Stem Cell Nanotechnology: Methods and Protocols (Methods in Molecular Biology, 2125)

Preface
Contents
Contributors
Design of a Versatile Sample Holder for Facile Culture of Cells on Electrospun Membranes or Thin Polymer Films Under Flow Cond
	1 Introduction
	2 Materials
		2.1 Insert Design and Preparation
		2.2 Cleaning and Sterilization of the Inserts
		2.3 Cell Culture Consumables
		2.4 Immunofluorescence Staining and Confocal Microscopy
		2.5 Scanning Electron Microscopy
	3 Methods
		3.1 Preparation of Inserts
		3.2 Cleaning and Sterilizing the Inserts
		3.3 Preparing Inserts with Electrospun Membranes
		3.4 Cell Culture on Inserts
		3.5 Cell Fixation for Immunofluorescence Staining and SEM (See Note 13)
		3.6 Immunofluorescence Staining
		3.7 Scanning Electron Microscopy
	4 Notes
	References
Single Cell Densitometry and Weightlessness Culture of Mesenchymal Stem Cells Using Magnetic Levitation
	1 Introduction
	2 Materials
		2.1 Microfluidic Setup
		2.2 Magnetic Levitation
	3 Methods
		3.1 Microfluidic Setup
		3.2 Sample Preparation
		3.3 Magnetic Levitation
		3.4 Imaging and Analysis
		3.5 Calibration of Setup
	4 Notes
	References
Fluorescent Nanoclusters for Imaging of Cells/Stem Cells
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Hemoglobin
		3.2 Clarify the Purity of Prepared Hemoglobin
		3.3 Synthesis of Hemoglobin-Capped Pt NCs
		3.4 Synthesis of HA-Conjugated Pt NCs
		3.5 Cell Culture
			3.5.1 Adherent Cells
				Replacement of Culture Medium
				Subculturing (Standard Passaging)
			3.5.2 Suspension Cells
		3.6 CD44 Flow Cytometry Assay
		3.7 Cytotoxicity
		3.8 In Vitro Cellular Imaging
	4 Notes
	References
Protocol for Determining the Induction of Human Embryonic Stem Cells into Myogenic Lineage Using Electrospun Nanofibers
	1 Introduction
	2 Materials
		2.1 Fabrication of Cell Culture Substrate
		2.2 Cells
		2.3 Culture Medium
		2.4 Stem Cell Seeding on Scaffolds
		2.5 Reverse Transcription Polymerase Chain Reaction
		2.6 Morphology Determination
		2.7 Immunochemistry and Flow Cytometry
	3 Methods
		3.1 Isolation of hESC from Embryos
		3.2 Myogenic Differentiation of Stem Cells
		3.3 Characterization of the Differentiated Cells
			3.3.1 Quantitative Reverse Transcription PCR
			3.3.2 Morphological Analysis of the Differentiated Stem Cells by SEM
			3.3.3 Immunocytochemistry
			3.3.4 Flow Cytometry Analysis
	4 Notes
	References
Preparation and Characterization of Simvastatin Nanocapsules: Encapsulation of Hydrophobic Drugs in Calcium Alginate
	1 Introduction
	2 Materials
		2.1 Synthesis of Sodium Alginate Modified with GICP Moieties (GICP-Alginate)
		2.2 Encapsulation of Simvastatin in GICP-Alginate (SIMNCs)
		2.3 Size Distribution Determination
		2.4 Transmission Electron Microscopy
		2.5 Encapsulation Efficiency
	3 Methods
		3.1 Synthesis of GICP-Alginate (Fig. 1a)
		3.2 Synthesis of SIMNCs (Fig. 1b)
		3.3 Characterization of SIMNCs
			3.3.1 Size Distribution Determination
			3.3.2 Nanocapsule´s Morphology by Transmission Electron Microscopy (TEM)
			3.3.3 The Drug Encapsulation Efficiency
		3.4 Results
			3.4.1 The Morphology of SIMNCs
			3.4.2 The Size Distribution of SIMNCs
			3.4.3 The Drug Encapsulation Efficiency
	4 Notes
	References
Multifunctional Magnetic Nanoparticles-Labeled Mesenchymal Stem Cells for Hyperthermia and Bioimaging Applications
	1 Introduction
	2 Materials
		2.1 Preparation of Fe3O4 Nanoparticles
		2.2 Preparation of CdTe Quantum Dots (QDs)
		2.3 Silica Coating and Functionalization of FMCNPs
		2.4 Primary Culture and Identification of Mouse Mesenchymal Stem Cells (MSCs)
			2.4.1 Mice
			2.4.2 Isolation, Culture, and Expansion of MSCs
