Preface
Contents
Contributors
Benchtop Bioreactors in Mammalian Cell Culture: Overview and Guidelines
1 Introduction
2 Materials
2.1 Equipment
2.2 Bioreactor
2.3 Cell Culture Media, Feeds, and Supplements
2.4 Antifoam
2.5 Controller
2.6 Probes
2.7 Other Supplements
3 Methods
3.1 Cell Expansion
3.2 Probe Calibration and Sterilization
3.3 Bioreactor Preparation
3.4 Bioreactor Setpoints
3.5 Media Preparation/Hold
3.6 Inoculation
3.7 Fed-Batch (Sampling and Feeding)
3.8 Harvest and Clarification
4 Notes
References
Volumetric Mass Transfer Coefficient Measurement in a Stirred Tank Reactor
1 Introduction
1.1 The Measurement of kLa
1.2 The Unsteady-State (Dynamic) Method
1.3 The Steady-State Method
2 Materials
2.1 Bioreactor
2.2 Microorganism
2.3 Growth Media
2.4 Spectrophotometer
2.5 Chronometer
3 Methods
3.1 Installation of the Bioreactor
3.2 Preparation of the Growth Media
3.3 Preparation of Microorganism
3.4 Operation of the STR
3.5 Measurement of the Oxygen Transfer Coefficient: kLa
4 Notes
References
Fabrication Protocol for Thermoplastic Microfluidic Devices: Nanoliter Volume Bioreactors for Cell Culturing
1 Introduction
2 Materials
3 Methods
3.1 Thermoplastic Chip Design and Preparing Its Mold
3.1.1 Photolithography
3.1.2 Electrochemical Wet Etching
3.2 Fabrication of Electrodes
3.2.1 Creating an Electrode Design
3.2.2 Chromium and Gold Deposition on Blank COP Substrate
3.2.3 Cr/Au Etching to Form Electrodes on Coated COP Sample
3.3 Drilling and Planarization
3.4 Hot Embossing
3.5 Thermo-Compression Bonding
3.6 Establishing the Experiment
3.6.1 Microfluidic Device Without Electrodes
3.6.2 Microfluidic Device with Electrodes
4 Notes
References
Expansion of Human Pluripotent Stem Cells in Stirred Tank Bioreactors
1 Introduction
2 Materials
2.1 L7 TFO2 hPSC Complete Medium
2.2 Passaging Solutions
2.3 Buffers and Reagents
2.4 Tubing
2.5 Bioreactor
3 Methods
3.1 Extracellular Matrix Coating of T-75 and 1-Layer Cell Stack
3.2 hPSC Culture on T-75 Flask
3.3 hPSC Culture on 2D Seed Train
3.4 Microcarrier Coating
3.5 Bioreactor Setup
3.6 Cell Inoculum Introduction
3.7 Cell Culture Medium Perfusion Setup
3.8 Cell Count during Expansion
3.9 Cell Harvest
4 Notes
References
High-Efficiency Differentiation of Human Pluripotent Stem Cells to Hematopoietic Stem/Progenitor Cells in Random Positioning M
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Cell Culture Medium and Reagents
2.3 Bioreactor Equipment
2.4 Immunostaining and Flow Cytometry
2.5 Other Material
3 Methods
3.1 Feeder-Free Expansion of Human PSCs
3.2 Mesoderm Induction from hPSCs
3.3 Generation of Hemogenic Endothelium Progenitor
3.4 Generation of Hematopoietic Progenitor Cells
3.5 Characterization of Differentiated Cells Derived from hESCs in Bioreactor
3.5.1 Immunofluorescence Staining
3.5.2 Flow Cytometry
4 Notes
References
Integrating Human-Induced Pluripotent Stem Cell Expansion Capability and Cardiomyocyte Differentiation Potential in a Microcar
1 Introduction
2 Materials
2.1 General Equipment and Consumables
2.2 Media and Consumables for hiPSC Cultures
2.3 Reagents and Consumables for Microcarrier Cultures
2.4 Media and Small Molecules for CM Differentiation
2.5 Antibodies and Reagents
