Bioreactors in Stem Cell Biology: Methods and Protocols (Methods in Molecular Biology, 1502)

✍ Scribed by Kursad Turksen (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 246
• Edition: 1st ed. 2016
• Category: Library

✦ Synopsis

This meticulous volume recognizes the need to translate what has been learned primarily in tissue culture dishes to approaches supporting scale-up studies, not only to large quantities of cells but also to heterogeneous cell constructs. Notable advances are being made in these latter approaches, prompting this collection of a variety of representative protocols that facilitate important modifications and novel approaches to bioreactors in stem cell research, contributed by both established and new investigators in this area. 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.
Practical and authoritative, Bioreactors in Stem Cell Biology: Methods and Protocols will serve as an ideal guide for scientists seeking to increase our understanding of stem cells and their potential to repair and regenerate tissues and organs.

✦ Table of Contents

Preface
Contents
Contributors
Multicompartmental Hollow-Fiber-Based Bioreactors for Dynamic Three-Dimensional Perfusion Culture
1 Introduction
2 Materials
3 Methods
3.1 Setup of a 4-Chamber Analytic Scale Bioreactor
3.1.1 Connecting the Tubing System for Culture Medium Recirculation
3.1.2 Connecting the Tubing System for Mixed Gas Perfusion
3.1.3 Final Preparations and Inspections
3.2 Pre-culture Bioreactor Perfusion System Preparation
3.2.1 General Bioreactor Perfusion System Preparations
3.2.2 Mixed Gas Supply Assembly
3.2.3 Mixed Gas Supply Settings and Adjustment
3.2.4 Final Preparations
3.2.5 Filling the System with Medium: General Preparations
3.2.6 Filling of the Tubing System
3.2.7 Filling of the Cell Compartment and Capillary Membrane Bundles
3.2.8 Initial Recirculation
3.3 Bioreactor Perfusion System Cell Injection
3.3.1 Pre-cell Injection Culture Management
3.3.2 Pre-cell Injection Air Removal Process
3.3.3 Cell Injection Procedure
3.4 Bioreactor Perfusion System Culture Management
3.4.1 Culture Management
3.4.2 Air Removal/Cell Compartment Flush Procedure
4 Notes
References
A Bioreactor to Apply Multimodal Physical Stimuli to Cultured Cells
1 Introduction
2 Materials
2.1 List of Materials and Equipment
2.1.1 Experimental Setup
2.1.2 Data Acquisition
2.2 General Description of the Setup
2.3 Stretching
2.4 Pacing
3 Methods
3.1 PDMS Membrane Preparation
3.1.1 Molding
3.1.2 Sterilization
3.1.3 Coating
3.2 Bioreactor Construction
3.3 Improvements from the Original Design
4 Notes
References
Aggregate and Microcarrier Cultures of Human Pluripotent Stem Cells in Stirred-Suspension Systems
1 Introduction
2 Materials
2.1 Static Culture of Pluripotent Stem Cells Using Vitronectin or Matrigel
2.2 Microcarrier Culture of Stem Cells in Spinner Flasks
2.3 Differentiation to Definitive Endoderm and Mesoderm Cells in Spinner Flask Cultures
2.4 Characterization
2.4.1 Total RNA Isolation, Reverse Transcription, and Quantitative PCR
2.4.2 Flow Cytometry
2.4.3 Immunohistochemistry
2.4.4 Karyotyping
2.4.5 Differentiation into Mesoderm
2.4.6 Differentiation to Ectoderm Cells
2.5 Antibodies
3 Methods
3.1 Static (Dish) Culture
3.2 Stirred-Suspension Culture in Spinner Flasks
3.2.1 Microcarrier Culture Coated with Vitronectin, HSA, and UV Radiation
3.2.2 Microcarrier Culture Using Matrigel
3.3 Aggregate Culture of hPSCs in Spinner Flasks
3.4 Differentiation to Definitive Endoderm and Mesoderm Cells
3.5 Characterization of Cultured Cells
3.5.1 Reverse Transcription-Quantitative PCR
