Cancer Cell Culture: Methods and Protocols (Methods in Molecular Biology, 2645)

Description

This volume explores the latest collection of cell models that are used in preclinical cancer research, and covers both two-dimensional and three-dimensional culturing techniques. The chapters in this book are divided into two parts. Part One discusses two-dimensional cancer cell culture, cell models at the Air-Liquid Interface, and the latest advancements in three-dimensional complex spheroid models and dedicated disease animal models. Part Two contains technical chapters that illustrate step-by-step methodologies for specific cancer cell culture methods. The methods discussed range from the generation of isogenic cancer cell lines, the use of serum-free growth conditions, and three-dimensional cell cultures and their specific assays for the efficacy assessment of new anticancer therapies. Written in the highly 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 laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and comprehensive, Cancer Cell Culture: Methods and Protocols is a valuable tool to help researchers involved in this important field to further improve or advance their models for cancer research. Preface Acknowledgments Contents Contributors Part I: Overview of the Existing Methods for Cancer Cell Culture Chapter 1: Cancer Cell Culture: The Basics and Two-Dimensional Cultures 1 Introduction 1.1 Cancer Cell Lines 1.1.1 Definitions 1.2 Primary Versus Immortalized Cell Lines 1.2.1 Primary Cell Culture 1.2.2 Immortalized Cell Lines 1.3 Techniques Used to Characterize/Authenticate Cancer Cell Lines 1.3.1 Cytogenetics 1.3.2 DNA Fingerprinting/Profiling 1.3.3 Flow Cytometry 1.4 Methods to Verify Species of Origin 1.4.1 Antibody Detection 1.4.2 Polymerase Chain Reaction (PCR) 1.4.3 Isoenzyme Analysis 1.5 General Characteristics of Cancer Cell Lines 1.5.1 Morphology 1.5.2 Colony Forming Efficiencies (CFE) and Cell Growth Rate 1.5.3 Cell Cycle Characteristics/Ploidy 1.5.4 Expression Profiles 1.6 Important Culturing Considerations 1.6.1 Species Type 1.6.2 Continuous or Finite Cell Lines 1.6.3 Functional Characteristics 1.6.4 Growth Characteristic and Conditions 1.6.5 Transformed or Normal Cell Type 1.6.6 Other Considerations 2 Two-Dimensional (2D) Cell Culture in Cancer Diagnosis 2.1 Liquid Biopsies/Blood Analysis 2.2 Imaging and Histology 2.3 Biopsies 2.4 Cytology/Smears 2.5 DNA/RNA Screening and Genetic Testing 3 Two-Dimensional (2D) Cell Culture in Cancer Prognosis 4 Two-Dimensional (2D) Cell Culture in Drug Screening 4.1 Clonogenic Assay 4.2 Cell Proliferation and Cytotoxicity Assays 4.2.1 Metabolic and Enzyme Activity: MTT, LDH, and Alamar Blue 4.2.2 ATP Quantification: CellTiter-Glo 4.2.3 Membrane Integrity: G6PD Release, Trypan Blue 4.3 Adhesion, Migration, Invasion, and Wound Healing Assays 4.3.1 Adhesion Assays 4.3.2 Migration and Invasion Assays 4.3.3 Wound Healing/Scratch Assays 4.4 Angiogenesis 5 Two-Dimensional (2D) Cell Culture in Cellular-Based Cancer Interventions 5.1 Adoptive Cell Transfer and T Cells 5.2 Induced Pluripotent Stem Cells (Leukemia) 5.3 NK Cells 5.4 Cell-Based Vaccines 5.4.1 Tumor Cell Vaccines 5.4.2 Dendritic Cell Vaccines 6 Overall Conclusion References Chapter 2: Cell Cultures at the Air-Liquid Interface and Their Application in Cancer Research 1 Introduction 2 ALI Cell Culture Models: State of the Art 2.1 Lung Barrier 2.2 Intestinal Barrier 2.3 Skin Barrier 3 Application of ALI