2.6 Preparation of Extracellular Matrices for hPSCs Cultures
2.7 Preparation of Cell Culture Medium
2.8 Preparation of Small Molecules
2.9 Preparation of Microcarriers
3 Methods
3.1 hiPSC Culture
3.2 Screening for High Cardiac Differentiation Potency hiPSC Lines (Fig. 1)
3.2.1 hiPSC Seeding for CM Differentiation
3.2.2 CM Differentiation (Monolayer Method)
3.2.3 Characterization of hiPSC-Derived CM from Monolayer by Flow Cytometry (Fig. 2)
3.3 Testing of Cell Expansion in Microcarrier Cultures
3.3.1 Preparation of GelTrex-Coated Microcarriers
3.3.2 Seeding Cells into Spinner Flask
3.3.3 Cell Counting and Imaging during Cultivation
3.3.4 Evaluation the Pluripotency of hiPSCs in Spinner Flask Cultures by Flow Cytometry (Fig. 3)
3.4 Cardiac Differentiation in Microcarrier Spinner Cultures (Fig. 4)
4 Notes
References
Chemically Defined, Xeno-Free Expansion of Human Mesenchymal Stem Cells (hMSCs) on Benchtop-Scale Using a Stirred Single-Use B
1 Introduction
1.1 Expansion of hMSCs in Stirred Single-Use Bioreactors
1.2 Chemically Defined, Serum-Free Expansion of hMSCs
1.3 General Procedure for and Results of the Serum- and Xeno-Free Expansion of Human Mesenchymal Stem Cells from Adipose Tissu...
2 Materials
2.1 hASC Inoculum Production in T75-Flasks
2.2 hASC Expansion in Spinner Flasks
2.3 hASC Expansion in the BioBLU
2.4 Process Analytics
3 Methods
3.1 T75-Flask-Based Inoculum Production
3.2 hASC Expansion in CorningΒ΄s Spinner Flasks
3.3 hASC Expansion in EppendorfΒ΄s Instrumented BioBLU 0.3c
3.4 Sampling and Quality Control
4 Notes
References
Large-Scale Expansion of Umbilical Cord Mesenchymal Stem Cells with Microcarrier Tablets in Bioreactor
1 Introduction
2 Materials
2.1 Cell Culture Reagents
2.2 3D TableTrix Microcarrier Tablets
2.3 3D FloTrix Digest Solution
2.4 Cell Quality Assessment-Related Reagents
2.5 Other Reagents
3 Methods
3.1 Setting up 3D FloTrix vivaSPIN Bioreactor on Day 1
3.2 Inoculation on Day 0
3.3 Cell Culture, Medium Replenishment, and Growth Monitoring on Days 1-3
3.4 Cell Harvesting on Day 4
4 Notes
References
Optimized Method to Improve Cell Activity in 3D Scaffolds Under a Dual Real-Time Dynamic Bioreactor System
1 Introduction
2 Materials
2.1 Bioreactor System
3 Methods
3.1 Sample Preparation
3.2 Sample Transfer and Chamber Assembly
3.3 Mechanical Compression Load and Fluid Flow Parameters
4 Notes
References
In Vitro 3D Mechanical Stimulation to Tendon-Derived Stem Cells by Bioreactor
1 Introduction
2 Materials
2.1 Isolation of Mice TDSCs
2.2 Mechanical Stimulation
2.2.1 Scaffold-Free Mechanical Stimulation
2.2.2 Scaffold-Based Mechanical Stimulation
2.3 Extraction of RNA for Validation of the System
3 Methods
3.1 Isolation of Mice TDSCs
3.2 Mechanical Stimulation
3.2.1 Scaffold-Free Mechanical Stimulation
3.2.2 Scaffold-Based Mechanical Stimulation
3.3 Extraction of RNA for Validation of the System
4 Notes
References
Microcarrier-Supported Culture of Chondrocytes in Continuously Rocked Disposable Bioreactor
1 Introduction
2 Materials
2.1 Microcarrier Conditioning
2.2 Preparation of CP5 Cell Inoculum
2.3 Cell Culture
2.4 Analytical Methods
2.4.1 Preparation of Samples for Analysis
2.4.2 Cell Staining
2.4.3 Activity of Intracellular Oxidoreductases
2.4.4 Glucose Consumption Rate