3.5.2 Flow Cytometry
3.5.3 Immunohistochemistry
3.5.4 Karyotyping
3.5.5 Differentiation of Harvested Cells into Mesoderm
3.5.6 Differentiation of Harvested Cells into Ectoderm
4 Notes
References
Expansion of Human Induced Pluripotent Stem Cells in Stirred Suspension Bioreactors
1 Introduction
2 Materials
2.1 hiPSC Culture Reagents
2.2 Cell Counting and Viability Assessment
2.3 Pluripotency Assessment
2.4 Anaerobic Energy Metabolism Assessment
2.5 Suspension Bioreactor Materials
3 Methods
3.1 Preparation of hiPSCs in Static Culture
3.2 Transferring hiPSCs from Static to Suspension Culture
3.3 Culturing hiPSCs in Suspension
3.4 Passaging of hiPSCs in Suspension
3.5 Expansion Rate Calculation
3.6 Assessment of Pluripotency and Anaerobic Energy Metabolism
4 Notes
References
Uniform Embryoid Body Production and Enhanced Mesendoderm Differentiation with Murine Embryonic Stem Cells in a Rotary Suspens
1 Introduction
2 Materials
2.1 Cell and Cell Lines
2.2 Medium and Supplements for Cell Culture
2.3 Bioreactor Equipment
2.4 Real-Time PCR
2.5 Immunostaining
3 Methods
3.1 Cultivation of mESCs on Inactivated MEF
3.2 RCCS Preparation Before Cell Culture Initiation
3.3 Single Cell Suspension Preparation for EB Induction in RCCS
3.4 EB Induction and Differentiation in RCCS Bioreactor
3.5 Analysis of EBs Formed in RCCS: Real-Time PCR Detection
3.6 Analysis of EBs Formed in RCCS: Preparation of Slides and Immunostaining
4 Notes
References
Expansion of Human Mesenchymal Stem Cells in a Microcarrier Bioreactor
1 Introduction
2 Materials
2.1 Preparing the Media
2.2 Preparing MSCs for Seeding in SFB
2.3 Preparing the Spinner Flask
2.4 Preparing the Microcarriers
2.5 Preparing the Sterile Culture System
2.6 Preparing MTT Assay
3 Methods
3.1 Seeding BMSCs into the SFB
3.2 Bioreactor Operation and Sample Collection
3.2.1 Day 1
3.2.2 Day 3
3.2.3 Day 5
3.2.4 Day 7
3.3 MTT Measurement
3.4 MTT Staining
3.5 Glucose and Lactate Analysis
4 Notes
References
Large-Scale Expansion and Differentiation of Mesenchymal Stem Cells in Microcarrier-Based Stirred Bioreactors
1 Introduction
2 Materials
3 Methods
3.1 MSC Extraction, Subculture, and Minimal Characterization
3.1.1 MSC Isolation from Tissue Explants
3.1.2 MSCs from a Cell Bank
3.1.3 MSC Subculture in Monolayer
3.2 Characterization of MSC Population
3.2.1 Analysis of Membrane Marker Expression
3.2.2 MSC Differentiation
3.2.3 Quantification of MSC Differentiation
3.2.4 Additional Characterization of the MSC Population
3.3 Spinner Flasks and Microcarrier Preparation: MSC Seeding in Spinner Flasks
3.3.1 Microcarrier (MC) Preparation
3.3.2 Spinner Flasks (SFs) Preparation
3.3.3 MSC Seeding on MCs in SF
3.4 Monitoring MSC Adhesion, Proliferation, and Phenotype in MC-Based Bioreactor
3.4.1 Monitoring MSC Adhesion on MCs
3.4.2 Monitoring Cell Proliferation and Metabolic Status in SFs
3.4.3 MSC Feeding in SFs
3.5 Large-Scale MSC Recovery from Microcarriers
3.6 MSC Differentiation in MC-Based Bioreactors
3.7 Up-Scaling MSC Culture in Stirred Bioreactors
4 Notes
References
Bioreactor Expansion of Skin-Derived Precursor Schwann Cells
1 Introduction
2 Materials
2.1 Medium Preparation
2.2 Bioreactor Culture
3 Methods
3.1 SKP-SC Medium
3.2 Siliconization of Flasks and Reactors
3.3 Microcarrier Preparation
3.3.1 Hydration
3.3.2 Sterilization
3.4 Bioreactor Set-Up
3.5 Bioreactor Inoculation and Culture
3.6 Bioreactor Sampling
3.6.1 Sampling for Counting
3.6.2 Sampling for Photomicrographs
3.7 Harvesting Cells from Bioreactors
4 Notes
References
Use of Stirred Suspension Bioreactors for Male Germ Cell Enrichment
1 Introduction
2 Materials
