for Imaging
3.2 MR Imaging
3.3 Image Postprocessing and Analysis
4 Notes
References
Chapter 8: MicroRNAs in Type 2 Diabetes: Focus on MicroRNA Profiling in Islets of Langerhans
1 Introduction
1.1 Insulin Secretion and Type 2 Diabetes
1.2 Islet MicroRNAs in Type 2 Diabetes
1.3 Measuring of MiRNA Expression Levels
1.4 Differential Expression Analysis
2 Materials
2.1 Materials/Reagents
2.2 Equipment
3 Methods
3.1 Extraction of Total RNA from Islets (See Note 1)
3.2 Preparation of MiRNA cDNA Libraries (See Notes 1, 3, and 9-15)
3.2.1 Prepare 100 ng RNA Samples
3.2.2 3′ Ligation
3.2.3 5′ Ligation
3.2.4 cDNA Synthesis
3.2.5 cDNA Cleanup (Including QIAseq MiRNA NGS RT Bead Preparation) (See Notes 1, 3, and 20-24)
3.2.6 Library Amplification (See Note 1)
3.3 Small Noncoding RNAseq
3.3.1 Perform MiRNA Library Presequencing QC
3.3.2 Determine Library Concentration and Read Allocation (See Note 29)
3.3.3 Prepare for Illumina NextSeq 500 System Sequencing by Creating a Custom Library Prep Kit for the QIAseq MiRNA Library Ki...
3.3.4 Perform MiRNA Sequencing (See Notes 30 and 31)
3.4 MiRNA Mapping and Quantification
3.5 Tutorial MiRNA Differential Expression Analysis in R (See Note 32)
3.5.1 Package Installation and Loading: Data Downloading
3.5.2 Data Pre-processing for Deseq2
3.5.3 Result Extraction
3.5.4 Result Visualization
4 Notes
References
Chapter 9: Protocol for Clinical GLP-1 Receptor PET/CT Imaging with [68Ga]Ga-NODAGA-Exendin-4
1 Introduction
2 Materials
2.1 Labeling of [68Ga]Ga-NODAGA-Exendin-4
2.1.1 Labeling Using a GRP Synthesizer Module
2.1.2 Labeling Using an MLPT Synthesis Module
2.2 Filtration and Sampling of [68Ga]Ga-NODAGA-Exendin-4
2.3 Quality Control
2.4 PET/CT Imaging with [68Ga]Ga-NODAGA-Exendin-4
3 Method
3.1 Preparation of Radioactive [68Ga]Ga-NODAGA-Exendin-4
3.1.1 Labeling Using a GRP Synthesizer Module
3.1.2 Labeling Using an MLPT Synthesis Module
3.1.3 Filtration and Sampling of [68Ga]Ga-NODAGA-Exendin-4
3.1.4 Quality Control
3.2 PET/CT Imaging with [68Ga]Ga-NODAGA-Exendin-4
4 Notes
References
Chapter 10: Hyperinsulinemic-Euglycemic Clamp in Conscious Rats Based on the Tail Artery and Vein Catheterization
1 Introduction
2 Materials
2.1 Catheterization
2.2 Hyperinsulinemic-Euglycemic Clamp
3 Methods
3.1 Preparation for HEC
3.2 Tail Artery Catheterization
3.3 Tail Vein Catheterization
3.4 Hyperinsulinemic-Euglycemic Clamp
4 Notes
References
11: Synthesis of siRNA-Conjugated Dextran-Coated Iron Oxide Nanoparticles for Islet Protection During Transplantation and Noni...