Cultures 3.1 Toxicity Testing 3.2 Cancer Research 3.3 Translational Research 4 Conclusions and Outlook References Chapter 3: Three-Dimensional Spheroids for Cancer Research 1 Introduction 2 The Tumor Microenvironment (TME) and Its Implications in Cancer 3 Characteristics of Tumors and Spheroids and Types of 3D Spheroid Cultures 4 Techniques for Generating 3D Spheroids 4.1 Scaffold-Free Methods 4.1.1 Ultra-Low Attachment (ULA) Plates 4.1.2 Hanging Drop 4.1.3 Rotary Cultures 4.1.4 Magnetic Levitation 4.2 Scaffold Formation Methods 4.2.1 Matrices and Hydrogels 4.2.2 Microcarrier Beads 4.2.3 Spinner Flasks 4.2.4 Microfluidic Devices 5 Experimental Tools Used with Tumor Spheroids 5.1 Optical and Electron Microscopy 5.2 Molecular Biology Methods and Biological Assays 5.3 Flow Cytometry 6 Applications of 3D Cultures in Cancer Research 6.1 Chemoresistance Assessment 6.2 Spheroids in the Study of Nanomedicines 6.3 Invasion and Migration 6.4 Current Outlook: The Application of Cancer Spheroids to Pharma, Personalized Medicine, and the 3Rs Concept 7 Concluding Remarks References Chapter 4: Disease Animal Models for Cancer Research 1 Introduction 2 Ethical Frame of Animal Use in Cancer Research 3 Refinement at Its Best: Securing Animal Welfare of In Vivo Cancer Models 4 Choice of the Animal Model 5 The Role of Pathology Misinterpretation in Failure of the Model 6 Think Outside the Box: Wild Animal Models 6.1 North American Woodchuck (Marmota monax) 6.2 Naked Mole-Rat (Heterocephalus glaber) 6.3 Chicken (Gallus domesticus) 6.4 Tasmanian Devil (Sarcophilus harrisii) 7 Pets as Cancer Animal Models 8 Conclusions References Part II: Specific Methods for Cancer Cell Culture: Step-by-Step Methodologies Chapter 5: Generation of Radioresistant Prostate Cancer Cells 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Irradiation 2.3 Clonogenic Assays 2.4 Flow Cytometry 3 Method 3.1 Cell Culture and Irradiation 3.2 Clonogenic Assay 3.3 Apoptosis Assay by Flow Cytometry 3.4 Senescence Assay by Flow Cytometry 3.5 Cell Cycle Analysis by Flow Cytometry 4 Notes References Chapter 6: Generation and Characterization of an Isogenic Cell Line Model of Radioresistant Esophageal Adenocarcinoma 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Cell Counting and Viability Assessment 2.3 X-Ray Irradiation 2.4 Clonogenic Assay 2.4.1 Colony Staining 2.4.2 Colony Counting 2.5 PI Cell Cycle Analysis 2.6 Annexin-V-FITC/PI Apoptosis Assay 2.7 Detection of ROS 2.8 Analysis of DNA Damage by γH2AX Detection 3 Methods 3.1 Subculturing of Cells 3.2 Generation of an Isogenic Model of Radioresistant EAC 3.3 Assessing Radioresistance: Clonogenic Assay 3.4 Assessing Molecular Features of an Isogenic Model of Radioresistant EAC 3.4.1 PI Cell Cycle Analysis 3.4.2 Annexin-V-FITC/PI Apoptosis Assay 3.4.3 Detection of ROS 3.4.4 Analysis of DNA Damage and Repair by γH2AX Detection 4 Notes References Chapter 7: Developing an In Vitro Isogenic Model of Chemotherapy-Resistant Lung Cancer 1 Introduction 2 Materials 2.1 Cell Line Generation 2.2 Mycoplasma and Agarose Gel Electrophoresis 2.3 MTT Assay 2.4 Apoptosis Assay 2.5 Clonogenic Assay 3 Methods 3.1 Mycoplasma Screen 3.2 Drug Solution Preparation 3.3 Cisplatin Dose-Response Studies 3.4 MTT Cell Viability Assay 3.5 Generation of Cisplatin-Resistant Cell Sublines 3.6 Apoptosis (Annexin V-Propidium Iodide) Assay by Flow Cytometry 3.7 Clonogenic Assay 4 Notes References Chapter 8: Culturing Human Lung Adenocarcinoma Cells