			2.4.3 Fluorescence-Activated Cell Sorting Analysis
		2.5 Primary Culture and Identification of Mouse MSCs
		2.6 MSCs Differentiation Assays
			2.6.1 Osteoblast Differentiation Culture Medium
			2.6.2 Adipocyte Differentiation Medium
			2.6.3 Chondrocyte Differentiation Culture Medium
			2.6.4 Staining
		2.7 Preparation of Amino-Modified Fluorescent Magnetic Nanoparticles (FMNP)-Labeled MSCs
		2.8 In Vitro Cell Viability Assay
		2.9 Fluorescence Imaging and MRI of Gastric Cancer Cells In Vivo
		2.10 Immunofluorescence Assay, Prussian Blue Staining, and ICP-MS Analysis of Major Organs
		2.11 Hyperthermia Therapy for Nude Mice with Gastric Cancer Cells
		2.12 Analysis of Chemokine Receptors in MSC Cells and Chemokine in MFC Cells
		2.13 Instrumentation
	3 Methods
		3.1 Preparation of Fe3O4 Nanoparticles
		3.2 Preparation of CdTe QDs
		3.3 Silica Coating and Functionalization of FMCNPs (See Note 3)
		3.4 Primary Culture and Identification of Mouse Mesenchymal Stem Cells (MSCs)
			3.4.1 Isolation, Culture, and Expansion of MSCs
				Bone Marrow (BM)
				Compact Bone
				Adipose Tissue (AT)
			3.4.2 Primary Culture and Identification of Mouse MSCs
		3.5 MSCs Differentiation Assays
			3.5.1 Osteoblast Differentiation Culture Medium
			3.5.2 Adipocyte Differentiation Medium
			3.5.3 Chondrocyte Differentiation Culture Medium
			3.5.4 Staining
		3.6 Preparation of Amino-Modified FMNP-Labeled MSCs
		3.7 In Vitro Cell Viability Assay
		3.8 Fluorescence Imaging and MRI of Gastric Cancer Cells In Vivo
		3.9 Immunofluorescence Assay, Prussian Blue Staining, and ICP-MS Analysis of Major Organs
		3.10 Hyperthermia Therapy for Nude Mice with Gastric Cancer Cells
		3.11 Analysis of Chemokine Receptors in MSC Cells and Chemokine in MFC Cells
	4 Notes
	References
Synthesis of Hybrid Gold Nanoparticle (AuNP) Functionalized Superparamagnetic Nanoparticles (SPMNPs) for Efficient Coupling of
	1 Introduction
	2 Materials
	3 Methods
		3.1 Synthesis of AuNP and Preparation of AuNP-SPMNPs
	4 Notes
	References
Experimental Protocol for Induction of Transgene Expression in Neural Stem Cells Through Polymeric Nanoparticles
	1 Introduction
	2 Materials
		2.1 Polymeric Nanoparticles
		2.2 Cells
		2.3 Cell Culture Media
		2.4 Phosphate Buffer Saline (PBS)
		2.5 Cytotoxicity Assay for the Nanoparticles
		2.6 In Vitro Transfection
	3 Methods
		3.1 pDNA-Loaded Chitosan-Tripolyphosphate/Hyaluronic Acid Nanoparticles
		3.2 The Encapsulation Efficiency of pDNA by Nanoparticles
		3.3 In Vitro pDNA Release from Nanoparticles
		3.4 Characterization
		3.5 Culturing Neural Stem Cells
			3.5.1 Preparing Well Plates
			3.5.2 Obtaining Cells
		3.6 Cryopreservation of the Neural Stem Cells
		3.7 MTT Assay for Cytotoxicity Evaluation
		3.8 Assay for Cellular Uptake of Nanoparticles
		3.9 In Vitro Transfection
		3.10 Determination of Transfection Efficiency
	4 Notes
	References
Methodology Involved in the Osteogenic Differentiation of Mesenchymal Stem Cells on Chitosan-Collagen Nanofibers Incorpo
	1 Introduction
	2 Materials
		2.1 Synthesis of Titanium Dioxide Nanoparticles and the Fabrication of Nanofibers
		2.2 Isolation of MSCs Derived from Bone Marrow
		2.3 Culture or Expansion of MSCs
		2.4 Cell Seeding on Hybrid Nanofibrous Scaffolds
		2.5 MTT Assay
		2.6 Differentiation of Isolated MSCs into Osteoblasts
		2.7 Immunofluorescence Staining
		2.8 Quantitative Real-Time PCR Assay
		2.9 Alkaline Phosphate Activity
	3 Methods
		3.1 Synthesis of Titanium Dioxide Nanoparticles
		3.2 Fabrication of Chitosan/Titanium Dioxide/Collagen Nanofibers
		3.3 Isolation and Culture of MSCs
		3.4 Culture or Expansion of MSCs
