Stem Cells and Good Manufacturing Practices: Methods, Protocols, and Regulations

Description

This detailed book explores the utilization and delivery of stem cells for therapeutic purposes in patients in the clinic and the tightly controlled Good Manufacturing Practices (GMP) that make these powerful techniques possible. Fully updated and expanded from the first edition, this collection features a new set of protocols to arm stem cell biologists with protocols that are currently being used in a number of well-established facilities around the world. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and up to date, Stem Cells and Good Manufacturing Practices: Methods, Protocols, and Regulations, Second Edition serves as an ideal guide for researchers aiming to drive continued improvements to GMP and accelerate new stem cell treatments for patients in need. Preface Contents Contributors Swiss Fetal Transplantation Program and Non-enzymatically Isolated Primary Progenitor Cell Types for Regenerative Medicine Abbreviations 1 Introduction 2 Materials 2.1 Biological Starting Materials 2.2 Raw Materials 2.3 Ancillary Materials 2.4 Contact Process Consumables 2.5 Non-contact Process Consumables 2.6 Product Container System Components 2.7 Critical Process Facility and Equipment 3 Methods 3.1 Generalities About Methods 3.2 Transplantation Program Organigram Setup 3.3 Validation of the Transplantation Program 3.4 Donor Population Screening 3.5 Identified Donor Consent and Screening 3.6 Donation Procurement 3.7 Donation Processing 3.8 Media Preparation 3.9 Non-enzymatic Primary Cell Isolation 3.10 Parental Cell Bank Establishment 3.11 Parental Cell Bank Testing 4 Notes References GMP Tiered Cell Banking of Non-enzymatically Isolated Dermal Progenitor Fibroblasts for Allogenic Regenerative Medicine Abbreviations 1 Introduction 2 Materials 2.1 Biological Starting Materials 2.2 Raw Materials 2.3 Ancillary Materials 2.4 Contact Process Consumables 2.5 Non-contact Process Consumables 2.6 Product Container System Components 2.7 Critical Process Facility and Equipment 3 Methods 3.1 Generalities About Methods 3.2 Complete Medium Preparation 3.3 Cryopreservation Medium Preparation 3.4 Pilot Study and Cell Type Validation 3.5 Master Cell Bank Establishment: Definition 3.6 MCB: Initiation from Liquid Nitrogen Storage 3.7 MCB: Maintenance of Cultures and Medium Exchanges 3.8 MCB: Harvest and Cryopreservation 3.9 Working Cell Bank Establishment 3.10 End of Production Cell Bank Establishment 3.11 Cell Recovery and Viability Studies 3.12 Product Release Testing 3.13 Product Characterization Testing 3.14 Safety Testing 3.15 Stability Testing of Cryopreserved Cell Banks 3.16 Banking Campaigns for Clinical Lot Productions 3.17 Production Lot Validation and Clinical Release 4 Notes References Progenitor Biological Bandages: An Authentic Swiss Tool for Safe Therapeutic Management of Burns, Ulcers, and Donor Site Graft Abbreviations 1 Introduction 2 Materials 2.1 Biological Starting Materials 2.2 Raw Materials 2.3 Ancillary Materials 2.4 Contact Process Consumables 2.5 Noncontact Process Consumables 2.6 Critical Process Facility and Equipment 3 Methods 3.1 Generalities About Methods 3.2 Media Preparation 3.3 Clinical Assessment and PBB Requirement Evaluation 3.4 Thawing of Cryopreserved Progenitor Fibroblasts 3.5 PBB Scaffold Preconditioning and Cell Seeding 3.6 PBB Construct Lot Preparation and Liberation 3.7 Clinical PBB Construct Application 3.8 PBB Construct Exchanges and