2.1 Testes Tissue Enzymatic Digestion Reagents
2.2 Stirred Suspension Bioreactor Culture
2.3 Stirred Suspension Bioreactor Followed by Differential Plating
3 Methods
3.1 Testes Tissue Enzymatic Digestion
3.2 Stirred Suspension Bioreactor Culture
3.3 Stirred Suspension Bioreactor Followed by Differential Plating
4 Notes
References
Generation of Neural Progenitor Spheres from Human Pluripotent Stem Cells in a Suspension Bioreactor
1 Introduction
2 Materials
2.1 Materials for hiPSC Expansion to Seed Bioreactor Culture
2.2 Materials for hiPSC Differentiation into NPCs in a Spinner Bioreactor
3 Methods
3.1 Expansion of Undifferentiated hiPSCs
3.1.1 Thaw the Vial of hiPSC from a Cell Bank
3.1.2 Undifferentiated hiPSC Culture
3.2 Differentiation of NPC Spheres from hiPSCs in a Suspension Bioreactor
3.2.1 Start the Suspension Culture with Single-Cell Inoculation on Day 0
3.2.2 NPC Sphere Formation from hiPSCs in a Spinner Bioreactor During Days 1-7
3.2.3 HiPSC-Derived NPC Sphere Culture in a Spinner Bioreactor During Days 7-20
3.2.4 Further Differentiation of hiPSC-Derived NPC Spheres After Day 20
4 Notes
References
Perfusion Stirred-Tank Bioreactors for 3D Differentiation of Human Neural Stem Cells
1 Introduction
2 Materials
2.1 General Equipment
2.2 Cell Culture and Characterization Supplies
2.3 Bioreactor and Related Equipment
2.4 Cell Culture Medium
2.5 Biological Material
3 Methods
3.1 STB Assembly and Preparation
3.2 Bioreactor Inoculation
3.3 Initiating Perfusion Mode
3.4 Bioreactor Disassembly
3.5 Culture Characterization
3.5.1 Cell Aggregation Monitoring
3.5.2 Cell Viability
4 Notes
References
Scalable Expansion of Human Pluripotent Stem Cell-Derived Neural Progenitors in Stirred Suspension Bioreactor Under Xeno-fr
1 Introduction
2 Materials
2.1 Chemicals
2.2 Disposables
2.3 Equipment
2.4 Reagent Setup
3 Methods
3.1 Thawing and Expansion of hNPCs in Adherent Culture Conditions
3.2 Passage of hNPCs in the Adherent Culture Condition
3.3 Transfer from the Static Suspension Culture to a Dynamic Suspension Culture System
3.3.1 Transfer from an Adherent Culture to a Static Suspension Culture System
3.3.2 Procedure for the Preparation of Glass Spinner Flask Before Use or at the End of Each Run
3.3.3 Transfer from Static Suspension Culture to Dynamic Suspension Culture in the Spinner Flask
3.3.4 Passaging hNPCs in the Dynamic Suspension Culture
3.4 Freezing of hNPCs Generated in the Adherent and Dynamic Culture Systems
3.5 hNPC Colony-Forming Assay
3.6 Anticipated Results
4 Notes
References
A Microfluidic Bioreactor for Toxicity Testing of Stem Cell Derived 3D Cardiac Bodies
1 Introduction
2 Materials
2.1 Photolithography
2.2 Soft Lithography
2.3 Cell Culture
2.3.1 Production of Cardiac Bodies
2.3.2 Cardiac Body Monitoring in a Microfluidic Bioreactor
3 Methods
3.1 Master Fabrication by Photolithography
3.2 Microfluidic Bioreactor Fabrication by Soft Lithography
3.3 Beating Frequency Monitoring of 3D CBs in a Microfluidic Bioreactor
4 Notes
References
Novel Bioreactor Platform for Scalable Cardiomyogenic Differentiation from Pluripotent Stem Cell-Derived Embryoid Bodies
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Chemicals and Culture-Grade Reagents
2.3 Disposables
2.4 Equipment
2.5 Reagent Setup
3 Methods (Fig.1)
3.1 Mouse PSC Lines Preparation (Day-7-Day 0)
3.2 STLV Bioreactor System Preparation and Sterilization (Day-3)
3.3 Culture Dish and Plate Preparation
3.4 EB Formation in the STLV Bioreactor (Day 0)
3.4.1 Passaging Mouse PSC Lines
3.4.2 Seeding mPSC Lines in STLV Bioreactor
3.5 Medium Renewal (Day 2)
3.6 Cardiomyogenic Differentiation (Day 3-21)