		3.5 Cell Seeding on Hybrid Chitosan/Titanium Dioxide/Collagen Scaffolds
		3.6 MTT Assay
		3.7 Differentiation of MSCs into Osteogenic Lineage
		3.8 Immunocytochemistry
		3.9 Quantitative Real-Time PCR Assay
		3.10 ALP Activity/Quantification Assay
	4 Notes
	References
Experimental Protocol for Culture and Differentiation of Osteoblasts on 3D Abode Using Nanofiber Scaffolds
	1 Introduction
	2 Materials
		2.1 Tissue Culture Media, Supplements, and Other Reagents
		2.2 Secondary Culture and Cell Culture on Nanofiber Scaffolds
		2.3 Cell Viability and Proliferation
		2.4 Phenotypic Characterization
	3 Methods
		3.1 Culture Preparation
		3.2 Thawing and Culturing
		3.3 Subculturing the Osteoblasts
		3.4 Preparation of Nanofibrous Scaffolds for Cell Culture
		3.5 Culture of Osteoblasts on Nanofiber Scaffolds
		3.6 Determination of Cell Viability/Proliferation
		3.7 In Vitro Differentiation and Mineralization of the Osteoblasts
		3.8 Phenotypic Characterization of Osteoblasts
	4 Notes
	References
Experimental Protocol of MSC Differentiation into Neural Lineage for Nerve Tissue Regeneration Using Polymeric Scaffolds
	1 Introduction
	2 Materials
		2.1 Isolation of MSCs and Culture
		2.2 Cell Seeding on Scaffolds
		2.3 Cell Viability Assay
		2.4 Differentiation of MSCs into Neural Lineage
		2.5 Immunocytochemistry
		2.6 Quantitative Real-Time PCR Assay
	3 Methods
		3.1 Isolation of MSCs and Culture
		3.2 Cell Seeding on Polymeric Scaffolds
		3.3 Viability/MTT Assay
		3.4 Differentiation of MSCs into Neural Lineage
		3.5 Immunocytochemistry
		3.6 Quantitative Real-Time PCR Assay
	4 Notes
	References
Fabrication and Electrospinning of 3D Biodegradable Poly-l-Lactic Acid (PLLA) Nanofibers
	1 Introduction
	2 Materials
		2.1 Electrospinning
		2.2 Physical/Mechanical Validation
		2.3 Biological Validation
	3 Methods
		3.1 Fabrication of PLLA Nanofibers (Safety Precautions; See Notes 7-11)
		3.2 Drug Loading on Nanofibers
		3.3 Validation Parameters
			3.3.1 Thickness
			3.3.2 Contact Angle
			3.3.3 Scanning Electron Microscopy (SEM)
			3.3.4 Mechanical Testing
			3.3.5 Atomic Force Microscopy (AFM)
			3.3.6 Drug Release Kinetics of Nanofibers
				Formula for Determination of Percentage of Release of Drug from In Vitro Dissolution Testing
			3.3.7 In Vitro Cytotoxicity Experiments
				Extract of Nanofibers
				Direct-Contact Cytotoxicity
				In Vitro Cell Culture Protocol
				MTT (3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium Bromide) Assay
				Data Analysis for Cell Proliferation Assays
				Data Analysis for Cell Cytotoxicity Assays
			3.3.8 In Vivo Biocompatibility Animal Experiments
	4 Notes
	References
Differentiation of Menstrual Blood Stem Cells into Keratinocyte-Like Cells on Bilayer Nanofibrous Scaffold
	1 Introduction
	2 Materials
		2.1 Study Population
		2.2 Collection of Menstrual Blood
			2.2.1 Materials
			2.2.2 Solutions
		2.3 Isolation and Culture of Menstrual Blood Stem Cells
			2.3.1 Materials
			2.3.2 Solutions
		2.4 Amniotic Membrane Procurement, Processing, and Decellularization
			2.4.1 Materials
			2.4.2 Solutions
		2.5 Histological Evaluation of Amniotic Membrane
		2.6 Fibroin Extraction
			2.6.1 Materials
			2.6.2 Solutions
		2.7 Characterization of Extracted Silk Fibroin
		2.8 Fabrication of Bilayer Scaffold
		2.9 Investigation of Nanofiber Morphology
		2.10 Isolation of Keratinocytes from Human Foreskin
			2.10.1 Materials
			2.10.2 Solutions
		2.11 MenSCs Differentiation into Keratinocytes
		2.12 Evaluation of Cell Differentiation
			2.12.1 Materials
			2.12.2 Solutions
	3 Methods
		3.1 Collection of Menstrual Blood
		3.2 Isolation and Culture of Menstrual Blood Stem Cells