Course of Treatment 3.9 Clinical Endpoints, Expected Therapeutic, and Adverse Effects 4 Notes References Validation of the Media Fill Method for Solid Tissue Processing in Good Manufacturing Practice-Compliant Cell Production 1 Introduction 2 Materials 2.1 Materials and Reagents 2.2 Equipment and Apparatus 3 Methods 3.1 Preparation of Process Simulation Group 3.2 Preparation of Positive and Negative Control Groups 3.3 Preparation of McFarland Concentration 3.4 Acceptance Criteria 3.5 Data Analyses 4 Notes References Isolation, Culture, Cryopreservation, and Preparation of Umbilical Cord-Derived Mesenchymal Stem Cells as a Final Cellular Pro 1 Introduction 2 Materials 2.1 Equipment 2.2 Reagents and Consumables 3 Methods 3.1 Isolation of Mesenchymal Stem Cells Derived from Human Umbilical Cord 3.1.1 Enzymatic Isolation of Mesenchymal Stem Cells Derived from Human Umbilical Cord 3.1.2 Mechanical Isolation (Explant Culture) of Mesenchymal Stem Cells Derived from Human Umbilical Cord 3.2 Subculturing of Mesenchymal Stem Cells 3.3 Cryopreservation of Subcultured Mesenchymal Stem Cells 3.4 Thawing Cryopreserved Mesenchymal Stem Cells 3.5 Preparation of Mesenchymal Stem Cells as a Final Product 4 Notes References Isolation, Culture, Cryopreservation, and Preparation of Skin-Derived Fibroblasts as a Final Cellular Product Under Good Manuf 1 Introduction 2 Materials 2.1 Equipment 2.2 Reagents and Consumables 3 Methods 3.1 Enzymatic Isolation of Fibroblasts Derived from Human Skin 3.2 Subculturing of Skin-Derived Fibroblasts 3.3 Cryopreservation of Subcultured Fibroblasts 3.4 Thawing Cryopreserved Fibroblasts 3.5 Preparation of Fibroblasts as a Final Product 4 Notes References The Isolation and Manufacture of GMP-Grade Bone Marrow Stromal Cells from Bone Specimens 1 Introduction 2 Materials 2.1 Solutions 2.2 Equipment and Supplies 2.3 Bone Specimens 3 Methods 3.1 Preparation of Bone Marrow Cell Suspensions 3.2 BMSCs Isolation 3.3 BMSCs Expansion 3.4 Cryopreservation 4 Notes References Scalable Manufacturing of Human Hematopoietic Stem/Progenitor Cells Exploiting a Co-culture Platform with Mesenchymal Stromal 1 Introduction 2 Materials 2.1 Cells 2.2 Solutions 2.3 Equipment and Supplies 3 Methods 3.1 BM MSC Thawing and Expansion Under Static Conditions 3.2 Expansion of BM MSC in the Spinner Flask 3.3 Monitoring the MSC Culture in the Spinner Flask 3.3.1 Cell Count and Viability 3.3.2 Metabolite Analysis 3.3.3 Cell Distribution on Microcarriers 3.3.4 Immunophenotypic Analysis of Expanded MSC 3.4 Isolation of UCB-Derived CD34+ HSPC 3.5 Ex Vivo Expansion of CD34+ HSPC Under Dynamic Conditions 3.6 Monitoring the HPSC Culture in the Spinner Flask 3.6.1 Cell Count and Viability 3.6.2 Colony-Forming Unit (CFU) Assay 3.6.3 Cobblestone Area-Forming Cell (CAFC) Assay 3.6.4 Immunophenotypic Analysis of HSPC Expanded Under Dynamic Conditions 4 Notes References GMP-Compatible, Xeno-Free Culture of Human Induced Mesenchymal Stem Cells 1 Introduction 2 Materials 2.1 Cells 2.2 Xeno-Free Medium 2.3 GMP-Compatible Medium 2.4 General Cell Culture Reagents and Appliances 2.5 Cell Passaging 2.6 Cell Storage 3 Methods 3.1 Before Starting 3.2 Medium Preparation 3.3 Thawing Cells 3.4 Counting Cells 3.5 Plating Cells 3.6 Passaging Cells 3.7 Storing Cells 3.8 Cell Monitoring and Quality Control of Culture 4 Notes References GMP-Grade Methods for Cardiac Progenitor Cells: Cell Bank Production and Quality Control 1 Introduction 2 Materials 2.1 Master Cell Bank Production 2.1.1 Cardiac Tissue