2.4.5 Activity of Lactate Dehydrogenase (LDH)
2.5 General Description of the Setup
2.5.1 ReadyToProcess WAVE 25 Bioreactor System
3 Methods
3.1 Rehydration, Conditioning, and Sterilization of Microcarriers
3.2 Preparation of CP5 Chondrocyte Inoculum
3.3 Maintaining CP5 Cells in Bioreactor System
3.4 Analytical Methods
3.4.1 Preparation of Samples for Analysis
3.4.2 Cell Density and Viability
3.4.3 Metabolic Activity of Cells Adhered to Microcarriers
3.4.4 Specific Glucose Consumption Rate
3.4.5 Activity of Lactate Dehydrogenase
3.4.6 DO and pH Level Measurement
4 Notes
References
Tracheal In Vitro Reconstruction Using a Decellularized Bio-Scaffold in Combination with a Rotating Bioreactor
1 Introduction
2 Materials
2.1 Porcine Trachea Collection
2.2 Generation of the Decellularized Tracheal ECM-Based Porcine Bio-Scaffold
2.3 Human Chondrocyte Propagation and Maintenance
2.4 Repopulation of the Decellularized Tracheal ECM-Based Porcine Bio-Scaffold with Human Chondrocytes and Bioreactor Setup
3 Methods
3.1 Porcine Trachea Collection
3.2 Generation of the Decellularized Tracheal ECM-Based Porcine Bio-Scaffold
3.3 Human Chondrocyte Propagation and Maintenance
3.4 Repopulation of the Decellularized Tracheal ECM-Based Porcine Bio-Scaffold with Human Chondrocytes and Bioreactor Setup
4 Notes
References
Bioreactor-Based De-epithelialization of Long-Segment Tracheal Grafts
1 Introduction
2 Materials
2.1 Bioreactor Main Components (Fig. 1)
2.2 Bioreactor Extra Components
2.3 Other Materials
2.4 Tools
2.5 Devices
2.6 Antibiotics, Media, and Detergents
3 Methods
3.1 Preparation of Antibiotics, Media, and Detergents (See Notes 1 and 2)
3.1.1 Cocktail of Antibiotics
3.1.2 DMEM + Antibiotics
3.1.3 1% Sodium Dodecyl Sulfate
3.1.4 1% Triton X-100
3.2 Graft Procurement and Preparation
3.3 Bioreactor Setup for De-epithelialization
3.3.1 Detergent Circuitries
3.3.2 Bioreactor Setup
3.3.3 Positioning the Trachea
3.3.4 Motor and Pumps Installation
3.4 Tracheal Graft De-epithelialization
3.5 Decontamination of the De-epithelialized Graft
4 Notes
References
Production of Extracellular Vesicles Using a CELLine Adherent Bioreactor Flask
1 Introduction
2 Materials
2.1 Cell Culture
2.2 EV Isolation Materials
2.3 Characterization Materials
3 Methods
3.1 Preparation of CELLine AD 1000 Bioreactor Flask (Figs. 1 and 2)
3.2 Inoculation of Cells (See Note 4)
3.3 Adapting Cells to Long-Term Bioreactor Media (See Note 6)
3.4 Continually Harvesting EVs and Monitoring Shed Cells from the Bioreactor (Fig. 2)
3.5 EV Isolation and Purification (See Note 10)
3.6 Characterizing EVs
3.7 Imaging the Bioreactor Growth Surface
3.8 EV-Associated RNA
4 Notes
References
Extracellular Vesicle Collection from Human Stem Cells Grown in Suspension Bioreactors
1 Introduction
2 Materials
2.1 Materials for hiPSC Differentiation in Planar and Bioreactor Cultures
2.2 Materials for hMSC Expansion in Planar and Bioreactor Cultures
2.3 Bioreactor Preparation
2.4 Materials for EV Isolation from Human Stem Cells Grown in Bioreactor Cultures
3 Methods
3.1 Culture and Expansion of hiPSCs in Planar Culture
3.2 Differentiation of NPC Organoid from hiPSCs in Spinner Flasks
3.3 Expansion of hMSCs in Planar Culture
3.4 Expansion of hMSCs in PBS-VW Bioreactors