		3.3 Amniotic Membrane Procurement, Processing, and Decellularization
		3.4 Histological Evaluation of Amniotic Membrane
		3.5 Fibroin Extraction
		3.6 Characterization of Extracted Silk Fibroin
		3.7 Fabrication of Bilayer Scaffold
		3.8 Investigation of Nanofiber Morphology
		3.9 Isolation of Keratinocytes from Human Foreskin
		3.10 MenSCs Seeding on Bilayer Scaffold
		3.11 MenSCs Differentiation into Keratinocytes
		3.12 Evaluation of Cell Differentiation
	4 Notes
	References
Dental Pulp Stem Cells in Customized 3D Nanofibrous Scaffolds for Regeneration of Peripheral Nervous System
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of the Spinning Solution
		3.2 Fabrication of Electrospinning Scaffold Around the Titanium Implant/3D Hollow/Solid Tubular Scaffold
		3.3 Isolation of DPSCs
		3.4 Purity and Undifferentiated Status of DPSCs
		3.5 In Vitro Neural Differentiation
	4 Notes
	References
Step-by-Step Protocol for Superparamagnetic Nanoparticle-Based Endosome and Lysosome Isolation from Eukaryotic Cell
	1 Introduction
	2 Materials
	3 Methods
		3.1 Surface Functionalization of SPMNP 1.0 via Ligand Exchange (SPMNP 2.0)
		3.2 Surface Functionalization of SPMNP 2.0 via Maleimide-SH Linker
		3.3 Endosome and Lysosome Isolation Using SPMNP 2.0 (Fig. 4)
	4 Notes
	References
Step-by-Step Protocol for Superparamagnetic Nanoparticle-Based Plasma Membrane Isolation from Eukaryotic Cell
	1 Introduction
	2 Materials
	3 Methods
		3.1 Synthesis of SPMNP 1.0
		3.2 Characterization of SPMNPs
			3.2.1 Transmission Electron Microscopy (TEM)
			3.2.2 Dynamic Light Scattering (DLS)
			3.2.3 Fourier-Transform Infrared (FTIR) Spectra Measurement
		3.3 Surface Functionalization of SPMNP 1.0 via Ligand Addition (SPMNP 2.0)
		3.4 SPMNP-Based Plasma Membrane Isolation (Fig. 5)
	4 Notes
	References
Nanoscaled Materials for Drug Delivery into Cells/Stem Cells
	1 Introduction
	2 Materials
		2.1 Synthesis of APTES-COF-1
		2.2 Synthesis of CCM-COOH
		2.3 Synthesis of PEG-CCM System
		2.4 Self-Assembly of APTES-COF-1 and PEG-CCM
		2.5 Self-Assembly of APTES-COF-1@DOX and PEG-CCM
		2.6 In Vitro Cellular Uptake
		2.7 Flow Cytometry Analysis
		2.8 In Vitro Cell Viability
		2.9 Immunohistochemistry
		2.10 Animals and Tumor Models
		2.11 Instrumentation
	3 Methods (Fig. 11)
		3.1 Synthesis of APTES-COF-1
		3.2 Synthesis of CCM-COOH
		3.3 Synthesis of PEG-CCM System
		3.4 Self-Assembly of APTES-COF-1 and PEG-CCM
		3.5 Encapsulation of Hydrophilic Molecules DOX in APTES-COF-1
		3.6 Self-Assembly of APTES-COF-1@DOX and PEG-CCM
		3.7 In Vitro Cellular Uptake (See Note 9)
		3.8 Flow Cytometry Analysis (See Note 10)
		3.9 In Vitro Cell Viability (see Note 11)
		3.10 Immunohistochemistry
		3.11 Animals and Tumor Models
	4 Notes
	References
Method for Evaluating Neuromodulatory Properties of Dental Pulp Stem Cell as an In Vitro Model for Parkinson´s Disease
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Detection and Monitoring of Stem Cell Differentiation Using Nanotechnology
	1 Introduction
	2 Materials
		2.1 Fabrication of 3D GO-Encapsulated GNPs on ITO Surface and Cell Chip Synthesis of Graphene-Encapsulated GNPs
		2.2 Fabrication of GO-GNP Nanoparticle-Modified Microgap
		2.3 Electrical and Electrochemical Measurement of Single mNSC
		2.4 Immunostaining of mNSCs
		2.5 Cell Culture and Induction of Stem Cell Differentiation
		2.6 Instrumentation
	3 Methods
		3.1 Fabrication of 3D GO-Encapsulated GNPs on ITO Surface and Cell Chip
		3.2 Fabrication of GO-GNP Nanoparticles-Modified Microgap
	4 Notes
	References
BacMam System for Rapid Recombinant Protein Expression in Mammalian Cells
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Index