Che Connon, is Professor of Tissue Engineering at the Institute of Genetic Medicine, University of Newcastle upon Tyne, UK; he is also a member of the Bioprocessing Research Industry Club (supported by the BBSRC and EPSRC). His research focuses on seeking to engineer functional replacement and temporary 'bridge' tissues using a modular approach while also developing model systems to study physiological and pathophysiological corneal tissue formation. He is the author of Corneal Regenerative Medicine (with Bernice Wright, 2013) and Hydrogels in Cell-Based Therapies (with Ian W Hamley, 2014), as well as numerous research papers.

• List of Contributors xiPreface xv1 Overview of the Cell Therapy Field 1Michael Whitaker, Lucy Foley and Stephen Ward1.1 The Context of Cell Therapies and Their Manufacturing Challenges 11.1.1 Regulation of Cell Therapies 41.1.2 Manufacturing Challenges in Cell Therapy 51.2 The Cell Therapy Landscape 51.2.1 Licensed Cell Therapy Products 71.2.2 Companies, Clinicians, Products and Procedures 81.2.3 Cell Therapy Clinical Trials 81.3 Operations in Cell Therapy Manufacture 111.3.1 Cells for Cell Therapy Production 121.3.1.1 Cell Source 121.4 Upstream Processing of Cellular Therapies 131.4.1 Cell Separation 131.4.2 Cell Expansion 131.4.3 Tissue Expansion 151.4.4 Adherent Cell Expansion 151.4.4.1 Multi-layer Reactors 151.4.4.2 Hollow Fibre Reactors 161.4.4.3 Scaffolds 171.4.5 Suspension Cell Expansion 181.4.5.1 Stirred Tank Bioreactors 181.4.5.2 Rocking Platforms 191.4.5.3 Perfusion Cell Culture 191.4.6 Differentiation 191.5 Downstream Processing of Cellular Therapies 201.5.1 Harvest, Washing and Concentration 201.5.1.1 Centrifugation 211.5.1.2 Filtration 211.5.2 Separation and Purification 221.5.2.1 Centrifugation 221.5.2.2 Magnetic Separation 241.6 Formulation, Fill and Finish of Cellular Therapies 241.6.1 Formulation 251.6.2 Fill and Finish 251.6.3 Preservation and Shipment 261.7 Administration of the Cell Therapy to a Patient 271.8 Cell Therapy Manufacturing Facilities of the Future 281.8.1 Factory of the Future Requirements 311.9 Conclusion 31References 322 Structured Methodology for Process Development in Scalable Stirred Tank Bioreactors Platforms 35Huaqing Wang, Daniel Kehoe, Julie Murrell and Donghui Jing2.1 Introduction 352.2 Understanding the Engineering of the Stirred Tank Bioreactors 362.2.1 Mixing Phenomena in Stirred Tank Bioreactors 372.2.2 Understanding Oxygen Transfer Rate (kLa) with Different Sparging Methodologies 402.2.3 Heat Transfer in STB (Minimum Volume, Sensor/Sensing Control) 422.2.4 How to Choose a Microcarrier for Adherent Cells (hMSCs) 432.3 Understanding the Biology of the Cells in Stirred Tank Bioreactors (STB) 452.3.1 Cell Types (Adherent and Suspension Cells) 452.3.2 Assays for Comparability, Nutrients, Senescence and Doubling Dime 462.4 Process Development of Adherent Cells in STB Platforms 472.4.1 Standard Comparison to Cell Factory 472.4.2 Methodology for Screening of Microcarriers 492.4.3 Process Development in Small-scale Bioreactors (3L) 502.4.3.1 Cell Seeding Density 512.4.3.2 Microcarrier Concentrations (Cells:Bead Ratio) 522.4.3.3 Operation Ranges of pH and Dissolved Oxygen 532.4.3.4 Feeding Strategy 542.4.4 Process Development in Medium-scale Bioreactors (50L) 552.4.5 Case Study for Expansion of Bone Marrow Derived MSCs in Stirred Tank Bioreactors 592.5 Future Directions 60References 613 The Effect of Scale-up on Cell Phenotype: Comparability Testing to Optimize Bioreactor Usage and Manufacturing Strategies 65Jason Hamilton and Bart Vaes3.1 Introduction 653.1.1 Cell Characterization in the Development Path 663.1.2 The MultiStem® Allogeneic Cell Therapy Product: Mechanisms of Benefit and Target Cells Numbers 693.2 Challenges in Cell Product Development 723.2.1 Effect of Large-scale Expansion on Stem Cell Properties 723.2.2 Serum-free and Xeno-free Media Development 743.3 Stem Cell Characterization 753.3.1 ISCT Requirements 753.3.2 Potency Assays 773.3.3 Omics Screens for Therapeutic Stem Cell Characterization 783.4 Next-generation Stem Cell Development 80References 834 The Scale-up of Human Mesenchymal