in a Serum-Free Environment 1 Introduction 2 Materials 2.1 Equipment 2.2 Culture and Cryopreservation Materials 3 Methods 3.1 A549 Cell Culture in DMEM 3.2 Method 1 - Reducing the Serum Content in the Cell Medium Step-by-Step 3.3 Method 2 - Sequential Adaption 3.4 Cell Cryopreservation in Serum-Free Conditions 4 Notes References Chapter 9: A Method for Culturing 3D Tumoroids of Lung Adenocarcinoma Cells at the Air-Liquid Interface 1 Introduction 2 Materials 2.1 Cell Culture Formation 2.1.1 Cell Components 2.1.2 Cell Substrate 2.1.3 Reagents 2.1.4 Equipment 2.2 Immunofluorescence Staining for Confocal Imaging Analysis 2.3 Quantitative Evaluation of Cell Viability by Flow Cytometry 3 Methods 3.1 Cell Culture Formation 3.1.1 Membrane Conditioning 3.1.2 Seeding of Human Lung Fibroblasts 3.1.3 Seeding of Lung Cancer Cells 3.1.4 Airlifting 3.1.5 Cell Culture Maintenance Overtime 3.2 Immunofluorescence Staining for Confocal Imaging 3.2.1 Fixation 3.2.2 Fluorescent Staining 3.2.3 Quantitative Evaluation of Cell Viability by Flow Cytometry 4 Notes References Chapter 10: Isolation and Cryopreservation of Mononuclear Cells from Peripheral Blood and Bone Marrow of Blood Cancer Patients 1 Introduction 2 Materials 2.1 Isolation of Patient Derived Mononuclear Cells 2.2 Freezing and Storage of Mononuclear Cells 2.3 Thawing of Mononuclear Cells 3 Method 3.1 Isolation of Mononuclear Cells 3.2 Cryopreservation of Mononuclear Cells 3.3 Thawing of Mononuclear Cells 4 Notes References Chapter 11: Serum-Free Production of Human Stem Cell-Derived Liver Spheres for Cancer Metastasis Research 1 Introduction 2 Materials 2.1 Culture Plates Preparation 2.2 Stage 1: Human Pluripotent Stem Cell (hPSC) Culture 2.3 Stage 2a: Hepatic Stellate Cell (HSC) Differentiation 2.4 Stage 2b: Hepatic Progenitor Cell (HPC) Differentiation 2.5 Stage 2c: Endothelial Cell (EC) Differentiation 2.6 Stage 3a: Liver Sphere Culture 2.7 Stage 3b: Formation and Characterization of Liver Cancer Co-culture Spheres 2.8 Quantification of CYP P450 Activity in Liver Spheres 2.9 Quantification of Human Serum Albumin or Alpha-Fetoprotein Secretion in Liver Spheres 3 Methods 3.1 Preparation of Cell Culture Substrates 3.1.1 Preparation of Laminin-521 (LN-521)-Coated 6-Well Plates/Petri-Dishes 3.1.2 Preparation of 2% Agarose Molds in 12-Well Plates 3.1.3 Preparation of Poly 2-Hydroxyethyl Methacrylate (Poly-HEMA)-Coated 48-Well Plates 3.2 Stage 1: Passaging Human Pluripotent Stem Cells (hPSC) for Differentiation 3.3 Stage 2a: Hepatic Stellate Cell (HSC) Differentiation and Preparation for Sphere Formation 3.4 Stage 2b: Hepatic Progenitor (HB) Cell Differentiation and Preparation for Sphere Formation 3.5 Stage 2c: Endothelial Cell (ELC) Differentiation and Preparation for Sphere Formation 3.6 Stage 3a: Aggregation and Maintenance of Liver Spheres 3.7 Stage 3b: Formation of Liver Cancer Co-culture Spheres 3.8 Quantification of CYP P450 Activity in Liver Spheres 3.9 Quantification of Human Serum Albumin or Alpha-Fetoprotein Secretion in Liver Spheres 4 Notes References Chapter 12: A Self-Assembly Method for Creating Vascularized Tumor Explants Using Biomaterials for 3D Culture 1 Introduction 2 Materials 2.1 Cellular Components 2.2 Mouse Tumor Explants 2.3 In Situ Immunofluorescence Staining and Confocal Imaging Analysis 2.3.1 Reagents 2.3.2 Equipment 3 Methods 3.1 Formation of Co-cultures of Endothelial Cells (ECs) and SMCs 3.2 Formation of Mu89, BT474, PANC-1 Vascularized Tumor