Collagen:
A polypeptide substance comprising about one third of the total protein in mammalian organisms. It is the main constituent of Skin; Connective tissue; and the organic substance of bones (Bone and bones) and teeth (Tooth).
JoVE Bioengineering
In vitro Synthesis of Native, Fibrous Long Spacing and Segmental Long Spacing Collagen
1Department of Chemistry, University of Toronto, 2Institute for Optical Sciences, University of Toronto
Simple and reproducible procedures are described for making three structurally distinct collagen assemblies from a common commercially available Type I collagen monomer. Native type, fibrous long spacing or segmental long spacing collagen can be constructed by varying the conditions to which the 300 nm long and 1.4 nm diameter monomer building block is exposed.
JoVE Clinical and Translational Medicine
MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression
1Department of Pharmacology, Wayne State University, 2Barbara Ann Karmanos Cancer Institute, Wayne State University
We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.
JoVE Bioengineering
Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context
1The Beatson Institute for Cancer Research, University of Glasgow, 2Section of Dermatology, School of Medicine, University of Glasgow
A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.
JoVE Bioengineering
Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells
1Institute of Biomaterials and Biomedical Engineering / Department of Chemical Engineering and Applied Chemistry, University of Toronto, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto
Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.
JoVE Neuroscience
Analyzing Murine Schwann Cell Development Along Growing Axons
1Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 2Department of Neuroanatomy, University of Heidelberg, 3FRIAS, University of Freiburg
Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.
JoVE General
Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons
1Department of Biological Sciences, University of Southern California, 2Neuroscience Graduate Program, University of Southern California
This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.
JoVE General
Isolation and Culture of Avian Embryonic Valvular Progenitor Cells
Department of Biomedical Engineering, Cornell University
This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.
JoVE Immunology and Infection
Generation of Organotypic Raft Cultures from Primary Human Keratinocytes
1Department of Microbiology & Immunology, University of North Carolina-Chapel Hill, 2Lineberger Cancer Center, University of North Carolina-Chapel Hill
An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.
JoVE Neuroscience
Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays
Explants from the midbrain dopamine system and striatum are used in a collagen matrix assay for the in vitro analysis of mesostriatal and striatonigral pathway development. In this assay axonal outgrowth and guidance can be manipulated and quantified. It can also be modified for assessing other regions or molecular cues.
JoVE Clinical and Translational Medicine
Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice
Department of Pathology, University of Kansas Medical Center
In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.
JoVE Clinical and Translational Medicine
Spheroid Assay to Measure TGF-β-induced Invasion
An assay to quantitatively measure Transforming Growth Factor (TGF)-β-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.
JoVE Bioengineering
Engineering a Bilayered Hydrogel to Control ASC Differentiation
1Department of Extremity Trauma Research and Regenerative Medicine, United States Army Institute of Surgical Research, 2Department of Biomedical Engineering, The University of Texas at Austin
This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.
JoVE General
Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography
1Breakthrough Breast Cancer Research Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, 2MRC Technology
We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.
JoVE Bioengineering
Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers
Department of Chemical Engineering, Stanford University
In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.
JoVE Immunology and Infection
Granulocyte-dependent Autoantibody-induced Skin Blistering
1Department of Dermatology, University of Freiburg, 2Kepler High School Freiburg, 3Centre for Biological Signalling Studies (BIOSS), University of Freiburg
In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human epidermolysis bullosa acquisita (EBA)1.
JoVE General
Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells
1Department of Medical Oncology, Dana-Farber Cancer Institute, 2Harvard Medical School, Boston, MA, 3Sheba Cancer Research Center, Chaim Sheba Medical Center, 4Department of Pathology, Brigham and Women's Hospital
The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.
JoVE Neuroscience
High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium
1Neural Development Group, Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee, UK, 2Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, UK
Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.
JoVE General
Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood
Herman B Wells Center for Pediatric Research, Indiana University School of Medicine
Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.
JoVE General
Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells
The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.
JoVE General
Isolating Stem Cells from Soft Musculoskeletal Tissues
1Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC, 2Department of Bioengineering, University of Pittsburgh, 3Department of Orthopedic Surgery, University of Pittsburgh, 4Department of Pathology, University of Pittsburgh, 5Department of Molecular Genetics & Biochemistry, University of Pittsburgh
Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.
JoVE General
Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors
Institute for Biological Interfaces, Forschungszentrum Karlsruhe
We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.
JoVE Bioengineering
Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres
Department of Regenerative Medicine, United States Army Institute of Surgical Research
A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.
JoVE Bioengineering
The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal Skin and Melanoma Progression
Molecular and Cellular Oncogenesis Program, The Wistar Institute
In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.