Stem Cell Expansion and Recovery 91Thomas R. J. Heathman, Qasim A. Rafiq, Karen Coopman, Alvin W. Nienow and Christopher J. Hewitt4.1 Introduction 914.2 Scale-up or Scale-out 934.3 Understanding the Small Scale 964.4 Microcarrier Screening 1034.5 Spinner Flask Culture 1084.6 Large-scale Expansion in Conventional Stirred Tank Bioreactors 1114.7 Cell Recovery from Microcarriers 1174.8 Conclusions 120References 1215 Challenges of Scale-up of Cell Separation and Purification Techniques 127Marieke A. Hoeve, Paul A. De Sousa and Nicholas A. Willoughby5.1 Introduction 1275.1.1 Cell Separation for Cell-based Therapeutics 1275.1.2 Separation Methodology Design 1285.1.3 Objective of this Chapter 1285.1.3.1 Cell Yield 1295.1.3.2 Standardisation 1295.1.3.3 Economical viability 1295.2 Scalable Cell Separation for Cell Therapy 1305.2.1 Label Requiring versus Label-free Separation 1305.2.2 Active versus Passive Method 1335.2.3 Isolated Purification (Including Off-the-Shelf) versus Embedded Integrated Process 1335.2.4 Low versus High Resolution 1335.2.5 Open versus Closed Systems 1345.2.6 Batch versus Continuous Separation 1345.3 Currently Developed Cell Separation Techniques 1355.3.1 Acoustophoresis 1355.3.2 Aqueous Two-Phase System (ATPS) 1375.3.3 Centrifugal Techniques 1385.3.3.1 Centrifugal Counterflow Elutriation (CCE) 1385.3.3.2 Centrifuge Systems with Integrated Filters 1395.3.4 Dielectrophoresis (DEP) 1405.3.5 Deterministic Lateral Displacement (DLD) 1415.3.6 Genetic Engineering 1415.3.7 Hydrodynamic Filtration (HDF) 1425.3.8 Immunoadsorption 1425.3.9 Immunomagnetic Cell Sorting 1455.3.10 Inertial Migration 1455.3.11 Magnetic Cell Sorting – Label-free 1475.3.11.1 Magnetophoretic Cell Separation 1475.3.11.2 Magnetic Solution-based Separation 1485.3.12 Microscale Vortices 1505.3.13 Normal Flow Filter (NFF) 1505.3.14 Optical – Label-free 1515.3.15 Tangential Flow Filters (TFF) 1555.3.16 Weir and Pillar 1565.4 Conclusion 157Acknowledgements 159References 1596 Fundamental Points to Consider in the Cryopreservation and Shipment of Cells for Human Application 167Glyn N. Stacey, Lyn Healy, Jennifer Man, Charles J. Hunt and John Morris6.1 Introduction 1676.2 The Role of Cryoprotective Agents (CPA) 1686.3 Vitrification versus Cryopreservation 1696.4 Points to Consider in the Development of Cryopreservation Protocols 1696.4.1 General Considerations 1696.4.2 Cellular Characteristics and Selection of Appropriate CPAs and Cooling Protocols 1706.4.3 Key Events in Cryopreservation 1726.4.3.1 Ice Nucleation 1746.4.3.2 CPAs: Concentration and Composition 1746.4.3.3 Cooling Rate 1746.4.3.4 Storage of Cryopreserved Cells 1766.4.3.5 Thawing and Recovery of Frozen Cells 1776.5 Large-volume Freezing 1786.6 Cryopreservation as Part of Manufacturing Processes 1796.6.1 Containers for Cryopreserved Cells 1796.6.2 Controlled Rate Freezers and Storage Systems 1796.6.3 The Cold-chain: Challenges and Solutions 1806.6.4 Biobanking and Regulatory Requirements 1816.7 Conclusions 181Acknowledgements 182References 1827 Short-term Storage of Cells for Application in Cell-based Therapies 187Stephen Swioklo and Che J. Connon7.1 Introduction 1877.1.1 Advances in Cell-based Therapies 1887.1.2 The Logistical Landscape for CTPs and the Requirement for Short-term Storage of Cells 1887.2 Hypothermia and Mammalian Cell Storage 1937.2.1 Hypothermic Storage of Mesenchymal Stem Cells (MSCs) 1947.2.2 Optimal Temperature for Cell Storage 2027.3 The Application of Hypothermic Storage in Cell-based Therapies 2047.4 Concluding Remarks 205References 2058 Cell Therapy in Practice 211Gustavo S. Figueiredo, Julian R. De Havilland, Majlinda Lako and Francisco C. Figueiredo8.1 Introduction 2118.2 The Classification of ATMPs 2128.3 European Regulations 2168.3.1 Hospital Exemption (HE) and “Specials” Manufacturing 2198.3.2 Orphan Medicinal Product Designation 2208.3.3 Committee for Advanced Therapies 2218.3.4 Good Manufacturing Practice (GMP) 2218.3.5 European Union Tissue and Cells Directives (EUTCD) 2238.4 ATMP Case Study: Autologous Limbal Stem Cell Therapy: the Newcastle Experience 2248.5 Conclusion 233References 234Index 237