Explant (VTE) Culture 3.3 Formation of Patient-Derived PDAC Vascularized Tumor Explant (VTE) Culture 3.4 Immunofluorescence Staining and Imaging of ECs-SMCs Co-cultures 3.5 Immunofluorescence Staining and Imaging of VTEs 3.6 Extracellular Matrix Quantification in EC-SMC Co-cultures 4 Notes References Chapter 13: A Step-by-Step Methodological Guide for Developing Zonal Multicellular Scaffold-Based Pancreatic Cancer Models 1 Introduction 2 Materials 3 Method 3.1 Development of Scaffolds for Cancer and Stromal Compartment of Hybrid Model 3.2 Cell Seeding on PU Scaffolds and Formation of Hybrid PDAC Model 3.3 Live-Dead Analysis of Hybrid Scaffolds 4 Notes References Chapter 14: Measuring Immune Cell Movement Toward the Soluble Microenvironment of Human Tissues Using a Boyden Chamber-Based M... 1 Introduction 2 Materials 2.1 Reagents 2.2 Beads and Antibodies 2.3 Plastics 2.4 Equipment 2.5 Media and Supplements 2.6 Buffers 3 Methods 3.1 Peripheral Blood Mononuclear Cell (PBMC) Isolation 3.2 Activation and Treatment of Lymphocytes for Boyden Chamber Assay 3.3 Visceral Adipose Tissue-Conditioned Media Generation 3.4 Liver Tissue-Conditioned Media Generation 3.5 Tumor-Conditioned Media Generation 3.6 Boyden Chamber Immune Cell Migration Assay 3.7 Flow Cytometric Staining 3.8 Flow Cytometric Acquisition and Analysis 4 Notes References Chapter 15: A Method for the In Vitro Cytotoxicity Assessment of Anti-cancer Compounds and Materials Using High Content Screen... 1 Introduction 2 Materials 2.1 Cell Culture Formation 2.1.1 Cell Components 2.1.2 Reagents 2.1.3 Equipment 2.2 Immunofluorescence Staining for High Content Screening Imaging Analysis 2.3 Quantitative Evaluation of Cell Viability by High Content Screening Imaging Analysis 3 Methods 3.1 Cell Culture Formation and Treatment with Compounds 3.1.1 Cell Plating 3.1.2 Treatment (or Exposure) of Cultures with Nanomaterials/Compounds/Controls 3.2 Immunofluorescence Staining of Cultures for Microscopy Analysis 3.2.1 Fluorescent Staining 3.2.2 Quantitative Evaluation of Cell Viability Using High Content Screening Analysis (See Note 3) 4 Notes References Chapter 16: Testing the Effects of Magnetic Hyperthermia in 2D Cell Culture 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Magnetic Hyperthermia 2.3 Cell Viability Assay 2.4 Data Analysis 3 Methods 3.1 In Vitro Magnetic Hyperthermia (MH) 3.2 Cell Viability Assessment 3.3 Analyzing Cell Viability Data with Flow Cytometry and Controlling for Interference 4 Notes References Chapter 17: Cell Viability Assay with 3D Prostate Tumor Spheroids 1 Introduction 2 Materials 2.1 PolyHEMA Microplate 2.2 Formation of Spheroids 2.3 Treatment of Spheroids and Determination of Their Viability 3 Methods 3.1 Preparation of PolyHEMA-Coated U-Bottom 96-Well Plates 3.2 Formation of Tumor Spheroids of PC-3 Prostate Cancer Cell Line 3.3 Drug Treatment 3.4 Application of WST-8/Medium Mixture 3.5 Measurement of the Absorbance 3.6 Analysis of Percentage (%) Cell Viability of Spheroids 4 Notes References Chapter 18: Analysis of Cancer Cell Line Secretomes: A Complementary Source of Disease-Specific Protein Biomarkers 1 Introduction 2 Materials 2.1 Solutions and Reagents 2.2 Equipment 3 Method 3.1 Conditioned Media Preparation 3.2 Protein Assay 3.3 Filter Aided Sample Preparation (FASP) of Conditioned Media 3.4 Peptide Clean-Up 3.5 Mass Spectrometry Analysis 3.6 Protein Identification, Quantification, and Statistical Analysis 4 Notes References Index