4 Notes
References
Whole-Heart Construct Cultivation Under 3D Mechanical Stimulation of the Left Ventricle
1 Introduction
2 Materials
2.1 Recellularization of the Coronary Vascular System
2.1.1 Recellularization
2.1.2 Solutions
2.1.3 Cell Population
2.2 Recellularization of the Stroma
2.2.1 Recellularization
2.2.2 Solutions
2.2.3 Cell Population
2.3 Bioreactor Hardware
2.3.1 Processing Flask (as Depicted in Fig.1)
2.3.2 Tubing System
2.3.3 Stimulation System
2.3.4 Perfusion System
2.3.5 Process Control/Field Control Level
2.3.6 Process Control System
2.4 Cellular Viability Assay
2.5 Staining of Nucleic Acids and Cytoskeleton
3 Methods
3.1 Acellular Whole-Heart Scaffold
3.2 Repopulation of the Whole-Heart Scaffold
3.2.1 Recellularization of the Coronary Vascular System
3.2.2 Recellularization of the Stroma
3.3 Preparation of the Hardware System
3.4 Setting the Control System
3.4.1 Setting of Stimulation Control
3.4.2 Setting of the Perfusion Control
3.5 Estimation of Cellular Viability
3.6 Determination of Repopulation and Cellular Constitution in the ECM
3.7 Quantification of Cellular Alignment
4 Notes
References
Tendon Differentiation on Decellularized Extracellular Matrix Under Cyclic Loading
1 Introduction
2 Materials
2.1 Tendon Decellularization
2.2 Bioreactor Culture
2.3 Sample Processing
3 Methods
3.1 Tendon Decellularization
3.1.1 Phase 1: Collection Day
3.1.2 Phase 2: Dermatome Day
3.1.3 Phase 3: Detergent Decellularization
3.2 Bioreactor Culture
3.3 Sample Processing
4 Notes
References
Bioengineered Models of Solid Human Tumors for Cancer Research
1 Introduction
2 Materials
2.1 Decellularized Bone Scaffold (Fig.2b)
2.2 Cells
2.3 Culture Media
2.4 PDMS Rings
2.5 TE-Tumor
2.6 Bioreactor: Design and Parts
2.7 Bioreactor: Mathematical Modeling
3 Methods
3.1 TE-Bone
3.2 Cancer Cell Aggregates (Fig.2e)
3.3 PDMS Rings
3.4 TE-Tumor (Fig.2c, f)
3.5 Bioreactor Stimulation (Fig.2g)
4 Notes
References
Development of a Bladder Bioreactor for Tissue Engineering in Urology
1 Introduction
2 Materials
2.1 Construction of Bioreactor
2.2 Culture and Expansion of UCs
2.3 Measurement of Urothelial Cell Viability
2.4 Scanning Electron Microscopy
2.5 4-6-Diamidino-2-phenylindole (DAPI) Staining
2.6 Measurement of Fluorescence Activity
3 Methods
3.1 Bioreactor and Physiological Intravesical Pressures
3.2 Urothelial Cell-Seeding Techniques in Control and Experimental Group
3.3 Measurement of Urothelial Cell Viability
3.4 Scanning Electron Microscopy
3.5 4-6-Diamidino-2-phenylindole Staining
3.6 Measurement of Fluorescence Activity
4 Notes
References
Use of Microfluidic Technology to Monitor the Differentiation and Migration of Human ESC-Derived Neural Cells
1 Introduction
2 Materials
2.1 Microfluidic Devices
2.2 Cell Lines
2.3 Preparation of MMC Treated-MEF Medium
2.4 Preparation of Human ESCs Medium
2.5 Preparation of PA6 Stromal Cell Medium
2.6 Neural Differentiation Medium
2.7 Immunocytochemistry
3 Methods
3.1 Preparation of Microfluidic Culture Platform
3.1.1 PDMS Mixing
3.1.2 Sample and Cover Glass Cleaning
3.1.3 Bonding Process
3.1.4 PLO/FN Double-Layer Coating
3.2 Human ESC Culture and Maintenance
3.2.1 Preparation of Mitomycin C (MMC)-Treated MEF Cells
3.2.2 Maintenance and Subculture of Human ESCs
3.3 Neural Differentiation by Coculturing with PA6 Stromal Cells
3.3.1 Prepare of PA6 Stromal Feeder Cells
3.3.2 Neural Differentiation of Human ESCs
3.4 Monitoring of Neurite Outgrowth in Microfluidic Devices
3.5 Immunocytochemistry
4 Notes
References
Chapter 20: Erratum to: Bioengineered Models of Solid Human Tumors for Cancer Research
Index