Manipulation 2.1.2 CPC Isolation and Culture 2.1.3 CPC Freezing 2.2 Master Cell Bank Quality Control 2.2.1 Thawing of Cell Samples for QC 2.2.2 Microbiological Control of Cellular Product 2.2.3 Endotoxin Assay 2.2.4 Mycoplasma Assay by NAT 2.2.5 Expression Analysis of Marker Genes 2.2.6 Cell Count and Viability 2.2.7 Identity Assay (Immunophenotype) 2.2.8 In Vitro Transformation Assay 2.2.9 In Vitro Senescence Assay 3 Methods 3.1 Master Cell Bank Production 3.1.1 Cardiac Tissue Manipulation 3.1.2 CPC Isolation and Culture 3.1.3 Master Cell Bank Freezing 3.1.4 Post Production Cell Bank Freezing 3.2 Master Cell Bank Quality Control 3.2.1 Thawing of Cell Samples for QC Controls 3.2.2 Microbiological Control of Cellular Product Procedure 3.2.3 Endotoxin Assay Procedure Acceptance Criteria 3.2.4 Mycoplasma Assay by NAT Sample Preparation PCR Mix Preparation PCR Reaction Assembly Agarose Gel Analysis Acceptance Criteria 3.2.5 Expression Study of Marker Genes Sample Collection and Preparation RNA Extraction and Quantification RNA Retrotranscription Gene Amplification Agarose Gel Analysis Acceptance Criteria 3.2.6 Cell Count and Viability Procedure Acceptance Criteria 3.2.7 Identity Assay (Immunophenotype) Procedure Acceptance Criteria 3.2.8 In Vitro Transformation Assay Preparation of Reagents Preparation of Base Agar Layer Preparation of Cell Agar Layer Quantitation of Anchorage-Independent Growth 3.2.9 In Vitro Senescence Assay Preparing Cell Suspension Loading Cells with FDG by Hypotonic Shock Flow Cytometer Set-Up and Calibration 4 Notes References Suspension Culture of Human Induced Pluripotent Stem Cells in Single-Use Vertical-Wheel Bioreactors Using Aggregate and 1 Introduction 2 Materials 2.1 Laboratory Equipment 2.2 Culture Media and Solutions 2.3 Dynamic Expansion of hiPSCs as Aggregates in PBS VWBRs 2.4 Dynamic Expansion of hiPSCs on Microcarriers in PBS VWBRs 3 Methods 3.1 Static Culture 3.2 Dynamic Expansion of hiPSCs as Aggregates in PBS VWBRs 3.2.1 Cell Seeding and Bioreactor Culture 3.2.2 Sampling and Cell Counts 3.2.3 Cell Harvest at the End of Culture 3.3 Dynamic Expansion of hiPSCs on Microcarriers in PBS VWBRs 3.3.1 Microcarrier Preparation 3.3.2 Cell Seeding and Bioreactor Culture 3.3.3 Sampling and Cell Counts 3.3.4 Cell Harvest at the End of Culture 4 Notes References Addressing Manufacturing Challenges for Commercialization of iPSC-Based Therapies 1 Introduction: Industrialization of Human Pluripotent Stem Cell-Based Therapies 2 Development of a Human iPSC Manufacturing Process Under Current Good Manufacturing Practices 2.1 Establishing an iPSC Generation Process Using a Non-integrating, Episomal-Based Technology 2.1.1 Tissue Acquisition 2.1.2 Reprogramming and Cell Culture 2.2 Performing Process Optimization and Protocol Development Based on Meeting Critical Quality Attributes of the Target Cells 2.3 Technology Transfer of the Optimized Manufacturing Process to the cGMP Manufacturing Suite 3 Analytical Methods and Characterization iPSC During the Manufacturing Process 4 Long-Term Stability of iPSC Master Cell Banks (MCBs) and Working Cell Banks (WCBs) 5 Scale-Up of iPSC Expansion and Differentiation in 3D Bioreactors References Standard Operating Procedure for the Good Manufacturing Practice-Compliant Production of Human Endometrial Stem Cells for Mult Abbreviations 1 Introduction 2 Materials 2.1 Human Endometrial Tissue Procurement 2.2 Endometrial Tissue Harvesting, Endometrial Stem Cell Isolation, Culture, Subculture, Cryopreservation, and