JoVE General
Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures
1Institute of Human Genetics and Northeast England Stem Cell Institute, Newcastle University, 2Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin
Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.
JoVE Neuroscience
Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays
Department of Biological Sciences, Purdue University
We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.
JoVE Bioengineering
Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion
School of Biomedical Engineering, Science and Health Systems, Drexel University
Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.
JoVE Bioengineering
Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation
Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.
JoVE Bioengineering
Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study
1Department of Biomedical Engineering, Virginia Commonwealth University, 2Department of Anatomy and Neurobiology, Virginia Commonwealth University, 3Department of Cardiovascular Surgery, University Hospital of Geneva
The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.
JoVE Bioengineering
Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening
1Department of Biomedical Engineering, University of Texas at San Antonio, 2Department of Biology, University of Texas at San Antonio
We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.
JoVE Bioengineering
Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix
University of California, Davis
Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.
JoVE Neuroscience
Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model
Klinik und Poliklinik für Anästhesiologie, University of Wurzburg
This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.
JoVE General
Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI
We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.
JoVE General
Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures
1Department of Pediatric Oncology, Dana Farber Cancer Institute, 2Department of Neurobiology, Harvard Medical School
Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.
JoVE General
Bioengineering Human Microvascular Networks in Immunodeficient Mice
Department of Cardiac Surgery, Children's Hospital Boston, Harvard Medical School
Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.
JoVE Bioengineering
Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering
1Department of Orthopaedics, The Warren Alpert Brown Medical School of Brown University and the Rhode Island Hospital, 2Center for Restorative and Regenerative Medicine, VA Medical Center, Providence, RI, 3University of Texas Southwestern Medical Center
We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.
JoVE Immunology and Infection
Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation
Department of Surgery, University of Wisconsin-Madison, School of Medicine and Public Health
We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.
JoVE Clinical and Translational Medicine
Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model
1Department of Orthopaedic Sports Medicine, Klinikum rechts der Isar der Technischen Universität München, 2Department of Radiology, Klinikum rechts der Isar der Technischen Universität München, 3Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar der Technischen Universität München, 4Department of Radiology, Uniklinik Köln
An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.
JoVE Immunology and Infection
A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation
1Department of Microbiology, New York University School of Medicine, 2Molecular Neurobiology Program, Skirball Institute for Biomolecular Medicine, New York University School of Medicine, 3Department of Otolaryngology, New York University School of Medicine, 4Department of Cell Biology, New York University School of Medicine, 5Department of Physiology and Neuroscience, New York University School of Medicine, 6Department of Psychiatry, New York University School of Medicine, 7Center for Neural Science, New York University School of Medicine
The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.
JoVE General
Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects
1Orthopaedics Research, University of Virginia, 2Biological Sciences, University of Delaware, 3Orthopaedic Surgery, University of Virginia
A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.
JoVE Clinical and Translational Medicine
The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice
Pharmazentrum Frankfurt / ZAFES, Goethe University Hospital Frankfurt
Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.
JoVE Neuroscience
Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells
1Department of Neurosurgery, University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences, 3STEMCELL Technologies, Inc.
This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.
JoVE General
Fabrication of Myogenic Engineered Tissue Constructs
1Department of Anesthesiology, Children's Hospital Boston and Harvard Medical School, 2Perioperative and Pain Medicine, Children's Hospital Boston and Harvard Medical School
Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].
JoVE General
Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy
The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.
JoVE General
Mechanical Stimulation of Chondrocyte-agarose Hydrogels
1Department of Mechanical and Materials Engineering, Queen's University, 2Department of Chemical Engineering, Queen's University
The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.
JoVE General
Evisceration of Mouse Vitreous and Retina for Proteomic Analyses
1Omics Laboratory, University of Iowa, 2Ophthalmology and Visual Sciences, University of Iowa, 3Harkness Eye Institute, Columbia University College of Physicians and Surgeons
The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.
JoVE Bioengineering
Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering
Biomedical Engineering Department, Worcester Polytechnic Institute
This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.
JoVE General
Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy
1Department of Biochemistry and Molecular Biology, LSU School of Medicine, 2Department of Oral Biology, LSU School of Dentistry, 3Stanley S. Scott Cancer Center, LSU School of Medicine
This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.
JoVE Immunology and Infection
Non-invasive Imaging of Leukocyte Homing and Migration in vivo
1Department of Pathology and Immunology, Washington University in St. Louis, 2National Institute of Neurological Disorders and Stroke, NINDS, NIH - National Institute of Health
Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.
JoVE Bioengineering
Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography
Biomedical Engineering, Tulane University
Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.
JoVE Clinical and Translational Medicine
Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface
1Department of Medicine, Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, 2Cellular and Molecular Pathology, School of Medicine, University of Pittsburgh
Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.
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