1 Introduction
2 Materials
3 Methods
3.1 Synthesis of Dextran-Coated Monocrystalline Iron Oxide Nanoparticles
3.2 Cross-Linking and Amination
3.3 Conjugation of Cy5.5 -NHS Ester to MN and Conjugation of siRNA to MN-Cy5.5
3.4 Characterization
4 Notes
References
Chapter 12: Micelle-Based Nanocarriers for Targeted Delivery of Cargo to Pancreas
1 Introduction
2 Materials
3 Methods
3.1 Step 1: Functional Group Activation
3.2 Measurement of the Degree of Activation of Pluronic
3.3 Measurement of Critical Micelle Concentration (CMC) of Activated Pluronic
3.4 Step 2: Quantum Dot Encapsulation in Pluronic F127-pNP (Schematic shown in Fig. 3)
3.5 Step 3: Antibody Conjugation (Schematic shown in Fig. 4)
References
Chapter 13: Magnetic Particle Imaging of Transplanted Human Islets Using a Machine Learning Algorithm
1 Introduction
2 Materials
2.1 Labeling of Human Islets
2.2 Transplanting of Human Islets
2.3 Imaging of Transplanted Islets
2.4 Unsupervised Machine Learning Analysis of MPI Images
3 Methods
3.1 Labeling of Human Islets for Transplant
3.2 Transplanting of Labeled Human Islets
3.3 Imaging of Transplanted Islets
3.4 Unsupervised Machine Learning Analysis of 2D MPI Using K-Means++ Clustering
4 Notes
References
Chapter 14: In Vivo Bioluminescence for the Detection of the Fate of Pancreatic Islet Organoids Post-transplantation
1 Introduction
2 Materials
2.1 Cell Transduction
2.2 BLI Detection of hiPSC/Luc-Expressing hiPSCs
2.3 Immunostaining of hiPSC/Luc-Expressing hiPSCs
2.4 Insulin-Producing Pancreatic Islet Organoid Differentiation
2.5 Kidney Capsule Islet Organoid Transplantation in Mouse Models
2.6 In Vivo BLI Detection of Islet Organoid Grafts Derived from Luc-Expressing hiPSCs
3 Methods
3.1 Cell Transduction
3.1.1 Plasmid Amplification
3.1.2 Purifying Plasmid DNA
3.1.3 Lentivirus Production and Purification
3.1.4 Lentiviral Transduction of hiPSCs
3.2 BLI Detection of Luc-Expressing hiPSCs
3.3 Immunostaining of Luc-Expressing hiPSCs
3.4 Islet Organoid Differentiation
3.5 Islet Organoid Transplantation
3.6 BLI Detection of Transplanted Islet Organoids in a Mouse Model
4 Notes
References
Chapter 15: miRNA Theranostic Nanoparticles Promote Pancreatic Beta Cell Proliferation in Type 1 Diabetes Model
1 Introduction
2 Materials
2.1 Reagents
2.2 Cell Lines and Animals
2.3 Equipment
3 Methods
3.1 Synthesis and Amination of Dextran-Coated Magnetic Nanoparticles
3.2 Conjugation of Fluorescent Probe
3.3 Conjugation of Oligonucleotides
3.4 Evaluation of miRNA Targets In Vitro
3.5 Evaluation of miRNA Targets Ex Vivo
3.6 Assessment of as-Prepared Nanodrugs In Vivo
4 Notes
References
Chapter 16: Isolation and Purification of Human Pancreatic Islets
1 Introduction
2 Materials
2.1 List of Laboratory Specialty Equipment for Human Islet Isolation
2.2 Reagents for Isolation and Purification of Human Islets
2.3 Materials for Testing Islets In Vivo
3 Methods
3.1 Acquisition of the Pancreas
3.2 Trimming and Perfusion of the Pancreas
3.3 Digestion of the Pancreas
3.4 Islet Purification
3.5 Detection of Islets After Purification
3.6 Viability and Function of Purified Islets
4 Notes
References
Chapter 17: Vascularized Islet Transplantation as Composite Islet-Kidney Grafts with Nanoparticle-Labeled Islets in Large Anim...
Abbreviations
1 Introduction
1.1 Strategy to Minimize Ischemic Injury and Promote Tolerance of Islets by Composite Islet-Kidney Grafts
1.2 Advanced Strategy to Minimize Extent of Donor Pancreas Resection Required for Composite I-K Preparation
2 Materials
2.1 Central Line Placement
2.2 Islet-Kidney (I-K) Preparation and Removal of Composite I-K in Donors
2.3 Composite Islet-Kidney Transplantation to Recipients
2.4 Islet Isolation
2.5 Prelabeling Islets In Vitro Followed by Islet Transplantation
2.6 C-Peptide Assay
3 Methods
3.1 Donor Surgical Procedure and Islet Isolation/Culture Including Nanoparticle
3.1.1 Distal Pancreatectomy for Isolation of Islets (Donor Survival Procedure)
3.1.2 Islet Isolation (NHP)
3.1.3 NHP Islet Labeling with MN-Cy5.5-siRNA Probe
3.1.4 Islet-Kidney Preparation (Yamada´s Islet-Kidney Procedure)
3.1.5 Islet-Kidney Procurement
3.2 Composite Islet-Kidney Transplantation (Recipient)
3.3 C-Peptide Assay (NHP)
3.4 Mixed Lymphocyte Reaction (MLR)
4 Notes
References
Index