Banking 2.2.1 Raw Materials 2.2.2 Equipment 3 Methods 3.1 Human Endometrial Tissue Procurement 3.2 Controls Prior to Endometrial Tissue Processing 3.3 Endometrial Tissue Preparation and Endometrial Stem Cell Isolation 3.4 Endometrial Stem Cell Culture and Subcultures 3.5 Characterization of Cultured Endometrial Stem Cell 3.6 Cryopreservation and Banking 4 Notes References GMP-Compliant Production of Human Placenta-Derived Mesenchymal Stem Cells Abbreviations 1 Introduction 2 Materials 2.1 Human Placenta Tissue Procurement 2.2 Placenta Tissue Harvesting, PLMSCs Manufacturing, and Banking 3 Methods 3.1 Human Placenta Tissue Procurement 3.2 Controls Prior to Placenta Tissue Processing 3.3 Placenta Tissue Processing and MSCs Isolation 3.4 PLMSCs Manufacturing 3.5 Characterization of PLMSCs 3.6 Cryopreservation and Banking 4 Notes References GMP-Compliant Human Schwann Cell Manufacturing for Clinical Application Abbreviations 1 Introduction 2 Materials 2.1 Human Peripheral Nerve Procurement 2.2 Human Schwann Cell Isolation, Culture, Subculture, Cryopreservation, and Banking 3 Methods 3.1 Peripheral Nerve Samples Preparation 3.2 Controls Prior to Processing and Schwann Cell Isolation 3.3 Human Schwann Cell Isolation and Culture 3.4 Schwann Cell Subculture 3.5 Characterization of Schwann Cells 3.6 Cryopreservation and Banking for Clinical Transplantation 4 Notes References GMP-Compliant Adenoviral Vectors for Gene Therapy Abbreviations 1 Introduction 2 Materials 2.1 Reagents 2.2 Laboratory Supplies 3 Methods 3.1 Producing Infectious Plasmids 3.2 Cell Procurement 3.2.1 Cell Culture for Transfection 3.2.2 293 Cells Suspension Preparation 3.3 Transfection for Rescue of Ad Vectors 3.4 Screening of Plaque Isolated Ad Vector 3.5 Ad Vectors Preparation 3.5.1 Preparation Using Adherent 293 Cells 3.5.2 Preparation Using Suspension Adapted 293 Cells 3.6 Purification of Vector 3.7 Characterization of Purified vectors 4 Notes References Use of Multipotent Mesenchymal Stromal Cells, Fibrin, and Scaffolds in the Production of Clinical Grade Bone Tissue Engineerin 1 Introduction 2 Materials 2.1 Equipment 2.2 Reagents/Solutions 2.3 Kits and Consumables 3 Methods 3.1 Human Multipotent Mesenchymal Stromal Cells 3.1.1 Cell Thawing 3.1.2 Cell Washing and Concentration 3.2 Manufacture of Combined Products with Colonization 3.2.1 Colonization of Bony Scaffolds 3.2.2 Incubation (Optional) 3.2.3 Washing 3.2.4 Final Conditioning 3.3 Combined Products with Cells in Suspension 3.3.1 Preparation of the Cellular Component 3.3.2 Preparation of 1% (v/v) Thrombin Solution 3.3.3 Preparation of the Final Product, Storage, and Release 3.4 Analytical Methods 3.4.1 Product Release 3.4.2 Further Analyses 4 Notes References Quality Management Systems (QMSs) of Human-Based Tissue and Cell Product Manufacturing Facilities 1 Introduction 2 The Leading Current Good Practices (cGxPs) in Biopharmaceutical Manufacturing 3 The Essentials of a Proper Biopharmaceutical Quality Management System (QMS) 3.1 Key Personnel 3.2 Quality Procedures 3.2.1 Quality Assurance (QA) 3.2.2 Quality Control (QC) 3.3 Process Validation 3.4 Risk Management 3.4.1 Risk Analysis and Risk Assessment 3.4.2 Corrective and Preventive Actions (CAPAs) 3.5 Donor Eligibility Assessment 3.6 Coding, Traceability, and Product Labeling 3.7 Storage and Distribution 3.8 Biovigilance, Product Recalls, and End User Complaints 4 Strategic Planning in Aid of a High-Performance QMS 5 The QMS Documentation Ladder 6 Notes References Index