3.5 EV Isolation from the Collected Media
4 Notes
References
Bacterial Nanocellulose-Based Grafts for Cell Colonization Studies: An In Vitro Bioreactor Perfusion Model
1 Introduction
2 Materials
2.1 Bioreactor Setup
2.2 Assays and Staining Solutions
2.3 Cell Culture
2.4 Antibodies
2.5 Chemicals and Solutions
2.6 Consumables
2.7 Hardware
3 Methods
3.1 General Considerations
3.2 Bioreactor Setup
3.3 Connection of Small Diameter Vascular Grafts to Bioreactor Chamber
3.4 Cell Seeding
3.5 Lactate Monitoring
3.6 WST-1 Proliferation Assay
3.6.1 Perform WST-1 Assay (Modified from ManufacturerΒ΄s Instructions)
3.7 Termination of Experiment
3.8 AcLDL Uptake Assay (Modified from ManufacturerΒ΄s Instructions)
3.9 Phalloidin-F-Actin Staining
3.10 Acridine Orange Staining
3.11 CD31 Immunofluorescence Staining
4 Notes
References
A Guideline to Set Up Cascaded Continuous Cultivation with E. coli Bl21 (DE3)
1 Introduction
2 Materials
2.1 Host Cells
2.2 Required Media
2.3 Required Devices for Cultivation
2.4 Required Equipment for Process Analysis
2.4.1 Biomass Determination
2.4.2 Viable Biomass Determination
2.4.3 Metabolite Determination
2.4.4 Product Determination
3 Methods
3.1 Cultivation Setup
3.2 Cultivation Scheme
3.2.1 Preculture and Batch Phase
3.2.2 Continuous Adaptation Phase
3.2.3 Induction Phase
3.3 Sampling and Analysis
3.3.1 Biomass Determination
3.3.2 Determination of Metabolite Accumulation
3.3.3 Product Determination
3.4 Calculation of Flow and Substrate Uptake Rates
3.4.1 Calculation of Flow Rates
3.4.2 Calculation of Substrate Uptake Rates
4 Notes
5 General Notes on Process Intensification for Cascaded Continuous Cultivation
5.1 Dilution Rate
5.2 Substrate Uptake Rate
5.3 Cultivation Temperature
References
Applying Stirred Perfusion to 3D Tissue Equivalents to Mimic the Dynamic In Vivo Microenvironment
1 Introduction
2 Materials
2.1 Cell Culture
2.1.1 HepG2 Tissue Model
2.1.2 Intestinal Tissue Model
Stromal Compartment
Epithelial Compartment
2.2 Processing and Analysis of Samples
3 Methods
3.1 Growth of HepG2 Liver Models for Drug Toxicity Testing
3.1.1 Revival of HepG2 Cells
3.1.2 Preparation of Alvetex Strata
3.1.3 Seeding HepG2 Cells onto the Alvetex Strata
3.1.4 Preparation of the Perfusion Bioreactors
3.1.5 Moving HepG2 Models into the Perfusion System
3.2 Creation of Perfused Intestinal Models
3.2.1 Revival of Cells
3.2.2 Preparation of Alvetex Scaffold
3.2.3 Seeding HDFn Cells onto the Alvetex Scaffold
3.2.4 Seeding Caco2 Cells onto the Stromal Compartments
3.2.5 Preparation of the Perfusion Bioreactors
3.2.6 Moving Intestinal Models into the Perfusion System
3.3 Processing and Analysis of the Perfused Models
3.3.1 Processing Samples for Paraffin Wax Embedding
3.3.2 Generation of Transverse Sections of Tissue Models
3.3.3 Histological Analysis
3.3.4 Immunofluorescent Analysis
3.3.5 MTT Assay
4 Notes
References
Bioreactor-Based Adherent Cells Harvesting from Microcarriers with 3D Printed Inertial Microfluidics
1 Introduction
2 Materials
2.1 3D Printing
2.2 PDMS Device Making (in Replacement of Direct-Printed Device)
2.3 Cell Harvesting
3 Methods
3.1 Device Design
3.2 Direct Fabrication
3.3 Mold Fabrication
3.4 Operation
4 Notes
References
Index